RU2817602C2 - Antibody molecules that bind cd137 and ox40 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are antibody molecules which bind to CD137 and OX40 and are capable of having an agonist effect on both CD137 and OX40. Said antibody molecules contain a CDR-based binding site for CD137 and an antigen-binding site for OX40, which is located in the constant domain of the antibody molecule. Also disclosed is a pharmaceutical composition containing said antibodies. Invention also relates to nucleic acids coding antibodies, expression vectors and host cells containing said nucleic acids, a method of producing the antibody.

EFFECT: invention provides highly effective activation of immune cells with reduced risk of toxicity and can be used in treating cancer.

25 cl, 15 dwg, 39 tbl, 28 ex

Description

Field of technology

The present invention relates to antibody molecules that bind to and are capable of exerting agonistic effects on both CD137 and OX40. These antibody molecules contain a complementarity determining region (CDR) binding site for CD137 and an antigen binding site for OX40, which is located in the constant domain of the antibody molecule. The antibody molecules of the present invention can be used, for example, in the treatment of diseases such as cancer and infectious diseases.

State of the art

The mammalian immune system is a delicately balanced system whose functions are sometimes disrupted by diseases such as various types of cancer. Checkpoint receptors play a critical role in the immune system's response to disease, exerting either costimulatory or coinhibitory effects, the balance of which determines the direction of the immune response (Pardoll, 2012). Co-inhibitors inhibit T cell proliferation and induce the release of anti-inflammatory cytokines. They suppress inflammation and prevent organ/tissue damage due to an excessive immune response. Costimulators, on the other hand, stimulate T cell clonal expansion, differentiation and survival of effector cells to facilitate the development of a protective immune response.

One proven approach to cancer immunotherapy triggers the recognition and destruction of tumor cells by the immune system by targeting these checkpoint receptors with antibodies that either block the function of coinhibitory receptors or induce the activity of costimulatory receptors (Pardoll, 2012). Antibodies that block the activity of coinhibitory receptors have shown good clinical activity and are currently approved for the treatment of cancer (Larkin et al. , 2015). Antibodies that induce co-stimulatory receptor activity have shown significant promise in preclinical model systems (Moran et al. , 2013; Schaer et al. , 2014), and several agents are currently in clinical trials (Mayes et al. , 2018; Melero et al., 2018). al. , 2013). These antibodies are also called agonistic antibodies because they are designed to mimic the ligands of such co-stimulatory receptors.

Several T cell co-stimulatory receptors are members of the TNF receptor superfamily, a large family of proteins involved in the functions of both immune and non-immune cells that are expressed on the cell surface (Bremer, 2013). Structural analysis of complexes formed between TNF family receptors and their cognate ligands suggests that in most cases there is a trimer-to-trimer stoichiometry, and TNFR family ligands are typically expressed on the cell surface as trimers (Wajant, 2015). A proposed model for TNFR activation is that interaction with a trimeric ligand induces trimerization of monomeric receptors and initiates signal transduction. This suggests a priori that members of the TNFR family are expressed as monomers and only interaction with a ligand induces the formation of receptor trimers. This model has recently been criticized (Vanamee & Faustman, 2018) and the association of these monomers into higher order structures in the absence of ligand interaction is still a matter of debate. The existence of preassembled receptor dimers or even inactive trimers that require additional clustering of multiple receptor complexes could explain the lower activity of some soluble, trimeric-only TNF ligands compared to their membrane-bound forms, which can form ligand superclusters and induce superclusters TNF receptors, which induces higher levels of receptor activation (Müller et al. , 2008). This theory is also consistent with the observation that TNFR-specific antibodies typically have no or only low agonist activity and require secondary cross-linking of antibody-TNF receptor complexes to induce sufficient receptor clustering and activation, thereby mimicking TNF ligand superclusters (Wajant, 2015).

Secondary cross-linking of antibody-TNF receptor complexes can be achievedin vitro using cross-linking agents such as protein A or G or secondary antibodies targeting the constant domains of TNF receptor-specific agonistic antibodies (Vanamee & Faustman, 2018; Wajant, 2015). Howeverin vivo for such secondary cross-linking requires interaction with Fc-gamma receptors present on the surface of immune cells such as macrophages, NK cells or B cells. The interaction of antibodies with Fc-gamma receptors is complex because humans have 6 Fc-gamma receptors with different expression profiles and affinities for the 4 human IgG isotypes (Bruhnset al., 2009). Fc-gamma receptors have been shown to be required for optimal antitumor activity of agonistic antibodies targeting targets of the TNF receptor superfamilyin vivo (Bulliardet al., 2013; Bulliardet al., 2014). However, the dependence of agonistic anti-TNFR antibodies on Fc-gamma receptor-mediated cross-linking to induce potent receptor activation is likely to limit their overall activityin vivo for several reasons: 1) antibody-bound cells will need to interact with Fc gamma receptor-expressing cells under trans conditions, and the frequency of this interaction will limit the activation of TNFR-expressing cells; 2) the affinity of Fc-gamma receptors for human IgG is generally significantly lower compared to the affinity of a typical therapeutic antibody for its target (micromolar range versus nanomolar range, respectively); and 3) Fc-gamma receptors mediate antibody effector functions such as ADCC (antibody-dependent cellular cytotoxicity) and ADCP (antibody-dependent cellular phagocytosis), and therefore have the potential to eliminate the very cells that agonistic antibodies are designed to activate (Mayeset al., 2018).

Bivalent bispecific antibodies, which use one of the cognate antigens to cross-link a TNF receptor agonist, provide an alternative option for Fc-gamma receptor-mediated cross-linking. The cross-linking effect of an antibody will result from binding to a member of the TNF receptor family and another receptor expressed on the cell surface, either on the same cell, in cis conditions, or on a different cell, in trans conditions. This antibody cross-linking mechanism will then lead to superclustering of the TNF receptor, provided that the second target is expressed at high levels, mimicking TNF ligand superclusters. The bispecific antibody approach to developing TNFR agonist antibodies has several theoretical advantages over monospecific agonist antibodies: 1) TNFR agonist action can be directed to specific immune cells in the tumor microenvironment and in the periphery by targeting a second antigen such as a receptor checkpoint or tumor-associated antigen, as the second type of specificity of the bispecific antibody; 2) the affinity of the cross-linking binding domains of a bispecific antibody can be designed to exceed the affinity of the antibody for Fc-gamma receptors, making cross-linking more efficient; 3) the effector functions of antibodies can be selectively disabled using mutations, which ensures that cells intended for activation are not depleted; 4) agonist action on two separate TNF receptors can be achieved in one dual agonist molecule by combining the activation of different immune cells to obtain a stronger stimulation of the immune response; 5) targeting co-expressed receptors can lead to activation of a single cell in cis conditions without the need for interaction between the two cells.

Several members of the TNF receptor family have overlapping expression profiles in immune cells. In particular, OX40, CD137, GITR and CD27 are expressed on activated T cells, and the co-expression of OX40 and CD137 has been confirmed experimentally (Ma et al. , 2005).

OX40 is predominantly expressed on activated T cells, including CD4 ⁺ T cells, CD8 ⁺ T cells, T helper cells type 1 and 2 (Th1 and Th2), and regulatory T cells (Treg), and is also expressed on activated natural cells killer cells (NK cells). The interaction of OX40 with its ligand, OX40 ligand (OX40L), expressed on antigen presenting cells (APCs), enhances T cell lineage, differentiation and survival and enhances the generation of memory T cells (Croft et al. , 2009). OX40 stimulation may have a direct effect on T cells by promoting their proliferation and survival, or an indirect effect through increased production of inflammatory cytokines such as IL2 and IFNγ. OX40 signaling can also modulate Treg function, although in these cells it abolishes their suppressor activity (Takeda et al. , 2004). In cancer, OX40 has been found to be expressed on tumor-infiltrating T cells from patients suffering from head and neck cancer, melanoma and various types of colorectal cancer, with high levels of OX40-positive lymphocytes correlating with better survival (Petty et al. , 2002; Vetto et al. , 1997). Preclinical studies of agonistic anti-OX40 antibodies in mice have demonstrated therapeutic efficacy in several syngeneic tumor models, but the effectiveness of targeting OX40 as monotherapy has been variable and appears to correlate with tumor immunogenicity (Kjærgaard et al. , 2000). This is consistent with the notion that OX40 expression on tumor-specific T cells would require sufficient priming that would likely not be provided by poorly immunogenic tumors. In certain syngeneic models, the antitumor activity of the anti-OX40 antibody, OX86, was found to be due to its ability to deplete intratumoral Tregs that express high levels of OX40 in an Fc-gamma receptor-dependent manner (Bulliard et al. , 2014).

OX40 agonistic antibodies are currently being tested in clinical trials for cancer treatment, with most showing good safety profiles but limited clinical activity (Curti et al. , 2013). The isotype chosen for these antibodies varies, but several investigational drugs are human IgG1 antibodies engaging the Fc-gamma receptor, which are designed to potentially deplete Tregs as a mechanism of action. The lack of clear clinical activity of these antibodies has prompted studies of combining agonistic anti-OX40 antibodies with several other therapies, including PD1/PD-L1 or CTLA4 inhibition, anti-VEGF therapy, and the tyrosine kinase inhibitor axitinib.

This Treg depletion-based mechanism of action has been demonstrated to be very effective in preclinical models, and several receptors may be targets for Treg depletion, such as GITR (Bulliard et al. , 2014) and CTLA4 (Simpson et al. , 2013). However, antibodies targeting equivalent receptors in humans have not been shown to have the same levels of antitumor efficacy in clinical settings (Glisson et al. , 2016; Tran et al. , 2017). The reasons for this are unclear, but the lower levels of Fc-gamma receptor-expressing cells, such as macrophages, in human tumors compared to mouse models of syngeneic tumors (Milas et al., 1987) may partly explain the lack of extrapolation of the mechanism of action of these antibodies to clinical clinical trials. conditions. Other reasons may be the different expression levels of these markers in human Tregs compared to mouse Tregs (Aspeslagh et al. , 2016).

CD137 is also expressed on activated T cells, including CD4 ⁺ , CD8 ⁺ , Th1, Th2 and Tregs, but its expression profile also includes B cells, natural killer cells (NK cells), natural killer T cells (NKT -cells) and dendritic cells (DCs) (Bartkowiak & Curran, 2015). As with OX40, the interaction of CD137 with its ligand triggers the activation of intracellular signaling pathways that lead to the survival, proliferation, and induction of cytotoxic activity of T cells. CD137 stimulation preferentially stimulates CD8 ⁺ T cells over CD4 ⁺ T cells and leads to their proliferation, survival and cytotoxic effector function through the production of inflammatory cytokines, and promotes the differentiation and maintenance of memory CD8 ⁺ T cells. CD137 has also been demonstrated to be specifically expressed on tumor-reactive tumor-infiltrating lymphocyte (TIL) subsets (Weigelin et al. , 2016), partly justifying its agonistic binding in vivo and its use in selecting TILs for adoptive transfer. CD137 monotherapy is effective in several preclinical models of immunogenic tumors, such as MC38, CT26 and B-cell lymphomas. However, in the case of even more effective treatment of established tumors, CD137 binding in combination with other agents such as chemotherapy, cytokines and other checkpoint regulators has shown more beneficial effects in reducing tumor growth (Bartkowiak & Curran, 2015). In preclinical models, targeting CD137 with agonistic antibodies is also associated with liver inflammation and transaminitis, which results from increased CD8 ⁺ T cell accumulation dependent on IL27 production by myeloid cells (Bartkowiak et al. , 2018).

Agonistic antibodies to CD137 are currently in clinical trials for the treatment of cancer, however clinical progress has been slowed by dose-limiting severe liver inflammation, likely similar to that observed in mice (Sanchez-Paulete et al. , 2016). Urelumab (BMS-663513) was the first CD137 agonist antibody to be included in clinical trials and showed signs of clinical activity before the studies were discontinued due to fatal hepatotoxicity at doses above 1 mg/kg (Segal et al. , 2017 ). It is a human IgG4 antibody that is capable of activating CD137 in the absence of cross-linking (US 8137667 B2), although activity is enhanced by cross-linking, as expected under the theory of full receptor activation mediated by superclustering. In contrast, no dose-limiting toxicities were detected with utomilumab (PF-05082566) when tested up to 10 mg/kg (Tolcher et al. , 2017). It is a human IgG2 antibody and is able to activate CD137 only when cross-linked (US 8337850 B2). Additional clinical studies for both antibodies are currently underway, with both antibodies being tested both as monotherapy and in combination with radiation and chemotherapy, as well as existing targeted and immuno-oncology therapies. Due to the hepatotoxicity observed with urelumab, this antibody had to be dosed at very low levels, and early signs of clinical activity have not yet been observed at these levels.

Several bispecific molecules targeting both CD137 and OX40 are in early stage development by a number of companies. Tumor targeting of CD137 stimulation is being tested by Macrogenics using HER2- and EphA2-targeting CD137 agonist molecules DART, Roche using FAPalpha- or CD20-targeting CD137 ligand fusion proteins, and Pieris Pharmaceuticals using HER2-targeting CD137 agonist molecules anticalins. Dual targeting of OX40 and CTLA4 is being tested by Aligator Biosciences to specifically deplete intratumoral Tregs, which are expected to express high levels of both targets.

Co-stimulation of OX40 and CD137 in vivo has been shown to stimulate both CD4 ⁺ and CD8 ⁺ T cells and induce cytotoxic function of both antigen-exposed and non-antigen-exposed bystander CD4 ⁺ T cells (Qui et al. , 2011). It is of interest that dual co-stimulation was able to induce engrafted CD4 ⁺ T cells with reduced tumor growth in immunodeficient mice inoculated with a syngeneic melanoma tumor model (B16-F10), highlighting the ability of this therapy to induce CD4 ⁺ T cell antitumor activity (Qui et al. , 2011). A phase I dose escalation clinical trial is currently underway to investigate the effect of combining an OX40 agonist (PF-04518600) with a CD137 agonist (utomilumab - PF-05082566) (NCT02315066) to evaluate the safety of this combination, and a phase I clinical trial is currently underway Ib/II combining the same TNFR agonists with PD-1 blockade by avelumab (NCT02554812). These studies will look at a combination of simple monospecific agonist antibodies that will require cross-linking of Fc-gamma receptors for their agonistic effects, and therefore may underestimate the clinical activity of targeting these receptors in combination.

Dual co-stimulation of OX40 and CD137 was also recently tested in mice using a bispecific antibody approach by chemically conjugating two existing antibodies against OX40 and CD137 (Ryan et al. , 2018). The molecule, named OrthomAb, was able to induce the proliferation of CD4 ⁺ and CD8 ⁺ T cells, as well as the production of the inflammatory cytokines IL-2 and IFNγ in vitro . OrthomAb was also able to reduce tumor growth in an in vivo syngeneic melanoma tumor model (B16-F10). The bivalent bispecific nature of OrthomAb is predicted to ensure efficient cross-linking of the molecules upon binding to both targets, leading to clustering of the OX40 and CD137 receptors and hence T cell activation. These results support a bispecific antibody approach to target OX40 and CD137 in a single molecule. The method of manufacturing the OrthomAb molecule results in multiple higher order molecules as well as targeted antibody dimers that must be further purified through multiple rounds of gel filtration steps. It is unlikely that this method of fabrication will make the approach competitive for anything other than a research tool for testing specific target combinations. Additionally, the structure of this bispecific antibody, in which two large macromolecules are held together by a small chemical linker, is likely to be unstable in vivo , and pharmacokinetic data are lacking to address this issue. Unfortunately, the antitumor effect of OrthomAb in vivo was compared only with the activity of agonistic antibodies to either OX40 or CD137, and not with their combination, making it unclear whether the molecule had an effect because of its bispecificity or because OrthomAb leads itself as a combination of agonistic antibodies to OX40 and CD137 as separate agents.

Thus, the feasibility of combining agonistic effects on members of the TNF receptor family, OX40 and CD137, in one divalent, bispecific and stable molecule has been established, which is promising for performing superclustering of OX40 and CD137, independent of the Fc-gamma receptor, thereby activating both CD4 ⁺ , and CD8 ⁺ T cells to induce an effective antitumor immune response. Based on preclinical combination data generated using monoclonal antibodies targeting either the OX40 or CD137 pathway, this molecule also holds promise as a combination partner to enhance the effect of standard cancer therapies to provide patient benefit.

Summary of the invention

The present inventors have found that antibody molecules that bind to both CD137 and OX40 and that are capable of inducing clustering and signaling of OX40 and/or CD137 upon binding to both targets are highly effective in activating immune cells, for example, in the tumor microenvironment . In addition, the present inventors have determined that restricting CD137 activation to sites where CD137 and OX40 are coexpressed will be highly effective in activating immune cells without causing the various toxicities associated with known CD137 agonist molecules. It is expected that this could be used, for example, in immunotherapy to treat cancer and other diseases.

As described above in the prior art section, it is believed that the initial ligation of an OX40 ligand or a CD137 ligand to OX40 or CD137, respectively, initiates a chain of events that leads to trimerization of the receptor, followed by clustering and activation of the receptor and subsequently initiation of effective antitumor T cell activity. Thus, it is expected that to achieve effective activation of OX40 or CD137 by a therapeutic agent, multiple receptor monomers must be linked together in a manner that mimics trimeric ligand binding.

The present inventors have isolated antibody molecules that contain a complementarity determining region (CDR)-based antigen binding site for CD137 and an antigen binding site for OX40 located in the constant domain of the antibody molecule. The present inventors have shown that such antibody molecules are capable of simultaneously binding to both targets when both targets are co-expressed. Co-expression here includes situations where CD137 and OX40 are expressed on the same cell, such as an immune cell, and situations where CD137 and OX40 are expressed on different cells, such as two different immune cells located next to each other in tumor microenvironment. Thus, it is believed that the antibody molecules of the present invention are capable of binding in cis to both targets expressed on the same cell, and are also capable of binding in trans to two targets expressed on different cells.

The present inventors have also shown that an antibody molecule that contains an antigen binding site for CD137 based on the CDR and an antigen binding site for OX40 located in the constant domain of the antibody molecule is capable of divalently binding to both targets. In particular, the present inventors showed that when such an antibody molecule was conditioned to bind to OX40 and CD137, and the resulting complexes were cross-linked and subjected to mass spectrometric analysis, 19% of the complexes were shown to contain two OX40 groups and two groups CD137, indicating that the antibody molecule was divalently bound to both targets.

In addition, we have shown that when these antibody molecules are bound to both targets, they are capable of inducing OX40 and CD137 clustering and signaling in vitro . Based on this mode of action, these antibody molecules have been termed “dual agonists,” i.e., the antibody molecules are capable of inducing receptor signaling through cross-linking by double binding to both OX40 and CD137.

As demonstrated in the examples, OX40 is predominantly expressed on CD4 ⁺ T cells, and CD137 is predominantly expressed on CD8 ⁺ T cells. The present inventors have demonstrated that antibody molecules are capable of inducing OX40 agonistic effects on CD4 ⁺ T cells. In these cases, the antibody molecule is believed to bind to CD137 through its CDR-based antigen binding domain to cross-link the antibody molecule, while the OX40 antigen binding domain is, at the same time, able to bind, cluster and activate OX40 expressed on CD4 ⁺ T -cells. Likewise, the present inventors have demonstrated that antibody molecules are capable of inducing CD137 agonistic effects on CD8 ⁺ T cells. In these cases, the antibody molecule is believed to bind to OX40 through its OX40 antigen binding domain to cross-link the antibody molecule, and the OX40 antigen binding domain is, at the same time, able to bind, cluster and activate CD137 expressed on CD8 ⁺ T- cells.

In addition, the present inventors showed that antibody molecules containing the two antigen binding sites detailed above and modified to reduce or eliminate binding to Fcγ receptors were able to induce receptor signaling when CD137 and OX40 were coexpressed, indicating that the agonistic effect occurred without the need for cross-linking by Fcγ receptors. Since the activity of the antibody molecule of the present invention does not require Fcγ receptor-mediated cross-linking, signaling through the OX40 or CD137 receptors is expected to be localized to sites where both targets are present, such as in the tumor microenvironment. Thus, the antibody molecule is capable of autonomously triggering an agonistic effect based on the expression of both specific targets and without the need for additional cross-linking agents.

In addition, since ADCC requires binding to the Fcγ receptor, it is expected that decreasing binding to Fcγ receptors will also reduce ADCC so that target immune cells are not depleted by the antibody molecules of the present invention. The present inventors considered this to be important because antibody molecules were designed to activate immune cells expressing CD137 and/or OX40 to stimulate an immune response. Thus, depletion of these immune cells is not desirable. The present inventors demonstrated that antibody molecules having the properties defined herein were able to activate and induce proliferation of immune cells, in particular T cells that express CD137 and/or OX40.

The present inventors also showed that antibody molecules containing the CD137 and OX40 antigen binding sites, as detailed above, were able to suppress tumor growth in vivo in mice. In addition, more effective suppression of tumor growth was observed with the use of bispecific antibody molecules compared to a combination of two monospecific antibody molecules, where one of the antibody molecules contained the antigen binding site for CD137 based on the CDR, and the other molecule contained the antigen binding site for OX40 based on the CDR, this suggests that simultaneous binding and agonist action on OX40 and CD137 leads to improved antitumor efficacy. In addition, the antibody molecules were shown to be able to induce complete tumor regression and the formation of protective immunological memory against repeated stimulation by tumor cells in the CT26 tumor model in mice. Thus, the antibody molecules of the present invention are expected to be effective in the treatment of cancer in human patients. Since these antibody molecules eliminated ADCC activity, they are thus expected to suppress tumor growth by agonistically acting on target immune cells without significantly depleting these beneficial T cells (memory and effector cells).

As observed in in vivo studies in mice, T cell activation and proliferation induced by the antibody molecules described herein was a systemic rather than tumor-localized effect. In addition, increased proliferation and activation of central memory CD4 ⁺ and CD8 ⁺ T cells in peripheral blood and effector memory CD4 ⁺ and CD8 ⁺ T cells were observed in a preliminary dose ranging study in cynomolgus monkeys administered an antibody molecule of the present invention . Thus, in addition to targeting T cells in the tumor microenvironment, the antibody molecule is expected to target memory T cells in the peripheral blood expressing OX40 and CD137 to trigger the proliferation of tumor-reactive T cells that will then feed them antitumor effect.

Thus, in addition to the actual site of the existing tumor, the anatomical site affected by the tumor may also be considered to include sites elsewhere in the body, such as lymph nodes in the periphery, at which tumor-specific immune responses arise.

As explained in the prior art section above, clinical development of CD137 agonist molecules has been stalled, at least in part, because the treatment was associated with either dose-limiting severe liver inflammation (urelumab) or poor clinical efficacy (utomilumab).

Without being limited to any theory, it is believed that T cells present in the liver may be capable of activation by CD137 agonist molecules, resulting in liver inflammation. CD8 ⁺ T cells have been shown to stimulate liver inflammation and apoptosis following sepsis/viral infection (Wesche-Soldato et al. , 2007). Treatment with an agonistic CD137 antibody in mice has been shown to result in CD137-dependent T cell infiltration into the liver (Dubrot J et al. , 2010). Taken together, the results of these studies indicate that highly potent agonistic CD137 antibodies, such as urelumab, may induce infiltration of activated CD8 ⁺ T cells into the liver, leading to liver inflammation. The activity of utomilumab may have been too low to detect this effect. Alternatively, the dose-limiting liver toxicity observed with urelumab treatment may be due to a specific epitope bound by this antibody.

The present inventors have conducted an extensive screening program to isolate antibody molecules that bind dimeric human CD137 with high affinity, i.e., they are expected to bind CD137 with high avidity. Given the selection protocol used, the antibody molecules are expected to bind to monomeric CD137 with lower affinity than that observed for dimeric CD137.

"Affinity" as used herein may refer to the strength of the binding interaction between an antibody molecule and its cognate antigen, as measured by _KD . As one skilled in the art will readily appreciate, if an antibody molecule is capable of forming multiple binding interactions with an antigen (for example, if the antibody molecule is capable of divalently binding an antigen and optionally the antigen is dimeric), the affinity measured by K _D may also be influenced by avidity, wherein avidity refers to the overall strength of the antibody-antigen complex.

Expression of CD137 by immune cells such as T cells is up-regulated upon activation. Without being limited to any theory, it is believed that due to the high expression of CD137 on activated immune cells, CD137 will be in the form of dimers, trimers and higher order multimers on the surface of such cells. In contrast, naïve immune cells, such as naïve T cells, express low or negligible levels of CD137 on their cell surface, and therefore any CD137 present is likely to be in monomeric form. Thus, antibody molecules that bind to CD137 with high avidity are expected to preferentially bind to activated immune cells, such as activated T cells, as opposed to naïve immune cells.

Thus, in view of the above, it is expected that the antibody molecules of the present invention will be substantially unable to activate CD137 in the absence of cross-linking by binding to OX40. In addition, as described above, the present inventors have developed antibody molecules in which Fcγ receptor-mediated cross-linking has been reduced or eliminated, which is expected to avoid activation of CD137 at sites where there is little or no co-expression of OX40 . It has been shown that eliminating Fcγ receptor binding does not affect the antitumor activity of the antibody molecule. Without being limited by any theory, it is believed that such antibody molecules will exhibit reduced toxicity when administered to patients. This is believed to be due to the significant restriction of CD137 activation to sites in which OX40 and CD137 are coexpressed at levels sufficient to drive CD137 clustering and activation. The present inventors have shown that in a preliminary dose ranging study in cynomolgus monkeys, doses of the antibody molecule of the present invention up to 30 mg/kg were well tolerated.

The present inventors have shown that the antibody molecules of the present invention are capable of inducing low levels of OX40 clustering and activation even in the absence of cross-linking. In contrast to CD137 agonistic antibodies, OX40 agonistic antibodies have not exhibited any dose-limiting toxicity (DLT) in clinical settings, and therefore OX40 agonistic activity in the absence of cross-linking is not expected to pose a problem for clinical treatment. In contrast, depending on the condition being treated, the low level of agonistic activity of antibody molecules against OX40 in the absence of cross-linking may represent an advantage. Without being bound by any theory, it is believed that antibody molecules containing an antigen-binding site for OX40 with this property can be used in the treatment of cancer by inducing limited activation and expansion of tumor-reactive T cells in the absence of cross-linking, resulting in to a larger pool of tumor-reactive T cells, which can then be further activated by cross-linked Fcab molecules in the tumor microenvironment.

Another advantage of antibody molecules of the present invention that have been modified to reduce or eliminate binding to Fcγ receptors is that these antibody molecules have antitumor activity that does not rely on depletion of OX40-expressing regulatory T cells (Tregs). Tregs are localized in the periphery, are potentially protective and may reduce the impact of autoimmunity, which can be caused by overstimulation of the immune system (Vignali DA et al. , 2008). Thus, it has been hypothesized that Treg depletion may have a significant effect on reducing tumor growth in mouse models (Bulliard et al. , 2014; Simpson et al. , 2013). However, there is limited evidence that Treg depletion in human tumors can be achieved by ADCC, and if Treg depletion does occur in humans, it does not appear to result in as significant antitumor activity as observed in mouse models (Powell et al. , 2007; Glisson B. et al . , 2017 ; Thus, if an antibody molecule does not significantly deplete Treg but still has antitumor activity, this may indicate that the antibody molecule has antitumor activity that is independent of Fcγ receptor-mediated Treg depletion.

In addition, the antibody molecules were shown to be able to bind with high affinity to both human and cynomolgus CD137 and human and cynomolgus OX40. This cross-reactivity is an advantage because it allows dosing and safety testing of antibody molecules in cynomolgus monkeys during preclinical development.

Another feature of the antibody molecules identified by the present inventors is that the antigen binding site for CD137 and the antigen binding site for OX40 are both contained within the antibody structure itself. In particular, antibody molecules do not need to hybridize other proteins to the antibody molecule through linkers or other means to produce a molecule that can bind divalently to both of its targets. This provides a number of benefits. In particular, the antibody molecules identified by the present inventors can be produced using methods similar to those used to produce standard antibodies, since they do not contain any additional hybridized moieties. The structure is also expected to result in improved antibody stability since linkers can degrade over time, resulting in a heterogeneous population of antibody molecules. Antibodies in a population that have only one fusion protein may be unable to act as a dual agonist and signal through receptors through cross-linking by binding to both OX40 and CD137. Cleavage or degradation of the linker may occur before or after administration of the therapeutic agent to the individual (eg, via enzymatic degradation or the individual's in vivo pH), resulting in a decrease in its effectiveness during circulation in the individual. Since there are no linkers in the antibody molecules identified by the present inventors, the antibody molecules are expected to retain the same number of binding sites both before and after administration. In addition, the structure of the antibody molecules identified by the present inventors is also advantageous from the point of view of the immunogenicity of the molecules, since the introduction of fusion proteins or linkers or both may induce immunogenicity when the molecules are administered to an individual, resulting in a decrease in the effectiveness of the therapeutic agent.

Thus, according to the present invention it is proposed:

[1] Antibody molecule that binds to CD137 and OX40 containing

(a) a complementarity determining region (CDR)-based antigen binding site for CD137; And

(b) an antigen binding site for OX40 located in the CH3 domain of the antibody molecule;

wherein said CDR-based antigen binding site comprises CDRs 1-6 presented in:

(i) SEQ ID NOs 1, 2, 3, 4, 5 and 6 , respectively [ FS30-10-16 ];

(ii) SEQ ID NOs 1, 2, 16, 4, 5 and 6 , respectively [ FS30-10-3 ];

(iii) SEQ ID NOs 1, 2, 21, 4, 5 and 6 , respectively [FS30-10-12] ;

(iv) SEQ ID NOs 25, 26, 27, 4, 5 and 28 , respectively [FS30-35-14] ; or

(v) SEQ ID NOs 33, 34, 35, 4, 5 and 36 , respectively [FS30-5-37] ; And

wherein said antigen binding site for OX40 comprises a first sequence, a second sequence and a third sequence located in the structural loops AB, CD and EF of the CH3 domain, respectively, wherein said first, second and third sequence have the sequence set forth in SEQ ID NOs 51, 52 and 53 , respectively [FS20-22-49] .

[2] Antibody molecule that binds to CD137 and OX40 containing

(a) a complementarity determining region (CDR)-based antigen binding site for CD137; And

(b) an antigen binding site for OX40 located in the CH3 domain of the antibody molecule;

wherein said CDR-based antigen binding site comprises CDRs 1-6 presented in:

(i) SEQ ID NOs 7, 8, 9, 10, 11 and 6 , respectively [ FS30-10-16] ;

(ii) SEQ ID NOs 7, 8, 17, 10, 11 and 6 , respectively [ FS30-10-3] ;

(iii) SEQ ID NOs 7, 8, 22, 10, 11 and 6 , respectively [FS30-10-12] ;

(iv) SEQ ID NOs 29, 30, 31, 10, 11 and 28 , respectively [FS30-35-14] ; or

(v) SEQ ID NOs 37, 38, 39, 10, 11 and 36 , respectively [FS30-5-37] ; And

wherein said antigen binding site for OX40 comprises a first sequence, a second sequence and a third sequence located in the structural loops AB, CD and EF of said CH3 domain, respectively, wherein the first, second and third sequence have the sequence set forth in SEQ ID NOs 51, 52 and 53 , respectively [FS20-22-49] .

[3] Antibody molecule according to claim [1] or [2], characterized in that:

(i) said first sequence is located at positions 14 to 18 of the CH3 domain of said antibody molecule;

(ii) said second sequence is located at positions 45.1 to 77 of the CH3 domain of said antibody molecule; and/or

(iii) said third sequence is located at positions 93 to 101 of the CH3 domain of said antibody molecule; And

wherein the specified numbering of amino acid residues corresponds to the IMGT numbering scheme.

[4] The antibody molecule according to any one of paragraphs. [1]-[3], characterized in that said antibody molecule contains the CH3 domain sequence indicated under SEQ ID NO: 54 [FS20-22-49] .

[5] The antibody molecule according to any one of paragraphs. [1]-[4], characterized in that said antibody molecule contains CDRs 1-6 presented in any of (i)-(iv) paragraphs. [1 or 2].

[6] The antibody molecule according to any one of paragraphs. [1]-[5], characterized in that said antibody molecule contains CDRs 1-6 presented in any of (i)-(iii) paragraphs. [1 or 2].

[7] The antibody molecule according to any one of paragraphs. [1]-[6], characterized in that said antibody molecule contains CDRs 1-6 presented in (i) paragraphs. [1 or 2].

[8] The antibody molecule according to any one of paragraphs. [1]-[7], characterized in that said antibody molecule contains a heavy chain variable domain (VH) and/or a light chain variable domain (VL), preferably a VH domain and a VL domain.

[9] The antibody molecule according to any one of paragraphs. [1]-[8], characterized in that said antibody molecule contains an immunoglobulin heavy chain and/or an immunoglobulin light chain, preferably an immunoglobulin heavy chain and an immunoglobulin light chain.

[10] Antibody molecule according to claims. [8] or [9], characterized in that said antibody molecule contains a VH domain and/or a VL domain, preferably a VH domain and a VL domain represented in:

(i) SEQ ID NOs 12 and 14 , respectively [ FS30-10-16] ;

(ii) SEQ ID NOs 18 and 14 , respectively [ FS30-10-3] ;

(iii) SEQ ID NOs 23 and 14 , respectively [FS30-10-12] ;

(iv) SEQ ID NOs 170 and 172 , respectively [FS30-35-14] ; or

(v) SEQ ID NOs 40 and 42 , respectively [FS30-5-37] ;

[11] The antibody molecule of claim [10], characterized in that said antibody molecule contains said VH domain and a VL domain represented in any one of (i) to (iv) of claim [10].

[12] Antibody molecule according to claims. [10] or [11], characterized in that the specified antibody molecule contains the specified VH and VL domain presented in any of (i)-(iii) of paragraph [10].

[13] The antibody molecule according to any one of paragraphs. [10]-[12], characterized in that the specified antibody molecule contains the specified VH domain and the VL domain presented in (i) paragraph [10].

[14] The antibody molecule according to any one of paragraphs. [1]-[13], characterized in that said antibody molecule is a human IgG1 molecule.

[15] The antibody molecule according to any one of paragraphs. [1]-[14], characterized in that said antibody molecule contains a heavy chain and a light chain of an antibody:

(i) FS20-22-49AA / FS30-10-16 , shown in SEQ ID NOs 95 and 97 , respectively;

(ii) FS20-22-49AA/FS30-10-3 presented in SEQ ID NOs 99 and 97 , respectively;

(iii) FS20-22-49AA/FS30-10-12 presented in SEQ ID NOs 103 and 97 , respectively;

(iv) FS20-22-49AA/FS30-35-14 , shown in SEQ ID NOs 105 and 107 , respectively; or

(v) FS20-22-49AA/FS30-5-37 shown in SEQ ID NOs 109 and 111 , respectively.

[16] The antibody molecule of claim [15], characterized in that said antibody molecule contains said light chain and heavy chain represented in any one of (i) to (iv) of claim [15].

[17] The antibody molecule of claim [15], characterized in that said antibody molecule contains said light chain and heavy chain represented in any one of (i) to (iii) of claim [15].

[18] The antibody molecule according to claim [15], characterized in that said antibody molecule contains the specified light chain and heavy chain presented in (i) of claim [15].

[19] The antibody molecule according to any one of paragraphs. [1]-[18], characterized in that said antibody molecule binds human CD137 and human OX40.

[20] The antibody molecule of claim [19], characterized in that said human CD137 consists of or contains the sequence set forth in SEQ ID NO: 127 .

[21] The antibody molecule of claim [19] or [20], characterized in that said human OX40 consists of or contains the sequence set forth in SEQ ID NO: 130 .

[22] The antibody molecule according to any one of paragraphs. [1]-[21], characterized in that said antibody molecule binds cynomolgus CD137 and cynomolgus OX40.

[23] The antibody molecule of claim [22], characterized in that said cynomolgus CD137 consists of or contains the sequence set forth in SEQ ID NO: 129 .

[24] The antibody molecule of claim [23] or [24], characterized in that said cynomolgus OX40 consists of or contains the sequence set forth in SEQ ID NO: 131 .

[25] The antibody molecule according to any one of paragraphs. [5]-[7], [11]-[13] and [16]-[18], characterized in that said antibody molecule binds human CD137 and human OX40, and the affinity (K _D ) with which said antibody molecule binds human CD137 is within the 2-fold affinity (K _D ) with which the antibody molecule binds human OX40.

[26] The antibody molecule according to any one of paragraphs. [19]-[25], characterized in that said antibody molecule is capable of simultaneously binding to human CD137 and human OX40.

[27] The antibody molecule according to any one of paragraphs. [1]-[26], characterized in that said antibody molecule is capable of activating OX40 on an immune cell in the presence of CD137 expressed on the cell surface.

[28] The antibody molecule according to any one of paragraphs. [1]-[27], characterized in that the binding of said antibody molecule to OX40 on an immune cell and to CD137 causes clustering of OX40 on said immune cell.

[29] The antibody molecule according to any one of paragraphs. [1]-[28], characterized in that said antibody molecule is capable of activating CD137 on an immune cell in the presence of OX40 expressed on the cell surface.

[30] The antibody molecule according to any one of paragraphs. [1]-[29], characterized in that the binding of said antibody molecule to CD137 on an immune cell and to OX40 causes clustering of CD137 on said immune cell, and wherein OX40 is expressed on the same immune cell or on a separate cell.

[31] The antibody molecule according to any one of paragraphs. [27]-[30], characterized in that said immune cell is a T cell.

[32] The antibody molecule according to any one of paragraphs. [1]-[31], characterized in that said antibody molecule has been modified to reduce or eliminate binding of the CH2 domain of said antibody molecule to one or more Fcγ receptors.

[33] The antibody molecule according to any one of paragraphs. [1]-[32], characterized in that said antibody molecule does not bind to one or more Fcγ receptors.

[34] The antibody molecule of claim [32] or [33], characterized in that said Fcγ receptor is selected from the group consisting of: FcγRI, FcγRIIa, FcγRIIb and FcγRIII.

[35] The antibody molecule according to any one of paragraphs. [1]-[34], characterized in that said antibody molecule is capable of inducing proliferation of T cells.

[36] A conjugate containing an antibody molecule according to any one of paragraphs. [1]-[35] and a bioactive molecule.

[37] A conjugate containing an antibody molecule according to any one of paragraphs. [1]-[36] and the detectable mark.

[38] A nucleic acid molecule or molecules encoding an antibody molecule according to any one of claims. [1]-[35].

[39] A nucleic acid molecule or molecules encoding an antibody molecule according to any one of claims. [1]-[4], [8]-[10], [14]-[15] and [19]-[35], which are characterized in that said nucleic acid molecule(s) contain(s) a nucleic acid sequence heavy chain acids and/or light chain nucleic acid sequence:

(i) FS20-22-49AA / FS30-10-16, shown in SEQ ID NOs 96 and 98 , respectively;

(ii) FS20-22-49AA/FS30-10-3, shown in SEQ ID NOs 100 and 102 , respectively;

(iii) FS20-22-49AA/FS30-10-12, shown in SEQ ID NOs 104 and 102 , respectively;

(iv) FS20-22-49AA/FS30-35-14, set forth in SEQ ID NOs 106 and 108 , respectively; or

(v) FS20-22-49AA/FS30-5-37 shown in SEQ ID NOs 110 and 112 , respectively.

[40] A vector or vectors containing a nucleic acid molecule or molecules according to any one of claims. [38]-[39].

[41] A recombinant host cell containing a nucleic acid molecule(s) according to any one of claims. [38]-[39], or vector(s) according to item [40].

[42] A method for producing an antibody molecule according to any one of claims. [1]-[35], comprising culturing a recombinant host cell according to claim [41] under conditions to obtain said antibody molecule.

[43] The method of claim [42], further comprising isolating and/or purifying said antibody molecule.

[44] A pharmaceutical composition containing an antibody molecule or conjugate according to any one of paragraphs. [1]-[37] and a pharmaceutically acceptable excipient.

[45] The antibody molecule or conjugate according to any one of paragraphs. [1]-[37] for use in a method of treating a human or animal body with therapy.

[46] A method of treating a disease or disorder in an individual, comprising administering to said individual a therapeutically effective amount of an antibody molecule or conjugate according to any one of claims. [1]-[37].

[47] An antibody molecule or conjugate for use according to claim [45], characterized in that said antibody molecule or conjugate is for use in the treatment of cancer or an infectious disease in an individual.

[48] The method of claim [46], which is characterized in that said disease or disorder is a cancer or infectious disease in the individual.

[49] Use of an antibody molecule or conjugate according to any one of paragraphs. [1]-[37] in obtaining a medicament for the treatment of cancer or an infectious disease.

[50] An antibody molecule or conjugate for use according to claim [47], a method according to claim [48], or use of an antibody molecule or conjugate according to claim [49], characterized in that said cancer is a solid cancer, optionally specified solid cancer selected from the group consisting of melanoma, bladder cancer, brain cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, pancreatic cancer, kidney cancer and stomach cancer.

[51] An antibody molecule or conjugate for use according to claim [47], a method according to claim [48] or use of an antibody molecule or conjugate according to claim [49], characterized in that said infectious disease is a persistent viral infection, wherein optionally, said persistent viral infection is selected from the group consisting of human immunodeficiency virus (HIV), Epstein-Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, varicella zoster virus.

[52] An antibody molecule or conjugate for use according to claim [47], a method according to claim [48] or use of an antibody molecule or conjugate according to claim [49], characterized in that said infectious disease is a persistent bacterial infection, wherein optionally, said persistent bacterial infection is a persistent infection of Staphylococcus aureus, Hemophilus influenza, Mycobacterium tuberculosis, Mycobacterium leprae, Helicobacter pylori, Treponema pallidum, Enterococcus faecalis or Streptococcus pneumoniae .

[53] An antibody molecule or conjugate for use according to claim [47], a method according to claim [48] or use of an antibody molecule or conjugate according to claim [49], characterized in that said infectious disease is a persistent fungal infection, wherein optionally, said persistent fungal injection is a persistent Candida infection, eg Candida albicans , Cryptococcus (gattii and neoformans) , Talaromyces (Penicillium) marneffe, Microsporum, eg Microsporum audouinii, and Trichophyton tonsurans .

[54] An antibody molecule or conjugate for use according to claim [47], a method according to claim [48], or use of an antibody molecule or conjugate according to claim [49], characterized in that said infectious disease is a persistent parasitic infection, wherein optionally said persistent parasitic injection is a persistent infection with Plasmodium , such as Plasmodium falciparum, or Leishmania , such as Leishmania donovani .

[55] An antibody molecule or conjugate for use according to any one of claims. [45], [47] and [50]-[54], characterized in that said treatment comprises administering said antibody molecule or conjugate to an individual in combination with a second therapeutic agent.

[56] Method according to claims. [46], [48] and [50]-[54], characterized in that the method further comprises administering a therapeutically effective amount of a second therapeutic agent to the individual.

[57] An antibody molecule or conjugate for use in a method for treating cancer in an individual according to claims. [47] or [50], characterized in that said method comprises administering to said individual said antibody molecule or conjugate in combination with an antibody that binds PD-1 or PD-L1.

Brief description of drawings

Figure 1 shows the sequence alignment of the CH3 domains of Fcab FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, FS20-22-85, FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108 and FS20-31-115, as well as wild-type (WT) Fcab. The positions of the AB, CD, and EF structural loops are indicated, as well as any amino acid substitutions, deletions (indicated by a tilde "~"), or insertions present in the CH3 domains of Fcab compared to the WT sequence. Residue numbers according to the IMGT, IMGT exon (sequential numbering), EC, and Kabat numbering systems are shown.

Figure 2 shows the activity of mAb to CD137 and mAb ² to OX40/CD137 in the activation assay of T cells expressing human CD137, in the presence and absence of cross-linking. Figures 2A and B show IL2 release in the presence of increasing concentrations of anti-CD137 mAbs and in the presence ( Figure 2A ) or absence ( Figure 2B ) of cross-linking antibody. G1AA/20H4.9 showed activity in the presence and absence of cross-linking antibody, while activity of antibodies G1AA/MOR7480.1 and G1AA/FS30-10-16 was detected only in the presence of cross-linking antibody. Figure 2C and D show the release of IL-2 in the presence of increasing concentrations of the anti-CD137 mAb FS30 in the mAb ² format containing anti-OX40 human Fcab (FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10- 3, FS20-22-49AA/FS30-10-12, FS20-22-49AA/FS30-10-16 and FS20-22-49AA/FS30-35-14) in the presence ( Figure 2C ) or absence ( Figure 2D ) cross-linking agent. The following controls were included: anti-CD137 G2/MOR7480.1 (positive control); MAT to OX40 G1/11D4 and MAT ² FS20-22-49AA/4420 (negative controls); MAb to FITC G1/4420 (negative isotype control). Figure 2C shows that there was a concentration-dependent increase in activation of DO11.10-hCD137 cells, as evidenced by increased murine IL-2 release in the presence of cross-linked positive control mAb (G2/MOR7480.1) and anti-CD137 mAb ² FS30 (FS20 -22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA/FS30-10-16 and FS20-22 -49AA/FS30-35-14), but not in the presence of MAT and MAT ² for negative control (G1/4420, FS20-22-49AA/4420 and G1/11D4). Figure 2D shows that in the absence of cross-linking, positive control G2/MOR7480.1, MAT ² FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30 -10-12, FS20-22-49AA/FS30-10-16 and FS20-22-49AA/FS30-35-14, as well as negative controls G1/4420, FS20-22-49AA/4420 and G1/11D4 showed no or poor T cell activation, as evidenced by measured low initial IL-2 levels.

Figure 3 shows the activity of mAb anti-CD137, Fcab anti-OX40 and mAb ² against OX40/CD137 in assays using staphylococcal enterotoxin A (SEA). IL-2 release was measured in the presence of the indicated MAbs/MAT ² and in the presence and absence of cross-linking agents (FITC-dextran for FITC MAbs and control mock OX40/FITC MAbs ² , and anti-human CH2 antibody for all others tested molecules). Figure 3A shows the release of IL-2 in the presence of mAbs G1/4420 (anti-FITC; isotype control), G1AA/MOR7480.1 (anti-CD137), G1AA/FS30-10-16 (anti-CD137), G1AA/20H4.9 (anti-CD137). CD137), G1AA/11D4 (against OX40), FS20-22-49AA/4420 (imitation MAT ² to OX40/FITC) and FS20-22-49AA/4420 plus G1AA/FS30-10-16 in combination, as well as MAT ² FS20-22-49AA/FS30-10-16, at a concentration of 3.7 nM. Results show that only mAb ² to OX40/CD137 increased T cell activation in the absence of artificial cross-linkers compared to isotype control, while OX40-targeting antibodies G1AA/11D4 and FS20-22-49AA/4420 and CD137 G1AA/20H4.9 showed an increase in T cell activation only in the presence of artificial cross-linking agents compared to isotype control, and antibodies to CD137 G1AA/MOR7480.1 and G1AA/FS30-10-16 did not show statistically significant activity even in the presence artificial cross-linking agent. Figure 3B shows the release of IL-2 in the presence of anti-OX40/CD137 mAb ² FS20-22-49AA/FS30-10-16 at increasing concentrations in the presence and absence of an artificial cross-linker (human CH2 antibody). The results show that T cell activation induced by anti-OX40/CD137 mAb ² in the absence of anti-human CH2 antibody was comparable to that tested in the presence of the indicated artificial cross-linking agent. Figure 3C and D show the release of IL-2 in the presence of increasing concentrations of MAT and MAT ² in the presence ( Figure 3D ) and absence ( Figure 3C ) of an artificial cross-linking agent (FITC-dextran for FITC Mab and control mock OX40 MAT ² ). FITC and anti-human CH2 antibody for all other molecules tested). Controls were: G1/4420 (anti-FITC), G1/11D4 (anti-OX40), G2/MOR7480.1 (anti-CD137), G1/11D4 plus G2/MOR7480.1 in combination and FS20-22-49AA/4420 ( imitation MAT ² to OX40/FITC). Results indicate that there was a concentration-dependent increase in T cell activation when OX40 was bound to G1/11D4 controls, both alone and when dosed in combination with anti-CD137 mAbs G2/MOR7480.1 and FS20-22-49A/ 4420 when they were cross-linked. Anti-OX40/CD137 mAb ² had comparable activity in the presence and absence of an artificial cross-linker, and activity was similar to that of the anti-OX40 cross-linked Fcab (FS20-22-49AA/4420 Xlink). Little activity was observed using the control anti-CD137 antibody (G2/MOR7480.1) alone, with or without cross-linking.

Figure 4 shows the activity of mAb anti-CD137, Fcab anti-OX40 and mAb ² against OX40/CD137 in assays for activation of a general population of human T cells. IL-2 release was measured in the presence of the indicated MAbs/MAT ² and in the presence and absence of cross-linking agents (FITC-dextran for FITC MAbs and control mock OX40/FITC MAbs ² , and anti-human CH2 antibody for all others tested molecules). Figure 4A shows the release of IL-2 in the presence of MAT and MAT ² at a concentration of 3.7 nM. The results show that only MAb ² to OX40/CD137 increased T cell activation in the absence of artificial cross-linking agents. The OX40-targeting antibodies G1AA/11D4 and FS20-22-49AA/4420 and the anti-CD137 antibody G1AA/20H4.9 showed increased T cell activation only in the presence of cross-linking agents. No activity was detected for anti-CD137 antibodies G1AA/MOR7480.1 and G1AA/FS30-10-16 even in the presence of an artificial cross-linker, confirming the results of the SEA assay presented in Figure 3A. Figure 4B shows IL-2 release induced by increasing concentrations of anti-OX40/CD137 mAb ² FS20-22-49AA/FS30-10-16 in the presence and absence of an artificial cross-linker (human CH2 antibody). Anti-OX40/CD137 mAb ² had comparable activity in the presence and absence of an artificial cross-linker. Figure 4C shows the release of IL-2 in the presence of increasing concentrations of mAb ² to OX40/CD137 and controls in the absence of artificial cross-linking agents, while Figure 4D shows the release of IL-2 in the presence of increasing concentrations of controls G1/4420, G1/11D4, G2/MOR7480.1 and FS20-22-49AA/4420 as single agents in the presence of an artificial cross-linker (FITC-dextran or anti-human CH2 antibody, as appropriate). The results show that anti-OX40/CD137 mAb ² had activity in the sub-nanomolar or single-digit nanomolar range in the absence of an artificial cross-linker. As expected, the G1/4420 control had no activity, regardless of the presence of the cross-linker. Without the presence of the cross-linker, controls G1/11D4, FS20-22-49AA/4420, G2/MOR7480.1, and the combination of G1/11D4 and G2/MOR7480.1 had little or no activity. When cross-linked with anti-human CH2 antibody or FITC-dextran, the anti-OX40 and anti-CD137 controls as separate agents exhibited a concentration-dependent increase in T cell activation, thus demonstrating that the assay was capable of detecting signaling through both OX40 receptors and and CD137 receptors on T cells.

Figure 5 shows the activity of MAb ² to human OX40/CD137 in CD4 ⁺ and CD8 ⁺ T cell activation assays. Figure 5A and B show the release of IL-2 in a CD4 ⁺ T cell activation assay in the presence of increasing concentrations of MAT and MAT ² as indicated. MAbs and MAbs ² were tested in the presence ( Figure 5B ) or absence ( Figure 5A ) of artificial cross-linkers (FITC-dextran for FITC MAbs and control mock OX40/FITC MAbs ² , and anti-human CH2 antibody for all others tested molecules). The results show that anti-OX40/CD137 mAb ² was able to activate CD4 ⁺ T cells in the absence of an artificial cross-linker. CD4 ⁺ T cells were activated by cross-linked anti-OX40 controls G1AA/11D4 and FS20-22-49AA/4420 (alone and in combination with G1AA/FS30-10-16), but not by anti-CD137 controls G1AA/MOR7480.1 and G1AA/FS30-10-16 as single agents. The anti-OX40 control FS20-22-49AA/4420 also showed low levels of activity in the presence of CD4 ⁺ T cells without cross-linking, which was significantly increased by cross-linking antibody. Thus, it was shown that Fcab to OX40, common to both mock mAb ² FS20-22-49AA/4420 and Mab ² FS20-22-49AA/FS30-10-16, was able to activate CD4 ⁺ T cells through agonistic effects on OX40 when antibodies were cross-linked using an artificial cross-linking agent or Fab binding to CD137. Figure 5C and D shows the release of IL-2 in a CD8 ⁺ T cell activation assay in the presence of increasing concentrations of MAT and MAT ² as indicated. MAT and MAT ² were tested in the presence ( Figure 5D ) or absence ( Figure 5C ) of artificial cross-linkers (see legend to Figures 5A and B for more details). The results show that anti-OX40/CD137 mAb ² was able to activate CD8 ⁺ T cells in the absence of an artificial cross-linker. CD8 ⁺ T cell activation was observed for both anti-CD137 controls G1AA/MOR7480.1 and G1AA/FS30-10-16 (alone and in combination with FS20-22-49AA/4420) and anti-OX40 controls FS20-22 -49AA/4420 and, to a lesser extent, G1AA/11D4 in the presence of an artificial cross-linker. Thus, it was shown that Fab arms to CD137, common to both the control mAb G1AA/FS30-10-16 and mAb ² FS20-22-49AA/FS30-10-16, are capable of exerting an agonistic effect on CD137 expressed on CD8 ⁺ T cells when antibodies were cross-linked either by an artificial cross-linking agent or Fcab binding to OX40, while Fcab to OX40 common to both mock mAb ² FS20-22-49AA/4420 and for mAb ² FS20-22-49AA/FS30-10-16, was able to activate CD8 ⁺ T cells through an agonistic effect on OX40 when the antibodies were cross-linked using an artificial cross-linking agent or Fab binding to CD137. Figure 5E and F show the release of IL-2 in a CD4 ⁺ and CD8 ⁺ T cell activation assay, respectively, in the presence of MAT/MAT ² at a concentration of 3.7 nM and in the presence or absence of an artificial cross-linker (for more details see caption for Figures 5A and B). Figure 5E shows that mAb ² to OX40/CD137 was able to activate CD4 ⁺ T cells in the absence of an artificial cross-linker. CD4 ⁺ T cells were activated by cross-linked anti-OX40 controls G1AA/11D4 and FS20-22-49AA/4420, but not by anti-CD137 controls G1AA/MOR7480.1 and G1AA/FS30-10-16 as single agents. The anti-OX40 control FS20-22-49AA/4420 also showed a low level of activity without cross-linking, which was significantly increased when the antibody was cross-linked. Thus, it was shown that Fcab to OX40, common to both mock mAb ² FS20-22-49AA/4420 and mAb ² FS20-22-49AA/FS30-10-16, was able to activate CD4 ⁺ T cells through agonistic effects on OX40 when antibodies were cross-linked using an artificial cross-linking agent or Fab binding to CD137. Figure 5F shows that mAb ² to OX40/CD137 was able to activate CD8 ⁺ T cells in the absence of an artificial cross-linker. CD8 ⁺ T cell activation was observed for anti-CD137 controls G1AA/20H4.9 and G1AA/FS30-10-16 (alone and in combination with FS20-22-49AA/4420) in the presence of an artificial cross-linker, but not for control against CD137 G1AA/MOR7480.1 or cross-linked controls against OX40 G1AA/11D4 and FS20-22-49AA/4420. CD8 ⁺ T cell activation was also observed for the anti-CD137 G1AA/20H4.9 control in the absence of an artificial cross-linker. Thus, it was shown that Fab arms to CD137, common to both the control mAb G1AA/FS30-10-16 and mAb ² FS20-22-49AA/FS30-10-16, were capable of exerting an agonistic effect on CD137. expressed on CD8 ⁺ T cells when antibodies were cross-linked either by an artificial cross-linking agent or Fcab binding to OX40.

Figure 6 shows that CD4 ⁺ T cells express lower levels of CD137 and higher levels of OX40 than CD8 ⁺ T cells. The graph shows the geometric mean fluorescence intensity (GMFI) of CD4 ⁺ or CD8 ⁺ T cells treated with G1AA/MOR7480.1 or G1AA/11D4. G1AA/MOR7480.1 binding to CD137 is a measure of CD137 expression, and G1AA/11D4 binding to OX40 is a measure of OX40 expression.

Figure 7 shows the activity of Mab and Mab ² to mouse CD137 in a T cell activation assay. Figure 7A and B show the release of IL-2 in the presence of increasing concentrations of mAb ² , which binds mouse OX40 and mouse CD137 receptors (FS20m-232-91AA/Lob12.3), as well as control antibodies, in the absence ( Figure 7A ) and in the presence ( Figure 7B ) artificial cross-linker (anti-human CH2 antibody or FITC-dextran, as appropriate). Controls were antibodies G1/4420 (anti-FITC), G1AA/OX86 (anti-mOX40), G1AA/Lob12.3 (anti-mCD137), G1AA/OX86 plus G1AA/Lob12.3 in combination and FS20m-232-91AA/4420 ( imitation MAT ² to mOX40/FITC). The results show that in the absence of cross-linking agent, the controls G1AA/OX86, FS20m-232-91AA/4420, G1AA/Lob12.3 and the combination of G1AA/OX86 and G1AA/Lob12.3 had no activity. When cross-linked with anti-human CH2 antibody or FITC-dextran, the controls G1AA/OX86, FS20m-232-91AA/4420, and G1AA/OX86 plus G1AA/Lob12.3 exhibited a concentration-dependent increase in T cell activation. A slight increase in activity was observed for the G1AA/Lob12.3 control upon cross-linking. MAb ² to OX40/CD137 showed good activity regardless of the presence of an artificial cross-linking agent. Figure 7C and D show the activity of various anti-mouse CD137 antibodies (G1AA/Lob12.3 and G1AA/3H3) in the absence ( Figure 7C ) or presence ( Figure 7D ) of cross-linking antibody (clone MK1A6) in CD3-stimulated DO11.10 cells -mCD137. G1AA/3H3 activity was observed in the presence and absence of cross-linking antibody, while G1AA/Lob12.3 antibody activity was observed only in the presence of cross-linking antibody. Thus, the G1AA/3H3 antibody is referred to as “cross-linking independent” and the G1AA/Lob12.3 antibody is referred to as “cross-linking dependent.”

Figure 8 shows a competition assay for testing the activity of the human OX40/CD137 Mab ² clone FS20-22-49AA/FS30-10-16 in the presence of a 100-fold excess of mock human OX40-targeting Mab ² (FS20-22-49AA/4420) , antibodies to human CD137 (G1AA/FS30-10-16) or combinations thereof. Data for two replicates are shown as the mean plus or minus the standard deviation of the mean (SD). Statistical analysis was performed using one-way analysis of variance and Dunnett's test for multiple comparisons. Asterisks above error bars represent significant differences compared to isotype control (G1/4420) treated samples (***p<0.0002). The results show that the activity of MAb ² to OX40/CD137 was significantly reduced by superior competition from both the mock MAb ² FS20-22-49AA/4420 for binding to OX40 and MAb G1AA/FS30-10-16 for binding to CD137. compared to when mAb ² to OX40/CD137 could bind to both receptors in the absence of anti-OX40 antibody and anti-CD137 antibody. The combination of the OX40-targeting mock mAb ² FS20-22-49AA/4420 and the anti-CD137 mAb G1AA/FS30-10-16 further reduced the activity of mAb ² to OX40/CD137. These results indicate that dual binding of Mab ² to both receptors is required for OX40/CD137 Mab ² to induce T cell activation through clustering and agonistic effects on OX40 and CD137.

Figure 9 shows a competition assay for testing the activity of mouse FS20m-232-91AA/Lob12.3 anti-OX40/CD137 mAb ² in the presence of a 100-fold excess of either OX40-targeting anti-FS20m-232-91AA/4420 mAb ² mock or anti-CD137 mAb 2 G1/Lob12.3, or negative control MAT G1AA/4420 (against FITC). The results show that MAT ² activity was significantly reduced when outcompeted by MAT G1/Lob12.3 for binding to CD137, and was also reduced to low levels when outcompeted by the mock MAT ² FS20m-232-91AA/4420 for binding to OX40 compared to when Mab ² could bind to both receptors in the absence of anti-OX40 antibody and anti-CD137 antibody. As expected, similar levels of activity were observed for Mab ² in the presence of excess negative control Mab, as in its absence, and anti-OX40 and anti-CD137 antibodies. These results indicate that dual binding of MAT ² to both receptors is required for MAT ² to induce T cell activation through clustering and agonistic effects on OX40 and CD137.

Figure 10 shows the antitumor activity of mouse mAb ² against OX40/CD137 in the CT26 syngeneic tumor model. Figure 10A shows the average CT26 tumor volumes (plus or minus standard error of the mean) of Balb/c mice treated with G1/OX86 (positive control against OX40 without LALA mutation), G1/Lob12.3 (positive control against CD137 without LALA mutation) , G1/4420 (IgG control), combination of G1/OX86 and G1/Lob12.3, combination of mAb to OX40 G1AA/OX86 and mAb to CD137 G1AA/Lob12.3 (both with LALA mutation), FS20m-232-91/Lob12 .3 (MAT ² to OX40/CD137 without the LALA mutation) and FS20m-232-91AA/Lob12.3 (MAT ² to OX40/CD137 with the LALA mutation). The results show that treatment with anti-OX40/CD137 mAbs ² with and without the LALA mutation (FS20m-232-91AA/Lob12.3 and FS20m-232-91/Lob12.3, respectively) resulted in decreased tumor growth compared with with treatment with anti-OX40 antibody G1/OX86, anti-CD137 antibody G1/Lob12.3, a combination of these two antibodies (G1/OX86 plus G1/Lob12.3) and a combination of LALA-containing antibodies to OX40 and anti-CD137 (G1AA/OX86 plus G1AA /Lob12.3). Figure 10B shows tumor volumes (over time) of individual CT26 tumor-bearing mice that were generated by intraperitoneal injection of 3 mg/kg isotype control (clone G1AA/4420), mOX40/FITC mAb ² mock (clone FS20m-232-91AA/ 4420), MAT to mCD137 (clone G1AA/Lob12.3), combinations of mock MAT ² to mOX40/FITC and MAT to mCD137 or MAT ² to mOX40/CD137 (clone FS20m-232-91AA/Lob12.3). The horizontal dotted lines indicate the position of 0 mm ³ on the y-axis. In a qualitative analysis, mOX40/CD137 mAb ² and the combination of mOX40/FITC mock mAb ² and mCD137 mAb inhibited CT26 tumor growth in a subset of animals. Figure 10C shows the average tumor volumes (plus or minus standard error of the mean) of mice bearing the CT26 tumor individually presented in Figure 10B. The mOX40/CD137 mAb ² group had delayed early phase tumor growth (days 10-22) compared to the isotype control group. Mab to mCD137 and mock mAb ² to mOX40/FITC did not affect the rates of early phase tumor growth, either as individual agents or in combination. Figure 10D shows a Kaplan-Meier survival plot of the same CT26 tumor-bearing mice shown in Figures 10B and 10C. Survival analysis shows that treatment with mOX40/CD137 mAb ² , but not anti-mCD137 mAb and mOX40/FITC mock mAb ² , either as single agents or in combination, resulted in a statistically significant increase in survival compared with isotype control. (Pairwise comparisons were performed using the log-rank (Mantel-Cox) test; ****p≤0.0001, ns = not statistically significant.)

Figure 11 shows the antitumor activity of mouse mAb ² against OX40/CD137 in the B16-F10 syngeneic tumor model. Mice received FS20m-232-91AA/Lob12.3 (Mab ² to OX40/CD137) or G1/4420 (IgG control). The mean tumor volume plus or minus the standard error of the mean is plotted. The results show that mAb ² to OX40/CD137 was able to significantly reduce tumor growth in the syngeneic B16-F10 model compared to mice treated with the control antibody G1/4420.

Figure 12 shows the activity of mAb ² to OX40/CD137 in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody in a SEA assay. MAT ² tested was FS20-22-49AA/FS30-10-16. Controls were G1/4420 (anti-FITC), G1AA/S1 (anti-PD-L1; Figure 12A ), G1AA/5C4 (anti-PD-1; Figure 12B ), tested either in the presence or absence of mAb ² FS20-22 -49AA/FS30-10-16. The results show a concentration-dependent increase in T cell activation in the presence of FS20-22-49AA/FS30-10-16 and that addition of G1AA/S1 or G1AA/5C4 to mAb ² FS20-22-49AA/FS30-10-16 increased IL-2 release (maximal response) compared to T cells treated with MAT ² alone. No activity was observed when T cells were treated with control antibodies alone. Statistical analysis between groups G1/4420 plus FS20-22-49AA/FS30-10-16 and G1AA/S1 plus FS20-22-49AA/FS30-10-16 ( Figure 12A ) or G1/4420 plus FS20-22-49AA/ FS30-10-16 and G1AA/5C4 plus FS20-22-49AA/FS30-10-16 ( Figure 12B ) were performed using two-way ANOVA and Tukey's test for multiple comparisons, with asterisks indicating p-value (*p<0.032 , **p<0.0021, ***p<0.0002, ****p<0.0001).

Figure 13 shows the antitumor activity of mouse anti-OX40/CD137 mAb ² and PD-1 antagonist in the CT26 tumor mouse model, which were tested individually and in combination. Shown are tumor volumes in mice bearing the CT26 tumor treated with ( Figure 13A ) a combination of isotype control antibodies (G1AA/4420 and mIgG1/4420), ( Figure 13B ) anti-mouse PD-1, ( Figure 13C ) anti-OX40 mAb ² / mouse CD137 (MAT ² FS20m-232-91AA/Lob12.3) or ( Figure 13D ) a combination of anti-mouse PD-1 antibody and anti-OX40/CD137 mouse MAT ² FS20m-232-91AA/Lob12.3. The proportion of mice with regressed tumors (defined as tumor volume less than or equal to 62.5 mm ³ ) at the end of the study, 60 days after cell inoculation, is shown for each treatment group. The results show that the combination of a PD-1 antagonist antibody and FS20m-232-91AA/Lob12.3 resulted in the highest proportion of animals, 7 of 15 (47%), responding with complete tumor regression ( Figure 13D ). Mice treated with anti-PD-1 ( Figure 13B ) or FS20m-232-91AA/Lob12.3 ( Figure 13C ) as single agents showed 0% and 7% tumor regression at the end of the study, respectively. Figure 13E shows a Kaplan-Meier survival plot of CT26 tumor-bearing mice treated as described for Figures 13A-D . Survival analysis showed that the combination of FS20m-232-91AA/Lob12.3 and anti-PD-1 antibody resulted in a statistically significant survival advantage compared with isotype control antibodies (log-rank test (Mantel-Cox), p<0. 0001). No significant differences in survival were observed for single agent treatments compared with isotype control antibodies.

Figure 14 shows the dose-dependent antitumor activity of mouse mAb ² against OX40/CD137 in the CT26 syngeneic tumor model. Figure 14A shows tumor volumes of CT26 tumor-bearing mice treated by intraperitoneal (IP) injection of either 10 mg/kg isotype control antibody (G1AA/4420) or 0.1, 0.3, 1, 3, or 10 mg. /kg FS20m-232-91AA/Lob12.3. The proportion of mice with regressed tumors (defined as tumor volume less than or equal to 62.5 mm ³ ) at the end of the study, 67 days after cell inoculation, is shown for each treatment group (see top right of each graph). Results show that 0.3, 1, 3 or 10 mg/kg FS20m-232-91AA/Lob12.3 resulted in tumor regression in 4% (1/25), 4% (1/25), 8% (2 /25) and 4% (1/25) of animals at the end of the study, respectively. Tumor regression was not observed in any of the animals in the isotype control and 0.1 mg/kg FS20m-232-91AA/Lob12.3 groups. Figure 14B shows a Kaplan-Meier survival plot of CT26 tumor-bearing mice treated as described for Figure 14A. Survival analysis showed that FS20m-232-91AA/Lob12.3 at all dose levels tested resulted in a statistically significant survival benefit compared to the isotype control. Comparison of the 1 and 3 mg/kg groups and the 3 and 10 mg/kg groups revealed no statistical difference in survival. Pairwise comparisons were performed between each group and the 10 mg/kg isotype control, unless otherwise stated, using the log-rank test (Mantel-Cox); *p≤0.05, ***p≤0.0005, ****p≤0.0001, ns = not statistically significant.

Figure 15 shows a comparison of the antitumor efficacy of anti-OX40/CD137 Mab ² antibodies containing various anti-CD137 Fab clones in the CT26 syngeneic tumor model. Figure 15A shows the average volumes of CT26 tumors in BALB/c mice treated with G1/4420 (IgG control), FS20m-232-91AA/Lob12.3 (Mab ² to OX40/CD137 with CD137 agonist clone Lob12.3 cross-linking dependent ) and FS20m-232-91AA/3H3 (MAT ² to OX40/CD137 with CD137 agonist clone 3H3, independent of cross-linking). Mean tumor volumes plus or minus standard error of the mean are shown. Results show that treatment with either of the anti-OX40/CD137 Mab ² antibodies (FS20m-232-91AA/Lob12.3 or FS20m-232-91AA/3H3) resulted in reduced tumor growth compared to treatment with the isotype control antibody (G1/4420 ) and that there is no difference in the level of reduction in mice treated with FS20m-232-91AA/Lob12.3 or FS20m-232-91AA/3H3. Figure 15B shows a Kaplan-Meier survival plot of CT26 tumor-bearing mice treated as described for Figure 15A. Survival analysis showed that treatment with any of the anti-OX40/CD137 mAbs ² (FS20m-232-91AA/Lob12.3 or FS20m-232-91AA/3H3) resulted in a statistically significant survival advantage compared with treatment with an isotype control antibody (log -rank test (Mantel-Cox); p<0.05), while no differences were observed between mice receiving any of the MAbs ² to OX40/CD137.

Detailed description

Aspects and embodiments of the present invention will now be described with reference to the accompanying drawings. Additional aspects and embodiments will be apparent to those skilled in the art. All documents referred to in this text are incorporated herein by reference.

The present invention relates to antibody molecules that bind to both CD137 and OX40. In particular, the antibody molecules of the present invention contain a CDR-based antigen binding site for CD137 and an antigen binding site for OX40 located in the constant domain of the antibody molecule. The terms “CD137” and “OX40” may refer to human CD137 and human OX40, mouse CD137 and mouse OX40, and/or cynomolgus CD137 and cynomolgus OX40, unless the context otherwise requires. Preferably, the terms “CD137” and “OX40” refer to human CD137 and human OX40 unless the context requires otherwise.

The term "antibody molecule" describes an immunoglobulin, whether natural or produced partially or entirely synthetically. The antibody molecule may be human or humanized, preferably human. The antibody molecule is preferably a monoclonal antibody molecule. Examples of antibodies are isotypes of immunoglobulins, such as immunoglobulin G, and isotypic subclasses thereof, such as IgG1, IgG2, IgG3 and IgG4, as well as fragments thereof. The antibody molecule may be isolated in the sense that it does not contain impurities, such as antibodies, capable of binding other polypeptides and/or serum components.

As used herein, the term “antibody molecule” thus includes antibody fragments, provided that said fragments contain a CDR-based antigen binding site for CD137 and an antigen binding site for OX40 located in the constant domain. Thus, unless the context otherwise requires, as used herein, the term “antibody molecule” is equivalent to “antibody molecule or fragment thereof.”

To produce other antibodies or chimeric molecules that retain the specificity of the original antibody, monoclonal and other antibodies can be used and recombinant DNA technology techniques can be used. Such techniques may involve introducing CDRs or variable regions and/or constant domain sequences providing an antigen binding site for OX40 into another immunoglobulin. The introduction of a CDR of one immunoglobulin into another immunoglobulin is described, for example, in EP-A-184187, GB 2188638A and EP-A-239400. Similar techniques can be used for corresponding constant domain sequences. Alternatively, the hybridoma or other cell producing the antibody molecule may be subject to genetic mutation or other changes that may or may not alter the binding specificity of the antibodies produced.

Because antibodies can be modified in a variety of ways, the term "antibody molecule" should be interpreted to include antibody fragments, derivatives, functional equivalents, and antibody homologues, including any polypeptide containing an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Thus, chimeric molecules containing an immunoglobulin binding domain, or equivalent thereof, hybridized to another polypeptide are included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.

An example of an antibody fragment containing both CDR sequences and a CH3 domain is a minibody that contains a scFv linked to a CH3 domain (Hu et al. , 1996).

The antibody molecule of the present invention binds to CD137 and OX40. Binding in this case may refer to specific binding. The term “specific” may refer to a situation in which an antibody molecule will not show any significant binding to molecules other than its specific binding partner(s), herein CD137 and OX40. The term "specific" also applies if the antibody molecule is specific for certain epitopes, such as epitopes on CD137 and OX40, which are carried by a number of antigens, in which case the antibody molecule will be able to bind to different antigens bearing the epitope. In a preferred embodiment, the antibody molecule of the present invention does not bind or show any significant binding to TNFRSF1A, TNFRSF1B, GITR, NGFR, CD40 and/or DR6.

Antibodies and methods for their construction and use are well known in the art and are described, for example, in Holliger and Hudson 2005. To produce other antibodies or chimeric molecules that retain the specificity of the original antibody, monoclonal and other antibodies can be used and recombinant DNA technology techniques can be used. Such techniques may involve introducing CDRs or variable regions of one antibody molecule into another antibody molecule (EP-A-184187, GB 2188638A and EP-A-239400).

A CDR-based antigen binding site is an antigen binding site in the variable region of an antibody. The CDR-based antigen binding site may be formed by three CDRs, such as three light chain variable domain (VL) CDRs or three heavy chain variable domain (VH) CDRs. Preferably, the CDR-based antigen binding site is formed by six CDRs, three VL CDRs and three VH CDRs. The contribution of different CDRs to antigen binding may vary at different antigen-binding sites.

The three CDR domains of the VH antigen binding site may be located within the VH domain of an immunoglobulin, and the three CDRs of the VL domain may be located within the VL domain of the immunoglobulin. For example, a CDR-based antigen binding site may be located in the variable region of an antibody.

The antibody molecule may have one, or preferably more than one, for example two, antigen binding sites based on the CDR for the first antigen. Thus, an antibody molecule may contain one VH domain and one VL domain, but preferably contains two VH domains and two VL domains, that is, two VH/VL domain pairs, as in the case of natural IgG molecules, by way of example.

The CDR-based antigen binding site may comprise three CDR VH or three CDR VL, preferably three CDR VH and three CDR VL antibodies FS30-10-16, FS30-10-3, FS30-10-12 or FS30-35-14, or FS30 -5-37 , preferably FS30-10-16 antibodies.

The sequences of the VH and VL domains of these antibodies are presented below:

(i) the VH and VL domain sequences for SEQ ID NO FS30-10-16 are shown in SEQ ID NOs 12 and 14 , respectively;

(ii) the VH and VL domain sequences for SEQ ID NO FS30-10-3 are shown in SEQ ID NOs 18 and 14 , respectively;

(iii) the VH and VL domain sequences for SEQ ID NO FS30-10-12 are shown in SEQ ID NOs 23 and 14 , respectively;

(iv) the VH and VL domain sequences for SEQ ID NO FS30-35-14 are shown in SEQ ID NO 170 and 172 , respectively; And

(v) the sequences of the VH and VL domains for SEQ ID NO FS30-5-37 are shown in SEQ ID NO 40 and 42 , respectively.

One of skill in the art can easily determine the CDR sequences from the VH and VL domain sequences of the antibodies presented above. CDR sequences can be determined, for example, according to Kabat (Kabat et al. , 1991) or the international information system ImMunoGeneTics (IMGT) (Lefranc et al. , 2015).

The CDR1, CDR2 and CDR3 sequences of the VH domain of the antibody molecule according to IMGT numbering may be the sequences located at positions 27-38, 56-65 and 105-117 of the VH domain of the antibody molecule, respectively.

The CDR1, CDR2 and CDR3 sequences of the VH domain of an antibody molecule according to Kabat numbering may be the sequences located at positions 31-35, 50-65 and 95-102 of the VH domain, respectively.

The CDR1, CDR2 and CDR3 sequences of the VL domain of an antibody molecule according to IMGT numbering may be the sequences located at positions 27-38, 56-65 and 105-117 of the VL domain, respectively.

The CDR1, CDR2 and CDR3 sequences of the VL domain of an antibody molecule according to Kabat numbering may be the sequences located at positions 24-34, 50-56 and 89-97 of the VL domain, respectively.

For example, an antibody molecule may contain the VH domain CDR1, CDR2 and CDR3 sequence from:

(i) SEQ ID NOs 1, 2 and 3 , respectively [ FS30-10-16 ];

(ii) SEQ ID NOs 1, 2 and 16 , respectively [ FS30-10-3 ];

(iii) SEQ ID NOs 1, 2 and 21 , respectively [ FS30-10-12 ];

(iv) SEQ ID NOs 25, 26 and 27 , respectively [ FS30-35-14 ]; or

(v) SEQ ID NOs 33, 34 and 35 , respectively [ FS30-5-37 ],

wherein said CDR sequences are defined in accordance with the ImMunoGeneTics (IMGT) numbering scheme.

The antibody molecule may contain the sequence of CDR1, CDR2 and CDR3 of the VH domain from:

(i) SEQ ID NOs 7, 8 and 9 , respectively [ FS30-10-16 ];

(ii) SEQ ID NOs 7, 8 and 17 , respectively [ FS30-10-3 ];

(iii) SEQ ID NOs 7, 8 and 22 , respectively [ FS30-10-12 ];

(iv) SEQ ID NOs 29, 30 and 31 , respectively [ FS30-35-14 ]; or

(v) SEQ ID NOs 37, 38 and 39 , respectively [ FS30-5-37 ],

wherein said CDR sequences are defined in accordance with the Kabat numbering scheme.

For example, an antibody molecule may contain the VL domain CDR1, CDR2 and CDR3 sequence from:

(i) SEQ ID NOs 4, 5 and 6 , respectively [ FS30-10-16 ];

(ii) SEQ ID NOs 4, 5 and 6 , respectively [ FS30-10-3 ];

(iii) SEQ ID NOs 4, 5 and 6 , respectively [ FS30-10-12 ];

(iv) SEQ ID NOs 4, 5 and 28 , respectively [ FS30-35-14 ]; or

(v) SEQ ID NOs 4, 5 and 36 , respectively [ FS30-5-37 ],

wherein said CDR sequences are defined in accordance with the ImMunoGeneTics (IMGT) numbering scheme.

For example, an antibody molecule may contain the VL domain CDR1, CDR2 and CDR3 sequence from:

(i) SEQ ID NOs 10, 11 and 6 , respectively [ FS30-10-16 ];

(ii) SEQ ID NOs 10, 11 and 6 , respectively [ FS30-10-3 ];

(iii) SEQ ID NOs 10, 11 and 6 , respectively [ FS30-10-12 ];

(iv) SEQ ID NOs 10, 11 and 28 , respectively [ FS30-35-14 ]; or

(v) SEQ ID NOs 10, 11 and 36 , respectively [ FS30-5-37 ],

wherein said CDR sequences are defined in accordance with the Kabat numbering scheme.

The VH and VL sequences of antibodies FS30-10-16 , FS30-10-3 and FS30-10-12 are identical except for the residue at position 109 VH according to the IMGT numbering scheme (residue 97 VH according to the Kabat numbering scheme). Thus, the antibody molecule may comprise VH domain CDR1, CDR2 and CDR3 sequences and/or VL domain CDR1, CDR2 and CDR3 sequences, a VH domain sequence and/or a VL domain sequence, FS30-10-16 antibody, wherein said antibody molecule optionally contains an amino acid substitution at position 109 of the heavy chain in accordance with the IMGT numbering scheme (residue 97 of the heavy chain in accordance with the Kabat numbering scheme), wherein said residue at said position is preferably selected from the group consisting of asparagine (N), threonine (T) and leucine (L).

The CDR-based antigen binding site may comprise VH or VL domains, preferably VH and VL domains, antibodies FS30-10-16 , FS30-10-3 , FS30-10-12 , FS30-35-14 or FS30-5-37 , preferably FS30-10-16 , FS30-10-3 , FS30-10-12 or FS30-35-14 antibodies, more preferably FS30-10-16 , FS30-10-3 or FS30-10-12 antibodies, most preferably FS30 antibodies -10-16 .

The VH domain of antibodies FS30-10-16 , FS30-10-3 , FS30-10-12 , FS30-35-14 and FS30-5-37 may have the sequence shown under SEQ ID NOs 12, 18, 23, 170 and 40 , respectively. The VL domain of antibodies FS30-10-16 , FS30-10-3 , FS30-10-12 , FS30-35-14 and FS30-5-37 may have the sequence shown under SEQ ID NOs 14, 14, 14, 172 and 42 , respectively.

The antibody molecule of the present invention contains an antigen binding site for OX40 located in the constant domain of the antibody molecule. The constant domain may be a CL, CH1, CH2, CH3 or CH4 domain, preferably the constant domain is a CH1, CH2 or CH3 domain, more preferably a CH2 or CH3 domain, most preferably a CH3 domain.

The amino acid residue positions of the constant domain are numbered herein in accordance with the ImMunoGeneTics (IMGT) numbering scheme unless otherwise noted. The IMGT numbering scheme is described in Lefranc et al. ,Dev. Comp. Immunol., 29, 185-203 (2005).

The antigen binding site for OX40 may comprise a first, second and third sequence located in the first, second and third structural loop of the constant domain, respectively. The construction of antibody constant domain structural loops to create antigen binding sites for target antigens is known in the art and is described, for example, in Wozniak-Knopp et al. , 2010, and patent publications No. WO2006/072620 and WO2009/132876. Preferably, the first, second and third structural loops are the AB, CD and EF structural loops of the CH3 domain of the antibody molecule, respectively. In the CH3 domain, the structural loops AB, CD, and EF are located at residues 11–18, 43–78, and 92–101 of the CH3 domain, respectively. Modification of structural loop sequences of antibody constant domains to create new antigen binding sites is described, for example, in WO2006/072620 and WO2009/132876.

In a preferred embodiment, the antigen binding site for the OX40 antibody molecule comprises a first, second and third sequence:

(i) FS20-22-49 , listed under SEQ ID NOs 51, 52 and 53 , respectively;

(ii) FS20-22-38 , listed under SEQ ID NOs 51, 59 and 60 , respectively;

(iii) FS20-22-41 listed under SEQ ID NOs 51, 52 and 60 , respectively;

(iv) FS20-22-47 , listed under SEQ ID NOs 51, 52 and 65 , respectively; or

(v) FS20-22-85 , listed under SEQ ID NOs 51, 52 and 68 , respectively.

The antigen binding site for OX40 may contain AB, CD and EF structural loop sequences FS20-22-49, FS20-22-38, FS20-22-41, FS20-22-47 or FS20-22-85 , wherein said structural loops AB, CD and EF are sequences located at residues 11-18, 43-78 and 92-101 of the CH3 domain, respectively, and the CH3 domain FS20-22-49, FS20-22-38, FS20-22-41, FS20-22 -47 or FS20-22-85 is shown in SEQ ID NO: 54, 61, 63, 66 and 69 , respectively.

In a more preferred embodiment, the antigen binding site for the OX40 antibody molecule comprises the first, second and third sequence FS20-22-49 set forth in SEQ ID NOs 51, 52 and 53 , respectively. For example, the antigen binding site for OX40 may contain the structural loop sequences AB, CD and EF FS20-22-49 listed under SEQ ID NOs 56, 57 and 58 , respectively.

If the antigen binding site for OX40 of the antibody molecule contains the first, second and third sequence FS20-22-38 , FS20-22-41 , FS20-22-47 , FS20-22-49 or FS20-22-85, said first, second and third the sequence is preferably located at positions 14 to 18, 45.1 to 77, and 93 to 101 of the CH3 domain of the antibody molecule, respectively.

If the antigen binding site for OX40 contains the AB, CD and EF structural loop sequences FS20-22-38 , FS20-22-41 , FS20-22-47 , FS20-22-49 or FS20-22-85 , said AB structural loop sequences, CD and EF are preferably located at positions 11 to 18, 43 to 78, and 92 to 101 of the CH3 domain of the antibody molecule, respectively.

The antibody molecule may further contain leucine (L) at position 91 of the CH3 domain of the antibody molecule. In particular, an antibody molecule containing an antigen binding site for OX40 containing the first, second and third sequence FS20-22-85 may contain a leucine at position 91 of the CH3 domain of the antibody molecule.

In an alternative embodiment, the antigen binding site for the OX40 antibody molecule comprises a first, second, and third sequence:

(i) FS20-31-58 , listed under SEQ ID NOs 71, 72 and 73 , respectively;

(ii) FS20-31-66 , listed under SEQ ID NOs 71, 72 and 76 , respectively;

(iii) FS20-31-94 , listed under SEQ ID NOs 79, 80 and 81 , respectively;

(iv) FS20-31-102 , listed under SEQ ID NOs 84, 85 and 76 , respectively;

(v) FS20-31-108 , listed under SEQ ID NOs 84, 88 and 89 , respectively; or

(vi) FS20-31-115 , listed under SEQ ID NOs 84, 92 and 89 , respectively.

The antigen binding site for OX40 may contain the structural loop sequences AB, CD and EF FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108 or FS20-31-115 . wherein said structural loops AB, CD and EF are sequences located at residues 11-18, 43-78 and 92-101 of the CH3 domain, respectively, and the CH3 domain FS20-31-58, FS20-31-66, FS20-31 -94, FS20-31-102, FS20-31-108 or FS20-31-115 is shown in SEQ ID NO: 54, 61, 63, 66 and 69 , respectively.

If the antigen binding site for OX40 of an antibody molecule contains the first, second and third sequence FS20-31-58 , FS20-31-66 , FS20-31-94 , FS20-31-102, FS20-31-108 or FS20-31-115 , said first, second and third sequences are preferably located at positions 14 to 18, 45.1 to 77 and 92 to 101 of the CH3 domain of the antibody molecule, respectively.

If the antigen binding site for OX40 contains the AB, CD and EF structural loop sequences FS20-31-58 , FS20-31-66 , FS20-31-94 , FS20-31-102, FS20-31-108 or FS20-31-115 , these structural loop sequences AB, CD and EF are preferably located at positions 11 to 18, 43 to 78 and 92 to 101 of the CH3 domain of the antibody molecule, respectively.

As an alternative to IMGT numbering, the positions of amino acid residues in the constant domain, including the positions of amino acid sequences, substitutions, deletions and insertions described herein, can be numbered according to IMGT exon numbering (also called sequential numbering), EC numbering or Kabat numbering. The correspondence between IMGT numbering, IMGT exon numbering, EC numbering, and Kabat numbering of CH3 domain residue positions is shown in Figure 1 .

Thus, for example, in the case where the present application relates to the first, second and third sequences located at positions 14 to 18, 45.1 to 77 and 93 to 101 of the CH3 domain of an antibody molecule, respectively, said residue positions being numbered in accordance with the IMGT numbering scheme, said first, second and third sequences are located at positions 18 to 22, 46 to 50 and 74 to 82 of the CH3 domain, said residue positions being numbered in accordance with the IMGT exon numbering scheme, as shown in Figure 1 .

In one embodiment, the antibody molecule comprises a CH3 domain that contains, has, or consists of the CH3 domain sequence FS20-22-38, FS20-22-41 , FS20-22-47 , FS20-22-49, FS20-22-85 . FS20-31-58 , FS20-31-66 , FS20-31-94 , FS20-31-102, FS20-31-108 or FS20-31-115 , wherein the CH3 domain sequence is FS20-22-38, FS20-22 -41 , FS20-22-47 , FS20-22-49, FS20-22-85 , FS20-31-58 , FS20-31-66 , FS20-31-94 , FS20-31-102, FS20-31-108 and FS20-31-115 are shown in SEQ ID NOs 54, 61, 63, 66, 69, 74, 77, 82, 86, 90 and 93 , respectively.

In a preferred embodiment, the antibody molecule comprises a CH3 domain that contains, has, or consists of the CH3 domain sequence FS20-22-49 set forth in SEQ ID NO 54 .

The CH3 domain of an antibody molecule may optionally contain an additional lysine (K) residue at the very C-terminus of the CH3 domain sequence.

In addition, the antibody molecule of the present invention may contain the CH2 domain of an immunoglobulin G molecule, such as the CH2 domain of an IgG1, IgG2, IgG3 or IgG4 molecule. Preferably, the antibody molecule of the present invention contains the CH2 domain of an IgG1 molecule. The CH2 domain may have the sequence set forth in SEQ ID NO: 48 .

The CH2 domain of an antibody molecule may contain one or more mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIII, and/or complement. The present inventors assume that reducing or eliminating binding to Fcγ receptors will reduce or eliminate ADCC mediated by the antibody molecule. Likewise, reduction or elimination of complement binding is expected to reduce or eliminate complement-dependent cytotoxicity (CDC) mediated by the antibody molecule. Mutations to reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors and/or complement are known in the art (Wang et al. , 2018). These mutations include the “LALA mutation” described in Bruhns et al. , 2009 and Hezareh et al. , 2001, which involves replacing leucine residues at positions 1.3 and 1.2 of the IMGT CH2 domain with alanine (L1.3A and L1.2A). Alternatively, generate α-glycosyl antibodies by mutating the conserved N-linked glycosylation site by mutating the asparagine (N) at position 84.4 of the IMGT CH2 domain to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q), as is also known to reduce IgG1 effector function (Wang et al. , 2018). As another alternative, complement activation (C1q binding) and ADCC are known to be reduced by mutation of the proline at position 114 of the IMGT CH2 domain to alanine or glycine (P114A or P114G) (Idusogie et al. , 2000; Klein et al. , 2016 ). These mutations can also be combined to produce antibody molecules with or without further reduced ADCC or CDC activity.

Thus, an antibody molecule may contain a CH2 domain, said CH2 domain comprising:

(i) alanine residues at positions 1.3 and 1.2; and/or

(ii) alanine or glycine at position 114; and/or

(iii) alanine, glutamine or glycine at position 84.4;

wherein the specified numbering of amino acid residues corresponds to the IMGT numbering scheme.

In a preferred embodiment, the antibody molecule comprises a CH2 domain, wherein said CH2 domain comprises:

(i) an alanine residue at position 1.3; And

(ii) an alanine residue at position 1.2;

wherein the specified numbering of amino acid residues corresponds to the IMGT numbering scheme.

For example, the CH2 domain may have the sequence shown under SEQ ID NO: 49 .

In an alternative preferred embodiment, the antibody molecule comprises a CH2 domain, wherein said CH2 domain comprises:

(i) an alanine residue at position 1.3;

(ii) an alanine residue at position 1.2; And

(iii) alanine at position 114;

wherein the specified numbering of amino acid residues corresponds to the IMGT numbering scheme.

For example, the CH2 domain may have the sequence shown under SEQ ID NO: 50 .

In a preferred embodiment, the antibody molecule that binds to CD137 and OX40 comprises

(a) antigen binding site based on the CDR for CD137; And

(b) an antigen binding site for OX40 located in the CH3 domain of the antibody molecule;

wherein said CDR-based antigen binding site contains three CDR VH and three CDR VL (CDR 1-6) antibodies FS30-10-16 , FS30-10-3 , FS30-10-12 , FS30-35-14 , or FS30-5 -37 , preferably FS30-10-16 , FS30-10-3 or FS30-10-12 , more preferably FS30-10-16 or FS30-10-3 , most preferably FS30-10-16 ; And

wherein said antigen binding site for OX40 comprises a first sequence, a second sequence and a third sequence located in the structural loops AB, CD and EF of the CH3 domain, respectively, wherein said first, second and third sequences have the sequence FS20-22-49 indicated under SEQ ID NO 51, 52 and 53 , respectively.

In another preferred embodiment, an antibody molecule that binds to CD137 and OX40 comprises

(a) antigen binding site based on the CDR for CD137; And

(b) a CH3 domain that contains, has, or consists of the sequence set forth in SEQ ID NO: 54 [FS20-22-49] ;

wherein said CDR-based antigen binding site contains three CDR VH and three CDR VL (CDR 1-6) antibodies FS30-10-16 , FS30-10-3 , FS30-10-12 , FS30-35-14 , or FS30-5 -37 , preferably FS30-10-16 , FS30-10-3 or FS30-10-12 , more preferably FS30-10-16 or FS30-10-3 , most preferably FS30-10-16 .

In another preferred embodiment, an antibody molecule that binds to CD137 and OX40 comprises

(a) a VH domain and a VL domain containing an antigen binding site based on the CDR for CD137; And

(b) a CH3 domain that contains, has, or consists of the sequence set forth in SEQ ID NO: 54 [FS20-22-49] ;

wherein said VH and VL domain contains, has or consists of a VH and VL antibody FS30-10-16 , FS30-10-3 , FS30-10-12 , FS30-35-14 or FS30-5-37 , preferably FS30-10 -16 , FS30-10-3 or FS30-10-12 , more preferably FS30-10-16 or FS30-10-3 , most preferably FS30-10-16 .

In another preferred embodiment, the antibody molecule that binds to CD137 and OX40 contains a heavy chain that contains, has, or consists of a heavy chain and a light chain of the antibody:

(i) FS20-22-49AA/FS30-10-16, set forth in SEQ ID NOs 95 and 97 , respectively;

(ii) FS20-22-49AA/FS30-10-3, shown in SEQ ID NOs 99 and 97 , respectively;

(iii) FS20-22-49AA/FS30-10-12, shown in SEQ ID NOs 103 and 97 , respectively;

(iv) FS20-22-49AA/FS30-35-14, shown in SEQ ID NOs 105 and 107 , respectively; or

(v) FS20-22-49AA/FS30-5-37, shown in SEQ ID NOs 109 and 111 , respectively;

wherein said antibody molecule preferably contains a light chain and a heavy chain presented in (i)-(iv), more preferably contains a light chain and a heavy chain presented in (i)-(iii), most preferably contains a light chain and a heavy chain, presented in (i).

The antibody molecules of the present invention may also contain variants of the first, second or third sequence, AB, CD or EF structural loop sequence, CH3 domain, CH2 domain, CH2 and CH3 domain, CDR, VH domain, VL domain, light chain sequences and/or heavy chain disclosed in this application. Suitable variants can be obtained using sequence alteration or mutation methods and screening. In a preferred embodiment, an antibody molecule comprising one or more sequence variants retains one or more functional characteristics of the original antibody molecule, such as binding specificity and/or binding affinity for CD137 and OX40. For example, an antibody molecule containing one or more sequence variants preferably binds to CD137 and/or OX40 with the same or higher affinity than the (original) antibody molecule. A parent antibody molecule is an antibody molecule that does not contain the amino acid substitution(s), deletion(s) and/or insertion(s) that were included in the variant antibody molecule.

For example, an antibody molecule of the present invention may comprise a first, second or third sequence, an AB, CD or EF structural loop sequence, a CH3 domain, a CH2 domain, a CH2 and CH3 domain, a CDR, a VH domain, a VL domain, a light chain sequence and/or heavy chain which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with structural loop, CH3 domain, CH2 domain, CH2 domain and CH3, CDR, VH domain, VL domain, light chain or heavy chain sequence disclosed herein.

In a preferred embodiment, the antibody molecule of the present invention contains a CH3 domain sequence that has at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least At least 99.9% sequence identity with the CH3 domain sequence presented in SEQ ID NO: 54 [FS20-22-49] .

In another preferred embodiment, the antibody molecule has or contains a CH2 domain sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99 ,1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7 %, at least 99.8%, or at least 99.9% sequence identity with the CH2 domain sequence set forth in SEQ ID NO: 48 or 49 .

Sequence identity is usually determined by reference to the GAP algorithm (Wisconsin GCG package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Typically the default parameters are used with a gap creation penalty of 12 and a gap expansion penalty of 4. Using GAP may be preferred, but other algorithms may be used, for example BLAST (which uses the method of Altschul et al. (1990)), FASTA ( which uses the method of Pearson and Lipman (1988)), or the Smith-Waterman algorithm (Smith and Waterman (1981), or the TBLASTN program, Altschul et al. 1990, above, usually using default parameters. In particular, the psi algorithm can be used -Blast (Altschul et al., 1997).

The antibody molecule of the present invention may also contain a first, second or third sequence, an AB, CD or EF structural loop sequence, a CH3 domain, a CH2 domain, a CH2 and CH3 domain, a CDR, a VH domain, a VL domain, a light chain and/or a heavy chain. which has one or more amino acid sequence changes (adding, deleting, substituting and/or insertion of an amino acid residue), preferably 20 changes or less, 15 changes or less, 10 changes or less, 5 changes or less, 4 changes or less, 3 changes or less, 2 changes or less, or 1 change from the first, second or third sequence, structural loop sequence AB, CD or EF, CH3 domain, CH2 domain, CH2 and CH3 domain, Fcab, CDR, VH domain, VL domain , the light chain or heavy chain sequence disclosed herein. In particular, changes may be made in one or more framework regions of the antibody molecule outside the VH and VL domain sequences and/or in one or more framework regions of the CH3 domain. For example, changes may be present in the CH3 domain beyond the sequences described herein as the first, second and third sequences or as the structural loop sequences AB, CD or EF.

In a preferred embodiment, the antibody molecule of the present invention may comprise a CH3 domain sequence with one or more amino acid sequence changes (adding, deleting, substituting and/or insertion of an amino acid residue), preferably 20 changes or less, 15 changes or less, 10 changes or less , 5 changes or less, 4 changes or less, 3 changes or less, 2 changes or less, or 1 change compared to the CH3 domain sequence shown in SEQ ID NO: 54, 61, 63, 66, 69, 74, 77, 82, 86, 90 or 93 .

In another preferred embodiment, the antibody molecule comprises a CH2 domain sequence with one or more amino acid sequence changes (adding, deleting, substituting and/or inserting an amino acid residue), preferably 20 changes or less, 15 changes or less, 10 changes or less, 5 changes or less, 4 changes or less, 3 changes or less, 2 changes or less, or 1 change compared to the CH2 domain sequence shown in SEQ ID NO: 48 or 49 .

In preferred embodiments in which one or more amino acids are replaced by another amino acid, the substitutions may be conservative substitutions, for example, in accordance with the following Table. In some embodiments, amino acids in the same category in the middle column are replaced with each other, i.e., for example, a non-polar amino acid is replaced with another non-polar amino acid. In some embodiments, the amino acids on the same line in the rightmost column are replaced with each other.

Aliphatic Non-polar G A P I L V Polar - uncharged C S T M NQ Polar - charged D E K R Aromatic H F W Y

In some embodiments, the replacement(s) may be functionally conservative(s). In other words, in some embodiments, the substitution may not affect (or may not significantly affect) one or more functional properties (eg, binding affinity) of the antibody molecule containing the substitution compared to an equivalent unsubstituted antibody molecule.

The antibody molecule preferentially binds to human CD137 and human OX40. Preferably, the antibody molecule is capable of simultaneously binding to human CD137 and human OX40, wherein human CD137 and human OX40 are co-expressed. Co-expression here includes situations in which CD137 and OX40 are expressed on the same cell, such as an immune cell such as a T cell, and situations in which CD137 and OX40 are expressed on different cells, such as two different cells. immune cells located next to each other in the tumor microenvironment. Thus, it is believed that the antibody molecules of the present invention are capable of binding to both targets on the same cell under cis conditions, and are also capable of binding to two targets expressed on different cells under trans conditions.

The antibody molecule preferentially binds to dimeric human CD137 with an affinity ( _KD ) of 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM, or 0. 3 nM or higher affinity. Preferably, the antibody molecule binds to human CD137 with an affinity ( _KD ) of 0.3 nM or higher affinity. The antibody molecule can bind dimeric CD137 with higher affinity than monomeric CD137. Human CD137 may have the sequence, for example, set forth under SEQ ID NO: 127 .

The antibody molecule preferentially binds to dimeric human OX40 with an affinity ( _KD ) of 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM, or 0. 3 nM or higher affinity. Preferably, the antibody molecule binds to human OX40 with an affinity ( _KD ) of 0.3 nM or higher affinity. The antibody molecule can bind dimeric OX40 with higher affinity than monomeric OX40. Human OX40 may have a sequence such as that shown under SEQ ID NO: 130 .

The antibody molecule preferentially binds to cynomolgus CD137 and cynomolgus OX40. Binding to cynomolgus CD137 and OX40, as well as human CD137 and OX40, is advantageous as it allows the efficacy and toxicity of the antibody molecule to be tested in cynomolgus monkeys before administration to humans. Preferably, the antibody molecule is capable of simultaneously binding to cynomolgus CD137 and cynomolgus OX40, wherein cynomolgus CD137 and cynomolgus OX40 are co-expressed.

The antibody molecule preferentially binds to dimeric cynomolgus CD137 with affinity ( _KD ) 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM, or 0.3 nM, or higher affinity. Preferably, the antibody molecule binds to dimeric cynomolgus CD137 with an affinity ( _KD ) of 0.3 nM or higher affinity. Cynomolgus CD137 may have a sequence such as that shown under SEQ ID NO: 129 .

The antibody molecule preferentially binds to dimeric cynomolgus OX40 with an affinity ( _KD ) of 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM or 1 nM or s higher affinity. Preferably, the antibody molecule binds to cynomolgus OX40 with an affinity ( _KD ) of 1 nM or higher affinity. Cynomolgus OX40 may have a sequence such as that shown under SEQ ID NO: 131 .

The antibody molecule preferentially binds to dimeric cynomolgus OX40 with an affinity ( _KD ) that is in the range of 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, or 5-fold affinity ( _KD ), with in which the antibody molecule binds to dimeric human OX40. Preferably, the antibody molecule binds to cynomolgus dimeric OX40 with an affinity ( _KD ) that is within 5-fold of the affinity ( _KD ) with which the antibody molecule binds to human dimeric OX40.

The antibody molecule preferentially binds to dimeric cynomolgus CD137 with an affinity (K _D ) that is in the range of 30-fold, 20-fold, 10-fold, 5-fold, 4-fold, 3-fold, or 2-fold affinity (K _D ), to which the antibody molecule binds to dimeric human CD137. Preferably, the antibody molecule binds to dimeric cynomolgus CD137 with an affinity (K _D ) that is within 2-fold of the affinity (K _D ) with which the antibody molecule binds to dimeric human CD137.

As described in the Examples herein, it is believed that the similarity in binding to human and cynomolgus antigens may be an advantage in that it is hoped that the behavior of Mab ² in cynomolgus studies can be extrapolated to humans. This is believed to be favorable for conducting efficacy and toxicity studies using the antibody molecule in cynomolgus monkeys, which may have predictive value regarding the efficacy and toxicity of the antibody molecule in humans.

The antibody molecule preferentially binds to dimeric human CD137 with an affinity ( _KD ) that is within the 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, or 5-fold affinity ( _KD ) with which the antibody molecule binds to dimeric human OX40. Preferably, the antibody molecule binds to dimeric human CD137 with an affinity (K _D ) that is within 2-fold the affinity (K _D ) with which the antibody molecule binds to dimeric human OX40.

The antibody molecule preferentially binds to dimeric cynomolgus CD137 with an affinity (K _D ) that is in the range of 10-fold, 9-fold, 8-fold, 7-fold, 6-fold or 5-fold affinity (K _D ), with in which the antibody molecule binds to the dimeric OX40 of the cynomolgus monkey.

As described in the Examples herein, it is believed that an antibody molecule having similar binding affinity to both targets, i.e., CD137 and OX40, may provide an advantage because the antibody molecule is more likely to bind to cells that express both targets.

The binding affinity of an antibody molecule to a cognate antigen such as human OX40, human CD137, cynomolgus OX40, or cynomolgus CD137 can be determined using surface plasmon resonance (SPR), such as Biacore, as an example. The binding affinity of an antibody molecule to OX40 or CD137 expressed on the cell surface can be determined using flow cytometry.

Antibody molecules have been shown to have several ligand binding activities. For example, the antibody molecule may be able to block, may not be able to block, or may be able to partially block the binding of CD137L to CD137.

Preferably, the antibody molecule may be able to block, may not be able to block, or may be able to partially block the binding of CD137L to CD137. More preferably, the antibody molecule is capable of partially blocking the binding of CD137L to CD137.

Preferably, the antibody molecule is capable of inducing OX40 and/or CD137 signaling through cross-linking by double binding to both OX40 and CD137 when the two targets are co-expressed. Based on this mode of action, such antibody molecules are called “dual agonists,” i.e., the antibody molecules are capable of inducing receptor signaling through cross-linking by double binding to both OX40 and CD137. Thus, preferably the antibody molecule is capable of causing a dual agonist effect when both OX40 and CD137 are co-expressed. As described herein, such dual agonists are expected to provide benefits. For example, it is believed that such a dual agonist may be able to produce a stronger stimulation of the immune response, since it can combine the activation of different immune cells, for example, combine the activity of CD8 ⁺ and CD4 ⁺ T cells by binding to both targets on different cells in trans - conditions. As another example, it is believed that such a dual agonist may be capable of leading to activation of a single cell co-expressing both targets, without the need for interaction between the two cells, by binding to both targets in cis conditions.

More preferably, the dual agonist should be capable of triggering agonist effects autonomously by simultaneously binding to its specific targets (OX40 and CD137) and without the need for additional cross-linking, such as cross-linking agents or Fcγ receptors. As described herein, such autonomous activity is expected to provide the benefit of being restricted to sites where both targets are co-expressed and is therefore expected to reduce the toxicity potentially associated with CD137 activation at sites where in which there is little or no OX40 co-expression.

The ability of an antibody molecule to activate T cells can be measured using a T cell activation assay. T cells release IL-2 when activated. Thus, a T cell activation assay can measure the release of IL-2 to determine the level of T cell activation induced by the antibody molecule.

For example, the ability of an antibody molecule to activate T cells is determined by measuring the concentration of the antibody molecule required to achieve half-maximal IL-2 release by T cells in a T cell activation assay. Hereinafter it is called EC ₅₀ .

In a preferred embodiment, the antibody molecule has an EC₅₀ in a T cell activation assay that is within 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, or 5x EC₅₀ FS20-22-49AA/FS30-10-16 in the same analysis, andFS20-22-49AA/FS30-10-16 consists of a heavy chain having SEQ ID NO:95, and a light chain having SEQ ID NO:97.

For example, an antibody molecule may have an EC ₅₀ in a T cell activation assay of 30 nM or less, 25 nM or less, 20 nM or less, 14 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1.5 nM, 1 nM or 0.5 nM or less, preferably 1.5 nM or less, more preferably 1 nM or less when cross-linked.

In addition or alternatively, the ability of an antibody molecule to activate T cells can be determined by measuring the maximum concentration of IL-2 released by T cells in a T cell activation assay in the presence of the antibody molecule.

In a preferred embodiment, the maximum concentration of IL-2 released by T cells in the T cell activation assay in the presence of the antibody molecule is within 20% or 10% of the maximum concentration of IL-2 released by T cells in the presence of FS20-22- 49AA / FS30-10-16 in the same assay, with FS20-22-49AA / FS30-10-16 consisting of a heavy chain having SEQ ID NO: 95 and a light chain having SEQ ID NO: 97 .

The T cell activation assay preferably contains T cells co-expressing OX40 and CD137. In a preferred embodiment, the T cell activation assay does not contain any agents capable of cross-linking antibody molecules other than CD137 and OX40.

The T cell activation assay may be a T cell assay described herein, such as a total T cell population assay, a CD4 ⁺ T cell assay, or a CD8 ⁺ T cell assay described in the Examples herein.

For example, the T cell activation assay may be an IL-2 release assay based on T cells isolated from peripheral blood mononuclear cells (PBMCs). The CD4 ⁺ T cell activation assay or the CD8 ⁺ T cell activation assay may be an IL-2 release assay based on CD4 ⁺ T cells or CD8 ⁺ T cells isolated from human PBMC, respectively. As explained in the Examples herein, an antibody molecule that is capable of activating T cells in both a CD4 ⁺ T cell assay and a CD8 ⁺ T cell assay is capable of activating both OX40 and CD137 (also called a “dual agonist”). "). For example, a T cell activation assay may involve isolating human PBMCs from cones to deplete leukocytes. Methods for isolating PBMCs are known in the art and are described in the examples in this application. T cells can then be isolated from the PBMCs. Methods for isolating T cells (total T cells, CD4 ⁺ T cells or CD8 ⁺ T cells) from PBMCs are also known in the art and are described in the Examples herein.

The activation assay may involve obtaining a desired number of T cells, for example, in experimental media such as T cell media. The required number of T cells can be obtained at a concentration of 1.0×10 ⁶ cells/ml. The T cells can then be stimulated using a suitable T cell activation reagent that provides the signals required for T cell activation. For example, the T cell activation reagent may be a reagent containing CD3 and CD28, such as beads containing CD3 and CD28. Isolated T cells can be incubated overnight with a T cell activation reagent to activate the T cells. The activated T cells can then be washed to separate the T cells from the T cell activation reagent and resuspended in T cell medium at a suitable concentration, such as 2.0 x 10 ⁶ cells/ml. The activated T cells can then be added to plates coated with anti-human CD3 antibody.

An appropriate dilution of each antibody molecule to be tested can be prepared and added to the wells. The T cells can then be incubated at 37°C, 5% CO ₂ for 24 hours with the antibody to be tested. Supernatants can be collected and analyzed to determine the concentration of IL-2 in the supernatant. Methods for determining the concentration of IL-2 in a solution are known in the art and are described in the examples in this application. The concentration of human IL-2 can be plotted against the logarithmic concentration of the antibody molecule. The resulting curves can be fitted using the log(agonist) response equation.

The antibody molecule may be conjugated to a bioactive molecule or detectable label. In this case, the antibody molecule may be called a conjugate. Such conjugates can be used in the treatment of diseases described in this application.

For example, the bioactive molecule may be an immune system modulator such as a cytokine, preferably a human cytokine. For example, the cytokine may be a cytokine that stimulates T cell activation and/or proliferation. Examples of cytokines for conjugation with the antibody molecule include IL-2, IL-10, IL-12, IL-15, IL-21, GM-CSF and IFN-gamma.

Alternatively, the bioactive molecule may be a ligand decoy, such as a cytokine ligand decoy, such as TGF-beta or IL-6.

Alternatively, the bioactive molecule may be a therapeutic radioisotope.

Radioimmunotherapy is used in the treatment of cancer, as an example. Therapeutic radioisotopes suitable for radioimmunotherapy are known in the art and include yttrium-90, iodine-131, bismuth-213, astatine-211, lutetium-177, rhenium-188, copper-67, actinium-225 and iodine-125, and terbium -161.

Suitable detectable labels that can be conjugated to antibody molecules are known in the art and include radioisotopes such as iodine-125, iodine-131, yttrium-90, indium-111 and technetium-99; fluorochromes such as fluorescein, rhodamine, phycoerythrin, Texas red and cyanine dye derivatives such as Cy7 and Alexa750; chromogenic dyes such as diaminobenzidine; latex granules; enzyme tags such as horseradish peroxidase; phosphor or laser dyes with spectrally isolated absorption or emission characteristics; and chemical groups, such as biotin, which can be detected by binding to a specific cognate detectable group, such as labeled avidin.

The antibody molecule can be conjugated to a bioactive molecule or detectable label using any suitable covalent or non-covalent bond, such as a disulfide or peptide bond. If the bioactive molecule is a cytokine, the cytokine can be attached to the antibody molecule using a peptide linker. Suitable peptide linkers are known in the art and may be 5 to 25, 5 to 20, 5 to 15, 10 to 25, 10 to 20, or 10 to 15 amino acids in length.

In some embodiments, the bioactive molecule can be conjugated to an antibody molecule via a cleavable linker. The linker may cause the bioactive molecule to be released from the antibody molecule at the site of therapy. Linkers may include amide bonds (eg, peptide linkers), disulfide bonds, or hydrazones. Peptide linkers, for example, can be cleaved by site-specific proteases, disulfide bonds can be cleaved by the reducing environment of the cytosol, and hydrazones can be cleaved by acid-mediated hydrolysis.

The present invention also provides an isolated nucleic acid molecule or molecules encoding an antibody molecule of the present invention. Such nucleic acid molecules can be easily prepared by one skilled in the art using methods well known in the art.

The nucleic acid molecule or molecules may contain, for example, the sequence shown under SEQ ID NO: 55 or 113 , 62 , 64 , 67 , 70 , 75 , 78 , 83 , 87 , 91 or 94 , which encode CH3 domains FS20-22- 49 , FS20-22-38 , FS20-22-41 , FS20-22-47 , FS20-22-85, FS20-31-58 , FS20-31-66 , FS20-31-94 , FS20-31-102, FS20-31-108 and FS20-31-115 , respectively. For example, the nucleic acid molecule or molecules may contain the sequence shown under SEQ ID NO: 55 or 113 , both of which encode the CH3 domain FS20-22-49 . In some embodiments, the nucleic acid molecule or molecules comprise the sequence set forth in SEQ ID NO: 113 , which encodes the CH3 domain FS20-22-49 . Preferably, the nucleic acid molecule or molecules contain the sequence shown under SEQ ID NO: 55 , which encodes the CH3 domain FS20-22-49 .

The nucleic acid molecule or molecules may encode a VH domain and/or a VL domain, preferably the VH domain and VL domain of the antibody FS30-10-16 , FS30-10-3 , FS30-10-12 , FS30-35-14 or FS30-5- 37 , preferably antibodies FS30-10-16 , FS30-10-3 , FS30-10-12 or FS30-35-14 , more preferably antibodies FS30-10-16 , FS30-10-3 or FS30-10-12 , most preferably antibodies FS30-10-16 . The present application describes the sequences of the VH and VL domains of these antibodies.

For example, the nucleic acid molecule(s) may contain:

(i) the nucleic acid sequence of the VH domain of the antibody FS30-10-16 indicated under SEQ ID NO: 13 , and/or the nucleic acid sequence of the VL domain of the antibody FS30-10-16 indicated under SEQ ID NO: 15 ; or

(ii) the nucleic acid sequence of the VH domain of the antibody FS30-10-3 indicated under SEQ ID NO: 19 , and/or the nucleic acid sequence of the VL domain of the antibody FS30-10-3 indicated under SEQ ID NO: 20 ;

(iii) the nucleic acid sequence of the VH domain of the antibody FS30-10-12 indicated under SEQ ID NO: 24 , and/or the nucleic acid sequence of the VL domain of the antibody FS30-10-12 indicated under SEQ ID NO: 20 ;

(iv) the nucleic acid sequence of the VH domain of the antibody FS30-35-14 indicated under SEQ ID NO: 171 , and/or the nucleic acid sequence of the VL domain of the antibody FS30-35-14 indicated under SEQ ID NO: 32 ; or

(v) the nucleic acid sequence of the VH domain of the antibody FS30-5-37 indicated under SEQ ID NO: 41 , and/or the nucleic acid sequence of the VL domain of the antibody FS30-5-37 indicated under SEQ ID NO: 43 .

The nucleic acid molecule or molecules may encode a heavy chain and/or a light chain, preferably the heavy chain and light chain of the antibody FS20-22-49AA/FS30-10-16 , FS20-22-49AA/FS30-10-3 , FS20-22- 49AA/FS30-10-12 , FS20-22-49AA/FS30-35-14 or FS20-22-49AA/FS30-5-37 , preferably antibodies FS20-22-49AA/FS30-10-16 , FS20-22- 49AA/FS30-10-3 , FS20-22-49AA/FS30-10-12 or FS20-22-49AA/FS30-35-14 , more preferably antibodies FS20-22-49AA/FS30-10-16 , FS20-22 -49AA/FS30-10-3 or FS20-22-49AA/FS30-10-12 , most preferably FS20-22-49AA/FS30-10-16 . The present application describes the sequences of the VH and VL domains of these antibodies.

For example, the nucleic acid molecule(s) may contain:

(i) the nucleic acid sequence of the heavy chain of the antibody FS20-22-49AA/FS30-10-16 specified under SEQ ID NO: 96 , and/or the nucleic acid sequence of the light chain of the antibody FS20-22-49AA/FS30-10-16 , indicated under SEQ ID NO: 98 ; or

(ii) the nucleic acid sequence of the heavy chain of the antibody FS20-22-49AA/FS30-10-3 indicated under SEQ ID NO: 100 , and/or the nucleic acid sequence of the light chain of the antibody FS20-22-49AA/FS30-10-3 , indicated under SEQ ID NO: 102 ;

(iii) the nucleic acid sequence of the heavy chain of the antibody FS20-22-49AA/FS30-10-12 indicated under SEQ ID NO: 104 , and/or the nucleic acid sequence of the light chain of the antibody FS20-22-49AA/FS30-10-12 , indicated under SEQ ID NO: 102 ;

(iv) the nucleic acid sequence of the heavy chain of the antibody FS20-22-49AA/FS30-35-14 specified under SEQ ID NO: 106 , and/or the nucleic acid sequence of the light chain of the antibody FS20-22-49AA/FS30-35-14 , indicated under SEQ ID NO: 108 ; or

(v) the nucleic acid sequence of the heavy chain of the antibody FS20-22-49AA/FS30-5-37 indicated under SEQ ID NO: 110 , and/or the nucleic acid sequence of the light chain of the antibody FS20-22-49AA/FS30-5-37 , listed under SEQ ID NO: 112 .

If the nucleic acid encodes the VH and VL domain, or the heavy and light chain, of the antibody molecules of the present invention, the two domains or chains may be encoded on two separate nucleic acid molecules.

The isolated nucleic acid molecule can be used to express an antibody molecule according to the present invention. The nucleic acid will typically be provided in the form of a recombinant expression vector. Thus, according to another aspect of the present invention, there is provided a vector containing the nucleic acid described above. Suitable vectors can be selected or constructed containing suitable regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences, as appropriate. Preferably, the vector contains appropriate regulatory sequences to control the expression of the nucleic acid in the host cell. The vectors may be plasmids, viral vectors such as phage, or phagemid, depending on the situation.

The nucleic acid molecule or vector described herein can be introduced into a host cell. Techniques for introducing nucleic acid or vectors into host cells are well established in the art, and any suitable technique can be used. A number of host cells suitable for the production of recombinant antibody molecules are known in the art and include bacterial, yeast, insect or mammalian host cells. A preferred host cell is a mammalian cell, such as a CHO, NS0 or HEK cell, for example a HEK293 cell.

According to another aspect of the present invention, there is provided a method for producing an antibody molecule according to the present invention, comprising expressing a nucleic acid encoding the antibody molecule in a host cell and optionally isolating and/or purifying the antibody molecule so produced. Methods for culturing host cells are well known in the art. The method may further include isolating and/or purifying the antibody molecule. Techniques for purifying recombinant antibody molecules are well known in the art and include, for example, HPLC, FPLC, or affinity chromatography, for example, using Protein A or Protein L. In some embodiments, purification can be accomplished using an affinity tag on the antibody molecule. The method may also include preparing the antibody molecule as a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance described below. As explained above, CD137 and OX40 are both expressed on cells of the immune system, including T cells. For example, OX40 is expressed on cells of the immune system, including activated T cells, particularly CD4 ⁺ T cells, CD8 ⁺ T cells, type 1 helper T cells (Th1), type 2 helper T cells (Th2), regulatory T cells (Treg) and tumor-infiltrating T cells, as well as activated natural killer cells (NK cells). CD137 is expressed on cells of the immune system, including T cells, particularly CD8 ⁺ T cells, B cells, NK cells, and tumor infiltrating lymphocytes (TILs). CD137 is expressed at lower levels on CD4 ⁺ T cells than on CD8 ⁺ T cells (see Example 14 and Figure 6 ), but has also been shown to be involved in the induction of proliferation and activation of certain CD4 ⁺ T cell subsets (Wen et al. , 2002).

OX40 activation has been shown to play a role in enhancing T cell activation, T cell clonal expansion, T cell differentiation and survival, and the production of memory T cells. Activation of CD137 has been shown to play a role in enhancing CD8 ⁺ T cell proliferation, survival, and cytotoxic effector function, as well as CD8 ⁺ T cell differentiation and maintenance of memory CD8 ⁺ T cells. Activation of CD137 has also been demonstrated to enhance NK cell-mediated ADCC as well as B cell proliferation, survival, and cytokine production.

Given the activity of OX40 and CD137 in enhancing the immune response, OX40 and CD137 agonist molecules have been studied for cancer treatment and are also expected to find use in the treatment of infectious diseases.

Thus, the antibody molecules described herein can be used for therapeutic applications, particularly in the treatment of cancer and infectious diseases.

The antibody molecule described herein can be used in a method of treating a human or animal body. According to related aspects of the present invention there is provided;

(i) an antibody molecule described in this application for use as a drug,

(ii) an antibody molecule described herein, for use in a method of treating a disease or disorder,

(iii) use of an antibody molecule described herein in the manufacture of a medicament for use in treating a disease or disorder; And,

(iv) a method of treating a disease or disorder in an individual, the method comprising administering to the individual a therapeutically effective amount of an antibody molecule described herein.

The individual may be a patient, preferably a human patient.

Treatment can be any treatment or therapy that achieves some targeted therapeutic effect, such as inhibiting or delaying the progression of a condition, and includes reducing the rate of progression, slowing the rate of progression, improving the condition, curing or remission (whether partial or complete) of the condition. preventing, ameliorating, delaying, reducing or stopping one or more symptoms and/or signs of a condition or prolonging the survival time of an individual or patient beyond what would be expected in the absence of treatment.

Treatment as a preventive measure (eg, prophylaxis) is also included. For example, an individual susceptible or at risk of developing or reoccurring a disease, such as cancer, can be treated as described herein. Such treatment may prevent or delay the onset or recurrence of the disease in an individual.

The described method of treatment may include administering at least one additional treatment to the individual in addition to the antibody molecule. Thus, the antibody molecule described herein can be administered to an individual alone or in combination with one or more other treatments. If the antibody molecule is administered to an individual in combination with another treatment, the additional treatment may be administered to the individual simultaneously, sequentially, or separately from the administration of the antibody molecule. If the additional treatment is administered simultaneously with the antibody molecule, the antibody molecule and the additional treatment can be administered to the individual as a combination preparation. For example, the additional therapy may be a known therapy or therapeutic agent for the disease being treated.

Although the antibody molecule may be administered separately, the antibody molecules will typically be administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the antibody molecule. Thus, according to another aspect of the present invention, there is provided a pharmaceutical composition comprising an antibody molecule described herein. A method is also proposed, which includes producing an antibody molecule in the form of a pharmaceutical composition.

Pharmaceutical compositions may contain, in addition to the antibody molecule, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and/or dosage forms that, within the bounds of sound medical judgment, are suitable for use in contact with tissue of a subject (e.g., human) without undue toxicity, irritation, allergic reaction or other problem or complication commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in terms of compatibility with the other ingredients of the formulation. The specific nature of the carrier or other material will depend on the route of administration, which may be infusion, injection, or any other suitable route, as discussed below.

For parenteral, eg subcutaneous or intravenous administration, eg by injection, the pharmaceutical composition containing the antibody molecule may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art will readily be able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's solution for injection, lactated Ringer's solution for injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives can be used, including buffers such as phosphate, citrate and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3'-pentanol and m-cresol); low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagines, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as Tween™, Pluronic Agents™ or polyethylene glycol (PEG).

In some embodiments, the antibody molecules may be provided in lyophilized form for reconstitution prior to administration. For example, lyophilized antibody molecules can be reconstituted in sterile water and mixed with physiological saline before administration to an individual.

Administration may be in a "therapeutically effective amount" that is sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of what is being treated, the particular individual being treated, the clinical condition of the individual, the cause of the disorder, the site of delivery of the composition, the type of antibody molecule, the route of administration, the schedule of administration, and others. factors known to practicing physicians. Treatment decisions, such as dosage decisions, etc., are the responsibility of GPs and other physicians and may depend on the severity of symptoms and/or progression of the disease being treated. Appropriate dosages of antibody molecules are well known in the art (Ledermann et al. , 1991; Bagshawe et al. , 1991). The specific dosages specified herein or in the Physician's Desk Reference (2003) appropriate to the antibody molecule administered may be used. A therapeutically effective amount or appropriate dose of an antibody molecule can be determined by comparing in vitro activity and in vivo activity in an animal model. Methods are known to extrapolate effective dosages from mice and other test animals to humans. The exact dose will depend on a number of factors, including the size and location of the area being treated, as well as the specific nature of the antibody molecule.

A typical antibody dose ranges from 100 μg to 1 g for various systemic applications and from 1 μg to 1 mg for various topical applications. A higher initial loading dose may be administered followed by one or more lower doses. This is a single dose dose for an adult, which can be adjusted proportionally for children and infants, and can also be adjusted for other antibody formats in proportion to molecular weight.

Treatment may be repeated at daily, twice weekly, weekly, or monthly intervals at the discretion of the physician. The treatment schedule for an individual may depend on the pharmacokinetic and pharmacodynamic properties of the antibody composition, the route of administration, and the nature of the condition being treated.

Treatment may be intermittent, and the period between administrations may be about two weeks or more, for example, about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more . For example, treatment can be administered every two to four weeks or every four to eight weeks. Suitable formulations and routes of administration are described above.

In a preferred embodiment, the antibody molecule described herein may be for use in a method of treating cancer.

Cancer may be characterized by abnormal proliferation of malignant cancer cells. When referring to a specific type of cancer, such as breast cancer, it refers to the abnormal proliferation of malignant cells of the corresponding tissue, such as breast tissue. Secondary cancer, which is localized in the breast but is the result of abnormal proliferation of malignant cells of another tissue, such as ovarian tissue, is not breast cancer referred to in this application, but ovarian cancer.

The cancer may be a primary or secondary cancer. Thus, the antibody molecule described herein may be intended for use in a method of treating cancer in an individual, wherein said cancer is a primary tumor and/or a metastatic tumor.

A cancer to be treated with an antibody molecule described herein may contain TILs that express OX40 and/or CD137, for example, on their cell surface. In one embodiment, a tumor could be defined as containing TILs that express one or both of OX40 and CD137. Methods for determining the expression of an antigen on a cell surface are known in the art and include, for example, flow cytometry.

For example, the cancer to be treated with the antibody molecule described herein may be selected from the group consisting of leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma; and various types of solid cancers, such as sarcomas (eg, soft tissue sarcomas), skin cancer (eg, Merkel cell carcinoma), melanoma, bladder cancer (eg, urothelial carcinoma of the bladder), brain cancer (eg, glioblastoma multiforme ), breast cancer, uterine/endometrial cancer, ovarian cancer (eg, ovarian serous cystadenoma), prostate cancer, lung cancer (eg, non-small cell lung carcinoma (NSCLC) such as squamous cell lung carcinoma, and small cell lung cancer (SCLC) ), colorectal cancer (eg, colorectal adenocarcinoma), cervical cancer (eg, squamous cell carcinoma of the cervix and cervical canal adenocarcinoma), liver cancer (eg, hepatocellular carcinoma), head and neck cancer (eg, squamous cell carcinoma of the head and neck) , esophageal cancer (eg, esophageal carcinoma), pancreatic cancer, kidney cancer (eg, renal cell carcinoma), adrenal cancer, stomach cancer (eg, gastric adenocarcinoma), testicular cancer (eg, testicular germ cell tumors), gallbladder cancer and biliary tract (eg, cholangiocarcinoma), thyroid cancer, thymic cancer, bone cancer and cerebral cancer.

In a preferred embodiment, the cancer to be treated with the antibody molecule described herein is a solid cancer.

More preferably, the cancer to be treated with the antibody molecule described herein is a solid cancer selected from the group consisting of melanoma, bladder cancer, brain cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, pancreatic cancer, kidney cancer and stomach cancer.

In another preferred embodiment, the cancer to be treated with the antibody molecule described herein may be a cancer that responds to treatment with one or more checkpoint inhibitors, such as an antibody that binds PD-1, PD-L1, or CTLA4 . Such tumors are believed to have higher TIL levels and/or a higher tumor mutational load than tumors that do not respond to checkpoint inhibitor therapy. Such tumors are also called “warm” or “hot” tumors.

Examples of such tumors include head and neck squamous cell carcinoma (HNSCC), melanoma, lung cancer (such as squamous cell lung cancer, lung adenocarcinoma, non-small cell lung carcinoma [NSCLC] or small cell lung carcinoma [SCLC]), prostate cancer, cervical cancer, bladder cancer, breast cancer, thyroid cancer, kidney cancer, colorectal cancer (MSI or MSS; e.g. colorectal adenocarcinoma), esophageal cancer, non-Hodgkin's lymphoma (NHL), stomach cancer, endometrial cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, mesothelioma and urothelial cancer. In a preferred embodiment, the cancer is gastric cancer. The cancer may also be a cancer that has not previously been treated with a chemotherapeutic or radiotherapeutic agent, i.e., the individual to be treated may be a patient suffering from cancer who has not been treated with a chemotherapeutic or radiotherapeutic agent to treat the cancer in question. In a preferred embodiment, the antibody molecule described herein is for use in a method of treating cancer that responds to one or more immune checkpoint inhibitors in an individual, the method comprising treating the patient with the antibody molecule in combination with an agent that inhibits the interaction between PD-1 and PD-L1.

Alternatively, the cancer to be treated with the antibody molecule described herein may be a cancer, such as pancreatic cancer or prostate cancer, that does not respond to treatment with one or more checkpoint inhibitors, such as an antibody that binds PD-1, PD-L1 or CTLA4. These tumors are also called “cold” tumors.

The present inventors have shown that tumors that did not respond to treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody alone responded to treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody in combination with the antibody molecule described herein . Thus, the antibody molecule of the present invention may be for use in a method of treating cancer in an individual, wherein said cancer does not respond or is refractory to treatment with one or more checkpoint inhibitors individually, and wherein said method comprises administering to the individual the antibody molecule in combination with an agent that inhibits the interaction between PD-1 and PD-L1. Also provided is a method of treating cancer in an individual, wherein said cancer does not respond or is refractory to treatment with one or more checkpoint inhibitors alone, wherein said method comprises administering an antibody molecule to the individual in combination with an agent that inhibits the interaction between PD-1 and PD -L1.

Without being limited to any theory, it is believed that treatment of a cancer that does not respond to treatment with one or more checkpoint inhibitors alone, chemotherapy, radiation therapy, an immunotherapeutic agent such as an immunostimulating agent, or a tumor vaccine will result in the death of the cancer cells , which in turn will lead to an increase in TILs in the tumor and higher expression of immunosuppressive receptors, which in turn will make the cancer susceptible to treatment with checkpoint inhibitors, i.e., turning a “cold” tumor into a “warm” tumor. Thus, the antibody molecule of the present invention may be for use in a method of treating cancer in an individual, wherein said cancer does not respond or is refractory to treatment with one or more checkpoint inhibitors individually, and wherein said method comprises administering to the individual the antibody molecule in combination with a chemotherapeutic, radiotherapeutic or immunostimulating agent or cancer vaccine, and optionally an agent that inhibits the interaction between PD-1 and PD-L1. Also provided is a method of treating cancer in an individual, wherein said cancer does not respond to or is refractory to treatment with one or more checkpoint inhibitors alone, wherein said method comprises administering an antibody molecule to the individual in combination with a chemotherapeutic, radiotherapeutic or immunostimulating agent or cancer vaccine, and optionally an agent that inhibits the interaction between PD-1 and PD-L1. In a preferred embodiment, the agent that inhibits the interaction between PD-1 and PD-L1 is an antibody that binds PD-1 or PD-L1.

In the context of cancer, treatment may include inhibition of cancer growth, including complete remission of cancer, and/or inhibition of cancer metastasis, as well as inhibition of cancer recurrence. Cancer growth usually refers to any of a number of indicators that indicate a change within the cancer to a more advanced form. Thus, indicators for measuring inhibition of cancer growth include reduction in cancer cell survival, reduction in tumor volume or morphology (eg, as determined using computed tomography (CT), sonography, or other imaging modality), delayed tumor growth, destruction of tumor vasculature, improvement skin test results for delayed hypersensitivity, increased activity of anticancer immune cells or other anticancer immune responses, and decreased levels of tumor-specific antigens. Activating or enhancing immune responses to cancer tumors in an individual may improve the individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and/or reduce the propensity for cancer growth in the individual.

In connection with the treatment of cancer, the antibody molecule described herein may be administered to an individual in combination with another anticancer therapy or therapeutic agent, such as an anticancer therapy or therapeutic agent that has been shown to be suitable, or potentially suitable, for the treatment of the subject. cancer. For example, the antibody molecule may be administered to an individual in combination with a chemotherapeutic agent, radiation therapy, a radionuclide, an immunotherapeutic agent, a tumor vaccine, an oncolytic virus, an adoptive cell transfer therapy (ACT) such as NK cell adoptive therapy or T cell therapy, expressing chimeric antigen receptor (CAR), autologous TIL or gamma/delta T cells, or a hormone therapy agent. The antibody molecule described herein can also be administered to an individual in combination with an adjuvant or neoadjuvant, such as neoadjuvant hormonal therapy, an anti-angiogenic agent such as an anti-VEGF antibody or an anti-VEGFR2 antibody, a cytotoxic agent.

Without being limited by any theory, it is believed that the antibody molecule described herein can act as an adjuvant in anticancer therapy. In particular, it is believed that administering an antibody molecule to an individual in combination with chemotherapy or radiation therapy, for example, will trigger a stronger immune response against cancer than that achieved by administering chemotherapy or radiation therapy alone.

The one or more chemotherapeutic agents to be administered in combination with the antibody molecule described herein may be selected from the group consisting of: taxanes, cytotoxic antibiotics, tyrosine kinase inhibitors, PARP inhibitors, B-Raf enzyme inhibitors, MEK inhibitors, c- MET, VEGFR inhibitors, PDGFR inhibitors, alkylating agents, platinum analogues, nucleoside analogues, antifolates, thalidomide derivatives, antineoplastic chemotherapeutic agents and others. Taxanes include docetaxel, paclitaxel and nab-paclitaxel; cytotoxic antibiotics include actinomycin, bleomycin and anthracyclines such as doxorubicin, mitoxantrone and valrubicin; tyrosine kinase inhibitors include erlotinib, gefitinib, axitinib, PLX3397, imatinib, cobemitinib and trametinib; PARP inhibitors include piraparib; B-Raf enzyme inhibitors include vemurafenib and dabrafenib; alkylating agents include dacarbazine, cyclophosphamide and temozolomide; platinum analogues include carboplatin, cisplatin and oxaliplatin; nucleoside analogs include azacitidine, capecitabine, fludarabine, fluorouracil and gemcitabine; antifolates include methotrexate and pemetrexed. Other chemotherapeutic agents suitable for use in the present invention include defactinib, entinostat, eribulin, irinotecan and vinblastine. The chemotherapeutic agent to be administered in combination with the antibody molecule described herein may be a fluoropyrimidine. For example, if the cancer being treated is a HER2-negative cancer, such as HER2-negative gastric cancer, the antibody molecule described herein can be administered in combination with platinum, a platinum analogue and a fluoropyrimidine. If the cancer to be treated is a HER2-positive cancer, for example, HER2-positive gastric cancer, the antibody molecule described herein can be administered in combination with platinum or a platinum analogue, fluoropyrimidine and trastuzumab.

Preferred therapeutic agents for administration with the antibody molecule described herein are doxorubicin, mitoxantrone, cyclophosphamide, cisplatin and oxaliplatin.

The radiation therapy to be administered in combination with the antibody molecule described herein may be external beam radiation therapy or brachytherapy.

The radionuclides for administration with the antibody molecule described herein may be selected from the group consisting of: yttrium-90, iodine-131, bismuth-213, astatine-211, lutetium-177, rhenium-188, copper-67, actinium -225, iodine-125 and terbium-161.

The immunotherapeutic agent to be administered in combination with the antibody molecule described herein may be a therapeutic antibody molecule, a nucleotide, a cytokine, or a cytokine-based therapy. For example, a therapeutic antibody molecule may bind to an immune regulatory molecule, such as a checkpoint inhibitor molecule or an immune co-stimulatory molecule, an innate immune system receptor, or a tumor antigen, such as a cell surface tumor antigen or a soluble tumor antigen. Examples of immune regulatory molecules that a therapeutic antibody molecule may bind to include CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, programmed cell death ligand 1 (PD-L1), programmed cell death protein 1 (PD-1 ), CD47, CD73, CSF-1R, KIR, CD40, HVEM, IL-10 and CSF-1. Examples of innate immune system receptors to which a therapeutic antibody molecule may bind include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (eg, RIG-I and MDA-5), and STING. Examples of tumor antigens to which a therapeutic antibody molecule may bind include HER2, EGFR, CD20, and TGF-beta.

The present inventors have shown that administration of an antibody molecule of the present invention in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody resulted in increased T cell activation and tumor regression in a mouse tumor model compared to either treatment or the antibody molecule of the present invention. the invention, either an anti-PD-1 antibody or an anti-PD-L1 antibody alone. Without being limited to any theory, these results indicate that administration of an antibody molecule of the present invention in combination with an agent capable of inhibiting the interaction between PD-1 and PD-L1 results in enhanced antitumor effects and that such combination administration may be appropriate for the treatment of tumors that are refractory or resistant to or have relapsed after anti-PD-1 antibody or anti-PD-L1 monotherapy.

Thus, the antibody molecule of the present invention may be for use in a method of treating cancer in an individual, the method comprising administering the antibody molecule in combination with an agent that is capable of inhibiting the interaction between PD-1 and PD-L1. Also provided is an agent capable of inhibiting the interaction between PD1 and PD-L1, such as an antibody molecule that binds PD-1 or PD-L1, for use in a method of treating cancer in an individual, the method comprising administering an agent that is capable of inhibiting the interaction between PD-1 and PD-L1, in combination with an antibody of the present invention. A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody molecule of the present invention and a therapeutically effective amount of an agent that is capable of inhibiting the interaction between PD-1 and PD-L1.

In a preferred embodiment, the agent that is capable of inhibiting the interaction of PD-1 and PD-L1 is an antibody molecule that binds PD-1 or PD-L1. Antibodies that bind to PD-1 are known in the art and include nivolumab (5C4) and pembrolizumab. Known antibodies that bind to PD-L1 include YW243.55.S1, durvalumab, atezolizumab, and avelumab. The antibody molecule of the present invention may be designed for administration with one of these known anti-PD-1 or anti-PD-L1 antibodies or with another anti-PD-1 or anti-PD-L1 antibody. Producing alternative antibodies that bind to PD-1 or PD-L1 is within the capabilities of the skilled artisan using conventional methods.

The nucleic acid to be administered in combination with the antibody molecule described herein may be siRNA.

Cytokines or cytokine-based therapy may be selected from the group consisting of: IL-2, conjugated IL2 prodrug, GM-CSF, IL-7, IL-12, IL-9, IL-15, IL-18, IL-21 and type I interferon.

Tumor vaccines for the treatment of cancer have been introduced into the clinic and are discussed in detail in the scientific literature (such as Rosenberg, 2000). The focus is on strategies to stimulate the immune system to respond to various cellular markers expressed by autologous or allogeneic cancer cells by using these cells as a means of vaccination, either with or without granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF produces a strong response upon antigen presentation and works particularly well when used with these strategies.

The antibody molecule described herein can also be administered to an individual suffering from cancer, in particular to an individual suffering from gastric cancer, in combination with ramucirumab and/or paclitaxel; irinotecan and docetaxel or paclitaxel; or pembrolizumab. Treatment with the antibody molecule described herein in combination with pembrolizumab is preferred in the treatment of MSI-H and/or dMMR gastric cancer.

Given the activity of OX40 and CD137 in enhancing the immune response, dual agonist molecules OX40 and CD137 are expected to find application in the treatment of infectious diseases. Thus, in another preferred embodiment, an antibody molecule described herein may be for use in a method of treating an infectious disease, such as an acute or persistent infectious disease.

Without being limited to any theory, it is believed that OX40 and CD137 agonist molecules may be able to enhance the immune response against acute infectious disease caused by a pathogen by inducing rapid infiltration and activation of innate immune cells such as neutrophils and monocytes, thereby facilitating clearance of the pathogen which caused an acute infectious disease. Thus, in another embodiment, an antibody molecule described herein may be for use in a method of treating an acute infectious disease, such as an acute bacterial disease. In a preferred embodiment, the acute infectious disease is an acute bacterial disease caused by infection with a gram-positive bacterium, such as a bacterium of the genus Listeria , Streptococcus pneumoniae , or Staphylococcus aureus .

Infectious diseases are usually cleared by the immune system, but some infections persist for long periods of time, such as months or years, and the immune system does not fight them effectively. Such infections are also called persistent or chronic infections.

Preferably, the antibody molecule described herein is used to treat a persistent infectious disease, such as a persistent viral, bacterial, fungal or parasitic infection, preferably a persistent viral or bacterial infection.

In a preferred embodiment, the persistent viral infection to be treated with the antibody molecule described herein is a persistent infection with: human immunodeficiency virus (HIV), Epstein-Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, or varicella zoster virus

In a preferred embodiment, the persistent bacterial infection to be treated with the antibody molecule described herein is a persistent infection of: Staphylococcus aureus, Hemophilus influenza, Mycobacterium tuberculosis, Mycobacterium leprae, Helicobacter pylori, Treponema pallidum, Enterococcus faecalis, or Streptococcus pneumoniae .

An agonistic effect on CD137 has been described as beneficial in the treatment of infections with Gram-positive bacteria. Thus, in a preferred embodiment, the persistent bacterial infection to be treated with the antibody molecule described herein is a persistent infection with a gram-positive bacterium. In a more preferred embodiment, the persistent bacterial infection is a persistent infection with a gram-positive bacterium selected from the group consisting of Staphylococcus aureus, Mycobacterium leprae, Enterococcus faecalis and Streptococcus pneumoniae .

In a preferred embodiment, the persistent fungal infection to be treated with the antibody molecule described herein is a persistent infection of: Candida , e.g. Candida albicans , Cryptococcus (gattii and neoformans) , Talaromyces (Penicillium) marneffe, Microsporum, e.g. Microsporum audouinii and Trichophyton tonsurans .

In a preferred embodiment, the persistent parasitic infection to be treated with the antibody molecule described herein is a persistent infection: Plasmodium , for example, Plasmodium falciparum, or Leishmania , for example, Leishmania donovani .

In relation to the treatment of a persistent infectious disease, the antibody molecule may be administered to an individual in combination with a second therapy or therapeutic agent that has been shown to be, or is expected to be suitable, for treating the subject. pathogen. For example, the antibody molecule may be administered to an individual in combination with an immunotherapeutic agent. The immunotherapeutic agent to be administered in combination with the antibody molecule described herein may be a therapeutic antibody molecule. For example, a therapeutic antibody molecule may bind to a receptor of the innate immune system. Examples of innate immune system receptors to which a therapeutic antibody molecule may bind include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (eg, RIG-I and MDA-5), and STING.

If the antibody molecule is used to prevent an infectious disease, the antibody molecule may be administered in combination with a vaccine against the pathogen in question. Without being limited by any theory, it is believed that the antibody molecule described herein can act as a vaccination adjuvant. In particular, it is believed that administration of an antibody molecule to an individual in combination with a vaccine will produce a stronger immune response against the pathogen than that achieved by administering the vaccine alone.

When applied to the treatment of a persistent infectious disease, treatment may include eliminating the infection, reducing the individual's pathogen load, and preventing recurrence of the infection. For example, treatment may include preventing, ameliorating, delaying, reducing, or controlling one or more symptoms and/or signs of persistent infection. Alternatively, treatment may involve preventing the infectious disease.

The features disclosed in the foregoing description, or in the accompanying claims, or in the accompanying drawings, expressed in their particular forms or in the context of means for performing the disclosed function, or the method or process for obtaining the disclosed results, as the case may be, may be applied, according to alone or in any combination of such features, to implement the present invention in its various forms.

Although the present invention has been described in connection with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art upon consideration of the present disclosure. Accordingly, the exemplary embodiments of the present invention set forth above are intended to be illustrative and non-limiting. Various changes may be made to the described embodiments without departing from the spirit and scope of the present invention.

For the avoidance of any doubt, any theoretical explanations presented in this application are intended to enhance the reader's understanding. The inventors of the present invention do not wish to be bound by any of these theoretical explanations.

Any section headings used in this application are for organizational purposes only and should not be construed as limiting the subject matter described.

Throughout the specification, including the appended claims, unless the context otherwise requires, the words “comprise” and “include,” and variations such as “comprises,” “comprising,” and “including,” are to be understood to imply the inclusion of the specified integer or a stage or group of integers or stages, but not to the exclusion of any other integer or stage or group of integers or stages.

It should be noted that in the specification and the accompanying claims, the singular forms (respectively, “a”, “an” and “the” in the English source text) include the plural unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and/or to "about" another particular value. When expressing such a range, another embodiment includes from one specific value and/or to another specific value. Likewise, when values are expressed as approximations, using the antecedent "approximately", it should be understood that the particular value constitutes another embodiment. The term “about” in relation to a numerical value is optional and means, for example, +/- 10%.

Examples

The aim of the present invention was to provide mAbs ² that were capable of exerting agonistic effects on both OX40 and CD137 in the absence of artificial cross-linking agents or Fcγ receptor-mediated cross-linking, and which were capable of inducing an enhanced immune response against diseases such as like cancer. In this case, MAT ² is an antibody molecule that contains a CDR-based antigen binding site that binds CD137 and a binding site for OX40 located in the CH3 domain of the antibody molecule.

To achieve this goal, we first used affinity selection and maturation methods to identify Fcabs that were capable of binding OX40 and inducing T cell activation in humans and mice, respectively (see Examples 2 and 3 ). We then introduced the antigen binding site for OX40 from these Fcabs into the MAb ² format and showed that several of these human OX40 MAb ² "mock" MAbs were able to bind human and cynomolgus OX40 with high affinity and activate T cells upon cross-linking. (see Example 4 ). Of these, clone FS20-22-49 showed the largest increase in agonist activity upon cross-linking and also had the lowest EC ₅₀ for its agonist activity in the presence of cross-linking and was therefore prioritized as the antigen binding site for OX40 for the development of the subject MAb ² .

In order to develop a CDR-based antigen binding site that binds and is capable of agonizing CD137, we used screening methods to identify monoclonal antibodies (MAbs) that could bind human CD137 and were capable of activating T cells only upon cross-linking (see Example 5 ). The CDRs from these identified MAbs were subsequently cloned into MAbs ² that contained the antigen binding site for OX40 FS20-22-49. The CDRs of these MAT ² were sequence optimized to obtain the following MAT ² : FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA/FS30-10-16 , FS20-22-49AA/FS30-35-14 and FS20-22-49AA/FS30-5-37 (see Example 6 ). All of these MAbs ² were demonstrated to have a high level of specificity for human CD137 and were able to activate CD137 upon cross-linking in a T cell activation assay (see Example 7 ). Neither mAb ² showed any significant ability to activate CD137 in the absence of cross-linking.

Once the antigen binding site for OX40 has been determined to be FS20-22-49AA in the selected mAb² was able to bind and activate OX40 upon cross-linking and that, separately, the antigen binding sites for CD137 based on the CDRs from FS30-10-3, FS30-10-12, FS30-10-16, FS30-35-14 and FS30-5-37 were able to bind and activate CD137 upon cross-linking, we have attempted to demonstrate that the mAb²containing these antigen-binding domains were able to activate both OX40 and CD137 (also called “dual agonist”). Such a dual agonist may be able to i) bind to OX40 to cross-link MAT² and bind, cluster and activate (exert agonistic effects) CD137, and ii) bind to CD137 to cross-link mAbs² and bind, cluster and activate (produce agonistic effects) OX40. Importantly, the dual agonist must be able to autonomously trigger dual agonist actions based on the expression of specific targets (OX40 and CD137) and without the need for additional cross-linking agents.

We demonstrated that the test MAT ² molecules were able to bind human CD137, human OX40, cynomolgus CD137 and cynomolgus OX40 (see Example 8 ) and that the test MAT ² molecules were able to simultaneously bind human CD137 and human OX40 (see Example 9 ). We have shown that the "LALA" mutation in the CH2 domain of MAT ² reduced their binding to Fcγ receptors and that the MAT ² clone FS20-22-49AA/FS30-10-16 was unable to induce ADCC activation in the ADCC bioassay (see Example 10 ).

We also showed that the tested anti-OX40/CD137 mAb ² molecules bound to human and cynomolgus monkey OX40 and CD137 expressed cells without observed non-specific binding (see Example 11 ).

We then demonstrated that test anti-OX40/CD137 mAb ² molecules containing the LALA mutation were able to induce T cell activation in the absence of artificial cross-linking agents in a T cell activation assay using staphylococcal enterotoxin A (SEA; see Example 12 ). We have also demonstrated that the test MAT ² molecules can induce T cell activation in the absence of artificial cross-linking agents in a general T cell population activation assay and that this activity is dependent on the simultaneous binding of MAT ² to both OX40 and CD137 ( see Examples 13 and 16 ). The present inventors further confirmed that MAb ² FS20-22-49AA/FS30-10-16 was able to activate these receptors in CD4 ⁺ and CD8 ⁺ T cells, respectively, in the absence of cross-linking (see Example 14 ).

Because human OX40/CD137 mAb ² did not bind to mouse proteins, to test the ability of anti-OX40/CD137 mAb ² to induce a T cell-mediated antitumor response, a similar mAb ² targeting mouse OX40 and mouse CD137 was prepared as with the LALA mutation. and without it (designated as FS20m-232-91AA/Lob12.3 and FS20m-232-91/Lob12.3, respectively). We have shown that MAT ² FS20m-232-91AA/Lob12.3 can induce T cell activation without any additional cross-linking agents and that this activity is dependent on the simultaneous binding of MAT ² to both OX40 and CD137 ( see Examples 15 and 16 ).

The present inventors demonstrate that MAbs ² FS20m-232-91AA/Lob12.3 and FS20m-232-91/Lob12.3 have in vivo antitumor efficacy in the CT26 syngeneic tumor model (see Example 17 ). We further demonstrate that Mab ² FS20m-232-91AA/Lob12.3 has an effect on circulating T cells, increasing the frequency of activated and proliferating T cells (see Examples 18 and 19 ). The present inventors have demonstrated that MAb ² FS20m-232-91AA/Lob12.3 has in vivo antitumor efficacy in the B16-F10 syngeneic tumor model (see Example 20 ).

The authors of the present invention carried out analytical characterization and preliminary assessment of the stability of MAT ² (see Example 21 ). All five MAbs ² tested showed favorable analytical performance and favorable stability.

The present inventors have demonstrated that combination of MAb ² FS20-22-49AA/FS30-10-16 with anti-PD-L1 antibody or anti-PD-1 antibody in a T cell activation assay using SEA can result in an increase in maximal T cell activity in vitro , above the activity observed for MAb ² to OX40/CD137 alone. We also demonstrated that treatment with a combination of mAb ² FS20m-232-91AA/Lob12.3 and anti-PD-1 in vivo in a CT26 tumor mouse model was able to result in increased antitumor activity, provide a survival benefit, and enhance pharmacodynamic modulation proliferating T cells and NK cells compared to treatment with either single agent (see Example 22 ).

The present inventors demonstrated that MAb ² FS20m-232-91AA/Lob12.3 had dose-dependent in vivo antitumor activity in the CT26 syngeneic tumor model up to a certain dose level and that this activity was maintained at higher dose levels. We have also shown that MAb ² FS20m-232-91AA/Lob12.3 can induce the formation of protective immunological memory in “complete response” mice and protect against reinoculation of CT26 cells (see Example 23 ). We have demonstrated that Mab ² FS20m-232-91AA/Lob12.3 has an effect on circulating T cells, significantly increasing the frequency of proliferating (Ki67 ⁺ ) CD4 ⁺ and CD8 ⁺ T cells at varying dose levels (see Example 24 ). The present inventors have also shown that MAb ² FS20m-232-91AA/Lob12.3 is able to increase the frequency of activated (CD69 ⁺ ) and proliferating (Ki67 ⁺ ) CD8 T cells and that depletion of CD4 T cells has an adverse effect on peripheral pharmacodynamic response , mediated by MAT ² FS20m-232-91AA/Lob12.3 (see Example 25 ). We have demonstrated that MAT ² FS20-22-49AA/FS30-10-16 had similar functional activity in a primary cynomolgus monkey PBMC assay compared to the equivalent human assay and that MAT ² was well tolerated in cynomolgus monkeys at doses up to 30 mg. /kg, and that it was able to induce a drug-associated increase in the proliferation and activation of central memory CD4 ⁺ and CD8 ⁺ T cells and effector CD4 ⁺ and CD8 ⁺ memory T cells and NK cells in cynomolgus monkeys (see Example 26 ).

We also showed that, in a study in BALB/c mice, mAb ² FS20m-232-91AA/Lob12.3 induced a modest and transient increase in the levels of T cell infiltration and proliferation in the liver compared to the cross-linking independent CD137 agonist. which induced increased and sustained T cell infiltration, proliferation and activation in the liver (see Example 27 ). Finally, in a mouse model of the syngeneic CT26 tumor, we showed no difference in tumor growth or survival between mice treated with either Mab ² FS20m-232-91AA/Lob12.3 or Mab ² against OX40/CD137 containing the same Fcab OX40 paired with a cross-linking-independent anti-CD137 Fab clone despite the ability of the cross-linking-independent anti-CD137 Fab clone to induce increased levels and proliferation of T cells compared to the cross-linking-dependent anti-CD137 Lob12.3 clone from MAT ² FS20m-232-91AA/Lob12.3 (see Example 28 ).

These experiments are described in more detail in the following Examples.

Example 1 - Antigen selection and characterization

The selection and screening methods used to identify mAbs ² that are able to bind and have agonistic effects on both OX40 and CD137 require the use of different OX40 and CD137 antigens. The preparation of these antigens is described in more detail below.

1.1 OX40 antigens

The OX40 antigens used to select human and mouse OX40-specific Fcabs and to test cross-reactivity of selected Fcabs with cynomolgus OX40 were either produced in-house or obtained from commercial sources as described below.

1.1.1 Production of recombinant soluble human, cynomolgus and mouse OX40 antigens

To produce recombinant soluble dimeric OX40 antigens, the extracellular domain of OX40 was hybridized with mouse Fc, which improved the solubility and stability of the antigen. Specifically, the extracellular domain of the corresponding OX40 (human, cynomolgus or mouse) was cloned into the vector pFUSE-mIgG2aFc2 (Invivogen, cat. no. pfuse-mg2afc2) using the restriction enzymes EcoRI-HF and BglII to obtain antigens with the Fc domain of mouse IgG2a at C -end. Recombinant OX40 antigens were then produced by transient expression in HEK293-6E cells (National Research Council of Canada) and purified using MabSelect SuRe™ Protein A columns (GE Healthcare, 11003494) followed by size exclusion chromatography (SEC) to ensure that the resulting antigen was a single molecule and did not contain aggregates.

To generate biotinylated versions of recombinant OX40 antigens, antigens were biotinylated using the EZ-Link™ Sulfo-NHS-SS-Biotin kit (Thermo Fisher Scientific, cat. no. 21331) according to the manufacturer's protocol. Biotinylated OX40 antigen was used for the selection experiments described below, but not for binding affinity measurements. Purification of biotinylated OX40 antigens was performed in two steps using a PD-10 desalting column (GE Healthcare, 17-0851-01 ) followed by an Amicon 30k spin column (Millipore, UFC903024 ) according to the manufacturer's instructions. The biophysical properties of the recombinant antigens were characterized by SE-HPLC analysis to ensure the absence of aggregates and by SDS-PAGE to confirm the molecular size. Size determination by PAGE indicated that the soluble antigens were dimeric, as their estimated molecular weight was twice the predicted molecular weight of the monomer. The recombinant antigens were also analyzed by gel mobility shift assay, which showed that the degree of biotinylation was greater than 90%. Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) were used to confirm that biotinylated recombinant human (hOX40-mFc), mouse (mOX40-mFc), and cynomolgus macaque (cOX40-mFc) OX40 antigens could be bound by OX40-specific antibodies (11D4 antibody [European Patent No. 2242771] for human and cynomolgus OX40; polyclonal sheep anti-human OX40 antibody for cynomolgus OX40 [R&D Systems, catalog number AF3388]; ACT35 antibody for human OX40 [Biolegend, catalog number 35002] and mouse OX40 antibody OX86 [Biolegend, catalog number 119408]). These antigens are listed in Table 2 below.

1.1.2 Generation of human, cynomolgus and mouse OX40-expressing cell lines

Human, cynomolgus and mouse OX40 (see Table 1 for sequences) was cloned into the pLVX-EF1a-IRES-puro vector (Clontech, catalog no. 631253) using the restriction enzymes SpeI-HF and NotI-HF. The vectors were then transformed into the Lenti-X 293T cell line (Clontech, cat. no. 632180) along with Lenti-X HTX packaging mix (Clontech, cat. no. 631249) to produce lentivirus. Lentivirus was then used to transduce DO11.10 cells (National Jewish Health). Cells overexpressing OX40 were selected by incubating the cells with 5 μg/ml puromycin (Life Technologies, cat. no. A11113803) for approximately 2 weeks, followed by cloning the cell line by serial dilution. Expression of OX40 by cell lines was tested by flow cytometry using fluorescently labeled OX40-specific antibodies (OX86; ACT35; and polyclonal sheep anti-human OX40 antibody as described in Example 1.1.1 and Table 2 ). Cell lines expressing human (DO11.10-hOX40), mouse (DO11.10-mOX40) or cynomolgus (DO11.10-cOX40) OX40 were selected, in which all cells showed at least 10-fold higher fluorescence values, than non-transduced cells in flow cytometry analysis. These cell lines are listed in Table 2 below.

Table 1: OX40 sequences

Gene of interest View Source Ident. clone
(catalog no.) Genbank accession number SEQ ID NO OX40 Human Thermo Fisher Scientific MHS6278-202858046 BC105070 132 OX40 Cynomolgus macaque Gene synthesis Not available XP_005545179 134 OX40 Mouse Gene synthesis Not available NM_011659.2 133

1.1.3 Commercially available OX40 antigens

Several commercially available OX40 antigens were tested.

Recombinant His-tagged human OX40 extracellular domain was obtained from SinoBiologicals (cat. no. 10481-H08H-50). However, SE-HPLC analysis of this antigen showed that less than 50% of the antigen was in monomeric, non-aggregated form. Therefore, this antigen was not used in subsequent analysis.

Recombinant human OX40/human Fc (hOX40-hFc) and recombinant mouse OX40/human Fc (mOX40-hFc), which contained the human IgG1 Fc domain at the C terminus, were obtained from R&D Systems (hOX40-hFc: catalog number 3388-OX -050; mOX40-hFc: catalog number 1256-OX-050) and biotinylated in-house. The biophysical properties of these soluble antigens were characterized by SE-HPLC analysis to ensure the absence of aggregates and by PAGE to check the molecular size. Size determination by PAGE indicated that the soluble antigens were dimeric, as their estimated molecular mass was twice the expected mass for a monomeric antigen. Soluble antigens were also analyzed by gel mobility shift assay, which showed that the degree of biotinylation was greater than 90%. ELISA and SPR were used to confirm that biotinylated recombinant human (hOX40-hFc) and mouse (mOX40-hFc) OX40 antigens could be bound by OX40-specific antibodies (11D4; ACT35; and OX86, as described in Example 1.1.1 and Table 2 below).

Table 2: OX40 Antigens

Antigen name Source (commercial/in-house) Is the biotinylated version obtained? View Soluble/cell expressed antigen Antigen format SEQ ID NO/ Antigen Source hOX40-mFc On your own Yes Human Soluble Dimeric 135 mOX40-mFc On your own Yes Mouse Soluble Dimeric 136 cOX40-mFc On your own Yes Cynomolgus macaque Soluble Dimeric 137 DO11.10-hOX40 On your own No Human Expressed by cells Natural conformation 132 DO11.10-mOX40- On your own No Mouse Expressed by cells Natural conformation 133 DO11.10-cOX40 On your own No Cynomolgus macaque Expressed by cells Natural conformation 134 hOX40-hFc Commercial Yes Human Soluble Dimeric Cat. No. 3388-OX-050 (R&D Systems) mOX40-hFc Commercial Yes Mouse Soluble Dimeric Cat. No. 1256-OX-050 (R&D Systems)

1.2 CD137 antigens

The CD137 antigens used to select MAbs specific for human CD137 and to test the cross-reactivity of the selected Fcabs with cynomolgus OX40 were either produced in-house or obtained from commercial sources as described below.

1.2.1 Preparation of recombinant soluble human and cynomolgus CD137 antigens

Since several commercially available recombinant antigens were found to be unsuitable for use, for example due to unacceptable levels of aggregates present when tested, the following recombinant dimeric and monomeric antigens ( Table 3 ) were prepared in-house for use in mAb selection, screening and further characterization to CD137.

Table 3: Recombinant human and cynomolgus CD137 antigens

Type Designation View Soluble or expressed by cells Is the biotinylated version obtained? Antigen format SEQ ID NO Recombinant hCD137-mFc-Avi Human Soluble Yes Dimer 138 and 141 Recombinant hCD137-Avi-His Human Soluble Yes Monomer 158 Recombinant cCD137-mFc-Avi Cynomolgus macaque Soluble Yes Dimer 140 and 141

Monomeric antigen was prepared by cloning DNA encoding the extracellular domain of human CD137 along with the Avi sequence and six C-terminal histidine residues into modified pFUSE vectors (Invivogen, cat. no. pfuse-mg2afc2) using the restriction enzymes EcoRI-HF and BamHI-HF. Vectors were transfected into HEK293-6E cells and expressed CD137 was purified using a HisTrap™ excel nickel column (GE Healthcare, 17-3712-06) and gel permeation chromatography (SEC) to ensure that the antigen was a single molecule and not contained aggregates.

To produce dimeric antigens, DNA constructs encoding the extracellular domain of human or cyno CD137 hybridized to the Fc domain of mIgG2a along with the Avi sequence were cloned into modified pFUSE vectors and transfected into HEK293-6E cells. Recombinant CD137 was purified using a MabSelect SuRe™ Protein A column (GE Healthcare, 11003494) and size exclusion chromatography (SEC) to ensure that the antigen was a single molecule and free of aggregates.

Biotinylated variants of dimeric and monomeric CD137 antigens were prepared using the BirA biotin-protein ligase reaction kit (Avidity LLC, BirA500) to produce monomeric CD137 antigen labeled with one biotin molecule and dimeric CD137 antigens labeled with two biotin molecules, one for each two monomers. Specifically, 3 mg of CD137 antigen was mixed with 7.8 μl of BirA enzyme mixture to a molar ratio of enzyme to substrate of 1:50. Supplements were then added according to the manufacturer's recommendations (142 μl Biomix A, 142 μl Biomix B, 142 μl biotin) and the reaction mixture was incubated for two hours at room temperature. To maintain the integrity of the biotinylated antigens, the buffer in the reaction mixture was immediately replaced with DFSB using Amicon 30 μm filters.

CD137 antigens were further purified by SEC to ensure removal of the BirA enzyme and a final high-quality, monodisperse protein preparation free of high molecular weight aggregates. Specifically, antigens from the same production batch were mixed and analyzed for stability and purity using gel permeation high-performance liquid chromatography (SE-HPLC), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and multi-angle gel permeation chromatography. by light scattering (SEC-MALS). Complete biotinylation of proteins was confirmed by SDS-PAGE with electrophoretic mobility shift upon binding to streptavidin. Binding of recombinant human CD137 antigens to the anti-human CD137 antibody, 20H4.9, as a positive control (US Patent No. 7288638) was confirmed in vitro using surface plasmon resonance (SPR), and binding to DO11.10 cells expressing human CD137 ligand , were confirmed using flow cytometry. Binding of recombinant cynomolgus CD137 antigen to DO11.10 cells expressing cynomolgus CD137 ligand was confirmed by flow cytometry. To ensure the highest possible purity of the CD137 antigens used in the selection protocols, careful protein characterization of the antigens was performed to ensure that the percentage of protein aggregates present did not exceed 2%.

1.2.2 Generation of cell lines expressing human, cynomolgus and mouse CD137

DO11.10 cells (National Jewish Health) expressing full-length human or cynomolgus CD137, designated "DO11.10-hCD137" and "DO11.10-cCD137", respectively (see Table 4 ), were prepared to present the antigen to its the most natural conformation during selection and for further characterization of the selected mAbs to CD137.

DO11.10 cells expressing full-length mouse CD137, designated "DO11.10-mCD137", were also prepared to determine the binding of mouse OX40/CD137 mAb ² to mouse CD137 expressed by the cells (see Example 11.2 ).

Lentiviral transduction was used to generate DO11.10 cells overexpressing human, cynomolgus or mouse CD137 receptors using the Lenti-X HTX packaging system (Clontech, 631249). Lenti-X expression vector (pLVX) (Clontech, 631253) containing cDNA encoding human CD137 (SEQ ID NO: 126), cynomolgus CD137 (SEQ ID NO: 128), or mouse CD137 (SEQ ID NO: 164) was cotransfected with Lenti-X HTX packaging mixture into the Lenti-X 293T cell line (Clontech, 632180) to produce virus. The DO11.10 cell line was then transduced with these lentiviral vectors.

Expression of human, cynomolgus and mouse CD137 on these cells was confirmed using flow cytometry based on cell binding of anti-CD137 antibodies (20H4.9, MOR7480.1 (Patent Publication No. US 2012/0237498 A1) and Lob12.3 (University of Southampton) , respectively) as a positive control.

Table 4: Human and Cynomolgus CD137 Antigens Expressed on the Cell Surface

Type Designation View Presentation SEQ ID Cell DO11.10-hCD137 Human Expressed on the cell surface 126 Cell DO11.10-cCD137 Cynomolgus macaque Expressed on the cell surface 128 Cell DO11.10-mCD137 Mouse Expressed on the cell surface 164

Example 2 - Selection and characterization of human Fcab to OX40

2.1 Naive selection of Fcab to human OX40

To select human OX40-specific Fcabs from naive phage libraries, both recombinant biotinylated soluble dimeric human OX40 (hOX40-mFc; see Table 2 ) and cell-expressed human OX40 (DO11.10-hOX40) were used as antigens. Cells expressing human OX40 were used in addition to recombinant biotinylated soluble dimeric human OX40 in some selection protocols to ensure that the selected Fcabs were capable of binding to OX40 in its native conformation on the cell surface.

Six naïve phage libraries were constructed displaying a CH3 domain (IMGT numbering 1.4-130) containing partially randomized AB loops (residues 14 to 18 according to the IMGT numbering scheme) and EF loops (residues 92 to 101 according to the IMGT numbering scheme ) in the CH3 domain. One of the six libraries additionally contained clones with an insertion of either two or four amino acids (encoded by two or four NNK codons) at position 101 in the EF loop of the CH3 domain (the inserted residues are numbered 101.1 to 101.4 according to the IMGT numbering scheme).

All six libraries were subjected to three rounds of selection using recombinant biotinylated soluble dimeric human OX40 (hOX40-mFc; see Table 2 ). All six libraries were also subjected to a subsequent selection campaign using hOX40-mFc in the first round of selection, followed by human OX40 expressed by the cells (DO11.10-hOX40 in two subsequent rounds of selection; see Table 2 ).

The 2133 clones identified after the third round of selection from six libraries were screened by ELISA to determine binding to human OX40. As a result, 32 unique positive binding elements were identified, which were subcloned and expressed as soluble Fcabs (consisting of a truncated hinge [SEQ ID NO: 101], CH2 and CH3 domain) in HEK Expi293 cells (Fcabs cloned into the pTT5 vector [National Research Council of Canada ] transfected using the ExpiFectamine 293 transfection kit [Life Technologies, A14524] into Expi293F cells [Life Technologies, A14527]).

The ability of 32 unique Fcabs to bind cell-expressed human OX40 (DO11.10-hOX40) was tested. 15 of 32 Fcabs screened showed binding to DO11.10-hOX40 cells, and EC₅₀ for these interactions ranged from 0.1 to 62 nM. 15 Fcabs that showed binding to DO11.10-hOX40 were tested using an in-house developed human NF-κB reporter assay that tests for activation of the NF-κB signaling pathway. Six of the 15 Fcabs showed increased activity when cross-linked with anti-human Fc antibody in a human NF-κB reporter assay, suggesting that these Fcabs would be capable of activating OX40 signaling. Fcabs, designated FS20-22 and FS20-31, showed high levels of activity in this assay, and their activity was increased when Fcab was cross-linked with the human anti-CH2 mAb (clone MK1A6 (Jefferiset al., 1985 and Jefferiset al., 1992), obtained in-house). They were selected for affinity maturation.

2.2 Affinity maturation of Fcab for human OX40

Affinity maturation libraries FS20-22 and FS20-31 were generated by randomizing five residues in the AB loop (residues 14 to 18) or five residues in the CD loop (residues 45.1 to 77) of the CH3 domain using randomized primers from ELLA Biotech using equimolar distribution of amino acids excluding cysteines, or by randomizing parts of the EF loop (residues 92 to 94 and 97 to 101 of the CH3 domain (all residues numbered according to the IMGT numbering scheme).

1410 Fcabs from affinity maturation outputs were screened by ELISA for binding to human OX40, and 204 unique positive binding elements were identified, subcloned, and expressed as soluble Fcabs in HEK Expi293 cells as described in Example 2.1 above.

Dissociation rates of soluble Fcabs upon binding to hOX40-mFc were measured using Biacore 3000 (GE Healthcare) in the absence and presence of anti-CH2 cross-linking using a human anti-CH2 mAb clone, MK1A6 (see Example 2.1 ). Fcabs with improved dissociation rates compared to the corresponding parent Fcab were further screened to determine binding to human cell-expressed OX40 and activity in an in-house developed human T-cell activation assay. All Fcabs bound human OX40 expressed by the cells. The 10 Fcabs from the FS20-22 line and the 18 Fcabs from the FS20-31 line that showed high levels of activity in the human T cell activation assay were selected for loop rearrangement as described below.

For line FS20-22, two loop-swapped libraries were generated by swapping three CD loops, six EF loops, and either the original AB loop or the affinity-matured AB loop. For line FS20-31, one loop-swapped library was obtained, containing four AB loops, seven CD loops, and seven EF loops.

The rearranged sequences were expressed as soluble Fcabs in HEK Expi293 cells as described inExample 2.1 above and screened for binding to biotinylated hOX40-mFc antigen using Dip and Read™ streptavidin biosensors (Pall FortéBio, 18-5050) on an Octet system QK^e (Pall FortéBio). Fcabs with improved dissociation rates upon binding to hOX40-mFc compared to the parent Fcabs were sequenced, yielding 35 unique Fcabs from the FS20-22 lineage and 62 from the FS20-31 lineage. Identified unique Fcabs were tested for binding to hOX40-mFc antigen in the presence and absence of CH2 cross-linking using the human CH2 mAb clone MK1A6 using a Biacore 3000 instrument (GE Healthcare).

For the FS20-22 line, 18 Fcabs were selected for expression in the mock (4420 LALA) MAT ² format and further characterization based on the lowest dissociation rate with CH2 cross-linking when bound to hOX40-mFc, the largest difference in dissociation rate between the dissociation rate without cross-linking and the rate of dissociation with CH2 cross-linking upon binding to hOX40-mFc, as well as the strength of binding to hOX40-mFc, as above. For line FS20-31, the nine Fcabs with the lowest dissociation rate when bound to hOX40-mFc with CH2 cross-linking and the nine Fcabs with the lowest dissociation rate when bound to hOX40-mFc without CH2 cross-linking were selected for expression and further characterization in a mock format ( 4420 LALA) MAT ² . Since several Fcabs were common to both groups of nine Fcabs, additional Fcabs were selected from the FS20-31 line that showed low dissociation rates when bound to hOX40-mFc in the absence of CH2 cross-linking to total Fcabs from this line for expression and further characterization in the mock MAT ² format was 18. Using data from the T cell activation assay, we identified a further six Fcabs from the FS20-22 lineage and eight Fcabs from the FS20-31 lineage that showed high activity in this assay and which were therefore also expressed in the mock format (4420 LALA) MAT ² and further characterized (see Example 4 ).

Example 3 - Selection and characterization of Fcab to mouse OX40

3.1. Naive selection of Fcab to mouse OX40

For selections, we used a naive yeast library exhibiting the CH1-CH3 domains of human IgG1, which contained randomizations in the AB loop (residues 11-18 in accordance with the IMGT numbering scheme) and the EF loop (residues 92-101 in accordance with the IMGT numbering scheme) of the CH3 domain and involved a randomized five-residue insertion between residues 16 and 17 (according to the IMGT numbering scheme) of the AB loop. Yeast was incubated with biotinylated recombinant mouse OX40 hybridized to the Fc domain of human IgG (mOX40-hFc; Table 2 ) and sorted by MACS using streptavidin-coated beads. Three rounds of FACS selections were then performed using decreasing concentrations of biotinylated mOX40-hFc in the presence of a five-fold molar excess of hFc. Cells were stained with streptavidin-allophycocyanin (APC) (BD Bioscience, 349024) or biotin-APC antibody (Miltenyi Biotec, 130-090-856) and sorted using a FACSAria cell sorter (BD Bioscience). 182 individual Fcabs from enriched populations were screened for antigen binding, and two unique positive binding elements were subcloned and expressed as soluble Fcabs as previously described in Example 2.1 . Fcab binding to mOX40-hFc was characterized by ELISA, and activity was characterized in an in-house developed mouse NF-κB reporter assay. Only one Fcab, FS20m-232, was active in the NF-κB reporter assay and showed binding to cells expressing mouse OX40, so this Fcab was selected for affinity maturation.

3.2 Affinity maturation of Fcab to mOX40

Three phage display libraries for affinity maturation were constructed by randomizing seven residues in the AB loop (residues 15-16.5 according to the IMGT numbering scheme) (Library 1), six residues in the CD loop (residues 45.1-78 according to the IMGT numbering scheme) ( Library 2) or five residues in the EF loop (residues 92-94 and 97-98 according to the IMGT numbering scheme) (Library 3) Fcab FS20m-232 using randomized primers from ELLA Biotech, using an equimolar distribution of amino acids except cysteine. Three rounds of selection were performed on affinity maturation libraries using recombinant biotinylated mOX40-mFc alternately captured on streptavidin-coated (ThermoFisher Scientific, 11205D) and neutravidin-coated (ThermoFisher Scientific, 14203 and A2666) Dynabeads. To identify high affinity binding elements, we used a decrease in antigen concentrations from 50 nM (Round 1) to 10 nM (Round 2) and to 1 nM (Round 3). The 1655 individual phages from the third round of selection were screened by phage ELISA for binding to mOX40-mFc, and 98 unique positive binding elements were identified, subcloned, and expressed as soluble Fcabs in HEK Expi293 cells as described in Example 2.1 . Fcab was further screened for cell binding and activity in a mouse NF-κB reporter assay. The most active Fcabs were selected for loop rearrangement.

A loop-swapped library was produced containing 27 CD loops (all 26 unique sequences identified by affinity maturation and the WT sequence) swapped with 37 EF loops (which have the best binding to mouse OX40 in the phage ELISA and the WT sequence), with all rearranged clones contained the AB loop of Fcab FS20m-232. The 750 rearranged sequences were expressed as soluble Fcabs (containing a truncated hinge) in HEK Expi293 cells as described above. HEK supernatants containing Fcab were screened to determine improved dissociation rates by measuring the binding of Fcab to biotinylated mOX40-mFc (table 2) using Dip and Read™ streptavidin biosensors (Pall FortéBio, 18-5050) on the Octet system QK^e (Pall FortéBio). 11 unique Fcabs with a randomized AB loop and 60 unique Fcabs with a randomized EF loop were subcloned and expressed as soluble Fcabs in HEK Expi293 cells as described above. These Fcabs were further screened along with 43 rearranged Fcabs with the lowest dissociation rates to determine cell binding and activity in a mouse T cell activation assay. Fcab FS20m-232-91 had the lowest dissociation rate when bound to biotinylated mOX40-mFc and the highest activity in the mouse T cell activation assay when cross-linked with the human CH2 mAb clone MK1A6 and was therefore selected as a (surrogate) mouse Fcab for use in subsequent experiments.

Example 4 - Construction, expression and characterization of Fcab to OX40 in MAT format ²

4.1 Construction and expression of mock MAT ²

"Imitation" MAT²containing anti-human OX40 and anti-mouse OX40 Fcabs identified above were prepared to allow characterization of these Fcabs in mAb format². Simulation MAT² prepared from Fcab to OX40 and variable regions of the antibody to FITC 4420 (Bedzyket al., 1989 and Bedzyket al., 1990) in the human IgG1 backbone (see SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116 for more details) or the variable regions of the hen egg white lysozyme antibody D1.3 (HEL) (Bradenet al., 1996) in the human IgG1 backbone (see SEQ ID NO: 117 and SEQ ID NO: 118 for more details) by replacing the anti-FITC and anti-HEL CH3 domains with the anti-OX40 Fcab CH3 domains at the XhoI and BamHI sites present in sequence of the unmodified CH3 domain of human IgG1. Simulation MAT² contained a light chain MAb to FITC 4420 (SEQ ID NO: 116) or a MAb to HEL D1.3 (SEQ ID NO: 118), respectively, and also contained a LALA mutation in the CH2 domain of the heavy chain to reduce interaction with the Fc-gamma receptor and potential Fc-gamma receptor-induced cross-linking. Presence of LALA mutation in mock MAbs² and MAT², mentioned in these examples, are indicated by the suffix "AA" at the end of the Fcab portion of their clone names.

Mock ² MAbs were produced by transient expression in HEK293-6E cells and purified using MabSelect SuRe™ Protein A columns.

4.2 Binding affinity of Fcab to human OX40 in the format of mock MAb ² with human and cynomolgus macaque OX40 expressed by cells

Affinity of Fcab for human OX40 in mock (4420 LALA) MAT ² format for binding to human or cynomolgus OX40 expressed in cells (DO11.10 cells expressing either human OX40 [DO11.10-hOX40] or cynomolgus OX40 [DO11. 10-cOX40]; see Table 2 ) was measured by flow cytometry. Nonspecific binding was also assessed by flow cytometry by testing binding to HEK cells not expressing OX40.

Dilutions of mock (4420 LALA) MAT ² and control MAT (2× final concentration) were prepared in triplicate in 1× DFSB (Gibco, 14190-094). Suspensions of DO11.10-hOX40 or DO11.10-cOX40 or HEK cells were prepared in PBS + 2% BSA (Sigma, A7906) and seeded at 4x10 ⁶ cells/ml with 50 μl/well in 96-well plates with V -shaped bottom (Costar, 3897). 50 μl dilutions of mock (4420 LALA) mAb ² or control mAb (human OX40 mAb, 11D4) were added to wells containing cells (final volume 100 μl) and incubated at 4°C for 1 hour. The plates were washed and then 100 μl/well of secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 in PBS + 2% BSA was added and incubated for 30 minutes at 4°C In the dark. The plates were washed and the contents were resuspended in 100 μl of PBS containing DAPI (Biotium, catalog number 40043) at 1 μg/ml. The plates were read using a Canto II flow cytometer (BD Bioscience). Dead cells were excluded and fluorescence was measured in the FITC channel (488 nm/530/30). Data were fitted using the log(agonist) response equation in GraphPad Prism software.

Fcab (all tested in mock [4420 LALA] MAb ² format) and a positive control MAb to human OX40, 11D4, in the human IgG1 backbone and containing a LALA mutation in the heavy chain CH2 domain (G1AA/11D4; SEQ ID NOs 173 and 175), bound to human OX40 with a range of affinities. The ability of five clones from the FS20-22 line and six clones from the FS20-31 line to bind human and cynomolgus monkey OX40 expressed by cells was tested; The binding affinities of these clones are presented in Table 5 .

Table 5: Binding affinity of Fcab to OX40 in mock (4420 LALA) MAT ² format with cell-expressed human or cynomolgus OX40

Imitation (4420 LALA) MAT ² /MAT Binding to DO11.10-hOX40, EC ₅₀ (nM) Binding to DO11.10-cOX40, EC ₅₀ (nM) FS20-22-38AA/4420 0.8315 0.5925 FS20-22-41AA/4420 0.2991 0.1821 FS20-22-47AA/4420 0.7655 0.5809 FS20-22-49AA/4420 0.7412 0.3197 FS20-22-85AA/4420 0.4486 1.058 FS20-31-58AA/4420 0.7466 1.454 FS20-31-66AA/4420 0.2677 2.038 FS20-31-94AA/4420 0.6132 3.52 FS20-31-102AA/4420 0.5366 0.3948 FS20-31-108AA/4420 0.6516 0.3716 FS20-31-115AA/4420 0.7853 1.235 G1AA/11D4 0.8143 0.2126

4.3 Activation of OX40 in vitro using Fcab to OX40 in mock MAT ² format

Activated T cells express OX40 on their cell surface. Binding of the trimeric OX40 ligand to OX40 results in trimerization of the receptor. Because OX40 ligand is expressed in clusters on the cell surface of antigen-presenting cells, the interaction between OX40 ligand and OX40 results in OX40 clustering, which is known to be essential for OX40 signaling and subsequent T cell activation. Antibodies that have an agonistic effect on OX40 should mimic this clustering activity of the OX40 ligand. In the case of monospecific OX40 antibodies, Fc-gamma receptors bind to and cross-link the Fc domains of the antibodies, resulting in OX40 clustering.

Anti-human OX40 and anti-mouse OX40 Fcabs in the mock (4420) MAb ² format containing the LALA mutation described above were tested in T cell activation assays to determine their ability to activate OX40 expressed on T cells when Fcab cross-linked in the presence cross-linking agent. The human T cell activation assay for testing human Fcab to OX40 in a mock (4420 LALA) MAT ² format involved isolating T cells from human peripheral blood mononuclear cells (PBMCs) and testing the release of IL-2, which is a marker of T cell activation. Assays were performed in a manner similar to that described below in Example 13 and involved the use of a human CH2 mAb clone, MK1A6, or FITC-dextran (Sigma) to cross-link the positive control antibody (11D4) or Fcab in mock format (4420 LALA ) MAT ² , respectively.

Anti-human OX40 Fcab in mock (4420 LALA) MAb ² format when cross-linked with a Fab target (FITC-dextran) showed a range of activity values in a T-cell activation assay. All Fcabs were able to co-stimulate T cells in the presence of anti-CD3 antibody and induce human IL2 production. Fcabs from lines FS20-22 and FS20-31 showed activity both with and without cross-linking. In particular, Fcabs from these lines had activity in the absence of a cross-linking agent that was significantly enhanced by cross-linking. Because these Fcabs have high cross-reactivity with cynomolgus OX40 (comparable to human OX40 binding), toxicology studies may be possible in this species. Of all the clones in the FS20-22 line, clones FS20-22-41, FS20-22-47, FS20-22-49 and FS20-22-85 had the lowest EC ₅₀ values for their cross-linking agonist activity and are therefore preferred clones from this line. Of these, clone FS20-22-49 showed the highest increase in agonist activity upon cross-linking and also had the lowest EC ₅₀ for its agonist activity in the presence of cross-linking and is therefore the preferred clone.

As described above, the aim of the present invention was to obtain MAbs ² that are capable of exerting agonistic effects on both OX40 and CD137 in the absence of additional cross-linking agents. The experiments mentioned above demonstrate that Fcab FS20-22-49 is capable of activating OX40 in the presence of an additional cross-linking agent. To obtain a dual agonist that does not require additional cross-linking agents, the present inventors decided to produce antibodies to CD137 with the intention of using the CDRs from these antibodies in the final mAb ² molecule targeting OX40 and CD137.

Example 5 - Selection and Characterization of Antibodies to Human CD137

Synthetic naïve phagemid libraries exhibiting the human germline Fab domain with randomization at CDR1, CDR2 and CDR3 (MSM Technologies) were used for naïve selection of human CD137 mAbs with recombinant and cell surface expressed CD137 antigens described in Example 1.2 .

Fab libraries were screened in three rounds using Dynabeads with streptavidin (Thermo Fisher Scientific, 11205D) and Dynabeads-associated binding protein, Neutravidin (Thermo Fisher Scientific, 31000) to isolate phage bound to biotinylated human CD137-mFc-Avi or CD137- Avi-His human. To ensure Fab binding to CD137 expressed on the cell surface, the outputs of the first round from selections using recombinant CD137 antigen were also subjected to two additional rounds of selections using DO11.10-hCD137 cells and a fourth round using DO11.10-cCD137 cells.

Approximately 2200 clones from rounds 3 and 4 outputs were screened by phage ELISA for binding to human and cynomolgus CD137-mFc-Avi. Biotinylated mFc was included as a negative control. The variable regions of positive clones (clones with a CD137 binding signal at least 4 times greater than the mFc binding signal) were sequenced, resulting in the identification of 36 unique VH/VL sequence combinations. The sequences identified were from both selections using recombinant CD137 antigen and cell surface expressed CD137 antigen, with several clones isolated using both selection strategies. Based on the phage ELISA, 22 of the 36 clones were cross-reactive against cyno, however, since the sensitivity of the phage ELISA might not be sufficient to detect weak binders cross-reactive against cyno, all 36 clones were prioritized for production in molecular format IgG1. For each clone, the VH and VL domains were individually cloned into the expression vector pTT5 (National Research Council of Canada) containing either the CH1, CH2 (with the LALA mutation in the CH2 domain), and CH3 domains or the CL domains, respectively. The resulting LALA (AA) mutant pTT5-FS30 VH and pTT5-FS30 VL vectors were transiently co-transfected into HEK293-6E cells. Twenty-eight clones were expressed as soluble IgG1 molecules. They were purified using MabSelect SuRe™ Protein A columns and subjected to further testing.

Mab binding to CD137 was analyzed by ELISA using human and cynomolgus CD137-mFc-Avi. Of the 28 clones tested, 10 showed dose-dependent binding to human CD137-mFc-Avi and no binding to human OX40-mFc-Avi, mFc, or streptavidin. Four clones in this group, FS30-5, FS30-10, FS30-15, and FS30-16, were cross-reactive against cynomolgus CD137-mFc-Avi. Due to the small number of clones obtained that were cross-reactive against cynomolgus monkey, additional clones were screened and expressed as described above. As a result, one additional binding element, FS30-35, was isolated that was cross-reactive against cynomolgus monkeys.

MAT to human CD137 FS30-5, FS30-10, FS30-15 and FS30-16 were tested to determine binding to cells expressing human or cynomolgus CD137 (DO11.10-hCD137 or DO11.10-cCD137) using flow cytometry. Non-specific binding was also assessed by testing binding to DO11.10 cells and HEK293 cells not expressing CD137. Binding affinities were compared to those of two positive control mAbs, MOR7480.1 (US Patent No. 2012/0237498) and 20H4.9 (US Patent No. 7288638), whose variable domains were cloned and expressed in human IgG1 format containing the LALA mutation in the CH2 domain (G1AA format).

Clones FS30-5, FS30-10, FS30-15 and FS30-16 were found to bind to human and cynomolgus CD137 cell surface expressed receptors with _EC50 values in the range of 0.15-0.57 nM, which comparable to MAT for positive control. No binding was detected to parental DO11.10 or HEK293 cells, indicating binding specificity. In these cells, the positive control, anti-CD137 antibody 20H4.9, was not detected to bind to cynomolgus CD137. Published data (US Patent No. 7288638) show that 20H4.9 in IgG1 format does bind to cynomolgus CD137 on cynomolgus PBMCs induced by PMA (phorbol myristate acetate). We showed that 20H4.9 in G1AA format bound to recombinant cynomolgus CD137, but the affinity was much lower than for human CD137 (data not shown), which may explain the observed lack of binding of the antibody to DO11.10-cCD137 cells .

To determine the biophysical characteristics of mAb FS30, they were subjected to gel permeation chromatography (SEC) and the percentage of monomer fraction was analyzed. All four FS30 mAbs tested showed a single peak profile and contained >97% monomers. The high level of monomeric protein allowed further testing of functional activity.

The functional activity of the anti-CD137 mAb was then analyzed in a primary T cell activation assay. Anti-CD137 mAbs induce agonistic effects in vivo by engaging Fcγ receptors, thereby causing clustering of the mAbs and the CD137 receptor. To mimic the maximal ability of the mAb to cluster CD137 receptor surface molecules, the FS30 mAb was cross-linked using an anti-human CH2 antibody (clone MK1A6, prepared in-house) prior to analysis. T cell activation was compared with non-crosslinked mAbs. Antibody D1.3 to hen egg white lysozyme (HEL) in the human IgG1 backbone with LALA mutation (G1AA/HelD1.3) was included as a negative control.

When cross-linked, mAbs FS30-5, FS30-10, FS30-15 and FS30-16 showed strong activity in the T cell activation assay with EC ₅₀ values less than 10 nM and maximum IL-2 levels (E _max ) similar to the positive control Anti-CD137 MAb (anti-CD137 MAb MOR7480.1, 5637 hIL-2 pg/ml; and anti-CD137 MAb 20H4.9, 10232 hIL-2 pg/ml). E _max of mAb FS30-6 (1512 hIL-2 pg/ml) was significantly lower than that of positive controls and other FS30 mAbs, indicating a lower overall level of T cell activation. In contrast to the positive control mAb to CD137 20H4.9, which was active in the absence of cross-linking (hIL-2 production 3174 pg/ml), mAb FS30 was not active (without cross-linking, as evidenced by measured background levels of the IL-2 response). 2).

Example 6 - Construction and Expression of MAT ² , targeting human OX40 and human CD137

MAbs ² containing Fcab to human OX40 paired with Fab to human CD137 were obtained. Fcab FS20-22-49, targeting human OX40, was selected to pair with Fabs targeting CD137 due to its higher activity in T cell assays (see Example 4.3 ).

6.1 Expression and characteristics of MAT in MAT ² format

MAT ² molecules were obtained, which consisted of an IgG1 molecule containing the CDR of clone FS30-5, FS30-10, FS30-15, FS30-16 or FS30-35 and containing the LALA mutation in the CH2 domain, as well as the human OX40 receptor binding site from FS20 -22-49 in the CH3 domain. These MAT ² molecules were prepared by replacing the VH domain of human OX40 MAT ² , FS20-22-49AA/HelD1.3, with the corresponding VH domains of FS30 clones and cotransfecting the resulting VH with the corresponding MAT FS30 light chain. The LALA mutation in the CH2 domain of the IgG1 molecule was retained in the resulting MAT ² molecules. These MAT ² molecules were designated FS20-22-49AA/FS30-5, FS20-22-49AA/FS30-10, FS20-22-49AA/FS30-15, FS20-22-49AA/FS30-16 and FS20-22-49AA /FS30-35. MAT ² was produced by transient expression in HEK293-6E cells and purified using MabSelect SuRe™ Protein A columns.

CD137 belongs to the tumor necrosis factor receptor superfamily (TNFRSF) of cytokine receptors (Moran et al. , 2013). To analyze the Fab binding site specificity for CD137 of the five MAT ² molecules, the binding of MAT ² to human CD137 and five closely related human TNFRSF members (TNFRSF1A, TNFRSF1B, GITR, NGFR and CD40) was tested using SPR. The goal was to demonstrate 1000-fold specificity by showing no binding of MAT ² to closely related antigens at a concentration of 1 μM, while showing binding to CD137 receptors at a concentration of 1 nM.

While MAT² FS20-22-49AA/FS30-5, FS20-22-49AA/FS30-10, FS20-22-49AA/FS30-16 and FS20-22-49AA/FS30-35 showed a high level of specificity (close to 1000-fold) , MAT² FS20-22-49AA/FS30-15 showed nonspecific binding to all five closely related TNFRSF members tested. The nonspecific binding exhibited by this clone was on average approximately 5-10-fold lower than binding to CD137 receptors at the same concentration and was concluded to be due to the Fab binding site of the MAT molecule², since mAb FS30-15 showed the same binding profile when tested binding to the same five TNFRSF members closely related to CD137. Based on these data, clone FS30-15 was excluded from further selection campaigns.

6.2 Sequence optimization of mAbs to CD137

Although the anti-CD137 mAbs, FS30-5, FS30-10, FS30-16 and FS30-35, showed high affinity and specificity for CD137, as well as activity in the T cell activation assay, they contained one or more potential post-translational modification (PTM) sites in CDR loops. The decision was made to further modify these clones to attempt to identify amino acid residues that could be changed at these sites to maintain or improve binding and activity. Identified potential PTM sites included methionine residues in CDR3 VH (position according to Kabat M100D and M100H in FS30-5, M97 in FS30-10, M100A in FS30-16 and M100F in FS30-35), a potential aspartate isomerization motif in CDR2 VH (position Kabat D54G55 in FS30-16) and a potential deamidation site in CDR3 VL (position Kabat Q90G91 in FS30-16).

Site-directed mutagenesis was carried out using five MAT ² clones FS20-22-49AA/FS30 as templates and primers that contained the degenerate codon NNK at sites encoding methionine, aspartate or glycine residues to ensure all possible amino acid substitutions. Cysteine residues and amino acids that could lead to the formation of new potential PTM motifs were excluded. Clones were expressed and screened for binding to DO11.10-hCD137 cells. Clones with similar (within two-fold) or improved binding at 10 nM compared to the original MAT ² clones were selected for expression on a 30-50 ml scale, purified on Protein A columns and screened in a T cell activation assay using cells DO11.10-hCD137 and anti-human CH2 antibody, MK1A6, as a cross-linking agent.

DO11.10-hCD137 cells were washed once in PBS and resuspended in DO11.10 cell medium (RPMI medium (Life Technologies) with 10% fetal bovine serum (FBS (Life Technologies)) and 5 μg/ml puromycin (Life Technologies) , A11113803)) at a concentration of 1.0×10 ⁶ cells/ml. 96-well flat-bottom plates were coated with anti-mouse CD3 antibody (Thermo Fisher Scientific, clone 17A2) by incubating with 0.1 μg/ml anti-mouse CD3 antibody diluted in PBS for 2 hours at 37°C, 5% CO ₂ and then washed twice with PBS. DO11.10-hCD137 cells were added to plates at 1×10 ⁵ cells/well. A 2 μM dilution of each antibody tested was prepared in DPSB (Gibco) and further diluted 1:10 in DO11.10 cell media (30 μL + 270 μL) to obtain a 200 nM dilution. The MK1A6 cross-linker was added to the wells in a 1:1 molar ratio with the test antibody samples to be cross-linked. Serial dilutions of each antibody or antibody/crosslinker mixture were prepared in a 96-well plate. 100 μl of diluted antibody or antibody/cross-linker mixture was added to DO11.10-hCD137 cells on the plate. Cells were incubated at 37°C, 5% CO ₂ for 72 hours. Supernatants were collected and analyzed using a mouse IL-2 ELISA kit (eBioscience or R&D Systems) according to the manufacturer's instructions. The plates were read at 450 nm using a plate reader with Gen5 software, BioTek. The 630 nm absorbance values were subtracted from the 450 nm absorbance values (Correction). The standard curve for calculating cytokine concentration was based on a four-parameter logistic curve fit (Gen5 software, BioTek). Mouse IL-2 (mIL-2) concentration was plotted against log antibody concentration, and the resulting curves were fitted using the log(agonist) response equation in GraphPad Prism.

For each of the clones, a limited number of amino acids that retained or improved binding to cell surface CD137 were identified to replace the methionine residue in the heavy chain CDR3. Clone MAT² FS20-22-49AA/FS30-16 contained three potential PTM sites, and mutation of each of them resulted in a small decrease in binding affinity. When they were combined in a single molecule, the reduction in binding was additive (data not shown) and therefore this clone was not used further. Several mutations were identified that improved CD137 binding and functional activity compared to the corresponding parent clone. It was found that three mutant clones of MAT², all derived from the MAT clone² FS20-22-49AA/FS30-10, have improved binding affinity and functional activity. These MAT² contained either an asparagine residue, a threonine residue, or a leucine residue replacing the methionine residue at position 97 in the original MAT² FS20-22-49AA/FS30-10, and were designated FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12 and FS20-22-49AA/FS30-10-16, respectively . Despite the fact that the EC values₅₀ for mutant clones derived from the original MAT clone² FS20-22-49AA/FS30-35, showed no improvement in functional activity compared to the parent clone, one mutant clone, designated FS20-22-49AA/FS30-35-14, which contained an alanine residue replacing a methionine residue at position 100F in original clone, but showed improved binding. In the case of the original MAT clone² FS20-22-49AA/FS30-5 both the methionine residue at position 100D and the methionine residue at position 100H were replaced, respectively, by an isoleucine residue and a leucine residue in the same molecule to obtain a mutant MAT clone², designated FS20-22-49AA/FS30-5-37. Clones FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA/FS30-10-16, FS20-22-49AA/FS30-35-14 and FS20 -22-49AA/FS30-5-37 was selected for further characterization.

6.3 Human CD137 ligand blocking assays

Activation of the CD137 receptor requires the CD137-CD137L interaction. Agonistic antibodies to CD137 can trigger CD137 activation by mimicking ligand interaction, potentially blocking ligand binding, or by triggering receptor clustering and activation without disrupting ligand binding. If the antibody potentially mimics CD137L, it could block receptor-ligand interaction. MOR7480.1 is known in the art to block the ligand/receptor interaction (US 2012/0237498), while the 20H4.9 antibody has previously been reported to not block the interaction between CD137 and its ligand (US Patent No. 7288638).

Ability of MAT ² clones to human CD137 FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA/FS30 -10-16 and FS20-22-49AA/FS30-35-14 block the CD137-CD137L interaction were tested using an ELISA-based method. Anti-OX40 mAb 11D4 (European Patent No. 2242771) in IgG1 format (G1/11D4; SEQ ID NOs 174 and 175) was used as isotype/negative control; MAB ² FS20-22-49AA/4420, containing the anti-OX40 Fcab clone FS20-22-49AA and the Fab region of the anti-FITC antibody 4420, was used as MAB ² as a negative control for OX40 binding; and anti-CD137 mAbs G1/MOR7480.1 (SEQ ID NOs 119 and 120) and G1/20H4.9 (SEQ ID NOs 121 and 122) were used as positive controls for CD137 binding and ligand blocking activity.

Specifically, Maxisorp 96-well plates were coated with recombinant human CD137-mFc-Avi antigen at a concentration of 1 μg/ml in PBS overnight at 4°C. The next day, plates were washed with PBS (PBS + 0.05% Tween20™) and blocked with PBS + 1% BSA (Sigma, A3059-500G) for 1 hour at room temperature with stirring. After blocking, the plates were washed again with PBST. A 100 nM dilution of each antibody tested was prepared in PBS + 1% BSA, added to CD137-coated plates, and incubated for 1 hour at room temperature with mixing. After this incubation, the plates were washed with PBST and then incubated with 20 ng/ml CD137L-His (R&D Systems, 2295-4L-025/CF) in PBS for 1 hour at room temperature with agitation. The plates were then washed with PBST and then incubated with a 1:1000 dilution of anti-His secondary antibody (R&D Systems, MAB050H) in PBS for 1 hour at room temperature with agitation. The plates were then washed with PBST and incubated with TMB detection reagent (Thermo Fisher Scientific, 002023) until a blue color appeared in the positive control wells, after which the reaction was stopped by adding 2 N H ₂ SO ₄ . The plates were read at 450 nm using a plate reader with Gen5 software, BioTek. The 630 nm absorbance values were subtracted from the 450 nm absorbance values (Correction). Subtracted absorbance values were plotted against log antibody concentration and the resulting curves were fitted using the log(inhibitor) response equation in GraphPad Prism. The values were normalized by setting the control MAbs G1/11D4 and G1/MOR7480.1 as 0% and 100% blocking values, respectively. Data were analyzed using one-way analysis of variance and the Holm-Sidak test for multiple comparisons using GraphPad Prism.

A range of blocking activities were observed for the five anti-human CD137 mAb ² clones tested. FS20-22-49AA/FS30-5-37 showed, like the positive control antibodies, complete inhibition of receptor-ligand interaction. All MAT ² clones containing Fab regions from the anti-CD137 MAT line FS30-10 (i.e. FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12 and FS20-22-49AA /FS30-10-16), inhibited the interaction between CD137 and CD137L by 49-54% and were therefore considered partial blockers. Because of only partial blocking of the interaction between CD137 and CD137L, it is possible that these mAbs may not completely inhibit the natural interaction of CD137L with its receptor such that some CD137 signaling may still occur through this mechanism even if one of these antibodies is bound. Clone FS20-22-49AA/FS30-35-14, like the negative control MAT ² molecule FS20-22-49AA/4420, was not able to significantly inhibit the receptor-ligand interaction and was therefore considered non-blocking.

In summary, the results of this ELISA-based assay showed that the panel of CD137 mAbs tested showed multiple patterns of ligand blocking activity, including complete, partial, and no blocking activity. Each of the clones FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA/FS30-10-16 and FS20-22-49AA/FS30-35-14 showed blocking activity that was different from that of anti-CD137 mAbs as positive controls. Since a number of ligand blocking activities have been identified, the functional activity of each antibody was tested.

Ability of clones FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12 and FS20-22-49AA/FS30-10-16 to block The CD137-CD137L interaction was further tested using a cell-based method. A range of blocking activities were observed, with FS20-22-49AA/FS30-5-37 showing, like the positive control antibody (G1/MOR7480.1) used in this assay, complete inhibition of receptor-ligand interaction. All three MAT ² clones containing Fab regions from the anti-CD137 MAT line FS30-10 (i.e. FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12 and FS20-22- 49AA/FS30-10-16), inhibited the interaction between CD137 and CD137L by 46-76% and were therefore considered partial blockers. In summary, the results of this assay were similar to the results of the ELISA blocking assay and showed that the panel of CD137 mAbs tested exhibited multiple ligand blocking abilities, ranging from complete to partial blocking activity. Each of the clones FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12 and FS20-22-49AA/FS30-10-16 showed blocking activity that was different from that of the positive control antibody .

Example 7 - Binding Specificity and Functional Activity of MAT and MAT Clones ² in an activation assay of T cells expressing human CD137

7.1 Binding specificity of MAT ² clones

CD137 and OX40 belong to the tumor necrosis factor receptor superfamily (TNFRSF) of cytokine receptors (Moran et al. , 2013). To analyze the specificity of Fab to CD137, as well as the binding site of Fcab to OX40 of five MAT ² molecules, binding of MAT ² FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA /FS30-10-16, FS20-22-49AA/FS30-35-14 and FS20-22-49AA/FS30-5-37 with human CD137, human OX40 and six closely related human TNFRSF members were tested using surface plasmon resonance (SPR ). The goal was to demonstrate 1000-fold specificity by showing no binding of MAT ² to closely related antigens at a concentration of 1 μM, while showing binding to the CD137 and OX40 receptors at a concentration of 1 nM. Anti-CD137 mAb MOR7480.1 and anti-OX40 mAb 11D4 were used as positive controls.

In general terms, human CD137-mFc-Avi ( Table 3 ), OX40-mFc ( Table 2 ), recombinant human TNFRSF1A-Fc, recombinant human TNFRSF1B-Fc, recombinant human GITR-Fc, recombinant human NGFR-Fc, recombinant CD40- Human Fc or recombinant human DR6-Fc was immobilized on flow cells on CM5 chips at ca. 1000 RU. Flow cell 1 was left to immobilize the blank sample. Five MAbs ² were diluted to 1 μM and 1 nM in 1× HBS-EP buffer (GE Healthcare, product code BR100188), passed over the chip for 3 min, and then allowed to dissociate for 4 min. For regeneration, a 30-second injection of 10 mM glycine, pH = 1.5, was used. Positive control mAbs were injected at 50–100 nM to demonstrate coverage of each antigen. Binding levels were determined at the end of the association phase and compared.

All of the selected mAbs ² showed high levels of specificity for human CD137 and OX40 receptors, similar to or higher than the positive controls MOR7480.1 and 11D4, respectively.

7.2 Functional activity of agonistic antibodies to CD137 in the activation assay of T cells expressing human CD137

To determine the activity of various CD137 agonistic antibodies, a T cell activation assay was used using DO11.10-hCD137 cells. Agonistic antibodies tested against CD137 G1AA/MOR7480.1 (SEQ ID NO: 125 and 120), G1AA/20H4.9 (SEQ ID NO: 165 and 122) and G1AA/FS30-10-16 (SEQ ID NO: 154 and 97 ), as well as anti-FITC 4420 in IgG1 format (G1/4420; SEQ ID NOs: 115 and 116) as a negative isotype control. Antibody molecules were tested both in the presence and absence of the human CH2 cross-linking antibody MK1A6 (see Example 2.1 ). Mouse IL-2 production was used as a measure of T cell activation.

DO11.10-hCD137 cells were washed once in PBS and resuspended in DO11.10 cell medium (RPMI medium (Life Technologies) with 10% PBS (Life Technologies) and 5 μg/ml puromycin (Life Technologies, A11113803)) in concentration 1.0×10 ⁶ cells/ml. 96-well flat-bottom plates were coated with anti-mouse CD3 antibody (Thermo Fisher Scientific, clone 17A2) by incubating with 0.1 μg/ml anti-mouse CD3 antibody diluted in PBS for 2 hours at 37°C, 5% CO ₂ and then washed twice with PBS. DO11.10-hCD137 cells were added to plates at 1×10 ⁵ cells/well. A 2 μM dilution of each antibody tested was prepared in DPSB (Gibco) and further diluted 1:10 in DO11.10 cell media (30 μL + 270 μL) to obtain a 200 nM dilution. MK1A6 cross-linker was added to wells in a 1:1 molar ratio with test antibodies where required. Serial dilutions of the antibody or antibody/cross-linking antibody mixture were prepared in a 96-well plate. 100 μl of diluted antibody or antibody/cross-linking antibody mixture was added to DO11.10-hCD137 cells on the plate. Cells were incubated at 37°C, 5% CO ₂ for 72 hours. Supernatants were collected and analyzed using a mouse IL-2 ELISA kit (eBioscience or R&D Systems) according to the manufacturer's instructions. The plates were read at 450 nm using a plate reader with Gen5 software, BioTek. The 630 nm absorbance values were subtracted from the 450 nm absorbance values (Correction). The standard curve for calculating cytokine concentration was based on a four-parameter logistic curve fit (Gen5 software, BioTek). Mouse IL-2 (mIL-2) concentration was plotted against log antibody concentration, and the resulting curves were fitted using the log(agonist) response equation in GraphPad Prism.

The results of the analysis are shown in Figure 2C and D. Anti-CD137 antibodies differed in their requirement for cross-linking antibody to exert their activity, with all three anti-CD137 antibodies showing a concentration-dependent increase in IL-2 production in the presence of cross-linking antibody, but only antibody G1AA/20H4.9 was active in the absence cross-linking antibodies. Thus, G1AA/MOR7480.1 and G1AA/FS30-10-16 required the addition of a cross-linking antibody, i.e. their activity was “cross-linking dependent”, while G1AA/20H4.9 showed activity as in the presence of , and in the absence of cross-linking antibody, i.e. its activity was “cross-linking independent”.

7.3 Functional activity of MAT ² clones in the activation assay of T cells expressing human CD137

Functional activity of selected MAT clones² FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12 and FS20-22-49AA/FS30-10-16 were tested in a T cell activation assay using DO11.10-hCD137 cells. Anti-FITC 4420 antibody in IgG1 format (G1/4420; SEQ ID NOs: 115 and 116) was used as a negative isotype control; MAT to OX40 G1/11D4 (SEQ ID NO 174 and 175) and clone MAT² FS20-22-49AA/4420 (SEQ ID NOs 123 and 116) were used as negative controls; and anti-CD137 antibody MOR7480.1 in both IgG1 (G1/MOR7480.1; SEQ ID NOs 119 and 120) and IgG2 (G2/MOR7480.1; SEQ ID NOs 124 and 120) formats, the IgG2 format being the , in which the antibody was tested in clinical studies (Gopalet al., 2017; Tolcheret al., 2017) were used as a positive control. MAT and MAT molecules² cross-linked with the anti-human CH2 antibody, MK1A6 (seeExample 2.1), and in one experiment the activity of the molecules MAT and MAT was studied², not cross-linked. Mouse IL-2 production was used as a measure of T cell activation. The experiment was performed as described inExample 7.2.

When cross-linked, all five selected MAT ² clones showed strong activity in the T cell activation assay with average EC ₅₀ values of less than 15 nM and average E _max values ranging from approximately 16,000-20,000 pg/ml IL-2 ( Table 6 and Figure 2A ) . In the absence of cross-linking, no activity was observed for the MAT ² clones tested ( Figure 2B ). Antibody activity for the positive control MOR7480.1 was observed only upon cross-linking (EC ₅₀ 3.3 nM and E _max 12575 pg/ml for G1/MOR7480.1 and EC ₅₀ 2.4 nM and E _max 8547 pg/ml for G2/ MOR7480.1). The combination of the lack of activity of the cross-linked anti-OX40 mAb (G1/11D4) and the low background signals observed for non-cross-linked mAb ² molecules containing anti-OX40 Fcab indicate that the results of this assay represent a readout of CD137 activity only, most likely due to for high levels of CD137 receptor expression and undetectable levels of OX40 receptor expression in DO11.10 cells (data not shown).

Table 6: Activity of MAT ² in the activation assay of T cells expressing human CD137

MAT/MAT ² Activity of non-crosslinked MAT/MAT ² (n=1) Activity of cross-linked MAT/MAT ²
(average n=2) EC ₅₀ (nM) E _max (mIL-2 pg/ml) EC ₅₀ (nM) E _max (mIL-2 pg/ml) G1/4420 Not applicable Not applicable Not applicable Not applicable G1/11D4 Not applicable Not applicable Not applicable Not applicable G1/MOR7480.1 Didn't measure Didn't measure 3.3 12575 G2/MOR7480.1 Not applicable Not applicable 2.4 8547 FS20-22-49AA/4420 Not applicable Not applicable Not applicable Not applicable FS20-22-49AA/FS30-5-37 Not applicable Not applicable 13.4 18129 FS20-22-49AA/FS30-10-3 Not applicable Not applicable 6.1 17049 FS20-22-49AA/FS30-10-12 Not applicable Not applicable 9.5 17183 FS20-22-49AA/FS30-10-16 Not applicable Not applicable 4.7 16310 FS20-22-49AA/FS30-35-14 Not applicable Not applicable 5.1 19837

N/A: not applicable, since the low signal did not allow reliable determination of EC ₅₀ /E _max

NM: not measured

Thus, MAT ² containing the CDRs of monoclonal antibodies to human CD137 FS30-5-37, FS30-10-3, FS30-10-12, FS30-10-16 and FS30-35-14 showed strong activity in terms of the ability to activate CD137 in the DO11.10-hCD137 cross-linking T cell activation assay. In the absence of cross-linking, no significant activity was observed. These MAbs ² contain the CH3 domain from Fcab to human OX40 FS20-22-49, which also showed high cross-linking activity in the T cell assay (see Example 4.3 ). MAbs ² obtained with the LALA mutation were designated FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA /FS30-10-16.

These mAbs ² were selected for further analysis to determine whether they are capable of acting as a dual agonist that can autonomously exert agonistic effects on both OX40 and CD137 based on the expression of specific targets and without the need for additional cross-linking agents.

Example 8 - MAT Binding Affinity ² against OX40 and CD137 in humans and cynomolgus macaques

To determine affinity for CD137, the CM5 Biacore chip (GE Healthcare) was coated with anti-human Fc antibody using the Human Antibody Capture kit (GE Healthcare) according to the manufacturer's specifications to a surface density of approximately 4000 RU. Samples of tested antibodies (MAT ² FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12 and FS20-22-49AA/FS30- 10-16, positive control to CD137 G1/MOR7480.1 and negative control to hOX40 G1/11D4) captured up to approximately 80 RU. Human or cynomolgus CD137 (hCD137-mFc-Avi or cCD137-mFc-Avi) was passed over the chip at a range of concentrations in a series of threefold dilutions starting at 200 nM at a flow rate of 70 μl/min. The association time was 2 min and the dissociation time was 8 min. The running buffer was HBS-EP (GE Healthcare, BR100188). The flow cells were regenerated by injecting 3 M magnesium chloride at a flow rate of 30 μL/min for 30 seconds.

To determine affinity for OX40, the CM5 Biacore chip was coated with anti-human Fab antibody using the Human Fab Capture kit (GE Healthcare, 28958325) according to the manufacturer's specifications to a surface density of approximately 8000 RU. Samples of tested antibodies (MAT ² FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12 and FS20-22-49AA/FS30- 10-16, G1/MOR7480.1 (negative control) and G1/11D4 (positive control)) were captured to approximately 80 RU, and then human or cynomolgus OX40 antigen (hOX40-mFc or cOX40-mFc) was passed over the chip in the range concentrations in a series of threefold dilutions, starting from 200 nM, at a flow rate of 70 μl/min. The association time was 2 min and the dissociation time was 8 min. The running buffer was HBS-EP. The flow cells were regenerated by injecting glycine-HCl at pH=2.1 at a flow rate of 30 μL/min for 30 seconds.

Data were analyzed by subtracting two standards against a flow cell that was intentionally left blank (without antibody binding). Binding kinetics were fitted using a 1:1 Langmuir model to obtain binding association (k _a ) and dissociation (k _d ) rates. Equilibrium binding constants ( _KD ) were calculated by dividing the dissociation rate by the association rate for each sample. Data analysis was performed using BiaEvaluation software, version 3.2. The results are presented in Table 7 .

Table 7: Binding affinity of MAT ² to human and cynomolgus CD137 and OX40 determined by SPR

CD137 OX40 MAT/MAT ² Human K _D (nM) K _D of cynomolgus macaque (nM) Human K _D (nM) K _D of cynomolgus macaque (nM) G1/MOR7480.1 0.127 Didn't measure No binding detected No binding detected G1/11D4 No binding detected No binding detected 0.0337 Didn't measure FS20-22-49AA/FS30-5-37 3.85 6.42 0.385 1.63 FS20-22-49AA/FS30-10-3 0.342 0.318 0.285 1.11 FS20-22-49AA/FS30-10-12 0.255 7.24 0.37 1.02 FS20-22-49AA/FS30-10-16 0.17 0.15 0.214 0.861

NB - no binding detected. NM - Not measured.

The binding affinity values for MAT ² to OX40/CD137 indicate that these molecules bind with high affinity to both receptors. The affinities of these molecules for human OX40 are similar, as would be expected since all of these molecules share a common Fcab for OX40. The affinity for cynomolgus OX40 is within 5-fold of the affinity for human OX40. Affinity for human CD137 ranges from 4–0.2 nM, and cross-reactivity with cynomolgus CD137 also varies because Fabs to CD137 differ in each molecule. FS20-22-49AA/FS30-10-16 has higher affinity for human CD137 and similar affinity for cynomolgus CD137. The similarity in binding to human and cynomolgus antigens may be an advantage in that it is hoped that the behavior of MAT ² in cynomolgus monkey studies can be extrapolated to humans.

FS20-22-49AA/FS30-10-16 also has similar affinity for human OX40 and human CD137, so MAT ² is expected to bind equally well to both targets when coexpressed.

MAT ² , which binds to OX40 and CD137 and simultaneously triggers clustering and activation of both targets, is expected to act as a dual agonist. Both OX40 and CD137 are known to be present on T cells (Ma, et al. , 2005). Without being limited to any theory, it is believed that MAT ² having similar binding affinities to both targets may provide an advantage as a dual agonist because MAT ² is more likely to bind to cells that express both targets. A mAb ² that preferentially bound one target with significantly higher affinity than the other may be unable to act as a dual agonist because it may preferentially bind to cells that do not express both targets.

Example 9 - Simultaneous MAT Binding ² with OX40 and CD137

9.1 Simultaneous binding of MAT ² to human OX40 and human CD137

MAT ability² to OX40/CD137 FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3 and FS20-22-49AA/FS30-10-16 simultaneously binding to OX40 and CD137 were tested using SPR on Biacore 3000. G1/MOR7480.1 was used as a control. Biotinylated human CD137 (hCD137-mFc-Avi-Bio) was diluted to 100 nM in HBS-EP buffer and immobilized on a streptavidin (SA) chip (GE Healthcare, BR100032) to a surface density of approximately 1000 RU, according to the manufacturer's instructions, and flow-through the cell was activated and deactivated without any protein immobilized for background subtraction. Antibodies diluted to 100 nM in HBS-EP buffer were co-injected with 100 nM human OX40 (hOX40-mFc) or HBS-EP buffer at a flow rate of 30 μl/min. For each binding step, dissociation was observed for 3 minutes. The sensor chip was regenerated after each cycle by injecting 15 μL of glycine 2.5 (GE Healthcare) at a flow rate of 30 μL/min. All tested MATs² were able to simultaneously bind to OX40 and CD137. The control mAb, G1/MOR7480.1, bound only to CD137.

9.2 Simultaneous binding of mouse receptor-targeted MAb ² to mouse OX40 and mouse CD137

MAb ² containing Fcab to mouse OX40 with Fab to mouse CD137 was prepared to test its ability to simultaneously bind to mouse OX40 and mouse CD137. The mouse OX40-targeting Fcab, FS20m-232-91, was selected for its higher activity in T cell assays, and the mouse CD137 antibody Fab Lob12.3 (Taraban et al. , 2002) was in human IgG1 isotype format ( G1/Lob12.3; University of Southampton) was selected to pair with Fcab FS20m-232-91 because it showed good cellular binding to mouse CD137-expressing cells and is widely used in the literature as an agonistic anti-CD137 antibody with in vitro and in vivo activity. . MAT ² containing the CH3 domain FS20m-232-91 and Fab anti-mouse CD137 antibody Lob12.3 and the LALA mutation were designated “FS20m-232-91AA/Lob12.3”, while MAT ² containing the CH3 domain FS20m-232 -91 and Fab anti-CD137 antibodies from Lob12.3 mice without the LALA mutation, designated “FS20m-232-91/Lob12.3.”

The ability of MAT ² FS20m-232-91AA/Lob12.3 to simultaneously bind to its two targets was tested using SPR on a BIAcore 3000 instrument (GE Healthcare). G1/Lob12.3 was used as a positive control. Recombinant mouse CD137 (mCD137-hFc; R&D Systems, catalog number 937-4B-050) was diluted to 200 nM in sodium acetate pH 5.0 (GE Healthcare) and immobilized on a CM5 Biacore chip to a surface density of approximately 1000 RU, according to the manufacturer's instructions, and the flow cell was activated and deactivated without any protein immobilized for background subtraction. MAT ² and positive control diluted to 100 nM in HBS-EP buffer were co-injected with 100 nM human OX40 (mOX40-mFc) or HBS-EP buffer at a flow rate of 30 μl/min. For each binding step, dissociation was observed for 3 minutes. The sensor chip was regenerated after each cycle using a 30-second injection of an aqueous glycine-HCl solution at pH=1.7 at a flow rate of 20 μL/min. MAT ² was able to simultaneously bind to OX40 and CD137. MAb G1/Lob12.3 bound only to CD137.

Example 10 - Binding of MAT ² to Fc γ receptors

As is known from the literature, agonistic antibodies targeting members of the TNFR family require cross-linking through Fcγ receptors to trigger clustering and activation of the target for activity in vivo (Wajant, 2015). However, this may not be desirable for an antibody that is intended to be a dual agonist. In this regard, it was decided to reduce the ability of MAT ² to bind to Fcγ receptors by inserting a LALA mutation.

Antibodies of the human IgG1 isotype are able to bind to Fcγ receptors. As a result, they can induce effector function, such as antibody-dependent cellular cytotoxicity (ADCC), of target-expressing cells when they bind to Fcγ receptors, resulting in cell lysis. Since the intended mechanism of MAT ² to OX40/CD137 is to activate cells expressing OX40 and CD137 without killing them, the goal is to reduce MAT ² -induced ADCC. Since MAbs ² to OX40/CD137 are also designed to function as dual agonists, their intended mechanism of action is receptor signaling through cross-linking by double binding to both OX40 and CD137 when they are co-expressed on the same and the same cell or are expressed on different cells and therefore do not require the ability to cross-link via Fcγ receptors for their function.

Additionally, CD137-targeting antibodies are known to be toxic to the liver in clinical settings (Segal et al. , 2017) and although the mechanism of toxicity is unknown, it may be based on FcγR-mediated cross-linking of CD137 antibodies and activation of CD137-expressing cells in the liver or periphery. Preventing the agonistic effect on CD137 by FcγR-mediated cross-linking may reduce the risk of any toxicity of Mab ² to OX40/CD137 according to the present invention, since these molecules will only cross-link by dual binding to OX40 and CD137.

SPR binding was used to confirm that the presence of the LALA mutation in MAT ² FS20-22-49AA/FS30-10-16 reduced binding affinity for Fcγ receptors, particularly hFcγR1 (R&D Systems, catalog number 1257-FC- 050/CF), hFcγR2a (R&D Systems, part number 1330-CD-050/CF), hFcγR2b (R&D Systems, part number 1460-CD-050/CF) and hFcγR3a (R&D Systems, part number 4325-FC -050/CF). Anti-hOX40 MAbs G1AA/11D4 and G1/11D4 (with and without the LALA mutation, respectively) and anti-CD137 MAbs G1AA/20H4.9 and G1/20H4.9 (with and without the LALA mutation, respectively), all in isotype format hIgG1, as well as mAb to hCD137 G4/20H4.9 in hIgG4 isotype format were used as control antibodies. Binding was tested on a Biacore 3000 instrument (GE Healthcare). The SA chip (GE Healthcare, catalog no. BR100398) was coated with biotinylated his-tagged human OX40 (BPS Bioscience, catalog no. 71310) and human CD137 (prepared in-house) antigens at a concentration of 2 μM. Human OX40 and human CD137 were applied to separate flow cells while the other flow cell was left blank for background subtraction. Regeneration conditions were defined as 12 μL of aqueous 10 mM glycine-HCl at pH = 2.0 at a flow rate of 20 μL/min. Antibodies (see Table 8 ) and human FcγR (see Table 8 ) were diluted to 100 nM (antibodies) or 500 nM (human FcγR) in HBS-P (0.01 M HEPES, pH=7.4, 0.15 M NaCl, 0.005% v/v Surfactant P20, GE Healthcare, BR-1003-68) and co-injected at a flow rate of 20 μl/min, and then monitored for dissociation for 5 min.

Data analysis was performed using BiaEvaluation software, version 3.2 RC1, by comparison with a blank flow cell and fitting curves after antibody association. Values for the binding response at the end of the association phase were obtained by subtracting the absolute response at the end of the FcγR association phase from the absolute response at the end of the antibody association phase to normalize the effect of antibody binding to the OX40 and CD137 receptors.

Measurement of binding response values at the end of the FcγRI dissociation phase was performed to demonstrate the effect of the LALA mutation in increasing the dissociation rate for FcγRI binding in the absence of complete elimination of binding to that FcγR. These values were obtained by subtracting the absolute response at the end of the FcγR association phase from the absolute response at the end of the FcγR dissociation phase. Values for anti-CD137 antibodies were obtained from a flow cell coated with CD137-his antigen, values for anti-OX40 antibodies were obtained from a flow cell coated with OX40-his antigen, and values for MAb ² to OX40/CD137 were obtained from both flow cells coated with OX40 antigen -his and from a flow cell coated with the CD137-his antigen. The results are presented in Table 8 .

Table 8: Antibody binding response to human Fcγ receptors according to SPR

MAT/MAT ² Antigen on a chip Binding response at the end of the FcγR association phase (RU) (forward reaction) Decreased binding response at the end of the dissociation phase of FcγR (RU) FcγRIIa FcγRIIb FcγRIIIa FcγRI FcγRI G1/11D4 OX40-his 123 89.7 142.7 370.3 46.4 G1AA/11D4 OX40-his 64.5 60.9 67.3 292.3 202.6 G1/20H4.9 CD137-his 224.8 158.5 297.4 741 -16.3 G1AA/20H4.9 CD137-his 97.5 95.4 129.4 504.6 380.6 G4/20H4.9 CD137-his 156 163.9 113.1 693.3 57.1 FS20-22-49AA/FS30-10-16 OX40-his 37.4 34.3 31.4 237.5 234.8 FS20-22-49AA/FS30-10-16 CD137-his 10.9 9 17.3 245.8 367.3

Mab ² and control antibodies without the LALA mutation all bound to each of the Fcγ receptors as expected in both IgG1 and IgG4 formats. MAb ² and control IgG1 antibodies containing the LALA mutation showed significantly reduced binding at the end of the association phase (forward reaction) to each of the Fcγ receptors tested, with the exception of FcγRI, compared to control IgG1 antibodies without the LALA mutation and control antibody in IgG4 format. The rate of direct binding of the high-affinity FcγR receptor, FcγRI, to LALA-containing hIgG1 antibodies was only slightly reduced compared with non-LALA IgG1 antibodies, and thus was not significantly altered by the introduction of the mutation. However, for FcγRI, the dissociation rate was higher for antibodies containing the LALA mutation than for antibodies without LALA, as evidenced by a greater decrease in the binding response at the end of the FcγRI dissociation phase (more than 200 RU for each of the LALA-containing antibodies compared with less than 60 RU for antibodies that do not contain LALA).

Overall, anti-OX40/CD137 mAb ² containing the LALA mutation reduced binding to Fcγ receptors compared to wild-type human IgG1, similarly to other LALA-containing hIgG1 antibodies, and at a lower level than the control IgG4 antibody. Because Fcγ receptor binding is required for ADCC activity, the reduction in Fcγ receptor binding caused by the LALA mutation is expected to also reduce ADCC so that target cells are not depleted by MAT ² binding. This is considered important because MAb ² to OX40/CD137 are agonistic antibodies and therefore depletion of target cells is undesirable since these are the cells that MAb ² is intended to stimulate.

FcγRIIIa is expressed on immune effector cells such as natural killer cells (NK cells) and has been shown to play an important role as a mediator of ADCC (Chan et al ., 2015). To determine whether the reduced binding of MAT ² FS20-22-49AA/FS30-10-16 to FcγRIIIa, confirmed by SPR data, would result in low or negligible activation of the ADCC pathway, an ADCC bioassay was performed using modified Jurkat cells expressing FcγRIIIa as effectors cells, and Raji cells overexpressing either human OX40 or human CD137 as target cells. MAT ² was not found to induce ADCC activation in either OX40-expressing or CD137-expressing Raji cells, compared with the responses observed for the negative and positive control antibodies used in the assay.

To exert their agonistic activity, other agonistic antibodies are known to rely on antibody cross-linking with the Fcγ receptor to form higher order structures (Stewart et al. , 2014; Wajant, 2015), which leads to clustering and activation of receptors on the cell surface. Since MAT ² activity of the present invention does not require Fcγ receptor-mediated cross-linking, the agonistic effect on cells will be localized to sites where both targets are present. Because the LALA mutation in MAb ² to OX40/CD137 results in reduced binding to Fcγ receptors, activation triggered by cross-linking to the Fcγ receptor is not expected to be possible through binding of CD137 alone. Therefore, it is unlikely that MAbs ² activate CD137-expressing cells in the absence of OX40 expression. Since there is a known risk of liver toxicity associated with targeting CD137 in humans (for example, as observed with urelumab (BMS-663513) treatment (Segal et al. , 2017), it is hoped that reducing the likelihood of Fcγ receptor-induced MAT ² cross-linking , containing the LALA mutation, will reduce the chances of such liver toxicity occurring after MAT ² treatment, since CD137 will only be activated at sites that also express OX40. The current theory of CD137-induced liver toxicity suggests that myeloid cells, expressing CD137 are a cell type responsible for the liver inflammation observed in mice treated with CD137 agonists (Bartkowiak, et al. , 2018).

Macrophages are known to express FcγRI, which could potentially mediate cross-linking of CD137-targeting antibody, but these cells are not known to express OX40. Thus, MAb ² of the present invention containing the LALA mutation should theoretically not be able to activate liver macrophages that express CD137 but not OX40. This is believed to reduce the risk of liver toxicity of the anti-OX40/CD137 mAb ² of the present invention compared to either a CD137 agonist, which requires cross-linking to the Fcγ receptor for activity, or a CD137 agonist, which does not require cross-linking for activity. In the case of OX40, although some residual Fcab activity was observed in the absence of cross-linking, which may result in some activation of OX40 in the absence of CD137 binding, as to date, dose-limiting toxicities have not been reported in clinical studies using OX40 agonists , this is not considered a risk.

Example 11 - MAT Binding ² with cells expressing OX40 or CD137

11.1. Binding of MAT ² to cells expressing human or cynomolgus OX40 or CD137

Binding affinity of MAT ² FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12 and FS20-22-49AA/FS30-10- 16 for human or cynomolgus macaque cell-expressed OX40 and CD137 were determined by flow cytometry. Dilutions (2× final concentration) of these MAT ² antibodies and control antibodies G1/4420 (FITC), G1/11D4 (OX40), G1/MOR7480.1 (CD137) and FS20-22-49AA/4420 (mock MAT ² to OX40 /FITC) (all in IgG1 isotype format) were prepared in 1×DFSB (Gibco, 14190-094). Suspensions of DO11.10-hOX40, DO11.10-cOX40, DO11.10-hCD137, DO11.10-cCD137 or HEK cells were prepared in PBS + 2% BSA (Sigma, A7906) and seeded at 4×10 ⁶ cells/ml with 50 µl/well in 96-well V-bottom plates (Costar, 3897). 50 μl of antibody dilutions were added to wells containing cells (final volume 100 μl) and incubated at 4°C for 1 hour. The plates were washed and then 100 μl/well of secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 in PBS + 2% BSA was added and incubated for 30 minutes at 4°C In the dark. The plates were washed and the contents were resuspended in 100 μl of PBS containing DAPI (Biotium, catalog number 40043) at 1 μg/ml. Plates were analyzed using a Canto II flow cytometer (BD Bioscience) and data were analyzed using FlowJo. Dead cells were identified by their higher fluorescence in the UV channel (405 nm/450/50) and were excluded from the analysis. Geometric mean fluorescence intensity (GMFI) in the FITC channel (488 nm/530/30) was used as a measure of antibody binding. GMFI data were fitted using the log(agonist) response equation (three parameters) in GraphPad Prism software to obtain _EC50 values.

Table 9: Mab ² binding affinity to OX40/CD137 for DO11.10 cells expressing human or cynomolgus OX40 or CD137 by flow cytometry.

OX40 human OX40 cynomolgus macaque Human CD137 CD137 cynomolgus macaque HEK MAT EK ₅₀
(nM) EK ₅₀
(nM) EK ₅₀
(nM) EK ₅₀
(nM) EK ₅₀
(nM) G1/4420 No binding detected No binding detected No binding detected No binding detected No binding detected G1/11D4 0.1248 0.09408 No binding detected No binding detected No binding detected G1/MOR7480.1 No binding detected No binding detected 0.07682 0.06119 No binding detected FS20-22-49AA/4420 0.1619 0.3262 No binding detected No binding detected No binding detected FS20-22-49AA/FS30-5-37 0.2007 0.3552 0.2578 0.1105 No binding detected FS20-22-49AA/FS30-10-3 0.175 0.394 0.1197 0.0682 No binding detected FS20-22-49AA/FS30-10-16 0.1566 0.3798 0.1291 0.08027 No binding detected FS20-22-49AA/FS30-10-12 0.1517 0.3684 0.2899 0.1074 No binding detected

NB: No binding detected.

The results confirm that the tested anti-OX40/CD137 MAbs ² bind to human and cynomolgus OX40 and CD137 expressed on DO11.10 cells. Mab ² and positive controls (human OX40 mAb, G1/11D4, in the human IgG1 backbone; and anti-human CD137 mAb, G1/MOR7480.1, in the human IgG1 backbone) bound to both OX40 and human and cynomolgus CD137 with a range of affinities (see Table 9 ). No cross-reactivity was observed with other proteins expressed on the surface of the HEK cell line, as none of the antibodies tested were found to bind to this cell line. Thus, mAb ² to OX40/CD137 specifically bound to human OX40 and human CD137, and no nonspecific binding was observed.

11.2 Binding of MAT ² FS20-22-49AA/FS30-10-16 and its constituent parts to cells expressing human or cynomolgus OX40 or CD137

To compare the affinity of MAT ² FS20-22-49AA/FS30-10-16 and its constituents, i.e. Fcab to OX40 (in mock MAT ² format to OX40/FITC; FS20-22-49AA/4420) and Fab to CD137 ( in IgG1 format; FS30-010-016), in relation to human or cynomolgus macaque expressed OX40 and CD137 cells, the same method was used as described in Example 11.1 . However, in this experiment, instead of using HEK cells for the nonspecific binding assay, untransduced DO11.10 cells were used. Anti-FITC antibody G1/4420 was used as a control. The experiment was repeated three times to increase the reliability of the calculated _EC50 values. The average EC ₅₀ values for the tested molecules are shown in Table 10 .

Table 10: Binding affinity of Mab ² to OX40/CD137 FS20-22-49AA/FS30-10-16 and its constituents to DO11.10 cells expressing human or cynomolgus OX40 or CD137, determined by flow cytometry.

OX40 human OX40 cynomolgus macaque Human CD137 CD137 cynomolgus macaque Untransduced DO11.10 MAT EK ₅₀
Avg. ± CO
(nM) EK ₅₀
Avg. ± CO
(nM) EK ₅₀
Avg. ± CO
(nM) EK ₅₀
Avg. ± CO
(nM) EK ₅₀
Avg. ± CO
(nM) G1/4420 No binding detected No binding detected No binding detected No binding detected No binding detected FS20-22-49AA/4420 0.23 ± 0.02 0.55 ± 0.14 No binding detected No binding detected No binding detected G1/FS30-10-16 No binding detected No binding detected 0.10 ± 0.05 0.09 ± 0.01 No binding detected FS20-22-49AA/FS30-10-16 0.22 ± 0.01 0.71 ± 0.21 0.11 ± 0.02 0.12 ± 0.01 No binding detected

Avg: average value; SD: standard deviation; NB: No binding detected.

The results confirm that MAb ² to OX40/CD137 (FS20-22-49AA/FS30-10-16) binds to human and cynomolgus OX40 and CD137 expressed on DO11.10 cells with subnanomolar affinity, and that the Fcab component to OX40 of MAT ² binds to human and cynomolgus OX40 with an affinity comparable to that of MAT ² to OX40/CD137, and that the Fab component to CD137 of MAT ² binds to human and cynomolgus CD137 with an affinity comparable to that of MAT ² to OX40/ CD137. Nonspecific binding to non-transduced DO11.10 cells was not observed for either MAb ² to OX40/CD137, its constituents, or the isotype control antibody (G1/4420). The results indicate that the affinity of Mab ² to OX40/CD137 FS20-22-49AA/FS30-10-16 and Fcab to OX40 FS20-22-49AA for cynomolgus OX40 expressed by cells is higher (as indicated by lower values EC ₅₀ ) than previously observed ( Example 11.1 and Table 9 ), and is similar to the SPR affinity results ( Example 8 and Table 7 ). Because the average EC ₅₀ values, presented in more detail in Table 10 , are obtained from three independent experiments, they better represent the affinity of the tested molecules for human and cynomolgus OX40 and CD137 expressed on DO11.10 cells.

11.3 Binding of MAT ² to cells expressing mouse OX40 or CD137

The binding affinity of MAT ² FS20m-232-91AA/Lob12.3 for mouse OX40 and CD137 expressed cells was determined using flow cytometry. Dilutions (2× final concentration) of FS20m-232-91AA/Lob12.3 and control antibodies G1/4420 (FITC), G1/Lob12.3 (CD137), G1/OX86 (OX40) and FS20m-232-91AA/HEL D1 .3 (mock MAT ² to OX40/HEL) was prepared in 1×DFSB (Gibco, 14190-094). Suspensions of DO11.10-mOX40, DO11.10-mCD137 or HEK cells were prepared in PBS + 2% BSA (Sigma, A7906) and seeded at 4 × 10 ⁶ cells/ml with 50 μl/well in 96-well plates with V- shaped bottom (Costar, 3897). 50 μl of antibody dilutions were added to wells containing cells (final volume 100 μl) and incubated at 4°C for 1 hour. The plates were washed and then 100 μl/well of secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 in PBS + 2% BSA was added and incubated for 30 minutes at 4°C. In the dark. The plates were washed and the contents were resuspended in 100 μl of PBS containing DAPI (Biotium, catalog number 40043) at 1 μg/ml. Plates were analyzed using a Canto II flow cytometer (BD Bioscience) and data were analyzed using FlowJo. Dead cells were identified by their higher fluorescence in the UV channel (405 nm/450/50) and were excluded from the analysis. Geometric mean fluorescence intensity (GMFI) in the FITC channel (488 nm/530/30) was used as a measure of antibody binding. GMFI data were fitted using the log(agonist) response equation (three parameters) in GraphPad Prism software to obtain _EC50 values. The results are presented in Table 11 .

Table 11: Mab ² binding affinity to mouse OX40/CD137 for DO11.10 cells expressing mouse OX40 or CD137 determined by flow cytometry.

OX40 mice CD137 mouse MAT EK ₅₀
(nM) EK ₅₀
(nM) G1/4420 No binding detected No binding detected G1/Lob12.3 No binding detected 0.1206 G1/OX86 0.5381 No binding detected FS20m-232-91AA/HEL D1.3 0.2677 No binding detected FS20m-232-91AA/Lob12.3 0.159 0.118

NB: No binding detected.

The results confirm that mAb ² FS20m-232-91AA/Lob12.3 binds to mouse OX40 and CD137 expressed on DO11.10 cells. Mab ² and positive controls (mouse OX40 mAb, OX86, in the human IgG1 backbone; and mouse CD137 mAb Lob12.3, in the human IgG1 backbone) bound to mouse OX40 and/or CD137 with a range of affinities (see Table 11 ). No cross-reactivity was observed with other proteins expressed on the surface of the HEK cell line, as none of the antibodies tested were found to bind to this cell line.

Thus, mouse OX40/CD137 mAb ² specifically bound to mouse OX40 and mouse CD137 without observed nonspecific binding.

Example 12 - MAT activity ² to OX40/CD137 targeting co-expressed receptors in a staphylococcal enterotoxin A (SEA) assay

Expression of OX40 on tumor-infiltrating lymphocytes is likely to be accompanied by expression of CD137, as these two molecules are often co-expressed on activated T cells (Ma et al. , 2005). Agonistic action on OX40 and CD137 by mAb ² targeting these two co-expressed receptors can induce proliferation and production of inflammatory cytokines by pre-activated T cells.

For full activation, T cells require two signals, the first signal, which is antigen specific and provided through the T cell receptor (TCR), which interacts with major histocompatibility complex (MHC) molecules displaying peptide antigen on the membrane of antigen presenting cells (APCs), and the second, antigen-nonspecific signal is a costimulatory signal, which is provided through the interaction between costimulatory molecules expressed on the membrane of APC and T cells.

To test the activity of mAb ² against OX40/CD137, a T cell activation assay was developed using the superantigen staphylococcal enterotoxin A (SEA) as the first signal. SEA cross-links MHC class II molecules on the surface of APCs and TCRs of T cells, thereby providing the first signal for T cell activation. To be fully activated, T cells must also receive a second, co-stimulatory signal from control molecules or MAT ² appropriately cross-linked. This assay is performed using isolated PBMCs from blood and should more accurately reflect events expected in vivo than an assay performed using isolated T cells.

The SEA stimulation assay was used to establish the activity of various agonistic anti-OX40 and anti-CD137 antibodies and the anti-OX40/CD137 MAT ² antibody in the presence or absence of artificial cross-linking agents to compare different anti-OX40/CD137 MAT ² clones and establish a typical EC value of ₅₀ for clone MAT ² to OX40/CD137 FS20-22-49AA/FS30-10-16 in a group of 10 PBMC donors.

12.1 Activity of agonistic antibodies to OX40 and CD137 against SEA-stimulated PBMCs

To establish the sensitivity of the SEA assay to various anti-OX40 and anti-CD137 agonistic antibodies, the activity of the MAT ² antibody (FS22-20-49AA/FS30-10-16) and the control antibodies listed in Table 12 was tested in this assay. G1/4420 (anti-FITC), G1AA/MOR7480.1 (anti-CD137), G1AA/FS30-10-16 (anti-CD137), G1AA/20H4.9 (anti-CD137), G1AA/11D4 (anti-OX40) and FS20- 22-49AA/4420 (mock MAT ² to OX40/FITC) was used as controls. IL-2 production was used as a measure of T cell activation.

Table 12: Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation Cross-linking agent SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No FITC-dextran 115 116 G1AA/MOR7480.1 hCD137 Absent hIgG1 Yes a-hCH2 125 120 G1AA/FS30-10-16 hCD137 Absent hIgG1 Yes a-hCH2 154 97 G1AA/20H4.9 hCD137 Absent hIgG1 Yes a-hCH2 165 122 G1AA/11D4 hOX40 Absent hIgG1 No a-hCH2 173 175 FS20-22-49AA/4420 FITC hOX40 hIgG1 Yes FITC-dextran 123 116 FS20-22-49AA/FS30-10-16 hCD137 hOX40 hIgG1 Yes a-hCH2 95 97

Peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte depletion cones (NHS Blood and Transplant service), a by-product of platelet donation. In general, the contents of the buffy cone were washed away with PBS and applied to a Ficoll gradient (GE Lifesciences, cat. no. 17144002). PBMCs were isolated by centrifugation and isolation of cells that did not cross the Ficoll gradient. PBMCs were further washed with PBS, and the remaining red blood cells were lysed by adding 10 ml of red blood cell lysis buffer (eBioscience) according to the manufacturer's instructions. PBMCs were counted and resuspended to 2.0 x 10 ⁶ cells/ml in T cell medium (RPMI medium (Life Technologies) with 10% FBS (Life Technologies), 1x penicillin/streptomycin (Life Technologies), sodium pyruvate ( Gibco), 10 mM Hepes (Gibco), 2 mM L-glutamine (Gibco), and 50 μM 2-mercaptoethanol (Gibco)). SEA (Sigma, cat. no. S9399) was then added to the PBMCs at 200 ng/ml, and cells were added to the plates at 2 x 10 ⁵ cells/well (100 μl/well).

2 μM dilutions of each antibody tested (see Table 12 for details) were prepared in DPSB (Gibco) and further diluted 1:10 in T cell media (30 μl + 270 μl) to obtain 200 nM dilutions. Artificial cross-linkers (anti-human CH2 antibody (clone MK1A6, prepared in-house) or FITC-dextran (Sigma) (see Table 12 ) were added to the wells in a 1:1 molar ratio with test antibodies, where necessary. Serial dilutions of the test antibodies were prepared in the well plate, and 100 μl of the diluted antibody mixture was added to the activated T cells on the plate.

Cells were incubated at 37°C, 5% CO ₂ for 120 hours. Supernatants were collected, and IL-2 release was measured using a human IL-2 ELISA kit (eBioscience or R&D Systems) according to the manufacturer's instructions. The plates were read at 450 nm using a plate reader with Gen5 software, BioTek. The 630 nm absorbance values were subtracted from the 450 nm absorbance values (Correction). The standard curve for calculating cytokine concentration was based on a four-parameter logistic curve fit (Gen5 software, BioTek). Human IL-2 (hIL-2) concentrations were plotted against the logarithmic concentration of test antibodies, and the resulting curves were fitted using the log(agonist) response equation in GraphPad Prism. Table 13 shows the EC ₅₀ values and maximum IL-2 release response observed in the SEA assay in the presence or absence of cross-linking with artificial cross-linking agents. Figure 3A shows the levels of IL-2 release induced by the test antibodies at one concentration (3.7 nM) in the SEA assay. For this analysis, the concentration at which these antibodies induced the highest levels of IL-2 production was selected. Statistical analysis was performed using two-way analysis of variance and Tukey's test for multiple comparisons. Asterisks above error bars represent significant difference compared to isotype control (G1/4420) treated samples (*p<0.032, **p<0.0021, ***p<0.0002, ****p<0 .0001). Figure 3B shows plots of IL-2 release induced by anti-OX40/CD137 mAb ² (FS20-22-49AA/FS30-10-16) in the presence or absence of an artificial cross-linker in the SEA assay.

Table 13: SEA assay using agonistic antibodies and MAbs ² to OX40 and CD137

No cross-linking Cross linking MAT/MAT ² EC ₅₀ (nM) Max. answer EC ₅₀ (nM) Max. answer (nM) 95% CI (hIL-2 pg/ml) 95% CI (nM) 95% CI (hIL-2 pg/ml) 95% CI G1/4420 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/MOR7480.1 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/FS30-10-16 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/20H4.9 No activity detected No activity detected No activity detected No activity detected 0.3062 0.1224-0.7376 4324 3904-4762 G1AA/11D4 No activity detected No activity detected No activity detected No activity detected 0.07163 0.01113-0.2910 4269 3693-4864 FS20-22-49AA/4420 No activity detected No activity detected No activity detected No activity detected 0.3364 0.1377-0.7743 8719 7943-9532 FS20-22-49AA/4420 + G1AA/FS30-10-16 No activity detected No activity detected No activity detected No activity detected 0.3793 0.1665-0.8578 7644 6964-8358 FS20-22-49AA/FS30-10-16 0.2548 0.1050-0.6082 8931 8028-9877 0.1877 0.06915-0.4945 9930 8920-10990

NAD = No activity detected.

The results show that only Mab ² to OX40/CD137 (FS20-22-49AA/FS30-10-16) was able to increase IL-2 levels in the absence of artificial cross-linkers and that the addition of an artificial cross-linker did not increase the activity of Mab ² to OX40/CD137, applied to both EC ₅₀ and maximum response. The activity of the OX40-targeting antibodies G1AA/11D4 and FS20-22-49AA/4420 and the anti-CD137 antibody G1AA/20H4.9 was observed only in the presence of artificial cross-linking agents, and for the anti-CD137 antibodies G1AA/MOR7480.1 and G1AA/FS30-10 -16 no statistically significant activity was detected compared to the isotype control, even in the presence of an artificial cross-linker. Anti-OX40 G1AA/11D4 induced higher levels of IL-2 than anti-CD137 G1AA/MOR7480.1 and G1AA/FS30-10-16, and levels of IL-2 comparable to those of anti-CD137 G1AA/20H4.9 , despite the observation that antibody G1AA/11D4 is more potent than antibody G1AA/20H4.9, as evidenced by its significantly lower EC ₅₀ value. These results suggest that the SEA assay is more sensitive to agonistic effects on OX40 than to agonistic effects on CD137. This may be due to the fact that OX40 is predominantly expressed on CD4 ⁺ T cells and CD137 is predominantly expressed on CD8 ⁺ T cells (Croft, 2014 and own data shown in Figure 6 ), and also because in human PBMCs, both generally more CD4 ⁺ T cells than CD8 ⁺ T cells.

12.2 Activity of various clones of MAb ² against OX40/CD137 against SEA-stimulated PBMCs

The activity of five different anti-OX40/CD137 mAb ² clones was tested in the SEA assay. Details of MAb ² and control antibodies used in the assay are presented in Table 14 . G1/4420 (anti-FITC), G1/11D4 (anti-OX40), G2/MOR7480.1 (anti-CD137), G1/11D4 plus G2/MOR7480.1 in combination and FS20-22-49AA/4420 (imitation MAT ² k OX40/FITC) were used as controls. The analysis was performed as described in Example 12.1 .

Table 14: Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation Cross-linking agent SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No FITC-dextran 115 116 G1/11D4 hOX40 Absent hIgG1 No a-hCH2 174 175 G2/MOR7480.1 hCD137 Absent hIgG2 No a-hCH2 124 120 FS20-22-49AA/4420 FITC hOX40 hIgG1 Yes FITC-dextran 123 116 FS20-22-49AA/FS30-5-37 hCD137 hOX40 hIgG1 Yes a-hCH2 109 111 FS20-22-49AA/FS30-10-3 hCD137 hOX40 hIgG1 Yes a-hCH2 99 97 FS20-22-49AA/FS30-10-12 hCD137 hOX40 hIgG1 Yes a-hCH2 103 97 FS20-22-49AA/FS30-10-16 hCD137 hOX40 hIgG1 Yes a-hCH2 95 97 FS20-22-49AA/FS30-35-14 hCD137 hOX40 hIgG1 Yes a-hCH2 105 107

Table 15 shows the EC ₅₀ values and maximum IL-2 release response observed in the SEA assay in the presence or absence of cross-linking with artificial cross-linking agents. Figure 3C and D show IL-2 release plots for the SEA assay.

Table 15: SEA assay using MAbs ² targeting OX40 and CD137

No cross-linking Cross linking MAT/MAT ² EC ₅₀ (nM) Max. answer EC ₅₀ (nM) Max. answer (nM) 95% CI (hIL-2 pg/ml) 95% CI (nM) 95% CI (hIL-2 pg/ml) 95% CI G1/4420 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1/11D4 No activity detected No activity detected No activity detected No activity detected 0.13 0.01-0.77 6614.00 4743-8561 G2/MOR7480.1* No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1/11D4 + G2/MOR7480.1 No activity detected No activity detected No activity detected No activity detected 0.11 No activity detected -0.79 9451.00 6633-12384 FS20-22-49AA/4420 8.53 0.83-58.93 808.70 328.7-1602 0.31 0.15-0.68 28603.00 24885-32460 FS20-22-49AA/FS30-5-37 0.51 0.26-0.97 29242.00 26131-32465 0.50 0.26-0.91 30748.00 27773-33822 FS20-22-49AA/FS30-10-3 0.29 0.15-0.55 34945.00 31391-38616 0.38 0.20-0.69 33919.00 30775-37170 FS20-22-49AA/FS30-10-12 1.36 0.73-2.65 24912.00 22201-27799 2.33 1.25-4.58 23721.00 21156-26587 FS20-22-49AA/FS30-10-16 0.14 0.077-0.25 35115.00 32213-38074 0.10 0.06-0.18 33761.00 31415-36145 FS20-22-49AA/FS30-35-14 0.09 0.021-0.30 32363.00 27164-37691 0.14 0.07-0.30 32212.00 28906-35597

NAD = no activity detected

Figure 3C and D and Table 15 show that IL-2 production was not observed when using the anti-FITC antibody G1/4420, either uncross-linked or cross-linked, or when using the non-cross-linked anti-OX40 antibody (G1/11D4 alone or in combination with G2/MOR7480.1), as expected. IL-2 was produced by T cells when OX40 was activated by binding anti-OX40 antibody, as a positive control, but only in the presence of an artificial cross-linker (EC ₅₀ 0.13 nM for G1/11D4 alone and EC ₅₀ 0.11 nM in combination with G2/MOR7480.1). OX40-targeting Fcab in mock MAb ² format (4420 LALA) FS20-22-49AA/4420 showed some agonist activity in the absence of cross-linking in this assay (EC ₅₀ 8.53 nM), but when cross-linking via Fab arm binding with FITC-dextran had increased activity, as evidenced by a decrease in EC ₅₀ (0.31 nM) and an increase in the maximum amount of IL-2 produced (maximum response), as evidenced by increased IL-2 production.

No activity was observed using cross-linked CD137-targeting antibody G2/MOR7480.1 alone, and the cross-linking activity of the combination of OX40-targeting antibody G1/11D4 and CD137-targeting antibody G2/MOR7480.1 was similar to that of cross-linking OX40- targeting G1/11D4 antibody alone.

In this SEA T cell activation assay, the activity of the five anti-OX40/CD137 mAb ² clones (see Table 15 ) was comparable, regardless of the presence of the artificial cross-linker. The activity of MAT ² against OX40/CD137 in the presence of an artificial cross-linker was also comparable to the activity of the cross-linked mock MAT ² FS20-22-49AA/4420. The results of the SEA assay indicate that anti-OX40/CD137 mAbs ² are capable of signaling through OX40, without the need for artificial cross-linking agents, as a result of the cross-linking provided by binding of the anti-CD137 Mab ² Fab arms.

Although this assay did not detect cross-linking activity for the CD137-targeting antibody G2/MOR7480.1, it is expected that CD137 was expressed on T cells at a level that allows mAb ² cross-linking to occur. It is hypothesized that expression was at a level at which each of the five MAT ² clones, when bound to CD137, could also bind to OX40 and trigger its activation to a much greater extent, compared with the low level of activity induced by the MAT ² mock FS20-22 -49AA/4420, not cross-linked.

The T cell activation observed with anti-OX40/CD137 mAb ² in the absence of an artificial cross-linker also suggests that these molecules will be able to activate T cells in which both OX40 and CD137 are expressed in vivo .

12.3 Activity of clone MAT ² against OX40/CD137 FS20-22-49AA/FS30-10-16 against SEA-stimulated PBMCs from 10 PBMC donors

The anti-OX40/CD137 mAb ² clone FS20-22-49AA/FS30-10-16 was tested in an SEA assay using PBMCs from 10 different donors to establish the precise EC ₂₀ , EC ₃₀ and EC ₅₀ values for its activity. The assay was performed as described in Example 12.1 in the absence of an artificial cross-linker. Means plus or minus the standard deviation of the mean (SD) were calculated from the raw data for each donor. To calculate EC ₅₀ values, the source data was approximated to a logistic function (4 parameters: maximum (top), minimum (bottom), Hill slope and EC ₅₀ (EC50):

The y-axis shows the measured response (IL-2 levels) as a function of _log10 (c), where c denotes the concentration of the test article.

Each parameter estimate from the approximation has a standard error, which indicates the accuracy of that estimate. Because different donors and/or technical replicates for a given experiment will produce different parameter estimates and different levels of precision (depending, for example, on the quality of the data in each case), parameters from each donor and/or technical replicate were included in the weighted average. Weights were determined as the reciprocal of the squared standard error of the parameter, assuming normal distribution of the parameter.

In addition, the values of log ₁₀ (EC ₂₀ ) and log ₁₀ (EC ₃₀ ) were calculated by fitting the data to similar equations:

All logistic fits were performed using GraphPad Prism, and weighted averaging was performed using Microsoft Excel. The formulas used to calculate the weighted mean and standard error of the weighted mean are given below:

and the weighted standard deviation was calculated as:

The EC ₂₀ , EC ₃₀ and EC ₅₀ values for IL-2 release observed for anti-OX40/CD137 mAb ² in the SEA assay are shown in Table 16 .

Table 16: EC ₂₀ , EC ₃₀ and EC ₅₀ values for MAT ² to OX40/CD137 in SEA analysis

EC ₅₀ (nM) EC ₃₀ (nM) EC ₂₀ (nM) Donor 1 0.21 0.09 0.05 Donor 2 0.38 0.14 0.08 Donor 3 0.34 0.18 0.12 Donor 4 0.36 0.18 0.11 Donor 5 0.30 0.11 0.06 Donor 6 0.41 0.17 0.10 Donor 7 0.44 0.21 0.13 Donor 8 0.29 0.15 0.10 Donor 9 0.17 0.10 0.07 Donor 10 0.04 0.02 0.02 Weighted average 0.32 0.14 0.09 95% CI 0.25-0.41 0.11-0.18 0.07-0.12

These results indicate that MAb ² to OX40/CD137 has comparable activity to PBMCs from different donors.

Example 13 - MAT activity ² to human OX40/CD137 in a general T cell population activation assay

The SEA T cell activation assay described in Example 12 used PBMCs and SEA superantigen to stimulate T cells. A T cell activation assay was developed to evaluate the effect of OX40 and CD137 agonists on isolated T cells. In this assay, T cells were isolated and stimulated using anti-CD3 antibody immobilized on a plastic surface. The immobilized anti-CD3 antibody was able to cluster the TCR of T cells, providing the first signal required for T cell activation, while the test molecules provided the second signal.

The T cell stimulation assay was used to establish the activity of various agonistic antibodies to OX40 and CD137 and MAT ² to OX40/CD137 in the presence or absence of cross-linking agents, to compare different clones of MAT ² to OX40/CD137, and to establish the typical value EC ₅₀ for clone MAT ² to OX40/CD137 FS20-22-49AA/FS30-10-16 in a group of nine PBMC donors.

13.1 Activity of agonistic antibodies to OX40 and CD137 in the overall T cell population activation assay

To establish the sensitivity of the T cell activation assay to various agonistic antibodies to OX40 and CD137, the activity of the mAb antibody was tested in this assay² (FS20-22-49AA/FS30-10-16) and control antibodies listed inTable 17. G1/4420 (anti-FITC), G1AA/MOR7480.1 (anti-CD137), G1AA/FS30-10-16 (anti-CD137), G1AA/20H4.9 (anti-CD137), G1AA/11D4 (anti-OX40) and FS20- 22-49AA/4420 (imitation MAT²To OX40/FITC) were used as controls. IL-2 production was used as a measure of T cell activation.

Table 17: Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation Cross-linking agent SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No FITC-dextran 115 116 G1AA/MOR7480.1 hCD137 Absent hIgG1 Yes a-hCH2 125 120 G1AA/FS30-10-16 hCD137 Absent hIgG1 Yes a-hCH2 154 97 G1AA/20H4.9 hCD137 Absent hIgG1 Yes a-hCH2 165 122 G1AA/11D4 hOX40 Absent hIgG1 No a-hCH2 173 175 FS20-22-49AA/4420 FITC hOX40 hIgG1 Yes FITC-dextran 123 116 FS20-22-49AA/FS30-10-16 hCD137 hOX40 hIgG1 Yes a-hCH2 95 97

Human PBMCs were isolated as described in Example 12.1 . T cells were then isolated from PBMCs using the Total T Cell Population Isolation Kit II (Miltenyi Biotec Ltd) according to the manufacturer's instructions.

Human CD3/CD28 T cell activator Dynabeads™ (Life Technologies, 11452D) were resuspended by vortexing. Beads were washed twice with T cell medium (RPMI medium (Life Technologies) with 10% FBS (Life Technologies), 1× penicillin/streptomycin (Life Technologies), sodium pyruvate (Gibco), 10 mM Hepes (Gibco), 2 mM L -glutamine (Gibco) and 50 μM 2-mercaptoethanol (Gibco)).

The required number of T cells at a concentration of 1.0 x 10 ⁶ cells/ml in T cell medium were stimulated with washed human T cell activator CD3/CD28 Dynabeads™ at a cell to bead ratio of 2:1 in a T-25 flask (Sigma) and incubated overnight at 37°C, 5% CO ₂ to activate T cells. Activated T cells were washed from Dynabeads and resuspended in T cell medium at a concentration of 2.0 x 10 ⁶ cells/ml. 96-well flat-bottom plates were coated with anti-human CD3 antibody by incubating with 2.5 μg/ml anti-human CD3 antibody (R&D Systems, clone UHCT1) diluted in PBS for 2 hours at 37°C, 5% CO ₂ and then washed twice with PBS. Activated T cells were added to the plates at 2×10 ⁵ cells/well.

2 μM dilutions of each antibody tested were prepared (see Table 17 for details) and added to the wells in a 1:1 molar ratio with cross-linking agent (human CH2 antibody (clone MK1A6, prepared in-house) or FITC-dextran (Sigma) (see Table 17 )), where necessary, as described above in Example 12.1 . Serial dilutions of the test antibodies were prepared in a 96-well plate, and 100 μl of the diluted antibody mixture was added to the activated T cells on the plate.

T cells were incubated at 37°C, 5% CO ₂ for 72 hours. Supernatants were then collected, IL-2 release measured and data obtained as described in Example 12.1 . Table 18 shows the EC ₅₀ values and maximum IL-2 release response observed in the T cell activation assay in the presence or absence of cross-linking by cross-linking agents. Figure 4A shows the levels of IL-2 release induced by the test antibodies at one concentration (3.7 nM) in a T cell activation assay. The concentration at which these antibodies induced the highest levels of IL-2 production was selected for this analysis. Statistical analysis was performed using two-way analysis of variance and Tukey's test for multiple comparisons. Asterisks above error bars represent significant difference compared to isotype control (G1/4420) treated samples (*p<0.032, **p<0.0021, ***p<0.0002, ****p<0 .0001). Figure 4B shows plots of IL-2 release induced by anti-OX40/CD137 mAb ² (FS20-22-49AA/FS30-10-16) in the presence or absence of a cross-linking agent in a T cell activation assay.

Table 18: T cell activation assay using agonistic antibodies and MAbs ² to OX40 and CD137

No cross-linking Cross linking MAT/MAT ² EC ₅₀ (nM) Max. answer EC ₅₀ (nM) Max. answer (nM) 95% CI (hIL-2 pg/ml) 95% CI (nM) 95% CI (hIL-2 pg/ml) 95% CI G1/4420 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/MOR7480.1 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/FS30-10-16 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/20H4.9 No activity detected No activity detected No activity detected No activity detected 0.234 0.04154-1.057 5303 4068-6620 G1AA/11D4 No activity detected No activity detected No activity detected No activity detected 0.1301 0.01642-0.6356 4130 3266-5037 FS20-22-49AA/4420 No activity detected No activity detected No activity detected No activity detected 0.278 0.1611-0.4790 12450 11440-13488 FS20-22-49AA/4420 + G1AA/FS30-10-16 No activity detected No activity detected No activity detected No activity detected 0.1746 0.05500-0.5209 15001 12556-17552 FS20-22-49AA/FS30-10-16 0.09306 0.03231-0.2430 16927 14533-19389 0.07916 0.01737-0.2851 14434 11757-17202

NAD - no activity detected.

The results show that only Mab ² to OX40/CD137 (FS20-22-49AA/FS30-10-16) was able to increase IL-2 levels in the absence of artificial cross-linkers and that the addition of an artificial cross-linker did not increase the activity of Mab ² to OX40/CD137 in relation to both EC ₅₀ and maximum response. The activity of the OX40-targeting antibodies G1AA/11D4 and FS20-22-49AA/4420 and the anti-CD137 antibody G1AA/20H4.9 was observed only in the presence of artificial cross-linking agents, and the activity of the anti-CD137 antibodies G1AA/MOR7480.1 and G1AA/FS30-10 -16 was not detected even in the presence of an artificial cross-linker. The OX40 agonist antibody FS20-22-49AA/4420 induced higher levels of IL-2 than all three CD137 agonist antibodies. Anti-OX40 G1AA/11D4 induced higher levels of IL-2 than anti-CD137 G1AA/MOR7480.1 and G1AA/FS30-10-16, and levels of IL-2 comparable to those of anti-CD137 G1AA/20H4.9 , despite the observation that antibody G1AA/11D4 has greater potency than antibody G1AA/20H4.9, as evidenced by its lower EC ₅₀ value. These results suggest that this T cell activation assay is more sensitive to agonistic effects on OX40 than to agonistic effects on CD137. As suggested in Example 12.1 , this may be due to the fact that OX40 is predominantly expressed on CD4 ⁺ T cells and CD137 is predominantly expressed on CD8 ⁺ T cells (Croft, 2014 and own data shown in Figure 6 ), as well as because human PBMCs tend to have more CD4 ⁺ T cells than CD8 ⁺ T cells.

13.2 Analysis of the activity of agonistic antibodies to OX40 and CD137 against multiple cytokines in a total T cell population activation assay

To better understand the effect of OX40 and CD137 stimulation on T cell activation assays, levels of multiple cytokines were analyzed. The antibodies and MAT ² antibody (FS20-22-49AA/FS30-10-16) were used, as well as the control antibodies listed in Table 19 . Control antibodies G1/4420 (anti-FITC), G1AA/FS30-10-16 (anti-CD137) and FS20-22-49AA/4420 (mock Mab ² to OX40/FITC) were tested in the presence of artificial cross-linkers, and Mab ² to OX40/CD137 was tested in the absence of an artificial cross-linker. All antibodies were used at the same concentration (10 nM). The analysis was performed as described in Example 13.1 .

Table 19: Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation Cross-linking agent SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No FITC-dextran 115 116 G1AA/FS30-10-16 hCD137 Absent hIgG1 Yes a-hCH2 154 97 FS20-22-49AA/4420 FITC hOX40 hIgG1 Yes FITC-dextran 123 116 FS20-22-49AA/FS30-10-16 hCD137 hOX40 hIgG1 Yes No 95 97

The levels of cytokines IL-2, IL-6, IL12p70, IL-13, TNFα, IFNγ and IL-10 in the supernatants collected after incubation were then determined using the Pro-inflammatory V-plex kit (MSD, K15049D-1) according to with the manufacturer's instructions. The results showed that mAb ² to OX40/CD137 (FS20-22-49AA/FS30-10-16) and a cross-linked antibody targeting OX40 (FS20-22-49AA/4420) increased the release of cytokines IL-2, IL- 6, IL-12p70, IL-13 and TNFα and decreased the release of IL-10 by T cells. Anti-CD137 antibody (G1AA/FS30-10-16) activity was not detected.

13.3 Activity of various clones of mAb ² to OX40/CD137 in the analysis of activation of the total population of T cells

Details of the molecules tested in this assay and their respective cross-linking agents, where applicable, are presented in Table 20 below. G1/4420 (anti-FITC), G1/11D4 (anti-OX40), G2/MOR7480.1 (anti-CD137), G1/11D4 plus G2/MOR7480.1 in combination and FS20-22-49AA/4420 (imitation MAT ² k OX40/FITC) were used as controls. All molecules were tested in the absence of an artificial cross-linker. Controls G1/4420, G1/11D4, G2/MOR7480.1 and FS20-22-49AA/4420 as single agents were further tested in the presence of an artificial cross-linker. The analysis was performed as described in Example 13.1 .

Table 20: Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation Cross-linking agent SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No FITC-dextran 115 116 G1/11D4 hOX40 Absent hIgG1 No Antibody to hCH2 174 175 G2/MOR7480.1 hCD137 Absent hIgG2 No Antibody to hCH2 124 120 FS20-22-49AA/4420 FITC hOX40 hIgG1 Yes FITC-dextran 123 116 FS20-22-49AA/FS30-5-37 hCD137 hOX40 hIgG1 Yes Not applicable 109 111 FS20-22-49AA/FS30-10-3 hCD137 hOX40 hIgG1 Yes Not applicable 99 97 FS20-22-49AA/FS30-10-12 hCD137 hOX40 hIgG1 Yes Not applicable 103 97 FS20-22-49AA/FS30-10-16 hCD137 hOX40 hIgG1 Yes Not applicable 95 97 FS20-22-49AA/FS30-35-14 hCD137 hOX40 hIgG1 Yes Not applicable 105 107

Table 21 shows the EC ₅₀ values and maximum IL-2 release response observed for all molecules tested in the T cell activation assay in the absence of cross-linking. Table 22 shows the EC ₅₀ values and maximum IL-2 release response observed for the controls G1/4420, G1/11D4, G2/MOR7480.1 and FS20-22-49AA/4420 as single agents, further tested in the presence of cross-linkers agents. Figure 4C and D show IL-2 release plots for the T cell activation assay.

Table 21: T cell activation assay using mAb ² targeting co-expressed receptors in the absence of cross-linking agent

No cross-linking agent MAT/MAT ² EC ₅₀ (nM) Max. answer (nM) 95% CI (hIL-2 pg/ml) 95% CI G1/4420 No activity detected No activity detected No activity detected No activity detected G1/11D4 No activity detected No activity detected No activity detected No activity detected G2/MOR7480.1 No activity detected No activity detected No activity detected No activity detected G1/11D4 + G2/MOR7480.1 No activity detected No activity detected No activity detected No activity detected FS20-22-49AA/4420 5.02 0.2478-2583 1508 926.2-26580 FS20-22-49AA/FS30-5-37 1.201 0.1358-15.06 3663 2817-4979 FS20-22-49AA/FS30-10-3 0.2905 0.01754-3.867 4219 3204-5408 FS20-22-49AA/FS30-10-12 0.845 0.01871-85.72 3939 2388-7001 FS20-22-49AA/FS30-10-16 0.2019 0.0108-3.071 3873 3012-4897 FS20-22-49AA/FS30-35-14 0.2285 Not defined-14.77 4379 2915-6181

NAD = no activity detected

ND = not defined

Table 22: Single Agent Controls in the Presence of Cross-Linking Agent

With cross-linking agent MAT/MAT ² EC ₅₀ (nM) Max. answer (nM) 95% CI (hIL-2 pg/ml) 95% CI G1/4420 No activity detected No activity detected No activity detected No activity detected G1/11D4 0.05132 Not defined -0.3545 6375 5385-7400 G2/MOR7480.1 2.38 1.231-4.754 2306 2090-2545 FS20-22-49AA/4420 0.06129 0.01408-0.1939 5806 5242-6386

NAD = no activity detected

ND = not defined

Table 21 and Figure 4C show that non-crosslinked OX40/CD137 mAbs ² were active ( _EC50 values ranging from 0.2019 to 1.201 nM) and were therefore able to bind to both targets, resulting in clustering one or both of them with induction of T cell activation. IL-2 production was not observed when using non-crosslinked or cross-linked anti-FITC antibody G1/4420, as expected, or using non-crosslinked anti-OX40 antibody (G1/11D4 alone or in combination with G2/MOR7480.1). T cells produced IL-2 when the OX40 receptor was targeted by an anti-OX40 antibody as a positive control in the presence of a cross-linking agent (EC ₅₀ 0.05 nM for G1/11D4 alone and EC ₅₀ 0.02 nM in combination with G2/ MOR7480.1).

OX40-targeting Fcab in the mock MAb ² format (4420 LALA) FS20-22-49AA/4420 had some agonist activity in the absence of cross-linking (EC ₅₀ 5.02 nM and max response 1508 pg/ml hIL-2), as was observed in the SEA assay, and activity was further enhanced when mock MAT ² was cross-linked by coupling its Fab arms to FITC-dextran.

No activity was observed using the uncrosslinked anti-CD137 antibody G2/MOR7480.1 alone, but when crosslinked it was able to induce T cell activation, suggesting that, unlike the T cell activation assay with SEA ( Example 12 ), this assay is capable of measuring CD137 signaling by this clone to CD137, as well as OX40 signaling confirmed above. The difference in activity observed for this cross-linking antibody compared to the same anti-CD137 clone in IgG1 format (G1AA/MOR7480.1) in Example 13.1 , for which no activity was detected either in the absence or presence of the artificial cross-linking agent, may be explained by T cell donor variability, where some donors may respond better to CD137 stimulation than others.

In the activation assay of human CD137-expressing T cells using DO11.10-hCD137 cells described in Example 7.1 , anti-OX40/CD137 MAbs ² tested (FS20-22-49AA/FS30-5-37, FS20-22-49AA /FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA/FS30-10-16 and FS20-22-49AA/FS30-35-14) and control G2/MOR7480.1 effectively induced IL-2 production. Thus, it is hypothesized that the anti-CD137 Fab arms of anti-OX40/CD137 mAb ² are also capable of exerting an agonistic effect on CD137 expressed by T cells to produce a detectable IL-2 signal in the primary T cell activation assay in the present example.

13.4 Activity of anti-OX40/CD137 mAb ² clone FS20-22-49AA/FS30-10-16 in a total T cell population activation assay using T cells from nine PBMC donors

The anti-OX40/CD137 mAb ² clone FS20-22-49AA/FS30-10-16 was tested in a T cell activation assay using PBMCs from nine different donors to establish the precise EC ₂₀ , EC ₃₀ and EC ₅₀ values for its activity. The assay was performed as described in Example 13.1 in the absence of an artificial cross-linker.

Means plus or minus the standard deviation of the mean (SD) were calculated from the raw data as described in Example 12.3 for each donor. The EC ₂₀ , EC ₃₀ and EC ₅₀ values for IL-2 release observed for anti-OX40/CD137 mAb ² (FS20-22-49AA/FS30-10-16) in the T cell assay were also calculated as described in Example 12.3 , and presented in Table 23 .

Table 23: EC ₂₀ , EC ₃₀ and EC ₅₀ values of anti-OX40/CD137 mAb ² in T-cell activation assay

EC ₅₀ (nM) EC ₃₀ (nM) EC ₂₀ (nM) Donor 1 0.170 0.203 0.280 Donor 2 0.067 0.103 0.153 Donor 3 0.167 0.158 0.193 Donor 4 0.251 0.226 0.223 Donor 5 0.175 0.182 0.222 Donor 6 0.116 0.177 0.264 Donor 7 0.114 0.297 0.467 Donor 8 0.121 0.283 0.448 Donor 9 0.199 0.174 0.194 Weighted average 0.179 0.067 0.040 95% CI 0.154-0.208 0.049-0.090 0.026-0.061

These results indicate that mAb ² to OX40/CD137 has comparable activity against T cells from different donors.

Example 14 - MAT activity ² to human OX40/CD137 in CD4 activation assays ⁺ and CD8 ⁺ T cells

T cells can be divided into CD4 ⁺ and CD8 ⁺ T cells according to their function in the immune system. CD4 ⁺ T cells are called helper T cells and produce cytokines that modulate the immune response, and CD8 ⁺ T cells are called killer T cells and directly eliminate target cells. On CD4 ⁺ T cells, OX40 expression was found to be higher than CD137 expression, and conversely, on CD8 ⁺ T cells, CD137 expression was higher than OX40 expression (Croft, 2014 and see Figure 6 ). Despite this difference in expression levels, both CD4 ⁺ and CD8 ⁺ T cells coexpress the two receptors (Ma et al. , 2005).

To further examine the activity of Mab ² to OX40/CD137 in these two T cell populations, CD4 ⁺ and CD8 ⁺ T cells were isolated to test the ability of the molecules listed in Table 24 below to activate each T cell population in separate CD4 ⁺ activation assays. and CD8 ⁺ T cells. In this analysis, co-expression of OX40 and CD137 was used to determine the cross-linking of Mab ² to OX40/CD137 FS20-22-49AA/FS30-10-16. G1/4420 (anti-FITC), G1AA/11D4 (anti-OX40), G1AA/MOR7480.1 (anti-CD137) G1AA/FS30-10-16 (anti-CD137), FS20-22-49AA/4420 (imitation MAT ² to OX40 /FITC) and FS20-22-49AA/4420 plus G1AA/FS30-10-16 in combination were used as controls. IL-2 production was used as a measure of T cell activation.

Table 24: Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation Cross-linking agent SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No FITC-dextran 115 116 G1AA/11D4 hOX40 Absent hIgG1 Yes Antibody to hCH2 173 175 G1AA/MOR7480.1 hCD137 Absent hIgG1 Yes Antibody to hCH2 125 120 G1AA/FS30-10-16 hCD137 Absent hIgG1 Yes Antibody to hCH2 154 97 G1AA/20H4.9 hCD137 Absent hIgG1 Yes Antibody to hCH2 165 122 FS20-22-49AA/4420 FITC hOX40 hIgG1 Yes FITC-dextran 123 116 FS20-22-49AA/FS30-10-16 hCD137 hOX40 hIgG1 Yes Antibody to hCH2 95 97

To isolate human CD4 ⁺ and CD8 ⁺ T cells, PBMCs were first isolated as described in Example 13.1 . CD4 ⁺ and CD8 ⁺ T cells were then separately isolated from PBMCs using, respectively, a CD4 ⁺ T Cell Isolation Kit (Human) (Miltenyi Biotec, 130-096-533) and a CD8 ⁺ T Cell Isolation Kit ( human) (Miltenyi Biotec, 130-096-495) in accordance with the manufacturer's instructions.

CD4 ⁺ or CD8 ⁺ T cells were activated overnight at the required concentration of 1.0 x 10 ⁶ cells/ml in T cell medium using human CD3/CD28 Dynabeads T cell activator as described in Example 13.1 .

Activated CD4 ⁺ or CD8 ⁺ T cells were washed from Dynabeads and resuspended in T cell medium at a concentration of 2.0 x 10 ⁶ cells/ml. 96-well flat-bottom plates were coated with anti-human CD3 antibody by incubation with 2.5 μg/ml (for CD4 ⁺ T cell activation assay) or 10 μg/ml (for CD8 ⁺ T cell activation assay) anti-human CD3 antibody (R&D Systems, clone UHCT1) diluted in PBS for 2 hours at 37°C, 5% CO ₂ and then washed twice with PBS. Activated CD4 ⁺ or CD8 ⁺ T cells were then added to the appropriate plates at 2×10 ⁵ cells/well.

2 μM dilutions of each test antibody were prepared (see Table 24 for details) and added to the wells in a 1:1 molar ratio with cross-linking agent (human CH2 antibody or FITC-dextran (Sigma) (see Table 24 )). where required, as described in Example 6 . Serial dilutions of test antibodies were prepared in a 96-well plate, and 100 μl of the diluted antibody mixture was added to activated CD4 ⁺ or CD8 ⁺ T cells on the appropriate plates.

T cells were incubated at 37°C, 5% CO ₂ for 72 hours. Supernatants were collected, IL-2 release was measured, and data were obtained as described in Example 12.1 . Table 25 shows the EC ₅₀ values and maximum IL-2 release response observed in individual T cell activation assays in the presence or absence of cross-linking using cross-linking agents. Figures 5A-C show IL-2 release plots for CD4 ⁺ or CD8 ⁺ T cell activation assays, respectively.

After collection of supernatants, T cells were washed in PBS and stained with Alexa Fluor 488-labeled secondary anti-human Fc antibody (Jackson Immunoresearch, cat. no. 109-546-098) diluted 1:1000 in PBS for 1 hour at 4°C. . Cells were then washed once with PBS and resuspended in 100 μl/well of PBS with DAPI (Biotium, cat. no. 89139-054) at 1 μg/ml. Cells were then analyzed on a BD FACSCanto II flow cytometer (BD Biosciences). Figure 6 shows the geometric mean fluorescence intensity in channel 488 of either CD4 ⁺ or CD8 ⁺ T cells treated with G1AA/MOR7480.1 or G1AA/11D4.

Table 25: CD4 ⁺ and CD8 ⁺ T cell activation assay using MAbs ² targeting co-expressed receptors

CD4 ⁺ T cells No cross-linking Cross linking EC ₅₀ (nM) Max. answer EC ₅₀ (nM) Max. answer MAT/MAT ² (nM) 95% CI (hIL-2 pg/ml) 95% CI (nM) 95% CI (hIL-2 pg/ml) 95% CI G1/4420 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/11D4 No activity detected No activity detected No activity detected No activity detected 0.0813 0.01883-0.2796 8817 7129-10561 G1AA/MOR7480.1 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/FS30-10-16 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected FS20-22-49AA/4420 0.5641 0.06536 - undefined 2242 1578-+infinity 0.1553 0.06145-0.3765 18872 16200-21634 FS20-22-49AA/4420 + G1AA/FS30-10-16 22.54 1.881-162.8 3820 2403-8413 0.181 0.08648-0.3728 18895 16953-20903 FS20-22-49AA/FS30-10-16 0.1131 0.04802-0.2529 16232 14326-18191 0.08334 0.03012-0.2113 16232 14031-18494 CD8 ⁺ T cells No cross-linking Cross linking EC ₅₀ (nM) Max. answer EC ₅₀ (nM) Max. answer MAT/MAT ² (nM) 95% CI (hIL-2 pg/ml) 95% CI (nM) 95% CI (hIL-2 pg/ml) 95% CI G1/4420 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/11D4 No activity detected No activity detected No activity detected No activity detected 0.09964 0.004042-1.04 387.6 306.3-474.2 G1AA/MOR7480.1 No activity detected No activity detected No activity detected No activity detected 1.011 0.2837-3.658 1066 849.8-1308 G1AA/FS30-10-16 No activity detected No activity detected No activity detected No activity detected 3.875 2.547-5.943 2560 2344-2796 FS20-22-49AA/4420 No activity detected No activity detected No activity detected No activity detected 0.268 0.1418-0.5024 1663 1510-1821 FS20-22-49AA/4420 + G1AA/FS30-10-16 No activity detected No activity detected No activity detected No activity detected 0.4312 0.1721-1.081 2534 2185-2905 FS20-22-49AA/FS30-10-16 0.1183 0.06831-0.2032 4915 4592-5246 1.98 0.3441-8.779 7397 6071-9139

NAD = no activity detected

ND = not defined

Table 25 and Figure 5B show that CD4 ⁺ T cells can be activated by cross-linked anti-OX40 controls G1AA/11D4 and FS20-22-49AA/4420 (either alone or in combination with G1AA/FS30-10- 16), but not by the anti-CD137 controls G1AA/MOR7480.1 and G1AA/FS30-10-16 as single agents. On the other hand, Figure 5C shows that CD8 ⁺ T cells were activated by both the cross-linked anti-CD137 controls G1AA/MOR7480.1 and G1AA/FS30-10-16, as well as the cross-linked anti-OX40 controls G1AA/11D4 and FS20- 22-49AA/4420, although the response rate to the single-agent anti-CD137 G1AA/FS30-10-16 control was higher than that to both single-agent anti-OX40 controls. As observed in the SEA assay ( Example 12.2 ) and the general human T cell population activation assay ( Example 13.3 ), the anti-OX40 Fcab in mock MAb ² format (FS20-22-49AA/4420) showed some activity in the absence of cross-linking in the presence CD4 ⁺ T cells, and this activity increased when the antibody was cross-linked. Anti-OX40/CD137 mAb ² (FS20-22-49AA/FS30-10-16) was active in the presence of both CD4 ⁺ and CD8 ⁺ T cells in the absence of cross-linking, as expected based on previous results (see Examples 12 and 13 ).

Figure 6 shows that CD4 ⁺ T cells express lower levels of CD137 and higher levels of OX40 than CD8 ⁺ T cells. G1AA/MOR7480.1 binding to CD137 is a measure of CD137 expression, and G1AA/11D4 binding to OX40 is a measure of OX40 expression.

The T cell assay with isolated CD4 ⁺ and CD8 ⁺ T cells was repeated according to the same protocol as described above, but with T cells isolated from PBMCs from a different donor and with the addition of anti-CD137 antibody G1AA/20H4. 9 (see Table 24 ). Consistent with the results shown in Figures 5A-D , Figures 5E and 5F show that CD8 ⁺ T cells respond more significantly to agonist action on CD137, and CD4 ⁺ T cells respond more significantly to agonist action on OX40. Antibodies to OX40 (G1AA/11D4 and Fcab to OX40 in the format of mock MAb ² FS20-22-49AA/4420) when cross-linked activated CD4 ⁺ T cells, but not CD8 ⁺ T cells, and antibodies to CD137 (G1AA/20H4 .9 and G1AA/FS30-10-16) when cross-linked activated CD8 ⁺ T cells but not CD4 ⁺ T cells. Antibody G1AA/20H4.9 also activated CD8 ⁺ T cells in the absence of cross-linking antibody, consistent with the results obtained in the DO11.10-hCD137 cell assay described in Example 7.1 . In this replicate experiment, antibody G1AA/MOR7480.1 did not activate CD8 ⁺ T cells upon cross-linking. Some PBMC donors may be more susceptible to CD137 costimulation than others, and the different results obtained in this experiment may be due to this natural variability.

These data indicate that CD4 ⁺ T cells are more sensitive to activation by agonistic action on OX40 than CD8 ⁺ T cells and, conversely, that CD8 ⁺ T cells are more sensitive to activation by agonistic action on CD137 than CD4 ⁺ T cells. This correlates with reported differences in the expression levels of OX40 and CD137 receptors on CD4 ⁺ T cells and CD8 ⁺ T cells, with the former expressing higher levels of OX40 than CD137 and the latter expressing higher levels of CD137 than OX40. The activity of the cross-linked control anti-CD137 antibody G1AA/FS30-10-16, whose Fab arms are present in Mab ² to OX40/CD137 FS20-22-49AA/FS30-10-16, in the presence of CD8 ⁺ T cells demonstrates that Mab ² has the ability to activate the CD137 receptor upon cross-linking by binding its Fcab to OX40. In addition, the activity of cross-linked Fcab to OX40 in the format of mock MAT ² (FS20-22-49AA/4420), which is also present in MAT ² to OX40/CD137 FS20-22-49AA/FS30-10-16, in the presence of CD4 ⁺ T cell assay shows that Mab ² has the ability to activate the OX40 receptor upon cross-linking by binding its Fab arms to CD137. Thus, it can be concluded that mAb ² FS20-22-49AA/FS30-10-16 can act as a dual agonist by activating CD4 ⁺ T cells through an agonistic effect on OX40 and CD8 ⁺ T cells through an agonistic effect on CD137 and to a lesser extent on OX40. OX40 activation by MAT ² occurs through its Fcab and is enhanced by MAT ² cross-linking upon binding to CD137 through its Fab arms, while CD137 activation occurs through its Fab arms binding to CD137 and MAT ² cross-linking upon binding with OX40 at the expense of his Fcab.

Example 15 - MAT activity ² anti-mouse OX40/CD137 and anti-mouse CD137 in T cell activation assays

Because human OX40/CD137 mAb ² does not bind to mouse proteins, to test the ability of anti-OX40/CD137 mAb ² to induce a T cell-mediated antitumor response, a similar reagent targeting mouse OX40 and mouse CD137 was prepared (see Example 8.2 ).

15.1 Activity of mAb ² to mouse OX40/CD137 in the analysis of activation of the total population of T cells

To test whether mouse anti-OX40/CD137 mAb ² (FS20m-232-91AA/Lob12.3), targeting these two co-expressed receptors, could induce the production of inflammatory cytokines by pre-activated T cells, a mouse T cell activation assay was developed. Antibodies G1/4420 (anti-FITC), G1AA/OX86 (anti-mOX40), G1AA/Lob12.30 (anti-mCD137), G1AA/OX86 and G1AA/Lob12.3 in combination, as well as FS20m-232-91AA/4420 (mock Anti-mOX40/FITC mAb ² ) were used as controls (see Table 26 for details) and IL-2 production was used as a measure of T cell stimulation.

Table 26: Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation Cross-linking agent SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No FITC-dextran 115 116 G1AA/OX86 mOX40 Absent hIgG1 Yes a-hCH2 155 156 G1AA/Lob12.3 mCD137 Absent hIgG1 Yes a-hCH2 University of Southampton FS20m-232-91AA/4420 FITC mOX40 hIgG1 Yes FITC-dextran 157 116 FS20m-232-91AA/Lob12.3 mCD137 mOX40 hIgG1 Yes a-hCH2 The receipt is described above in Example 9.2

To isolate T cells, spleens were collected from female Balb/C mice aged 4–8 weeks (Charles River). Mice were humanely sacrificed, and spleens were isolated by dissection. Splenocytes were isolated by pushing spleens through a 70-μm cell strainer (Corning) using the inside of a 5-ml plastic syringe. The cell strainer was washed 10 times with 1 mL of Dulbecco's phosphate-buffered saline (DPBS) (Gibco), and the eluate was collected in a 50-mL tube. Red blood cells present in the eluate were lysed by adding 10 ml of red blood cell lysis buffer (eBioscience) according to the manufacturer's instructions. T cells were isolated from splenocytes present in the eluate using the Total T Cell Population Isolation Kit II (Mouse) (Miltenyi Biotec Ltd) according to the manufacturer's instructions and then activated and used in a protocol essentially similar to the T activation assay -human cells as described in Example 13.1 , but using mouse CD3/CD28 Dynabeads T cell activator (Life Technologies) for T cell activation, anti-mouse CD3 antibody (Biolegend, clone 145-2C11) for plate coating and ELISA kit Mouse IL-2 (eBioscience or R&D systems) to measure IL-2 release.

Table 27 shows the EC ₅₀ values and the maximum IL-2 release response observed in the T cell activation assay in the presence of MAT ² and MAT tested. Figure 7A and B show typical IL-2 release plots for a T cell activation assay.

Table 27: T cell activation assay using MAbs ² targeting co-expressed receptors

No cross-linking Cross linking MAT/MAT ² EC ₅₀ (nM) Max. answer EC ₅₀ (nM) Max. answer (nM) 95% CI (mIL-2 pg/ml) 95% CI (nM) 95% CI (mIL-2 pg/ml) 95% CI G1/4420 No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected No activity detected G1AA/OX86 No activity detected No activity detected No activity detected No activity detected 2,413 1,730-3,365 14544 13647-15441 G1AA/Lob12.3 No activity detected No activity detected No activity detected No activity detected 1.179 0.001061-1309 373.6 139.3-607.8 G1AA/OX86 + G1AA/Lob12.3 No activity detected No activity detected No activity detected No activity detected 1.722 0.9596-3.090 12834 11531-14138 FS20m-232-91AA/4420 No activity detected No activity detected No activity detected No activity detected 0.2568 0.1181-0.5585 14672 13279-16065 FS20m-232-91AA/Lob12.3 0.1141 0.01023-1.273 8750 6614-10885 0.1011 0.04358-0.2346 13563 12485-14640

NAD = no activity detected

Table 27 and Figure 7B show that increased T cell activation occurs when the OX40 receptor is targeted and OX40 antibodies are cross-linked. T cell activation was not observed using cross-linked or non-cross-linked anti-FITC G1/4420, as expected, or using non-cross-linked anti-OX40 (G1AA/OX86 alone or in combination with G1AA/Lob12.3) . T cells produced IL-2 when the OX40 receptor was targeted by the anti-OX40 antibody G1AA/OX86 in the presence of a cross-linking agent (EC ₅₀ 2.41 nM for G1AA/OX86 alone and EC ₅₀ 1.72 nM in combination with G1AA/Lob12 .3).

OX40-targeted Fcab in mock MAb ² format (FS20m-232-91AA/4420) had no agonist activity in the absence of cross-linking, but when cross-linked by binding Fab arms to FITC-dextran showed effective T cell activation. When the OX40-targeting Fcab was paired with an anti-CD137 Fab (Lob12.3), MAb ² exhibited activity against T cells in the absence of any additional cross-linking agents. This indicates that MAT ² is cross-linked by binding to co-expressed receptors on the surface of the same cell.

Minimal activity was observed using cross-linked CD137-targeting antibody G1AA/Lob12.3 alone, and the cross-linking activity of the combination of OX40-targeting antibody G1AA/OX86 and CD137-targeting antibody G1AA/Lob12.3 was comparable to that of cross-linking OX40- targeting antibody G1AA/OX86, indicating low sensitivity of the assay for detecting CD137 agonistic effects by Lob12.3. These data are in contrast to the human T cell assay described in Example 13.3 , in which a stronger CD137-specific signal (maximal IL-2 release response) was observed for the cross-linked control against CD137 G2/MOR7480.1. This difference in functional activity observed for control anti-mouse CD137 and anti-human CD137 antibodies may be due to their different affinities for their respective CD137 targets. It may also reflect the cell source (human PBMCs versus mouse splenocytes) or subtle differences between target biology in mouse and human systems.

These data indicate that anti-OX40/CD137 mAb ² FS20m-232-91AA/Lob12.3 can induce T cell activation without any additional cross-linking agents by simultaneously binding to both receptors.

Since human anti-OX40/CD137 mAb ² molecules are not cross-reactive with mouse proteins and mouse anti-OX40/CD137 mAb ² molecules are functionally comparable to candidate human compounds in parallel in vitro experimental systems, anti-mouse protein molecules are considered suitable surrogates for inference about the ability of MAb ² to OX40/CD137 to induce antitumor immunity in vivo .

15.2 Activity of anti-mouse CD137 antibodies in the activation assay of T cells expressing mouse CD137

Because there was little or no activity of the anti-CD137 Fab clone (Lob12.3) of the mouse anti-OX40/CD137 mAb ² in the assay with the total T cell population of Example 15.1 , a T cell activation assay was performed to determine the activity of the various anti-CD137 agonist antibodies using DO11.10-mCD137 cells. The agonist antibodies to CD137 G1AA/Lob12.3 (see Table 26 ) and G1AA/3H3 (SEQ ID NOs: 166 and 167) were tested, as well as the antibody to FITC 4420 in IgG1 format (G1/4420; SEQ ID NOs 115 and 116 ) as a negative isotype control. Mab molecules were tested both in the presence and absence of the human CH2 cross-linking antibody, MK1A6 (see Example 2.1 ). Mouse IL-2 production was used as a measure of T cell activation.

The assay was performed as described in Example 6.2 , but using DO11.10-mCD137 cells instead of DO11.10-hCD137 cells. The plates were read at 450 nm using a plate reader with Gen5 software (BioTek). The 630 nm absorbance values were subtracted from the 450 nm absorbance values (Correction). The standard curve for calculating cytokine concentration was based on a four-parameter logistic curve fit (Gen5 software, BioTek). Mouse IL-2 (mIL-2) concentration was plotted against log antibody concentration, and the resulting curves were fitted using the log(agonist) response equation in GraphPad Prism.

The results are shown in Figure 7C and D. Antibodies to CD137 varied in their requirement for cross-linking antibody to induce activity. While G1AA/Lob12.3 was found to require the addition of a cross-linking antibody for activity, i.e. its activity was dependent on cross-linking, G1AA/3H3 was active in both the presence and absence of a cross-linking antibody and, thus had cross-linking-independent activity.

Example 16 - For MAT activity ² OX40/CD137 requires dual binding of OX40 and CD137.

16.1 MAT ² to human OX40/CD137

MAb ² , targeting OX40/CD137, was active in the absence of additional cross-linking agents in assays using SEA ( Example 12 ), the total population of human T cells ( Example 13 ), and human CD4 ⁺ and CD8 ⁺ T cells ( Example 14 ) in which T cells coexpressed OX40 and CD137. To test whether simultaneous binding to two receptors is required for the activity of Mab ² to OX40/CD137, a T cell competition assay was performed to assess the ability of Mab ² FS20-22-49AA/FS30-10-16 to activate isolated T cells in the presence of 100-fold excess of OX40-targeting mock MAT ² FS20-22-49AA/4420, anti-CD137 MAT G1AA/FS30-10-16, combinations of mock MAT ² FS20-22-49AA/4420 plus MAT G1AA/FS30-10-16, or MAT for isotype control G1/4420. IL-2 production was used as a measure of T cell activation.

T cells were isolated as described in Example 13 . The isolated T cells were then activated and the plates were coated with anti-CD3 antibody as described in Example 13 . 2 nM mAb ² to OX40/CD137 (FS20-22-49AA/FS30-10-16) was added to activated T cells and added to plates at 2×10 ⁵ cells/well in 100 μl. Thus, the final concentration of MAb ² to OX40/CD137 was 1 nM.

2 μM dilutions of each antibody tested were prepared in DFSB (Gibco) and further diluted 1:10 in T cell media (30 μL + 270 μL) to produce 200 nM dilutions, and 100 μL of each diluted antibody was added to activated T cells on the tablet.

T cells were incubated, supernatants were collected and IL-2 release was measured as described in Example 13 . The standard curve for calculating cytokine concentration was based on a four-parameter logistic curve fit (Gen5 software, BioTek). Statistical analysis was performed using one-way analysis of variance and Dunnett's test for multiple comparisons using the GraphPad Prism software package.

Figure 8 shows the release of IL-2 for the competition assay. The activity of MAT ² was significantly reduced when there was superior competition from both the mock MAT ² FS20-22 - 49AA/4420 for binding to OX40 and MAT G1AA/FS30-10-16 for binding to CD137, compared with when MAT ² was capable of binding to both receptors in the absence of antibodies to OX40 and CD137. The combination of the OX40-targeting mock mAb ² FS20-22-49AA/4420 and the anti-CD137 mAb G1AA/FS30-10-16 further reduced the activity of mAb ² to OX40/CD137. These results indicate that dual binding of MAT ² to both receptors is required for MAT ² to induce T cell activation through clustering and agonistic effects on OX40 and CD137.

16.2 MAT ² to OX40/CD137 mice

Mab ² , which targets mouse OX40/CD137, is active in the absence of additional cross-linking agents in a T cell assay when T cells co-express the two receptors. To test whether simultaneous binding to two receptors is required for the activity of Mab ² against OX40/CD137, a competition assay was performed to evaluate the ability of Mab ² FS20m-232-91AA/Lob12.3 to activate isolated T cells in the presence of a 100-fold excess of either OX40- targeted mock mAb ² FS20m-232-91AA/4420, or anti-CD137 mAb G1/Lob12.3, or mAb G1AA/4420 (FITC) as a negative control. T cells were isolated as described in Example 15.1 . Isolated T cells were then activated and plates were coated with anti-CD3 antibody as described in Example 13.1 (General Human T Cell Population Activation Assay), but CD3/CD28 Dynabeads Mouse T Cell Activator (Life Technologies) was used to activate T cells and anti-mouse CD3 antibody (Biolegend, clone 145-2C11) for coating plates. 2 nM mAb ² to OX40/CD137 (FS20m-232-91AA/Lob12.3) was added to activated T cells and added to the plates at 2×10 ⁵ cells/well.

2 μM dilutions of each antibody tested were prepared in DFSB (Gibco) and further diluted 1:10 in T cell media (30 μL + 270 μL) to produce 200 nM dilutions, and 100 μL of each diluted antibody was added to activated T cells on the tablet.

T cells were incubated, supernatants were collected, and IL-2 release was measured as described in Example 12.1 , but a mouse IL-2 ELISA kit (eBioscience or R&D systems) was used to measure IL-2 release. The standard curve for calculating cytokine concentration was based on a four-parameter logistic curve fit (Gen5 software, BioTek). Statistical analysis was performed using one-way analysis of variance and Dunnett's test for multiple comparisons using the GraphPad Prism software package. Figure 9 shows a typical IL-2 release schedule for a competition assay.

Figure 9 shows that there is a decrease in the amount of IL-2 production induced by anti-OX40/CD137 mAb ² when antibodies competing for OX40 or CD137 binding are administered in excess. Constituents of MAT ² (Fcab in mock MAT ² format (4420) and Fab without Fcab) were used as competing antibodies to ensure targeting of the same epitope. The addition of these competing antibodies reduced the magnitude of IL-2 release induced by mAb ² to OX40/CD137, suggesting that the molecule requires double binding to exert its activity. This shows that the activity of mAb ² against OX40/CD137 depends on the simultaneous binding of both OX40 and CD137, which leads to clustering and agonistic effects on both receptors.

Example 17 - MAT activity ² to OX40/CD137 in the CT26 syngeneic tumor model

17.1 Comparison of the antitumor activity of mAb ² to OX40/CD137 with or without the LALA mutation

The CT26 syngeneic colorectal tumor model in Balb/c mice was used to test the antitumor activity of mouse mAb ² against OX40/CD137 in vivo . The CT26 tumor model has previously been shown to be sensitive to agonistic antibodies to both OX40 and CD137 (Sadun et al. , 2008), and tumor infiltrating lymphocytes (TILs) isolated from CT26 tumors are expected to express both OX40 and and CD137. The antibodies tested are detailed in Table 28 .

Table 28: Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No 115 116 G1/OX86 mOX40 Absent hIgG1 No 159 156 G1AA/OX86 mOX40 Absent hIgG1 Yes 155 156 G1/Lob12.3 mCD137 Absent hIgG1 No University of Southampton G1AA/Lob12.3 mCD137 Absent hIgG1 Yes University of Southampton FS20m-232-91/Lob12.3 mCD137 mOX40 hIgG1 No The receipt is described above in Example 9.2 FS20m-232-91AA/Lob12.3 mCD137 mOX40 hIgG1 Yes

The ability of mAb ² , with or without the LALA mutation (FS20m-232-91AA/Lob12.3 and FS20m-232-91/Lob12.3, respectively), to inhibit tumor growth was compared with the control isotype Mab G1/4420 (anti-FITC), MAb G1/OX86 (control against OX40 without LALA mutation) or G1/Lob12.3 (control against CD137 without LALA mutation) as single agents, combination G1/OX86 plus G1/Lob12.3 or combination G1AA/OX86 (MAB against OX40 with LALA mutation) plus G1AA/Lob12.3 (anti-CD137 mAb with LALA mutation).

Female BALB/c (Charles River) mice, 8-10 weeks old and weighing approximately 20 g each, were acclimatized for one week before the start of the study. All animals were microchipped and assigned a unique identifier. There were 12 mice in each cohort. The colon carcinoma cell line CT26 (ATCC, CRL-2638) was expanded, banked, and then prescreened at IDEXX Bioresearch for pathogen detection using the IMPACT I protocol, and the line was shown to be pathogen-free. CT26 cells (approximately 3-5 x 10 ⁶ ) were thawed from storage at -150°C and added to 20 ml DMEM (Gibco, 61965-026) with 10% fetal calf serum (FCS) (Gibco, 10270-106) in a flask for tissue culture T175. Mice were anesthetized using isoflurane (Abbott Laboratories) and each animal received 1 x 10 ⁶ cells injected subcutaneously into the left flank to generate tumors. At day 10 after tumor cell inoculation, tumors were measured and mice were randomized into study cohorts based on tumor volume. Any mice that did not have tumors at this point were excluded from the study.

Within 24 hours prior to injection, antibodies were analyzed by SEC-HPLC profiling and checked for contaminants. Antibodies were diluted to a final concentration of 0.1 mg/ml in PBS and administered by intraperitoneal injection (i.p.) at 200 μl/mouse to produce a final dose of 1 mg/kg for a 20 g mouse. Injections were performed on days 13, 15, and 17 (three doses every two days) after tumor inoculation. The health of the animals was checked under anesthesia three times a week, during which time precise measurements of the tumors were taken. Tumor volume measurements were performed using calipers to determine the longest axis and shortest axis. To calculate tumor volume, the following formula was used:

L×(S ² )/2

(Where L = longest axis; S = shortest axis)

The study was stopped at day 27 when tumor volume reached the humane endpoint in accordance with the UK Animals (Scientific Procedures) Act and EU Directive EU86/609.

For statistical analysis, tumor volumes are analyzed on a logarithmic scale using a mixed model. Each pair of treatments of interest was fitted using a separate model. The model is:

Log ₁₀ ( volume )=A+B×( start day-day)+ ε

A and B represent the intercept and slope, respectively; these are different for each mouse and include a fixed effect for group and a random effect for animal:

T is a dummy variable representing treatment group, with a value of 0 in one group and 1 in the other. Random effects are distributed according to the normal distribution:

Where And represent the standard deviations of between-animal variability at the intercept and slope, respectively. Variation between animals also typically has a normal distribution with standard deviation σ:

For each pair of treatments, the model above was fitted to the data. For A ₁ and B _1, a (two-sided) p-value was calculated for difference from zero; A p-value below 0.05 is statistically significant evidence of a difference between treatment groups.

The results are shown in Figure 10A . Average CT26 tumor volumes plus or minus standard error of the mean are plotted. The results show that treatment with mAb ² to OX40/CD137 both with and without the LALA mutation (FS20m-232-91AA/Lob12.3 and FS20m-232-91/Lob12.3, respectively) led to a decrease in tumor growth compared to treated with anti-OX40 control (G1/OX86), anti-CD137 control (G1/Lob12.3), a combination of these two antibodies (G1/OX86 + G1/Lob12.3) or a combination of LALA-containing anti-OX40 and anti-CD137 antibodies (G1AA /OX86 + G1AA/Lob12.3).

The results indicate a statistically significant antitumor effect of mAb ² to OX40/CD137 (FS20m-232-91AA/Lob12.3 and FS20-232-91/Lob12.3) compared to the control antibody (G1/4420). The activity of a combination of OX40 and CD137-targeting antibodies (G1/OX86 plus G1/Lob12.3 or G1AA/OX86 plus G1AA/Lob12.3) did not significantly suppress tumor growth, nor did single agent controls (G1/OX86 or G1 /Lob12.3).

Introduction of the LALA mutation into the Fc region of the human IgG1 backbone of Mab ² to OX40/CD137 is expected to prevent ADCC and ADCC of OX40- or CD137-expressing cells, as well as Fcγ receptor-mediated cross-linking of Mab ² when binding to either OX40 or CD137 on cells expressing these receptors. Therefore, it is believed that the activity of MAT ² FS20m-232-91AA/Lob12.3 will be triggered by cooperative binding of OX40 and CD137, resulting in signal transduction through one or both receptors, rather than through Fc-mediated effector function or crosstalk. Fcγ receptor-mediated binding. This is expected to subsequently lead to activation of OX40- and CD137-expressing T cells, ultimately leading to T cell-mediated antitumor activity.

These results demonstrate that anti-OX40/CD137 MAb ² has antitumor efficacy in vivo against tumors expected to contain TILs expressing OX40 and CD137, suggesting that activation of OX40 and CD137 in vivo is mediated by bispecific binding of OX40 and CD137 using MAb ² to OX40/CD137, effectively controls tumor growth.

As described in the prior art section above, liver toxicity has been observed with the use of an agonistic CD137 antibody in clinical settings (Segal et al. , 2017). The mechanism of this toxic effect is not fully defined, but studies in preclinical models have highlighted the role of CD137-expressing myeloid cells that produce IL-27 in response to agonistic CD137 antibodies (Bartkowiak et al. , 2018). The role of Fcγ receptors in the mechanism of liver toxicity has not been investigated, but one possible explanation for the observed toxicity is that coexpression of CD137 and Fcγ receptors on myeloid cells may result in cross-binding of agonistic anti-CD137 antibodies on these cells, triggering the production of inflammatory cytokines. Thus, in the event that Fcγ receptor-mediated cross-linking of the molecule could result in any activation of cells expressing CD137 in the absence of OX40, in locations remote from the tumor microenvironment or periphery, inclusion of the LALA mutation in the anti-OX40/CD137 antibody molecule was considered desirable. being a dual agonist according to the present invention. Thus, by designing a dual agonist antibody molecule that stimulates a T cell expressing both OX40 and CD137 by simultaneously binding to both targets, but which does not activate CD137-expressing cells through Fcγ receptor-mediated cross-linking in the absence of OX40 due to the presence of the LALA mutation in the molecule, it is believed that the antibody molecule of the present invention is likely to have reduced potential toxicity in a clinical setting.

Another reason for including the LALA mutation in the antibody molecule of the present invention is that it avoids the Fcγ receptor-mediated killing of OX40- and CD137-expressing cells that the molecule is designed to activate to suppress tumor growth. The mechanism of action of agonistic antibodies to OX40 in some preclinical tumor models has been described and involves Fcγ receptor-mediated depletion of Tregs in the tumor microenvironment, and introducing mutations into these molecules that suppress Fcγ receptor function impairs their antitumor activity (Bulliard et al. , 2014 ). Although the effect of the LALA mutation may be to preserve the beneficial immune cells that the antibody molecule of the present invention is designed to activate, coupled with not depleting Treg cells, it should be noted that OX40-targeting human IgG1 antibodies designed to do the same mechanism of tumor depletion, Tregs, as observed in preclinical tumor models, did not exhibit a similar ability to control tumor growth (Glisson et al. , 2016). Other molecules developed to deplete Tregs have also failed to show high levels of clinical activity (Powell et al. , 2007; Tran et al. , 2017). The lack of clinical extrapolation of the effects of Treg depletion observed in mouse models of syngeneic tumors may be due to lower levels of Fcγ receptor-expressing cells in the tumor microenvironment (Milas et al., 1987), differences in Treg biology between humans and mice (Liu et al. , 2016) or other unknown factors (Stewart et al. , 2014).

The inclusion of the LALA mutation in mAb ² FS20m-232-91AA/Lob12.3 unexpectedly did not impair its antitumor activity in the CT26 model, suggesting that it has an Fcγ receptor-independent mechanism of action that does not rely on interaction with receptor-expressing cells Fcγ. The lack of observed depletion of tumor Tregs and the induction of robust T cell proliferation in the blood by this LALA mutation-containing mAb ² in the “mechanism of action” study described in Example 19 provides further support for an Fcγ receptor-independent mechanism of action of the dual agonist Mab ² k OX40/CD137 described in this application. Given the low clinical activity observed with antibodies that rely on interaction with the Fcγ receptor for their activity, the Fcγ receptor-independent mechanism of action of the antibody molecule of the present invention is expected to result in greater efficacy in the clinical setting.

17.2 Comparison of the antitumor activity of mAb ² to OX40/CD137 and its components Fcab and Fab

In a general mouse T cell population activation assay ( Example 15 ), anti-mouse OX40/CD137 mAb ² (FS20m-232-91AA/Lob12.3) was active in vitro in the absence of additional cross-linking agents, in contrast to the monospecific control antibodies G1AA/ Lob12.3 (MAT to mCD137) and FS20m-232-91AA/4420 (mock MAT ² to mOX40/FITC), by simultaneously binding to both CD137 and OX40 receptors ( Example 16.2 ). After analyzing the activation of the total population of T cells, the antitumor activity of FS20m-232-91AA/Lob12.3 was compared with the activity of its components, that is, Fcab FS20m-232-91AA in the format of a mock (anti-FITC) MAT ² (FS20m-232-91AA/ 4420) and a monospecific mouse mAb to CD137 without Fcab (G1AA/Lob12.3) as single agents or in combination, or isotype control (G1AA/4420) in the CT26 tumor model.

Following the same method as described in Example 17.1 , CT26 tumors were formed subcutaneously in female BALB/c mice. At day 10 after CT26 cell inoculation, tumor-bearing mice were randomized into study cohorts of 25 mice per group and administered antibodies.

Antibodies were diluted to a final concentration of 0.3 mg/ml in PBS and administered by intraperitoneal injection in a volume of 200 μl to each mouse, resulting in a final dose of 3 mg/kg for a 20 g mouse (fixed dose of 60 μg of each antibody). Injections were performed once every two days (Q2D) for a total of three doses starting at day 10 after tumor inoculation. Tumor volumes were determined using caliper measurements as previously described. The study was stopped 64 days after cell inoculation, and animals were removed from the study when humane endpoints based on tumor volume and condition were met.

Tumor volume data over time for individual animals is shown in Figure 10B , and the average results shown in Figure 10C indicate that mAb ² FS20m-232-91AA/Lob12.3 inhibited CT26 tumor growth rate at an earlier stage (between days 10 and 22) compared with the isotype control antibody (G1AA/4420). No apparent tumor growth inhibition was observed in cohorts treated with anti-mouse CD137 mAb, mock anti-OX40/FITC mouse mAb ² , or a combination thereof.

Following the same mixed model approach as previously described, analysis of tumor volume data up to day 22 (after cell inoculation, Table 29 ) showed that FS20m-232-91AA/Lob12.3 resulted in a statistically significant (p=0.003) reduction average tumor growth rate compared to isotype control. In comparison, treatment with mouse anti-CD137 mAb, anti-OX40/FITC murine mAb ² , or a combination of both did not result in significantly different tumor growth rates compared with isotype controls. Comparison of tumor growth rates throughout the study period (64 days) using a mixed model method showed statistically significant reductions in tumor growth rates in all treatment groups compared with isotype controls (analysis not shown).

Table 29: Pairwise comparison of mean CT26 tumor growth rates using mixed-effects model analysis

Pairwise comparison of A with B Mean log(TGR) [lower, upper 95% CI] P-value Bottom line A B A B Isotype control FS20m-232-91AA/4420 0.310
[0.279, 0.340] 0.291
[0.244, 0.339] > 0.05 Unreliable Isotype control G1AA/Lob12.3 0.310
[0.279, 0.340] 0.281
[0.235, 0.327] > 0.05 Unreliable Isotype control FS20m-232-91AA/4420 + G1AA/Lob12.3 0.310
[0.279, 0.340] 0.277
[0.237, 0.316] > 0.05 Unreliable Isotype control FS20m-232-91AA/Lob12.3 0.310
[0.279, 0.340] 0.205
[0.164, 0.247] 0.003 ***

ns = not statistically significant; TGR = tumor growth rate; CI = confidence interval

NOTE: To compare early tumor growth rates, tumor volume data for the first 22 days after inoculation were used in the mixed-effects model. For each pairwise comparison, at least one of the groups included in the p-value calculation contained more than 50% of tumor growth rates that did not significantly follow a lognormal distribution.

Survival analysis using the log-rank (Mantel-Cox) test showed that FS20m-232-91AA/Lob12.3 resulted in a statistically significant improvement in survival compared to isotype control (p≤0.0001) ( Figure 10D ). Tumor-bearing mice treated with either mouse anti-CD137 mAb or anti-mouse OX40 mAb ² /FITC, or a combination of both, had no statistically significant difference in survival compared with isotype controls.

In summary, the results demonstrate that MAT ² FS20m-232-91AA/Lob12.3 had greater and non-equivalent antitumor activity compared to the combination of its Fcab and Fab moieties or either moiety alone.

Example 18 - Pharmacodynamic response of MAT ² to OX40/CD137 in the CT26 syngeneic tumor model

18.1 Comparison of the pharmacodynamic response of mAb ² to OX40/CD137 and control mAbs to OX40 and CD137

The pharmacodynamic response of surrogate mAb ² to OX40/CD137 was assessed in mice bearing syngeneic CT26 tumors. To this end, mice carrying CT26 were inoculated with mAb ² FS20m-232-91AA/Lob12.3, isotype control (G1/4420), anti-mouse OX40 control (G1/OX86) as a single agent, anti-mouse CD137 control ( G1/Lob12.3) as a single agent or a combination of these anti-OX40 and anti-CD137 controls (G1/OX86 plus G1/Lob12.3), blood samples were collected over time and analyzed by flow cytometry to determine T cell activation and proliferation markers.

Following the same protocol as described in Example 17 , female BALB/c mice (Charles River), 8-10 weeks old and weighing approximately 20 g each, were prepared to begin the study and inoculated with the CT26 colon carcinoma cell line (ATCC, CRL- 2638). At day 10 after tumor cell inoculation, tumors were measured and mice were randomized into study cohorts of 10 mice per group based on tumor volume. Any mice that did not have tumors at this point were excluded from the study.

Antibodies were assayed and screened for contaminants as previously described, diluted to a final concentration of 0.1 mg/ml in PBS, and injected at 200 μl/mouse to produce a final dose of 1 mg/kg for a 20 g mouse. Antibodies were administered to mice by intraperitoneal (IP) injection on days 10, 12 and 14 after tumor inoculation.

Blood was collected from the tail vein into tubes containing EDTA 1 hour before dosing on day 10, on day 11 (24 hours after the first dose), on day 15 (24 hours after the third dose), and by cardiac puncture in day 17 and day 24. Uncoagulated red blood cells were lysed twice in red blood cell lysis buffer (eBioscience, catalog number 00-4300-54) according to the manufacturer's instructions. Cells were stained for flow cytometry using either dye 1 (CD4-E450 (clone GK1.5), Ki67-FITC (clone SolA15), Foxp3-PE (clone FJK-16s), CD69-PECy5 (clone H1.2F3), CD3 -PECy7 (clone 145-2C11), CD8-APC (clone 53-6.7), fixable viability dye 780, all supplied by eBioscience; and CD45-V500 (clone 30-F11), supplied by BD Bioscience), or dye 2 ( CD49b-E450 (clone DX5), F4/80-PE (clone 6F12), CD69-PECy5 (clone H1.2F3), CD19-PECy7 (clone 1D3), CD3-APC (clone 145-2C11) and fixative dye for evaluation viability 780, all supplied by eBioscience; CD45-V500 (clone 30-F11), supplied by BD Bioscience; and anti-hFc-488 (polyclonal), supplied by Jackson ImmunoResearch) in the presence of Fc block (eBioscience, catalog number 14-0161-86). at 1:100). Cells were then washed once with PBS, and Dye 2-stained samples were resuspended in 200 μl of PBS and analyzed on a FACS Canto II. For samples stained with dye 1, cells were initially stained with 100 μl of antibody mixture 1 (all except anti-Ki67 and anti-Foxp3) for 30 min at 4°C. Cells were then fixed and permeabilized using the eBioscience Foxp3 staining kit (eBioscience, cat. no. 00-5523-00) according to the manufacturer's instructions. In general, 200 μl of fixation solution was added to each well and left overnight in the dark at 4°C. Cells were then washed in 200 μl of permeabilization buffer. Cells were then centrifuged again and resuspended in 100 μl of permeabilization buffer with anti-Ki67 and anti-Foxp3 in the presence of Fc block (all diluted 1:100) and incubated for 30 min in the dark at 4°C. Cells were then washed once with permeabilization buffer and resuspended in 200 μl PBS. The cells were then analyzed on a BD FACS CantoII cytometer. Data were analyzed using FlowJoX, Excell and GraphPad Prism. T cell activation and proliferation observed over time was determined for all T cells as well as CD4 ⁺ and CD8 ⁺ subsets.

This experiment showed that mAb ² to OX40/CD137 had an effect on circulating T cells, increasing the frequency of activated T cells (CD45 ⁺ CD3 ⁺ CD69 ⁺ ) and CD4 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD4 ⁺ CD69 ⁺ ), as well as proliferating T cells (CD45 ⁺ CD3 ⁺ Ki67 ⁺ ), CD4 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD4 ⁺ Ki67 ⁺ ) and CD8 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ Ki67 ⁺ ) compared to all control groups, as well as increasing the frequency of activated CD8 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ CD69 ⁺ ) compared to treatment with either the anti-OX40 control, the anti-CD137 control alone, or the isotype control. A similar increase in the frequency of activated CD8 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ CD69 ⁺ ) was observed for the control group receiving a combination of control mAbs to OX40 and to CD137. These results are consistent with those observed in vitro , in which anti-OX40/CD137 mAb ² also showed an increase in T cell activation as measured by the production of IL-2, which is also known as a cytokine involved in T cell proliferation.

18.2 Comparison of the pharmacodynamic response of Mab ² to OX40/CD137 and its constituents Fcab and Fab

The peripheral pharmacodynamic response of Mab ² to mouse OX40/CD137 (FS20m-232-91AA/Lob12.3) was compared with that of its constituent parts, in particular, Fcab FS20m-232-91AA in the format of a mock (4420) MAT ² (FS20m-232- 91AA/4420) and a monospecific mouse mAb to CD137 without Fcab (G1AA/Lob12.3) as single agents or in combination, or isotype control (G1AA/4420) in the CT26 tumor model.

In a similar study described in Example 17.2 , on day 16 after inoculation of CT26 cells, blood samples were taken from the tail veins of 10 mice per group and collected in tubes containing EDTA. Following the same methods as described in Example 18.1 , red blood cells were lysed, the remaining cells were then stained with viability dye followed by surface staining with the reagents listed in Example 22.2.2 (except for the use of the mouse anti-CD4 antibody clone GK1 in this study .5 (BD Bioscience, catalog number 563790) instead of the anti-CD4 antibody clone RM4-5), excluding the anti-Ki67 antibody and the anti-Foxp3 antibody, in the presence of the Fc block. Cells were then fixed and permeabilized overnight using the eBioscience Foxp3 staining kit (eBioscience) according to the manufacturer's instructions. The cells were then intracellularly stained with anti-Ki67 antibody and anti-Foxp3 antibody. After washing, the cells were then analyzed using a BD Fortessa flow cytometer. Data analysis was performed using FlowJo, Excel, and GraphPad Prism 7 software.

FS20m-232-91AA/Lob12.3 was found to significantly increase the proportions of Ki67 ⁺ CD4 ⁺ effector (% of total CD4 ⁺ Foxp3 ^- cells) and Ki67 ⁺ CD8 ⁺ peripheral T cells (% of total CD8 ⁺ cells) in the blood compared to isotype control treatment. Murine anti-CD137 mAb and mock mAb ² FS20m-232-91AA/4420, either as single agents or in combination, were also able to induce significant increases in the levels of proliferating Ki67 ⁺ CD4 ⁺ effector T cells and Ki67 ⁺ CD8 ⁺ T cells compared with mice treated with isotype control. However, the increase in levels of proliferating Ki67 ⁺ CD8 ⁺ T cells following FS20m-232-91AA/Lob12.3 dosing was significantly greater than that observed for mouse anti-CD137 mAb alone, mock mAb ² FS20m-232-91AA/4420 alone, or their combinations.

In summary, these results demonstrate that mAb ² FS20m-232-91AA/Lob12.3 was able to induce an enhanced peripheral pharmacodynamic response in terms of increasing the frequency of proliferating Ki67 ⁺ CD8 ⁺ T cells compared to the combination of its Fcab and Fab moieties or either moiety partly separately.

Example 19 - Mechanism of action of MAT ² to OX40/CD137 in the CT26 syngeneic tumor model

The CT26 syngeneic tumor model was used to determine the mechanism of action (MOA) of the antitumor activity of mouse anti-OX40/CD137 mAb ² in vivo . The syngeneic CT26 tumor model has previously been shown to be sensitive to agonistic anti-OX40 and anti-CD137 antibodies (Sadun et al. , 2008), and tumor infiltrating lymphocytes (TILs) isolated from CT26 tumors are expected to express both OX40 and CD137 . The antibodies tested are detailed in Table 30 .

Table 30. Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No 115 116 G1/OX86 mOX40 Absent hIgG1 No 159 156 G1/Lob12.3 mCD137 Absent hIgG1 No University of Southampton G1AA/OX86 mOX40 Absent hIgG1 Yes 155 156 G1AA/Lob12.3 mCD137 Absent hIgG1 Yes The receipt is described above in Example 9.2 FS20m-232-91/Lob12.3 mCD137 mOX40 hIgG1 No FS20m-232-91AA/Lob12.3 mCD137 mOX40 hIgG1 Yes

The ability of mAb ² , with or without the LALA mutation (FS20m-232-91AA/Lob12.3 and FS20m-232-91/Lob12.3, respectively), to activate and induce proliferation of T cells in the blood and tumor was compared with mAb control isotype G1/4420 (anti-FITC), mAb G1/OX86 (control against OX40 without LALA mutation) or G1/Lob12.3 (control against CD137 without LALA mutation) as single agents, combination G1/OX86 plus G1/Lob12.3 or a combination of G1AA/OX86 (mAb to OX40 with the LALA mutation) plus G1AA/Lob12.3 (mAb to CD137 with the LALA mutation).

Female BALB/c (Charles River) mice, 8-10 weeks old and weighing approximately 20 g each, were allowed a rest period of one week before the start of the study. All animals were microchipped and assigned a unique identifier. There were 5 mice in each cohort. The colon carcinoma cell line CT26 (ATCC, CRL-2638) was initially expanded, maintained, and then prescreened at IDEXX Bioresearch for pathogen detection using the IMPACT I protocol, and the line was shown to be pathogen-free. CT26 cells (approximately 3-5 x 10 ⁶ ) were thawed from -150°C storage and added to 20 ml DMEM (Gibco, 61965-026) with 10% FST (Gibco, 10270-106) in a T175 tissue culture flask. Mice were anesthetized using isoflurane (Abbott Laboratories) and each animal received 1 x 10 ⁶ cells administered by subcutaneous injection into the left flank. At day 10 after tumor cell inoculation, the health status of the mice was monitored, tumors were measured using calipers, and mice were randomized into study cohorts based on tumor volume. Any mice that did not have tumors at this point were excluded from the study.

Antibodies administered by injection were analyzed within 24 hours prior to injection using SEC-HPLC profiling and checked for contaminants. Antibodies were diluted to a final concentration of 0.1 mg/ml in PBS and administered by injection at 200 μl/mouse to produce a final dose of 1 mg/kg for a 20 g mouse. Antibodies were administered to mice by intraperitoneal (IP) injection on days 10 , 12 and 14 after tumor inoculation. Tumor volume measurements were taken three times a week using calipers to determine the longest axis and shortest axis of the tumor. Seven days after the third dose (day 21 after tumor inoculation), mice were sacrificed, tumors were isolated by dissection, and blood was collected by cardiac puncture.

Tumors were dissociated using a tumor dissociation kit, mouse (Miltenyi 130-096-730), according to the manufacturer's instructions. In general terms, the enzyme mixture was prepared by adding 2.35 ml RPMI 1640, 100 µl enzyme D, 50 µl enzyme R and 12.5 µl enzyme A to the tumors, and each tumor was placed in a gentleMACS™ C tube, this tube was placed on a dissociator gentleMACS™ and ran the program m_TDK_1, and then incubated for 1 hour at 37°C with shaking (200 rpm). The resulting cell suspension was passed through a sieve using a 70 μm cell sieve (Corning, part number 352350), centrifuged (10 minutes at 1500 rpm), washed once in PBS, and resuspended in 5 ml of PBS.

Blood was collected by cardiac puncture into tubes containing EDTA. Red blood cells from uncoagulated blood were lysed twice in red blood cell lysis buffer (eBioscience, catalog number 00-4300-54) according to the manufacturer's instructions.

Cells isolated from tumors and blood were stained for flow cytometry using the following panel of antibodies and reagents (Dye 1): CD4-E450 (clone GK1.1), Ki67-FITC (clone SolA15), Foxp3-PE (FJK-16s) , CD69-PECy5 (clone H1.2F3), CD3-PECy7 (clone 145-2C11), CD8-APC (clone 53-6.7), fixable viability dye 780 and Fc block (clone 93), all supplied by eBioscience; and CD45-V500 (clone 30-F11) supplied by BD Bioscience. Cells were washed in PBS and then incubated with 100 μl of antibody mixture 1 (all except anti-Ki67 and anti-Foxp3) for 30 minutes at 4°C. Cells were then washed with PBS and then fixed and permeabilized using the eBioscience Foxp3 staining kit (eBioscience, cat. no. 00-5523-00) according to the manufacturer's instructions. In general, 200 μl of fixation solution was added to each well and left overnight in the dark at 4°C. Cells were then washed in 200 μl of permeabilization buffer. Cells were then centrifuged again and resuspended in 100 μl of permeabilization buffer with anti-Ki67 and anti-Foxp3 in the presence of Fc block (all diluted 1:100) and incubated for 30 min in the dark at 4°C. The cells were then washed once with permeabilization buffer and resuspended in 200 μl of PBS. The cells were then analyzed on a BD FACS CantoII cytometer.

Data were analyzed using FlowJoX, Excell and GraphPad Prism. Statistical analysis for group comparisons was performed using one-way analysis of variance followed by Tukey's test for multiple comparisons of each pair using the GraphPad Prism software package.

The frequency of T cells (CD45 ⁺ CD3 ⁺ ), proliferating T cells (CD45 ⁺ CD3 ⁺ Ki67 ⁺ ) and regulatory T cells (CD45 ⁺ CD3 ⁺ CD4 ⁺ Foxp3 + ) in the blood or tumors of mice inoculated with CT26 cells was determined. after treatment with MAT ² to OX40/CD137 or controls. MAT ² FS20m-232-91AA/Lob12.3 showed a statistically significant increase in proliferating T cells, as well as an increase in Tregs in the blood compared to the isotype control (G1/4420). MAb ² FS20m-232-91AA/Lob12.3 generally increased the frequency of T cells in the tumor.

A statistically significant decrease in tumor Treg levels was observed in mice treated with anti-CD137 G1/Lob12.3 and the combination of this anti-CD137 antibody with anti-OX40 G1/OX86 compared with isotype control treatment. However, when the LALA mutation was introduced into the anti-OX40 antibody and anti-CD137 antibody, treatment with a combination of these antibodies (G1AA/OX86 plus G1AA/Lob12.3) no longer reduced Treg levels in the tumors. MAT ² to OX40/CD137 containing the LALA mutation (FS20m-232-91AA/Lob12.3) did not reduce Treg levels, while the wild-type human IgG1 variant MAT ² to OX40/CD137 without the LALA mutation (FS20m-232- 91/Lob12.3) did show a statistically significant decrease in Treg levels in the tumor.

These data demonstrate that introduction of the LALA mutation into human IgG1 eliminates the ability of anti-OX40/CD137 mAb ² to deplete Tregs and, therefore, that the antitumor activity observed with the LALA variant of anti-OX40/CD137 mAb ² in human IgG1 format (FS20m-232- 91AA/Lob12.3), is independent of Treg depletion. In addition, MAb ² FS20m-232-91AA/Lob12.3 was found to induce proliferation of T cells in the periphery at the assessed time point, which is expected to expand the pool of T cells that induce an antitumor immune response. These data suggest that LALA-containing anti-OX40/CD137 mAb ² in human IgG1 format may have antitumor activity in a variety of cancers in the absence of Mab ² binding to Fcγ receptors, which may or may not be abundant in the tumor.

Example 20 - MAT activity ² to OX40/CD137 mouse in the B16-F10 syngeneic tumor model

The B16-F10 syngeneic tumor model was used to test the antitumor activity of mouse mAb ² against OX40/CD137 (FS20m-232-91AA/Lob12.3) in vivo . Antibody G1/4420 was used as a control in the study. The B16-F10 syngeneic tumor model has not previously been shown to be sensitive to agonistic antibodies to OX40 or CD137 (Hirschhorn-Cymerman et al. , 2009; Wilcox et al. , 2002). However, tumor infiltrating lymphocytes (TILs) isolated from B16-F10 tumors are expected to express both OX40 and CD137.

Female C57BL/6 mice (Charles River), 8-10 weeks old and weighing approximately 20 g each, were acclimatized for one week before the start of the study. All animals were microchipped and assigned a unique identifier. There were 10 mice in each cohort. The colon carcinoma cell line B16-F10 (ATCC, catalog number CRL-6475) was initially expanded, maintained, and then prescreened at IDEXX Bioresearch for pathogen detection using the IMPACT I protocol, and the line was shown to be pathogen-free.

B16-F10 cells were thawed from storage at -150°C and added to 20 ml DMEM (Gibco, 61965-026) with 10% FST (Gibco, 10270-106) in a T175 tissue culture flask. Each animal received 1×10 ⁶ cells by subcutaneous injection into the left flank. 7-8 days after inoculation of tumor cells, mice that did not have tumors at this point were excluded from the study. Antibodies were assayed and checked for contaminants as previously described before injection at a final concentration of 0.1 mg/ml in PBS in a volume of 200 μl/mouse, resulting in a final dose of 1 mg/kg for a 20 g mouse. Each mouse received antibodies by intraperitoneal (i.p.) injection on days 8, 10, and 12 after tumor inoculation. Tumor volumes were determined by measuring using calipers (as described in Example 17 ) and dosing of any drug prescribed for the day in question was performed.

Mice were sacrificed when humane endpoints based on tumor volume and condition were reached. Statistical analysis of tumor growth was performed using the mixed model statistical analysis described in Example 17 . The results of the study are shown in Figure 11 .

Anti-OX40/CD137 mAb ² (FS20m-232-91AA/Lob12.3) exhibited significant antitumor activity compared to control animals injected with control antibody (G1/4420). This result was unexpected as this model has previously been shown to be insensitive to OX40 or CD137 stimulation (Hirschhorn-Cymerman et al. , 2009; Wilcox et al. , 2002). It is important to note that the activity observed for mAb ² to OX40/CD137 was in the presence of the LALA mutation and was therefore independent of depletion of tumor Tregs. This suggests that MOA mAb ² to OX40/CD137 results in antitumor activity in a variety of syngeneic tumor models, even those that have lower levels of immune infiltrate, such as B16-F10.

Example 21 - Analytical characterization and preliminary assessment of MAT stability ² to OX40/CD137

21.1 Expression, purification and analytical characterization of MAT ²

MAT ² FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA/FS30-10-16 and FS20-22-49AA/FS30-35-14 was prepared at laboratory scale and characterized using standard analytical methods using SE-HPLC and SDS-PAGE.

DNA sequences encoding MAT ² were transiently expressed in HEK293-6E (National Research Council of Canada). After 5 days, culture fluids were collected and purified on MabSelect prefilled Protein A columns using an AKTAxpress instrument (both from GE Healthcare). Columns were equilibrated in 50 mM Tris-HCl, 250 mM NaCl, pH=7.0, followed by loading with the collected culture fluid. The resin was then washed with 50 mM Tris-HCl, 250 mM NaCl at pH=7.0, and then eluted with MAT ² using buffer at pH=3.5. MAT ² buffer was replaced with formulation prebuffer using PD-10 desalting columns (GE Healthcare, product number 17085101).

SE-HPLC was performed on an Agilent 1100 series HPLC system (Agilent) equipped with a 4.6 mm × 30.0 cm i.d. TSK-GEL SUPERSW3000 column (Tosoh Bioscience), using 20 mM sodium phosphate, 200 mM sodium chloride, pH=6.8 as mobile phase. Quantification of monomer percentage was performed using Chemstation software (Agilent). The results of the SE-HPLC analysis are summarized in Table 31 .

Table 31 . Analytical characterization using SE-HPLC

MAT ² Monomer by SE-HPLC (%) FS20-22-49AA/FS30-5-37 98.4% FS20-22-49AA/FS30-10-3 97.4% FS20-22-49AA/FS30-10-12 95.9% FS20-22-49AA/FS30-10-16 97.5% FS20-22-49AA/FS30-35-14 97.3%

SDS-PAGE analysis was performed using NuPAGE® Novex® 4-12% Bis-Tris protein gels and 1× MOPS separation buffer (Thermo Fisher Scientific), essentially according to the manufacturer's instructions. For non-reducing SDS-PAGE, samples were exposed to the alkylating reagent N-ethylmaleimide (Sigma-Aldrich) before the denaturation step, and 2-mercaptoethanol was omitted from the denaturation mixture. Protein bands were visualized using Coomassie InstantBlue (Expedeon).

All five MAbs ² showed favorable analytical performance after purification on Protein A with monomer purity above 95% when determined by SE-HPLC. SDS-PAGE analysis revealed protein band profiles typical of recombinant IgG1. Thus, under non-reducing conditions, one band migrated to the region corresponding to the expected molecular weight, and under reducing conditions, two bands migrated close to the 51 kDa and 28 kDa molecular weight markers, corresponding to the heavy chain and light chain, respectively. No fragmentation was observed (data not shown).

21.2 Preliminary assessment of the stability of MAT ² to OX40/CD137

A preliminary assessment of the stability of MAT ² FS20-22-49AA/FS30-5-37, FS20-22-49AA/FS30-10-3, FS20-22-49AA/FS30-10-12, FS20-22-49AA/FS30- was performed 10-16 and FS20-22-49AA/FS30-35-14. Before MAT ² was subjected to preliminary stability assessment, it was further purified by size exclusion chromatography (SEC) using a Superdex HiLoad 26/600 200 pg column (GE Healthcare) equilibrated with formulation pre-buffer. Samples for stability assessment were stored at 5°C and analyzed after 2 and 4 weeks using standard analytical methods using SE-HPLC and capillary electrophoresis with sodium dodecyl sulfate (CE-SDS).

SE-HPLC was performed on an Agilent 1100 series HPLC system (Agilent) equipped with a TSK-GEL SUPERSW3000 4.6 mm × 30.0 cm i.d. column (Tosoh Bioscience), using 20 mM sodium phosphate, 200 mM sodium chloride, pH =6.8 as the mobile phase. Data acquisition and quantification of monomer content were performed using Chemstation software (Agilent). The results are summarized in Table 32 .

After storage at 5°C for 4 weeks, the monomer content determined by SE-HPLC for all MAbs ² tested remained comparable (within ±0.9%) to the starting material (T = 0). Thus, all MAT ² tested exhibited a favorable stability profile.

Table 32: Stability Analysis by SE-HPLC

MAT ² Monomer (%)
T=0 Monomer (%)
T= 2 weeks at 5°C Monomer (%)
T= 4 weeks at 5°C FS20-22-49AA/ FS30-5-37 100.0 99.2 99.1 FS20-22-49AA/FS30-10-3 100.0 100.0 99.9 FS20-22-49AA/FS30-10-12 100.0 100.0 100.0 FS20-22-49AA/FS30-10-16 100.0 100.0 100.0 FS20-22-49AA/FS30-35-14 99.5 99.2 99.3

CE-SDS analysis was performed on a Bioanalyzer 2100 capillary electrophoresis system (Agilent) according to the manufacturer's recommendations. To create reducing conditions in CE-SDS, DTT was added and samples were denatured at 70°C for 5 min. Data collection and quantification of percentages of heavy chain and light chain material were performed using Expert 2100 software (Agilent). Percent purity was calculated as the sum of the percentage of heavy chain material and the percentage of light chain material. The results of the analysis are summarized in Table 33 .

The purity of all MAbs ² tested, determined as the sum of the percentage of heavy chain material and light chain material by CE-SDS under reducing conditions, also remained comparable (within ± 1.0%) to the starting material. Thus, all MAT ² tested also showed favorable stability.

Table 33. Stability analysis using CE-SDS

MAT ² Purity (%)
T=0 Purity (%)
T= 2 weeks at 5°C Purity (%)
T= 4 weeks at 5°C FS20-22-49AA/ FS30-5-37 99.6 99.7 99.1 FS20-22-49AA/FS30-10-3 99.5 99.6 99.5 FS20-22-49AA/FS30-10-12 98.8 99.2 99.5 FS20-22-49AA/FS30-10-16 99.5 99.1 98.5 FS20-22-49AA/FS30-35-14 99.6 99.0 100.0

Example 22 - MAT activity ² anti-OX40/CD137 in combination with anti-PD-1 antibody or anti-PD-L1 antibody

Expression of PD-L1 on antigen-presenting cells (eg, dendritic cells, macrophages, B cells), tumor cells, and cells in the tumor microenvironment is known to inhibit T cell activation, proliferation, and effector and cytotoxic functions through interaction with PD-L1. 1. Blocking this interaction using anti-PD-1 or anti-PD-L1 monoclonal antibodies has been shown to improve survival rates in patients with several types of cancer.

However, in some tumors, anti-PD-L1 and anti-PD-1 antibodies have little or no effect. The present inventors tested the combination of anti-OX40/CD137 mAb ² with anti-PD-L1 antibody or anti-PD-1 antibody in in vitro and in vivo studies to determine whether the use of the combination could lead to an improved effect compared to the use of anti-OX40 mAb ² /CD137, anti-PD-L1 antibodies, or anti-PD-1 antibodies alone.

22.1 Activity of mAb ² against OX40/CD137 in combination with PD-1 or PD-L1 blockade in the staphylococcal enterotoxin A (SEA) assay

The activity of mAb ² to OX40/CD137 was tested in a T cell activation assay using staphylococcal enterotoxin A (SEA) superantigen as the first signal, as described in Example 12 above. To test the effect of anti-OX40/CD137 mAb ² on T cell stimulating activity in combination with blocking the interaction between PD-1 and PD-L1, anti-PD-1 or anti-PD-L1 blocking antibodies were combined with anti-OX40/CD137 mAb ² in an assay with SEA.

Antibodies and MAbs ² used in the SEA assay are listed in Table 34 below. Tested G1/4420 (anti-FITC) in combination with MAT ² FS20-22-49AA/FS30-10-16, G1AA/S1 (anti-PD-L1), G1AA/5C4 (anti-PD-1) individually or in combination with MAT ² FS20-22-49AA/FS30-10-16. Interleukin-2 (IL-2) production was used as a measure of T cell activation.

Table 34: Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No 115 116 FS20-22-49AA/
FS30-10-16 hCD137 hOX40 hIgG1 Yes 95 14 G1AA/S1 PD-L1 Absent hIgG1 Yes 162 163 G1AA/5C4 PD-1 Absent hIgG1 Yes 160 161

The sequences of the variable domains of antibodies 5C4 and YW243.55.S1 (S1) are also disclosed in US 8008449 B2 and US 2013/0045202 A1, respectively.

PBMCs were isolated and SEA analysis was performed essentially as described in Example 12.1 above. G1/4420 was used as an isotype control and no cross-linking agents were used in the assays.

The activity of mAb ² to OX40/CD137 (FS20-22-49AA/FS30-10-16) in combination with either an anti-PD-L1 antibody (G1AA/S1) or an anti-PD-1 antibody (G1AA/5C4) was compared with the activity MAT ² FS20-22-49AA/FS30-10-16 plus isotype control (G1/4420) or with anti-PD-L1 antibody activity (G1AA/S1) or anti-PD-1 antibody (G1AA/5C4), or isotype control ( G1/4420) separately. The EC ₅₀ values and maximum IL-2 release response observed in the SEA assay are shown in Table 35 . Figure 12A and B show IL-2 release plots for the SEA assay.

Table 35: SEA assay using mAbs ² targeting OX40 and CD137 in combination with antibodies blocking the interaction between PD-1 or PD-L1

MAT/MAT ² EC ₅₀ (nM) Max. answer (nM) 95% CI (hIL-2 pg/ml) 95% CI G1/4420 No activity detected No activity detected No activity detected No activity detected FS20-22-49AA/FS30-10-16 + G1/4420 0.1483 0.04556-0.4517 2741 2394-3103 G1AA/5C4 No activity detected No activity detected No activity detected No activity detected G1AA/S1 No activity detected No activity detected No activity detected No activity detected FS20-22-49AA/FS30-10-16 + G1AA/5C4 0.5939 0.1964-1.732 5326 4599-6116 FS20-22-49AA/FS30-10-16 + G1AA/S1 0.2399 0.1478-0.3970 5325 5022-5640

NAD = no activity detected

As expected, no activity was detected using the isotype control (G1/4420). Likewise, blocking the interaction between PD-1 and PD-L1 alone was not active in this assay. However, combining stimulation of OX40 and CD137 receptors (using MAb ² to OX40/CD137) with blockade of the interaction between PD-1 and PD-L1 (using either an anti-PD-L1 antibody or an anti-PD-1 antibody) resulted in an increase in maximum activity T cells, as measured by maximal IL-2 production, greater than that observed with anti-OX40/CD137 mAb ² alone. The increase in maximal T cell activity observed when anti-OX40/CD137 mAb ² was combined with anti-PD-L1 or anti-PD-1 was similar.

22.2 Antitumor activity and pharmacodynamic response of mouse OX40/CD137 mAb ² and PD-1 antagonist in the CT26 tumor mouse model

The CT26 tumor mouse model was used to establish the antitumor activity and pharmacodynamic response of the combination of FS20m-232-91AA/Lob12.3 and an anti-PD-1 antagonist antibody (mouse IgG1 clone RMP1-14) compared with either agent alone.

22.2.1 Evaluation of antitumor activity

Following the same protocol as described in Example 17 , female BALB/c mice (Charles River), 8-10 weeks old and weighing approximately 20 g, were prepared for the start of the study and inoculated with the CT26 colon carcinoma cell line. 10 days after tumor cell inoculation, tumors were measured, any mice that did not have tumors were excluded from the study, and the remaining mice were randomized into 4 treatment groups ( Table 36 ) of 15 animals per group. Animals were administered by intraperitoneal injection: (1) a combination of isotype control antibodies 1 mg/kg G1AA/4420 and 10 mg/kg mIgG1/4420 (Absolute Antibodies, clone 4420, catalog number Ab00102-1.1), (2) 10 mg/kg kg anti-mouse PD-1 antibody (Absolute Antibodies, mouse IgG1 clone RMP1-14, catalog number Ab00813-1.1), (3) 1 mg/kg MAT ² FS20m-232-91AA/Lob12.3, or (4) 10 mg/kg antibody to mouse PD-1 and 1 mg/kg MAb ² FS20m-232-91AA/Lob12.3 in PBS. Animals received intraperitoneal (IP) injections of G1AA/4420 or FS20m-232-91AA/Lob12.3 once every 2 days for a total of 3 doses, starting on day 10 after tumor inoculation. mIgG1/4420 or mouse anti-PD-1 was dosed IP once every 4 days for a total of 4 doses starting at day 10 after tumor inoculation. Tumor volumes were determined using caliper measurements (as described in Example 17 ). The study was stopped 60 days after tumor cell inoculation, and animals were removed from the study when humane endpoints based on tumor volume and condition were met. Treatment groups, molecules tested, doses, and dosing schedule are summarized in Table 36 .

Table 36. Summary of treatment groups and molecules tested

Group Group name Injected MAT and/or MAT ² Dose (mg/kg) Dosage schedule 1 Isotype controls G1AA/4420, mIgG1/4420 1, 10 Q2D,
Q4D 2 Antibody to PD-1 Anti-mouse PD-1 antibody mIgG1 (RMP1-14) 10 Q4D 3 FS20m-232-91AA/Lob12.3 FS20m-232-91AA/Lob12.3 1 Q2D 4 FS20m-232-91AA/Lob12.3 + anti-PD-1 antibody FS20m-232-91AA/Lob12.3,
Anti-mouse PD-1 antibody mIgG1 1, 10 Q2D,
Q4D

As shown in Figure 13A-D , the combination of PD-1 antagonist antibody and 1 mg/kg FS20m-232-91AA/Lob12.3 resulted in the highest proportion of animals, 7 of 15 (47%), responding with complete regression tumor (defined as tumor volume ≤62.5 mm ³ ) at the end of the study ( Figure 13D ). Isotype control antibodies ( Figure 13A ), anti-PD-1 antibody ( Figure 13B ) as a single agent, and 1 mg/kg FS20m-232-91AA/Lob12.3 ( Figure 13C ) showed 0%, 0%, and 7% regression tumors at the end of the study, respectively.

Survival analysis showed that the combination of FS20m-232-91AA/Lob12.3 with anti-PD-1 antibody resulted in a statistically significant survival advantage compared with isotype control antibodies (log-rank test (Mantel-Cox), p<0. 0001) ( Figure 13E ). No significant differences in survival were observed between treatments with either agent alone compared with isotype control antibodies. These results demonstrate that in this model, blockade of the PD-1/PD-L1 inhibitory pathway with an antagonist and dual agonist action on OX40 and CD137 using anti-OX40/CD137 mAb ² was able to enhance antitumor activity and provide a survival benefit compared with by separate agents.

22.2.2 Assessment of peripheral pharmacodynamic response

The study described in Example 22.2.1 also examined the ability of a PD-1 antagonist to modulate the pharmacodynamic response to FS20m-232-91AA/Lob12.3 and compared it to single agent treatment. 6 days after the start of dosing (16 days after tumor cell inoculation), blood from the tail veins of 6 randomly selected CT26 tumor-bearing mice from treatment groups 1, 2, 3 and 5 ( Table 36 ) was collected into tubes containing EDTA. Red blood cells from uncoagulated blood were lysed twice in red blood cell lysis buffer (Miltenyi Biotech, #130-094-183) according to the manufacturer's instructions. Cells were stained for flow cytometry analysis using the reagents CD4-BUV395 (clone RM4-5), CD8-BUV737 (clone 53-6.7), CD44-BV510 (clone IM7) and CD3e-BV786 (clone 145-2C11), all supplied BD Bioscience; CD69-FITC (clone H1.2F3), NKp46-PE (clone 29A1.4), PD-1-APC (clone J43), CD45-Alexa700 (clone 30-F11) and viability fixative dye 780, all supplied by eBioscience ; and CD62L-BV421 (clone MEL-14), supplied by Biolegend, in the presence of Fc block (eBioscience, catalog number 14-0161-86 at 1:50) for 30 minutes at 4°C. Cells were then fixed and permeabilized overnight using the eBioscience Foxp3 staining kit (eBioscience, cat. no. 00-5523-00) according to the manufacturer's instructions. Cells were resuspended in 100 μl permeabilization buffer with anti-Ki67 antibody and anti-Foxp3 antibody (Ki67-PE-Cy7 (clone SolA15) and Foxp3-PerCP-Cy5.5 (clone FJK-16s), both supplied by eBioscience) and incubated for 30 minutes at room temperature in the dark. Cells were then washed twice with permeabilization buffer and resuspended in PBS + 0.5% BSA. The cells were then analyzed on a BD Fortessa flow cytometer. Data analysis was performed using FlowJo, Excel, and GraphPad Prism 7 software.

Frequencies of proliferating Ki67 ⁺ CD4 ⁺ T cells (of total CD45 ⁺ CD3 ⁺ CD4 ⁺ ), Ki67 ⁺ CD8 ⁺ T cells (of total CD45 ⁺ CD3 ⁺ CD8 ⁺ ), and Ki67 ⁺ NKp46 ⁺ NK cells (of total CD45 ⁺ CD3 ⁻ NKp46 ⁺ ) counts were determined by flow cytometry analysis as described above. Compared to isotype controls, FS20m-232-91AA/Lob12.3 induced a statistically significant increase in proliferating Ki67 ⁺ CD4 ⁺ and Ki67 ⁺ CD8 ⁺ T cells, confirming previous results ( Example 18 ), and proliferating Ki67 ⁺ NK cells (non-parametric Mann-Whitney test for pairwise comparison; p≤0.005 for all three immune cell populations). The PD-1 antagonist antibody as a single agent had no detectable effect on the three immune cell populations compared with isotype controls. The combination of 1 mg/kg FS20m-232-91AA/Lob12.3 and anti-PD-1 antibody resulted in statistically significantly higher levels of proliferating Ki67 ⁺ CD4 ⁺ T cells, Ki67 ⁺ CD8 ⁺ T cells and Ki67 ⁺ NKp46 ⁺ NK- cells compared to both single agent and isotype controls (p≤0.005 for all statistically significant comparisons except the effect of the combination on levels of proliferating Ki67 ⁺ CD4 ⁺ T cells compared to FS20m-232-91AA/Lob12.3 by separately, for which p≤0.05).

Effects of anti-PD-1 antibody, MAT ² FS20m-232-91AA/Lob12.3 as individual agents and a combination of anti-PD-1 antibody and FS20m-232-91AA/Lob12.3 on PD-1-expressing peripheral blood T cells (CD4 ⁺ and CD8 ⁺ T cells) and NK cells were also determined by flow cytometry analysis as described above. Anti-PD-1 and FS20m-232-91AA/Lob12.3 as single agents increased the proportion of PD-1-expressing CD4 ⁺ and CD8 ⁺ T cells compared to isotype control with a higher median frequency of PD-1 ⁺ cells after treatment with FS20m-232-91AA/Lob12.3 compared with anti-PD-1 antibody alone. FS20m-232-91AA/Lob12.3 alone increased the frequency of PD-1 ⁺ NK cells compared to isotype controls. The combination resulted in statistically significantly higher levels of PD-1-expressing CD4 ⁺ and CD8 ⁺ T cells (but not NK cells) compared with both the single agent and isotype controls (nonparametric Mann-Whitney test for pairwise comparison; p ≤0.005 for all statistically significant comparisons, except for the effect of the combination on the frequency of PD-1-expressing CD4 ⁺ T cells compared with FS20m-232-91AA/Lob12.3 alone, for which p≤0.05).

In accordance with the results of the assessment of antitumor activity, simultaneous blockade of the PD-1/PD-L1 inhibitory pathway with an antagonist and dual agonist action on OX40 and CD137 using MAb ² to OX40/CD137 led to increased pharmacodynamic modulation of proliferating T cells and NK cells , which supports the use of a combination approach to trigger antitumor immunity.

In summary, blocking the PD-1/PD-L1 axis with simultaneous agonistic effects on OX40 and CD137 results in increased effect compared to blocking PD-1/PD-L1 alone. In particular, the combination of an anti-PD-1 antibody or an anti-PD-L1 antibody with anti-OX40/CD137 mAb ² resulted in improved response compared with the response using either antibody alone. In the SEA assay described in Example 22.1 , none of the anti-PD-1 or anti-PD-L1 antibodies tested showed any activity compared to anti-OX40/CD137 mAb ² , which had an EC ₅₀ of 0.1474 nM. However, when anti-OX40/CD137 mAb ² was tested in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody, the EC ₅₀ values were 0.2373 nM and 0.5961 nM, respectively. In addition, the maximum IL-2 response induced by either combination was more than twice that of Mab ² to OX40/CD137 alone. These in vitro data demonstrate that in a system in which anti-PD-L1 antibody or anti-PD-1 antibody is not found to be active, combining either antibody with anti-OX40/CD137 mAb ² results in a significant improvement in activity.

This in vitro activity was further confirmed by in vivo testing of mouse anti-OX40/CD137 mAb ² , alone or in combination with anti-PD-1 antibody, in the CT26 tumor model as described in Example 22.2 . The results of this study showed that at the end of the study, more animals were tumor free in the group receiving the combination of anti-PD-1 antibody with anti-OX40/CD137 mAb ² , compared with anti-OX40/CD137 mAb ² or anti-PD-1 antibody alone (when no tumor was found in any of the animals). In addition, statistically significant survival benefits were also observed ( Figure 13E ), and pharmacodynamic modulation of proliferating T cells and NK cells was enhanced by combination treatment compared to both Mab ² and anti-PD-1 antibody alone.

Since anti-PD-1 or anti-PD-L1 antibody activity was not observed in either in vitro or in vivo studies, but significant improvements were observed when either was dosed with anti-OX40/CD137 mAb ² , this may indicate that Anti-OX40/CD137 mAb ² in combination with such an antibody will result in enhanced antitumor efficacy, and that such a combination may be suitable for the treatment of tumors that are refractory, e.g., refractory or resistant to anti-PD-1 or anti-PD-1 monotherapy L1 or recurred after it.

Example 23 - Dose-dependent antitumor activity of MAT ² to OX40/CD137 mice in the CT26 syngeneic tumor model and the formation of protective immunological memory against repeated stimulation by CT26 tumor cells

To evaluate the relationship between dose and antitumor activity of surrogate mAb ² to OX40/CD137 in the CT26 syngeneic colorectal tumor mouse model, five different dose levels ranging from 0.1 to 10 mg/kg were evaluated.

Following the same protocol as described in Example 17 , female BALB/c mice (Charles River), 8-10 weeks old and weighing approximately 20 g each, were injected subcutaneously with CT26 colon carcinoma cells into the left flank of each animal. Ten days after tumor cell inoculation, tumors were measured and animals without developing tumors were excluded from the study. The remaining mice were randomized into six experimental groups of 25 animals per group.

Isotype control antibody (G1AA/4420) and surrogate mAb ² to OX40/CD137 (FS20m-232-91AA/Lob12.3) were filtered and diluted in PBS before injection. Each animal was administered by intraperitoneal injection of diluted antibody in a volume of 200 μl per injection, resulting in a final dose of 10 mg/kg G1AA/4420 or 0.1, 0.3, 1, 3 or 10 mg/kg FS20m-232-91AA/Lob12 .3 per administration for a 20 g mouse. Injections were performed once every two days (Q2D) for a total of three doses starting at day 10 after tumor inoculation. Tumor volumes were determined using caliper measurements as described in Example 17 . The study was stopped 67 days after tumor cell inoculation, and animals were removed from the study when humane endpoints based on tumor volume and condition were met.

Tumor volumes over time for individual animals treated with either G1AA/4420 or FS20m-232-91AA/Lob12.3 at various dose levels are shown in Figure 14A . Dose levels of 0.3, 1, 3, or 10 mg/kg FS20m-232-91AA/Lob12.3 resulted in complete tumor regression (defined as ≤62.5 mm ³ at day 60) in 4% (1/25), 4% (1/25), 8% (2/25) and 4% (1/25) animals per group, respectively. None of the animals in the isotype control and 0.1 mg/kg surrogate MAT ² groups had complete tumor regression.

Pairwise comparisons of mean tumor growth rates between the FS20m-232-91AA/Lob12.3 and G1AA/4420 treated groups were performed using mixed model statistical analysis as described in Example 17 and for all dose levels tested (0.1, 0 .3, 1, 3 and 10 mg/kg) observed statistically significant differences (p<0.01) compared to the isotype control ( Table 37 ). FS20m-232-91AA/Lob12.3 reduced mean tumor growth rate (TGR) in a dose-dependent manner at doses ranging from 0.1 to 3 mg/kg (mean log(TGR) 0.255529 to 0.156767, respectively). The mean TGR at 3 mg/kg FS20m-232-91AA/Lob12.3 was not statistically different from that at the 1 mg/kg dose level (p = 0.18). However, increasing the dose level to 10 mg/kg resulted in a higher TGR compared with the 3 mg/kg dose group (p<0.001).

Table 37: Pairwise comparison of mean CT26 tumor growth rates using mixed model statistical analysis

Pairwise comparison of A with B Mean log(TGR) [lower, upper CI] P-value Bottom line A > or ≈ or < B (average log(TGR)) A B A B Isotype control FS20m-232-91AA/Lob12.3 0.1 mg/kg 0.314856 [0.288207, 0.339504] 0.255529 [0.227356, 0.283703] 3.63×10 ^-4 **** A>B Isotype control FS20m-232-91AA/Lob12.3 0.3 mg/kg 0.313856 [0.288207, 0.339504] 0.252407 [0.215534, 0.289281] 2.24×10 ^-7 **** A>B Isotype control FS20m-232-91AA/Lob12.3 1 mg/kg 0.313856 [0.288207, 0.339504] 0.219461 [0.186503, 0.252419] 1.94×10 ^-7 **** A>B Isotype control FS20m-232-91AA/Lob12.3 3 mg/kg 0.313856 [0.288207, 0.339504] 0.156767 [0.120418, 0.193116] 2.23×10 ^-14 **** A>B Isotype control FS20m-232-91AA/Lob12.3 10 mg/kg 0.313856 [0.288207, 0.339504]] 0.197003 [0.155898, 0.238107] 7.50×10 ^-21 **** A>B FS20m-232-91AA/Lob12.3 1 mg/kg FS20m-232-91AA/Lob12.3 3 mg/kg 0.219461 [0.186503, 0.252419] 0.156767 [0.120418, 0.193116] 1.83×10 ^-1 Unreliable A ≈ B FS20m-232-91AA/Lob12.3 3 mg/kg FS20m-232-91AA/Lob12.3 10 mg/kg 0.156767 [0.120418, 0.193116] 0.197003 [0.155898, 0.238107] 4.72×10 ^-7 **** A < B

Abbreviations: ns = not statistically significant; TGR = tumor growth rate

Note: For each pairwise comparison, at least one of the groups included in the calculation of p-values contains more than 50% of tumor growth rates not significantly following a lognormal distribution.

Survival analysis using the log-rank (Mantel-Cox) test showed that FS20m-232-91AA/Lob12.3 at all dose levels tested resulted in a statistically significant survival benefit compared to the isotype control ( Figure 14B ). Comparison of the 1 mg/kg and 3 mg/kg groups showed no statistical difference in survival.

In summary, the tumor volume and survival data shown in Figures 14A and B and Table 37 confirm the results of Example 17 that OX40/CD137 surrogate mAb ² can induce antitumor activity in vivo in the CT26 tumor mouse model. In addition, the observed antitumor activity increased in a dose-dependent manner from 0.1 mg/kg to 1 mg/kg and was maintained at the higher dose levels tested (3 mg/kg and 10 mg/kg).

To test whether surrogate mAb ² to OX40/CD137 could induce protective immunological memory against CT26 tumor cells, animals that had complete tumor regression (complete response animals) from the dose-ranging study of the present example described above were reinoculated subcutaneously with 1 ×10 ⁵ CT26 cells on day 84 after the first cell inoculation. Untreated tumor-free BALB/c mice were also inoculated with CT26 cells as a control group. Tumor volumes were monitored as described above. The study was stopped at day 137 after the first cell inoculation, and animals were removed from the study when humane endpoints based on tumor volume and condition were met. At the end of the study, 0% (0/4) of mice in the control group survived, while, in contrast, 100% (4/4) of the animals with a complete response survived. These results indicate that in a subset of mice, surrogate mAb ² to OX40/CD137 can induce complete tumor regression and the formation of protective immunological memory against rechallenge with CT26 cells.

Example 24 - Dose-dependent pharmacodynamic response of MAT ² to OX40/CD137 mice in the CT26 tumor mouse model

The relationship between dose levels, dosing frequency, and peripheral pharmacodynamic response of OX40/CD137 surrogate mAb ² (FS20m-232-91AA/Lob12.3) was assessed using the CT26 syngeneic colorectal tumor mouse model. Comparisons were made of single intraperitoneal (IP) injections of FS20m-232-91AA/Lob12.3 at different dose levels of 1, 3, 10 or 30 mg/kg or three IP injections of FS20m-232-91AA/Lob12.3 at 1 mg /kg administered once every 2 days (Q2D). The pharmacodynamic response of FS20m-232-91AA/Lob12.3, specifically the effect of surrogate mAb ² on circulating T cells, was assessed using flow cytometry analysis of immune cell subsets in the blood as described in Example 18 .

Following the same protocol as described in Example 17 , female BALB/c mice (Charles River), 8-10 weeks old and weighing approximately 20 g each, were injected subcutaneously with CT26 colon carcinoma cells into the left flank of each animal. Ten days after tumor cell inoculation, tumors were measured and animals without tumor development were excluded from the study. The remaining mice were randomized into six treatment groups of six animals per group.

Isotype control antibody (G1AA/4420) and FS20m-232-91AA/Lob12.3 were filtered and diluted in PBS before injection. Each animal was injected intraperitoneally with diluted antibody in a volume of 200 μl per injection, resulting in a final dose of 30 mg/kg G1AA/4420 or 1, 3, 10 or 30 mg/kg FS20m-232-91AA/Lob12.3 per injection for a 20 g mouse Animals received either a single IP injection of G1AA/4420 (at 30 mg/kg) or FS20m-232-91AA/Lob12.3 (at 1, 3, 10 or 30 mg/kg), or a total of 3 doses. FS20m-232-91AA/Lob12.3 (at 1 mg/kg per dose), administered once every two days (Q2D), starting on day 10 after tumor inoculation. Tumor volumes were determined using caliper measurements as described in Example 17 . Animals were excluded from the study six days after dosing began (16 days after cell inoculation).

Blood was collected into tubes containing EDTA by cardiac puncture. Red blood cells from uncoagulated blood were lysed twice in red blood cell lysis buffer (Miltenyi Biotech, #130-094-183) according to the manufacturer's instructions. Cells were stained for flow cytometry analysis using the reagents CD4-BUV395 (clone RM4-5), CD8-BUV737 (clone 53-6.7), CD44-BV510 (clone IM7) and CD3e-BV786 (clone 145-2C11), all supplied BD Bioscience); CD69-FITC (clone H1.2F3), NKp46-PE (clone 29A1.4), CD45-Alexa700, and viability fixative dye 780, all supplied by eBioscience; and CD62L-BV421 (clone MEL-14), supplied by Biolegend, in the presence of an Fc block (eBioscience, catalog number 14-0161-86). Cells were then fixed and permeabilized overnight using the eBioscience Foxp3 staining kit (eBioscience, cat. no. 00-5523-00) according to the manufacturer's instructions. Cells were resuspended in 100 μl of permeabilization buffer with anti-Gzmb antibody, anti-Ki67 antibody, and anti-Foxp3 antibody (Gzmb-AF647 (clone GB11) supplied by Biolegend, and Ki67-PE-Cy7 (clone SolA15) and Foxp3-PerCP-Cy5. 5 (clone FJK-16s), both supplied by eBioscience) and incubated for 30 min in the dark at room temperature. Cells were then washed twice with permeabilization buffer and resuspended in PBS plus 0.5% BSA. The cells were then analyzed on a BD Fortessa flow cytometer. Data analysis was performed using FlowJo, Excel, and GraphPad Prism 7 software.

The frequencies of Ki67 ⁺ CD8 ⁺ (of total CD8 ⁺ ) and Ki67 ⁺ CD4 ⁺ (of total CD4 ⁺ ) proliferating T cells in peripheral blood six days after the first dose were determined by flow cytometry analysis. A statistically significant increase in the frequencies of proliferating Ki67 ⁺ CD4 ⁺ T cells was observed at single doses of 1 and 10 mg/kg FS20m-232-91AA/Lob12.3 compared to the isotype control. A statistically significant increase in the frequencies of proliferating Ki67 ⁺ CD8 ⁺ T cells was observed at single doses of 1, 3 and 10 mg/kg FS20m-232-91AA/Lob12.3 compared to the isotype control.

Proliferating Ki67 ⁺ CD8 ⁺ T cells tended to peak at the 1 mg/kg single dose level, while proliferating Ki67 ⁺ CD4 ⁺ T cells tended to peak at the 1 and 10 mg/kg dose levels . Increasing the dose level to 30 mg/kg did not result in a significant effect on Ki67 ⁺ CD8 ⁺ and Ki67 ⁺ CD4 ⁺ T cells compared with isotype control. It should be noted that no apparent clinical observations or weight loss were observed at any dose level.

Comparison of the multiple-dose group (FS20m-232-91AA/Lob12.3 at 1 mg/kg Q2D, three doses) and the single-dose 1 mg/kg group did not reveal statistical significance in the levels of Ki67 ⁺ CD8 ⁺ and Ki67 ⁺ CD4 ⁺ T cells (unpaired Mann-Whitney test, p = 0.4848 and p = 0.0931, respectively). These data suggest that repeated dosing, at least over the six-day period assessed in this study, did not provide additional effects on peripheral pharmacodynamic modulation of Ki67 ⁺ .

Consistent with the results of Example 18 , this experiment shows that anti-OX40/CD137 surrogate mAb ² has an effect on circulating T cells, significantly increasing the frequency of proliferating (Ki67 ⁺ ) CD8 ⁺ T cells at dose levels from 1 mg/kg to 10 mg /kg and proliferating (Ki67 ⁺ ) CD4 ⁺ T cells at dose levels of 1 mg/kg and 10 mg/kg.

Example 25 - Effect of CD4 T Cell Depletion on the Pharmacodynamic Response of Mab ² to OX40/CD137 mice in the CT26 tumor mouse model

In mice, a combination of co-stimulatory antibodies targeting CD137 and OX40 was previously shown to synergistically enhance clonal expansion of specific CD8 ⁺ T cells, compared with either agent alone, following administration of staphylococcal enterotoxin A (Lee et al. , 2004). Regarding the mechanism of action, Lee et al. demonstrated that CD4 T cells play a role in triggering enhanced specific CD8 ⁺ T cell responses. To test whether host CD4 T cells are required for or contribute to these processes for the activation and proliferation of peripheral CD8 ⁺ T cells in response to treatment with the OX40/CD137 surrogate mAb ² , we used the CT26 tumor model in mice and an antibody murine CD4 T cell depletion.

Following the same protocol as described in Example 17 , female BALB/c mice (Charles River), 8-10 weeks old and weighing approximately 20 g, were injected subcutaneously into the left flank of each animal with CT26 colon carcinoma cells. On the seventh day, animals were randomized into treatment groups, with five animals per group at each time point.

Antibodies were analyzed and checked for contaminants as previously described. Isotype control antibody (G1/4420) and surrogate mAb ² to OX40/CD137 (FS20m-232-91AA/Lob12.3) were diluted to a final concentration of 0.1 mg/ml in PBS. Anti-mouse CD4 antibody (GK1.5; BioXCell, catalog number BE0003-1) was diluted to a final concentration of 1 mg/ml in PBS. Each animal received diluted antibody in a volume of 200 μl per administration, resulting in a final dose of either 1 mg/kg (G1/4420 or FS20m-232-91AA/Lob12.3) or 10 mg/kg (GK1.5) for a 20-weight mouse. G1/4420 and FS20m-232-91AA/Lob12.3 were administered to animals by intraperitoneal (i.p.) injection on days 10, 12, and 14 after cell inoculation. IP injections of GK1.5 were given on days 8, 9, 11, 13, and 15.

Animals were removed from the study at day 16 after cell inoculation, and tissues were collected for flow cytometry analysis. Blood was collected into tubes containing EDTA by cardiac puncture. Following the same protocol as described in Example 19 , red blood cells from uncoagulated blood were lysed twice in red blood cell lysis buffer (Miltenyi Biotech, #130-094-183) according to the manufacturer's instructions, and tumors were dissociated using a red blood cell lysis kit. tumor dissociator, mouse (Miltenyi Biotech, 130-096-730), and gentleMACS dissociator (Miltenyi Biotech) according to the manufacturer's instructions. The resulting tumor cell suspension was passed through a sieve using a 70 μm cell sieve (Corning, part number 352350), washed, and resuspended in PBS. Cell suspensions from spleens were prepared by pushing the spleens through a 70-μm cell strainer (Corning), lysing the red blood cells by incubating in red blood cell lysis buffer (Milteny Biotech), washing the remaining splenocytes, and resuspending them in PBS.

Cells were first stained with reagents CD4-E450 (clone GK1.5), CD69-PE-Cy5 (clone H1.2F3), CD3-PE-Cy7 (clone 145-2C11), CD8-APC (clone 53-6.7) and a fixative dye for viability assessment 780, all supplied by eBioscience; and CD45-V500 (clone 30-F11), supplied by BD Bioscience, in the presence of Fc block (eBioscience, catalog number 14-0161-86). Cells were then fixed and permeabilized using the eBioscience Foxp3 staining kit (eBioscience, cat. no. 00-5523-00) according to the manufacturer's instructions. Cells were resuspended in 100 μl permeabilization buffer with anti-Ki67 antibody and anti-Foxp3 antibody (Ki67-FITC (clone SolA15) and Foxp3-PE (clone FJK-16s), both supplied by eBioscience) in the presence of Fc block (all 1:100) and incubated for 30 minutes in the dark at 4°C. The cells were then washed once with permeabilization buffer and resuspended in 200 μl of PBS. Cells were analyzed on a BD FACSCanto II cytometer. Data analysis was performed using FlowJo, Excel, and GraphPad Prism software. Pairwise comparisons between treatment groups were performed using a two-tailed Mann-Whitney test in GraphPad Prism software.

Treatment with FS20m-232-91AA/Lob12.3 alone induced a statistically significant increase in the proportion of activated CD69 ⁺ and proliferating Ki67 ⁺ CD8 ⁺ T cells in the blood and spleen, as well as proliferating Ki67 ⁺ CD8 ⁺ T cells in the tumor compared to animals who received isotype control.

Combining FS20m-232-91AA/Lob12.3 with the CD4 ⁺ T cell depleting antibody GK1.5 also resulted in a statistically significant increase in proliferating Ki67 ⁺ CD8 ⁺ T cells in the blood compared to isotype control, but this increase was significant lower than that observed in animals treated with FS20m-232-91AA/Lob12.3 as a single agent. There were no statistically significant differences in the levels of proliferating CD8 ⁺ T cells in the spleen and tumor tissues after treatment with FS20m-232-91AA/Lob12.3 alone compared to treatment with FS20m-232-91AA/Lob12.3 plus GK1.5 antibody , depleting CD4 ⁺ T cells.

The FS20m-232-91AA/Lob12.3-induced increase in activated CD69 ⁺ CD8 ⁺ T cells in the blood was inhibited by the GK1.5 antibody, as no statistically significant differences were found between the isotype control group and the FS20m-232-91AA/Lob12.3 plus group CD4 depletion (median 1.6% and 2.33% of total CD8 T cells, respectively). Comparison of the FS20m-232-91AA/Lob12.3 group as a single agent and the FS20m-232-91AA/Lob12.3 plus CD4 depletion group showed that the frequency of activated CD8 ⁺ T cells was significantly reduced in the spleen (median frequency 29.3 % compared with 6.45% without and with exhaustion, respectively) and in the tumor (median frequency 86.4% compared with 66.5%, without and with exhaustion, respectively).

Consistent with previous results described in Example 18 , anti-OX40/CD137 surrogate mAb ² increased the frequency of activated (CD69 ⁺ ) and proliferating (Ki67 ⁺ ) CD8 T cells, and the results of the present study indicate that depletion of CD4 ⁺ T cells had negative effect on the peripheral pharmacodynamic response mediated by MAb ² to OX40/CD137. Moreover, data suggest the potential interaction of CD4 ⁺ and CD8 ⁺ T cells as mediators of anti-OX40/CD137 mAb ² activity in vivo , and that CD4 ⁺ T cells may be required for optimal co-stimulation of CD8 ⁺ T cell immunity in vivo when applying MAT ² to OX40/CD137.

Example 26 - Functional activity of MAT ² to OX40/CD137 in an assay on cynomolgus macaque cells and pharmacodynamic response to MAT ² to OX40/CD137 and its tolerance in cynomolgus macaques

26.1 Functional activity of mAb ² against OX40/CD137 in an assay on cynomolgus macaque cells

An assay of primary PBMCs similar to the primary T cell assay described in Example 13 , but using PBMCs instead of isolated activated T cells, was performed to determine the relative potency of mAb ² FS20-22-49AA/FS30-10-16 against human proteins in in relation to endogenously expressed receptors in humans and cynomolgus macaques. In general, cynomolgus or human PBMCs were isolated and stimulated with anti-CD3 antibody in the presence of increasing concentrations of mAb ² FS20-22-49AA/FS30-10-16 or isotype control for three (cynomolgus) or four (human) periods. days, with IL-2 release serving as a measure of T cell activation.

The functional activity of MAT ² on cynomolgus monkey PBMC (mean _EC50 = 0.28 ± 0.15 nM) was found to be similar to that observed in the equivalent human assay (mean _EC50 = 0.26 ± 0.1 nM; IL-2). Thus, cynomolgus macaques are considered a pharmacologically viable species for MAT toxicity studies ² .

26.2 Tolerance of MAT ² to OX40/CD137 and pharmacodynamic response to it in cynomolgus monkeys

A preliminary dose ranging study was conducted to evaluate the tolerability of mAb ² to human OX40/CD137 FS20-22-49AA/FS30-10-16 and to evaluate potential pharmacodynamic changes in the proportions of major leukocyte populations, as well as induction of proliferation and activation of specific T cell subsets in response to FS20-22-49AA/FS30-10-16 in cynomolgus macaques.

In general terms, MAT ² FS20-22-49AA/FS30-10-16 was administered to cynomolgus monkeys by intravenous infusion as a single dose or in repeated doses. Standard toxicological parameters such as body weight, food intake, clinical observations, hematology and blood chemistry were assessed throughout the study to assess tolerability.

MAT ² FS20-22-49AA/FS30-10-16 was well tolerated up to 30 mg/kg weekly dosing as determined by clinical biochemistry and histopathology.

Consistent with the results of a study assessing the effect of mouse anti-OX40/CD137 mAb ² on circulating T cells in the CT26 syngeneic tumor mouse model ( Example 18 ), drug-associated increases in cell proliferation and activation were observed in CD4 ⁺ and CD8 ⁺ T cells. -central memory cells and effector memory CD4 ⁺ and CD8 ⁺ T cells, as well as NK cells, as measured by increased expression of Ki67 and, to some extent, CD69.

Taken together, these results strongly indicate that anti-human protein mAb ² FS20-22-49AA/FS30-10-16 has potent in vivo pharmacological activity in cynomolgus monkeys and is well tolerated up to 30 mg/kg. In addition, the pharmacodynamic data obtained in this study are consistent with those observed for the OX40/CD137 surrogate mAb ² in the murine pharmacodynamic study described in Example 18 and provide further evidence for the expected antitumor efficacy and tolerability of the OX40-CD137 binding mAb ² , such as MAT ² FS20-22-49AA/FS30-10-16, in human patients with cancer.

Example 27 - Pharmacology of MAT ² to OX40/CD137 in the liver of BALB/c mice

In preclinical mouse models, agonistic CD137 antibodies have been shown to induce increased T cell infiltration into the liver, and one agonistic CD137 antibody induced liver toxicity at doses above 1 mg/kg in clinical settings (Dubrot et al. , 2010; Segal et al. , 2017). Therefore, the effects of murine anti-OX40/CD137 mAb ² were examined in BALB/c mice to determine whether there is increased T cell infiltration into the liver compared with agonistic anti-CD137 antibodies. Blood and spleen tissue were used as controls and T cell levels and T cell proliferation and activation were examined. Details of the antibodies tested are presented in Table 38 .

Table 38 : Details of antibodies and MAT ² tested

MAT/MAT ² Binding Fab to Linking Fcab with Isotype LALA mutation SEQ ID heavy chain SEQ ID light chain G1/4420 FITC Absent hIgG1 No 115 116 G1/OX86 mOX40 Absent hIgG1 No 159 156 G1/Lob12.3 mCD137 Absent hIgG1 No University of Southampton G1/3H3 mCD137 Absent hIgG1 No 168 167 FS20m-232-91AA/Lob12.3 mCD137 mOX40 hIgG1 Yes The receipt is described above in Example 9.2

The ability of mAb ² (FS20m-232-91AA/Lob12.3) to increase, activate and induce proliferation of T cells in the blood, spleen and liver was compared with control mAbs in the form of single agents (G1/OX86, G1/Lob12.3, G1/ 3H3 and G1/4420) and control combination (G1/OX86 and G1/Lob12.3). Female BALB/c (Charles River) mice, 8-10 weeks old and weighing approximately 20 g each, were allowed a rest period of one week before the start of the study. All animals were microchipped and assigned a unique identifier. There were 6 mice in each cohort.

Within 24 hours prior to injection, antibodies were analyzed by SEC-HPLC profiling and checked for contaminants. Antibodies were diluted to a final concentration of 1 mg/ml in PBS and administered by intraperitoneal (ip) injection at 200 μl/mouse, resulting in a final dose of 10 mg/kg for mice weighing 20 g. Injections were performed on days 0, 2 and 4 ( one dose every two days) studies. Seven and fourteen days after the third dose, 3 mice per group were sacrificed, the spleen and liver were isolated by dissection, and blood was collected by cardiac puncture.

Livers and spleens were dissociated using Miltenyi dissociation kits (liver - Miltenyi, 130-105-807; spleen - Miltenyi, 130-095-926) according to the manufacturer's instructions. The resulting cell suspension was passed through a sieve using a 70 μm cell sieve (Corning, part number 352350), centrifuged (10 minutes at 1500 rpm), washed once in PBS, and resuspended in 5 ml of PBS.

Blood was collected into tubes containing EDTA by cardiac puncture. Red blood cells from uncoagulated blood were lysed twice in red blood cell lysis buffer (eBioscience, catalog number 00-4300-54) according to the manufacturer's instructions.

Cells isolated from tumors and blood were stained for flow cytometry using the panel of antibodies and reagents detailed in Example 19 (Stain 1). Cells were washed in PBS and then incubated with 100 μl of antibody mixture 1 (all except anti-Ki67 and anti-Foxp3) for 30 minutes at 4°C. Cells were then washed with PBS and subsequently fixed and permeabilized using the eBioscience Foxp3 staining kit (eBioscience, catalog number 00-5523-00) according to the manufacturer's instructions. In general, 200 μl of fixation solution was added to each well and left overnight in the dark at 4°C. Cells were then washed in 200 μl of permeabilization buffer. Cells were then centrifuged again and resuspended in 100 μl of permeabilization buffer with anti-Ki67 and anti-Foxp3 in the presence of Fc block (all diluted 1:100) and incubated for 30 min in the dark at 4°C. Cells were then washed once with permeabilization buffer and resuspended in 200 μl of PBS. The cells were then analyzed on a BD FACSCanto II flow cytometer.

Data were analyzed using FlowJoX, Excel and GraphPad Prism software. Statistical analysis for group comparisons was performed using one-way analysis of variance followed by Tukey's test for multiple comparisons of each pair using the GraphPad Prism software package. Data were expressed as a percentage of the original population.

The results showed that a cross-linking-independent agonistic anti-CD137 antibody (G1/3H3) induced increased levels of T cells in the liver, spleen and blood on both days 7 and 14, and that these T cells showed increased levels of proliferation and activation compared to the isotype control antibody (G1/4420). A cross-linking-dependent agonistic anti-CD137 antibody (G1/Lob12.3) did not show significant increases in T cell levels, proliferation, or activation in the liver, spleen, or blood. An agonistic antibody to OX40 (G1/OX86) did not induce increased levels of T cells in any of the tissues, but showed increased levels of T cell proliferation in the liver, spleen and blood on day 7 of the study, which returned to isotype control levels by day 14. The combination of an OX40 agonist antibody and a cross-linking dependent CD137 agonist antibody (G1/OX86 and G1/Lob12.3) showed increased levels of T cell infiltration in the liver at day 7, increased T cell proliferation in the liver at day 7 and in the spleen (not significant) and blood on days 7 and 14, and increased T cell activation in the liver and blood on days 14 and in the spleen on days 7 and 14. Mab ² to OX40/CD137 showed increased levels of T cell infiltration in liver (not significant) and T cell levels in the blood on day 7, which returned to isotype control levels by day 14, and increased T cell proliferation in the liver (not significant), spleen and blood on day 7, which also returned to levels for isotype control by day 14. These results indicate that only the cross-linking independent CD137 agonist (G1/3H3) induced increased and sustained T cell infiltration, proliferation, and activation in the liver, as well as in the spleen and blood, and suggest that that the OX40/CD137-targeting antibody molecules of the present invention may have a lower risk of hepatotoxicity compared to cross-linking-independent CD137 agonist antibodies. These results suggest the possibility of an association between the cross-linking-independent agonistic effect on CD137 induced by clone 3H3 and the increased T cell-induced liver inflammation observed for this cross-linking-independent clone in the present study.

Example 28 - Comparison of MAb Antibodies ² anti-OX40/CD137 containing various anti-CD137 Fab clones in the CT26 syngeneic tumor model

In Example 27 , a cross-linking-independent agonist anti-CD137 antibody (G1/3H3) was observed to induce increased and sustained levels of T cell infiltration, proliferation and activation in BALB/c mice. To test whether this increased activity has beneficial antitumor activity in the case of mAb ² to OX40/CD137, the syngeneic tumor model CT26 was used to compare the in vivo activity of two different mAb ² to OX40/CD137 mice, one of which is a CD137 agonist, which is a cross-linking dependent clone Lob1.23, and the other is the agonist CD137, which is a cross-linking independent clone 3H3. The syngeneic CT26 tumor model has previously been shown to be sensitive to agonistic anti-OX40 and anti-CD137 antibodies, and tumor-infiltrating lymphocytes (TILs) isolated from CT26 tumors express both OX40 and CD137.

28.1 Antitumor activity of MAT ² antibodies to OX40/CD137 containing various Fab clones to CD137 in the CT26 syngeneic tumor model

The antitumor activity of two different mAbs ² to OX40/CD137, FS20m-232-91AA/3H3 (SEQ ID NO: 169 and 167) and FS20m-232-91AA/Lob12.3 (see Table 38 ), was determined in vivo in a syngeneic model CT26 tumors in mice and compared with the activity of an isotype control antibody (G1/4420; see Table 38 ). In addition, the levels of T cell proliferation and activation induced in the blood by the two anti-OX40/CD137 mAbs ² were analyzed and compared with those induced by the isotype control antibody. Female BALB/c (Charles River) mice, 8-10 weeks old and weighing approximately 20 g each, were allowed a rest period of one week before the start of the study. All animals were microchipped and assigned a unique identifier. There were 10 mice in each cohort. The colon carcinoma cell line CT26 (ATCC, CRL-2638) was initially expanded, maintained, and then prescreened at IDEXX Bioresearch for pathogen detection using the IMPACT I protocol, and the line was shown to be pathogen-free. CT26 cells (approximately 3-5 x 10 ⁶ ) were thawed from storage at -150°C and added to 20 ml DMEM (Gibco, 61965-026) with 10% FST (Gibco, 10270-106) in a T175 tissue culture flask. Mice were anesthetized using isoflurane (Abbott Laboratories) and each animal received 1 x 10 ⁶ cells administered by subcutaneous injection into the left flank. At day 10 after tumor cell inoculation, mice were monitored for health status and tumor growth, sorted, and randomized into study cohorts. Any mice that did not have tumors at this point were excluded from the study.

Within 24 hours prior to injection, antibodies were analyzed by SEC-HPLC profiling and checked for contaminants. Antibodies were diluted to a final concentration of 0.1 mg/ml in PBS and administered by intraperitoneal (ip) injection at 200 μl/mouse to produce a final dose of 1 mg/kg for a 20 g mouse. Injections were performed on days 10, 12, and 14 (one dose every two days) after tumor inoculation. Animals were screened for health status under blinded anesthesia three times a week, at which time precise tumor measurements were taken. Tumor volumes were determined using caliper measurements (as described in Example 17 ). The study was stopped 35 days after tumor cell inoculation, and animals were removed from the study when humane endpoints based on tumor volume and condition were met. Treatment groups, molecules tested, doses, and dosing schedule are summarized in Table 39 . Tumor volumes at day 21 were statistically analyzed using two-way analysis of variance and Tukey's test for multiple comparisons using GraphPad Prism software. Statistical analysis of survival was performed using the log-rank (Mantel-Cox) test using GraphPad Prism software.

Table 39: Summary of data by treatment group and molecules tested

Group Group name Injected MAT and/or MAT ² Dose (mg/kg) Dosage schedule 1 Isotype control G1/4420 1 Q2D 3 FS20m-232-91AA/Lob12.3 FS20m-232-91AA/Lob12.3 1 Q2D 4 FS20m-232-91AA/3H3 FS20m-232-91AA/3H3 1 Q2D

As shown in Figures 15A and 15B , treatment with one of the two anti-OX40/CD137 Mab ² antibodies delayed tumor growth and increased survival compared with treatment with an isotype control antibody. There were no differences in tumor growth or survival between mice treated with MAT ² FS20m-232-91AA/3H3 and MAT ² FS20m-232-91AA/Lob12.3, respectively. These data indicate that, despite the increased T cell activation and proliferation observed for the cross-linking independent CD137 agonist (G1/3H3) as described in Example 27 , increased antitumor activity of anti-OX40/CD137 mAb ² was not observed. in which the anti-CD137 Fab clone is a cross-linking-independent clone 3H3 (FS20m-232-91AA/3H3), compared to anti-OX40/CD137 MAT ² , in which the anti-CD137 Fab clone is a cross-linking-dependent Lob12.3 clone (FS20m-232-91AA/Lob12.3).

28.2 Evaluation of the peripheral pharmacodynamic response of mAb ² to OX40/CD137 containing various Fab clones to CD137 in the CT26 syngeneic tumor model

In an extension of the study described in Example 28.1 above, five days after the third dose (ie, day 19 after tumor inoculation), blood from the tail vein of five mice was collected into tubes containing EDTA. Red blood cells from uncoagulated blood were lysed twice in red blood cell lysis buffer (eBioscience, catalog number 00-4300-54) according to the manufacturer's instructions.

Cells isolated from blood were stained for flow cytometry using the panel of antibodies and reagents detailed in Example 19 (Stain 1). Cells were washed in PBS and then incubated with 100 μl of antibody mixture 1 (all except anti-Ki67 and anti-Foxp3) for 30 minutes at 4°C. Cells were then washed with PBS and then fixed and permeabilized using a Foxp3 staining kit (eBioscience, cat. no. 00-5523-00) according to the manufacturer's instructions. In general, 200 μl of fixation solution was added to each well and left overnight in the dark at 4°C. Cells were then washed in 200 μl of permeabilization buffer. Cells were then centrifuged again and resuspended in 100 μl of permeabilization buffer with anti-Ki67 and anti-Foxp3 in the presence of Fc block (all at 1:100 dilution) and incubated for 30 min in the dark at 4°C. The cells were then washed once with permeabilization buffer and resuspended in 200 μl of PBS. The cells were then analyzed on a BD FACS CantoII cytometer.

Data were analyzed using FlowJoX, Excel and GraphPad Prism software. Statistical analysis for group comparisons was performed using one-way analysis of variance followed by Tukey's test for multiple comparisons of each pair using the GraphPad Prism software package.

FS20m-232-91AA/3H3 induced a statistically significant increase in blood T cell levels compared to both the isotype control antibody (G1/4420) and FS20m-232-91AA/Lob12.3. Increased levels of T cells induced by FS20m-232-91AA/3H3 were accompanied by a statistically significant decrease in the relative percentage of CD4 ⁺ T cells and a statistically significant increase in the relative percentage of CD8 ⁺ T cells compared with the relative percentages of these cell types observed for isotype control G1/4420 and MAT ² FS20m-232-91AA/Lob12.3. Both Mab ² antibodies to OX40/CD137 also induced proliferation of CD4 ⁺ and CD8 ⁺ T cells, but the levels induced by FS20m-232-91AA/3H3 were significantly higher than those induced by Mab ² FS20m-232-91AA/Lob12. 3. Mab ² FS20m-232-91AA/3H3 induced increased levels of activated CD4 ⁺ T cells compared to isotype control. Changes in the levels of activated T cells and activated CD8 ⁺ T cells in mice treated with FS20m-232-91AA/Lob12.3 or FS20m-232-91AA/3H3 compared with the isotype control cohort were small and not statistically significant. as were changes in levels of activated CD4 ⁺ T cells in mice treated with FS20m-232-91AA/Lob12.3. These results indicate that the cross-linking-independent CD137 agonist clone 3H3 is active on the OX40/CD137 mAb ² and is capable of inducing increased levels and proliferation of T cells compared to the cross-linking-dependent CD137 agonist clone Lob12.3 on the mAb ² to OX40/CD137, and are therefore consistent with the increased levels and proliferation of T cells induced by monoclonal antibody (Mab) clone 3H3 as observed in the BALB/c mouse study described in Example 27 .

Together with the antitumor activity data, these results suggest that there is no additional benefit in terms of antitumor response from the increased levels and proliferation of T cells induced by the crosslinking-independent CD137 agonist when applied to anti-OX40/CD137 mAb ² . These results, together with the results of Example 27 , in which increased T cell-induced liver inflammation was observed for a cross-linking independent agonist effect on CD137 induced by clone 3H3, suggest that the use of mAb ² to OX40/CD137 agonist effect on CD137, which is dependent on binding to OX40, may provide a safe and effective way to stimulate the immune system to fight cancer.

List of sequences

Amino acid sequences of CDR MAT FS30-10-16 (IMGT)

CDR1 VH - GFTFSSYD (SEQ ID NO: 1)

CDR2 VH - IDPTGSKT (SEQ ID NO: 2)

CDR3 VH - ARDLLVYGFDY (SEQ ID NO: 3)

CDR1 VL - QSVSSSY (SEQ ID NO: 4)

CDR2 VL - GAS (SEQ ID NO: 5)

CDR3 VL - QQSYSYPVT (SEQ ID NO: 6)

Amino acid sequences of CDR MAT FS30-10-16 (Kabat)

CDR1 VH - SYDMS (SEQ ID NO: 7)

CDR2 VH - DIDPTGSKTDYADSVKG (SEQ ID NO: 8)

CDR3 VH - DLLVYGFDY (SEQ ID NO: 9)

CDR1 VL - RASQSVSSSYLA (SEQ ID NO: 10)

CDR2 VL - GASSRAT (SEQ ID NO: 11)

CDR3 VL - QQSYSYPVT (SEQ ID NO: 6)

Amino acid sequence of the heavy chain variable domain of MAT FS30-10-16 (SEQ ID NO: 12)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EVQLLESGGGLVQPGGSLRLSCAAS GFTFS SYD MS WVRQAPGKGLEWVS D IDPTGSKT DYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR DLLVYGFDY WGQGTLVTVSS

Nucleic acid sequence of the heavy chain variable domain of MAT FS30-10-16 (SEQ ID NO: 13)

GAAGTTCAGCTGCTGGAATCTGGCGGCGGATTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCTTCCGGCTTCACCTTCCAGCTACGACATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTGTCCGACATCGACCCCACCGGCTCTAAGACCGACTACGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACTCCAAGAACACCCTGTACC TGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAGATCTGCTGGTGTACGGCTTCGACTATTGGGGCCAGGGCACACTGGTCACCGTGTCCTCT

Amino acid sequence of the light chain variable domain of MAT FS30-10-16 (SEQ ID NO: 14)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EIVLTQSPGTLSLSPGERATLSC RAS QSVSSSY L.A. WYQQKPGQAPRLLIY G.A.S. SRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQSYSYPVT FGQGTKVEIK

Nucleic acid sequence of the light chain variable domain of MAT FS30-10-16 (SEQ ID NO: 15)

GAGATCGTGCTGACCCAGTCTCCTGGCACACTGTCACTGTCTCCAGGCGAGAGCTACCCTGTCCTGTAGAGCCTCTCAGTCCGTGTCCTCCTCTTACCTGGCCTGGTATCAGCAGAAGCCTGGACAGGCTCCCCGGCTGTTGATCTACGGCGCTTCTTCTAGAGCCACAGGCATCCCTGACCGGTTCTCCGGATCTGGCTCTGGCACCGATTTCACCCTGACCATCTCTCGGCTGGA ACCCGAGGATTTCGCCGTGTACTACTGCCAGCAGTCCTACAGCTACCCCGTGACCTTTGGCCAGGGCACCAAGGTGGAAATCAAG

Amino acid sequences of CDR MAT FS30-10-3 (IMGT)

CDR1 VH - GFTFSSYD (SEQ ID NO: 1)

CDR2 VH - IDPTGSKT (SEQ ID NO: 2)

CDR3 VH - ARDLNVYGFDY (SEQ ID NO: 16)

CDR1 VL - QSVSSSY (SEQ ID NO: 4)

CDR2 VL - GAS (SEQ ID NO: 5)

CDR3 VL - QQSYSYPVT (SEQ ID NO: 6)

Amino acid sequences of CDR MAT FS30-10-3 (Kabat)

CDR1 VH - SYDMS (SEQ ID NO: 7)

CDR2 VH - DIDPTGSKTDYADSVKG (SEQ ID NO: 8)

CDR3 VH - DLNVYGFDY (SEQ ID NO: 17)

CDR1 VL - RASQSVSSSYLA (SEQ ID NO: 10)

CDR2 VL - GASSRAT (SEQ ID NO: 11)

CDR3 VL - QQSYSYPVT (SEQ ID NO: 6)

Amino acid sequence of the heavy chain variable domain of MAT FS30-10-3 (SEQ ID NO: 18)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EVQLLESGGGLVQPGGSLRLSCAAS GFTFS SYD MS WVRQAPGKGLEWVS D IDPTGSKT DYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR DLNVYGFDY WGQGTLVTVSS

Nucleic acid sequence of the heavy chain variable domain of MAT FS30-10-3 (SEQ ID NO: 19)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTAGTTACGATATGAGCTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTGATCCGACTGGTAGCAAGACCGACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGC TGTACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAGACCTCAATGTGTACGGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT

Amino acid sequence of the light chain variable domain of MAT FS30-10-3 (SEQ ID NO: 14)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EIVLTQSPGTLSLSPGERATLSC RAS QSVSSSY L.A. WYQQKPGQAPRLLIY G.A.S. SRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQSYSYPVT FGQGTKVEIK

Nucleic acid sequence of the light chain variable domain of MAT FS30-10-3 (SEQ ID NO: 20)

GAAATTGTGCTGACCCAGTCTCCGGGCACGTTATCTCTGAGCCCTGGTGAGCGCGCCACTCTGTCATGCCGGGCTTCTCAAAGTGTTAGCAGTAGCTACCTGGCGTGGTATCAGCAAAAACCGGGCCAGGCCCCGCGTCTGCTGATTTACGGTGCATCCAGCCGTGCCACCGGCATTCCAGATCGTTTTTCCGTAGTGGTTCTGGACGGACTTCACTCTGACAATCTCACGCC TGGAACCGGAGGATTTTGCGGTGTATTACTGCCAGCAATCTTATTCTTATCCTGTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

Amino acid sequences of CDR MAT FS30-10-12 (IMGT)

CDR1 VH - GFTFSSYD (SEQ ID NO: 1)

CDR2 VH - IDPTGSKT (SEQ ID NO: 2)

CDR3 VH - ARDLTVYGFDY (SEQ ID NO: 21)

CDR1 VL - QSVSSSY (SEQ ID NO: 4)

CDR2 VL - GAS (SEQ ID NO: 5)

CDR3 VL - QQSYSYPVT (SEQ ID NO: 6)

Amino acid sequences of CDR MAT FS30-10-12 (Kabat)

CDR1 VH - SYDMS (SEQ ID NO: 7)

CDR2 VH - DIDPTGSKTDYADSVKG (SEQ ID NO: 8)

CDR3 VH - DLTVYGFDY (SEQ ID NO: 22)

CDR1 VL - RASQSVSSSYLA (SEQ ID NO: 10)

CDR2 VL - GASSRAT (SEQ ID NO: 11)

CDR3 VL - QQSYSYPVT (SEQ ID NO: 6)

Amino acid sequence of the heavy chain variable domain of MAT FS30-10-12 (SEQ ID NO: 23)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EVQLLESGGGLVQPGGSLRLSCAAS GFTFS SYD MS WVRQAPGKGLEWVS D IDPTGSKT DYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR DLTVYGFDY WGQGTLVTVSS

Nucleic acid sequence of the heavy chain variable domain of MAT FS30-10-12 (SEQ ID NO: 24)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTAGTTACGATATGAGCTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTGATCCGACTGGTAGCAAGACCGACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGC TGTACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAGACCTCACGGTGTACGGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT

Amino acid sequence of the light chain variable domain of MAT FS30-10-12 (SEQ ID NO: 14)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EIVLTQSPGTLSLSPGERATLSC RAS QSVSSSY L.A. WYQQKPGQAPRLLIY G.A.S. SRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQSYSYPVT FGQGTKVEIK

Nucleic acid sequence of the light chain variable domain of MAT FS30-10-12 (SEQ ID NO: 20)

GAAATTGTGCTGACCCAGTCTCCGGGCACGTTATCTCTGAGCCCTGGTGAGCGCGCCACTCTGTCATGCCGGGCTTCTCAAAGTGTTAGCAGTAGCTACCTGGCGTGGTATCAGCAAAAACCGGGCCAGGCCCCGCGTCTGCTGATTTACGGTGCATCCAGCCGTGCCACCGGCATTCCAGATCGTTTTTCCGTAGTGGTTCTGGACGGACTTCACTCTGACAATCTCACGCC TGGAACCGGAGGATTTTGCGGTGTATTACTGCCAGCAATCTTATTCTTATCCTGTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

Amino acid sequences of CDR MAT FS30-35-14 (IMGT)

CDR1 VH - GFTFSAYN (SEQ ID NO: 25)

CDR2 VH - ISPYGGAT (SEQ ID NO: 26)

CDR3 VH - ARNLYELSAYSYGADY (SEQ ID NO: 27)

CDR1 VL - QSVSSSY (SEQ ID NO: 4)

CDR2 VL - GAS (SEQ ID NO: 5)

CDR3 VL - QQYYYSSPIT (SEQ ID NO: 28)

Amino acid sequences of CDR MAT FS30-35-14 (Kabat)

CDR1 VH - AYNIH (SEQ ID NO: 29)

CDR2 VH - DISPYGGATNYADSVKG (SEQ ID NO: 30)

CDR3 VH - NLYELSAYSYGADY (SEQ ID NO: 31)

CDR1 VL - RASQSVSSSYLA (SEQ ID NO: 10)

CDR2 VL - GASSRAT (SEQ ID NO: 11)

CDR3 VL - QQYYYSSPIT (SEQ ID NO: 28)

Amino acid sequence of the heavy chain variable domain of MAT FS30-35-14 (SEQ ID NO: 170)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EVQLLESGGGLVQPGGSLRLSCAAS GFTFS AYN IH WVRQAPGKGLEWVS D ISPYGGAT NYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR NLYELSAYSYGADY WGQGTLVTVSS

Nucleic acid sequence of the heavy chain variable domain of MAT FS30-35-14 (SEQ ID NO: 171)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTGCCTATAATATCCATTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTTCTCCGTATGGTGGCGCGACCAACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGCTGTA CCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAAACCTCTACGAGTTGAGCGCTTACTCTTACGGGGCGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGTCG

Amino acid sequence of the light chain variable domain of MAT FS30-35-14 (SEQ ID NO: 172)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EIVLTQSPGTLSLSPGERATLSC RAS QSVSSSY L.A. WYQQKPGQAPRLLIY G.A.S. SRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYYYSSPIT FGQGTKVEIK

Nucleic acid sequence of the light chain variable domain of MAT FS30-35-14 (SEQ ID NO: 32)

GAAATTGTGCTGACCCAGTCTCCGGGCACGTTATCTCTGAGCCCTGGTGAGCGCGCCACTCTGTCATGCCGGGCTTCTCAAAGTGTTAGCAGTAGCTACCTGGCGTGGTATCAGCAAAA ACCGGGCCAGGCCCCGCGTCTGCTGATTTACGGTGCATCCAGCCGTGCCACCGGCATTCCAGATCGTTTTTCCGTAGTGGTTCTGGACGGACTTCACTCTGACAATCTCACGCCTGGA ACCGGAGGATTTTGCGGTGTATTACTGCCAGCAATATTATTATTCTTCTCCTATCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

Amino acid sequences of CDR MAT FS30-5-37 (IMGT)

CDR1 VH - GFTFSSYA (SEQ ID NO: 33)

CDR2 VH - ISGSGGST (SEQ ID NO: 34)

CDR3 VH - ARSYDKYWGSSIYSGLDY (SEQ ID NO: 35)

CDR1 VL - QSVSSSY (SEQ ID NO: 4)

CDR2 VL - GAS (SEQ ID NO: 5)

CDR3 VL - QQYYSYYPVT (SEQ ID NO: 36)

Amino acid sequences of CDR MAT FS30-5-37 (Kabat)

CDR1 VH - SYAMS (SEQ ID NO: 37)

CDR2 VH - AISGSGGSTYYADSVKG (SEQ ID NO: 38)

CDR3 VH - SYDKYWGSSIYSGLDY (SEQ ID NO: 39)

CDR1 VL - RASQSVSSSYLA (SEQ ID NO: 10)

CDR2 VL - GASSRAT (SEQ ID NO: 11)

CDR3 VL - QQYYSYYPVT (SEQ ID NO: 36)

Amino acid sequence of the heavy chain variable domain of MAT FS30-5-37 (SEQ ID NO: 40)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EVQLLESGGGLVQPGGSLRLNCAAS GFTFS S.Y.A. MS WVRQAPGKGLEWVS A ISGSGGST YYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR SYDKYWGSSIYSGLDY WGQGTLVTVSS

Nucleic acid sequence of the heavy chain variable domain of MAT FS30-5-37 (SEQ ID NO: 41)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAATTGCGCGGCCAGTGGCTTTACCTTCAGTAGCTATGCCATGAGCTGGGTGCGTCAGGCGCCGGGCAAAGGTCTGGAATGGGTTAGCGCGATTAGCGGTAGTGGCGGTAGCACGTACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGC TGTACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGATCTTACGACAAATACTGGGGTTCTTCTATTTACTCTGGCTTGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT

Amino acid sequence of the light chain variable domain of MAT FS30-5-37 (SEQ ID NO: 42)

CDR according to IMGT numbering (bold italics), CDR according to Kabat numbering (underlined italics)

EIVLTQSPGTLSLSPGERATLSC RAS QSVSSSY L.A. WYQQKPGQAPRLLIY G.A.S. SRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYYSYYPVT FGQGTKVEIK

Nucleic acid sequence of the light chain variable domain of MAT FS30-5-37 (SEQ ID NO: 43)

GAAATTGTGCTGACCCAGTCTCCGGGCACGTTATCTCTGAGCCCTGGTGAGCGCGCCACTCTGTCATGCCGGGCTTCTCAAAGTGTTAGCAGTAGCTACCTGGCGTGGTATCAGCAAAA ACCGGGCCAGGCCCCGCGTCTGCTGATTTACGGTGCATCCAGCCGTGCCACCGGCATTCCAGATCGTTTTTCCGTAGTGGTTCTGGACGGACTTCACTCTGACAATCTCACGCCTGGA ACCGGAGGATTTTGCGGTGTATTACTGCCAGCAATATTATTCTTATTATCCTGTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

Amino acid sequences of the structural loops of the WT CH3 domain

Hinge WT AB - RDELTKNQ (SEQ ID NO: 44)

Loop WT CD - SNGQPENNY (SEQ ID NO: 45)

Hinge WT EF - DKSRWQQGNV (SEQ ID NO: 46)

Amino acid sequence of WT CH3 domain (SEQ ID NO: 47)

Loops AB, CD and EF are underlined

GQPREPQVYTLPPS RDELTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNY KTTP

PVLDSDGSFFLYSKLTV DKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of the CH2 domain (SEQ ID NO: 48)

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

Amino acid sequence of CH2 domain with LALA mutation (SEQ ID NO: 49)

LALA mutation is underlined

APE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

Amino acid sequence of CH2 domain with LALA mutation and P114A mutation (SEQ ID NO: 50)

The LALA mutation is underlined; P114A mutation is shown in bold and underlined

APE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL A APIEKTISKAK

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-22-49

First sequence FS20-22-49 - YWDQE (SEQ ID NO: 51)

Second sequence FS20-22-49 - DEQFA (SEQ ID NO: 52)

Third sequence FS20-22-49 - QYRWNPADY (SEQ ID NO: 53)

Amino acid sequence of the CH3 domain of Fcab FS20-22-49 (SEQ ID NO: 54)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE YWDQE VSLTCLVKGFYPSDIAVEWESNG DEQFA YKTT

PPVLDSDGSFFLYSKLTVD QYRWNPADY FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-22-49 (SEQ ID NO: 55)

GGCCAGCCTAGGGAACCCCAGGTTTACACCTTGCCTCCAAGCCGGGACGAGTACTGGGATCAAGAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCGACGAGCAGTTCGCCTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACCAGTACAGATGGAACCCCGCCG ACTACTTCTCTTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCCCTGTCTCTGTCCCCTGGC

Amino acid sequences of the loop sequences AB, CD and EF of the CH3 domain Fcab FS20-22-49

Hinge AB FS20-22-49 - RDEYWDQE (SEQ ID NO: 56)

Hinge CD FS20-22-49 - SNGDEQFAY (SEQ ID NO: 57)

Hinge EF FS20-22-49 - DQYRWNPADY (SEQ ID NO: 58)

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-22-38

First sequence FS20-22-38 - YWDQE (SEQ ID NO: 51)

Second sequence FS20-22-38 - AEKYQ (SEQ ID NO: 59)

Third sequence FS20-22-38 - QYRWNPGDY (SEQ ID NO: 60)

Amino acid sequence of the CH3 domain of Fcab FS20-22-38 (SEQ ID NO: 61)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE YWDQE VSLTCLVKGFYPSDIAVEWESNG AEKYQ YKTT

PPVLDSDGSFFLYSKLTVD QYRWNPGDY FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-22-38 (SEQ ID NO: 62)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGCAGAAAAATACCAGTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAACCCAGGCGACT ATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-22-41

First sequence FS20-22-41 - YWDQE (SEQ ID NO: 51)

Second sequence FS20-22-41 - DEQFA (SEQ ID NO: 52)

Third sequence FS20-22-41 - QYRWNPGDY (SEQ ID NO: 60)

Amino acid sequence of the CH3 domain of Fcab FS20-22-41 (SEQ ID NO: 63)

The first, second and third sequences are underlined

GQPREPQVYTLPPS RDEYWDQE VSLTCLVKGFYPSDIAVEWE SNGDEQFAY KTT

PPVLDSDGSFFLYSKLTV DQYRWNPGDY FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-22-41 (SEQ ID NO: 64)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAACCCAGGCGACT ATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGA

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-22-47

First sequence FS20-22-47 - YWDQE (SEQ ID NO: 51)

Second sequence FS20-22-47 - DEQFA (SEQ ID NO: 52)

Third sequence FS20-22-47 - QYRWSPGDY (SEQ ID NO: 65)

Amino acid sequence of the CH3 domain of Fcab FS20-22-47 (SEQ ID NO: 66)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE YWDQE VSLTCLVKGFYPSDIAVEWESNG DEQFA YKTT

PPVLDSDGSFFLYSKLTVD QYRWSPGDY FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-22-47 (SEQ ID NO: 67)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAGTCCGGGTGA TTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-22-85

First sequence FS20-22-85 - YWDQE (SEQ ID NO: 51)

Second sequence FS20-22-85 - DEQFA (SEQ ID NO: 52)

Third sequence FS20-22-85 - QYRWNPFDD (SEQ ID NO: 68)

Amino acid sequence of the CH3 domain of Fcab FS20-22-85 (SEQ ID NO: 69)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE YWDQE VSLTCLVKGFYPSDIAVEWESNG DEQFA YKTT

PPVLDSDGSFFLYSKLTLD QYRWNPFDD FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-22-85 (SEQ ID NO: 70)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCTTGGATCAGTATAGGTGGAATCCGTTTGAT GATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-31-58

First sequence FS20-31-58 - YYSGE (SEQ ID NO: 71)

Second sequence FS20-31-58 - QPEND (SEQ ID NO: 72)

Third sequence FS20-31-58 - PYWRWGSPRT (SEQ ID NO: 73)

Amino acid sequence of the CH3 domain of Fcab FS20-31-58 (SEQ ID NO: 74)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE YYSGE VSLTCLVKGFYPSDIAVEWESNG QPEND YKTTP

PVLDSDGSFFLYSKLTV PYWRWGSPRT FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-31-58 (SEQ ID NO: 75)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTACTCTGGTGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACGACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGCCTTATTGGAGGTGGGGTAGTCCGCG TACTTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-31-66

First sequence FS20-31-66 - YYSGE (SEQ ID NO: 71)

Second sequence FS20-31-66 - QPEND (SEQ ID NO: 72)

Third sequence FS20-31-66 - PYWRWGVPRT (SEQ ID NO: 76)

Amino acid sequence of the CH3 domain of Fcab FS20-31-66 (SEQ ID NO: 77)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE YYSGE VSLTCLVKGFYPSDIAVEWESNG QPEND YKTTP

PVLDSDGSFFLYSKLTV PYWRWGVPRT FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-31-66 (SEQ ID NO: 78)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTACTCTGGTGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACGACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGCCGTATTGGAGGTGGGGTGTTCCGC GTACTTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-31-94 Fcab

First sequence FS20-31-94 - WEHGE (SEQ ID NO: 79)

Second sequence FS20-31-94 - IREHD (SEQ ID NO: 80)

Third sequence FS20-31-94 - PYWRWGGPGT (SEQ ID NO: 81)

Amino acid sequence of the CH3 domain of Fcab FS20-31-94 Fcab (SEQ ID NO: 82)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE WEHGE VSLTCLVKGFYPSDIAVEWESNG IREHD YKTTP

PVLDSDGSFFLYSKLTV PYWRWGGPGT FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of the CH3 domain of Fcab FS20-31-94 Fcab (SEQ ID NO: 83)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTGGGAACATGGTGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGATCAGAGAACATGATTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGCCATATTGGAGGTGGGGCGGCCCAGGCA CCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-31-102

First sequence FS20-31-102 - WASGE (SEQ ID NO: 84)

Second sequence FS20-31-102 - QPEVD (SEQ ID NO: 85)

Third sequence FS20-31-102 - PYWRWGVPRT (SEQ ID NO: 76)

Amino acid sequence of the CH3 domain of Fcab FS20-31-102 (SEQ ID NO: 86)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE WASGE VSLTCLVKGFYPSDIAVEWESNG QPEVD YKTTP

PVLDSDGSFFLYSKLTV PYWRWGVPRT FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-31-102 (SEQ ID NO: 87)

GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTGGGCATCTGGTGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCAGAAGTTGATTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGCCGTATTGGAGGTGGGGTGTTCC GCGTACTTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-31-108

First sequence FS20-31-108 - WASGE (SEQ ID NO: 84)

Second sequence FS20-31-108 - EKEID (SEQ ID NO: 88)

Third sequence FS20-31-108 - PYWRWGAKRT (SEQ ID NO: 89)

Amino acid sequence of the CH3 domain of Fcab FS20-31-108 (SEQ ID NO: 90)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE WASGE VSLTCLVKGFYPSDIAVEWESNG EKEID YKTTP

PVLDSDGSFFLYSKLTV PYWRWGAKRT FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-31-108 (SEQ ID NO: 91)

GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTGGGCATCTGGTGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGAAAAAGAAATCGATTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGCCGTATTGGAGGTGGGGTGCTAAG CGTACTTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT

Amino acid sequences of the structural loop sequences of the CH3 domain Fcab FS20-31-115

First sequence FS20-31-115 - WASGE (SEQ ID NO: 84)

Second sequence FS20-31-115 - EQEFD (SEQ ID NO: 92)

Third sequence FS20-31-115 - PYWRWGAKRT (SEQ ID NO: 89)

Amino acid sequence of the CH3 domain of Fcab FS20-31-115 (SEQ ID NO: 93)

The first, second and third sequences are underlined

GQPREPQVYTLPPSRDE WASGE VSLTCLVKGFYPSDIAVEWESNG EQEFD YKTTP

PVLDSDGSFFLYSKLTV PYWRWGAKRT FSCSVMHEALHNHYTQKSLSLSPG

Nucleic acid sequence of CH3 domain Fcab FS20-31-115 (SEQ ID NO: 94)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTGGGCATCTGGTGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGAACAGGAATTCGATTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGCCGTATTGGAGGTGGGGTGCTA AGCGTACTTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

Heavy chain amino acid sequence FS20-22-49AA/FS30-10-16 with LALA mutation (SEQ ID NO: 95)

Variable domain (italics), LALA mutation (bold underlined)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSDIDPTGSKTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVYGFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN AA FAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy chain nucleic acid sequence FS20-22-49AA/FS30-10-16 with LALA mutation (SEQ ID NO: 96)

GAAGTTCAGCTGCTGGAATCTGGCGGCGGATTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCTTCCGGCTTCACCTTCTCCAGCTACGACATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTGTCCGACATCGACCCCACCGGCTCTAAGACCGACTACGCCGATTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAGAGATCTGCTGGTGTACGGCTTCGACTATTGGGGCCAGGGCACACTGGTCACCGTGTCCTCTGCTTCTACCAAGGGACCCAGCGTGTTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCAGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGACCCAGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACCTTGCCTCCAAGCCGGGACGAGTACTGGGATCAAGAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCGACGAGCAGTTCGCCTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACCAGTACAGATGGAACCCCGCCGACTACTTCTCTTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGTCCCTGTCTCTGTCCCCTGGC

Light chain amino acid sequence FS30-10-16 (SEQ ID NO: 97)

Variable domain (italics)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSYSYPVTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

Light chain nucleic acid sequence FS30-10-16 (SEQ ID NO: 98)

GAGATCGTGCTGACCCAGTCTCCTGGCACACTGTCACTGTCTCCAGGCGAGAGCTACCCTGTCCTGTAGAGCCTCTCAGTCCGTGTCCTCCTCTTACCTGGCCTGGTATCAGCAGAAGCCTGGACAGGCTCCCCGGCTGTTGATCTACGGCGCTTCTTCTAGAGCCACAGGCATCCCTGACCGGTTCTCCGGATCTGGCTCTGGCACCGATTTCACCCTGACCATCTCTCGGCTGGA ACCCGAGGATTTCGCCGTGTACTACTGCCAGCAGTCCTACAGCTACCCCGTGACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCAAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAG CCAGGAGACGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC

Heavy chain amino acid sequence of FS20-22-49AA/FS30-10-3 with LALA mutation (SEQ ID NO: 99)

Variable domain (italics), LALA mutation (bold underlined)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSDIDPTGSKTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLNVYGFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN AA FAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy chain nucleic acid sequence FS20-22-49AA/FS30-10-3 with LALA mutation (SEQ ID NO: 100)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTAGTTACGATATGAGCTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTGATCCGACTGGTAGCAAGACCGACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGCTG TACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAGACCTCAATGTGTACGGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCTAGCACTAAGGGCCCGTCGGTGTTCCCGCTGGCCCCATCGTCCAAGAGCACATCAGGGGGGTACCGCCGCCCTGGGCTGCCTTGTGAAGGATTACTTTCCCGAGCCCGTCACAGTGTCCTGGAACA GCGGAGCCCTGACCTCCGGAGTGCATACTTTCCCGGCTGTGCTTCAGTCCTCTGGCCTGTACTCATTGTCCTCCGTGGTCACCGTCCCTTCGTCCTCCCTGGGCACCCAGACCTATATCTGTAATGTCAACCATAAGCCCTCGAACACCAAGGTCGACAAGAAGGTCGAGCCGAAGTCGTGCGACAAGACTCACACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTG TTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGAT GGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAG TGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCGGA

Amino acid sequence of FS30-10-3 light chain (SEQ ID NO: 97)

Variable domain (italics)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSYSYPVTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

FS30-10-3 Light Chain Nucleic Acid Sequence (SEQ ID NO: 102)

GAAATTGTGCTGACCCAGTCTCCGGGCACGTTATCTCTGAGCCCTGGTGAGCGCGCCACTCTGTCATGCCGGGCTTCTCAAAGTGTTAGCAGTAGCTACCTGGCGTGGTATCAGCAAAAACCGGGCCAGGCCCCGCGTCTGCTGATTTACGGTGCATCCAGCCGTGCCACCGGCATTCCAGATCGTTTTTCCGTAGTGGTTCTGGACGGACTTCACTCTGACAATCTCACGCCTG GAACCGGAGGATTTTGCGGTGTATTACTGCCAGCAATCTTATTCTTATCCTGTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACTGTGGCCGCTCCTAGCGTGTTCATTTTTCCGCCATCCGACGAGCAGCTCAAGTCCGGCACCGCCTCCGTGGTCTGCCTGCTCAACAACTTCTACCCTCGCGAAGCTAAGGTCCAGTGGAAGGTCGACAATGCCCTGCAGTCCGGA AACTCGCAGGAAAGCGTGACTGAACAGGACTCCAAGGACTCCACCTATTCACTGTCCTCGACTCTGACCCTGAGCAAGGCGGATTACGAAAAGCACAAAGTGTACGCATGCGAAGTGACCCACCAGGGTCTTTCGTCCCCCGTGACCAAGAGCTTCAACAGAGGAGAGTGT

Heavy chain amino acid sequence FS20-22-49AA/FS30-10-12 with LALA mutation (SEQ ID NO: 103)

Variable domain (italics), LALA mutation (bold underlined)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSDIDPTGSKTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLTVYGFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN AA FAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy chain nucleic acid sequence FS20-22-49AA/FS30-10-12 with LALA mutation (SEQ ID NO: 104)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTAGTTACGATATGAGCTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTGATCCGACTGGTAGCAAGACCGACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGCTG TACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAGACCTCACGGTGTACGGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCTAGCACTAAGGGCCCGTCGGTGTTCCCGCTGGCCCCATCGTCCAAGAGCACATCAGGGGGTACCGCCGCCCTGGGCTGCCTTGTGAAGGATTACTTTCCCGAGCCCGTCACAGTGTCCTGGAACA GCGGAGCCCTGACCTCCGGAGTGCATACTTTCCCGGCTGTGCTTCAGTCCTCTGGCCTGTACTCATTGTCCTCCGTGGTCACCGTCCCTTCGTCCTCCCTGGGCACCCAGACCTATATCTGTAATGTCAACCATAAGCCCTCGAACACCAAGGTCGACAAGAAGGTCGAGCCGAAGTCGTGCGACAAGACTCACACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTGGTCCTTCGGTG TTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGAT GGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAG TGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

Light chain amino acid sequence FS30-10-12 (SEQ ID NO: 97)

Variable domain (italics)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSYSYPVTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

Light chain nucleic acid sequence FS30-10-12 (SEQ ID NO: 102)

GAAATTGTGCTGACCCAGTCTCCGGGCACGTTATCTCTGAGCCCTGGTGAGCGCGCCACTCTGTCATGCCGGGCTTCTCAAAGTGTTAGCAGTAGCTACCTGGCGTGGTATCAGCAAAAACCGGGCCAGGCCCCGCGTCTGCTGATTTACGGTGCATCCAGCCGTGCCACCGGCATTCCAGATCGTTTTTCCGTAGTGGTTCTGGACGGACTTCACTCTGACAATCTCACGCCTG GAACCGGAGGATTTTGCGGTGTATTACTGCCAGCAATCTTATTCTTATCCTGTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACTGTGGCCGCTCCTAGCGTGTTCATTTTTCCGCCATCCGACGAGCAGCTCAAGTCCGGCACCGCCTCCGTGGTCTGCCTGCTCAACAACTTCTACCCTCGCGAAGCTAAGGTCCAGTGGAAGGTCGACAATGCCCTGCAGTCCGGA AACTCGCAGGAAAGCGTGACTGAACAGGACTCCAAGGACTCCACCTATTCACTGTCCTCGACTCTGACCCTGAGCAAGGCGGATTACGAAAAGCACAAAGTGTACGCATGCGAAGTGACCCACCAGGGTCTTTCGTCCCCCGTGACCAAGAGCTTCAACAGAGGAGAGTGT

Heavy chain amino acid sequence of FS20-22-49AA/FS30-35-14 with LALA mutation (SEQ ID NO: 105)

Variable domain (italics), LALA mutation (bold underlined)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYNIHWVRQAPGKGLEWVSDISPYGGATNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNLYELSAYSYGADYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWES NGDEQFAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy chain nucleic acid sequence FS20-22-49AA/FS30-35-14 with LALA mutation (SEQ ID NO: 106)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTGCCTATAATATCCATTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTTCTCCGTATGGTGGCGCGACCAACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGCTG TACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAAACCTCTACGAGTTGAGCGCTTACTCTTACGGGGCGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGTCGGCTAGCACTAAGGGCCCGTCGGTGTTCCCGCTGGCCCCATCGTCCAAGAGCACATCAGGGGGTACCGCCGCCCTGGGCTGCCTTGTGAAGGATTACTTTCCCGAGCCCGTCACAG TGTCCTGGAACAGCGGAGCCCTGACCTCCGGAGTGCATACTTTCCCGGCTGTGCTTCAGTCCTCTGGCCTGTACTCATTGTCCTCCGTGGTCACCGTCCCTTCGTCCTCCCTGGGCACCCAGACCTATATCTGTAATGTCAACCATAAGCCCTCGAACACCAAGGTCGACAAGAAGGTCGAGCCGAAGTCGTGCGACAAGACTCACACTTGCCCGCCTTGCCCAGCCCCGGAAGCTGCCGGTG GTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGG GAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCG TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCGGA

Amino acid sequence of light chain FS30-35-14 (SEQ ID NO: 107)

Variable domain (italics)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYYYSSPITFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

Light chain nucleic acid sequence FS30-35-14 (SEQ ID NO: 108)

GAAATTGTGCTGACCCAGTCTCCGGGCACGTTATCTCTGAGCCCTGGTGAGCGCGCCACTCTGTCATGCCGGGCTTCTCAAAGTGTTAGCAGTAGCTACCTGGCGTGGTATCAGCAAAAACCGGGCCAGGCCCCGCGTCTGCTGATTTACGGTGCATCCAGCCGTGCCACCGGCATTCCAGATCGTTTTTCCGTAGTGGTTCTGGACGGACTTCACTCTGACAATCTCACGCC TGGAACCGGAGGATTTTGCGGTGTATTACTGCCAGCAATATTATTATTCTTCTCCTATCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACTGTGGCCGCTCCTAGCGTGTTCATTTTTCCGCCATCCGACGAGCAGCTCAAGTCCGGCACCGCCTCCGTGGTCTGCCTGCTCAACAACTTCTACCCTCGCGAAGCTAAGGTCCAGTGGAAGGTCGACAATGCCCTGCAGTCCG GAAACTCGCAGGAAAGCGTGACTGAACAGGACTCCAAGGACTCCACCTATTCACTGTCCTCGACTCTGACCCTGAGCAAGGCGGATTACGAAAAGCACAAAGTGTACGCATGCGAAGTGACCCACCAGGGTCTTTCGTCCCCCGTGACCAAGAGCTTCAACAGAGGAGAGTGT

Heavy chain amino acid sequence of FS20-22-49AA/FS30-5-37 with LALA mutation (SEQ ID NO: 109)

Variable domain (italics), LALA mutation (bold underlined)

EVQLLESGGGLVQPGGSLRLNCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSYDKYWGSSIYSGLDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT AA WESNGDEQFAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy chain nucleic acid sequence FS20-22-49AA/FS30-5-37 with LALA mutation (SEQ ID NO: 110)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAATTGCGCGGCCAGTGGCTTTACCTTCAGTAGCTATGCCATGAGCTGGGTGCGTCAGGCGCCGGGCAAAGGTCTGGAATGGGTTAGCGCGATTAGCGGTAGTGGCGGTAGCACGTACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGC TGTACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGATCTTACGACAAATACTGGGGTTCTTCTATTTACTCTGGCTTGGACTACTGGGGCCAGGGAACC CTGGTCACCGTCTCGAGTGCTAGCACTAAGGGCCCGTCGGTGTTCCCGCTGGCCCCATCGTCCAAGAGCACATCAGGGGTACCGCCGCCCTGGGCTGCCTTGTGAAGGATTACTTTCCCGAG CCCGTCACAGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGAGTGCATACTTTCCCGGCTGTGCTTCAGTCCTCTGGCCTGTACTCATTGTCCTCCGTGGTCACCGTCCCTTCGTCCTCCCTGGGCACCCAGACCTATATCTGTAATGTCAACCATAAGCCCTCGAACACCAAGGTCGACAAGAAGGTCGAGCCGAAGTCGTGCGACAAGACTCACACTTGCCCGCCTTGCCCAGCCCCGGAAGC TGCCGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGA AATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGGGCTGCACCAAGACT CTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCGGA

Amino acid sequence of light chain FS30-5-37 (SEQ ID NO: 111)

Variable domain (italics)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYYSYYPVTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

Light chain nucleic acid sequence FS30-5-37 (SEQ ID NO: 112)

GAAATTGTGCTGACCCAGTCTCCGGGCACGTTATCTCTGAGCCCTGGTGAGCGCGCCACTCTGTCATGCCGGGCTTCTCAAAGTGTTAGCAGTAGCTACCTGGCGTGGTATCAGCAAAAACCGGGCCAGGCCCCGCGTCTGCTGATTTACGGTGCATCCAGCCGTGCCACCGGCATTCCAGATCGTTTTTCCGTAGTGGTTCTGGACGGACTTCACTCTGACAATCTCACGCC TGGAACCGGAGGATTTTGCGGTGTATTACTGCCAGCAATATTATTCTTATTATCCTGTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACTGTGGCCGCTCCTAGCGTGTTCATTTTTCCGCCATCCGACGAGCAGCTCAAGTCCGGCACCGCCTCCGTGGTCTGCCTGCTCAACAACTTCTACCCTCGCGAAGCTAAGGTCCAGTGGAAGGTCGACAATGCCCTGCAGTC CGGAAACTCGCAGGAAAGCGTGACTGAACAGGACTCCAAGGACTCCACCTATTCACTGTCCTCGACTCTGACCCTGAGCAAGGCGGATTACGAAAAGCACAAAGTGTACGCATGCGAAGTGACCCACCAGGGTCTTTCGTCCCCCGTGACCAAGAGCTTCAACAGAGGAGAGTGT

Alternative CH3 Domain Nucleic Acid Sequence Fcab FS20-22-49 (SEQ ID NO: 113)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATT ATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

Amino acid sequence of the heavy chain of the anti-FITC mAb G1AA/4420 containing the LALA mutation (SEQ ID NO: 114)

The provisions of the CDR are underlined. The position of the LALA mutation is shown in bold.

EVKLDETGGGLVQPGRPMKLSCVAS GFTFSDYW MNWVRQSPEKGLEWVAQ IRNKPYNYET YYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYC TGSYYGMDY WGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG AA DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of the heavy chain of mAb to FITC G1/4420 without the LALA mutation (SEQ ID NO: 115)

The provisions of the CDR are underlined.

EVKLDETGGGLVQPGRPMKLSCVAS GFTFSDYW MNWVRQSPEKGLEWVAQ IRNKPYNYET YYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYC TGSYYGMDY WGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of MAT 4420 light chain (SEQ ID NO: 116)

VL domain (italics)

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Amino acid sequence of the heavy chain of the G1AA/HelD1.3 antibody with the LALA mutation (SEQ ID NO: 117)

QVQLQESGPGLVRPSQTLSLTCTVSGSTFSGYGVNWVRQPPGRGLEWIGMIWGDGNTDYNSALKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCARERDYRLDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Amino acid sequence of light chain of MAT HelD1.3 (SEQ ID NO: 118)

VL domain (italics)

DIQMTQSPASLSSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPRTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC

Amino acid sequence of G1/MOR7480.1 heavy chain (SEQ ID NO: 119)

VH domain (italics)

EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of the light chain of MAT G1/MOR7480.1, G1AA/MOR7480.1 and G2/MOR7480.1 (SEQ ID NO: 120)

VL domain (italics)

SYELTQPPSVSVSPGQTASITCSGDNIGDQYAHWYQQKPGQSPVLVIYQDKNRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCATYTGFGSLAVFGGGTKLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS

Amino acid sequence of G1/20H4.9 heavy chain (SEQ ID NO: 121)

VH domain (italics)

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of light chain of MAT G1/20H4.9 and G1AA/20H4.9 (SEQ ID NO: 122)

VL domain (italics)

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Heavy chain amino acid sequence FS20-22-49AA/4420 (with LALA mutation) (SEQ ID NO: 123)

VH domain (italics); LALA mutation (bold and underlined)

EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWES NGDEQFAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of G2/MOR7480.1 heavy chain (SEQ ID NO: 124)

VH domain (italics)

EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDH KPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Heavy chain amino acid sequence of G1AA/MOR7480.1 (SEQ ID NO: 125)

Domain VH (italics) LALA (bold and underlined)

EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN AA PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of human CD137 (SEQ ID NO: 126)

Extracellular domain (italics); transmembrane and intracellular domains (bold)

LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ IISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Amino acid sequence of the extracellular domain of human CD137 (SEQ ID NO: 127)

LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ

Amino acid sequence of cynomolgus CD137 (SEQ ID NO: 128)

Extracellular domain (italics); transmembrane and intracellular domains (bold)

LQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQRTCDICRQCKGVFKTRKECSSTSNAECDCISGYHCLGAECSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSATPPAPAREPGHSPQ IIFFLALTSTVVLFLLFFLVLRFSVVKRSRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Amino acid sequence of the extracellular domain of cynomolgus CD137 (SEQ ID NO: 129)

LQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQRTCDICRQCKGVFKTRKECSSTSNAECDCISGYHCLGAECSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSATPPAPAREPGHSPQ

Amino acid sequence of human OX40 extracellular domain (SEQ ID NO: 130)

LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRA

Amino acid sequence of the cynomolgus monkey OX40 extracellular domain (SEQ ID NO: 131)

LHCVGDTYPSNDRCCQECRPGNGMVSRCNRSQNTVCRPCGPGFYNDVVSAKPCKACTWCNLRSGSERKQPCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPPTQPQETQGPPARPTTVQPTEAWPRTSQRPSTRPVEVPRGPA

Amino acid sequence of DO11.10-hOX40 and human OX40 receptor (SEQ ID NO: 132)

LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALY LLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

Amino acid sequence of DO11.10-mOX40 and mouse OX40 receptor (SEQ ID NO: 133)

VTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDTLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPAFAVLLGLGLGLLA PLTVLLALYLLRKAWRLPNTPKPCWGNSFRTPIQEEHTDAHFTLAKI

Amino acid sequence of DO11.10-cOX40 and cynomolgus OX40 receptor (SEQ ID NO: 134)

KLHCVGDTYPSNDRCCQECRPGNGMVSRCNRSQNTVCRPCGPGFYNDVVSAKPCKACTWCNLRSGSERKQPCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPPTQPQETQGPPARPTTVQPTEAWPRTSQRPSTRPVEVPRGPAVAAILGLGLALGLLGPLAMLLALL LLRRDQRLPPDAPKAPGGGSFRTPIQEEQADAHSALAKI

Amino acid sequence of human OX40-mFc (SEQ ID NO: 135)

IL-2 leader sequence (underlined), OX40 extracellular domain (italics), mouse IgG2a Fc domain (bold)

MYRMQLLSCIALSLALVTNS LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVA GSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

Amino acid sequence of mouse-mFc OX40 (SEQ ID NO: 136)

IL-2 leader sequence (underlined), OX40 extracellular domain (italics), mouse IgG2a Fc domain (bold)

MYRMQLLSCIALSLALVTNS VTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDTLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPA GSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

Amino acid sequence of OX40 cynomolgus macaque-mfc (SEQ ID NO: 137)

IL-2 leader sequence (underlined), OX40 extracellular domain (italics), mouse IgG2a Fc domain (bold)

MYRMQLLSCIALSLALVTNS LHCVGDTYPSNDRCCQECRPGNGMVSRCNRSQNTVCRPCGPGFYNDVVSAKPCKACTWCNLRSGSERKQPCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPPTQPQETQGPPARPTTVQPTEAWPRTSQRPSTRPVEVPRGPAVAAI GSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

Amino acid sequence of human CD137 sequence for use with recombinant CD137-mFc-Avi antigen (SEQ ID NO: 138)

SLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ

Amino acid sequence of cynomolgus CD137 sequence for use with recombinant CD137-mFc-Avi and CD137-Avi-His antigens (SEQ ID NO: 139)

SLQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQRTCDICRQCKGVFKTRKECSSTSNAECDCISGYHCLGAECSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSATPPAPAREPGHSPQ

Amino acid sequence of mouse CD137 sequence for use with recombinant CD137-mFc-Avi antigen (SEQ ID NO: 140)

AVQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPNCNICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCTRCEKDCRPGQELTKQGCKTCSLGTFNDQNGTGVCRPWTNCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTTISVTPEGGPGGHSLQVL

Amino acid sequence of mFc-Avi for use with recombinant CD137-mFc-Avis antigens (SEQ ID NO: 141)

Mouse Fc domain (italics) Avi label (bold)

PRGPTIKPCPPCKCPAPNLEGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKAFACAVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYS KLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK GGGLNDIFEAQKIEWHE

Amino acid sequence of the truncated Fcab hinge region (SEQ ID NO: 101)

TCPPCP

Alternative CH3 Domain Nucleic Acid Sequence Fcab FS20-22-49 (SEQ ID NO: 143)

GGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATT ATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

Heavy Chain Amino Acid Sequence FS20-22-49/FS30-5-37 (no LALA) (SEQ ID NO: 144)

EVQLLESGGGLVQPGGSLRLNCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSYDKYWGSSIYSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWES NGDEQFAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy Chain DNA Sequence FS20-22-49/FS30-5-37 (no LALA) (SEQ ID NO: 145)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAATTGCGCGGCCAGTGGCTTTACCTTCAGTAGCTATGCCATGAGCTGGGTGCGTCAGGCGCCGGGCAAAGGTCTGGAATGGGTTAGCGCGATTAGCGGTAGTGGCGGTAGCACGTACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGC TGTACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGATCTTACGACAAATACTGGGGTTCTTCTATTTACTCTGGCTTGGACTACTGGGGCCAGGGAACC CTGGTCACCGTCTCGAGTGCTAGCACTAAGGGCCCGTCGGTGTTCCCGCTGGCCCCATCGTCCAAGAGCACATCAGGGGTACCGCCGCCCTGGGCTGCCTTGTGAAGGATTACTTTCCCGAG CCCGTCACAGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGAGTGCATACTTTCCCGGCTGTGCTTCAGTCCTCTGGCCTGTACTCATTGTCCTCCGTGGTCACCGTCCCTTCGTCCTCCCTGGGCACCCAGACCTATATCTGTAATGTCAACCATAAGCCCTCGAACACCAAGGTCGACAAGAAGGTCGAGCCGAAGTCGTGCGACAAGACTCACACTTGCCCGCCTTGCCCAGCCCCGGAACT GCTGGGTGGTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGA AATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGGGCTGCACCAAGACT CTGAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCGGA

Heavy Chain Amino Acid Sequence FS20-22-49/FS30-10-3 (no LALA) (SEQ ID NO: 146)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSDIDPTGSKTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLNVYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWESNGDEQFAY KTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy Chain DNA Sequence FS20-22-49/FS30-10-3 (no LALA) (SEQ ID NO: 147)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTAGTTACGATATGAGCTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTGATCCGACTGGTAGCAAGACCGACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGCTG TACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAGACCTCAATGTGTACGGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCTAGCACTAAGGGCCCGTCGGTGTTCCCGCTGGCCCCATCGTCCAAGAGCACATCAGGGGGGTACCGCCGCCCTGGGCTGCCTTGTGAAGGATTACTTTCCCGAGCCCGTCACAGTGTCCTGGAACA GCGGAGCCCTGACCTCCGGAGTGCATACTTTCCCGGCTGTGCTTCAGTCCTCTGGCCTGTACTCATTGTCCTCCGTGGTCACCGTCCCTTCGTCCTCCCTGGGCACCCAGACCTATATCTGTAATGTCAACCATAAGCCCTCGAACACCAAGGTCGACAAGAAGGTCGAGCCGAAGTCGTGCGACAAGACTCACACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTG TTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGAT GGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAG TGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCGGA

Heavy Chain Amino Acid Sequence FS20-22-49/FS30-10-12 (no LALA) (SEQ ID NO: 148)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSDIDPTGSKTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLTVYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWESNGDEQFAY KTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy chain DNA sequence FS20-22-49/FS30-10-12 (no LALA) (SEQ ID NO: 149)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTAGTTACGATATGAGCTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTGATCCGACTGGTAGCAAGACCGACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGCTG TACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAGACCTCACGGTGTACGGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCTAGCACTAAGGGCCCGTCGGTGTTCCCGCTGGCCCCATCGTCCAAGAGCACATCAGGGGGTACCGCCGCCCTGGGCTGCCTTGTGAAGGATTACTTTCCCGAGCCCGTCACAGTGTCCTGGAACA GCGGAGCCCTGACCTCCGGAGTGCATACTTTCCCGGCTGTGCTTCAGTCCTCTGGCCTGTACTCATTGTCCTCCGTGGTCACCGTCCCTTCGTCCTCCCTGGGCACCCAGACCTATATCTGTAATGTCAACCATAAGCCCTCGAACACCAAGGTCGACAAGAAGGTCGAGCCGAAGTCGTGCGACAAGACTCACACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTG TTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGAT GGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAG TGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

Heavy Chain Amino Acid Sequence FS20-22-49/FS30-10-16 (no LALA) (SEQ ID NO: 150)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSDIDPTGSKTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWESNGDEQFAY KTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy Chain DNA Sequence FS20-22-49/FS30-10-16 (no LALA) (SEQ ID NO: 151)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTAGTTACGATATGAGCTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTGATCCGACTGGTAGCAAGACCGACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGCTG TACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAGACCTCTTGGTGTACGGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCTAGCACTAAGGGCCCGTCGGTGTTCCCGCTGGCCCCATCGTCCAAGAGCACATCAGGGGGGTACCGCCGCCCTGGGCTGCCTTGTGAAGGATTACTTTCCCGAGCCCGTCACAGTGTCCTGGAACA GCGGAGCCCTGACCTCCGGAGTGCATACTTTCCCGGCTGTGCTTCAGTCCTCTGGCCTGTACTCATTGTCCTCCGTGGTCACCGTCCCTTCGTCCTCCCTGGGCACCCAGACCTATATCTGTAATGTCAACCATAAGCCCTCGAACACCAAGGTCGACAAGAAGGTCGAGCCGAAGTCGTGCGACAAGACTCACACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTGGTCCTTCGGTG TTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGAT GGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGGGAAGGAGTACAAG TGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCCGGA

Heavy Chain Amino Acid Sequence FS20-22-49/FS30-35-14 (no LALA) (SEQ ID NO: 152)

EVQLLESGGGLVQPGGSLRLSCAASGTFFSAYNIHWVRQAPGKGLEWVSDISPYGGATNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNLYELSAYSYGADYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEYWDQEVSLTCLVKGFYPSDIAVEWESNGDE QFAYKTTPPVLDSDGSFFLYSKLTVDQYRWNPADYFSCSVMHEALHNHYTQKSLSLSPG

Heavy Chain DNA Sequence FS20-22-49/FS30-35-14 (no LALA) (SEQ ID NO: 153)

GAAGTGCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGTGGTTCTCTGCGTCTGAGTTGCGCGGCCAGTGGCTTTACCTTCAGTGCCTATAATATCCATTGGGTGCGTCAGGCTCCGGGCAAAGGTCTGGAATGGGTTAGCGATATTTCTCCGTATGGTGGCGCGACCAACTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCTCGCGACAACAGCAAGAACACGCTG TACCTGCAGATGAACTCACTGCGTGCCGAAGATACGGCCGTGTATTACTGTGCGAGAAACCTCTACGAGTTGAGCGCTTACTCTTACGGGGCGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGTCGGCTAGCACTAAGGGCCCGTCGGTGTTCCCGCTGGCCCCATCGTCCAAGAGCACATCAGGGGGTACCGCCGCCCTGGGCTGCCTTGTGAAGGATTACTTTCCCGAGCCCGTCACAG TGTCCTGGAACAGCGGAGCCCTGACCTCCGGAGTGCATACTTTCCCGGCTGTGCTTCAGTCCTCTGGCCTGTACTCATTGTCCTCCGTGGTCACCGTCCCTTCGTCCTCCCTGGGCACCCAGACCTATATCTGTAATGTCAACCATAAGCCCTCGAACACCAAGGTCGACAAGAAGGTCGAGCCGAAGTCGTGCGACAAGACTCACACTTGCCCGCCTTGCCCAGCCCCGGAACTGCTGGGTG GTCCTTCGGTGTTCCTCTTCCCGCCCAAGCCGAAGGATACCCTGATGATCTCACGGACCCCCGAAGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCGGAAGTGAAATTCAATTGGTACGTGGATGGAGTGGAAGTGCACAACGCCAAGACCAAGCCACGGGAAGAACAGTACAACTCTACCTACCGCGTGGTGTCCGTGCTCACTGTGCTGCACCAAGACTGGCTGAACGG GAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCGCTGCCTGCCCCAATTGAGAAAACTATCTCGAAAGCCAAGGGACAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTACTGGGACCAGGAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGGATGAACAGTTCGCATACAAGACCACGCCTCCCG TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGATCAGTATAGGTGGAATCCTGCTGATTATTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCTTGTCCCTGTCGCCGGA

Heavy chain amino acid sequence of MAT G1AA/FS30-10-16 (with LALA) (SEQ ID NO: 154)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSDIDPTGSKTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLLVYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of MAT light chain G1AA/FS30-10-16 (SEQ ID NO: 97)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSYSYPVTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

Amino acid sequence of MAT G1AA/OX86 heavy chain (with LALA) (SEQ ID NO: 155)

QVQLKESGPGLVQPSQTLSLTCTVSGFSLTGYNLHWVRQPPGKGLEWMGRMRYDGDTYYNSVLKSRLSISRDTSKNQVFLKMNSLQTDDTAIYYCTRDGRGDSFDYWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of light chain of MAT G1/OX86 and G1AA/OX86 (SEQ ID NO: 156)

DIVMTQGALPNPVPSGESASITCRSSQSLVYKDGQTYLNWFLQRPGQSPQLLTYWMSTRASGVSDRFSGSGGTYFTLKISRVRAEDAGVYYCQQVREYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy Chain Amino Acid Sequence FS20m-232-91AA/4420 (with LALA) (SEQ ID NO: 157)

EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELFDPMYYYNQVSLTCLVKGFYPSDI AVEWESNGEPLWDYKTTPPVLDSDGSFFLYSKLTVWRDRWEDGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain amino acid sequence FS20m-232-91AA/4420 (SEQ ID NO: 116)

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Amino acid sequence of human CD137-Avi-His (SEQ ID NO: 158)

CD137 extracellular domain (bold); Avi label (italics); His mark (underlined)

SLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQGS GGGLNDIFEAQKIEWHE HHHHHH

Amino acid sequence of MAT G1/OX86 heavy chain (without LALA) (SEQ ID NO: 159)

QVQLKESGPGLVQPSQTLSLTCTVSGFSLTGYNLHWVRQPPGKGLEWMGRMRYDGDIYYNSVLKSRLSISRDTSKNQVFLKMNSLQTDDTAIYYCTRDGRGDSFDYWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Anti-PD-1 mAb heavy chain amino acid sequence G1AA/5C4 (SEQ ID NO: 160)

Variable domain (bold)

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-PD-1 mAb light chain amino acid sequence G1AA/5C4 (SEQ ID NO: 161)

Variable domain (bold)

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Anti-PD-L1 mAb heavy chain amino acid sequence G1AA/S1 (SEQ ID NO: 162)

Variable domain (bold)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-PD-L1 mAb light chain amino acid sequence G1AA/S1 (SEQ ID NO: 163)

Variable domain (bold)

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLFTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

Amino acid sequence of mouse CD137 (SEQ ID NO: 164)

Extracellular domain (italics); transmembrane and intracellular domains (bold)

VQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPNCNICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCTRCEKDCRPGQELTKQGCKTCSLGTFNDQNGTGVCRPWTNCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTTISVTPEGGPGGHSLQVL TLFLALTSALLLALIFITLLFSVLKWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGGGGYEL

Amino acid sequence of the heavy chain of MAT G1AA/20H4.9 (SEQ ID NO: 165)

VH domain (italics)

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of the heavy chain of MAT G1AA/3H3 (SEQ ID NO: 166)

VH domain (italics)

EMQLVESGGGLVQPGRSMKLSCAGSGFTLSDYGVAWVRQAPKKGLEWVAYISYAGGTTYYRESVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCTIDGYGGYSGSHWYFDFWGPGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Amino acid sequence of the light chain of MAT G1AA/3H3 and G1/3H3 and MAT ² FS20m-232-91AA/3H3 (SEQ ID NO: 167)

VL domain (italics)

DIQMTQSPSLLSASVGDRVTLNCRTSQNVYKNLAWYQQQLGEAPKLLIYNANSLQAGIPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQYYSGNTFGAGTNLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

Amino acid sequence of MAT G1/3H3 heavy chain (SEQ ID NO: 168)

VH domain (italics)

EMQLVESGGGLVQPGRSMKLSCAGSGFTLSDYGVAWVRQAPKKGLEWVAYISYAGGTTYYRESVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCTIDGYGGYSGSHWYFDFWGPGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Heavy Chain Amino Acid Sequence FS20m-232-91AA/3H3 (with LALA) (SEQ ID NO: 169)

VH domain (italics)

EMQLVESGGGLVQPGRSMKLSCAGSGFTLSDYGVAWVRQAPKKGLEWVAYISYAGGTTYYRESVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCTIDGYGGYSGSHWYFDFWGPGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELFDPMYYYNQVSLTCLVKGFYPS DIAVEWESNGEPLWDYKTTPPVLDSDGSFFLYSKLTVWRDRWEDGNVFSCSVMHEALHNHYTQKSLSLSPGK

Amino acid sequence of the heavy chain of the anti-OX40 mAb G1AA/11D4 (SEQ ID NO: 173)

VH domain (italics)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIDYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARESGWYLFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELRFYQVSLTCLVKGFYPSDIAVEWESNGQPDIFPN GLNYKTTPPVLDSDGSFFLYSKLTVPYPSWLMGTRFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of the heavy chain of MAb to OX40 G1/11D4 (SEQ ID NO: 174)

VH domain (italics)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIDYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARESGWYLFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELRFYQVSLTCLVKGFYPSDIAVEWESNGQPDIFPN GLNYKTTPPVLDSDGSFFLYSKLTVPYPSWLMGTRFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of the light chain of MAbs to OX40 G1AA/11D4 and G1/11D4 (SEQ ID NO: 175)

VL domain (italics)

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

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Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23(9), 1126-36 (2005).

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Idusogie EE, Presta LG, Gazzano-Santoro H, Totpal K, Wong PY, Ultsch M, Meng YG, Mulkerrin MG. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J. Immunol. 164(8), 4178-84 (2000).

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Lee SJ, Myers L, Muralimohan G, Dari J, Qiao Y, Li Z, Mittler RS, Vella AT. 4-1BB and OX40 dual costimulation synergistically stimulate primary specific CD8 T cells for robust effector function. J. Immunol. 173(5), 3002-12 (2004).

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Lefranc MP, Giudicelli V, Duroux P, Jabado-Michaloud J, Folch G, Aouinti S, Carillon E, Duvergey H, Houles A, Paysan-Lafosse T, Hadi-Saljoqi S, Sasorith S, Lefranc G, Kossida S. IMGT® , the international ImMunoGeneTics information system® 25 years on. Nucleic Acids Res. 43(Database issue), D413-22 (2015).

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Ma BY, Mikolajczak SA, Danesh A, Hosiawa KA, Cameron CM, Takaori-Kondo A, Uchiyama T, Kelvin DJ, Ochi A. The expression and the regulatory role of OX40 and 4-1BB heterodimer in activated human T cells. Blood 106(6), 2002-10 (2005).

Mayes PA, Hance KW, Hoos A. The promise and challenges of immune agonist antibody development in cancer. Nat. Rev. Drug Discov. 17, 509-27 (2018).

Melero I, Hirschhorn-Cymerman D, Morales-Kastresana A, Sanmamed MF, Wolchok JD. Agonist antibodies to TNFR molecules that costimulate T and NK cells. Clin. Cancer Res. 19(5), 1044-53 (2013).

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Miller L, Rymarchyk N, Kelly T, Crocenzi T, Bernstein E, Sanborn R, Urba WJ, Weinberg AD. OX40 is a potent immune-stimulating target in late-stage cancer patients. Cancer Res. 73(24), 7189-98 (2013).

Moran AE, Kovacsovics-Bankowski M, Weinberg AD. The TNFRs OX40, 4-1BB, and CD40 as targets for cancer immunotherapy. Curr. Opin. Immunol. 25(2), 230-7 (2013).

Müller N, Wyzgol A, Münkel S, Pfizenmaier K, Wajant H. Activity of soluble OX40 ligand is enhanced by oligomerization and cell surface immobilization. FEBS J. 275(9), 2296-304 (2008).

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List of sequences

<110> F-STAR BETA LIMITED

<120> Antibody molecules that bind to CD137 and OX40

<130> 007508237

<140> PCT/EP2019/068796

<141> 2019-07-12

<150> GB 1811407.4

<151> 2018-07-12

<150> GB 1818281.6

<151> 2018-11-09

<150> GB 1902598.0

<151> 2019-02-26

<160> 175

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 VH from MAT FS30-10-16 (IMGT)

<400> 1

Gly Phe Thr Phe Ser Ser Tyr Asp

15

<210> 2

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 VH MAT FS30-10-16 (IMGT)

<400> 2

Ile Asp Pro Thr Gly Ser Lys Thr

15

<210> 3

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-10-16 (IMGT)

<400> 3

Ala Arg Asp Leu Leu Val Tyr Gly Phe Asp Tyr

1 5 10

<210> 4

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 VL MAT FS30-10-16 (IMGT)

<400> 4

Gln Ser Val Ser Ser Ser Tyr

15

<210> 5

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 VL MAT FS30-10-16 (IMGT)

<400> 5

Gly Ala Ser

1

<210> 6

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VL MAT FS30-10-16 (IMGT)

<400> 6

Gln Gln Ser Tyr Ser Tyr Pro Val Thr

15

<210> 7

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 VH MAT FS30-10-16 (Kabat)

<400> 7

Ser Tyr Asp Met Ser

15

<210> 8

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 VH MAT FS30-10-16 (Kabat)

<400> 8

Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 9

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-10-16 (Kabat)

<400> 9

Asp Leu Leu Val Tyr Gly Phe Asp Tyr

15

<210> 10

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 VL MAT FS30-10-16 (Kabat)

<400> 10

Gly Ala Ser Ser Arg Ala Thr

15

<210> 11

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 VL MAT FS30-10-16 (Kabat)

<400> 11

Gln Gln Ser Tyr Ser Tyr Pro Val Thr

15

<210> 12

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-10-16

<400> 12

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Leu Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 13

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-10-16

<400> 13

gaagttcagc tgctggaatc tggcggcgga ttggttcaac ctggcggctc tctgagactg

60

tcttgtgccg cttccggctt caccttctcc agctacgaca tgtcctgggt ccgacaggct

120

cctggcaaag gactggaatg ggtgtccgac atcgacccca ccggctctaa gaccgactac

180

gccgattctg tgaagggcag attcaccatc agccgggaca actccaagaa caccctgtac

240

ctgcagatga actccctgag agccgaggac accgccgtgt actactgtgc cagagatctg

300

ctggtgtacg gcttcgacta ttggggccag ggcacactgg tcaccgtgtc ctct

354

<210> 14

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Variable domain light chain MAT FS30-10-16

<400> 14

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Tyr Pro

85 90 95

Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Variable domain light chain MAT FS30-10-16

<400> 15

gagatcgtgc tgacccagtc tcctggcaca ctgtcactgt ctccaggcga gagagctacc

60

ctgtcctgta gagcctctca gtccgtgtcc tcctcttacc tggcctggta tcagcagaag

120

cctggacagg ctccccggct gttgatctac ggcgcttctt ctagagccac aggcatccct

180

gaccggttct ccggatctgg ctctggcacc gatttcaccc tgaccatctc tcggctggaa

240

cccgaggatt tcgccgtgta ctactgccag cagtcctaca gctaccccgt gacctttggc

300

caggcacca aggtggaaat caag

324

<210> 16

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-10-3 (IMGT)

<400> 16

Ala Arg Asp Leu Asn Val Tyr Gly Phe Asp Tyr

1 5 10

<210> 17

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-10-3 (Kabat)

<400> 17

Asp Leu Asn Val Tyr Gly Phe Asp Tyr

15

<210> 18

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-10-3

<400> 18

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Asn Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 19

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-10-3

<400> 19

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt agttacgata tgagctgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat attgatccga ctggtagcaa gaccgactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagagacctc

300

aatgtgtacg ggttcgacta ctggggccag ggaaccctgg tcaccgtctc gagt

354

<210> 20

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain variable domain of MAT FS30-10-3

<400> 20

gaaattgtgc tgacccagtc tccggggcacg ttatctctga gccctggtga gcgcgccact

60

ctgtcatgcc gggcttctca aagtgttagc agtagctacc tggcgtggta tcagcaaaaa

120

ccgggccagg ccccgcgtct gctgatttac ggtgcatcca gccgtgccac cggcattcca

180

gatcgttttt ccggtagtgg ttctgggacg gacttcactc tgacaatctc acgcctggaa

240

ccggaggatt ttgcggtgta ttactgccag caatcttatt cttatcctgt cacgttcggc

300

caagggacca aggtggaaat caaa

324

<210> 21

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-10-12 (IMGT)

<400> 21

Ala Arg Asp Leu Thr Val Tyr Gly Phe Asp Tyr

1 5 10

<210> 22

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-10-12 (Kabat)

<400> 22

Asp Leu Thr Val Tyr Gly Phe Asp Tyr

15

<210> 23

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-10-12

<400> 23

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Thr Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 24

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-10-12

<400> 24

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt agttacgata tgagctgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat attgatccga ctggtagcaa gaccgactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagagacctc

300

acggtgtacg ggttcgacta ctggggccag ggaaccctgg tcaccgtctc gagt

354

<210> 25

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 VH MAT FS30-35-14 (IMGT)

<400> 25

Gly Phe Thr Phe Ser Ala Tyr Asn

15

<210> 26

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 VH MAT FS30-35-14 (IMGT)

<400> 26

Ile Ser Pro Tyr Gly Gly Ala Thr

15

<210> 27

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-35-14 (IMGT)

<400> 27

Ala Arg Asn Leu Tyr Glu Leu Ser Ala Tyr Ser Tyr Gly Ala Asp Tyr

1 5 10 15

<210> 28

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VL MAT FS30-35-14 (IMGT)

<400> 28

Gln Gln Tyr Tyr Tyr Ser Ser Pro Ile Thr

1 5 10

<210> 29

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 VH MAT FS30-35-14 (Kabat)

<400> 29

Ala Tyr Asn Ile His

15

<210> 30

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 VH MAT FS30-35-14 (Kabat)

<400> 30

Asp Ile Ser Pro Tyr Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 31

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-35-14 (Kabat)

<400> 31

Asn Leu Tyr Glu Leu Ser Ala Tyr Ser Tyr Gly Ala Asp Tyr

1 5 10

<210> 32

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<223> Variable domain of light chain MAT FS30-35-14

<400> 32

gaaattgtgc tgacccagtc tccggggcacg ttatctctga gccctggtga gcgcgccact

60

ctgtcatgcc gggcttctca aagtgttagc agtagctacc tggcgtggta tcagcaaaaa

120

ccgggccagg ccccgcgtct gctgatttac ggtgcatcca gccgtgccac cggcattcca

180

gatcgttttt ccggtagtgg ttctgggacg gacttcactc tgacaatctc acgcctggaa

240

ccggaggatt ttgcggtgta ttactgccag caatattatt attcttctcc tatcacgttc

300

ggccaaggga ccaaggtgga aatcaaa

327

<210> 33

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 VH MAT FS30-5-37 (IMGT)

<400> 33

Gly Phe Thr Phe Ser Ser Tyr Ala

15

<210> 34

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 VH MAT FS30-5-37 (IMGT)

<400> 34

Ile Ser Gly Ser Gly Gly Ser Thr

15

<210> 35

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-5-37 (IMGT)

<400> 35

Ala Arg Ser Tyr Asp Lys Tyr Trp Gly Ser Ser Ile Tyr Ser Gly Leu

1 5 10 15

Asp Tyr

<210> 36

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VL MAT FS30-5-37 (IMGT)

<400> 36

Gln Gln Tyr Tyr Ser Tyr Tyr Pro Val Thr

1 5 10

<210> 37

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 VH MAT FS30-5-37 (Kabat)

<400> 37

Ser Tyr Ala Met Ser

15

<210> 38

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 VH MAT FS30-5-37 (Kabat)

<400> 38

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 39

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 VH MAT FS30-5-37 (Kabat)

<400> 39

Ser Tyr Asp Lys Tyr Trp Gly Ser Ser Ile Tyr Ser Gly Leu Asp Tyr

1 5 10 15

<210> 40

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-5-37

<400> 40

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Asn Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Tyr Asp Lys Tyr Trp Gly Ser Ser Ile Tyr Ser Gly Leu

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 41

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-5-37

<400> 41

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

aattgcgcgg ccagtggctt taccttcagt agctatgcca tgagctgggt gcgtcaggcg

120

ccgggcaaag gtctggaatg ggttagcgcg attagcggta gtggcggtag cacgtactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagatcttac

300

gacaaatact ggggttcttc tatttactct ggcttggact actggggcca gggaaccctg

360

gtcaccgtct cgagt

375

<210> 42

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Variable domain of the light chain of MAT FS30-5-37

<400> 42

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Tyr

85 90 95

Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 43

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain variable domain of MAT FS30-5-37

<400> 43

gaaattgtgc tgacccagtc tccggggcacg ttatctctga gccctggtga gcgcgccact

60

ctgtcatgcc gggcttctca aagtgttagc agtagctacc tggcgtggta tcagcaaaaa

120

ccgggccagg ccccgcgtct gctgatttac ggtgcatcca gccgtgccac cggcattcca

180

gatcgttttt ccggtagtgg ttctgggacg gacttcactc tgacaatctc acgcctggaa

240

ccggaggatt ttgcggtgta ttactgccag caatattatt cttattatcc tgtcacgttc

300

ggccaaggga ccaaggtgga aatcaaa

327

<210> 44

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CH3 WT domain AB loop

<400> 44

Arg Asp Glu Leu Thr Lys Asn Gln

15

<210> 45

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CH3 WT CD domain loop

<400> 45

Ser Asn Gly Gln Pro Glu Asn Asn Tyr

15

<210> 46

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CH3 WT domain EF loop

<400> 46

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

1 5 10

<210> 47

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CH3 WT domain

<400> 47

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 48

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CH2 domain

<400> 48

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105 110

<210> 49

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CH2 domain with LALA mutation

<400> 49

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105 110

<210> 50

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CH2 domain with LALA mutation and P114A mutation

<400> 50

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105 110

<210> 51

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-22-49

<400> 51

Tyr Trp Asp Gln Glu

15

<210> 52

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-22-49

<400> 52

Asp Glu Gln Phe Ala

15

<210> 53

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-22-49

<400> 53

Gln Tyr Arg Trp Asn Pro Ala Asp Tyr

15

<210> 54

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-49

<400> 54

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Asp Glu Gln

35 40 45

Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr Arg Trp Asn Pro Ala

65 70 75 80

Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 55

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-49

<400> 55

ggccagccta gggaacccca ggtttacacc ttgcctccaa gccgggacga gtactgggat

60

caagaggtgt ccctgacctg cctcgtgaag ggcttctacc cttccgatat cgccgtggaa

120

tggggagagca atggcgacga gcagttcgcc tacaagacaa cccctcctgt gctggactcc

180

gacggctcat tctttctgta ctccaagctg acagtggacc agtacagatg gaaccccgcc

240

gactacttct cttgctccgt gatgcacgag gccctgcaca accactacac acagaagtcc

300

ctgtctctgt cccctggc

318

<210> 56

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> AB loop domain CH3 Fcab FS20-22-49

<400> 56

Arg Asp Glu Tyr Trp Asp Gln Glu

15

<210> 57

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD domain loop CH3 Fcab FS20-22-49

<400> 57

Ser Asn Gly Asp Glu Gln Phe Ala Tyr

15

<210> 58

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CH3 domain EF loop Fcab FS20-22-49

<400> 58

Asp Gln Tyr Arg Trp Asn Pro Ala Asp Tyr

1 5 10

<210> 59

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-22-38

<400> 59

Ala Glu Lys Tyr Gln

15

<210> 60

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-22-38

<400> 60

Gln Tyr Arg Trp Asn Pro Gly Asp Tyr

15

<210> 61

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-38

<400> 61

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Ala Glu Lys

35 40 45

Tyr Gln Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr Arg Trp Asn Pro Gly

65 70 75 80

Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 62

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-38

<400> 62

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtactgggac

60

caggaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atggggcaga aaaataccag tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtggatc agtataggtg gaacccaggc

240

gactatttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc

300

ctctccctgt ctccggggt

318

<210> 63

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-41

<400> 63

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Asp Glu Gln

35 40 45

Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr Arg Trp Asn Pro Gly

65 70 75 80

Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 64

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-41

<400> 64

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtactgggac

60

caggaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atggggatga acagttcgca tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtggatc agtataggtg gaacccaggc

240

gactatttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc

300

ctctccctgt ctccggga

318

<210> 65

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-22-47

<400> 65

Gln Tyr Arg Trp Ser Pro Gly Asp Tyr

15

<210> 66

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-47

<400> 66

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Asp Glu Gln

35 40 45

Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr Arg Trp Ser Pro Gly

65 70 75 80

Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 67

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-47

<400> 67

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtactgggac

60

caggaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atggggatga acagttcgca tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtggatc agtataggtg gagtccgggt

240

gattatttct catgctccgt gatgcatgag gctctgcaca accactacac tcagaagagc

300

ttgtccctgt cgcccgga

318

<210> 68

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-22-85

<400> 68

Gln Tyr Arg Trp Asn Pro Phe Asp Asp

15

<210> 69

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-85

<400> 69

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Asp Glu Gln

35 40 45

Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Leu Asp Gln Tyr Arg Trp Asn Pro Phe

65 70 75 80

Asp Asp Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 70

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-85

<400> 70

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtactgggac

60

caggaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atggggatga acagttcgca tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accttggatc agtataggtg gaatccgttt

240

gatgatttct catgctccgt gatgcatgag gctctgcaca accactacac tcagaagagc

300

ttgtccctgt cgcccgga

318

<210> 71

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-58

<400> 71

Tyr Tyr Ser Gly Glu

15

<210> 72

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-58

<400> 72

Gln Pro Glu Asn Asp

15

<210> 73

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-58

<400> 73

Pro Tyr Trp Arg Trp Gly Ser Pro Arg Thr

1 5 10

<210> 74

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 FS20-31-58

<400> 74

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Tyr Tyr Ser Gly Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asp Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Trp Arg Trp Gly Ser Pro

65 70 75 80

Arg Thr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 75

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 FS20-31-58

<400> 75

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtactactct

60

ggtgaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atgggcagcc ggagaacgac tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtgcctt attggaggtg gggtagtccg

240

cgtactttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc

300

ctctccctgt ctccggggt

318

<210> 76

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-66

<400> 76

Pro Tyr Trp Arg Trp Gly Val Pro Arg Thr

1 5 10

<210> 77

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 FS20-31-66

<400> 77

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Tyr Tyr Ser Gly Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asp Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Trp Arg Trp Gly Val Pro

65 70 75 80

Arg Thr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 78

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 FS20-31-66

<400> 78

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtactactct

60

ggtgaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atgggcagcc ggagaacgac tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtgccgt attggaggtg gggtgttccg

240

cgtactttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc

300

ctctccctgt ctccggggt

318

<210> 79

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-94 Fcab

<400> 79

Trp Glu His Gly Glu

15

<210> 80

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-94 Fcab

<400> 80

Ile Arg Glu His Asp

15

<210> 81

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-94 Fcab

<400> 81

Pro Tyr Trp Arg Trp Gly Gly Pro Gly Thr

1 5 10

<210> 82

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-31-94 Fcab

<400> 82

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Trp Glu His Gly Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Ile Arg Glu

35 40 45

His Asp Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Trp Arg Trp Gly Gly Pro

65 70 75 80

Gly Thr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 83

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-31-94 Fcab

<400> 83

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtgggaacat

60

ggtgaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atgggatcag agaacatgat tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtgccat attggaggtg gggcggccca

240

ggcaccttct catgctccgt gatgcatgag gctctgcaca accactacac tcagaagagc

300

ttgtccctgt cgcccgga

318

<210> 84

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-102

<400> 84

Trp Ala Ser Gly Glu

15

<210> 85

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-102

<400> 85

Gln Pro Glu Val Asp

15

<210> 86

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-31-102

<400> 86

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Trp Ala Ser Gly Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Val Asp Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Trp Arg Trp Gly Val Pro

65 70 75 80

Arg Thr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 87

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-31-102

<400> 87

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtgggcatct

60

ggtgaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atgggcagcc agaagttgat tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtgccgt attggaggtg gggtgttccg

240

cgtactttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc

300

ctctccctgt ctccggggt

318

<210> 88

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-108

<400> 88

Glu Lys Glu Ile Asp

15

<210> 89

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-108

<400> 89

Pro Tyr Trp Arg Trp Gly Ala Lys Arg Thr

1 5 10

<210> 90

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-31-108

<400> 90

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Trp Ala Ser Gly Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Glu Lys Glu

35 40 45

Ile Asp Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Trp Arg Trp Gly Ala Lys

65 70 75 80

Arg Thr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 91

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-31-108

<400> 91

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtgggcatct

60

ggtgaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atggggaaaa agaaatcgat tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtgccgt attggaggtg gggtgctaag

240

cgtactttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc

300

ctctccctgt ctccggggt

318

<210> 92

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fcab CH3 domain structural loop sequence

FS20-31-115

<400> 92

Glu Gln Glu Phe Asp

15

<210> 93

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-31-115

<400> 93

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Trp Ala Ser Gly Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Glu Gln Glu

35 40 45

Phe Asp Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Trp Arg Trp Gly Ala Lys

65 70 75 80

Arg Thr Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 94

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-31-115

<400> 94

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtgggcatct

60

ggtgaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atggggaaca ggaattcgat tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtgccgt attggaggtg gggtgctaag

240

cgtactttct catgctccgt gatgcatgag gctctgcaca accactacac tcagaagagc

300

ttgtccctgt cgcccgga

318

<210> 95

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-10-16 with LALA mutation

<400> 95

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Leu Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Asp Glu Gln Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr

405 410 415

Arg Trp Asn Pro Ala Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 96

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-10-16 with LALA mutation

<400> 96

gaagttcagc tgctggaatc tggcggcgga ttggttcaac ctggcggctc tctgagactg

60

tcttgtgccg cttccggctt caccttctcc agctacgaca tgtcctgggt ccgacaggct

120

cctggcaaag gactggaatg ggtgtccgac atcgacccca ccggctctaa gaccgactac

180

gccgattctg tgaagggcag attcaccatc agccgggaca actccaagaa caccctgtac

240

ctgcagatga actccctgag agccgaggac accgccgtgt actactgtgc cagagatctg

300

ctggtgtacg gcttcgacta ttggggccag ggcacactgg tcaccgtgtc ctctgcttct

360

accaagggac ccagcgtgtt ccctctggct ccttccagca agtctacctc tggcggaaca

420

gctgctctgg gctgcctggt caaggactac tttcctgagc ctgtgaccgt gtcttggaac

480

tctggcgctc tgacatctgg cgtgcacacc tttccagcag tgctgcagtc ctccggcctg

540

tactctctgt cctctgtcgt gaccgtgcct tccagctctc tgggaaccca gacctacatc

600

tgcaatgtga accacaagcc ttccaacacc aaggtggaca agaaggtgga acccaagtcc

660

tgcgacaaga cccacacctg tcctccatgt cctgctccag aagctgctgg cggcccttcc

720

gtgtttctgt tccctccaaa gcctaaggac accctgatga tctctcggac ccctgaagtg

780

acctgcgtgg tggtggatgt gtctcacgag gacccagaag tgaagttcaa ttggtacgtg

840

gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc

900

tacagagtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac

960

aagtgcaagg tgtccaacaa ggccctgcct gctcctatcg aaaagaccat ctccaaggcc

1020

aagggccagc ctagggaacc ccaggtttac accttgcctc caagccggga cgagtactgg

1080

gatcaagagg tgtccctgac ctgcctcgtg aagggcttct acccttccga tatcgccgtg

1140

gaatgggaga gcaatggcga cgagcagttc gcctacaaga caacccctcc tgtgctggac

1200

tccgacggct cattctttct gtactccaag ctgacagtgg accagtacag atggaacccc

1260

gccgactact tctcttgctc cgtgatgcac gaggccctgc acaaccacta cacacagaag

1320

tccctgtctc tgtcccctgg c

1341

<210> 97

<211> 215

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain FS30-10-16 (

<400> 97

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Tyr Pro

85 90 95

Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 98

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain FS30-10-16 (

<400> 98

gagatcgtgc tgacccagtc tcctggcaca ctgtcactgt ctccaggcga gagagctacc

60

ctgtcctgta gagcctctca gtccgtgtcc tcctcttacc tggcctggta tcagcagaag

120

cctggacagg ctccccggct gttgatctac ggcgcttctt ctagagccac aggcatccct

180

gaccggttct ccggatctgg ctctggcacc gatttcaccc tgaccatctc tcggctggaa

240

cccgaggatt tcgccgtgta ctactgccag cagtcctaca gctaccccgt gacctttggc

300

cagggcacca aggtggaaat caagcgtacg gtggccgctc ccagcgtgtt catcttcccc

360

ccaagcgacg agcagctgaa gagcggcacc gccagcgtgg tgtgtctgct gaacaacttc

420

taccccaggg aggccaaggt gcagtggaag gtggacaacg ccctgcagag cggcaacagc

480

caggagagcg tcaccgagca ggacagcaag gactccacct acagcctgag cagcaccctg

540

accctgagca aggccgacta cgagaagcac aaggtgtacg cctgtgaggt gacccaccag

600

ggcctgtcca gccccgtgac caagagcttc aacagggcg agtgc

645

<210> 99

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-10-3 with LALA mutation

<400> 99

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Asn Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Asp Glu Gln Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr

405 410 415

Arg Trp Asn Pro Ala Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 100

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-10-3 with LALA mutation

<400> 100

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt agttacgata tgagctgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat attgatccga ctggtagcaa gaccgactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagagacctc

300

aatgtgtacg ggttcgacta ctggggccag ggaaccctgg tcaccgtctc gagtgctagc

360

actaagggcc cgtcggtgtt cccgctggcc ccatcgtcca agagcacatc aggggtacc

420

gccgccctgg gctgccttgt gaaggattac tttcccgagc ccgtcacagt gtcctggaac

480

agcggagccc tgacctccgg agtgcatact ttcccggctg tgcttcagtc ctctggcctg

540

tactcattgt cctccgtggt caccgtccct tcgtcctccc tgggcaccca gacctatatc

600

tgtaatgtca accataagcc ctcgaacacc aaggtcgaca agaaggtcga gccgaagtcg

660

tgcgacaaga ctcacacttg cccgccttgc ccagccccgg aagctgccgg tggtccttcg

720

gtgttcctct tcccgcccaa gccgaaggat accctgatga tctcacggac ccccgaagtg

780

acctgtgtgg tggtggacgt gtcccacgag gacccggaag tgaaattcaa ttggtacgtg

840

gatggagtgg aagtgcacaa cgccaagacc aagccacggg aagaacagta caactctacc

900

taccgcgtgg tgtccgtgct cactgtgctg caccaagact ggctgaacgg gaaggagtac

960

aagtgcaaag tgtccaacaa ggcgctgcct gccccaattg agaaaactat ctcgaaagcc

1020

aagggacagc ctcgagaacc acaggtgtac accctgcccc catcccggga tgagtactgg

1080

gaccaggaag tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg

1140

gagtggggaga gcaatgggga tgaacagttc gcatacaaga ccacgcctcc cgtgctggac

1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg atcagtatag gtggaatcct

1260

gctgattatt tctcatgctc cgtgatgcat gaggctctgc acaaccacta cactcagaag

1320

agcttgtccc tgtcgcccgg a

1341

<210> 101

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Truncated hinge region Fcab

<400> 101

Thr Cys Pro Pro Cys Pro

15

<210> 102

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain FS30-10-3

<400> 102

gaaattgtgc tgacccagtc tccggggcacg ttatctctga gccctggtga gcgcgccact

60

ctgtcatgcc gggcttctca aagtgttagc agtagctacc tggcgtggta tcagcaaaaa

120

ccgggccagg ccccgcgtct gctgatttac ggtgcatcca gccgtgccac cggcattcca

180

gatcgttttt ccggtagtgg ttctgggacg gacttcactc tgacaatctc acgcctggaa

240

ccggaggatt ttgcggtgta ttactgccag caatcttatt cttatcctgt cacgttcggc

300

caagggacca aggtggaaat caaacgtact gtggccgctc ctagcgtgtt catttttccg

360

ccatccgacg agcagctcaa gtccggcacc gcctccgtgg tctgcctgct caacaacttc

420

taccctcgcg aagctaaggt ccagtggaag gtcgacaatg ccctgcagtc cggaaactcg

480

caggaaagcg tgactgaaca ggactccaag gactccacct attcactgtc ctcgactctg

540

accctgagca aggcggatta cgaaaagcac aaagtgtacg catgcgaagt gacccaccag

600

ggtctttcgt cccccgtgac caagagcttc aacagaggag agtgt

645

<210> 103

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-10-12 with LALA mutation

<400> 103

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Thr Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Asp Glu Gln Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr

405 410 415

Arg Trp Asn Pro Ala Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 104

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-10-12 with LALA mutation

<400> 104

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt agttacgata tgagctgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat attgatccga ctggtagcaa gaccgactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagagacctc

300

acggtgtacg ggttcgacta ctggggccag ggaaccctgg tcaccgtctc gagtgctagc

360

actaagggcc cgtcggtgtt cccgctggcc ccatcgtcca agagcacatc aggggtacc

420

gccgccctgg gctgccttgt gaaggattac tttcccgagc ccgtcacagt gtcctggaac

480

agcggagccc tgacctccgg agtgcatact ttcccggctg tgcttcagtc ctctggcctg

540

tactcattgt cctccgtggt caccgtccct tcgtcctccc tgggcaccca gacctatatc

600

tgtaatgtca accataagcc ctcgaacacc aaggtcgaca agaaggtcga gccgaagtcg

660

tgcgacaaga ctcacacttg cccgccttgc ccagccccgg aagctgccgg tggtccttcg

720

gtgttcctct tcccgcccaa gccgaaggat accctgatga tctcacggac ccccgaagtg

780

acctgtgtgg tggtggacgt gtcccacgag gacccggaag tgaaattcaa ttggtacgtg

840

gatggagtgg aagtgcacaa cgccaagacc aagccacggg aagaacagta caactctacc

900

taccgcgtgg tgtccgtgct cactgtgctg caccaagact ggctgaacgg gaaggagtac

960

aagtgcaaag tgtccaacaa ggcgctgcct gccccaattg agaaaactat ctcgaaagcc

1020

aagggacagc ctcgagaacc acaggtgtac accctgcccc catcccggga tgagtactgg

1080

gaccaggaag tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg

1140

gagtggggaga gcaatgggga tgaacagttc gcatacaaga ccacgcctcc cgtgctggac

1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg atcagtatag gtggaatcct

1260

gctgattatt tctcatgctc cgtgatgcat gaggctctgc acaaccacta cactcagaag

1320

agcttgtccc tgtcgcccgg a

1341

<210> 105

<211> 452

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-35-14 with LALA mutation

<400> 105

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Ser Pro Tyr Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Leu Tyr Glu Leu Ser Ala Tyr Ser Tyr Gly Ala Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

225 230 235 240

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Tyr Trp Asp Gln Glu

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Asp Glu Gln Phe Ala Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Gln Tyr Arg Trp Asn Pro Ala Asp Tyr Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 106

<211> 1356

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-35-14 with LALA mutation

<400> 106

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt gcctataata tccattgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat atttctccgt atggtggcgc gaccaactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagaaacctc

300

tacgagttga gcgcttactc ttacggggcg gactactggg gccagggaac cctggtcacc

360

gtctcgtcgg ctagcactaa gggcccgtcg gtgttcccgc tggccccatc gtccaagagc

420

acatcagggg gtaccgccgc cctgggctgc cttgtgaagg attactttcc cgagcccgtc

480

acagtgtcct ggaacagcgg agccctgacc tccggagtgc atactttccc ggctgtgctt

540

cagtcctctg gcctgtactc attgtcctcc gtggtcaccg tcccttcgtc ctccctgggc

600

acccagaacct atatctgtaa tgtcaaccat aagccctcga acaccaaggt cgacaagaag

660

gtcgagccga agtcgtgcga caagactcac acttgcccgc cttgcccagc cccggaagct

720

gccggtggtc cttcggtgtt cctcttcccg cccaagccga aggataccct gatgatctca

780

cggacccccg aagtgacctg tgtggtggtg gacgtgtccc acgaggaccc ggaagtgaaa

840

ttcaattggt acgtggatgg agtggaagtg cacaacgcca agaccaagcc acgggaagaa

900

cagtacaact ctacctaccg cgtggtgtcc gtgctcactg tgctgcacca agactggctg

960

aacgggaagg agtacaagtg caaagtgtcc aacaaggcgc tgcctgcccc aattgagaaa

1020

actatctcga aagccaaggg acagcctcga gaaccacagg tgtacaccct gcccccatcc

1080

cgggatgagt actgggacca ggaagtcagc ctgacctgcc tggtcaaagg cttctatccc

1140

agcgacatcg ccgtggagtg ggagagcaat ggggatgaac agttcgcata caagaccacg

1200

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggatcag

1260

tataggtgga atcctgctga ttatttctca tgctccgtga tgcatgaggc tctgcacaac

1320

cactacactc agaagagctt gtccctgtcg cccgga

1356

<210> 107

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain FS30-35-14

<400> 107

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Tyr Ser Ser

85 90 95

Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 108

<211> 648

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain FS30-35-14

<400> 108

gaaattgtgc tgacccagtc tccggggcacg ttatctctga gccctggtga gcgcgccact

60

ctgtcatgcc gggcttctca aagtgttagc agtagctacc tggcgtggta tcagcaaaaa

120

ccgggccagg ccccgcgtct gctgatttac ggtgcatcca gccgtgccac cggcattcca

180

gatcgttttt ccggtagtgg ttctgggacg gacttcactc tgacaatctc acgcctggaa

240

ccggaggatt ttgcggtgta ttactgccag caatattatt attcttctcc tatcacgttc

300

ggccaaggga ccaaggtgga aatcaaacgt actgtggccg ctcctagcgt gttcattttt

360

ccgccatccg acgagcagct caagtccggc accgcctccg tggtctgcct gctcaacaac

420

ttctaccctc gcgaagctaa ggtccagtgg aaggtcgaca atgccctgca gtccggaaac

480

tcgcaggaaa gcgtgactga acaggactcc aaggactcca cctattcact gtcctcgact

540

ctgaccctga gcaaggcgga ttacgaaaag cacaaagtgt acgcatgcga agtgacccac

600

cagggtcttt cgtcccccgt gaccaagagc ttcaacagag gagagtgt

648

<210> 109

<211> 454

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-5-37 with LALA mutation

<400> 109

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Asn Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Tyr Asp Lys Tyr Trp Gly Ser Ser Ile Tyr Ser Gly Leu

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Tyr Trp Asp

355 360 365

Gln Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Asp Glu Gln Phe Ala Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Gln Tyr Arg Trp Asn Pro Ala Asp Tyr Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly

450

<210> 110

<211> 1362

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/FS30-5-37 with LALA mutation

<400> 110

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

aattgcgcgg ccagtggctt taccttcagt agctatgcca tgagctgggt gcgtcaggcg

120

ccgggcaaag gtctggaatg ggttagcgcg attagcggta gtggcggtag cacgtactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagatcttac

300

gacaaatact ggggttcttc tatttactct ggcttggact actggggcca gggaaccctg

360

gtcaccgtct cgagtgctag cactaagggc ccgtcggtgt tcccgctggc cccatcgtcc

420

aagagcacat cagggggtac cgccgccctg ggctgccttg tgaaggatta ctttcccgag

480

cccgtcacag tgtcctggaa cagcggagcc ctgacctccg gagtgcatac tttcccggct

540

gtgcttcagt cctctggcct gtactcattg tcctccgtgg tcaccgtccc ttcgtcctcc

600

ctgggcaccc agacctatat ctgtaatgtc aaccataagc cctcgaacac caaggtcgac

660

aagaaggtcg agccgaagtc gtgcgacaag actcacactt gcccgccttg cccagccccg

720

gaagctgccg gtggtccttc ggtgttcctc ttcccgccca agccgaagga taccctgatg

780

atctcacgga cccccgaagt gacctgtgtg gtggtggacg tgtcccacga ggacccggaa

840

gtgaaattca attggtacgt ggatggagtg gaagtgcaca acgccaagac caagccacgg

900

gaagaacagt acaactctac ctaccgcgtg gtgtccgtgc tcactgtgct gcaccaagac

960

tggctgaacg ggaaggagta caagtgcaaa gtgtccaaca aggcgctgcc tgccccaatt

1020

gagaaaacta tctcgaaagc caagggacag cctcgagaac cacaggtgta caccctgccc

1080

ccatcccggg atgagtactg ggaccaggaa gtcagcctga cctgcctggt caaaggcttc

1140

tatcccagcg acatcgccgt ggagtgggag agcaatgggg atgaacagtt cgcatacaag

1200

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg

1260

gatcagtata ggtggaatcc tgctgattat ttctcatgct ccgtgatgca tgaggctctg

1320

cacaaccact acactcagaa gagcttgtcc ctgtcgcccg ga

1362

<210> 111

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain FS30-5-37

<400> 111

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Tyr

85 90 95

Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 112

<211> 648

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain FS30-5-37

<400> 112

gaaattgtgc tgacccagtc tccggggcacg ttatctctga gccctggtga gcgcgccact

60

ctgtcatgcc gggcttctca aagtgttagc agtagctacc tggcgtggta tcagcaaaaa

120

ccgggccagg ccccgcgtct gctgatttac ggtgcatcca gccgtgccac cggcattcca

180

gatcgttttt ccggtagtgg ttctgggacg gacttcactc tgacaatctc acgcctggaa

240

ccggaggatt ttgcggtgta ttactgccag caatattatt cttattatcc tgtcacgttc

300

ggccaaggga ccaaggtgga aatcaaacgt actgtggccg ctcctagcgt gttcattttt

360

ccgccatccg acgagcagct caagtccggc accgcctccg tggtctgcct gctcaacaac

420

ttctaccctc gcgaagctaa ggtccagtgg aaggtcgaca atgccctgca gtccggaaac

480

tcgcaggaaa gcgtgactga acaggactcc aaggactcca cctattcact gtcctcgact

540

ctgaccctga gcaaggcgga ttacgaaaag cacaaagtgt acgcatgcga agtgacccac

600

cagggtcttt cgtcccccgt gaccaagagc ttcaacagag gagagtgt

648

<210> 113

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Alternative domain CH3 Fcab FS20-22-49

<400> 113

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtactgggac

60

caggaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atggggatga acagttcgca tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtggatc agtataggtg gaatcctgct

240

gattatttct catgctccgt gatgcatgag gctctgcaca accactacac tcagaagagc

300

ttgtccctgt cgcccgga

318

<210> 114

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain of MAT to FITC G1AA/4420 containing the LALA mutation

<400> 114

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 115

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain MAb to FITC G1/4420 without LALA mutation

<400> 115

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 116

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain MAT 4420

<400> 116

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 117

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Antibody heavy chain G1AA/HelD1.3 with LALA mutation

<400> 117

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Thr Phe Ser Gly Tyr

20 25 30

Gly Val Asn Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Trp Gly Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Arg Asp Tyr Arg Leu Asp Tyr Trp Gly Gln Gly Ser Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 118

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain MAT HelD1.3

<400> 118

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 119

<211> 445

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain G1/MOR7480.1

<400> 119

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 120

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain MAT G1/MOR7480.1, G1AA/MOR7480.1 and G2/MOR7480.1

<400> 120

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu

85 90 95

Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 121

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain G1/20H4.9

<400> 121

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 122

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain MAT G1/20H4.9 and G1AA/20H4.9

<400> 122

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 123

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain FS20-22-49AA/4420 (with LALA mutation)

<400> 123

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Asp Glu Gln Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr

405 410 415

Arg Trp Asn Pro Ala Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 124

<211> 441

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain G2/MOR7480.1

<400> 124

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe

180 185 190

Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro

210 215 220

Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 125

<211> 445

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain G1AA/MOR7480.1

<400> 125

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 126

<211> 232

<212> PROTEIN

<213> CD137 Homo sapiens

<400> 126

Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro

130 135 140

Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu

165 170 175

Leu Phe Leu Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val Lys Arg

180 185 190

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

195 200 205

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

210 215 220

Glu Glu Glu Gly Gly Cys Glu Leu

225 230

<210> 127

<211> 163

<212> PROTEIN

<213> Extracellular domain of CD137 Homo sapiens

<400> 127

Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro

130 135 140

Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln

<210> 128

<211> 231

<212> PROTEIN

<213> CD137 cynomolgus macaque

<400> 128

Leu Gln Asp Leu Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Ser Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Lys Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Ile Ser Gly Tyr His Cys Leu Gly Ala Glu Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro

130 135 140

Gly Ala Ser Ser Ala Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln Ile Ile Phe Phe Leu Ala Leu Thr Ser Thr Val Val Leu

165 170 175

Phe Leu Leu Phe Phe Leu Val Leu Arg Phe Ser Val Val Lys Arg Ser

180 185 190

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

195 200 205

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

210 215 220

Glu Glu Gly Gly Cys Glu Leu

225 230

<210> 129

<211> 163

<212> PROTEIN

<213> Extracellular domain of cynomolgus CD137

<400> 129

Leu Gln Asp Leu Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Ser Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Lys Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Ile Ser Gly Tyr His Cys Leu Gly Ala Glu Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro

130 135 140

Gly Ala Ser Ser Ala Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln

<210> 130

<211> 186

<212> PROTEIN

<213> Extracellular domain OX40 Homo sapiens

<400> 130

Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His

1 5 10 15

Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln

20 25 30

Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val

35 40 45

Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly

50 55 60

Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg

65 70 75 80

Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp

85 90 95

Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala

100 105 110

Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln

115 120 125

Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro

130 135 140

Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr

145 150 155 160

Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr

165 170 175

Arg Pro Val Glu Val Pro Gly Gly Arg Ala

180 185

<210> 131

<211> 186

<212> PROTEIN

<213> Cynomolgus macaque OX40 extracellular domain

<400> 131

Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys Gln

1 5 10 15

Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Asn Arg Ser Gln

20 25 30

Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val

35 40 45

Ser Ala Lys Pro Cys Lys Ala Cys Thr Trp Cys Asn Leu Arg Ser Gly

50 55 60

Ser Glu Arg Lys Gln Pro Cys Thr Ala Thr Gln Asp Thr Val Cys Arg

65 70 75 80

Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp

85 90 95

Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala

100 105 110

Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln

115 120 125

Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro

130 135 140

Pro Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Thr Thr

145 150 155 160

Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Arg Pro Ser Thr

165 170 175

Arg Pro Val Glu Val Pro Arg Gly Pro Ala

180 185

<210> 132

<211> 249

<212> PROTEIN

<213> Artificial sequence

<220>

<223>DO11.10-hOX40 and human OX40 receptor

<400> 132

Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His

1 5 10 15

Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln

20 25 30

Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val

35 40 45

Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly

50 55 60

Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg

65 70 75 80

Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp

85 90 95

Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala

100 105 110

Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln

115 120 125

Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro

130 135 140

Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr

145 150 155 160

Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr

165 170 175

Arg Pro Val Glu Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly

180 185 190

Leu Gly Leu Val Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala

195 200 205

Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys

210 215 220

Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala

225 230 235 240

Asp Ala His Ser Thr Leu Ala Lys Ile

245

<210> 133

<211> 253

<212> PROTEIN

<213> Artificial sequence

<220>

<223>DO11.10-mOX40 and mouse OX40 receptor

<400> 133

Val Thr Ala Arg Arg Leu Asn Cys Val Lys His Thr Tyr Pro Ser Gly

1 5 10 15

His Lys Cys Cys Arg Glu Cys Gln Pro Gly His Gly Met Val Ser Arg

20 25 30

Cys Asp His Thr Arg Asp Thr Leu Cys His Pro Cys Glu Thr Gly Phe

35 40 45

Tyr Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gln Cys Thr Gln Cys

50 55 60

Asn His Arg Ser Gly Ser Glu Leu Lys Gln Asn Cys Thr Pro Thr Gln

65 70 75 80

Asp Thr Val Cys Arg Cys Arg Pro Gly Thr Gln Pro Arg Gln Asp Ser

85 90 95

Gly Tyr Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro Gly His Phe

100 105 110

Ser Pro Gly Asn Asn Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu

115 120 125

Ser Gly Lys Gln Thr Arg His Pro Ala Ser Asp Ser Leu Asp Ala Val

130 135 140

Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu Thr Gln Arg

145 150 155 160

Pro Thr Phe Arg Pro Thr Thr Val Gln Ser Thr Thr Val Trp Pro Arg

165 170 175

Thr Ser Glu Leu Pro Ser Pro Pro Thr Leu Val Thr Pro Glu Gly Pro

180 185 190

Ala Phe Ala Val Leu Leu Gly Leu Gly Leu Gly Leu Leu Ala Pro Leu

195 200 205

Thr Val Leu Leu Ala Leu Tyr Leu Leu Arg Lys Ala Trp Arg Leu Pro

210 215 220

Asn Thr Pro Lys Pro Cys Trp Gly Asn Ser Phe Arg Thr Pro Ile Gln

225 230 235 240

Glu Glu His Thr Asp Ala His Phe Thr Leu Ala Lys Ile

245 250

<210> 134

<211> 250

<212> PROTEIN

<213> Artificial sequence

<220>

<223> DO11.10-cOX40 and cynomolgus OX40 receptor

<400> 134

Lys Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys

1 5 10 15

Gln Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Asn Arg Ser

20 25 30

Gln Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val

35 40 45

Val Ser Ala Lys Pro Cys Lys Ala Cys Thr Trp Cys Asn Leu Arg Ser

50 55 60

Gly Ser Glu Arg Lys Gln Pro Cys Thr Ala Thr Gln Asp Thr Val Cys

65 70 75 80

Arg Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val

85 90 95

Asp Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln

100 105 110

Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu

115 120 125

Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro

130 135 140

Pro Pro Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Thr

145 150 155 160

Thr Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Arg Pro Ser

165 170 175

Thr Arg Pro Val Glu Val Pro Arg Gly Pro Ala Val Ala Ala Ile Leu

180 185 190

Gly Leu Gly Leu Ala Leu Gly Leu Leu Gly Pro Leu Ala Met Leu Leu

195 200 205

Ala Leu Leu Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala Pro

210 215 220

Lys Ala Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln

225 230 235 240

Ala Asp Ala His Ser Ala Leu Ala Lys Ile

245 250

<210> 135

<211> 442

<212> PROTEIN

<213> OX40-mFc Homo sapiens

<400> 135

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp

20 25 30

Arg Cys Cys His Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys

35 40 45

Ser Arg Ser Gln Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr

50 55 60

Asn Asp Val Val Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn

65 70 75 80

Leu Arg Ser Gly Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp

85 90 95

Thr Val Cys Arg Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys

100 105 110

Pro Gly Val Asp Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly

115 120 125

Asp Asn Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys

130 135 140

His Thr Leu Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp

145 150 155 160

Arg Asp Pro Pro Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala

165 170 175

Arg Pro Ile Thr Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln

180 185 190

Gly Pro Ser Thr Arg Pro Val Glu Val Pro Gly Gly Arg Ala Val Ala

195 200 205

Gly Ser Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

210 215 220

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

260 265 270

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

275 280 285

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

290 295 300

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

305 310 315 320

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

325 330 335

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

340 345 350

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

355 360 365

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

370 375 380

Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe

385 390 395 400

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

405 410 415

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

420 425 430

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440

<210> 136

<211> 447

<212> PROTEIN

<213> OX40 mouse-mFc

<400> 136

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Val Thr Ala Arg Arg Leu Asn Cys Val Lys His Thr

20 25 30

Tyr Pro Ser Gly His Lys Cys Cys Arg Glu Cys Gln Pro Gly His Gly

35 40 45

Met Val Ser Arg Cys Asp His Thr Arg Asp Thr Leu Cys His Pro Cys

50 55 60

Glu Thr Gly Phe Tyr Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gln

65 70 75 80

Cys Thr Gln Cys Asn His Arg Ser Gly Ser Glu Leu Lys Gln Asn Cys

85 90 95

Thr Pro Thr Gln Asp Thr Val Cys Arg Cys Arg Pro Gly Thr Gln Pro

100 105 110

Arg Gln Asp Ser Gly Tyr Lys Leu Gly Val Asp Cys Val Pro Cys Pro

115 120 125

Pro Gly His Phe Ser Pro Gly Asn Asn Gln Ala Cys Lys Pro Trp Thr

130 135 140

Asn Cys Thr Leu Ser Gly Lys Gln Thr Arg His Pro Ala Ser Asp Ser

145 150 155 160

Leu Asp Ala Val Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp

165 170 175

Glu Thr Gln Arg Pro Thr Phe Arg Pro Thr Thr Val Gln Ser Thr Thr

180 185 190

Val Trp Pro Arg Thr Ser Glu Leu Pro Ser Pro Pro Thr Leu Val Thr

195 200 205

Pro Glu Gly Pro Ala Gly Ser Pro Arg Gly Pro Thr Ile Lys Pro Cys

210 215 220

Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser

245 250 255

Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp

260 265 270

Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln

275 280 285

Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser

290 295 300

Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys

305 310 315 320

Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile

325 330 335

Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro

340 345 350

Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met

355 360 365

Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn

370 375 380

Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn

405 410 415

Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu

420 425 430

His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 137

<211> 444

<212> PROTEIN

<213> OX40 Cynomolgus-mFc

<400> 137

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp

20 25 30

Arg Cys Cys Gln Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys

35 40 45

Asn Arg Ser Gln Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr

50 55 60

Asn Asp Val Val Ser Ala Lys Pro Cys Lys Ala Cys Thr Trp Cys Asn

65 70 75 80

Leu Arg Ser Gly Ser Glu Arg Lys Gln Pro Cys Thr Ala Thr Gln Asp

85 90 95

Thr Val Cys Arg Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys

100 105 110

Pro Gly Val Asp Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly

115 120 125

Asp Asn Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys

130 135 140

His Thr Leu Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp

145 150 155 160

Arg Asp Pro Pro Pro Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala

165 170 175

Arg Pro Thr Thr Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln

180 185 190

Arg Pro Ser Thr Arg Pro Val Glu Val Pro Arg Gly Pro Ala Val Ala

195 200 205

Ala Ile Gly Ser Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys

210 215 220

Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile

260 265 270

Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr

275 280 285

His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro

290 295 300

Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val

305 310 315 320

Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro

325 330 335

Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu

340 345 350

Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp

355 360 365

Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr

370 375 380

Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu

405 410 415

Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His

420 425 430

His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440

<210> 138

<211> 164

<212> PROTEIN

<213> Homo sapiens CD137 sequence for use with

recombinant antigen CD137-mFc-Avi

<400> 138

Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp

1 5 10 15

Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser

20 25 30

Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly

35 40 45

Val Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys

50 55 60

Asp Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys

65 70 75 80

Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys

85 90 95

Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg

100 105 110

Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly

115 120 125

Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser

130 135 140

Pro Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly

145 150 155 160

His Ser Pro Gln

<210> 139

<211> 164

<212> PROTEIN

<213> Cynomolgus CD137 sequence for use with

recombinant antigens CD137-mFc-Avi and CD137-Avi-His

<400> 139

Ser Leu Gln Asp Leu Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp

1 5 10 15

Asn Asn Arg Ser Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser

20 25 30

Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly

35 40 45

Val Phe Lys Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys

50 55 60

Asp Cys Ile Ser Gly Tyr His Cys Leu Gly Ala Glu Cys Ser Met Cys

65 70 75 80

Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys

85 90 95

Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg

100 105 110

Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly

115 120 125

Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser

130 135 140

Pro Gly Ala Ser Ser Ala Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly

145 150 155 160

His Ser Pro Gln

<210> 140

<211> 165

<212> PROTEIN

<213> Mouse CD137 sequence for use with

recombinant antigen CD137-mFc-Avi

<400> 140

Ala Val Gln Asn Ser Cys Asp Asn Cys Gln Pro Gly Thr Phe Cys Arg

1 5 10 15

Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro Pro Ser Thr Phe Ser Ser

20 25 30

Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys Arg Val Cys Ala Gly Tyr

35 40 45

Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr His Asn Ala Glu Cys Glu

50 55 60

Cys Ile Glu Gly Phe His Cys Leu Gly Pro Gln Cys Thr Arg Cys Glu

65 70 75 80

Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr Lys Gln Gly Cys Lys Thr

85 90 95

Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn Gly Thr Gly Val Cys Arg

100 105 110

Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg Ser Val Leu Lys Thr Gly

115 120 125

Thr Thr Glu Lys Asp Val Val Cys Gly Pro Pro Val Val Ser Phe Ser

130 135 140

Pro Ser Thr Thr Ile Ser Val Thr Pro Glu Gly Gly Pro Gly Gly His

145 150 155 160

Ser Leu Gln Val Leu

165

<210> 141

<211> 249

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mFc-Avi for use with recombinant antigens

CD137-mFc-Avi

<400> 141

Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala

1 5 10 15

Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile

20 25 30

Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val

35 40 45

Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val

50 55 60

Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln

85 90 95

Asp Trp Met Ser Gly Lys Ala Phe Ala Cys Ala Val Asn Asn Lys Asp

100 105 110

Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val

115 120 125

Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr

130 135 140

Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu

145 150 155 160

Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr

165 170 175

Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr

180 185 190

Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr

195 200 205

Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys

210 215 220

Ser Phe Ser Arg Thr Pro Gly Lys Gly Gly Gly Leu Asn Asp Ile Phe

225 230 235 240

Glu Ala Gln Lys Ile Glu Trp His Glu

245

<210> 142

<400> 142

000

<210> 143

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Domain CH3 Fcab FS20-22-49

<400> 143

ggacagcctc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gtactgggac

60

caggaagtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag

120

tggggagagca atggggatga acagttcgca tacaagacca cgcctcccgt gctggactcc

180

gacggctcct tcttcctcta cagcaagctc accgtggatc agtataggtg gaatcctgct

240

gattatttct catgctccgt gatgcatgag gctctgcaca accactacac tcagaagagc

300

ttgtccctgt cgcccgga

318

<210> 144

<211> 454

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-5-37 (without LALA)

<400> 144

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Asn Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Tyr Asp Lys Tyr Trp Gly Ser Ser Ile Tyr Ser Gly Leu

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Tyr Trp Asp

355 360 365

Gln Glu Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Asp Glu Gln Phe Ala Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Gln Tyr Arg Trp Asn Pro Ala Asp Tyr Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly

450

<210> 145

<211> 1362

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-5-37 (without LALA)

<400> 145

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

aattgcgcgg ccagtggctt taccttcagt agctatgcca tgagctgggt gcgtcaggcg

120

ccgggcaaag gtctggaatg ggttagcgcg attagcggta gtggcggtag cacgtactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagatcttac

300

gacaaatact ggggttcttc tatttactct ggcttggact actggggcca gggaaccctg

360

gtcaccgtct cgagtgctag cactaagggc ccgtcggtgt tcccgctggc cccatcgtcc

420

aagagcacat cagggggtac cgccgccctg ggctgccttg tgaaggatta ctttcccgag

480

cccgtcacag tgtcctggaa cagcggagcc ctgacctccg gagtgcatac tttcccggct

540

gtgcttcagt cctctggcct gtactcattg tcctccgtgg tcaccgtccc ttcgtcctcc

600

ctgggcaccc agacctatat ctgtaatgtc aaccataagc cctcgaacac caaggtcgac

660

aagaaggtcg agccgaagtc gtgcgacaag actcacactt gcccgccttg cccagccccg

720

gaactgctgg gtggtccttc ggtgttcctc ttcccgccca agccgaagga taccctgatg

780

atctcacgga cccccgaagt gacctgtgtg gtggtggacg tgtcccacga ggacccggaa

840

gtgaaattca attggtacgt ggatggagtg gaagtgcaca acgccaagac caagccacgg

900

gaagaacagt acaactctac ctaccgcgtg gtgtccgtgc tcactgtgct gcaccaagac

960

tggctgaacg ggaaggagta caagtgcaaa gtgtccaaca aggcgctgcc tgccccaatt

1020

gagaaaacta tctcgaaagc caagggacag cctcgagaac cacaggtgta caccctgccc

1080

ccatcccggg atgagtactg ggaccaggaa gtcagcctga cctgcctggt caaaggcttc

1140

tatcccagcg acatcgccgt ggagtgggag agcaatgggg atgaacagtt cgcatacaag

1200

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg

1260

gatcagtata ggtggaatcc tgctgattat ttctcatgct ccgtgatgca tgaggctctg

1320

cacaaccact acactcagaa gagcttgtcc ctgtcgcccg ga

1362

<210> 146

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-10-3 (without LALA)

<400> 146

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Asn Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Asp Glu Gln Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr

405 410 415

Arg Trp Asn Pro Ala Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 147

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-10-3 (without LALA)

<400> 147

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt agttacgata tgagctgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat attgatccga ctggtagcaa gaccgactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagagacctc

300

aatgtgtacg ggttcgacta ctggggccag ggaaccctgg tcaccgtctc gagtgctagc

360

actaagggcc cgtcggtgtt cccgctggcc ccatcgtcca agagcacatc aggggtacc

420

gccgccctgg gctgccttgt gaaggattac tttcccgagc ccgtcacagt gtcctggaac

480

agcggagccc tgacctccgg agtgcatact ttcccggctg tgcttcagtc ctctggcctg

540

tactcattgt cctccgtggt caccgtccct tcgtcctccc tgggcaccca gacctatatc

600

tgtaatgtca accataagcc ctcgaacacc aaggtcgaca agaaggtcga gccgaagtcg

660

tgcgacaaga ctcacacttg cccgccttgc ccagccccgg aactgctggg tggtccttcg

720

gtgttcctct tcccgcccaa gccgaaggat accctgatga tctcacggac ccccgaagtg

780

acctgtgtgg tggtggacgt gtcccacgag gacccggaag tgaaattcaa ttggtacgtg

840

gatggagtgg aagtgcacaa cgccaagacc aagccacggg aagaacagta caactctacc

900

taccgcgtgg tgtccgtgct cactgtgctg caccaagact ggctgaacgg gaaggagtac

960

aagtgcaaag tgtccaacaa ggcgctgcct gccccaattg agaaaactat ctcgaaagcc

1020

aagggacagc ctcgagaacc acaggtgtac accctgcccc catcccggga tgagtactgg

1080

gaccaggaag tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg

1140

gagtggggaga gcaatgggga tgaacagttc gcatacaaga ccacgcctcc cgtgctggac

1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg atcagtatag gtggaatcct

1260

gctgattatt tctcatgctc cgtgatgcat gaggctctgc acaaccacta cactcagaag

1320

agcttgtccc tgtcgcccgg a

1341

<210> 148

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-10-12 (without LALA)

<400> 148

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Thr Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Asp Glu Gln Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr

405 410 415

Arg Trp Asn Pro Ala Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 149

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-10-12 (without LALA)

<400> 149

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt agttacgata tgagctgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat attgatccga ctggtagcaa gaccgactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagagacctc

300

acggtgtacg ggttcgacta ctggggccag ggaaccctgg tcaccgtctc gagtgctagc

360

actaagggcc cgtcggtgtt cccgctggcc ccatcgtcca agagcacatc aggggtacc

420

gccgccctgg gctgccttgt gaaggattac tttcccgagc ccgtcacagt gtcctggaac

480

agcggagccc tgacctccgg agtgcatact ttcccggctg tgcttcagtc ctctggcctg

540

tactcattgt cctccgtggt caccgtccct tcgtcctccc tgggcaccca gacctatatc

600

tgtaatgtca accataagcc ctcgaacacc aaggtcgaca agaaggtcga gccgaagtcg

660

tgcgacaaga ctcacacttg cccgccttgc ccagccccgg aactgctggg tggtccttcg

720

gtgttcctct tcccgcccaa gccgaaggat accctgatga tctcacggac ccccgaagtg

780

acctgtgtgg tggtggacgt gtcccacgag gacccggaag tgaaattcaa ttggtacgtg

840

gatggagtgg aagtgcacaa cgccaagacc aagccacggg aagaacagta caactctacc

900

taccgcgtgg tgtccgtgct cactgtgctg caccaagact ggctgaacgg gaaggagtac

960

aagtgcaaag tgtccaacaa ggcgctgcct gccccaattg agaaaactat ctcgaaagcc

1020

aagggacagc ctcgagaacc acaggtgtac accctgcccc catcccggga tgagtactgg

1080

gaccaggaag tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg

1140

gagtggggaga gcaatgggga tgaacagttc gcatacaaga ccacgcctcc cgtgctggac

1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg atcagtatag gtggaatcct

1260

gctgattatt tctcatgctc cgtgatgcat gaggctctgc acaaccacta cactcagaag

1320

agcttgtccc tgtcgcccgg a

1341

<210> 150

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-10-16 (without LALA)

<400> 150

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Leu Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Tyr Trp Asp Gln Glu Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Asp Glu Gln Phe Ala Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Gln Tyr

405 410 415

Arg Trp Asn Pro Ala Asp Tyr Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 151

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-10-16 (without LALA)

<400> 151

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt agttacgata tgagctgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat attgatccga ctggtagcaa gaccgactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagagacctc

300

ttggtgtacg ggttcgacta ctggggccag ggaaccctgg tcaccgtctc gagtgctagc

360

actaagggcc cgtcggtgtt cccgctggcc ccatcgtcca agagcacatc aggggtacc

420

gccgccctgg gctgccttgt gaaggattac tttcccgagc ccgtcacagt gtcctggaac

480

agcggagccc tgacctccgg agtgcatact ttcccggctg tgcttcagtc ctctggcctg

540

tactcattgt cctccgtggt caccgtccct tcgtcctccc tgggcaccca gacctatatc

600

tgtaatgtca accataagcc ctcgaacacc aaggtcgaca agaaggtcga gccgaagtcg

660

tgcgacaaga ctcacacttg cccgccttgc ccagccccgg aactgctggg tggtccttcg

720

gtgttcctct tcccgcccaa gccgaaggat accctgatga tctcacggac ccccgaagtg

780

acctgtgtgg tggtggacgt gtcccacgag gacccggaag tgaaattcaa ttggtacgtg

840

gatggagtgg aagtgcacaa cgccaagacc aagccacggg aagaacagta caactctacc

900

taccgcgtgg tgtccgtgct cactgtgctg caccaagact ggctgaacgg gaaggagtac

960

aagtgcaaag tgtccaacaa ggcgctgcct gccccaattg agaaaactat ctcgaaagcc

1020

aagggacagc ctcgagaacc acaggtgtac accctgcccc catcccggga tgagtactgg

1080

gaccaggaag tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg

1140

gagtggggaga gcaatgggga tgaacagttc gcatacaaga ccacgcctcc cgtgctggac

1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg atcagtatag gtggaatcct

1260

gctgattatt tctcatgctc cgtgatgcat gaggctctgc acaaccacta cactcagaag

1320

agcttgtccc tgtcgcccgg a

1341

<210> 152

<211> 452

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-35-14 (without LALA)

<400> 152

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Ser Pro Tyr Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Leu Tyr Glu Leu Ser Ala Tyr Ser Tyr Gly Ala Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Tyr Trp Asp Gln Glu

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Asp Glu Gln Phe Ala Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Gln Tyr Arg Trp Asn Pro Ala Asp Tyr Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 153

<211> 1356

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence

FS20-22-49/FS30-35-14 (without LALA)

<400> 153

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt gcctataata tccattgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat atttctccgt atggtggcgc gaccaactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagaaacctc

300

tacgagttga gcgcttactc ttacggggcg gactactggg gccagggaac cctggtcacc

360

gtctcgtcgg ctagcactaa gggcccgtcg gtgttcccgc tggccccatc gtccaagagc

420

acatcagggg gtaccgccgc cctgggctgc cttgtgaagg attactttcc cgagcccgtc

480

acagtgtcct ggaacagcgg agccctgacc tccggagtgc atactttccc ggctgtgctt

540

cagtcctctg gcctgtactc attgtcctcc gtggtcaccg tcccttcgtc ctccctgggc

600

acccagaacct atatctgtaa tgtcaaccat aagccctcga acaccaaggt cgacaagaag

660

gtcgagccga agtcgtgcga caagactcac acttgcccgc cttgcccagc cccggaactg

720

ctgggtggtc cttcggtgtt cctcttcccg cccaagccga aggataccct gatgatctca

780

cggacccccg aagtgacctg tgtggtggtg gacgtgtccc acgaggaccc ggaagtgaaa

840

ttcaattggt acgtggatgg agtggaagtg cacaacgcca agaccaagcc acgggaagaa

900

cagtacaact ctacctaccg cgtggtgtcc gtgctcactg tgctgcacca agactggctg

960

aacgggaagg agtacaagtg caaagtgtcc aacaaggcgc tgcctgcccc aattgagaaa

1020

actatctcga aagccaaggg acagcctcga gaaccacagg tgtacaccct gcccccatcc

1080

cgggatgagt actgggacca ggaagtcagc ctgacctgcc tggtcaaagg cttctatccc

1140

agcgacatcg ccgtggagtg ggagagcaat ggggatgaac agttcgcata caagaccacg

1200

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggatcag

1260

tataggtgga atcctgctga ttatttctca tgctccgtga tgcatgaggc tctgcacaac

1320

cactacactc agaagagctt gtccctgtcg cccgga

1356

<210> 154

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain MAT G1AA/FS30-10-16 (with LALA)

<400> 154

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asp Pro Thr Gly Ser Lys Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Leu Leu Val Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 155

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain MAT G1AA/OX86 (with LALA)

<400> 155

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Asn Leu His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Met Arg Tyr Asp Gly Asp Thr Tyr Tyr Asn Ser Val Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Thr

85 90 95

Arg Asp Gly Arg Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Val Met

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 156

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain MAT G1/OX86 and G1AA/OX86

<400> 156

Asp Ile Val Met Thr Gln Gly Ala Leu Pro Asn Pro Val Pro Ser Gly

1 5 10 15

Glu Ser Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Val Tyr Lys

20 25 30

Asp Gly Gln Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Thr Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Arg Ala Glu Asp Ala Gly Val Tyr Tyr Cys Gln Gln Val

85 90 95

Arg Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 157

<211> 453

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain FS20m-232-91AA/4420 (with LALA)

<400> 157

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Phe Asp Pro Met Tyr Tyr Tyr Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Glu Pro Leu Trp Asp Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Trp Arg Asp Arg Trp Glu Asp Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 158

<211> 189

<212> PROTEIN

<213> Homo sapiens CD137-Avi-His

<400> 158

Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp

1 5 10 15

Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser

20 25 30

Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly

35 40 45

Val Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys

50 55 60

Asp Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys

65 70 75 80

Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys

85 90 95

Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg

100 105 110

Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly

115 120 125

Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser

130 135 140

Pro Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly

145 150 155 160

His Ser Pro Gln Gly Ser Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala

165 170 175

Gln Lys Ile Glu Trp His Glu His His His His His

180 185

<210> 159

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain MAT G1/OX86 (without LALA)

<400> 159

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Asn Leu His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Met Arg Tyr Asp Gly Asp Ile Tyr Tyr Asn Ser Val Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Thr

85 90 95

Arg Asp Gly Arg Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Val Met

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 160

<211> 443

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-PD-1 MAT heavy chain G1AA/5C4

<400> 160

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

340 345 350

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 161

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain MAT to PD-1 G1AA/5C4

<400> 161

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 162

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain MAT to PD-L1 G1AA/S1

<400> 162

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 163

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain MAT to PD-L1 G1AA/S1

<400> 163

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Phe Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 164

<211> 233

<212> PROTEIN

<213> CD137 mouse

<400> 164

Val Gln Asn Ser Cys Asp Asn Cys Gln Pro Gly Thr Phe Cys Arg Lys

1 5 10 15

Tyr Asn Pro Val Cys Lys Ser Cys Pro Pro Ser Thr Phe Ser Ser Ile

20 25 30

Gly Gly Gln Pro Asn Cys Asn Ile Cys Arg Val Cys Ala Gly Tyr Phe

35 40 45

Arg Phe Lys Lys Phe Cys Ser Ser Thr His Asn Ala Glu Cys Glu Cys

50 55 60

Ile Glu Gly Phe His Cys Leu Gly Pro Gln Cys Thr Arg Cys Glu Lys

65 70 75 80

Asp Cys Arg Pro Gly Gln Glu Leu Thr Lys Gln Gly Cys Lys Thr Cys

85 90 95

Ser Leu Gly Thr Phe Asn Asp Gln Asn Gly Thr Gly Val Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Arg Ser Val Leu Lys Thr Gly Thr

115 120 125

Thr Glu Lys Asp Val Val Cys Gly Pro Pro Val Val Ser Phe Ser Pro

130 135 140

Ser Thr Thr Ile Ser Val Thr Pro Glu Gly Gly Pro Gly Gly His Ser

145 150 155 160

Leu Gln Val Leu Thr Leu Phe Leu Ala Leu Thr Ser Ala Leu Leu Leu

165 170 175

Ala Leu Ile Phe Ile Thr Leu Leu Phe Ser Val Leu Lys Trp Ile Arg

180 185 190

Lys Lys Phe Pro His Ile Phe Lys Gln Pro Phe Lys Lys Thr Thr Gly

195 200 205

Ala Ala Gln Glu Glu Asp Ala Cys Ser Cys Arg Cys Pro Gln Glu Glu

210 215 220

Glu Gly Gly Gly Gly Gly Tyr Glu Leu

225 230

<210> 165

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain MAT G1AA/20H4.9

<400> 165

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 166

<211> 454

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain MAT G1AA/3H3

<400> 166

Glu Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Gly Ser Gly Phe Thr Leu Ser Asp Tyr

20 25 30

Gly Val Ala Trp Val Arg Gln Ala Pro Lys Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Tyr Ala Gly Gly Thr Thr Tyr Tyr Arg Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Ile Asp Gly Tyr Gly Gly Tyr Ser Gly Ser His Trp Tyr Phe Asp

100 105 110

Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 167

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain MAT G1AA/3H3 and G1/3H3 and MAT2 FS20m-232-91AA/3H3

<400> 167

Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Leu Asn Cys Arg Thr Ser Gln Asn Val Tyr Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Gln Leu Gly Glu Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Asn Ser Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Asn Thr

85 90 95

Phe Gly Ala Gly Thr Asn Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 168

<211> 454

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain MAT G1/3H3

<400> 168

Glu Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Gly Ser Gly Phe Thr Leu Ser Asp Tyr

20 25 30

Gly Val Ala Trp Val Arg Gln Ala Pro Lys Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Tyr Ala Gly Gly Thr Thr Tyr Tyr Arg Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Ile Asp Gly Tyr Gly Gly Tyr Ser Gly Ser His Trp Tyr Phe Asp

100 105 110

Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 169

<211> 459

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain FS20m-232-91AA/3H3 (with LALA)

<400> 169

Glu Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Gly Ser Gly Phe Thr Leu Ser Asp Tyr

20 25 30

Gly Val Ala Trp Val Arg Gln Ala Pro Lys Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Tyr Ala Gly Gly Thr Thr Tyr Tyr Arg Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Ile Asp Gly Tyr Gly Gly Tyr Ser Gly Ser His Trp Tyr Phe Asp

100 105 110

Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Phe Asp Pro

355 360 365

Met Tyr Tyr Tyr Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Glu Pro Leu

385 390 395 400

Trp Asp Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Lys Leu Thr Val Trp Arg Asp Arg Trp Glu Asp Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 170

<211> 123

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-35-14

<400> 170

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Ser Pro Tyr Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Leu Tyr Glu Leu Ser Ala Tyr Ser Tyr Gly Ala Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 171

<211> 369

<212> DNA

<213> Artificial sequence

<220>

<223> Heavy chain variable domain of MAT FS30-35-14

<400> 171

gaagtgcaac tgctggagtc cggtggtggt ctggtacagc cgggtggttc tctgcgtctg

60

agttgcgcgg ccagtggctt taccttcagt gcctataata tccattgggt gcgtcaggct

120

ccgggcaaag gtctggaatg ggttagcgat atttctccgt atggtggcgc gaccaactat

180

gcggatagcg tgaaaggccg ttttaccatt tctcgcgaca acagcaagaa cacgctgtac

240

ctgcagatga actcactgcg tgccgaagat acggccgtgt attactgtgc gagaaacctc

300

tacgagttga gcgcttactc ttacggggcg gactactggg gccagggaac cctggtcacc

360

gtctcgtcg

369

<210> 172

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Variable domain light chain MAT FS30-35-14

<400> 172

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Tyr Ser Ser

85 90 95

Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 173

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<223> Variable domain of light chain MAT FS30-35-14

<400> 173

gaaattgtgc tgacccagtc tccggggcacg ttatctctga gccctggtga gcgcgccact

60

ctgtcatgcc gggcttctca aagtgttagc agtagctacc tggcgtggta tcagcaaaaa

120

ccgggccagg ccccgcgtct gctgatttac ggtgcatcca gccgtgccac cggcattcca

180

gatcgttttt ccggtagtgg ttctgggacg gacttcactc tgacaatctc acgcctggaa

240

ccggaggatt ttgcggtgta ttactgccag caatattatt attcttctcc tatcacgttc

300

ggccaaggga ccaaggtgga aatcaaa

327

<210> 174

<211> 452

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain MAT to OX40 G1/11D4

<400> 174

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ser Gly Trp Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Arg Phe Tyr Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Asp Ile Phe Pro Asn Gly Leu Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Pro Tyr Pro Ser Trp Leu Met Gly Thr Arg Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 175

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain MAT to OX40 G1AA/11D4 and G1/11D4

<400> 175

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<---

Claims (61)

1. Antibody molecule that binds to CD137 and OX40, containing: (a) a complementarity determining region (CDR)-based antigen binding site for CD137; And (b) an antigen binding site for OX40 located in the CH3 domain of the antibody molecule; wherein said CDR-based antigen binding site comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, defined according to the ImMunoGeneTics (IMGT) numbering scheme presented in: (i) SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively; (ii) SEQ ID NO: 1, 2, 16, 4, 5 and 6 respectively; (iii) SEQ ID NO: 1, 2, 21, 4, 5 and 6 respectively; (iv) SEQ ID NO: 25, 26, 27, 4, 5 and 28, respectively; or (v) SEQ ID NO: 33, 34, 35, 4, 5 and 36, respectively; or wherein said CDR-based antigen binding site comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, defined according to the Kabat numbering scheme presented in: (vi) SEQ ID NO: 7, 8, 9, 10, 11 and 6, respectively; (vii) SEQ ID NO: 7, 8, 17, 10, 11 and 6, respectively; (viii) SEQ ID NO: 7, 8, 22, 10, 11 and 6, respectively; (ix) SEQ ID NO: 29, 30, 31, 10, 11 and 28 respectively; or (x) SEQ ID NO: 37, 38, 39, 10, 11 and 36, respectively; And wherein said antigen binding site for OX40 comprises a first sequence, a second sequence and a third sequence located in the structural loops AB, CD and EF of the CH3 domain, respectively, wherein said first, second and third sequences have the sequences listed under SEQ ID NO: 51, 52 and 53 respectively. 2. Antibody molecule according to claim 1, characterized in that: (i) said first sequence is located at positions 14 to 18 of the CH3 domain of said antibody molecule; (ii) said second sequence is located at positions 45.1 to 77 of the CH3 domain of said antibody molecule; and/or (iii) said third sequence is located at positions 93 to 101 of the CH3 domain of said antibody molecule; And wherein the specified numbering of amino acid residues corresponds to the IMGT numbering scheme. 3. Antibody molecule according to any one of paragraphs. 1 or 2, wherein said antibody molecule contains the CH3 domain sequence set forth in SEQ ID NO: 54. 4. The antibody molecule according to claim 3, characterized in that said CH3 domain additionally contains a lysine residue (K) directly at the C-terminus of the CH3 domain sequence. 5. Antibody molecule according to any one of paragraphs. 1-4, characterized in that said antibody molecule contains a VH domain and a VL domain, presented in: (i) SEQ ID NO: 12 and 14 respectively; (ii) SEQ ID NO: 18 and 14 respectively; (iii) SEQ ID NO: 23 and 14 respectively; (iv) SEQ ID NO: 170 and 172, respectively; or (v) SEQ ID NO: 40 and 42 respectively. 6. Antibody molecule according to any one of paragraphs. 1-5, characterized in that said antibody molecule contains the sequences of the heavy chain and the light chain of the antibody: (i) according to SEQ ID NO: 95 and 97, respectively; (ii) according to SEQ ID NO: 99 and 97, respectively; (iii) according to SEQ ID NO: 103 and 97, respectively; (iv) according to SEQ ID NO: 105 and 107, respectively; or (v) according to SEQ ID NO: 109 and 111, respectively. 7. The antibody molecule of claim 6, wherein said CH3 domain in the heavy chain contains a lysine residue (K) immediately at the C-terminus of the CH3 domain sequence. 8. Antibody molecule according to any one of paragraphs. 1-7, characterized in that said antibody molecule contains: (i) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, defined according to the IMGT numbering scheme, listed under SEQ ID NO: 1, 2, 3, 4, 5 and 6, respectively; (ii) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, defined according to the Kabat numbering scheme, listed under SEQ ID NO: 7, 8, 9, 10, 11 and 6, respectively; (iii) the VH domain and the VL domain indicated under SEQ ID NO: 12 and 14, respectively; and/or (iv) a heavy chain and a light chain shown under SEQ ID NO: 95 and 97, respectively. 9. The antibody molecule according to claim 8, characterized in that said antibody molecule contains a heavy chain and a light chain specified under SEQ ID NO: 95 and 97, respectively. 10. The antibody molecule of claim 9, wherein the CH3 domain of the heavy chain contains a lysine residue (K) immediately at the C-terminus of the CH3 domain sequence. 11. Antibody molecule according to any one of paragraphs. 1-10, wherein said antibody molecule binds human CD137 and human OX40, wherein optionally said antibody molecule is capable of simultaneously binding to human CD137 and human OX40. 12. Antibody molecule according to any one of paragraphs. 1-11, characterized in that: (i) said antibody molecule is capable of activating OX40 on an immune cell in the presence of CD137 expressed on the cell surface, and/or said antibody molecule is capable of activating CD137 on an immune cell in the presence of OX40 expressed on the cell surface; (ii) binding of said antibody molecule to OX40 on an immune cell and to CD137 causes clustering of OX40 on said immune cell and/or binding of said antibody molecule to CD137 on an immune cell and to OX40 causes clustering of CD137 on said immune cell; and/or (iii) said antibody molecule has been modified to reduce or eliminate binding of the CH2 domain of said antibody molecule to one or more Fcγ receptors. 13. A nucleic acid molecule encoding an antibody molecule according to any one of claims. 1-12. 14. A combination of nucleic acid molecules encoding an antibody molecule according to any one of claims. 1-12. 15. A vector for expressing a nucleic acid molecule or molecules in a host cell, comprising a nucleic acid molecule according to claim 13 or a combination of nucleic acid molecules according to claim 14. 16. A combination of vectors for expressing a nucleic acid molecule or molecules in a host cell, comprising a nucleic acid molecule according to claim 13 or a combination of nucleic acid molecules according to claim 14. 17. Recombinant host cell for producing an antibody molecule according to any one of paragraphs. 1-12, containing a nucleic acid molecule according to claim 13, or a combination of nucleic acid molecules according to claim 14, or a vector according to claim 15, or a combination of vectors according to claim 16. 18. A method for producing an antibody molecule according to any one of claims. 1-12, comprising culturing the recombinant host cell of claim 17 under conditions to obtain said antibody molecule, further comprising isolating said antibody molecule. 19. The method of claim 18, further comprising purifying said antibody molecule. 20. Pharmaceutical composition for the treatment of a disease or disorder associated with the expression of CD137 and OX40, containing an antibody molecule according to any one of paragraphs. 1-12 in a therapeutically effective amount and a pharmaceutically acceptable excipient. 21. Use of an antibody molecule according to any one of paragraphs. 1-12 for the treatment of cancer in an individual. 22. The use of claim 21, wherein said treatment comprises administering said antibody molecule to said individual in combination with a second therapeutic agent. 23. The use of claim 22, wherein said second therapeutic agent is an antibody that binds PD-1 or PD-L1. 24. An antibody molecule that binds to CD137 and OX40, wherein the antibody molecule contains the light chain sequence set forth in SEQ ID NO: 97 and the heavy chain sequence set forth in SEQ ID NO: 95. 25. An antibody molecule that binds to CD137 and OX40, wherein the antibody molecule contains the light chain sequence set forth in SEQ ID NO: 97 and the heavy chain sequence set forth in SEQ ID NO: 95, and wherein the heavy chain CH3 domain comprises a residue lysine (K) immediately at the C-terminal end of the CH3 domain sequence.