RU2782381C2 - High-affinity anti-pd-1 and anti-lag-3 antibodies and bispecific binding proteins obtained from them - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to bispecific polyvalent binding protein capable of binding PD-1 and LAG-3, as well as to an anti-LAG-3 antibody or its antigen-binding part. A composition containing the above-mentioned protein or antibody, as well as a method for the manufacture of a drug are also disclosed.

EFFECT: invention is effective for the treatment of a disorder, in which activity mediated by PD-1 and/or mediated by LAG-3 is harmful.

31 cl, 13 dwg, 55 tbl, 16 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to novel antibodies recognizing programmed cell death protein 1 (PD-1), novel antibodies recognizing the lymphocyte activation gene 3 (LAG-3) protein, and bispecific PD-1/LAG-3 binding proteins derived from these antibodies, such as FIT-Ig binding proteins. Antibodies and bispecific binding proteins are useful in the treatment of immunological diseases and hematological malignancies.

BACKGROUND OF THE INVENTION

Programmed cell death protein 1 (PD-1)

Programmed cell death protein 1 (PD-1, CD279) is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Expression of PD-1 is often found in immune cells such as T cells, B cells, monocytes, and natural killer (NK) cells. PD-1 and similar family members are type I transmembrane glycoproteins containing an immunoglobulin-like domain resembling an Ig variable domain that is responsible for ligand binding and a cytoplasmic tail that is responsible for binding signaling molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling motifs, ITIM (tyrosine-based immunoreceptor inhibitory motif) and ITSM (tyrosine-based immunoreceptor switching motif). Vivier et al., Immunol. Today, 18:286-291 (1997) and Chemnitz et al., J. Immunol., 173:945-954 (2004).

Two cell surface glycoprotein ligands of the PD-1 protein, programmed death ligand 1 (PD-L1, CD274, B7-H1) and PD-L2 (CD273, B7-DC), have been identified and shown to induce intracellular signaling that suppresses CD3- and CD28-mediated activation of T cells. Riley, Immunol. Rev. 229:114-125 (2009). This suppression of T cell activation in turn leads to a decrease in T cell proliferation, IL-2 secretion, IFN-γ secretion, and secretion of other growth factors and cytokines. Freeman et al., J. Exp. Med. 192:1027-1034 (2000); Latchman et al., Nat. Immunol. 2:261-8 (2001); Carter et al., Eur. J. Immunol., 32:634-43 (2002); Ohigashi et al., Clin. Cancer Res. 11:2947-53 (2005). It is suggested that signaling through the PD-1/PD-L1 interaction performs important, irreplaceable functions in the immune system, providing a negative regulation of T-cell responses. This regulation is associated with the development of T cells in the thymus, the regulation of chronic inflammatory responses, and the maintenance of both peripheral tolerance and immune privilege. The key role of these functions is illustrated in PD-1 deficient mice with a manifest autoimmune phenotype. PD-1 deficiency in C57BL/6 mice leads to the development of chronic progressive lupus-like glomerulonephritis and arthritis. In Balb/c mice, PD-1 deficiency leads to the development of severe cardiomyopathy due to the presence of autoreactive antibodies specific to cardiac tissue.

Upon stimulation of T cells, PD-1 recruits tyrosine phosphatase SHP-2 to the ITSM motif located in the cytoplasmic tail, which leads to dephosphorylation of effector molecules such as CD3-ζ, PKC-θ, and ZAP70 involved in the CD3 T-cell signaling cascade. The mechanism by which PD-1 suppresses T cell responses is similar to, but different from, that of CTLA-4 because both molecules regulate an overlapping set of signaling proteins. Parry et al., Mol. Cell Biol., 25:9543-9553 (2005). In general, the inhibitory signal mediated by PD-1 plays an important role in immune tolerance. Bour-Jordan et al., Immunol. Rev. 241:180-205 (2011).

Increased expression of PD-1 is found in tumor-infiltrating lymphocytes (TIL), and according to published reports, the expression of PD-1 ligands in tumor cells is observed in various cancers of various tissues, including lung, liver, stomach, kidney, breast, ovary , pancreas, melanocytes and esophagus. In general, for many types of tumors, PD-1 ligand expression on tumor cells correlates with a poor prognosis for cancer patients. Okazaki and Honjo, Int. Immunol. 19:813-824 (2007).

Blockade of the PD-1/PD-L1 interaction may lead to an increase in tumor-specific T-cell immunity and, therefore, be useful for the destruction of tumor cells by the immune system. In a mouse model of aggressive pancreatic cancer, T. Nomi et al demonstrated the therapeutic efficacy of PD-1/PD-L1 blockade, showing that administration of an anti-PD-1 or anti-PD-L1 antibody leads to a significant suppression of tumor growth. Nomi et al., Clin. Cancer Res. 13:2151-2157 (2007). Antibody blockade was effective against infiltration of tumor CD8+ T cells into the tumor, leading to an increase in the activity of antitumor effectors, including IFN-γ, granzyme B, and perforin. In another study in a mouse model of squamous cell carcinoma, blockade of anti-PD-1 or anti-PD-L1 antibodies significantly suppressed tumor growth. Tsushima et al., Oral Oncol. 42:268-274 (2006).

It has recently been shown that PD-1 is highly expressed in T cells of HIV-infected humans, and that expression of this receptor is correlated with T cell dysfunction and disease progression. Day et al., Nature, 443:350-354 (2006); Trautmann et al., Nat. Med. 12:1198-1202 (2006). In both studies, blockade of the PD-L1 ligand significantly increased the proliferation of HIV-specific, IFN-γ-producing cells in vitro .

Accordingly, therapeutic modulation of PD-1 signaling by antagonist molecules can reverse the development of immune cells from tolerance and reactivate them to kill cancer and eliminate chronic viral infections.

Lymphocyte activation gene 3 (LAG-3)

Lymphocyte activation gene 3 protein (LAG-3, CD223) is a negatively regulated co-stimulatory receptor that modulates T cell homeostasis, proliferation, and activation. Sierro et al., Expert Opin. Ther. Targets, 15:91-101 (2010). A member of the immunoglobulin superfamily, LAG-3 is a CD4-like protein that, like CD4, binds to MHC class II molecules, but with twice the affinity and in a different region than CD4. Huard et al., Proc. Natl Acad. sci. USA 94:5744-9 (1997). LAG-3 is expressed on activated CD8+ T cells, γδ T cells, natural killer cells, B cells, plasma dendritic cells and regulatory T cells (Treg). The role of LAG-3 as a negative regulator of T-cell responses was determined based on studies in LAG-3 knockout mice and the use of blocking anti-LAG-3 antibodies in in vitro and in vivo model systems. Sierro et al. (2010), op. cit.; Hannier et al., J. Immunol., 161:4058-65 (1998); Macon-Lemaitre et al., Immunology, 115:170-8 (2005); Workman et al., Eur. J. Immunol., 33:970-9 (2003). Both natural and induced Tregs express elevated levels of LAG-3, which are required to maximize their inhibitory function. Camisaschi et al., J. Immunol., 184:6545-6551 (2010); Huang, et al., Immunity, 21:503-513 (2004). In addition, ectopic expression of LAG-3 on effector CD4+ T cells reduces their proliferative capacity and confers on them a regulatory capacity directed against third-party T cells. Huang, see ibid. Recent studies have also shown that high levels of LAG-3 expression on depleted T cells specific for lymphocytic choriomeningitis virus (LCMV) promote their transition to a non-responsive state and limit antitumor responses of CD8+ T cells. Blackburn et al., Nat. Immunol., 10:29-37 (2009) and Grosso et al., J. Clin. Invest., 117:3383-3392 (2007).

The important role of LAG-3 in the antitumor immune response and the immune response to infection makes it a target for immunotherapy. For the treatment of certain cancers and chronic viral infections, it has been proposed to block LAG-3 with antagonists, including monoclonal antibodies. Turnis et al., Eur. J. Immunol., 45:1892-1905 (2015).

As the importance of PD-1 and LAG-3 mediated signaling becomes clear, the need for new inhibitory anti-PD-1 and anti-LAG-3 antibodies that could effectively alter T cell functionality or increase tumor cell reactivity to immune effector cells, is becoming more and more relevant. Moreover, the development of bispecific molecules combining the inhibitory effects of PD-1 and LAG-3 could lead to desirable improvements in cancer therapeutic approaches as well.

SUMMARY OF THE INVENTION

The present invention relates to novel antibodies that bind to PD-1 with high affinity and novel antibodies that bind to LAG-3 with high affinity. The invention also relates to PD-1/LAG-3 bispecific Fabs-in-Tandem (FIT-Ig) immunoglobulins that react with both PD-1 and LAG-3. Antibodies and bespecifically binding proteins of the present invention can block LAG-3 on TIL to reduce the population of tumor-infiltrated Treg cells or to restore TIL to a cytotoxic phenotype. In addition, the antibodies and bispecific binding proteins of the invention can be used to suppress PD-1/PD-L1 signaling to reactivate tumor-infiltrated cytotoxic T cells. The bispecific polyvalent binding proteins disclosed in this application will be useful as bispecific PD-1/LAG-3 inhibitors to provide a synergistic combined effect to overcome the suppression of the antitumor immune response and thus improve outcomes even in those patients who do not respond. or have ceased responding to either anti-PD-1 therapy or anti-LAG-3 therapy provided separately.

The invention also relates to methods for preparing and using anti-PD-1 and anti-LAG-3 antibodies and bispecific PD-1/LAG-3 binding proteins of the present invention, as well as various compositions that can be used in methods for detecting PD-1 and/or LAG-3 in a sample or in methods for treating or preventing a disorder associated with PD-1 and/or LAG-3 activity in an individual.

In a further embodiment, the invention relates to a bispecific Fabs-in-Tandem binding immunoglobulin (FIT-Ig) protein comprising first, second and third polypeptide chains,

moreover, the specified first polypeptide chain contains, in the direction from the amino end to the carboxyl end, (i) VL _A -CL-VH _B -CH1-Fc, where CL is fused directly to VH _B , or (ii) VH _B -CH1-VL _A - CL-Fc, where CH1 is fused directly to VL _A ;

moreover, the specified second polypeptide chain contains, in the direction from the amino end to the carboxyl end, VH _A -CH1; and

moreover, the specified third polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _B -CL;

wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, A is a PD-1 or LAG epitope -3, and B is a PD-1 or LAG-3 epitope, provided that A and B are different. In accordance with the present invention, such FIT-Ig binding proteins bind to both PD-1 and LAG-3.

In preferred embodiments, the Fab fragments of such FIT-Ig binding proteins comprise the VL _A -CL and VH _A -CH1 domains of the parent antibody binding to one of the target PD-1 or LAG-3 antigens and include the VL _B -CL and VH domains. _B -CH1 of another parental antibody that binds to another target antigen of PD-1 and LAG-3. Thus, VH-CH1/VL-CL pairing results in the formation of tandem Fab fragments that recognize PD-1 and LAG-3.

In accordance with the present invention, the PD-1/LAG-3 binding FIT-Ig protein may advantageously comprise first, second and third polypeptide chains, wherein said first polypeptide chain contains, in the direction from the amino terminus to the carboxyl terminus, VL _PD-1 -CL -VH _LAG-3 -CH1-Fc, where CL is fused directly to VH _LAG-3 , wherein said second polypeptide chain contains, in the direction from the amino to the carboxyl end, VH _PD-1 -CH1; and moreover, the specified third polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _LAG-3 -CL; wherein VL _PD-1 is the light chain variable domain of an anti-PD-1 antibody, CL is the light chain constant domain, VH _PD-1 is the heavy chain variable domain of an anti-PD-1 antibody, CH1 is the heavy chain constant domain , VL _LAG-3 is the light chain variable domain of an anti-LAG-3 antibody, VH _LAG-3 is the heavy chain variable domain of an anti-LAG-3 antibody, and Fc is the Fc region of an immunoglobulin. Advantageously, in the first polypeptide chain, the VL domains _{of PD-1} -CL are the same as the light chain of the parental anti-PD-1 antibody, the VH domains _{of PD-1} -CH1 are the same as the heavy chain variable domain and the heavy chain constant domain of the parent anti-PD-1 antibodies, the VL domains _{of LAG-3} -CL are the same as the light chain of the parent anti-LAG-3 antibody, and the VH domains _{of LAG-3} -CH1 are the same as the heavy chain variable domain and heavy chain constant domain parental anti-LAG-3 antibody chain.

In alternative embodiments, the PD-1/LAG-3 FIT-Ig binding protein may advantageously comprise first, second, and third polypeptide chains, wherein said first polypeptide chain comprises, in the amino-to-carboxyl-terminal direction, VL _LAG-3 -CL- VH _PD-1 -CH1-Fc, where CL is fused directly to VH _PD-1 , and the specified second polypeptide chain contains, in the direction from the amino end to the carboxyl end, VH _LAG-3 -CH1; and moreover, the specified third polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _PD-1 -CL; wherein VL _PD-1 is the light chain variable domain of an anti-PD-1 antibody, CL is the light chain constant domain, VH _PD-1 is the heavy chain variable domain of an anti-PD-1 antibody, CH1 is the heavy chain constant domain , VL _LAG-3 is the light chain variable domain of an anti-LAG-3 antibody, VH _LAG-3 is the heavy chain variable domain of an anti-LAG-3 antibody, and Fc is the Fc region of an immunoglobulin. Preferably, in the first polypeptide chain, the VL domains _{of LAG-3} -CL are the same as the light chain of the parent anti-LAG-3 antibody, the VH domains _{of LAG-3} -CH1 are the same as the heavy chain variable domain and the heavy chain constant domain of the parent anti-LAG-3 antibodies, the VL domains _{of PD-1} -CL are the same as the light chain of the parent anti-PD-1 antibody, and the VH domains _{of PD-1} -CH1 are the same as the heavy chain variable domain and heavy chain constant domain chain of the parent anti-PD-1 antibody.

In alternative embodiments, the PD-1/LAG-3 FIT-Ig binding protein may advantageously comprise first, second, and third polypeptide chains, wherein said first polypeptide chain comprises, in the amino-to-carboxyl-terminal direction, VH _LAG-3 -CH1- VL _PD-1 -CL-Fc, where CH1 is fused directly to VL _PD-1 , and the specified second polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _LAG-3 -CL; and in which the specified third polypeptide chain contains, in the direction from the amino end to the carboxyl end, VH _PD-1 -CH1; wherein VL _PD-1 is the light chain variable domain of an anti-PD-1 antibody, CL is the light chain constant domain, VH _PD-1 is the heavy chain variable domain of an anti-PD-1 antibody, CH1 is the heavy chain constant domain , VL _LAG-3 is the light chain variable domain of an anti-LAG-3 antibody, VH _LAG-3 is the heavy chain variable domain of an anti-LAG-3 antibody, and Fc is the Fc region of an immunoglobulin. Preferably, in the first polypeptide chain, the VL domains _{of LAG-3} -CL are the same as the light chain of the parent anti-LAG-3 antibody, the VH domains _{of LAG-3} -CH1 are the same as the heavy chain variable domain and the heavy chain constant domain of the parent anti-LAG-3 antibodies, the VL domains _{of PD-1} -CL are the same as the light chain of the parent anti-PD-1 antibody, and the VH domains _{of PD-1} -CH1 are the same as the heavy chain variable domain and heavy chain constant domain chain of the parent anti-PD-1 antibody.

In alternative embodiments, the PD-1/LAG-3 FIT-Ig binding protein may advantageously comprise first, second, and third polypeptide chains, wherein said first polypeptide chain comprises, in the amino to carboxyl direction, VH _PD-1 -CH1- VL _LAG-3 -CL-Fc, where CH1 is fused directly to VL _LAG-3 , and said second polypeptide chain contains, in the direction from the amino to the carboxyl end, VL _PD-1 -CL; and moreover, the specified third polypeptide chain contains, in the direction from the amino end to the carboxyl end, VH _LAG-3 -CH1; wherein VL _PD-1 is the light chain variable domain of an anti-PD-1 antibody, CL is the light chain constant domain, VH _PD-1 is the heavy chain variable domain of an anti-PD-1 antibody, CH1 is the heavy chain constant domain , VL _LAG-3 is the light chain variable domain of an anti-LAG-3 antibody, VH _LAG-3 is the heavy chain variable domain of an anti-LAG-3 antibody, and Fc is the Fc region of an immunoglobulin. Preferably, in the first polypeptide chain, the VL domains _{of LAG-3} -CL are the same as the light chain of the parent anti-LAG-3 antibody, the VH domains _{of LAG-3} -CH1 are the same as the heavy chain variable domain and the heavy chain constant domain of the parent anti-LAG-3 antibodies, the VL domains _{of PD-1} -CL are the same as the light chain of the parent anti-PD-1 antibody, and the VH domains _{of PD-1} -CH1 are the same as the heavy chain variable domain and heavy chain constant domain chain of the parent anti-PD-1 antibody.

In the above formulas of the first polypeptide chain of the FIT-Ig binding protein, the Fc region may be a natural or altered Fc region. In specific embodiments, the Fc region is a human Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In specific embodiments, the Fc is a human Fc from IgG1 or a modified human Fc such as shown in Table 6 below (SEQ ID NO: 28).

In one embodiment, the FIT-Ig binding proteins of the present invention retain one or more of the properties of the parent antibodies from which the Fab fragment sequences are derived and incorporated into the FIT-Ig structure. In preferred embodiments, the FIT-Ig retains a binding affinity for target antigens (e.g., LAG-3 and PD-1) comparable to the binding affinity of the parent antibodies, meaning that the binding affinity of the FIT-Ig binding protein for the target antigens PD-1 and LAG-3 does not differ more than 10-fold from the binding affinity of the parent antibodies to the respective target antigens as measured by surface plasmon resonance or biolayer interferometry.

In one embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-679 of SEQ ID NO: 78; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence 20-240 of SEQ ID NO: 83; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-236 of SEQ ID NO: 86 (see Table 27).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-684 of SEQ ID NO: 88; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence 20-235 of SEQ ID NO: 91; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-236 of SEQ ID NO: 93 (see Table 28).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-679 of SEQ ID NO: 95; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence 20-242 of SEQ ID NO: 98; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-236 of SEQ ID NO: 100 (see Table 29).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-684 of SEQ ID NO: 102; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence 20-235 of SEQ ID NO: 105; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-236 of SEQ ID NO: 107 (see Table 30).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-679 of SEQ ID NO: 140; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 144; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 146 (see FIT107-1-6a-1; Table 41).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-684 of SEQ ID NO: 147; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 151; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 153 (see FIT-107-1-6b-1; Table 42).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-679 of SEQ ID NO: 154; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 158; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 160 (see FIT-107-1-6a-2; Table 43).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-684 of SEQ ID NO: 161; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 165; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 167 (see FIT-107-1-6b-2; Table 44).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-679 of SEQ ID NO: 168; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 172; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 174 (see FIT-107-1-6a-3; Table 45).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-684 of SEQ ID NO: 175; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 179; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 181 (see FIT-107-1-6b-3; Table 46).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-679 of SEQ ID NO: 182; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 186; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 188 (see FIT-107-1-7a-1; Table 47).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-687 of SEQ ID NO: 189; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 193; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 195 (see FIT-107-1-7b-1; Table 48).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-679 of SEQ ID NO: 196; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 200; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 202 (see FIT-107-1-7a-2; Table 49).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-687 of SEQ ID NO: 203; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 207; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 209 (see FIT-107-1-7b-2; Table 50).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-679 of SEQ ID NO: 210; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 214; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 216 (see FIT-107-1-7a-3; Table 51).

In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-687 of SEQ ID NO: 217; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 221; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence of SEQ ID NO: 223 (see FIT-107-1-7b-3; Table 52).

The invention also relates to novel antibodies capable of binding human PD-1, wherein the antigen-binding domain of said antibodies contains a set of six CDRs, i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from the group of CDR sets defined below:

CDR set no. CDR CDR
Amino acid sequence SEQID NO: one CDR-H1 SYMMS residues 31-35 SEQ ID NO:4 CDR-H2 SMSGGGRDTYYPDSVKG residues 50-66 SEQ ID NO:4 CDR-H3 RGTYAMDY residues 99-106 SEQ ID NO:4 CDR-L1 LASQTIGTWLT residues 24-34 SEQ ID NO:5 CDR-L2 AATSLAD residues 50-56 SEQ ID NO:5 CDR-L3 QQLYSTPWT residues 89-97 SEQ ID NO:5 2 CDR-H1 TGYYWN residues 31-36 SEQ ID NO:6 CDR-H2 YMSYDGNNNYNPSLKN residues 51-66 SEQ ID NO:6 CDR-H3 DRGTTILGGTMDY residues 99-111 SEQ ID NO:6 CDR-L1 KASQSVSNDVA residues 24-34 SEQ ID NO:7 CDR-L2 YAFYRYT residues 50-56 SEQ ID NO:7 CDR-L3 QQDYSSPWT residues 89-97 SEQ ID NO:7 3 CDR-H1 FYTMS residues 31-35 SEQ ID NO:8 CDR-H2 TISGGGRDTYYPDSVKG residues 50-66 SEQ ID NO:8 CDR-H3 QGGNYLFAY residues 99-107 SEQ ID NO:8 CDR-L1 KASQDVNTVVA residues 24-34 SEQ ID NO:9 CDR-L2 WASTRHT residues 50-56 SEQ ID NO:9 CDR-L3 QQHYTTPYT residues 89-97 SEQ ID NO:9 four CDR-H1 DYGMH residues 31-35 SEQ ID NO:10 CDR-H2 YISSGSYTIYYADTVKG residues 50-66 SEQ ID NO:10 CDR-H3 RGGSSHVNVMDY residues 99-110 SEQ ID NO:10 CDR-L1 KASDHINNWLA residues 24-34 SEQ ID NO:11 CDR-L2 GATSLET residues 50-56 SEQ ID NO:11 CDR-L3 QQYWSPPYT residues 89-97 SEQ ID NO:11 5 CDR-H1 DNNVE residues 31-35 SEQ ID NO:12 CDR-H2 DINPNNGDTLYSQYFKD residues 50-66 SEQ ID NO:12 CDR-H3 GKSDQFDY residues 99-106 SEQ ID NO:12 CDR-L1 LASQTIGTWLA residues 24-34 SEQ ID NO:13 CDR-L2 AATSLAD residues 50-56 SEQ ID NO:13 CDR-L3 QQLYSSPWT residues 89-97 SEQ ID NO:13 6 CDR-H1 SYAMS residues 31-35 SEQ ID NO:14 CDR-H2 TISGGGRDTYYPDSVKG residues 50-66 SEQ ID NO:14 CDR-H3 QGGTYLFAS residues 99-107 SEQ ID NO:14 CDR-L1 KASQDVNTAVA residues 24-34 SEQ ID NO:15 CDR-L2 WASTRHT residues 50-56 SEQ ID NO:15 CDR-L3 QQHYTTPYT residues 89-97 SEQ ID NO:15 7 CDR-H1 DYEMH residues 31-35 SEQ ID NO:16 CDR-H2 VIEPESGGTVYNQKFKG residues 51-66 SEQ ID NO:16 CDR-H3 EGFNSDHYFDY residues 99-109 SEQ ID NO:16 CDR-L1 RSSQNIVHSNGNTYLE residues 24-39 SEQ ID NO:17 CDR-L2 KVFNRFS residues 55-61 SEQ ID NO:17 CDR-L3 FQGSHVPYT residues 94-102 SEQ ID NO:17 eight CDR-H1 SHLMS residues 31-35 SEQ ID NO:18 CDR-H2 AISGGGADTYYPDSVKG residues 50-66 SEQ ID NO:18 CDR-H3 QILAFDS residues 99-105 SEQ ID NO:18 CDR-L1 HASQNIYVWLN residues 24-34 SEQ ID NO:19 CDR-L2 KASNLHT residues 50-56 SEQ ID NO:19 CDR-L3 QQGQSYPWT residues 89-97 SEQ ID NO:19 9 CDR-H1 SHLMS residues 31-35 SEQ ID NO:53 CDR-H2 AISGGGADTYYPASVKG residues 50-66 SEQ ID NO:53 CDR-H3 QILAFDA residues 99-105 SEQ ID NO:53 CDR-L1 HASQNIYVWLN residues 24-34 SEQ ID NO:19 CDR-L2 KASNLHT residues 50-56 SEQ ID NO:19 CDR-L3 QQGQSYPWT residues 89-97 SEQ ID NO:19

The invention also relates to novel antibodies capable of binding human LAG-3, in which the antigen-binding domain of said antibodies contains a set of six CDRs, i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from the group of CDR sets defined below:

CDR set no. CDR CDR
Amino acid sequence SEQID NO: ten CDR-H1 DDYMH residues 31-35 SEQ ID NO:60 CDR-H2 WIVPENGNTEYASKFQG residues 50-66 SEQ ID NO:60 CDR-H3 YGDY residues 99-102 SEQ ID NO:60 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:61 CDR-L2 AASTLDS residues 50-56 SEQ ID NO:61 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:61 eleven CDR-H1 DDYMH residues 31-35 SEQ ID NO:60 CDR-H2 WIVPENGNTEYASKFQG residues 50-66 SEQ ID NO:60 CDR-H3 YGDY residues 99-102 SEQ ID NO:60 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:62 CDR-L2 AASTLDS residues 50-56 SEQ ID NO:62 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:62 12 CDR-H1 DYEMH residues 31-35 SEQ ID NO:63 CDR-H2 AIDPETGGTAYNQKFKG residues 50-66 SEQ ID NO:63 CDR-H3 WGSTVFPY residues 101-108 SEQ ID NO:63 CDR-L1 KSTKSLLNSDGFTYLD residues 24-39 SEQ ID NO:64 CDR-L2 LVSNRFS residues 55-61 SEQ ID NO:64 CDR-L3 FQSNYLPWT residues 94-102 SEQ ID NO:64 13 CDR-H1 DYEMH residues 31-35 SEQ ID NO:65 CDR-H2 AIDPATGGTAYNQKFKG residues 50-66 SEQ ID NO:65 CDR-H3 WGTTVFPY residues 99-106 SEQ ID NO:65 CDR-L1 KSTKSLLNSDGFTYLD residues 24-39 SEQ ID NO:66 CDR-L2 LVSNRFS residues 55-61 SEQ ID NO:66 CDR-L3 FQSNYLPWT residues 94-102 SEQ ID NO:66 fourteen CDR-H1 DDYMH residues 31-35 SEQ ID NO:67 CDR-H2 WIDPENGDTEYASKFQG residues 50-66 SEQ ID NO:67 CDR-H3 FDY residues 99-101 SEQ ID NO:67 CDR-L1 KSSQSLLDSDGKTYLN residues 24-39 SEQ ID NO:68 CDR-L2 LVSKLDS residues 55-61 SEQ ID NO:68 CDR-L3 WQGSHFPQT residues 94-102 SEQ ID NO:68 fifteen CDR-H1 DDYVH residues 31-35 SEQ ID NO:69 CDR-H2 WIDPENGDTEYASKFQG residues 50-66 SEQ ID NO:69 CDR-H3 WDAEENY residues 99-105 SEQ ID NO:69 CDR-L1 RSSKSLLHSNGNTYLY residues 24-39 SEQ ID NO:70 CDR-L2 RMSNLAS residues 55-61 SEQ ID NO:70 CDR-L3 MQHLEYPFT residues 94-102 SEQ ID NO:70 16 CDR-H1 DDYIH residues 31-35 SEQ ID NO:71 CDR-H2 WIDPENGDTEYASKFQG residues 50-66 SEQ ID NO:71 CDR-H3 DYRNWY residues 100-105 SEQ ID NO:71 CDR-L1 KSSQSLLDSDGKTYLN residues 24-39 SEQ ID NO:68 CDR-L2 LVSKLDS residues 55-61 SEQ ID NO:68 CDR-L3 WQGSHFPQT residues 94-102 SEQ ID NO:68 17 CDR-H1 DFNIKDDYMH residues 26-35 SEQ ID NO:114 CDR-H2 WIVPENGNTEYASKFQG residues 50-66 SEQ ID NO:114 CDR-H3 YGDY residues 99-102 SEQ ID NO:114 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:117 CDR-L2 AASTLDS residues 50-56 SEQ ID NO:117 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:117 eighteen CDR-H1 DDYMH residues 31-35 SEQ ID NO:72 CDR-H2 WIVPENGNTEYASKFQG residues 50-66 SEQ ID NO:72 CDR-H3 YGDY residues 99-102 SEQ ID NO:72 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:77 CDR-L2 AASTLDS residues 50-56 SEQ ID NO:77 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:77 19 CDR-H1 DDYMH residues 30-34 SEQ ID NO:119 CDR-H2 WIVPENGNTVYASKFQG residues 48-64 SEQ ID NO:119 CDR-H3 YGDY residues 95-98 SEQ ID NO:119 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:120 CDR-L2 AASALDS residues 50-56 SEQ ID NO:120 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:120 twenty CDR-H1 DDYMH residues 31-35 SEQ ID NO:121 CDR-H2 WIVPENGNTEYASKFQG residues 50-66 SEQ ID NO:121 CDR-H3 YGDY residues 99-102 SEQ ID NO:121 CDR-L1 RAMQEISGYLS residues 24-34 SEQ ID NO:122 CDR-L2 AASTLDS residues 50-56 SEQ ID NO:122 CDR-L3 LQYAYYPLT residues 89-97 SEQ ID NO:122 21 CDR-H1 DDYMH residues 31-35 SEQ ID NO:123 CDR-H2 WIVPENGNTEYASKFQG residues 50-66 SEQ ID NO:123 CDR-H3 YGDY residues 99-102 SEQ ID NO:123 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:124 CDR-L2 AASHLDS residues 50-56 SEQ ID NO:124 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:124 22 CDR-H1 DDYMH residues 31-35 SEQ ID NO:125 CDR-H2 WIVPENGLTEYASKFQG residues 50-66 SEQ ID NO:125 CDR-H3 YGDY residues 99-102 SEQ ID NO:125 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:126 CDR-L2 ATSTLDS residues 50-56 SEQ ID NO:126 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:126 23 CDR-H1 DDYMH residues 31-35 SEQ ID NO:127 CDR-H2 WIVPENGKTEYASKFQG residues 50-66 SEQ ID NO:127 CDR-H3 YGDY residues 99-102 SEQ ID NO:127 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:128 CDR-L2 AAMTLDS residues 50-56 SEQ ID NO:128 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:128 24 CDR-H1 DDYMH residues 31-35 SEQ ID NO:129 CDR-H2 WIVPENGNTHYASKFQG residues 50-66 SEQ ID NO:129 CDR-H3 YGDY residues 99-102 SEQ ID NO:129 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:130 CDR-L2 EASTLDS residues 50-56 SEQ ID NO:130 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:130 25 CDR-H1 DDYMH residues 31-35 SEQ ID NO:131 CDR-H2 WIVPRNGNTMYASKFQG residues 50-66 SEQ ID NO:131 CDR-H3 YGDY residues 99-102 SEQ ID NO:131 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:132 CDR-L2 AASTLDL residues 50-56 SEQ ID NO:132 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:132 26 CDR-H1 DDYMH residues 31-35 SEQ ID NO:135 CDR-H2 WIVPENANTVYASKFQG SEQ ID NO:224 CDR-H3 YGDY residues 99-102 SEQ ID NO:135 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:138 CDR-L2 AASALDS residues 50-56 SEQ ID NO:138 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:138 27 CDR-H1 DDYMH residues 31-35 SEQ ID NO:136 CDR-H2 WIVPRNANTVYASKFQG SEQ ID NO:225 CDR-H3 YGDY residues 99-102 SEQ ID NO:136 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:139 CDR-L2 AASALDL residues 50-56 SEQ ID NO:139 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:139 28 CDR-H1 DDYMH residues 31-35 SEQ ID NO:136 CDR-H2 WIVPRNANTVYASKFQG SEQ ID NO:225 CDR-H3 YGDY residues 99-102 SEQ ID NO:136 CDR-L1 RASQEISGYLS residues 24-34 SEQ ID NO:117 CDR-L2 AASTLDS residues 50-56 SEQ ID NO:117 CDR-L3 LQYASYPLT residues 89-97 SEQ ID NO:117

In one embodiment, a binding protein of the invention is a bispecific, polyvalent immunoglobulin binding protein comprising two or more antigen-binding sites, wherein at least one antigen-binding site contains a CDR set selected from CDR sets 1, 2, 3, and 4 above, and at least one antigen-binding site contains the above set of 5 CDRs.

In one embodiment, an anti-PD-1 antibody of the invention comprises VH and VL domains, wherein the two variable domains comprise amino acid sequences selected from the group consisting of:

SEQ ID NO:4 and SEQ ID NO:5 SEQ ID NO:6 and SEQ ID NO:7 SEQ ID NO:8 and SEQ ID NO:9 SEQ ID NO:10 and SEQ ID NO:11 SEQ ID NO:12 and SEQ ID NO:13 SEQ ID NO:14 and SEQ ID NO:15 SEQ ID NO:16 and SEQ ID NO:17 SEQ ID NO:18 and SEQ ID NO:19 SEQ ID NO:20 and SEQ ID NO:23 SEQ ID NO:21 and SEQ ID NO:23 SEQ ID NO:22 and SEQ ID NO:23 SEQ ID NO:20 and SEQ ID NO:24 SEQ ID NO:21 and SEQ ID NO:24 SEQ ID NO:22 and SEQ ID NO:24 SEQ ID NO:20 and SEQ ID NO:25 SEQ ID NO:21 and SEQ ID NO:25 SEQ ID NO:22 and SEQ ID NO:25 SEQ ID NO:20 and SEQ ID NO:26 SEQ ID NO:21 and SEQ ID NO:26 SEQ ID NO:22 and SEQ ID NO:26 SEQ ID NO:20 and SEQ ID NO:27 SEQ ID NO:21 and SEQ ID NO:27 SEQ ID NO:22 and SEQ ID NO:27 SEQ ID NO:30 and SEQ ID NO:34 SEQ ID NO:31 and SEQ ID NO:34 SEQ ID NO:32 and SEQ ID NO:34 SEQ ID NO:33 and SEQ ID NO:34 SEQ ID NO:30 and SEQ ID NO:35 SEQ ID NO:31 and SEQ ID NO:35 SEQ ID NO:32 and SEQ ID NO:35 SEQ ID NO:33 and SEQ ID NO:35 SEQ ID NO:30 and SEQ ID NO:36 SEQ ID NO:31 and SEQ ID NO:36 SEQ ID NO:32 and SEQ ID NO:36 SEQ ID NO:33 and SEQ ID NO:36 SEQ ID NO:30 and SEQ ID NO:37 SEQ ID NO:31 and SEQ ID NO:37 SEQ ID NO:32 and SEQ ID NO:37 SEQ ID NO:33 and SEQ ID NO:37 SEQ ID NO:38 and SEQ ID NO:43 SEQ ID NO:39 and SEQ ID NO:43 SEQ ID NO:40 and SEQ ID NO:43 SEQ ID NO:41 and SEQ ID NO:43 SEQ ID NO:42 and SEQ ID NO:43 SEQ ID NO:38 and SEQ ID NO:44 SEQ ID NO:39 and SEQ ID NO:44 SEQ ID NO:40 and SEQ ID NO:44 SEQ ID NO:41 and SEQ ID NO:44 SEQ ID NO:42 and SEQ ID NO:44 SEQ ID NO:38 and SEQ ID NO:45 SEQ ID NO:39 and SEQ ID NO:45 SEQ ID NO:40 and SEQ ID NO:45 SEQ ID NO:41 and SEQ ID NO:45 SEQ ID NO:42 and SEQ ID NO:45 SEQ ID NO:38 and SEQ ID NO:46 SEQ ID NO:39 and SEQ ID NO:46 SEQ ID NO:40 and SEQ ID NO:46 SEQ ID NO:41 and SEQ ID NO:46 SEQ ID NO:42 and SEQ ID NO:46 SEQ ID NO:38 and SEQ ID NO:47 SEQ ID NO:39 and SEQ ID NO:47 SEQ ID NO:40 and SEQ ID NO:47 SEQ ID NO:41 and SEQ ID NO:47 SEQ ID NO:42 and SEQ ID NO:47 SEQ ID NO:48 and SEQ ID NO:55 SEQ ID NO:49 and SEQ ID NO:55 SEQ ID NO:50 and SEQ ID NO:55 SEQ ID NO:51 and SEQ ID NO:55 SEQ ID NO:52 and SEQ ID NO:55 SEQ ID NO:53 and SEQ ID NO:55 SEQ ID NO:54 and SEQ ID NO:55 SEQ ID NO:48 and SEQ ID NO:56 SEQ ID NO:49 and SEQ ID NO:56 SEQ ID NO:50 and SEQ ID NO:56 SEQ ID NO:51 and SEQ ID NO:56 SEQ ID NO:52 and SEQ ID NO:56 SEQ ID NO:53 and SEQ ID NO:56 SEQ ID NO:54 and SEQ ID NO:56 SEQ ID NO:48 and SEQ ID NO:57 SEQ ID NO:49 and SEQ ID NO:57 SEQ ID NO:50 and SEQ ID NO:57 SEQ ID NO:51 and SEQ ID NO:57 SEQ ID NO:52 and SEQ ID NO:57 SEQ ID NO:53 and SEQ ID NO:57 SEQ ID NO:54 and SEQ ID NO:57.

In a further embodiment, an anti-LAG-3 antibody of the invention comprises VH and VL domains, wherein the two variable domains comprise amino acid sequences selected from the group consisting of:

SEQ ID NO:60 and SEQ ID NO:61 SEQ ID NO:60 and SEQ ID NO:62 SEQ ID NO:63 and SEQ ID NO:64 SEQ ID NO:65 and SEQ ID NO:66 SEQ ID NO:67 and SEQ ID NO:68 SEQ ID NO:69 and SEQ ID NO:70 SEQ ID NO:71 and SEQ ID NO:68 SEQ ID NO:74 and SEQ ID NO:75 SEQ ID NO:74 and SEQ ID NO:76 SEQ ID NO:74 and SEQ ID NO:77 SEQ ID NO:72 and SEQ ID NO:75 SEQ ID NO:72 and SEQ ID NO:76 SEQ ID NO:72 and SEQ ID NO:77 SEQ ID NO:73 and SEQ ID NO:75 SEQ ID NO:73 and SEQ ID NO:76 SEQ ID NO:73 and SEQ ID NO:77 SEQ ID NO:121 and SEQ ID NO:122 SEQ ID NO:123 and SEQ ID NO:124 SEQ ID NO:125 and SEQ ID NO:126 SEQ ID NO:127 and SEQ ID NO:128 SEQ ID NO:129 and SEQ ID NO:130 SEQ ID NO:131 and SEQ ID NO:132 SEQ ID NO:135 and SEQ ID NO:138 SEQ ID NO:136 and SEQ ID NO:139 SEQ ID NO:136 and SEQ ID NO:117 SEQ ID NO:226* and SEQ ID NO:138 SEQ ID NO:227* and SEQ ID NO:139 SEQ ID NO:227* and SEQ ID NO:117

* where SEQ ID NO: 226 is the same as SEQ ID NO: 135, except for the amino acid at position 56, which is Ala (A) instead of Gly (G) (replacing G55A by Kabat numbering); and SEQ ID NO: 227 is the same as SEQ ID NO: 136 except for the amino acid at position 56 which is Ala (A) instead of Gly (G) (replacing G55A with Kabat numbering).

In another embodiment, an anti-PD-1 antibody or an anti-LAG-3 antibody can be used to derivatize binding proteins that recognize the same target antigen using techniques well known in the art. Such a derivative may be, for example, a single chain antibody (scFv), Fab fragment (Fab), Fab' fragment, F(ab') ₂ , Fv and disulfide-linked Fv.

In another aspect of the invention, the antibody or bispecific binding protein of the present invention is capable of modulating the biological function of PD-1, LAG-3, or both. In another aspect, the pnti-PD-1 antibody disclosed herein is capable of suppressing PD-1/PD-L1 signaling. Signal suppression can be measured using a mixed lymphocyte culture response assay, such as described below in the Working Examples. In another aspect, an anti-LAG-3 antibody disclosed herein is capable of inhibiting an MHC class II/LAG-3 interaction. Such suppression can be measured using the PBMC activation analysis using SEB, such as described below in the working examples. In another aspect, the PD-1/LAG-3 bispecific FIT-Ig binding protein disclosed herein is capable of suppressing both PD-1/PD-L1 signaling and MHC class II/LAG-3 interaction.

In one embodiment, an anti-PD-1 antibody disclosed herein, or an antigen-binding fragment thereof, has an association rate constant (k _on ) with human PD-1 of at least 1×10 ⁴ M ^-1 s ^-1 . at least 3×10 ⁴ M ^-1 s ^-1 , at least 5×10 ⁴ M ^-1 s ^-1 , at least 7×10 ⁴ M ^-1 s ^-1 , at least 9×10 ⁴ M ^-1 s ^-1 , at least 1×10 ⁵ M ^-1 s ^-1 , at least 1.1×10 ⁵ M ^-1 s ^-1 , at least 1×10 ⁵ M ^-1 s ^-1 , at least 1.25×10 ⁵ M ^-1 s ^-1 , at least 1.4×10 ⁵ M ^-1 s ^-1 , at least 1.5×10 ⁵ M ^-1 s ^-1 , at least least 3×10 ⁵ M ^-1 s ^-1 or more, measured by surface plasmon resonance or biolayer interferometry.

In another embodiment, the anti-PD-1 antibody or antigen-binding fragment disclosed herein has a dissociation rate constant (k _off ) with human PD-1 of less than 1×10 ^-3 s ^-1 , less than 5×10 ^-4 s ^-1 , less than 3×10 ^-4 s ^-1 , less than 1×10 ^-4 s ^-1 , less than 8×10 ^-5 s ^-1 , less than 6×10 ^-5 s ^-1 , less than 4×10 ^-5 s ^-1 , less than 3×10 ^-5 s ^-1 or less than 1×10 ^-5 s ^-1 measured by surface plasmon resonance or biolayer interferometry.

In another embodiment, an anti-PD-1 antibody or antigen-binding fragment disclosed herein has a dissociation constant (K _D ) with human PD-1 of less than 1×10 ^-8 M, less than 5×10 ^-9 M, less than 3×10 ^-9 M, less than 1×10 ^-9 M, less than 8×10 ^-10 M, less than 6×10 ^-10 M, less than 4×10 ^-10 M, less than 2×10 ^-10 M or less than 1×10 ^-10 M.

In one embodiment, an anti-LAG-3 antibody disclosed herein, or an antigen-binding fragment thereof, has an association rate constant (k _on ) with human LAG-3 of at least 5×10 ³ M ^-1 s ^-1 . at least 7×10 ³ M ^-1 s ^-1 , at least 1×10 ⁴ M ^-1 s ^-1 , at least 3×10 ⁴ M ^-1 s ^-1 , at least 5×10 ⁴ M ^-1 s ^-1 , at least 7×10 ⁴ M ^-1 s ^-1 , at least 1×10 ⁵ M ^-1 s ^-1 or at least 2×10 ⁵ M ^-1 s ^-1 or more, measured by surface plasmon resonance or biolayer interferometry.

In another embodiment, the anti-LAG-3 antibody or antigen-binding fragment disclosed herein has a dissociation rate constant (k _off ) with human LAG-3 of less than 1.5×10 ^-3 s ^-1 , less than 1×10 ^-3 s ^-1 , less than 8×10 ^-4 s ^-1 , less than 6×10 ^-4 s ^-1 , less than 4×10 ^-4 s ^-1 , less than 2×10 ^-4 s ^-1 , less than 1×10 ^-4 s ^-1 , less than 9×10 ^-5 s ^-1 , less than 8×10 ^-5 s ^-1 , less than 7×10 ^-5 s ^-1 , less than 5×10 ^-5 s ^-1 , less than 4×10 ^-5 s ^-1 less than 2×10 ^-5 s ^-1 or less than 1×10 ^-5 s ^-1 measured by surface plasmon resonance or biolayer interferometry.

In another embodiment, the anti-LAG-3 antibody or antigen-binding fragment disclosed herein has a dissociation constant (K _D ) with LAG-3 of less than 5×10 ^-7 M, less than 2×10 ^-7 M, less than 1×10 ^{- 7} M, less than 8×10 ^-8 M, less than 6×10 ^-8 M, less than 4×10 ^-8 M; less than 2×10 ^-8 M; less than 1×10 ^-8 M; less than 8×10 ^-9 M; less than 6×10 ^-9 M, less than 4×10 ^-9 M; less than 2×10 ^-9 M; or less than 1×10 ^-9 M.

In one embodiment, the FIT-Ig bispecific binding protein of the invention capable of binding PD-1 and LAG-3 has an association rate constant (k _on ) with human PD-1 of at least 5×10 ³ M ^-1 s ^-1 , at least 1×10 ⁴ M ^-1 s ^-1 , at least 5×10 ⁴ M ^-1 s ^-1 , at least 1×10 ⁵ M ^-1 s ^-1 , at least 3× 10 ⁵ M ^-1 s ^-1 or at least 5×10 ⁵ M ^-1 s ^-1 or more, and the same binding protein has an association rate constant (k _on ) with human LAG-3 of at least 5× 10 ³ M ^-1 s ^-1 , at least 1×10 ⁴ M ^-1 s ^-1 , at least 5×10 ⁴ M ^-1 s ^-1 , at least 1×10 ⁵ M ^-1 s ^-1 , at least 3×10 ⁵ M ^-1 s ^-1 or at least 5×10 ⁵ M ^-1 s ^-1 or more, measured by surface plasmon resonance or biolayer interferometry. In additional embodiments, the FIT-Ig bispecific binding protein of the invention capable of binding PD-1 and LAG-3 has association rate constants (k _on ) with human PD-1 and LAG-3 that are no more than 10 times less than the values k _on for PD-1 and LAG-3 parental anti-PD-1 antibody and parental anti-LAG-3 antibody, respectively, from which the corresponding anti-PD-1 and anti-LAG-3 specificities of the FIT-Ig binding protein were derived . In other words, the retained association rate constant of the FIT-Ig binding protein with each antigen (PD-1 or LAG-3) is greater than, equal to, or no more than one order of magnitude less than the association rate constant (k _on ) observed in the parent antibodies reacting with corresponding PD-1 or LAG-3 antigens. As used herein, a PD-1/LAG-3 FIT-Ig binding protein may have an improved k _on for one or both antigens compared to the k _on for the corresponding antigens seen in parental antibodies, or a k _on value for one or both antigens may remain substantially the same as that of the parent antibodies, respectively, or, in the case of a decrease in the k _on value for one or both antigens observed in the FIT-Ig binding protein relative to the k _on values of the parent antibodies, such a decrease is no more than than 10 times. Preferably, the reduction in k _on for a FIT-Ig for a particular antigen compared to _that for that antigen in the parent antibody is less than 50%, more preferably less than 25%. Such high k _on values of the bispecific FIT-Ig compared to the k _on of parental antibodies are an unexpected development in the art.

In one embodiment, the FIT-Ig bispecific binding protein of the invention capable of binding PD-1 and LAG-3 has a dissociation rate constant (k _off ) with human PD-1 of less than 2×0 ^-4 s ^-1 , less than 1× 10 ^-4 s ^-1 , less than 5×10 ^-5 s ^-1 , less than 3×10 ^-5 s ^-1 , less than 2×10 ^-5 s ^-1 , less than 1×10 ^-5 s ^-1 or less than 8× 10 ^-6 s ^-1 , and the same binding protein has a dissociation rate constant (k _off ) with human LAG-3 less than 2×10 ^-4 s ^-1 , less than 1×10 ^-4 s ^-1 , less than 5×10 ^{- 5} s ^-1 , less than 3×10 ^-5 s ^-1 , less than 2×10 ^-5 s ^-1 , less than 1×10 ^-5 s ^-1 or less than 8×10 ^{-6 s -1} measured by surface plasmon resonance or biolayer interferometry.

In another embodiment, the FIT-Ig bispecific binding protein of the invention capable of binding PD-1 and LAG-3 has a dissociation constant (K _D ) with PD-1 of less than 2×10 ^-8 M, less than 1×10 ^-8 M, less than 5×10 ^-9 M, less than 1×10 ^-9 M, less than 6×10 ^-10 M, less than 5×10 ^-10 M, less than 3×10 ^-10 M, less than 2×10 ^-10 M, less than 1 ×10 ^-10 M, less than 8×10 ^-11 M, less than 6×10 ^-11 M, less than 4×10 ^-11 M, or less than 1×10 ^-11 M, and the same binding protein has a dissociation constant (K _D ) with human LAG-3 less than 2×10 ^-8 M, less than 1×10 ^-8 M, less than 5×10 ^-9 M, less than 1×10 ^-9 M, less than 6×10 ^-10 M, less than 5×10 ^{- 10} M, less than 3×10 ^-10 M, less than 2×10 ^-10 M, less than 1×10 ^-10 M, less than 8×10 ^-11 M, less than 6×10 ^-11 M, less than 4×10 ^-11 M or less than ^{1x10 -11} M. In further embodiments, the FIT-Ig bispecific binding protein of the invention capable of binding PD-1 and LAG-3 has dissociation constants (K _D ) for human PD-1 and LAG-3 that are no more than 10 times different from the K _D values for PD-1 and for LAG-3 parental th anti-PD-1 antibody and parental anti-LAG-3 antibody, respectively, from which the corresponding anti-PD-1 and anti-LAG-3 specificities of the FIT-Ig binding protein were derived. In other words, the FIT-Ig binding protein retains the binding affinity of the parent antibodies for each antigen (PD-1 or LAG-3), as indicated by a dissociation constant (K _D ) that is within the same order of magnitude as the K _D observed in parental antibodies reactive with PD-1 or LAG-3 antigens, respectively. As used herein, a PD-1/LAG-3 FIT-Ig binding protein may have an improved K _D (i.e., may have a lower K _D value; i.e., stronger binding) for one or both of the antigens compared to the K _D for the corresponding antigen observed in the parent antibodies, or may have substantially the same K _D for one or both antigens as in the parent antibodies, respectively, or observed in the FIT-Ig binding protein K _D for one or both antigens can be reduced (i.e. have a higher K _D value, i.e. stronger binding) relative to the K _D of the parent antibody, but in the case of a difference in K _D values between the FIT-Ig binding white and the parent antibody, such a difference is no more than 10 times. Preferably, the PD-1/LAG-3 binding FIT-Ig protein has a lower K _D value (binds more strongly) for one or both antigens compared to the K _D values for the corresponding antigens seen in one or both of the parental antibodies. Maintaining the binding affinity of parental anti-PD-1 and anti-LAG-3 antibodies within ±10-fold change in K _D is an unexpected achievement in the art.

The invention also relates to pharmaceutical compositions containing at least one anti-PD-1 antibody or antigen-binding fragment disclosed in the present description, and a pharmaceutically acceptable carrier. The invention also relates to pharmaceutical compositions containing at least one anti-LAG-3 antibody or antigen-binding fragments thereof and a pharmaceutically acceptable carrier. The invention also relates to pharmaceutical compositions containing a combination of anti-PD-1 and anti-LAG-3 antibodies disclosed in the present description, or their antigennegative fragments, and a pharmaceutically acceptable carrier. The invention also relates to bispecific, multivalent binding immunoglobulin proteins reactive with both PD-1 and LAG-3, these binding proteins including VH/VL binding sites derived from anti-PD-1 and anti-LAG-3 antibodies. disclosed in the present description. In particular, the invention relates to pharmaceutical compositions comprising at least one FIT-Ig binding protein capable of binding PD-1 and LAG-3 and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention may further contain at least one additional active ingredient. In one embodiment, such additional ingredient includes, without limitation, a therapeutic agent, an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule blocker, an adhesion molecule blocker, an antibody with a different specificity or a functional fragment thereof, a detectable label or reporter; specific cytokine(s) agonist or antagonist, narcotic, non-steroidal anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antimicrobial agent, corticosteroid, anabolic steroid, erythropoietin, immunogen, immunosuppressive agent, growth hormone, drug hormone replacement substance, radiopharmaceutical, antidepressant, neuroleptic, stimulant (eg, amphetamine, caffeine, etc.), beta-agonist, inhaled steroid, adrenaline or analog, cytokine.

In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent for treating a disorder in which PD-1 and/or LAG-3 mediated signaling activity is detrimental.

In a further embodiment, the invention provides isolated nucleic acids encoding one or more amino acid sequences of an anti-PD-1 antibody of the invention, or an antigen-binding fragment thereof; isolated nucleic acids encoding one or more amino acid sequences of an anti-LAG-3 antibody of the invention or an antigen-binding fragment thereof; and isolated nucleic acids encoding one or more amino acid sequences of a bispecific Fabs-in-Tandem binding immunoglobulin (FIT-Ig) protein capable of binding to both PD-1 and LAG-3. Such nucleic acids can be inserted into a vector to perform various genetic assays or to express, characterize, or improve one or more properties of an antibody or binding protein disclosed herein. The vector may contain one or more nucleic acid molecules encoding one or more amino acid sequences of the antibody or binding protein disclosed herein, wherein the one or more nucleic acid molecules are operably linked to appropriate transcriptional and/or translational sequences that allow expression of the antibody or binding protein. protein in the specific host cell carrying the vector. Examples of vectors for cloning or expression of nucleic acids encoding the amino acid sequences of the binding proteins disclosed herein include, without limitation, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ and their derivatives.

The invention also relates to a host cell containing a vector containing a nucleic acid encoding one or more amino acid sequences of an antibody or binding protein disclosed in the present description. The host cells used in the invention may be prokaryotic or eukaryotic. An example of a prokaryotic host cell is Escherichia coli . Eukaryotic cells useful as host cells in the invention include protist cells, animal cells, plant cells, and fungal cells. An example of a fungal cell is a yeast cell, including Saccharomyces cerevisiae . An exemplary animal cell used as a host cell of the invention includes, without limitation, a mammalian cell, an avian cell, and an insect cell. Preferred mammalian cells include, without limitation, CHO cells, HEK cells, and COS cells. The insect cell used as the host cell of the invention is an Sf9 insect cell.

In another aspect, the invention relates to a method for producing an anti-PD-1 antibody or functional fragment thereof, comprising culturing a host cell containing an expression vector encoding the antibody or functional fragment in a culture medium under conditions sufficient to induce cell expression - the host of an antibody or fragment capable of binding PD-1. In another aspect, the invention relates to a method for producing an anti-LAG-3 antibody or functional fragment thereof, comprising culturing a host cell containing an expression vector encoding the antibody or functional fragment in a culture medium under conditions sufficient to induce cell expression - the host of an antibody or fragment capable of binding LAG-3. In another aspect, the invention relates to a method for producing a bispecific, multivalent binding protein capable of binding PD-1 and LAG-3, in particular, a FIT-Ig binding protein that binds to PD-1 and LAG-3, comprising culturing a host cell, containing an expression vector encoding a FIT-Ig binding protein in a culture medium under conditions sufficient to cause the host cell to express a binding protein capable of binding PD-1 and LAG-3. Thus obtained proteins can be isolated and used in various compositions and methods disclosed in the present description.

In one embodiment, the present invention relates to a method of treating cancer in a subject in need, comprising administering to the subject an anti-PD-1 antibody or PD-1 binding fragment disclosed herein, said antibody or binding fragment being capable of binding PD -1 and inhibit PD-1/PD-L1 or PD-1/PD-L2-mediated signaling in a cell expressing PD-1. In another embodiment, the present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-LAG-3 antibody or LAG-3 binding fragment disclosed herein, wherein said antibody or binding fragment is capable of binding LAG- 3 and inhibit MHC class II/LAG-3 signaling in a cell expressing LAG-3. In another embodiment, the present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a FIT-Ig bispecific binding protein disclosed herein capable of binding LAG-3 and PD-1, wherein the binding protein is capable of binding LAG-3 and PD-1 and inhibit MHC class II/LAG-3 signaling in a cell expressing LAG-3 and inhibit PD-1/PD-L1 or PD-1/PD-L2-mediated signaling in a cell expressing PD-1. In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-684 of SEQ ID NO: 102; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence 20-235 of SEQ ID NO: 105; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-236 of SEQ ID NO: 107 (see Table 30). In a further embodiment, the FIT-Ig binding protein of the present invention binds PD-1 and LAG-3 and consists of a first polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-687 of SEQ ID NO: 189; a second polypeptide chain containing, essentially consisting of or consisting of the amino acid sequence 20-235 of SEQ ID NO: 192; and a third polypeptide chain comprising, essentially consisting of, or consisting of the amino acid sequence 23-236 of SEQ ID NO: 194 (see Table 48).

In another embodiment, the present invention relates to a method of treating an autoimmune disease or cancer in a subject in need thereof, wherein the binding protein is capable of binding LAG-3 and PD-1, and wherein the autoimmune disease or cancer is an autoimmune disease or cancer normally responsive to immunotherapy . In another embodiment, the cancer is a non-immunotherapy related cancer. In another embodiment, the cancer is a cancer that is a refractory or recurrent malignancy. In another embodiment, the binding protein inhibits the growth or survival of tumor cells. In another embodiment, the cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone resistant prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (eg, non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other malignancies.

The methods of treatment disclosed herein may further comprise administering to a subject in need thereof an immunostimulatory adjuvant such as a CpG oligodeoxynucleotide (CpG ODN) containing all or part of a phosphodiester or phosphorothioate backbone. For example, in the method of treatment of the present invention, an immunostimulatory adjuvant may be included in a composition containing an antibody or FIT-Ig binding protein of the present invention, the composition being administered to a subject in need of treatment. In another embodiment, the method of treatment of the present invention may include the step of administering to a subject in need of treatment an antibody or FIT-Ig binding protein of the present invention and a separate step of administering to the subject an immunostimulatory adjuvant before, simultaneously with, or after the step of administering to the subject the antibody or FIT-Ig binding protein according to invention.

BRIEF DESCRIPTION OF THE FIGURES

In FIG. 1A and 1B are bar graphs showing IL-2 production levels in a mixed lymphocyte culture reaction to evaluate the performance of various anti-PD-1 antibodies disclosed herein compared to two recombinant anti-PD-1 antibodies derived from published sequences ( nivolumab and pembrolizumab) and control human and mouse antibodies directed against irrelevant antigens ("hIgG4" and "mIgG"). In FIG. 1A and 1B show separate MLR tests using reactive lymphocytes obtained from different donors.

In FIG. 2A and 2B are bar graphs showing levels of interferon gamma (IFN-gamma) production in a mixed lymphocyte culture reaction to evaluate the effects of various anti-PD-1 antibodies disclosed herein compared to two recombinant anti-PD-1 antibodies, derived from published sequences (nivolumab and pembrolizumab) and control human and mouse antibodies directed against irrelevant antigens ("hIgG4" and "mIgG"). In FIG. 2A and 2B show separate MLR tests using reactive lymphocytes obtained from different donors.

In FIG. 3 shows histograms of IL-2 production levels in a mixed lymphocyte culture reaction to evaluate the performance of various humanized anti-PD-1 antibodies disclosed herein compared to a recombinant therapeutic anti-PD-1 antibody derived from published sequences (nivolumab), and a control human antibody directed against an irrelevant antigen ("HuF0323-1").

In FIG. 4 shows histograms of IL-2 production levels in a mixed lymphocyte culture reaction to evaluate the performance of various humanized anti-PD-1 antibodies disclosed herein compared to the parent mouse mAb709, a recombinant therapeutic anti-PD-1 antibody derived from the published sequence. (nivolumab), and a control human antibody directed against an irrelevant antigen ("HuF0323-1").

In FIG. 5 shows histograms of IL-2 production levels in a mixed lymphocyte culture reaction to assess the effects of various humanized anti-PD-1 antibodies disclosed herein compared to a murine mAb713 parental variable domain chimera (mAb713c), a recombinant therapeutic anti-PD- 1 antibody derived from published sequences (nivolumab) and a control human antibody directed against an irrelevant antigen (“HuF0323-1”). In FIG. 3, 4 and 5 show separate MLR tests using reactive lymphocytes obtained from different donors.

In FIG. 6 is a histogram of IL-2 production levels in an SEB-mediated T cell activation assay comparing the reversibility of T cell suppression at various concentrations of the two mouse anti-LAG-3 antibodies disclosed herein. The functionality of the anti-LAG-3 antibodies of the invention ("3502-mAb746" and 3502-mAb747") is compared to a recombinant anti-LAG-3 mAb derived from a published sequence ("BMS-986016"), a recombinant mouse anti-LAG-3 an antibody derived from a published sequence ("BAP050") and control human and mouse antibodies directed against irrelevant antigens ("hIgG4" and "mIgG").

In FIG. 7 is a bar graph showing IL-2 production in a SEB-mediated T cell activation assay comparing reversibility of T cell suppression at different concentrations of several of the presently disclosed bispecific FIT-Ig binding proteins, FIT107-1-2a . The functionality of FIT107-1-2a was compared to the combination of recombinant anti-LAG-3 mAb with known sequence (BMS-986016) and recombinant anti-PD-1 mAb with known sequence (nivolumab), and only one anti-PD-1 mAb ( disclosed in the present description "PD-1", mAb709).

In FIG. 8 are curves showing the relative levels of interferon-gamma (IFN-g) production in a mixed lymphocyte culture reaction to evaluate the effects of the FIT-Ig bispecific binding protein FIT107-1-2a at various concentrations compared to the combination of recombinant anti-LAG-3 mAb with a known sequence (BMS-986016) and a recombinant anti-PD-1 mAb with a known sequence (nivolumab), and a humanized anti-PD-1 antibody disclosed herein, HumAb709-8.

Figure 9 is a bar graph showing IL-2 production in an SEB-mediated T cell activation assay comparing reversibility of T cell suppression at different concentrations of mAb747c chimeric anti-LAG-3 antibody and humanized anti-LAG-3 antibodies HumAb747-60. See Example 13. The functionality of a humanized anti-LAG-3 antibody of the invention (HumAb747-60) is compared to a chimeric anti-LAG-3 mAb made using the mouse variable domains disclosed herein and a human antibody directed against an irrelevant antigen. ("hIgG4", control).

Figure 10 is a bar graph showing IL-2 production in an SEB-mediated T cell activation assay comparing reversibility of T cell suppression at different concentrations of mAb747c chimeric anti-LAG-3 antibody and high affinity humanized anti-LAG variants. -3 HumAb747-60 antibodies containing mutations indicated after affinity maturation experiments. See Example 14. The functionality of the altered anti-LAG-3 antibodies of the invention (HumAb747V-66 to HumAb747V-73) is compared to a chimeric anti-LAG-3 mAb made using the mouse variable domains disclosed herein and a human antibody, directed against an irrelevant antigen ("hIgG4", control).

Figure 11 is a bar graph showing IL-2 production in an SEB-mediated T cell activation assay comparing reversibility of T cell suppression at different concentrations of FIT-Ig binding protein specific for both target LAG-3 and and for target PD-1. See example 16.2. The functionality of PD-1/LAG-3 bispecific FIT-Ig antibodies of the invention is compared to a combination of recombinant monoclonal anti-PD-1 and anti-LAG-3 antibodies derived from published sequences ("nivolumab+BMS 986016") and a human antibody directed against an irrelevant antigen ("hIgG", control).

12 is a bar graph showing the results of a receptor blocking assay showing the ability of an anti-LAG-3 antibody of the invention (HumAb747V-67) and a PD-1/LAG-3 binding FIT-Ig protein of the invention (FIT107-1-7b -1) block the interaction between human LAG-3 and fibrinogen-like protein 1 (FGL1). See example 16.5.

13 is a series of graphs obtained from the evaluation of cell surface binding to PD-1 and LAG-3 expressed on T cells. The results show that the FIT-Ig bispecific protein FIT107-1-7b-1 recognizes both PD-1 and LAG-3 surface proteins on T cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel anti-PD-1 antibodies, novel anti-LAG-3 antibodies, antigen-binding portions thereof, and polyvalent bispecific binding proteins such as Fabs-in-Tandem immunoglobulins (FIT-Ig) that bind to both PD-1 , and LAG-3. Various aspects of the invention relate to anti-PD-1 and anti-LAG-3 antibodies and antibody fragments, FIT-Ig binding proteins that bind to human PD-1 and human LAG-3, and pharmaceutical compositions thereof, as well as nucleic acids. , recombinant expression vectors and host cells to produce such antibodies, functional antibody fragments and binding proteins. Also included within the scope of the present invention are methods of using the antibodies, functional antibody fragments, and bispecific binding proteins of the invention to detect human PD-1, human LAG-3, or both; to inhibit the activity of human PD-1 and/or human LAG-3 either in vitro or in vivo ; and for the treatment of diseases, especially cancer, which are mediated by the binding of PD-1 and/or LAG-3 to their respective ligands, ie. PD-1 and MHC class II.

Unless otherwise indicated, scientific and technical terms used in the context of the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms should be clear, however, in the event of any ambiguity, the definitions provided herein take precedence over any dictionary definition or definition found outside of this document. In addition, unless the context requires otherwise, terms in the singular include the plural, and terms in the plural include the singular. In this application, the use of the union "or" means "and/or", unless otherwise indicated. In addition, the use of the term "including", as well as other forms such as "includes" and "included", is not limiting. Similarly, terms such as "element" or "component" cover both elements and components containing a single unit, and elements and components that contain more than one subunit, unless specifically indicated otherwise.

As a rule, the notations and methods of cell and tissue culture, molecular biology, immunology, microbiology, genetics and chemistry of proteins and nucleic acids presented in the present description, as well as hybridization methods, are well known and widely used in this field. The methods and methods of the present invention are generally performed in accordance with conventional methods well known in the art and described generally and more specifically in the various documents cited and discussed herein, unless otherwise indicated. Enzymatic reactions and purification methods are performed according to the manufacturer's specifications, as is commonly done in the art or as disclosed herein. The designations used in connection with laboratory procedures and methods of analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry used in the present description are well known and widely used in this field. Standard methods are used for chemical synthesis, chemical analysis, pharmaceutical formulation, formulation and delivery, and patient treatment.

For a more complete understanding of the present invention, some terms are defined below.

The term "polypeptide" refers to any polymeric amino acid chain. The terms "peptide" and "protein" are used interchangeably with the term "polypeptide" and also refer to a polymeric amino acid chain. The term "polypeptide" embraces naturally occurring or artificial proteins, protein fragments, and polypeptide analogues of the amino acid sequence of a protein. The term "polypeptide" embraces fragments and variants thereof (including fragments of variants) unless the context dictates otherwise. For an antigenic polypeptide, a polypeptide fragment optionally contains at least one continuous or non-linear polypeptide epitope. The precise boundaries of at least one fragment of an epitope can be confirmed by one of ordinary skill in the art. The fragment contains at least about 5 contiguous amino acids, such as at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids. The polypeptide variant is as described in this application.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its origin or source of origin, is not associated with naturally associated components that accompany it in its native state; practically does not contain other proteins of the same species; expressed by a cell of another species; or not found in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the cell from which it naturally originates will be "isolated" from the components naturally associated with it. A protein can also be made substantially free of its naturally associated components using protein purification techniques well known in the art.

The term "recovery" refers to the process of freeing a chemical, such as a polypeptide, from its naturally associated components by isolation, for example, using protein purification methods well known in the art.

The term "biological activity" refers to all biological properties disclosed in the present description of anti-PD-1 or anti-LAG-3 antibodies. The biological properties of anti-PD-1 antibodies include, without limitation, binding to the PD-1 protein; the biological properties of anti-LAG-3 antibodies include, without limitation, binding to MHC class II proteins.

The term "specific binding" or "specifically binding" in relation to the interaction of an antibody, binding protein or peptide with a second chemical means that the interaction depends on the presence of a particular structure (for example, an antigenic determinant or epitope) in the second chemical. For example, an antibody recognizes and binds to a specific protein structure, and not to proteins in general. If the antibody is specific for an "A" epitope, the presence of a molecule containing the A epitope (or free unlabeled A) in a reaction containing the labeled "A" and the antibody reduces the amount of labeled A bound to the antibody.

The term "antibody" in the broad sense refers to any immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative thereof, which retains important epitope binding features inherent in the Ig molecule. Such mutant, variant or derivative antibody formats are known in the art. Their non-limiting embodiments are discussed below.

In a full length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single CL domain. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) that alternate with more conserved regions called framework regions (FRs). Each of the VH and VL contains three CDRs and four FRs arranged in the amino to carboxy direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The first, second, and third CDRs of the VH domain are commonly referred to as CDR-H1, CDR-H2, and CDR-H3; likewise, the first, second, and third CDRs of the VL domain are commonly referred to as CDR-L1, CDR-L2, and CDR-L3. Immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which can be formed when an intact antibody is digested with papain. The Fc region may be a native sequence Fc region or an altered Fc region. The Fc region of an immunoglobulin typically includes two constant domains, ie. a CH2 domain and a CH3 domain, and optionally includes a CH4 domain, such as the Fc regions of IgM and IgE antibodies. The Fc region of IgG, IgA, and IgD antibodies includes a hinge region, a CH2 domain, and a CH3 domain. In contrast, the Fc region of IgM and IgE antibodies does not have a hinge region, but contains a CH2 domain, a CH3 domain, and a CH4 domain. Altered Fc regions having substitutions of amino acid residues in the Fc portion to alter the effector function of an antibody are known in the art (see, for example, Winter et al. US Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, eg, cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC), and antibody half-life/clearance and antigen-antibody complex formation. Depending on the therapeutic goals, in some cases these effector functions are desirable for a therapeutic antibody, but in other cases they may be unnecessary or even detrimental. Certain isotypes of human IgG, in particular IgG1 and IgG3, mediate ADCC and CDC by binding to FcgR and complement C1q, respectively. In yet another embodiment, at least one amino acid residue is changed in the constant region of an antibody, eg, in the Fc region of an antibody, to change the effector functions of the antibody. The dimerization of two identical immunoglobulin heavy chains is mediated by the dimerization of the CH3 domains and is stabilized by disulfide bonds in the hinge region that connects the CH1 constant domains to the Fc constant domains (eg, CH2 and CH3). The anti-inflammatory activity of IgG is entirely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. Precise requirements for glycans to provide anti-inflammatory activity have been defined, allowing the creation of an appropriate IgG1 Fc fragment, which in turn allows the creation of a fully recombinant sialylated IgG1 Fc with significantly increased efficiency (see Anthony et al., Science, 320:373-376 (2008)).

The terms "antigen-binding portion" and "antigen-binding fragment" or "functional fragment" of an antibody are used interchangeably and refer to one or more antibody fragments that retain the ability to specifically bind to an antigen, i. the same antigen (eg, PD-1, LAG-3) that binds to the full-length antibody from which the specified part or fragment is derived. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Such embodiments of antibodies can also be in a bispecific, dual specific, or polyspecific format; specific binding to two or more different antigens (eg PD-1 and another antigen such as LAG-3). Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragment, divalent fragment containing two fragments of Fab linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one antibody arm, (v) a dAb fragment (Ward et al., Nature, 341:544-546 (1989); PCT Publication No. WO 90/05144) which contains a single variable domain; and (vi) a dedicated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be combined using recombinant methods through a synthetic linker that provides a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single-stranded Fv (scFv), see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single chain antibodies are also encompassed by the term "antigen-binding portion" of an antibody and the equivalent terms given above. Also included are other forms of single chain antibodies such as diabodies. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on the same polypeptide chain, but using a linker that is too short to allow pairing between the two domains of one chain, thereby resulting in domain pairing with complementary domains of the other chain and the formation of two antigen binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)). Such antibody binding sites are known in the art (Kontermann and Dübel eds., Antibody Engineering (Springer-Verlag, New York, 2001), p. 790 (ISBN 3-540-41354-5)). In addition, single chain antibodies also include "linear antibodies" containing a pair of tandem Fv segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., Protein Eng., 8 (10):1057-1062 (1995) and US Pat. No. 5,641,870).

The constant (C) domain of an immunoglobulin refers to a heavy (CH) or light (CL) chain constant domain. The amino acid sequences of the mouse and human IgG heavy chain and light chain constant domains are known in the art.

The term "monoclonal antibody" or "mAb" refers to an antibody derived from a population of substantially homogeneous antibodies, i. the individual antibodies that make up a population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic determinant (epitope). Moreover, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The "monoclonal" modifier should not be construed as requiring the production of the antibody by any particular method.

The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis, or in vivo somatic mutations), for example, in CDRs and, in particular, in CDR3. However, the term "human antibody" does not include antibodies in which human framework sequences are grafted with CDR sequences derived from the germline of another mammalian species, such as a mouse.

The term "recombinant human antibody" includes all human antibodies that are produced, expressed, generated or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial library of human antibodies (Hoogenboom , HR, Trends Biotechnol., 15:62-70 (1997); Azzazy and Highsmith, Clin. Biochem., 35:425-445 (2002); Gavilondo and Larrick, BioTechniques, 29:128-145 (2000); Hoogenboom and Chames, Immunol. Today, 21:371-378 (2000)), antibodies isolated from an animal (e.g., mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al., Nucl. Acids Res ., 20:6287-6295 (1992); Kellermann and Green, Curr. Opin. Biotechnol., 13:593-597 (2002); Little et al., Immunol. Today, 21:364-370 (2000)); or antibodies produced, expressed, constructed or isolated by any other means which involve splicing sequences of the human immunoglobulin gene with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or in vivo somatic mutagenesis when an animal transgenic for human Ig sequences is used) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not exist naturally in the in vivo human germline antibody repertoire.

The term "chimeric antibody" refers to antibodies that contain heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

The term "CDR-grafted antibody" refers to antibodies that contain the heavy and light chain variable region sequences of one species, but in which the sequences of one or more of the VH and/or VL CDR regions are replaced by CDR sequences of a different species, for example, antibodies having variable regions human heavy and light chains in which one or more human CDRs are replaced with murine CDR sequences.

The term "humanized antibody" refers to antibodies that contain heavy and light chain variable region sequences from a non-human species (e.g. mouse), but in which at least a portion of the VH and/or VL sequence has been altered "humanoid" , i.e. more similar to the human germline variable region sequences. One type of humanized antibody is a CDR-grafted antibody in which CDR sequences from a non-human species (eg mouse) are introduced into human VH and VL framework sequences. A humanized antibody is an antibody, or variant, derivative, analog, or fragment thereof, that binds immunospecifically to an antigen of interest and that contains framework regions and constant regions having essentially the amino acid sequence of a human antibody, and which contains complementarity determining regions (CDRs), having essentially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in relation to a CDR refers to a CDR having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the CDR of a non-human antibody. A humanized antibody comprises substantially all of at least one, and usually two, variable domains (Fab, Fab', F(ab') ₂ , Fv) in which all or substantially all of the CDR regions correspond to a CDR of a non-human immunoglobulin (t .e., donor antibody), and all or substantially all of the framework regions are regions from the human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. In some embodiments, the humanized antibody contains both a light chain and at least a heavy chain variable domain. The antibody may also include heavy chain CH1, hinge, CH2, CH3, and CH4 regions. In some embodiments, the implementation of the humanized antibody contains only a humanized light chain. In some embodiments, the implementation of the humanized antibody contains only a humanized heavy chain. In specific embodiments, the humanized antibody contains only a humanized light chain variable domain and/or a humanized heavy chain.

The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, without limitation, IgG1, IgG2, IgG3, and IgG4. A humanized antibody may contain sequences from more than one class or isotype, and specific constant domains may be selected to optimize the desired effector functions using techniques well known in the art.

The framework and CDRs of a humanized antibody do not have to exactly match the parent sequences, such as the CDRs of the donor antibody, or the acceptor framework can be modified by substitution, insertion, and/or deletion of at least one amino acid residue such that the CDR or framework residue in that site will match neither the donor antibody nor the consensus framework. However, in a typical embodiment, such mutations will not be extensive. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will match the original FR and CDR sequence residues. In order to maintain a particular loop structure, or to properly orient the CDR sequences required for contact with the target antigen, backmutation at a particular position in the framework is often used to restore the same amino acid that is contained at that position in the donor antibody.

The term "CDR" refers to complementarity determining regions in the sequences of the variable domains of an antibody. Each of the heavy chain and light chain variable regions contains three CDRs, which are referred to as CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3. In the context of the present description, the term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding an antigen. The precise boundaries of these CDRs vary between different definition systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any antibody variable region, but also allows you to determine the exact boundaries of the residues that define the three CDRs.

The term "Kabat numbering", which is known in the art, refers to a numbering system for amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof. See Kabat et al., Ann. NY Acad. Sci., 190: 382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

The growth and analysis of extensive public databases of amino acid sequences of heavy and light chain variable regions over the past twenty years has led to an understanding of the typical boundaries between framework regions (FR) and CDR sequences in variable region sequences and has allowed those skilled in the art to more accurately define CDRs according to with Kabat numbering, Chothia numbering or other systems. See, for example, Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains", In Kontermann and Dübel, eds., Antibody Engineering (Springer-Verlag, Berlin, 2001), chapter 31, pp. 432-433.

The term "polyvalent binding protein" means a binding protein containing two or more antigen-binding sites. The polyvalent binding protein is preferably designed to have three or more antigen-binding sites and is generally not a naturally occurring antibody. The term "bispecific binding protein" refers to a binding protein capable of binding two targets with different specificities. The Fabs-in-Tandem immunoglobulin (FIT-Ig) binding proteins of the invention contain two or more antigen-binding sites and are typically tetravalent binding proteins. FIT-Ig may be monospecific, ie. capable of binding one antigen, or polyspecific, i.e. able to bind two or more antigens. The preferred FIT-Ig of the present invention binds both PD-1 and LAG-3 and is therefore bispecific. The FIT-Ig binding protein, containing two long (heavy) chain V-C-V-C-Fc polypeptides and four short (light) chain V-C polypeptides, forms a hexamer displaying four Fab antigen-binding sites (VH-CH1 paired with VL-CL, sometimes referred to as as VH-CH1::VL-CL). Each half of the FIT-Ig contains a heavy chain polypeptide and two light chain polypeptides, and the pairing of complementary immunoglobulins, the three chain VH-CH1 and VL-CL elements, results in two Fab-structured antigen-binding sites arranged in tandem. In the present invention, it is preferred that the immunoglobulin domains containing Fab elements are directly fused to the heavy chain polypeptide without the use of interdomain linkers. In other words, the N-terminal V-C element of long (heavy) polypeptide chains is directly fused at its C-terminus to the N-terminus of another V-C element, which in turn is linked to the C-terminal Fc region. In bispecific FIT-Ig binding proteins, tandem Fab elements will react with different antigens. Each Fab antigen binding site includes a heavy chain variable domain and a light chain variable domain, for a total of six CDRs per antigen binding site.

A description of the construction, expression and characterization of FIT-Ig molecules is provided in PCT Publication WO 2015/103072. A preferred example of such FIT-Ig molecules includes a heavy chain and two different light chains. The heavy chain has the structural formula VL _A -CL-VH _B -CH1-Fc where CL is fused directly to VH _B , or VH _B -CH1-VL _A -CL-Fc where CH1 is fused directly to VL _A where VL _A is is a light chain variable domain from a parent antibody that binds antigen A, VH _B is a heavy chain variable domain from a parent antibody that binds antigen B, CL is a light chain constant domain, CH1 is a heavy chain constant domain, and Fc is is the Fc region of an immunoglobulin (eg, the C-terminal hinge-CH2-CH3 portion of the heavy chain of an IgG1 antibody). The two light chain FIT-Ig polypeptides have the formulas VH _A -CH1 and VL _B -CL, respectively. In embodiments of the bispecific FIT-Ig, antigen A and antigen B are different antigens or different epitopes of the same antigen. In the present invention, one of A and B is PD-1 and the other is LAG-3.

The term "activity" includes properties such as the ability to specifically bind a target antigen, the affinity (affinity) of an antibody for an antigen, the ability to neutralize the biological activity of the target antigen, the ability to inhibit the interaction of the target antigen with its natural receptor(s), and the like. Preferred antibodies and binding proteins of the present invention have the ability to inhibit the binding of PD-1 to the PD-L1 ligand, the ability to inhibit the binding of LAG-3 to the MHC class II ligand, or both in the case of the bispecific binding proteins disclosed herein.

The term " _kon " (also " _Kon ", " _kon ") as used herein refers to the rate constants of association of a binding protein (e.g., antibody) with an antigen to form an association complex, e.g., an antibody/antigen complex known in the art. "K _on " is also known by the term "association rate constant" or "k _a ", which is used interchangeably herein. This value indicates the rate at which an antibody binds to its target antigen, or the rate at which an antibody-antigen complex is formed, as shown by the equation below:

Antibody ("Ab") + Antigen ("Ag") → AAb-Ag.

The term “k _off ” (also “K _off ”, “k _off ”) as used herein refers to the dissociation rate constants or “dissociation rate constants” of a binding protein (e.g. antibody) from an association complex (e.g. antibody/antigen complex). ) known in the art. This value indicates the rate of dissociation of the antibody and its target antigen, or separation of the Ab-Ag complex over time into free antibody and antigen, as shown by the equation below:

Ab+Ag ← Ab-Ag.

The term “K _D ” (also “K _d ”) as used herein means “equilibrium dissociation constants” and refers to the value obtained by measuring a titration at equilibrium or by dividing the dissociation rate constant (k _off ) by the association rate constant (k _on ). The association rate constant (k _on ), the dissociation rate constant (k _off ) and the equilibrium dissociation constant (K _D ) are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based methods provides high sensitivity and the ability to study samples in physiological buffers at equilibrium. Other experimental approaches and tools may be used, such as the BIAcore® (biomolecular interaction assay) assay (eg, using the instrument available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). Biolayer interferometry (BLI) using, for example, the Octet® RED96 system (Pall FortéBio LLC) is another method for analyzing affinity. In addition, the KinExA® assay (kinetic exclusion assay) available from Sapidyne Instruments (Boise, Idaho) can also be used.

The term "isolated nucleic acid" means a polynucleotide (eg, genomic, cDNA, or synthetic origin, or some combination thereof) that, as a result of human intervention, is not associated with all or part of the polynucleotides with which it exists in nature; operably linked to a polynucleotide with which it is not naturally linked; or does not occur naturally as part of a larger sequence.

The term "vector" in the context of the present description refers to a nucleic acid molecule capable of transferring another nucleic acid to which it is associated. One type of vector is a "plasmid", which refers to a circular double-stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). When introduced into a host cell, other vectors (eg, non-episomal mammalian vectors) can be integrated into its genome and thus replicated along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors used in recombinant DNA techniques are often in the form of plasmids. In the present description, "plasmid" and "vector" can be used interchangeably, since the plasmid is the most commonly used form of the vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses), that perform equivalent functions.

The term "operably linked" refers to the placement next to or adjacent position, in which the components thus described are in a relationship that allows them to function as intended. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence occurs under conditions compatible with the control sequence. "Operably linked" sequences include both expression control sequences that are adjacent to the gene of interest and expression control sequences that provide trans or act at a distance to control the gene of interest. Used in the present description, the term "control expression sequence" refers to polynucleotide sequences that are necessary for the implementation of the expression and processing of the coding sequences with which they are ligated. Expression control sequences include appropriate initiation, transcription termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase the efficiency of translation (ie the Kozak consensus sequence); sequences that increase protein stability; and, if necessary, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences typically include promoters and a transcription termination sequence. The term "control sequences" is intended to include components whose presence is necessary for expression and processing, and may also include additional components whose presence is beneficial, such as leader or signal sequences and fusion partner sequences.

"Transformation" in the context of the present description refers to any process by which exogenous DNA enters the host cell. Transformation can occur in natural or artificial conditions using various methods well known in this field. Transformation may be based on any known method for inserting foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is chosen depending on the host cell to be transformed, which may include, but is not limited to, transfection, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host's chromosome. They also include cells that transiently express inserted DNA or RNA for limited periods of time.

The term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which exogenous DNA has been introduced. In one embodiment, the host cell contains two or more (eg, multiple) antibody-encoding nucleic acids, such as the host cells described, for example, in US Pat. No. 7,262,028. Such terms are intended to refer not only to the particular cell in question, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In one embodiment, the host cells include prokaryotic and eukaryotic cells selected from any of the Natural Kingdoms. In another embodiment, eukaryotic cells include protozoan, fungal, plant, and animal cells. In another embodiment, the host cells include, without limitation, the Escherichia coli prokaryotic cell line; mammalian cell lines CHO, HEK 293, COS, NS0, SP2 and PER.C6; insect cell line Sf9; and cells of the fungus Saccharomyces cerevisiae .

For recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection), standard methods can be used. Enzymatic reactions and purification methods may be performed according to the manufacturer's specifications, or according to conventional methods used in the art, or provided herein. The above methods and procedures can generally be performed in accordance with conventional methods well known in the art and described in general terms and more specifically in the references that are cited and discussed in the present description. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

The term "agonist" as used herein refers to a modulator that, when contacted with a molecule of interest, causes an increase in the specific activity or function of the molecule compared to the amount of activity or function observed in the absence of the agonist. The terms "antagonist" and "inhibitor" in the context of the present description refer to modulators that, when contacted with a molecule of interest, cause a decrease in a certain activity or function of the molecule compared to the amount of activity or function observed in the absence of an antagonist. Particular antagonists of interest include antagonists that block or reduce the biological or immunological activity of human PD-1 and human LAG-3.

As used herein, the term "effective amount" refers to an amount of therapeutic agent that is sufficient to reduce or alleviate the severity and/or duration of a disorder or one or more of its symptoms; preventing the development of the disorder; triggering a regression disorder; preventing the recurrence, development, or progression of one or more of the symptoms associated with the disorder; detection of the disorder; or enhancing or improving the prophylactic or therapeutic effect(s) provided by other therapeutic agents (eg, prophylactic or therapeutic agents).

Production of anti-PD-1 and anti-LAG-3 antibodies

Anti-PD-1 and anti-LAG-3 antibodies of the present invention can be obtained by any of several methods known in this field. For example, expression from host cells, wherein the expression vector(s) encoding the heavy and light chains are transfected into the host cell by standard methods. The various forms of the term "transfection" are intended to encompass a wide range of methods commonly used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. While expression of an antibody of the present invention is possible in prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferred and most preferred in mammalian host cells because such eukaryotic cells (and in particular mammalian cells) are more suitable than prokaryotic cells. cells to assemble and secrete correctly folded and immunologically active antibodies.

Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980 )) used with a DHFR selectable marker, for example, as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982)), NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, antibodies are obtained by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody into a culture medium in which grow host cells. Antibodies can be isolated from the culture medium using standard protein purification methods.

Host cells can also be used to generate functional antibody fragments such as Fab fragments or scFv molecules. It should be understood that modifications to the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding functional light chain and/or heavy chain fragments of an antibody of the present invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that is not necessary for binding to antigens of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, by cross-linking an antibody of the present invention with a second antibody by standard chemical cross-linking techniques, bifunctional antibodies can be obtained in which one heavy and one light chain is an antibody of the invention and the other heavy and light chain are specific for an antigen that is different. from the antigens of interest.

In an exemplary system for recombinant expression of an antibody or antigen-binding portion thereof of the invention, a recombinant expression vector encoding both an antibody heavy chain and an antibody light chain is introduced into dhfr-CHO cells via calcium phosphate mediated transfection. In the recombinant expression vector, each of the antibody heavy and light chain genes is operably linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of gene transcription. The recombinant expression vector also carries the DHFR gene, which allows selection of CHO cells transfected with the vector by selection/methotrexate-induced amplification. Selected transfected host cells are cultured to allow expression of the heavy and light chains of the antibody, and intact antibody is recovered from the culture medium. Obtaining a recombinant expression vector, transfection of host cells, selection of transfectants, cultivation of host cells and extraction of antibodies from the culture medium is carried out using standard methods of molecular biology. In addition, the invention relates to a method for producing a recombinant anti-PD-1 or anti-LAG-3 antibody of the present invention by culturing a transfected host cell of the present invention in a suitable culture medium until a recombinant antibody of the present invention is produced. The method may further include isolating the recombinant antibody from the culture medium.

Obtaining bispecific FIT-Igs that bind PD-1 and LAG-3

Clinical trials using immune checkpoint inhibitors such as antibodies targeting PD-1, PD-L1 and CTLA-4 have shown promising results, however it has been observed that only a subset of patients initially respond to these inhibitors and growing clinical evidence indicates that a significant proportion of patients who initially respond to treatment eventually relapse after a few months or years with a fatal outcome, the development of disease resistance to the drug. Syn et al., Lancet Oncology, 18(12):e731-e741 (2017). Both LAG-3 and PD-1 are coexpressed on tolerized tumor-infiltrated lymphocytes (TILS), which contributes to immune suppression in tumors; and dual blockade of LAG-3 and PD-1 has been proposed as a means of restoring the antitumor function of CD8+ T cells. Matsuzaki et al., Proc. Natl. Acad. sci. USA, 107(17):7875-7880 (2010). Accordingly, the development of bispecific LAG-3/PD-1 binding proteins that can simultaneously block both targets on immunosuppressive T cells may provide advances in this therapeutic area.

The present invention relates to Fabs-in-Tandem binding immunoglobulin (FIT-Ig) proteins that bind to both PD-1 and LAG-3. An exemplary embodiment of such FIT-Ig molecules includes (1) a heavy polypeptide chain that contains either structural formula (i) VL _A -CL-VH _B -CH1-Fc, where CL is fused directly to VH _B , or structural formula (ii) VH _B -CH1-VL _A -CL-Fc, where CH1 is fused directly to VL _A ; (2) a light chain of the formula VH _A -CH1; and (3) another light chain of the formula VL _B -CL,

where VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, A is a PD-1 or LAG epitope -3, and B is a PD-1 or LAG-3 epitope, provided that A and B are different. In accordance with the present invention, such FIT-Ig binding proteins bind to both PD-1 and LAG-3.

FIT-Ig may contain two such heavy chains (1), two such light chains (2), and two such light chains (3), forming a six-chain binding protein monomer having four functional antigen-binding Fab regions. Such a FIT-Ig binding protein contains two identical subunits, with each subunit comprising one heavy chain (1), one light chain (2) and one light chain (3), which together form a pair of Fab binding regions arranged in tandem. Pairing of the Fc regions of two such subunits produces a six-strand bispecific FIT-Ig binding protein of the invention having a total of four functional Fab binding units.

A heavy chain peptide linker can be used to separate tandem-linked Fab fragments, however, for the bispecific FIT-Igs of the invention, the exclusion of such linker sequences is preferred. However, it is believed in the art that in immunoglobulins designed in multivalent formats having tandem binding sites, adjacent binding sites will interfere with each other unless a flexible linker is used to spatially separate the binding sites. However, with respect to the PD-1/LAG-3 FIT-Ig of the present invention, it has been found that the arrangement of the immunoglobulin domains according to the above chain formulas provides polypeptide chains that are well expressed in transfected mammalian cells, are assembled appropriately and secreted as bispecific , polyvalent immunoglobulin-like binding proteins that bind the target antigens PD-1 and LAG-3. See example 10 below. Despite the absence of any linker sequences between Fab binding sites, the PD-1/LAG-3 FIT-Igs of the present invention retain binding affinity for target antigens, showing a binding affinity comparable to that of the parent mAbs. In addition, the exclusion of synthetic linker sequences from binding proteins avoids the creation of antigenic sites recognized by the mammalian immune system, and thus the exclusion of linkers reduces the potential immunogenicity of FIT-Ig and provides a circulating half-life that is similar to that of a natural antibody, i.e. e. FIT-Ig is not rapidly cleared through immune opsonization and hepatic uptake.

Each variable domain (VH or VL) in FIT-Ig can be derived from one or more "parent" monoclonal antibodies that bind one of the target antigens, ie. PD-1 or LAG-3. FIT-Ig binding proteins are advantageously prepared using the variable domain sequences of the monoclonal anti-PD-1 and anti-LAG-3 antibodies disclosed herein. Preferably, the parental antibodies are humanized antibodies.

In one aspect, the present invention relates to the selection of parental antibodies with at least one or more of the properties required for a FIT-Ig molecule. In one embodiment, antibody properties are selected from the group consisting of antigen specificity, antigen affinity, activity, biological function, epitope recognition, stability, solubility, production efficiency, lack of immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and orthologous antigen binding. PD-1 and LAG-3 are cell surface proteins and interaction with the respective ligands PD-L1 (cell surface receptor) and MHC class II (surface proteins on antigen presenting cells) results in intracellular signaling associated with T cell suppression and immune answer. Accordingly, the ability of anti-PD-1 antibodies, anti-LAG-3 antibodies and FIT-Ig PD-1/LAG-3 binding proteins of the invention to inhibit PD-1/PD-L1 and/or MHC class II/LAG interaction -3 makes them potent regulators of immune cell activation and immune effector cell activity.

Antibodies, functional fragments thereof, and binding proteins of the invention may be purified (for their intended use) using one or more of a variety of methods and materials available in the art for purifying antibodies and binding proteins. Such methods and materials include, without limitation, affinity chromatography (for example, using resins, particles, or membranes conjugated to protein A, protein G, protein L, or a specific ligand of an antibody, its functional fragment, or binding protein), ion exchange chromatography (for example, using ion exchange particles or membranes), hydrophobic interaction chromatography ("HIC"; e.g. using hydrophobic particles or membranes), ultrafiltration, nanofiltration, diafiltration, size exclusion chromatography ("SEC"), low pH treatment (to inactivate contaminating viruses) and combinations thereof to obtain an acceptable purity for the intended use. A non-limiting example of a low pH treatment to inactivate contaminating viruses includes lowering the pH of a solution or suspension containing an antibody, functional fragment thereof, or binding protein of the invention to pH 3.5 with 0.5M phosphoric acid at 18°C-25°C within 60-70 minutes.

Use of Antibodies and Binding Proteins of the Invention

Given the ability to bind to human PD-1 and/or LAG-3, the antibodies disclosed herein, their functional fragments and bispecific polyvalent binding proteins can be used to detect PD-1 or LAG-3, or both, for example, in a biological sample, containing cells that express one or both of these target antigens. The antibodies, functional fragments and binding proteins of the invention can be used in conventional immunoassays such as enzyme immunoassay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry. The invention relates to a method for detecting PD-1 or LAG-3 in a biological sample, which includes bringing the biological sample into contact with an antibody, its antigen-binding portion or binding protein according to the invention and determining whether binding to the target antigen occurs, thereby performing detection of the presence or the absence of a target in a biological sample. Detection of bound or unbound antibody/fragment/binding protein can be accomplished by direct or indirect labeling of the antibody, functional fragment, or binding protein with a detectable substance. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho or ¹⁵³ Sm.

The antibodies, functional fragments thereof and binding proteins of the invention are preferably capable of neutralizing human PD-1 and/or human LAG-3 activity both in vitro and in vivo . Accordingly, the antibodies, their functional fragments and binding proteins of the invention can be used to inhibit the activity of human PD-1 and/or human LAG-3, for example, inhibition of cell signaling mediated by PD-1/PD-L1 (or PD-1 /PD-L2), and/or MHC class II/LAG-3 interactions in cell culture containing cells expressing PD-1 and/or LAG-3 in human subjects or other mammalian subjects having PD-1 or LAG-3, with which the antibody, functional fragment or binding protein of the invention cross-reacts. In one embodiment, the invention relates to a method for restoring the activity of activated T cells (reversing suppression), comprising contacting cells expressing human PD-1 with an anti-PD-1 antibody or PD-1 binding protein of the present invention, thus inhibiting PD-1 activity. In another embodiment, the invention relates to a method for restoring the activity of activated T cells (reversing suppression), comprising contacting cells expressing human LAG-3 with an anti-LAG-3 antibody or LAG-3 binding protein of the invention, thereby suppressing the activity LAG-3.

In another embodiment, the invention relates to a method of treating a subject suffering from a disease or disorder in which PD-1 and/or LAG-3 activity is detrimental, such method comprising administering to the subject an antibody or binding protein of the present invention in an effective amount, thereby reducing in a subject, activity mediated by PD-1/PD-L1 or PD-1/PD-L2 binding and/or MHC class II/LAG-3 binding.

As used herein, the term "disorder in which PD-1 and/or LAG-3 activity is detrimental" includes diseases and other disorders in which the interaction of PD-1 with one or both of its ligands (PD-L1, PD-L2) or the interaction of LAG-3 with its ligand (MHC class II) in a subject suffering from the disorder is either responsible for the pathophysiology of the disorder or is a factor that contributes to the worsening of the disorder. Accordingly, a disorder in which PD-1 and/or LAG-3 activity is detrimental is a disorder in which suppression of PD-1 and/or LAG-3 activity is expected to alleviate the symptoms and/or progression of the disorder.

In another embodiment, the present invention relates to a method of treating an autoimmune disease or cancer in a subject in need thereof, comprising administering to the subject an antibody, a functional fragment thereof, or a binding protein disclosed herein that is capable of binding LAG-3, PD-1, or both LAGs. -3 and PD-1, wherein an autoimmune disease or cancer is a disease that responds to immunotherapy. In another embodiment, the method of the present invention is used to treat an autoimmune disease or cancer that has not been associated with immunotherapy. In another embodiment, the method of the invention is used to treat cancer that is a refractory or recurrent malignancy. In another embodiment, an anti-LAG-3 or anti-PD-1 antibody, a functional fragment thereof, or a LAG-3/PD-1 bispecific binding protein of the invention is used in a method that inhibits the growth or survival of tumor cells.

In another embodiment, the invention relates to a method of treating cancer in a subject, comprising the step of administering to the subject an anti-PD-1 or anti-LAG-3 antibody of the present invention, a functional fragment thereof, or a LAG-3/PD-1 bispecific binding protein of the present of the invention, such as, for example, a Fabs-in-tandem binding immunoglobulin (FIT-Ig) protein, wherein the cancer is selected from the group consisting of: melanoma (eg, metastatic malignant melanoma), renal cancer (eg, clear cell carcinoma), prostate cancer (eg, hormone resistant prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (eg, non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer glands, glioblastomas, gliomas, leukemias, lymphomas, primary bone cancers (eg, osteosarcomas, Ewing's sarcomas, malignant fibrous histiocytoma, and chondrosarcoma), metastatic cancer and other malignant neoplasms.

The invention also relates to pharmaceutical compositions containing an antibody, or an antigen-binding portion thereof, or a bispecific polyvalent binding protein of the invention (ie, the main active ingredient) and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing the proteins of the invention are intended for use, without limitation, in the diagnosis, detection, or monitoring of a disorder; treating, controlling or alleviating the disorder or one or more of its symptoms; and/or research. In a specific embodiment, the composition contains one or more antibodies or binding proteins according to the invention. In another embodiment, the pharmaceutical composition comprises one or more antibodies or binding proteins of the invention and one or more prophylactic or therapeutic agents, other than antibodies or binding proteins of the invention, for the treatment of a disorder in which PD-1 and/or LAG- activity is detrimental. 3. In one embodiment, prophylactic or therapeutic agents are known to be useful or have been or are currently being used to prevent, treat, control, or alleviate a disorder or one or more symptoms. According to these embodiments, the composition may further comprise a carrier, diluent, or excipient. An excipient is typically any compound or combination of compounds that imparts a desired characteristic to the composition that is different from that of the main active ingredient (i.e., different from an antibody, functional moiety, or binding protein of the invention).

Antibodies (including functional fragments thereof) and binding proteins of the invention may be included in pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition contains an antibody or binding protein of the invention and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity regulating agents and absorption delaying agents, and the like, which are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases it is preferable to include isotonicity adjusting agents in the composition, for example sugars, polyalcohols (such as mannitol or sorbitol) or sodium chloride. Pharmaceutically acceptable carriers may additionally contain minor amounts of adjuvants such as wetting or emulsifying agents, preservatives or buffers that increase the shelf life or effectiveness of the antibody or binding protein present in the composition.

The composition of the pharmaceutical composition according to the invention is selected so that it is compatible with the intended route of administration. Examples of routes of administration include, without limitation, parenteral (eg, intravenous, intradermal, subcutaneous, intramuscular), oral, intranasal (eg, inhalation), transdermal (eg, topical), intratumoral, transmucosal, and rectal administration. In a particular embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic to relieve pain at the injection site, such as lidocaine (xylocaine, lignocaine).

The method of the present invention may include administering the composition formulated for parenteral administration by injection (eg, by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (eg, in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulation agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the primary active ingredient may be in the form of a powder, intended to be mixed with a suitable carrier (eg, sterile, pyrogen-free water) prior to use.

The methods of the invention may further include administering compositions formulated as depot formulations. Such long acting formulations may be administered by implantation (eg subcutaneously or intramuscularly) or by intramuscular injection. Thus, compositions, for example, can be formulated with suitable polymeric or hydrophobic materials (eg, as an emulsion in a suitable oil) or ion exchange resins, or as sparingly soluble derivatives (eg, as a sparingly soluble salt).

An antibody, functional fragment thereof, or binding protein of the invention may also be administered with one or more additional therapeutic agents useful in the treatment of various diseases. Antibodies, their functional fragments and binding proteins disclosed in the present description, can be used alone or in combination with an additional agent, such as a therapeutic agent, and the specified additional agent is selected by a qualified specialist depending on the intended purpose. For example, the additional agent may be a therapeutic agent that is considered useful in the art for the treatment of the disease or condition being treated with the antibody or binding protein of the present invention. The additional agent may also be an agent that imparts beneficial properties to the therapeutic composition, such as an agent that affects the viscosity of the composition.

The present invention will be better understood upon reading the detailed description with reference to the following examples, which are included for the purpose of illustration only and are not intended to limit the invention.

Examples

Example 1: Creation of monoclonal antibodies to human PD-1.

Monoclonal antibodies to human PD-1 were prepared as follows:

Example 1.1: Immunization with human PD-1 antigen

50 μg of recombinant purified human PD-1 extracellular domain (ECD) polypeptide mixed with Freund's complete adjuvant was administered intraperitoneally to five 6-8 week old Balb/C mice and five SJL mice on day 1. On days 16 and 26, the same mice were administered intraperitoneally 25 μg of recombinant purified human immunogen PD-1 ECD mixed with incomplete Freund's adjuvant. A final booster immunization with 25 μg of the immunogen was performed 3-4 days before the fusion.

Example 1.2: Creating a hybridoma

To create hybridomas, splenocytes from the immunized mice described in Example 1.1 were fused with SP2/0-Ag-14 cells at a ratio of 5:1 according to the established method described in Kohler and Milstein, Nature, 256:495-497 ( 1975). The fusion products were placed in 96-well plates, in selection medium containing hypoxanthine-aminopterin-thymidine (HAT) at a density of 1×10 ⁵ spleen cells per well. Seven to ten days after fusion, macroscopic hybridoma colonies were observed.

Example 1.3 Evaluation of PD-1 Binding Activity by ELISA and FACS

The presence of PD-1-specific antibodies was determined by enzyme immunoassay (ELISA) as follows:

First, synthetic targets for human PD-1, cynomolgus PD-1, and murine PD-1 were made by order of Synbio Technologies (Suzhou, China). Each target consisted of a human, cynomolgus or mouse PD-1 polypeptide segment of the extracellular domain (ECD) fused to a human IgG Fc region. Synthetic genes encoding each ECD-Fc fusion protein were subcloned into the pCP expression vector (Chempartner, Shanghai, China) and expression plasmids transiently transfected into HEK 293E cells in 1-3 liters of medium and cultured for seven days in a CO ₂ shaker . The ECD sequences used for each fusion protein are shown in Table 1 below. The PD-1 ECD portion of each fusion protein is underlined.

Table 1: Amino acid sequences for PD-1 ECD-Fc target fusion proteins

SEQID NO. Source PD-1 Amino acid sequences
1234567890123456789012345678901234567890 one human ldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2 Javanese macaque lespdrpwnaptfspalllvtegdnatftcsfsnasesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtrlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3 mouse levpngpwrsltfypawltvseganatftcslsnwsedlmlnwnrlspsnqtekqaafcnglsqpvqdarfqiiqlpnrhdfhmnildtrrndsgiylcgaislhpkakieespgaelvvteriletstrypspspkpegrfqIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

HEK 293E transfectant supernatants containing recombinant ECD/Fc fusion proteins were harvested by centrifugation at 4000×g for 30 minutes followed by protein A purification using MabSelect SuRe™ affinity resin (GE Healthcare). The fusion products were dialyzed against phosphate buffered saline (PBS) at pH 7.4 and stored at -80°C.

ELISA plates were incubated overnight at 4° C. with 50 μl synthetic targets of the above described PD-1 ECD/Fc fusion protein, diluted in PBS buffer, pH 7.4, at a concentration of 1 μg/ml. The plates were washed four times in wash buffer (PBS containing 0.05% Tween 20) and blocked for 1 hour at 37°C using 200 µl per well of blocking buffer (1% BSA in PBS containing 0.05% Tween 20 ). After the blocking buffer was removed, the hybridoma supernatant (or later diluted purified mAbs) was added to the wells at 100 μl per well and incubated at 37° C. for 1 hour. The wells were washed four times with wash buffer and anti-mouse HRP (Sigma) for characterization of mouse anti-human PD-1 antibody was diluted 1:5000 and added to the wells at 100 μl per well. The plates were incubated for 1 hour at 37°C and washed four times with wash buffer. 100 μl of a chromogenic solution of tetramethylbenzidine (TMB) was added to the well. After color appeared, the reaction was stopped with 1N HCl and absorbance measured at 450 nm on a SpectraMax® M5e plate reader (Molecular Devices; San Jose, California, US).

Example 1.4: Generation of cell lines expressing PD-1 and FACS analysis

Stable cell lines overexpressing human PD-1 or cynomolgus PD-1 were obtained by transfection of CHO-K1 cells (obtained from ATCC) with pLvx lentiviral plasmid vectors (Clontech) containing inserted genes encoding human PD-1 or cynomolgus PD-1. toque. Single clones were isolated by the limiting dilution method. Clones were screened for expression level by FACS assay using anti-PD-1 antibodies recombinantly derived from known antibody sequences (Chempartner) and clones having the highest level of PD-1 expression were selected for use in FACS binding assays and functional assays. 1 as described below.

Binding Assay for Cell Surface Targets: The ability of purified antibodies to bind to human cell membrane PD-1 or cynomolgus monkey PD-1 was determined by FACS analysis. CHO-K1 cells stably expressing human PD-1 (CHO-K1-hPD-1 cells) or cynomolgus monkey PD-1 (CHO-K1-cynoPD-1) were resuspended in PBS containing 2% FBS (FACS buffer), and seeded at a density of 2×10 ⁴ cells/well in 96-well round bottom plates (Corning; cat. no. 3799). Supernatants of hybridomas producing anti-PD-1 antibodies were added to the wells and detected with high cross-adsorption donkey anti-mouse IgG (H+L) AlexaFluor® 488 (Invitrogen; cat. no. A-21202) secondary antibody, followed by analytical the plate was read on a flow cytometer. Hybridomas generating supernatants signaling positive for human PD-1 expressing targets were further characterized using CHO-K1/cynoPD-1 cells to determine cross-reactivity of antibodies to cynomolgus monkey PD-1.

Example 1.5 Receptor Blocking Assay (RBA)

Supernatants demonstrating PD-1 specific activity were tested for their ability to block PD-1 binding to PD-L1 and PD-L2 ligands using immobilized human PD-1 ECD/Fc protein and PD-L1/Fc fusion proteins as targets. and PD-L2/Fc prepared in the same manner as the PD-1 ECD/Fc binding proteins described in Example 1.3 above. To determine the relative effectiveness of antibody-containing supernatants, their ability to inhibit the binding of human PD-1 ligand (PD-L1 or PD-L2) to human PD-1 protein was evaluated. ELISA plates were coated with 100 μl of 50 ng/ml huPD-1/Fc (i.e., the extracellular domain of PD-1 grafted onto the N-terminus of a homodimer-isolated human Fc region) in PBS and incubated overnight at 4° C. The plates were washed four times in wash buffer (PBS containing 0.05% Tween 20) and blocked for 1 hour at 37°C using 200 µl per well of blocking buffer (1% BSA in PBS containing 0.05% Tween twenty). After blocking buffer was removed, hybridoma supernatant (50 μl) mixed with either 50 μl of biotinylated human PD-L1/Fc (final concentration 1.0 mg/ml) in blocking buffer or 50 μl of biotinylated human PD-L2/Fc was added to the wells. Fc (final concentration 50 μg/ml) in blocking buffer, then incubated at 37°C for 1 hour. The signal was manifested by adding streptavidin-HRP (Sigma, cat. no. S2468) (100 μl/well streptavidin-HRP at a dilution of 1:5000) and incubation for 40 minutes at 37°C and washing four times in wash buffer. 100 μl of TMB solution was added per well. After the appearance of color, the reaction was stopped with 1 normal HCl solution and the absorbance was measured at 450 nm.

Example 1.6 Expression and Purification of Anti-PD-1 Monoclonal Antibodies

Hybridoma cells producing mouse monoclonal antibodies were cultured in FreeStyle™ 293 expression medium (Gibco/Life Technologies) in a CO ₂ shaker at 37° C. for 5-7 days. The conditioned medium was collected by centrifugation at 4000×g for 30 minutes to remove all cells and cell debris, then filtered through a 0.22 μm membrane before purification. Mouse antibodies were loaded and bound through a column packed with MabSelect™ SuRe (GE Healthcare) protein A resin according to manufacturer's instructions, washed with PBS, eluted with buffer containing 20 mM citrate, 150 mM NaCl, pH 3.5. The eluted materials were immediately neutralized with 1M Tris at pH 8.0 and dialyzed against PBS. One-step purified antibodies typically had a purity greater than 90% as determined by SEC-HPLC. Protein concentrations were determined by measuring absorbance at 280 nm or using a NanoDrop™ microvolume spectrophotometer (Thermo Scientific). Purified antibodies were stored as aliquots in a freezer at -80°C.

Example 2 Binding Activity of Purified Anti-PD-1 Antibodies

Examples 2.1: Characterization by ELISA

Binding ELISA was performed in the same manner as described in Example 1.3 above. Each purified antibody was subjected to 10-fold serial dilution and duplicated. After blocking a 96-well assay plate with wells containing immobilized PD-1 ECD/Fc fusion protein targets, serial dilutions of purified antibody samples were added to assay plate wells. HRP-linked anti-mouse IgG (A0168, Sigma) and TMB reagent were used to detect and display the ELISA signal, which was read on a SpectraMax® M5e plate reader at 450 nm. Curve fitting was performed using GraphPad software and EC50 was calculated. Receptor blocking assay (RBA) was performed in the same manner as described in Example 1.5 with titrated purified antibodies and upper block percentages and IC50 values were determined.

Example 2.2: Characterization with FACS

CHO-K1/huPD-1 or CHO-K1/cynoPD-1 cells described above were plated in 96-well round bottom assay plates (Cat. No. 3799; Corning) at 2 x 10 ⁴ cells per well and stained with purified anti- PD-1 antibodies. Anti-PD-1 antibodies were detected with high cross-adsorption donkey anti-mouse IgG (H+L) AlexaFluor® (Cat. No. A21202; Invitrogen) and cell fluorescence monitored with a flow cytometer. Data was processed with GraphPad software and EC50 values were calculated.

The results of these binding characterization assays are shown in Table 2 below.

Table 2: Binding activity of purified anti-PD-1 antibodies

ELISA FACS Receptor Blocking Assay (RBA) mAb ID huPD-1/Fc coating CHO-K1/huPD-1 CHO-K1/cynoPD-1 ligand 1
(huPD-L1) ligand 2
(huPD-L2) EC50 (nM) EC50 (nM) EC50
(nM) inhibition
TOP (%) IC50 (nM) TOP inhibition (%) IC50 (nM) mAb701 0.10 3.0 3.8 87.1 5.32 94.1 2.67 mAb703 0.05 0.5 0.7 96.0 3.00 95.7 2.02 mAb707 0.10 27.4 4.3 87.7 13.94 23.1 9.91 mAb709 0.01 0.2 0.2 91.6 0.80 93.7 0.52 mAb711 0.03 18.6 3.0 89.3 11.56 27.4 6.77 mAb713 0.08 1.1 0.9 94.4 2.95 93.5 2.52 mAb714 0.05 1.6 0.8 92.3 2.95 93.1 2.24 mAb715 0.04 1.1 0.9 86.9 2.91 88.0 2.17 mAb716 0.02 0.7 0.7 95.8 1.56 96.9 1.05 mAb718 0.02 3.2 4.2 96.6 4.19 96.6 1.91 mAb719 0.02 1.7 2.1 96.5 3.39 95.2 1.85 human IgG1 (control) 63.95 0.0 NA 9.9 NA

Example 2.3: Affinity Measurement by Surface Plasmon Resonance (SPR)

The binding kinetics of purified antibodies were measured by surface plasmon resonance using a Biacore T200 instrument (GE Healthcare) using standard procedures. Briefly, goat anti-mouse IgG Fc polyclonal antibodies (Genway) were immobilized directly on the biosensor chip and antibody samples were injected into the reaction matrices at a flow rate of 5 μl/min. The rate constants of association and dissociation, k _on (M ^-1 s ^-1 ) and k _off (s ^-1 ), respectively, were determined at a continuous flow rate of 30 μl/min. Rate constants were obtained by measuring kinetic binding at five different concentrations of human PD-1/Fc fusion protein. Then, from the kinetic rate constants, the equilibrium dissociation constant K _D (M) of the reaction between antibodies and related target proteins was calculated using the formula: K _D =k _off /k _on . Affinity for eleven mouse anti-PD-1 antibodies was measured as shown in Table 3 below.

Table 3: Affinity measurement of 11 monoclonal anti-PD-1 antibodies

mAb ID k _on (1/ms) k _off (1/s) K _D (M) mAb701 7.52×10 ⁴ 5.12×10 ^-4 6.81×10 ^-9 mAb703 3.47×10 ⁵ 8.50×10 ^-4 2.45×10 ^-9 mAb707 5.26×10 ⁴ 3.10×10 ^-4 5.89×10 ^-9 mAb709 1.11×10 ⁵ 1.04×10 ^-4 9.39×10 ^-9 mAb711 4.80×10 ⁴ 2.52×10 ^-4 5.24×10 ^-9 mAb713 1.45×10 ⁵ 2.85×10 ^-4 1.96×10 ^-9 mAb714 9.94×10 ⁴ 2.10×10 ^-4 2.11×10 ^-9 mAb715 1.58×10 ⁵ 2.37×10 ^-4 1.50×10 ^-9 mAb716 1.26×10 ⁵ 1.40×10 ^-4 1.11×10 ^-9 mAb718 5.84×10 ⁴ 2.83×10 ^-4 4.84×10 ^-9 mAb719 7.15×10 ⁴ 2.15×10 ^-4 3.00×10 ^-9

Example 3 Functional Activity of Anti-PD-1 Antibodies

Mixed lymphocyte response (MLR) analysis was performed using dendritic cells derived from monocytes from one donor and allogeneic CD4+ T cells from another donor. Whole blood samples were collected from healthy donors and PBMCs were isolated from whole blood by Ficoll-Pague gradient centrifugation. On day 1, PBMCs were obtained from a single donor, which were diluted with RPMI 1640 serum-free medium to a concentration of 1×10 ⁶ cells/ml. The diluted PBMCs were plated in a 6-well tissue culture plate at 3 ml/well and incubated for 3 hours. The supernatant was removed and non-immobilized cells were washed away. Immobilized monocytes were polarized into dendritic cells with 250 U/ml IL-4 and 500 U/ml GM-CSF in RPMI 1640 with 10% FBS. On the fourth day, the medium was replaced with fresh medium containing IL-4 and GM-CSF. On day seven, immature dendritic cells were harvested and treated with 1 μg/ml bacterial lipopolysaccharide (LPS) (Sigma) in RPMI 1640 with 10% FBS for an additional 24 hours to maturation. On day 8, CD4+ T cells were isolated from PBMCs obtained from another donor by negative selection and adjusted to a final concentration of 2×10 ⁶ cells/ml. Mature dendritic cells were treated with mitomycin C at 37°C for 1.5 hours, then the dendritic cells were washed with PBS and brought to a final concentration of 1×10 ⁶ cells/ml. CD4+ T cells (responder cells) were added to 96-well plates at 100 µl/well and pre-treated with test antibody at a diluted concentration for 30 minutes. Mature dendritic cells (stimulator cells) were added to the wells at 100 μl/well. The final volume in each well is 200 μl. The mixed culture of lymphocytes was incubated at 37°C. The production of IL-2 was measured after 72 hours (see Fig. 1A and 1B); IFN-γ was measured after 120 hours (see FIGS. 2A and 2B).

Example 4: Cloning and sequence analysis of anti-PD-1 mAb

The total RNA of each hybridoma clone was isolated from >5×10 ⁶ cells using the TRIzol reagent (Cat. No. 15596; Invitrogen). cDNA was synthesized with the SuperMix SuperScript™ III First Strand Synthesis Kit (Cat. No. 18080; Invitrogen) and used as a PCR template for the Mouse Ig Primer Kit (Cat. No. 69831-3; Novagen). PCR products were analyzed by electrophoresis on 1.2% agarose gel with gel staining with DNA dye SYBR™ Safe (Invitrogen). DNA fragments of the correct size were purified using the NucleoSpin® Gel and PCR Clean-up Kit (Cat. No. 740609; Macherey-Nagel GmbH) according to the manufacturer's instructions and individually subcloned into the pMD18-T vector (Sino Biological Inc.). Fifteen colonies were selected from each transformation and the sequences of the insertion fragments were analyzed by DNA sequencing. Sequences were confirmed with at least 8 matches to consensus sequences for VH and VL. The protein sequences of the variable regions of the mouse anti-PD-1 mAb were analyzed by alignment with the homologous sequences shown in Table 4. Complementarity determining regions (CDRs) were identified according to the Kabat numbering shown in Table 4 by underlining.

Table 4: Amino acid sequences of 8 mouse monoclonal anti-PD-1 antibodies

antibody domain SEQID NO. Amino acid sequences
1234567890123456789012345678901234567890 mAb701 vh four EVLLVESGGGLVKPGGSLKLSCAASGFTFSSYMMSWIRQTPERRLEWVASMSGGGRDTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTALYYCARRGTYAMDYWGQGTSVTVSS VL 5 DIQMTQSPASQSASLGESVTITCLASQTIGTWLTWYQQKPGKSPQLLIYAATSLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYSTPWTFGGGTKLEIK mAb703 vh 6 DVQLQESGPGLVKPSQSLSLTCSVTGYSITTGYYWNWIRQFPGNKLEWMGYMSYDGNNNYNPSLKNRISITRDTSKNQFLLRLNSVTTEDTATYFCARDRGTTILGGTMDYWGQGTSVTVS VL 7 SIVMTQTPKFLFVSAGDRVTIACKASQSVSNDVAWYQQKPGQSPKLLIYYAFYRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPWTFGGGTKLEIK mAb709 vh eight EVKLVESGGGLVKPGGSLKLSCAASGFTFSFYTMSWVRQTPEKRLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNTLYLHMSSLRSEDTALYYCAGQGGNYLFAYWGQGTLVTVSA VL 9 DIVMTQSHKFMSTSVGDSVTITCKASQDVNTVVAWYQQKPGQSLKVLISWASTRHTGVPARFTGSGSGTDYTLTISSVQAEDLALYYCQQHYTTPYTFGGGTQLEIK mAb713 vh ten EVKLVESGGGLVKPGGSLELSCAASGFTSSDYGMHWVRQAPEKGLEWVAYISSGSYTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAKRGGSSHVNVMDYWGQGTSVTVSS VL eleven DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSPPYTFGGGTKLEIK mAb714 vh 12 EVHLQQSGPELVKPGASVKIFCKASGYTFTDNNVEWVKQSHGKSLEWIGDINPNNGDTLYSQYFKDKATLTVDKSSTTAYMELRSLTSEDTGLYYCARGKSDQFDYWGQGTTLTVSS VL 13 DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATSLADGVPSRFSGSGSGTKFSFKISSLQGEDFVSYYCQQLYSSPWTFGGGTKLEIK mAb715 vh fourteen EVMLVESGGGLLKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNTLYLQMTSLRSEDTAFYYCAGQGGTYLFASWGQGTLVTVSA VL fifteen DIVMTQSHKFMSTSVGDSVTITCKASQDVNTAVAWYQQKPGQPPKVLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYTTPYTFGGGTKLEIK mAb718 vh 16 QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWAKQTPVHGLEWIGVIEPESGGTVYNQKFKGKAKLTADKSSRTAYMELRSLTSEDSAVYYCTREGFNSDHYFDYWGQGTTLTVSS VL 17 DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVFNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK mAb719 vh eighteen EVKLVESGGGLVKPGGSLKLSCTASGFSFSSHLMSWVRQTPEKRLEWVAAISGGGADTYYPDSVKGRFTISRDNAKNTLYLQMRSLRSEDTALYYCTRQILAFDSWGQGTTLTVSS VL 19 DIQMNQSPSSLSVSLGDTITITCHASQNIYVWLNWYQQKPGNIPKLLIYKASNLHTGVPSRFSGGGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGGGTKLEIK

Example 5 Humanization of Mouse Anti-PD-1 Antibodies

Based on human PD-1 binding activity, similar human-like cross-reactivity with cynomolgus PD-1, nearly 100% blocking activity in RBA assay, functional activity in MLR, and at least nanomolar affinity as measured by Biacore for humanization four anti-PD-1 antibodies, mAb709, mAb713, mAb703 and mAb719 were selected.

Example 5.1: Humanization of mouse antibody mAb709

mAb709 variable region genes were used to generate a humanized antibody. In the first step of this process, the mAb709 VH and VL amino acid sequences were compared with the available human Ig V gene sequence database to find the overall most suitable germline human Ig V gene sequences. In addition, segment 4 of the VH or VL framework was compared to a database of J regions to find the human framework having the highest homology to the murine VH and VL regions, respectively. The O12 gene showed the closest match to the human light chain V gene; and the closest match to the human heavy chain gene was shown by the VH3-7 gene. Humanized variable domain sequences were then constructed where mAb709 light chain CDR-L1, CDR-L2 and CDR-L3 were grafted onto the O12 gene framework sequence with JK4 framework sequence 4 after CDR-L3; and mAb709 heavy chain CDR-H1, CDR-H2, and CDR-H3 were grafted onto VH3-7 framework sequences with JH1 framework sequence 4 after CDR-H3. A 3D Fv model of mAb709 was then created to determine if there were any positions in the framework where mouse amino acids are critical for maintaining loop structures or the VH/VL interface. These residues in the humanized sequences should be backmutated to mouse residues in the same position in order to maintain affinity/activity. In the case of the light chain, Phe backmutation to Tyr at position 71 (F71Y, Kabat numbering), Tyr backmutation to Ser at position 49 (Y49S, Kabat numbering), Gln backmutation to Val at position 3 were identified as desirable backmutations. (Q3V, Kabat numbering), Leu backmutation to Val at position 46 (L46V, Kabat numbering), Ser to Thr at position 63 (S63T Kabat numbering), Ala backmutation to Ser at position 43 (A43S Kabat numbering), and reverse mutation of Pro to Leu at position 44 (P44L Kabat numbering). In the case of the heavy chain, an Arg mutation in Gly at position 98 (R94G Kabat numbering) and a Gly mutation in Arg at position 44 (G44R Kabat numbering) were identified as desirable backmutations. Mutated variable domains were constructed containing one or more of these back mutations. See table 5 below. (Back-mutated amino acid residues in the framework region are double underlined; mouse CDRs of the original parental antibody are simply underlined.)

Table 5: Humanized VH/VL design for mAb709 with w/backmutations to mouse residues

Humanized VH or VL mAb709 ID SEQID NO. Amino acid sequences
1234567890123456789012345678901234567890 mAb709 VH.1 twenty evqlvesggglvqpggslrlscaasgftfsFYTMSwvrqapgkglewvaTISGGGRDTYYPDSVKGrftisrdnaknslylqmnslraedtavyycarQGGNYLFAYWGQGTLVTVSS mAb709 VH.1A 21 evqlvesggglvqpggslrlscaasgftfsFYTMSwvrqapgkglewvaTISGGGRDTYYPDSVKGrftisrdnaknslylqmnslraedtavyycaGQGGNYLFAYWGQGTLVTVSS mAb709 VH.1B 22 evqlvesggglvqpggslrlscaasgftfsFYTMSwvrqapgkRlewvaTISGGGRDTYYPDSVKGrftisrdnaknslylqmnslraedtavyycaGQGGNYLFAYWGQGTLVTVSS mAb709 VK.1A 23 DIQMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIK mAb709 VK.1B 24 DIQMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKLLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIK mAb709 VK.1C 25 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIK mAb709 VK.1D 26 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFTGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIK mAb709 VK.1E 27 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKSLKVLISWASTRHTGVPSRFTGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIK

Humanized VH and VK genes were generated synthetically and then respectively cloned into vectors containing human IgG1 and human kappa chain constant domains (see Table 6 below.)

Table 6: Human Constant Region Sequence Used to Humanize Antibodies

Constant domain SEQID NO. Amino acid sequences
1234567890123456789012345678901234567890 Human Ig gamma constant region mutant 1 28 astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk Human kappa constant region 29 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Pairing of humanized VH and humanized VK chains resulted in 15 humanized antibodies named HumAb709-1 - HumAb709-15 (Table 7). A chimeric antibody with parental mouse VH/VL and human constant region sequences (mAb709c) was also generated as a positive control for affinity comparison. All recombinant mAbs were expressed and purified.

Table 7: List of generated humanized anti-PD-1 antibodies mAb709

Antibody ID VH region in the heavy chain VL region in the κ light chain HumAb709-1 mAb709 VH.1 mAb709 VK.1A HumAb709-2 mAb709 VH.1A mAb709 VK.1A HumAb709-3 mAb709 VH.1B mAb709 VK.1A HumAb709-4 mAb709 VH.1 mAb709 VK.1B HumAb709-5 mAb709 VH.1A mAb709 VK.1B HumAb709-6 mAb709 VH.1B mAb709 VK.1B HumAb709-7 mAb709 VH.1 mAb709 VK.1C HumAb709-8 mAb709 VH.1A mAb709 VK.1C HumAb709-9 mAb709 VH.1B mAb709 VK.1C HumAb709-10 mAb709 VH.1 mAb709 VK.1D HumAb709-11 mAb709 VH.1A mAb709 VK.1D HumAb709-12 mAb709 VH.1B mAb709 VK.1D HumAb709-13 mAb709 VH.1 mAb709 VK.1E HumAb709-14 mAb709 VH.1A mAb709 VK.1E HumAb709-15 mAb709 VH.1B mAb709 VK.1E HumAb709c SEQ ID NO:8 SEQ ID NO:9

The 15 humanized antibodies and the chimeric antibody (mAb709c) were characterized by binding ELISA and cellular RBA methods. For cellular RBA, CHO-K1-huPD1 cells were added to a pre-blocked 96-well round bottom plate at 2×10 ⁵ cells/well, and after washing, 50 μl of diluted antibodies ranging from 0.064 nM to 200 nM were added to each well. Then, 50 μl of biotinylated PD-L1/Fc protein or biotinylated PD-L2/Fc protein was added at a concentration of 60 μg/ml. After gentle mixing and incubation at 4° C., cells were washed and stained with Alexa Fluor™ 488 streptavidin solution (1:1000, ThermoFisher Scientific; cat. no. S32354). Signals were read using FACS and curves were plotted using GraphPad software. The calculated IC50 values are shown in Table 8 below. Antibodies having positive (low) IC50 values (ie, below about 1.0 nM for at least one PD-1 ligand) were further analyzed for binding affinity by surface plasmon resonance using a Biacore T200 instrument. Briefly, a goat anti-human IgG Fc polyclonal antibody was immobilized directly on the biosensor chip and samples of the humanized anti-PD-1 antibody or chimeric antibody were injected into the reaction matrices at a flow rate of 5 μl/min. Association and dissociation rate constants, k _on (M ^-1 s ^-1 ) and k _off (s ^-1 ), respectively, were determined by performing a kinetic binding measurement at five different concentrations of human PD-1-His protein at a continuous flow rate of 30 μl/ min. The equilibrium dissociation constant K _D (M) of the reaction between antibodies and related target proteins was calculated from the kinetic rate constants according to the formula: K _D =k _off /k _on . Affinities for five of the humanized anti-PD-1 mAb709 derivatives are shown in Table 8. HumAb709-8 had minimal backmutations and maximally conserved affinity for the parental variable domains of the chimeric mAb709c.

Table 8: RBA values and binding affinities of humanized anti-PD-1 antibodies mAb709

Humanized antibody ID PD-L1 RBA
IC50 (nM) PD-L2 RBA
IC50 (nM) k _on (1/ms) k _off (1/s) K _D (M) HumAb709-1 1.02 1.64 1.95×10 ⁵ 2.145×10 ^-3 1.10×10 ^-8 HumAb709-2 0.47 0.99 8.03×10 ⁴ 5.5×10 ^-5 6.84×10 ^-10 HumAb709-3 1.25 1.64 HumAb709-4 0.78 1.68 HumAb709-5 0.67 0.97 HumAb709-6 1.23 1.26 HumAb709-7 0.40 0.84 1.41×10 ⁵ 3.36×10 ^-4 2.36×10 ^-9 HumAb709-8 0.44 1.00 1.27×10 ⁵ 4.69×10 ^-5 3.68×10 ^-10 HumAb709-9 1.04 1.76 HumAb709-10 0.29 0.80 1.46×10 ⁵ 2.97×10 ^-4 2.04×10 ^-9 HumAb709-11 0.55 0.92 HumAb709-12 0.45 1.35 HumAb709-13 0.50 0.78 HumAb709-14 0.51 0.92 HumAb709-15 0.90 1.21 mAb709c 0.62 0.56 1.21×10 ⁵ 6.88×10 ^-5 5.67×10 ^-10

Example 5.2: Humanization of mouse antibody mAb713

mAb713 anti-PD-1 variable region genes were used to generate a humanized antibody. The mAb713 VH and VK amino acid sequences were compared with the available human Ig V gene sequence database to find the overall most suitable germline human Ig V gene sequences. In addition, segment 4 of the VH or VL framework was compared to a database of J regions to find the framework having the highest homology to the murine VH and VL regions, respectively. The O18 gene showed the closest match to the human light chain V gene; and the closest match to the human heavy chain gene was shown by the VH3-48 gene. Humanized variable domain sequences were then constructed, where mAb713 light chain CDR-L1, CDR-L2, and CDR-L3 were grafted onto the O18 gene framework sequences with JK4 framework sequence 4 after CDR-L3; and mAb713 heavy chain CDR-H1, CDR-H2, and CDR-H3 were grafted onto VH3-48 framework sequences with JH6 framework sequence 4 after CDR-H3. A 3D Fv model of mAb709 was then created to determine if there were any positions in the framework where mouse amino acids are critical for maintaining loop structures or the VH/VL interface. These residues in the humanized sequences should be backmutated to mouse residues in the same position in order to maintain affinity/activity. In the case of the light chain, a Phe backmutation to Tyr at position 71 (F71Y, Kabat numbering), a Tyr backmutation to Ser at position 49 (Y49S, Kabat numbering), and a Thr backmutation to Lys at position 69 (T69K) were identified as desirable backmutations. , Kabat numbering). In the case of the heavy chain, an Arg mutation to Lys at position 98 (R94K, Kabat numbering), a Phe backmutation to Ser at position 29 (F29S, Kabat numbering), and a Ser backmutation to Ala at position 49 (S49A, Kabat numbering). Mutated variable domains were constructed containing one or more of these back mutations. See table 9 below. (Back-mutated amino acid residues in the framework region are double underlined; mouse CDRs of the original parental antibody are simply underlined.)

Table 9: mAb713 VH and VL variable domain sequence variants

VH/VL options mAb713 SEQID NO. Amino acid sequences
1234567890123456789012345678901234567890 mAb713 VH.1 thirty EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARRGGSSHVNVMDYWGQGTTVTVSS mAb713 VH.1A 31 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSS mAb713 VH.1B 32 EVQLVESGGGLVQPGGSLRLSCAASGFTsSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSS mAb713 VH.1C 33 EVQLVESGGGLVQPGGSLRLSCAASGFTsSDYGMHWVRQAPGKGLEWVaYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSS mAb713 VK.1 34 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIK mAb713 VK.1A 35 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIK mAb713 VK.1B 36 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIK mAb713 VK.1C 37 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGKDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIK

Humanized VH and VK genes were obtained synthetically and then separately cloned into vectors containing the constant domains of human IgG1 and human kappa chain (see table 6 above). Pairing of human VH variants and human VK variants resulted in 16 humanized antibodies named HumAb713-1 through HumAb713-16 (Table 10). a chimeric antibody (mAb713c) with parental mouse VH/VL and human constant sequences was also generated as a positive control for affinity comparison.

Table 10: List of mAb713 humanized anti-PD-1 antibodies obtained

Antibody ID VH region in the heavy chain VL region in the κ light chain HumAb713-1 mAb713 VH.1 mAb713 VK.1 HumAb713-2 mAb713 VH.1A mAb713 VK.1 HumAb713-3 mAb713 VH.1B mAb713 VK.1 HumAb713-4 mAb713 VH.1C mAb713 VK.1 HumAb713-5 mAb713 VH.1 mAb713 VK.1A HumAb713-6 mAb713 VH.1A mAb713 VK.1A HumAb713-7 mAb713 VH.1B mAb713 VK.1A HumAb713-8 mAb713 VH.1C mAb713 VK.1A HumAb713-9 mAb713 VH.1 mAb713 VK.1B HumAb713-10 mAb713 VH.1A mAb713 VK.1B HumAb713-11 mAb713 VH.1B mAb713 VK.1B HumAb713-12 mAb713 VH.1C mAb713 VK.1B HumAb713-13 mAb713 VH.1 mAb713 VK.1C HumAb713-14 mAb713 VH.1A mAb713 VK.1C HumAb713-15 mAb713 VH.1B mAb713 VK.1C HumAb713-16 mAb713 VH.1C mAb713 VK.1C mAb713c SEQ ID NO:10 SEQ ID NO:11

All 16 humanized antibodies and chimeric antibody (mAb713c) were characterized by binding ELISA, cellular RBA and Biacore affinity testing methods. The results obtained are summarized in Table 11.

Table 11: RBA values and binding affinities of humanized anti-PD-1 antibodies mAb713

Humanized antibody ID PD-L1 RBA
IC50 (nM) PD-L2 RBA
IC50 (nM) k _on (1/ms) k _off (1/s) K _D (M) HumAb713-1 0.50 1.20 ^9.010 ×104 1.003×10 ^-3 1.113×10 ^-8 HumAb713-2 1.71 3.13 HumAb713-3 0.77 1.24 ^8.447 ×104 2.082×10 ^-4 2.465×10 ^-9 HumAb713-4 1.06 2.24 HumAb713-5 0.91 2.95 HumAb713-6 1.04 1.46 HumAb713-7 0.76 1.40 1.237× ¹⁰⁵ 3,500×10 ^-4 2.829×10 ^-9 HumAb713-8 1.05 1.91 HumAb713-9 1.20 2.00 HumAb713-10 0.80 1.23 HumAb713-11 0.51 0.97 1.591× ¹⁰⁵ 3.776×10 ^-4 2.373×10 ^-9 HumAb713-12 0.94 1.59 HumAb713-13 0.70 2.13 HumAb713-14 0.91 1.45 HumAb713-15 0.88 1.65 HumAb713-16 0.65 1.63 mAb713c 0.91 2.20 ^2.182 ×105 2.839×10 ^-4 1.301×10 ^-9

HumAb713-7 had a minimal number of backmutations while maintaining the affinity characteristics of the parental variable domains of the mAb713c chimeric antibody. The functional activity of the humanized mAb713 antibodies was confirmed in the MLR assay as described in Example 3. As seen in FIG. 5, HumAb713-7 exhibited activity comparable to that of the chimeric mAb713c antibody in MLR, consistent with its retained binding properties.

Example 5.3: Humanization of mouse antibody mAb703

Following the same procedure as in Examples 5.1 and 5.2, the mouse anti-PD-1 antibody mAb703 was selected and humanized. Humanized variable domains were constructed, some of which contained one or more back mutations, the amino acid sequences of which are shown in Table 12 below. (Back-mutated amino acid residues in the framework region are double underlined; mouse CDRs of the original parental antibody are simply underlined.)

Table 12: mAb703 VH and VL variable domain sequence variants

mAb703 VH/VL options SEQID NO. Amino acid sequence
1234567890123456789012345678901234567890 mAb703 VH.1A 38 EVQLQESGPGLVKPSETLSLTCAVSGYSISTGYYYWNWIRQPPGKGLEWIGYMSYDGNNNYNPSLKNRVTISRDTSKNQFSLKLSSVTAADTAVYYCARDRGTTILGGTMDYWGQGTTVTVSS mAb703 VH.1B 39 EVQLQESGPGLVKPSETLSLTCAVSGYSISTGYYYWNWIRQPPGKGLEWIGYMSYDGNNNYNPSLKNRiTISRDTSKNQFSLKLSSVTAADTAVYYCARDRGTTILGGTMDYWGQGTTVTVSS mAb703 VH.1C 40 EVQLQESGPGLVKPSETLSLTCAVSGYSISTGYYWNWIRQPPGKGLEWmGYMSYDGNNNYNPSLKNRiTISRDTSKNQFSLKLSSVTAADTAVYYCARDRGTTILGGTMDYWGQGTTVTVSS mAb703 VH.1D 41 EVQLQESGPGLVKPSETLSLTCAVSGYSItTGYYWNWIRQPPGKGLEWmGYMSYDGNNNYNPSLKNRiTISRDTSKNQFSLKLSSVTAADTAVYYCARDRGTTILGGTMDYWGQGTTVTVSS mAb703 VH.1E 42 EVQLQESGPGLVKPSETLSLTCAVSGYSItTGYYWNWIRQPPGKkLEWmGYMSYDGNNNYNPSLKNRiTISRDTSKNQFSLKLSSVTAADTAVYfCARDRGTTILGGTMDYWGQGTTVTVSS mAb703 VK.1 43 DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYAFYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGGGTKVEIK mAb703 VK.1A 44 DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYAFYRYTGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGGGTKVEIK mAb703 VK.1B 45 DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYAFYRYTGVPDRFSGSGYGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGGGTKVEIK mAb703 VK.1C 46 DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKSPKLLIYYAFYRYTGVPDRFSGSGYGTDFTLTISSLQPEDFATYFCQQDYSSPWTFGGGTKVEIK mAb703 VK.1D 47 SIVMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKSPKLLIYYAFYRYTGVPDRFTGSGYGTDFTLTISSLQPEDFATYFCQQDYSSPWTFGGGTKVEIK

Humanized VH and VK genes were obtained synthetically and then separately cloned into vectors containing the constant domains of human IgG1 and human kappa chain (see table 6 above). Pairing of human VH variants and human VK variants resulted in 25 humanized antibodies named HumAb703-1 through HumAb703-25 (Table 13). A chimeric antibody with parental mouse VH/VL and human constant sequences was also generated as a positive control for affinity comparison.

Table 13: List of obtained humanized anti-PD-1 antibodies mAb703

Antibody ID VH region in the heavy chain VL region in the κ light chain HumAb703-1 mAb703 VH.1A mAb703 VK.1 HumAb703-2 mAb703 VH.1B mAb703 VK.1 HumAb703-3 mAb703 VH.1C mAb703 VK.1 HumAb703-4 mAb703 VH.1D mAb703 VK.1 HumAb703-5 mAb703 VH.1E mAb703 VK.1 HumAb703-6 mAb703 VH.1A mAb703 VK.1A HumAb703-7 mAb703 VH.1B mAb703 VK.1A HumAb703-8 mAb703 VH.1C mAb703 VK.1A HumAb703-9 mAb703 VH.1D mAb703 VK.1A HumAb703-10 mAb703 VH.1E mAb703 VK.1A HumAb703-11 mAb703 VH.1A mAb703 VK.1B HumAb703-12 mAb703 VH.1B mAb703 VK.1B HumAb703-13 mAb703 VH.1C mAb703 VK.1B HumAb703-14 mAb703 VH.1D mAb703 VK.1B HumAb703-15 mAb703 VH.1E mAb703 VK.1B HumAb703-16 mAb703 VH.1A mAb703 VK.1C HumAb703-17 mAb703 VH.1B mAb703 VK.1C HumAb703-18 mAb703 VH.1C mAb703 VK.1C HumAb703-19 mAb703 VH.1D mAb703 VK.1C HumAb703-20 mAb703 VH.1E mAb703 VK.1C HumAb703-21 mAb703 VH.1A mAb703 VK.1D HumAb703-22 mAb703 VH.1B mAb703 VK.1D HumAb703-23 mAb703 VH.1C mAb703 VK.1D HumAb703-24 mAb703 VH.1D mAb703 VK.1D HumAb703-25 mAb703 VH.1E mAb703 VK.1D mAb703c SEQ ID NO:6 SEQ ID NO:7

All 25 humanized antibodies and the chimeric antibody (mAb703c) were characterized by binding ELISA and Biacore affinity testing methods. The results of the evaluation of the affinity observed in positive binding agents are summarized in table 14.

Table 14: Binding affinity of selected humanized anti-PD-1 antibodies mAb703

Humanized antibody ID k _on (1/ms) k _off (1/s) K _D (M) HumAb703-11 1.874× ¹⁰⁵ 1.757×10 ^-3 9.374×10 ^-9 HumAb703-12 1.770× ¹⁰⁵ 1.594×10 ^-3 9.003×10 ^-9 HumAb703-13 1.454× ¹⁰⁵ 1.537×10 ^-3 1.057×10 ^-8 HumAb703-18 6.572×10 ⁴ 1.242×10 ^-3 1.890×10 ^-8 HumAb703-22 2.294× ¹⁰⁵ 1.593×10 ^-3 6.942×10 ^-9 mAb703c ^3.594 ×105 9.664×10 ^-4 2.684×10 ^-9

Functional activity of humanized mAb703 antibodies was confirmed in MLR assays, which were performed as described in Example 3 as shown in FIG. 2A and 3.

Example 5.4 Humanization of Mouse Antibody mAb719

Following the same procedure as in Examples 5.1 and 5.2, the mouse anti-PD-1 antibody mAb719 was selected and humanized. Designed humanized variable domains, and some of them contained one or more reverse mutations; amino acid sequences are presented in table 15 below. (Reverse mutated amino acid residues in the framework region are double underlined; mouse CDRs of the original parental antibody are simply underlined.) In addition, the Asp → Ala substitution in CDR-H2 and Ser → Ala in CDR-H3 were performed to avoid the possibility of Asp isomerization often observed in recombinant antibodies. (See mAb719 VH.1E and mAb719 VH.1F sequences in Table 15.)

Table 15: mAb719 VH and VL variable domain sequence variants

mAb719 VH/VL options SEQID NO. Amino acid sequence
1234567890123456789012345678901234567890 mAb719 VH.1 48 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHLMSWVRQAPGKGLEWVSAISGGGADTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQILAFDSWGQGTTVTVSS mAb719 VH.1A 49 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHLMSWVRQAPGKGLEWVSAISGGGADTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQILAFDSWGQGTTVTVSS mAb719 VH.1B fifty EVQLLESGGGLVQPGGSLRLSCAASGFSFSSHLMSWVRQAPGKGLEWVSAISGGGADTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQILAFDSWGQGTTVTVSS mAb719 VH.1C 51 EVQLLESGGGLVQPGGSLRLSCAASGFSFSSHLMSWVRQAPGKGLEWVAAISGGGADTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQILAFDSWGQGTTVTVSS mAb719 VH.1D 52 EVQLLESGGGLVQPGGSLRLSCAASGFSFSSHLMSWVRQAPGKRLEWVAAISGGGADTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQILAFDSWGQGTTVTVSS mAb719 VH.1E 53 EVQLLESGGGLVQPGGSLRLSCAASGFSFSSHLMSWVRQAPGKGLEWVAAISGGGADTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQILAFDAWGQGTTVTVSS mAb719 VH.1F 54 EVQLLESGGGLVQPGGSLRLSCAASGFSFSSHLMSWVRQAPGKRLEWVAAISGGGADTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQILAFDAWGQGTTVTVSS mAb719 VK.1 55 DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGQSYPWTFGGGTKVEIK mAb719 VK.1A 56 DIQMNQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGQSYPWTFGGGTKVEIK mAb719 VK.1B 57 DIQMNQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKIPKLLIYKASNLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGQSYPWTFGGGTKVEIK

Humanized VH and VK genes were obtained synthetically and then separately cloned into vectors containing the constant domains of human IgG1 and human kappa chain (see table 6 above). Pairing of human VH variants and human VK variants resulted in 21 humanized antibodies named HumAb719-1 through HumAb719-21 (Table 16). A chimeric antibody with parental mouse VH/VL and human constant sequences was also generated as a positive control for affinity comparison. All recombinant mAbs were expressed and purified.

Table 16: List of mAb719 humanized anti-PD-1 antibodies obtained

Antibody ID VH region in the heavy chain VL region in the κ-light chain HumAb719-1 mAb719 VH.1 mAb719 VK.1 HumAb719-2 mAb719 VH.1A mAb719 VK.1 HumAb719-3 mAb719 VH.1B mAb719 VK.1 HumAb719-4 mAb719 VH.1C mAb719 VK.1 HumAb719-5 mAb719 VH.1D mAb719 VK.1 HumAb719-6 mAb719 VH.1E mAb719 VK.1 HumAb719-7 mAb719 VH.1F mAb719 VK.1 HumAb719-8 mAb719 VH.1 mAb719 VK.1A HumAb719-9 mAb719 VH.1A mAb719 VK.1A HumAb719-10 mAb719 VH.1B mAb719 VK.1A HumAb719-11 mAb719 VH.1C mAb719 VK.1A HumAb719-12 mAb719 VH.1D mAb719 VK.1A HumAb719-13 mAb719 VH.1E mAb719 VK.1A HumAb719-14 mAb719 VH.1F mAb719 VK.1A HumAb719-15 mAb719 VH.1 mAb719 VK.1B HumAb719-16 mAb719 VH.1A mAb719 VK.1B HumAb719-17 mAb719 VH.1B mAb719 VK.1B HumAb719-18 mAb719 VH.1C mAb719 VK.1B HumAb719-19 mAb719 VH.1D mAb719 VK.1B HumAb719-20 mAb719 VH.1E mAb719 VK.1B HumAb719-21 mAb719 VH.1F mAb719 VK.1B mAb719c SEQ ID NO:18 SEQ ID NO:19

All 21 humanized antibodies and chimeric antibodies (mAb719c) were characterized by binding ELISA and affinity determination using the Octet® RED96 biolayer interferometry system (Pall FortéBio LLC) using a biosensor with immobilized human PD-1/Fc fusion protein as the target. antibodies. Rate constants were obtained by measuring the kinetic binding at five different antibody concentrations. The affinity was higher than in previous testing with Biacore, which was the result of using a target with divalent binding. The affinity scores observed for positive binders are summarized in Table 17.

Table 17: Binding affinity of selected humanized anti-PD-1 antibodies mAb719

Humanized antibody ID k _on (1/ms) k _off (1/s) K _D (M) HumAb719-8 1.066× ¹⁰⁵ 4.905×10 ^-5 4.602×10 ^-10 HumAb719-11 ^5.944 ×104 2.270×10 ^-4 3.819×10 ^-9 HumAb719-12 ^6.882 ×104 5.805×10 ^-5 8.435×10 ^-10 HumAb719-21 1.042× ¹⁰⁵ 6.256×10 ^-5 6.005×10 ^-10 mAb719c 9.735× ¹⁰⁴ <1.00×10 ^-5 <1.027×10 ^-10

Functional activity of humanized mAb719 antibodies was confirmed in MLR assays as shown in FIG. 2b and 3.

Example 6: Pharmacokinetic properties of leading anti-PD-1 antibodies.

The pharmacokinetic properties of HumAb709-8 and HumAb713-7 were evaluated using male Sprague-Dawley (SD) rats. Antibodies were administered to male SD rats as a single intravenous dose of 5 mg/kg. Serum samples were collected at different time points over 28 days: 0, 5, 15 and 30 minutes; 1, 2, 4, 8 and 24 hours; and 2, 4, 7, 10, 14, 21 and 28 days by serial tail vein blood sampling and analyzed by total ELISA. Briefly, ELISA plates were coated with 125 ng/well goat anti-human IgG Fc (Rockland, cat. no. 609-101-017) at 4° C. overnight, blocked with 1X PBS/1% BSA/0.05% Tween- 20/0.05% ProClin™ 300. All serum samples were first diluted 20-fold with blocking buffer. An additional dilution was performed using 5% pooled rat serum and incubated on the plate for 60 minutes at 37°C. Detection was performed with peroxidase-conjugated anti-human IgG (Fab fragment) (Sigma; Cat. No. A0293) and concentrations were determined using standard curves by fitting a four parameter logistic curve. Pharmacokinetic parameter values were determined using a non-compartmental model using WinNonlin software (Pharsight Corporation, Mountain View, CA). According to the results obtained (Table 18), HumAb09-8 and HumAb13-7 show stable properties.

Table 18: Pharmacokinetic properties of HumAb709-8 and HumAb713-7

Parameters P.K. CL Vss beta t _1/2 AUC MRT Antibody ml/day/kg ml/kg day day*mcg/ml day HumAb709-8 8.6 129.6 10.9 594.2 15.4 HumAb713-7 6.4 114.4 12.7 789.3 18.1

Example 7 Generation of Anti-LAG-3 Monoclonal Antibodies

Monoclonal anti-LAG-3 antibodies (mAb) were generated by hybridoma fusion.

Example 7.1: Immunization, hybridoma fusion and cloning.

Immunization of Balb/C mice was performed in the same manner as described above for the production of anti-PD-1 antibodies (Example 1), except for the homodimer used as immunogen: D1-D2 human LAG-3/mouse Fc. Immunized animals were re-vaccinated 2-4 times with an interval of 2-3 weeks. Three days after the final immunization, splenocytes were isolated from immunized mice and fused with the SP2/0 mouse myeloma cell line using standard methods.

Example 7.2: Identification and characterization of anti-LAG-3 antibodies

Synthetic targets for human LAG-3 and LAG-3 of the cynomolgus macaque were prepared by order of Synbio Technologies (Suzhou, China). Each target consisted of a polypeptide segment of the extracellular domain of the human or cynomolgus macaque LAG-3 protein fused to the Fc region of human IgG. Synthetic genes encoding each LAG-3 ECD/Fc fusion protein were subcloned into the pCP expression vector (Chempartner, Shanghai, CN) and expression plasmids transiently transfected into HEK 293E cells in 1-3 liters of medium and cultured for seven days in a shaker with CO2 _. The ECD sequences used for each merge are shown in Table 19 below. The ECD portion of the LAG-3 of each fusion protein is underlined.

Table 19: Amino acid sequences of target LAG-3 ECD/Fc fusion proteins

SEQID NO. Source LAG-3 Amino acid sequences
1234567890123456789012345678901234567890 58 human LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 59 Javanese macaque PQPGAEISVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAXAPGHPPVPGHRPAAPYSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRATVHLRDRALSCRLRLRVGQASMTASPPGSLRTSDWVILNCSFSRPDRPASVHWFRSRGQGRVPVQGSPHHHLAESFLFLPHVGPMDSGLWGCILTYRDGFNVSIMYNLTVLGLEPATPLTVYAGAGSRVELPCRLPPAVGTQSFLTAKWAPPGGGPDLLVAGDNGDFTLRLEDVSQAQAGTYICHIRLQGQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPASGQEHFVWSPLNTPSQRSFSGPWLEAQEAQLLSQPWQCQLHQGERLLGAAVYFTELSSPGAQRSGRAPGALRAGHLIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Hybridoma clone supernatants were first checked by ELISA. Briefly, 1 μg/ml human LAG-3 ECD/Fc in NaHCO ₃ was applied to each well of a 96-well plate at 50 μl/well left overnight. The plates were washed 3 times with 1X PBST at 300 µl per well. After blocking with 1% BSA in PBST at 250 μl per well and incubation at room temperature for 1 hour, hybridoma supernatants at 50 μl per well were added and incubated at 37°C for 1 hour. After washing, HRP-conjugated secondary goat anti-mouse IgG Fc (Cat. No. A0168, Sigma) was added at 100 μl/well, and the plates were incubated at room temperature for 1 hour. The TMB reagent (InnoReagents) was used to detect and develop the ELISA signal at 100 μl/well for 15 minutes, the reaction was stopped with 1 normal HCl. Plates were read using a plate reader (SpectraMax® M5e, Molecular Devices, USA) at 450 nm. ELISA positive antibody producing clones were further tested by FACS analysis using methods similar to those described in Example 1.4 above, except using stable HEK 293F cell lines expressing human LAG-3 or cynomolgus LAG-3. Hybridomas demonstrating LAG-3 binding activity were selected and further characterized in a receptor blocking assay (RBA).

Example 7.3 Receptor Blocking Assay (RBA)

Supernatants showing LAG-3-specific activity were tested for their ability to block the binding of the LAG-3 receptor to MHC class II. Raji human B cell lymphoblasts express high levels of MCH class II and have been used as binding targets for the LAG-3 ECD/Fc proteins described above. Briefly, Raji cells were harvested, resuspended in FACS buffer and plated in 96-well plates (2×10 ⁵ cells/well). Anti-LAG-3 hybridoma supernatants were mixed with soluble LAG-3 ECD/Fc and the mixture was added to the wells to a final volume of 100 μl/well. After adding the mixture to the cells, the plates were incubated at room temperature for 30 minutes. After washing twice with PBS, the cells were incubated with a secondary antibody to human IgG Alexa Fluor® 488 at 4°C for 1 hour, washed twice with PBS, and then fluorescence was measured on a flow cytometer.

Example 7.4 Expression and Purification of Anti-LAG-3 Monoclonal Antibodies

Hybridoma cells producing mouse monoclonal antibodies were cultured in FreeStyle™ 293 expression medium (Gibco/Life Technologies) in a CO ₂ shaker at 37° C. for 5-7 days. The conditioned medium was collected by centrifugation at 4000×g for 30 minutes to remove all cells and cell debris, then filtered through a 0.22 μm membrane before purification. Mouse antibodies were applied and bound through a column packed with MabSelect™ SuRe (GE Healthcare) protein A resin according to manufacturer's instructions, washed with PBS, eluted with buffer containing 20 mM citrate, 150 mM NaCl, pH 3.5. The eluted materials were immediately neutralized with 1M Tris at pH 8.0 and dialyzed against PBS. One-step purified antibodies typically had a purity greater than 90% as determined by SEC-HPLC. Protein concentrations were determined by measuring absorbance at 280 nm or using a NanoDrop™ microvolume spectrophotometer (Thermo Scientific). Purified antibodies were stored as aliquots in a freezer at -80°C.

Example 7.5 Binding Activity of Purified Anti-LAG-3 Antibodies

Characterization by ELISA

Binding ELISA was performed in the same manner as described in Example 7.2 above. Each purified antibody was subjected to a 10-fold serial dilution. After blocking a 96-well assay plate with wells containing immobilized LAG-3 ECD/Fc fusion protein targets, purified diluted antibody samples were added to the wells of the assay plates. HRP-linked anti-mouse IgG (A0168, Sigma) and TMB reagent were used to detect and display the ELISA signal, which was read on a SpectraMax® M5e plate reader at 450 nm. Curves were fitted using GraphPad software and EC50 values were calculated. Similarly, RBA was performed as described in Example 7.3 using purified antibody titers and upper block percentages and IC50 values were determined.

Characterization with FACS

FACS analysis was performed using methods similar to those described in Example 1.4 above, except using stable HEK 293F cell lines expressing either human LAG-3 or cynomolgus monkey LAG-3. LAG-3 expressing cells were loaded at 2×10 ⁴ cells/well into 96-well round bottom assay plates (Cat. No. 3799; Corning) and stained with purified anti-LAG-3 antibodies. Anti-LAG-3 antibodies were detected with a donkey anti-mouse IgG (H+L) highly cross-adsorbing AlexaFluor® secondary antibody (Cat. No. A21202; Invitrogen) and cell fluorescence monitored with a flow cytometer. Data was processed with GraphPad software and EC50 values were calculated.

The results of these binding characterization assays are shown in Table 20 below.

Table 20: Binding activity of purified mouse anti-LAG-3 antibodies

mAb ID Binding to human LAG-3 Binding to LAG-3 of the cynomolgus macaque ELISA FACS ELISA FACS EC50 (nM) EC50 (nM) Max MFI EC50 (nM) EC50 (nM) Max MFI mAb742 0.22 2.8 102.9 0.32 56.4 48.5 mAb743 0.26 30.4 115.7 0.26 111.5 36.9 mAb744 0.19 9.5 135.0 0.21 67.0 37.8 mAb745 0.27 32.2 54.8 0.30 224.3 26.0 mAb746 0.31 1.3 120.8 0.21 4.1 66.2 mAb747 0.25 1.1 104.4 0.34 3.1 65.2 mAb748 0.24 13.4 73.1 0.25 79.6 31.9 mAb749 0.55 3.3 123.6 0.33 14.3 67.4 mAb750 0.25 24.1 88.7 0.32 113.6 36.7 mAb751 0.22 26.2 88.9 0.27 79.3 33.1 mAb757 0.23 25.3 91.8 0.30 77.2 35.1 mAb758 0.87 9.8 64.8 3.18 15.0 17.3 mAb759 0.43 3.0 60.2 0.53 6.3 18.6 mAb760 N/A N/A 12.3 N/A N/A 4.9 mAb761 0.1.18 17.8 105.2 1.24 34.6 39.5 human IgG1 (control) 0.13 0.9 190.6 78.23 63.7 28.1

In this table, "N/A" means non-measurable binding activity.

Example 7.6: Characterization by RBA Method and Antigen-Dependent Activation Assay

Purified anti-LAG-3 antibodies were also tested by the RBA method in the same way as described in example 7.3. Antibodies were also tested in an antigen-specific T cell activation assay as shown below: a mouse T hybridoma cell line expressing huLAG-3 was obtained according to ChemPartner (Shanghai, CN). Splenocytes from mice of the same strain were used as effector cells. The hybridoma expressing the huLAG-3 receptor protein is able to bind to MHC-class II positive mouse splenocytes with an inhibitory effect due to interaction with class II. Assays for LAG-3 antibody-mediated reversibility of the inhibitory effect were measured by increasing the level of IL-2 production. Mouse splenocytes were collected from 6-8 week old female C57BL/6 mice, erythrocytes were lysed with red blood cell lysis buffer (Sigma-Aldrich; R7757) according to the manufacturer's instructions. Then, 50 μl of T-cell hybridoma-huLAG-3 cells (2×10 ⁶ cells/ml) were seeded in each well of a 96-well culture plate, and then serial dilutions of monoclonal anti-LAG-3 antibodies were added at a concentration of 50 μl/well and incubated at 37°C for 30 min. Mouse splenocytes (4×10 ⁶ cells/ml) and antigen (20 μg/ml) were mixed and incubated at 37°C for 30 minutes. The mixture (100 μl/well) was added to each well that had already been seeded with T-cell hybridoma-huLAG-3 cells and anti-LAG-3 mAb. The mixture of antibodies, T-cell hybridoma-huLAG-3 cells, mouse splenocytes and antigen were cultured for 3 days. After 72 hours, 100 µl of the cell culture supernatant was aspirated and diluted to appropriate concentrations to perform a quantitative ELISA for mouse IL-2 using an ELISA kit (R&D Systems) according to the manufacturer's protocol. The ELISA plate was read on a SpectraMax M5 plate reader (Molecular Devices) using the ELISA-Endpoint-TMB and HRP protocol. The results of RBA analysis and antigen-dependent activation are shown in Table 21.

Table 21: Characterization of mouse anti-LAG-3 antibodies

FACS RBA Antigen dependent activation Raji 1 µg/ml HuLAG-3 ECD/Fc mouse IL-2 mAb ID IC50 (nM) Max. inhibition (%) EC50 (nM) Max. IL-2 (pg/ml) mAb742 3.84 96.5 ++ 732.8 mAb743 11.66 96.6 ++ 583.2 mAb744 10.77 96.5 ++ 612.3 mAb745 12.38 95.0 + 357.1 mAb746 2.92 96.2 1.28 653.5 mAb747 2.84 96.3 1.27 729.2 mAb748 4.80 96.1 ++ 539.2 mAb749 4.73 93.9 +++ 513.0 mAb750 7.15 96.6 +++ 552.5 mAb751 6.59 96.0 ++ 447.0 mAb757 7.80 96.8 ++ 570.9 mAb758 N/A 7.9 - 182.9 mAb759 86.46 34.2 mAb760 N/A 3.4 mAb761 17.98 96.0 human IgG1 (control) 2.30 94.7 0.71 785.7

Example 7.7: Determination of Binding Affinity with Biacore

For antibodies with high binding affinity in ELISA and FACS assays, as well as with high functional activity, binding affinity was determined based on the measurement of kinetic binding constants in real-time binding reactions by surface plasmon resonance with Biacore. Briefly, antibody-antigen binding assay was performed with the Biacore T200 system using the antibody capture method. An anti-mouse IgG Fc antibody was immobilized on a CM5 sensor chip according to the manufacturer's instructions. The mouse monoclonal anti-LAG-3 antibody to be tested was injected and captured with the immobilized anti-mouse IgG Fc antibody. Serial dilutions of the LAG-3 antigen were then added separately and the binding profile was recorded for each dilution of the analyzed antigen. The assay temperature was 25° C. and the association and dissociation times were 180 and 1200 seconds, respectively. Biacore data were fitted with Biacore T200 scoring software 1.0 according to a 1:1 binding model to calculate association (k _on ) and dissociation (k _off ) rate constants, and the equilibrium dissociation constant (K _D ) was determined from these calculations. Affinity values (K _D ) for four selected anti-LAG-3 antibodies are shown in Table 22 below.

Table 22: Binding affinity of selected anti-LAG-3 antibodies

Antibody ID Affinity for LAG-3 antigen (K _D ) mAb746 3.774×10 ^-8M mAb747 5.201×10 ^-8M mAb749 1.893×10 ^-7M mAb750 7.506×10 ^-8M

Example 7.8 Comparison of Function of Anti-LAG-3 Antibodies in PBMC Assay

To further test the function of anti-LAG-3 antibodies in human PBMCs, a stimulation assay was performed with a bacterial toxin, superantigenic Staphylococcus aureus enterotoxin B (SEB). SEB is a known superantigen for activating the immune system by stimulating human T cells, which in turn causes the overproduction of several cytokines. PBMCs were isolated from a blood sample from a healthy human donor. PBMCs were seeded in a 96-well assay plate at 2×10 ⁵ cells/well, then various anti-LAG-3 antibodies to be tested were added to the plates and incubated with PBMC at 37°C for 30 minutes. The SEB solution was added to a final concentration of 10 ng/ml. The plates were then incubated for 96 hours. At the end of this incubation, 100 μl of cell culture supernatant was collected and the level of IL-2 production was measured using an IL-2 ELISA kit (R&D Systems; Cat. No. DY202). The results are shown in FIG. 6.

Example 8: Variable Region Sequencing of Mouse Anti-LAG-3 Antibodies

To amplify the variable regions of the heavy and light chains, total RNA was isolated from >5×10 ⁶ cells of selected hybridoma clones using the TRIzol® RNA Isolation Reagent (Invitrogen; cat. no. 15596). cDNA was synthesized with the SuperMix SuperScript™ III primary chain synthesis kit (Invitrogen; cat. no. 18080) and used as a PCR template for the mouse Ig primer kit (Novagen; cat. no. 69831-3). PCR products were analyzed by electrophoresis on 1.2% agarose gel with gel staining with DNA dye SYBR™ Safe (Invitrogen). DNA fragments of the correct size were purified using NucleoSpin® Gel and PCR Clean-up kits (Macherey-Nagel GmbH; Cat. No. 740609) according to the manufacturer's instructions and subcloned separately into pMD18-T cloning vectors. Fifteen colonies were selected from each transformation and the sequences of the insertion fragments were analyzed by DNA sequencing. Sequences were confirmed if at least 8 matches were found with consensus sequences for VH and VL. The variable region sequences of the seven mouse mAbs analyzed by homologous sequence alignment are shown in Table 23. Complementarity determining regions (CDRs) were identified according to Kabat numbering.

Table 23: VH/VL amino acid sequences of 7 mouse anti-LAG-3 antibodies

ID α-LAG-3 mAb domain SEQID NO. Amino acid sequences
1234567890123456789012345678901234567890 mAb746 vh 60 EVQLQQSGAELVRPGASVKLSCTASDFNIKDDYMHWVKQRPEQGLDWIGWIVPENGNTEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTVYGDYWGQGTTLTVSS VL 61 DIQMTQSPSSLSASLGERVSLNCRASQEISGYLSWLQQKSDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPLTFGAGTKLELK mAb747 vh 60 EVQLQQSGAELVRPGASVKLSCTASDFNIKDDYMHWVKQRPEQGLDWIGWIVPENGNTEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTVYGDYWGQGTTLTVSS VL 62 DIQMTQSPSSLSASLGERVSLNCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFAAYYCLQYASYPLTFGAGTKLELK mAb742 vh 63 QGQLQQSGAELVRPGASVTLSCKASGYTFNDYEMHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCIRWGSTVFPYWGQGTLVTVS VL 64 DGVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPWTFGGGTKLEIK mAb744 vh 65 QVQLQQSGAELVRPGTSVTLSCKASGYTFTDYEMHWMKQTPVHGLEWIGAIDPATGGTAYNQKFKGKAILTADKSSSTAYMDFRSLTSEDSAVYYCIRWGTTVFPYWGQGTLVTVS VL 66 DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSNYLPWTFGGGTKLEIK mAb748 vh 67 EVQMQQSGAELVRPGASVKLSCTVSGFNIKDDYMHWVKQRPEQGLEWIGWIDPENGDTEYASKFQGKATITADTSSNTAYLQLNSLTSEDTAVYYCTYFDYWGQGTTLTVSS VL 68 DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGSHFPQTFGGGTKLEIK mAb749 vh 69 EVQLQQSGAELVRPGASVKVSCTASDFNIKDDYVHWVKQRPEQGLEWIGWIDPENGDTEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYFCSTWDAEENYWGQGTTLSVSS VL 70 DIVLTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPVRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK mAb750 vh 71 EVQLQQSGAELVRPGASVKLSCTPSGLNIKDDYIHWVKQRPEQGLEWIGWIDPENGDTEYASKFQGKATITADTSSNTAYLQLSSLTSEDSAVYYCCTADYRNWYWGQGTTLTVSS VL 68 DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGSHFPQTFGGGTKLEIK

Example 9 Humanization of Mouse Anti-LAG-3 Antibody mAb747

mAb747 was selected for humanization based on antigen-binding activity, cross-reactivity with cynomolgus monkey LAG-3 protein, functional activity, and affinity.

Example 9.1 Humanization of mAb747

Anti-LAG-3 mAb747 variable region genes were used to create a humanized antibody. In the first step of this process, the VH and VL amino acid sequences of mAb747 (SEQ ID NO:60 and SEQ ID NO:62) were compared with the available human Ig V gene sequence database to find the overall best matching human Ig germline V gene sequences. . In addition, the 4 VH or VL framework sequence was compared to a database of J regions to find the human framework having the highest homology to the murine VH and VL regions, respectively. The closest match to the human light chain V gene was shown by the A1 gene; and the closest match to the human heavy chain was shown by the VH1-f gene. Humanized variable domain sequences were then constructed in which mAb747 light chain CDR-L1, CDR-L2 and CDR-L3 were grafted onto A1 gene framework sequences with JK4 framework sequence 4 after CDR-L3; and mAb747 heavy chain CDR-H1, CDR-H2 and CDR-H3 were grafted onto VH1-f framework sequences with JH4 framework sequence 4 after CDR-H3. A 3D Fv model of mAb747 was then created to determine if there were any positions in the framework where murine amino acids are critical for maintaining loop structures or the VH/VL interface. Such residues in humanized sequences should be backmutated to mouse residues in the same position to maintain affinity/activity. Several desirable backmutations were shown for the VH and VL of mAb747, and three alternative VH and VL constructs were generated, shown in Table 24 below. (Back-mutated amino acid residues in the framework region are double underlined; mouse CDRs of the original parental antibody are simply underlined.)

Table 24: Design of humanized VH/VL mAb747 - backmutations on mouse residues

Humanized VH/VL ID SEQID NO. Amino acid sequences
1234567890123456789012345678901234567890 mAb747 VH.2A 72 EVQLVQSGAEVKKPGASVKVSCKASDFNIKDDYMHWVRQAPGQGLEWIGWIVPENGNTEYASKFQGRVTITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSS mAb747 VH.2B 73 EVQLVQSGAEVKKPGASVKVSCKASDFNIKDDYMHWVRQAPGQGLEWIGWIVPENGNTEYASKFQGKATITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSS mAb747 VH.1G 74 EVQLVQSGAEVKKPGATVKISCKaSDFNIKDDYMHWVQQAPGKGLEWIGWIVPENGNTEYASKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS mAb747 VK.1E 75 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPGKTIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIK mAb747 VK.2A 76 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEKTIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIK mAb747 VK.2B 77 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIK

Humanized VH and VK genes were obtained synthetically and then cloned into vectors containing the constant domains of the human kappa chain, respectively. Pairing of human VH and human VK resulted in 9 humanized anti-LAG-3 antibodies named HumAb747-34-42 (Table 25). As a positive control for affinity comparison, a (mAb747c) chimeric antibody with parental mouse VH/VL and human constant sequences was also generated.

Table 25: List of generated humanized anti-LAG-3 antibodies mAb747

Antibody ID VH region in the heavy chain VL region in the κ light chain HumAb747-34 mAb747 VH.1G mAb747 VK.1E HumAb747-35 mAb747 VH.1G mAb747 VK.2A HumAb747-36 mAb747 VH.1G mAb747 VK.2B HumAb747-37 mAb747 VH.2A mAb747 VK.1E HumAb747-38 mAb747 VH.2A mAb747 VK.2A HumAb747-39 mAb747 VH.2A mAb747 VK.2B HumAb747-40 mAb747 VH.2B mAb747 VK.1E HumAb747-41 mAb747 VH.2B mAb747 VK.2A HumAb747-42 mAb747 VH.2B mAb747 VK.2B

All 9 humanized antibodies (Table 25) and a chimeric antibody having parental mouse VH and VL domains (mAb747c) were ranked by dissociation rate constant (k _off ). Briefly, antibodies were characterized for affinity and binding kinetics by biolayer interferometry performed on an Octet® RED96 device (Pall FortéBio LLC). Antibodies were captured by Anti-HIgG Fc Capture (AHC) biosensors (Pall) at a concentration of 100 nM for 30 seconds. The sensors were then immersed in running buffer (1X pH 7.2 PBS, 0.05% Tween 20, 0.1% BSA) for 60 seconds to check baseline. Binding was measured by immersing the sensors in one concentration of recombinant human protein LAG-3-his (Novoprotein). Dissociation was carried out by immersing the sensors in the working buffer for 1200 seconds. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using FortéBio Data Analysis software (Pall). The results are shown in table 26.

Table 26: Ranking of Humanized Anti-LAG-3 Antibodies by Dissociation Rate

Antibody Dissociation rate (k _off ) (1/s) mAb747c 3.77×10 ^-4 HumAb747-42 8.30×10 ^-4 HumAb747-39 1.14×10 ^-3

HumAb747-42 had a dissociation rate constant that was only 2.2-fold higher than that of the control chimeric parental variable domain antibody.

Example 10 Preparation of PD-1/LAG-3 polyvalent FIT (Fabs-in-tandem) immunoglobulins (FIT-Ig)

Bispecific Fabs-in-Tandem binding immunoglobulin proteins were designed to recognize both human PD-1 and human LAG-3.

For each of the FIT-Ig constructs described in the tables below, the signal sequence used in the expression vector for each of the three-component polypeptide chains is shown. In the preparation of the FIT-Ig proteins described below, either MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 79) or MEFGLSWLFLVAILKGVQC (SEQ ID NO: 84) was used, although many alternative signal peptides are generally known to those skilled in the art that may also be used. It will be understood that such signal sequences are cleaved during secretion of the polypeptides by the expression host cell, and thus the signal sequences are not part of the final FIT-Ig binding proteins.

Example 10.1: FIT107-1-2a

PD-1/LAG-3 FIT-Ig, designated FIT107-1-2a, was constructed using coding sequences for immunoglobulin domains derived from the parent antibodies mAb709 (mouse anti-PD-1, see Table 4 above) and mAb746 (mouse anti-LAG-3, see table 23 above). FIT-Ig FIT107-1-2a is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: mAb709 VL-CL fused directly to mAb709 VH-CH1 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region (see Table 6 above);

Polypeptide chain #2 has the domain formula: VH-CH1 mAb709; and

Polypeptide chain #3 has the domain formula: mAb709 light chain (VL-CL).

The amino acid sequences of the three expressed FIT107-1-2a polypeptide chains are shown in Table 27 below.

Table 27: Amino acid chain sequences of FIT107-1-2a components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig FIT107-1-2a 78 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSHKFMSTSVGDSVTITCKASQDVNTVVAWYQQKPGQSLKVLISWASTRHTGVPARFTGSGSGTDYTLTISSVQAEDLALYYCQQHYTTPYTFGGGTQLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLQQSGAELVRPGASVKLSCTASDFNIKDDYMHWVKQRPEQGLDWIGWIVPENGNTEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTVYGDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
mouse mAb709
(VL underlined) 80 DIVMTQSHKFMSTSVGDSVTITCKASQDVNTVVAWYQQKPGQSLKVLISWASTRHTGVPARFTGSGSGTDYTLTISSVQAEDLALYYCQQHYTTPYTFGGGTQLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSSADYSFEKHKVY VH-CH1 mAb
mouse mAb746
(VH underlined) 81 EVQLQQSGAELVRPGASVKLSCTASDFNIKDDYMHWVKQRPEQGLDWIGWIVPENGNTEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTVYGDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-2a 83 MEFGLSWLFLVAILKGVQCEVKLVESGGGLVKPGGSLKLSCAASGFTFSFYTMSWVRQTPEKRLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNTLYLHMSSLRSEDTALYYCAGQGGNYLFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
mouse mAb709
(VH underlined) 85 EVKLVESGGGLVKPGGSLKLSCAASGFTFSFYTMSWVRQTPEKRLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNTLYLHMSSLRSEDTALYYCAGQGGNYLFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK Polypeptide chain #3 FIT-Ig FIT107-1-2a 86 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASLGERVSLNCRASQEISGYLSWLQQKSDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
mouse mAb746
(VL underlined) 87 DIQMTQSPSSLSASLGERVSLNCRASQEISGYLSWLQQKSDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLECPVSGNSFACEVQ

Example 10.2: FIT107-1-2b

Another bispecific Fabs-in-Tandem immunoglobulin was constructed that recognizes both human PD-1 and human LAG-3. This PD-1/LAG-3 FIT-Ig is designated FIT107-1-2b. The FIT107-1-2b binding protein was constructed using immunoglobulin domain coding sequences derived from mAb709 and mAb746 parental mouse antibodies, but in this construct the FIT-Ig LAG-3-binding domain was in the N-terminal (outer) position and PD- The 1-binding domain was in an internal position, fused at the C-terminus with the VL-CL domains of the LAG-3-binding region. FIT-Ig FIT107-1-2b is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: VL-CL mAb746 fused directly to VH-CH1 mAb746 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region (see Table 6 above);

Polypeptide chain #2 has the domain formula: VH-CH1 mAb746; and

Polypeptide chain #3 has the domain formula: mAb709 light chain (VL-CL).

The amino acid sequences of the three expressed FIT107-1-2b polypeptide chains are shown in Table 28 below:

Table 28: Amino acid chain sequences of FIT107-1-2b components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig FIT107-1-2b 88 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASLGERVSLNCRASQEISGYLSWLQQKSDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVKLVESGGGLVKPGGSLKLSCAASGFTFSFYTMSWVRQTPEKRLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNTLYLHMSSLRSEDTALYYCAGQGGNYLFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
mouse mAb746
(VL underlined) 89 DIQMTQSPSSLSASLGERVSLNCRASQEISGYLSWLQQKSDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLECPVSGNSFACEVQ VH-CH1
mouse mAb709
(VH underlined) 90 EVKLVESGGGLVKPGGSLKLSCAASGFTFSFYTMSWVRQTPEKRLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNTLYLHMSSLRSEDTALYYCAGQGGNYLFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-2b 91 MEFGLSWLFLVAILKGVQCEVQLQQSGAELVRPGASVKLSCTASDFNIKDDYMHWVKQRPEQGLDWIGWIVPENGNTEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTVYGDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1 murine mAb746
(VH underlined) 92 EVQLQQSGAELVRPGASVKLSCTASDFNIKDDYMHWVKQRPEQGLDWIGWIVPENGNTEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTVYGDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK Polypeptide chain #3 FIT-Ig FIT107-1-2b 93 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSHKFMSTSVGDSVTITCKASQDVNTVVAWYQQKPGQSLKVLISWASTRHTGVPARFTGSGSGTDYTLTISSVQAEDLALYYCQQHYTTPYTFGGGTQLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
mouse mAb709
(VL underlined) 94 DIVMTQSHKFMSTSVGDSVTITCKASQDVNTVVAWYQQKPGQSLKVLISWASTRHTGVPARFTGSGSGTDYTLTISSVQAEDLALYYCQQHYTTPYTFGGGTQLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSSADYSFEKHKVY

Example 10.3: FIT107-1-5a

FIT-Ig PD-1/LAG-3, designated FIT107-1-5a, was constructed using coding sequences for immunoglobulin domains derived from parental humanized antibodies HumAb709-8 (anti-PD-1, SEQ ID NO: 21 and SEQ ID NO : 25) and HumAb747-42 (SEQ ID NO: 73 and SEQ ID NO: 77). FIT-Ig FIT107-1-5a is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb709-8 VL-CL fused directly to HumAb747-42 VH-CH1 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region (see Table 6 above);

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb709-8; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb747-42.

The amino acid sequences of the three FIT107-1-5a expressed polypeptide chains are shown in Table 29 below:

Table 29: Amino acid chain sequences of FIT107-1-5a components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig FIT107-1-5a 95 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVQSGAEVKKPGASVKVSCKASDFNIKDDYMHWVRQAPGQGLEWIGWIVPENGNTEYASKFQGKATITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb709-8
(VL underlined) 96 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VH-CH1 mAb
HumAb747-42
(VH underlined) 97 EVQLVQSGAEVKKPGASVKVSCKASDFNIKDDYMHWVRQAPGQGLEWIGWIVPENGNTEYASKFQGKATITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVNTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVKSC hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig FIT107-1-5a 98 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb709-8
(VH underlined) 99 evqlvesggglvqpggslrlscaasgftfsFYTMSwvrqapgkglewvaTISGGGRDTYYPDSVKGrftisrdnaknslylqmnslraedtavyycaGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYNHSSKNTVICVSLGTQTYKD Polypeptide chain #3 FIT-Ig FIT107-1-5a 100 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb747-42
(VL underlined) 101 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKTHNKSSVY

Example 10.4: FIT107-1-5b

Another bispecific Fabs-in-Tandem immunoglobulin was constructed that recognizes both human PD-1 and human LAG-3. This PD-1/LAG-3 FIT-Ig is designated FIT107-1-5b. The FIT107-1-5b binding protein was created using the coding sequences of immunoglobulin domains derived from the parental humanized antibodies HumAb709-8 and HumAb747-42, but in this FIT-Ig construct, the LAG-3 binding domain is in the N-terminal (outer) position , and the PD-1 binding domain is internally fused at the C-terminus to the VL-CL domains of the N-terminal LAG-3 binding site. FIT-Ig FIT107-1-5b is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb747-42 VL-CL fused directly to HumAb709-8 VH-CH1 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region (see Table 6 above);

Polypeptide chain #2 has the domain formula: VH-CH1 mAb747-42; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) of HumAb709-8.

The amino acid sequences of the three expressed FIT107-1-5b polypeptide chains are shown in Table 30 below:

Table 30: Amino acid chain sequences of FIT107-1-5b components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig FIT107-1-5b 102 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECevqlvesggglvqpggslrlscaasgftfsFYTMSwvrqapgkglewvaTISGGGRDTYYPDSVKGrftisrdnaknslylqmnslraedtavyycaGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb747-42
(VL underlined) 103 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKTHNKSSVY VH-CH1
HumAb709-8
(VH underlined) 104 evqlvesggglvqpggslrlscaasgftfsFYTMSwvrqapgkglewvaTISGGGRDTYYPDSVKGrftisrdnaknslylqmnslraedtavyycaGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYNHSSKNTVICVSLGTQTYKD hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig FIT107-1-5b 105 MEFGLSWLFLVAILKGVQCEVQLVQSGAEVKKPGASVKVSCKASDFNIKDDYMHWVRQAPGQGLEWIGWIVPENGNTEYASKFQGKATITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb747-42
(VH underlined) 106 EVQLVQSGAEVKKPGASVKVSCKASDFNIKDDYMHWVRQAPGQGLEWIGWIVPENGNTEYASKFQGKATITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVNTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVKSC Polypeptide chain #3 FIT-Ig FIT107-1-5b 107 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL of mAb
HuMAb709-8
(VL underlined) 108 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

Example 10.5: Expression and purification of FIT-Ig

The four PD-1/LAG-3 FIT-Ig constructs, FIT107-1-2a, FIT107-1-2b, FIT107-1-5a, and FIT107-1-5b, are a type of bispecific polyvalent binding protein known as Fabs-In -Tandem immunoglobulin (or FIT-Ig) as described in general in WO 2015/103072 and WO 2017/136820. Binding proteins were obtained by co-expression of three-component polypeptide chains in a mammalian host cell transfected with expression vectors for all three chains. The design of the binding protein requires that the long polypeptide chain (Chain #1) be paired with both short polypeptide chains (Chain #2 and #3) to form functional tandem Fab fragments, and the long chain is intended to dimerize through the Fc region. (hinge-CH2-CH3), so that a six-strand binding protein is formed, showing four intact Fab-binding sites. In the FIT107-1-2a and FIT107-1-5a binding proteins, N-terminal or "outer" Fab binding sites bind to PD-1, and adjacent "inner" Fab binding sites bind to LAG-3. The outer Fab fragment (anti-PD-1) of FIT107-1-2a and FIT107-1-5a is attached to the inner Fab fragment (anti-LAG-3) only through a long chain (Chain #1) by a direct fusion of VL-CLmAb709 or VL-CLHumAb709-8, since in this case it can be located at its C-terminus with the N-terminus of VH-CH1mAb746 or VH-CH1HumAb747-42, respectively, without the use of linkers connecting immunoglobulin domains. Similarly, the outer Fab fragment (anti-LAG-3) of FIT107-1-2b and FIT107-1-5b is attached to the inner Fab fragment (anti-PD-1) only through a long chain (Chain #1) by a direct VL fusion. -CLmAb746 or VL-CLmAb747-42, since in this case it can be located at its C-terminus with the N-terminus of VH-CH1mAb709 or VH-CH1mAb709-8, respectively, without the use of linkers connecting immunoglobulin domains.

Expression vectors encoding polypeptide chains #1, #2 and #3 of each FIT-Ig (FIT107-1-2a, FIT107-1-2b, FIT107-1-5a and FIT107-1-5b) were transiently co-expressed, using polyethyleneimine (PEI) as a transfection agent in human embryonic kidney 293E cells. Briefly, DNA in FreeStyle™ 293 Expression Medium was mixed with PEI at a 1:2 final concentration of DNA to PEI, incubated for 15-20 minutes at room temperature, and then added to HEK293E cells (1.0-1.2× 10 ⁶ /ml, cell viability >95%) at 60 μg DNA/120 ml culture. After 6-24 hours of cultivation in a shaker, peptone was added to the transfected cells to a final concentration of 5% with shaking at 125 rpm at 37°C and ±8% CO ₂ . On days 6-7, the supernatant was collected by centrifugation and filtration, and the FIT-Ig protein was purified by Protein A chromatography (Pierce, Rockford, IL) according to the manufacturer's instructions.

For expression, FIT107-1-2a, FIT107-1-2b, FIT107-1-5a and FIT107-1-5b DNA encoding for the expression of strands #1, #2 and #3 was transfected using the molar ratio Strand #1: Strand #2: Chain #3, equal to 1:3:3. This was designed to proportionally increase the expression of short chain #2 and #3 compared to the long chain (Chain #1), which in turn would reduce the appearance of VL-CL and VH-CH1 segments in the long chain (Chain #1) , which were not paired with the corresponding light chains and thus would not be able to form a functional Fab fragment. FIT-Ig protein expression products were purified with gjvjom. protein A chromatography. The composition and purity of the purified FIT-Ig were analyzed by size exclusion chromatography (SEC). Purified FIT-Ig in PBS was applied to a TSKgel SuperSW3000, 300×4.6 mm (TOSOH) column. A model U3000 HPLC instrument (DIONEX) was used for SEC using UV detection at 280 nm and 214 nm. See table 31 below.

Table 31: Expression and SEC analysis of PD-1/LAG-3 binding FIT-Ig proteins

FIT-Ig protein Molar ratio of DNA:strand #1:#2:#3 Expression level (mg/l) % peak monomer fraction according to SEC FIT107-1-2a 1:3:3 5.40 >90% FIT107-1-2b 1:3:3 5.04 >80% FIT107-1-5a 1:3:3 7.95 >80% FIT107-1-5b 1:3:3 8.52 >80%

The lower content of the monomer fraction for FIT107-1-2b, -5a and -5b indicates some possible aggregation.

Example 11 FIT-Ig Binding Affinity for Target Antigens

The binding kinetics of FIT-Ig to PD-1 and LAG-3 targets was determined using SPR Biacore measurements. The binding affinities of FIT107-1-2a and FIT107-1-2b for both PDL-1 and LAG-3 target antigens are shown in Table 32 below.

Table 32: Binding affinity of FIT107-1-2a and FIT107-1-2b

Target immobilized on a sensor chip analyte Analyte specificity k _on (1/ms) k _off (1/s) K _D (M) HuPD-1 ECD/Fc and
HuLAG-3ECD/Fc FIT107-1-2a external: PD-1
internal: LAG-3 6.97×10 ⁴ 1.17×10 ^-5 1.68×10 ^-10 FIT107-1-2b external: LAG-3
internal: PD-1 1.68×10 ⁵ 8.76×10 ^-5 5.23×10 ^-10 mAb709c PD-1 2.88×10 ⁵ 9.72×10 ^-6 3.37×10 ^-11 mAb746c LAG-3 9.51×10 ³ 1.54×10 ^-4 1.62×10 ^-8 mAb709c+mAb746c PD-1 and LAG-3 2.82×10 ⁵ 2.08×10 ^-5 7.35×10 ^-11 HuPD-1ECD/Fc mAb709c PD-1 2.52×10 ⁵ 8.82×10 ^-6 3.50×10 ^-11 HuLAG-3ECD/Fc mAb746c LAG-3 5.58×10 ³ 1.81×10 ^-4 3.25×10 ^-8

Example 12: Determination of specificity and function of FIT-Ig

Anti-PD1 and anti-LAG-3 bispecificity and biological activity of FIT107-1-2a binding protein were tested in a PBMC activation assay using staphylococcal enterotoxin B (SEB) as superantigen (see Example 7.8). Briefly, PBMC were isolated from a healthy donor, then seeded into a 96-well plate at 50 μl/well at a density of 2×10 ⁵ cells/well. Binding proteins to be tested (e.g., FIT107-1-2a, a combination of commercially available anti-PD-1 and anti-LAG-3 monoclonal antibodies, or an anti-PD-1 monoclonal antibody) were added to plates and incubated with PBMC at 37° C. within 30 min. The SEB solution was added to a final concentration of 10 ng/ml. Plates were incubated for 96 hours, then 100 μl of cell culture supernatant was collected and IL-2 production was measured using an IL-2 ELISA kit (R&D Systems; Cat. No. DY202). The results are shown in FIG. 7. The FIT-Ig bispecific protein FIT107-1-2a is able to enhance T cell activation as indicated by the level of IL-2 production compared to anti-PD-1 antibody alone or a mixture of anti-LAG-3 and anti-PD- 1 antibody.

In addition, a mixed lymphocyte reaction (MLR) assay was performed in the same manner as described in Example 3 to further test the anti-PD-1 and anti-LAG-3 function of FIT107-1-2a. The mixed lymphocyte culture response is an ex vivo cellular immune response that occurs between two populations of allogeneic lymphocytes when mixed with each other. Allogeneic lymphocytes undergo blast transformation, DNA synthesis, and cell proliferation in response to differences in major histocompatibility antigens (MHC Class I and II) between two cell populations, which are designated responder and stimulator cells. At MLR testing for FIT107-1-2a on day 1, PBMCs from healthy donors were purified and CD14+ monocytes isolated. Monocytes were seeded in 6-well plates and treated with 35 ng/ml IL-4 (R&D Systems) and 50 ng/ml GM-CSF (R&D Systems) in RPMI 1640 plus 10% FBS. The medium was changed after 4 days. On the seventh day, monocytes differentiated into immature dendritic cells were harvested and further treated for two days in maturation medium with 20 ng/ml TNF-α (R&D Systems), 50 μg/ml Poly I:C (Sigma), 35 n /ml IL-4 and 50 ng/ml GM-CSF. Serum-free X-VIVO™ 15 medium was used for MLR assays in co-culture to avoid serum interference with antibody performance. 96-well U-bottom plates were seeded with allogeneic CD4+ T cells (responder cells) at 1 x 10 ⁵ cells/well and pre-treated with the binding protein to be tested (i.e., FIT107-1-2a, a combination of commercially available monoclonal anti-PD-1 and anti-LAG-3 antibodies, or monoclonal anti-PD-1 antibodies) for 30 min. Then mature dendritic cells (stimulator cells) were seeded in wells at a density of 1×10 ⁴ cells/well and co-cultured with responder cells for five days, during which time 100 μl of supernatant was collected and the level of IFN-γ production was measured by ELISA . The results are shown in FIG. 8. The FIT-Ig bispecific binding protein showed an EC50 of 0.5084 nM compared to an EC50 of 16.84 nM for a combination of anti-PD-1 and anti-LAG-3 antibodies or anti-PD-1 antibody alone. . Thus, the FIT-Ig binding protein enhanced the production of IFN-γ (interferon gamma) in MLR at a concentration that is more than 30 times lower than the concentration of a single antibody or a combination of antibodies.

Example 13: Humanization of mAb747 from a new batch

Anti-LAG-3 mAb747 variable region genes were used to generate a humanized antibody. In the first step of this process, the VH and VL amino acid sequences of mAb747 (SEQ ID NO:60 and SEQ ID NO:62) were compared with the available human Ig V gene sequence database to find the overall best matching human Ig germline V gene sequences. . In addition, the VH or VL framework 4 (FW4) sequence was compared to a database of J regions to find the human framework having the highest homology to the murine VH and VL regions, respectively. The closest match to the human light chain V gene was shown by the A30 gene; and the closest match to the human heavy chain was shown by the VH1-69-2 gene. Humanized variable domain sequences were then constructed in which mAb747 light chain CDR-L1, CDR-L2 and CDR-L3 were grafted onto the A30 gene framework sequences with JK4 framework sequence 4 after CDR-L3; and mAb747 heavy chain CDR-H1, CDR-H2 and CDR-H3 were grafted onto VH1-69-2 framework sequences with JH6 framework sequence 4 after CDR-H3. A 3D Fv model of mAb747 was then created to determine if there were any scaffold positions where murine amino acids are critical for maintaining loop structures or the VH/VL interface. Such residues in humanized sequences should be backmutated to mouse residues in the same position to maintain affinity/activity. Several desirable backmutations for VH and VL mAb747 were identified, and three alternative VH and VL constructs were generated, shown in Table 33 below. (Back-mutated framework amino acid residues are double underlined; CDRs are underlined according to the Kabat numbering system, except for VH CDR1, defined using the ABM numbering system.)

Table 33: Design of humanized VH/VL for mAb747 - backmutations on mouse residues

Humanized VH/VL ID SEQID NO. Amino acid sequences
1234567890123456789012345678901234567890 huEpi001-VHv1 109 EVQLVQSGAEVKKPGATVKISCKVSDFNIKDDYMHWVQQAPGKGLEWIGWIVPENGNTEYASKFQGRVTITADTSTDTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001-VHv2 110 EVQLVQSGAEVKKPGATVKLSCKASDFNIKDDYMHWVQQAPGKGLEWIGWIVPENGNTEYASKFQGRVTITADTSTDTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001-VHv3 111 EVQLVQSGAEVKKPGATVKLSCTASDFNIKDDYMHWVQQAPGKGLEWIGWIVPENGNTEYASKFQGRVTITADTSTDTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001-VHv4 112 EVQLVQSGAEVKKPGATVKLSCTASDFNIKDDYMHWVKQAPGKGLEWIGWIVPENGNTEYASKFQGRATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001-VHv5 113 EVQLVQSGAEVKKPGATVKLSCTASDFNIKDDYMHWVKQRPEQGLEWIGWIVPENGNTEYASKFQGRATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001-VHv6 114 EVQLVQSGAEVKKPGATVKLSCTASDFNIKDDYMHWVKQRPEQGLDWIGWIVPENGNTEYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001 VLv1 115 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGKAIKSLIYAASTLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQYASYPLTFGQGTKLEIK huEpi001 VLv2 116 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGKAIKRLIYAASTLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQYASYPLTFGQGTKLEIK huEpi001 VLv3 117 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELK huEpi001 VLv4 118 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLEPEDFADYYCLQYASYPLTFGQGTKLELK

Humanized VH and VK genes were obtained synthetically and then cloned into vectors containing the constant domains of the human kappa chain, respectively. Pairing of human VH and human VK resulted in 9 humanized anti-LAG-3 antibodies, named HumAb747-43 - HumAb747-60 (Table 60). For affinity comparison, a chimeric antibody with parental mouse VH/VL and human constant sequences described above (mAb747c) was also used as a positive control.

Table 34: List of generated humanized anti-LAG-3 mAb747 antibodies

Antibody ID VH region in the heavy chain VL region in the κ light chain HumAb747-43 huEpi001-VHv1 huEpi001 VLv1 HumAb747-44 huEpi001-VHv2 huEpi001 VLv1 HumAb747-45 huEpi001-VHv3 huEpi001 VLv1 HumAb747-46 huEpi001-VHv4 huEpi001 VLv1 HumAb747-47 huEpi001-VHv1 huEpi001 VLv2 HumAb747-48 huEpi001-VHv2 huEpi001 VLv2 HumAb747-49 huEpi001-VHv3 huEpi001 VLv2 HumAb747-50 huEpi001-VHv4 huEpi001 VLv2 HumAb747-51 huEpi001-VHv1 huEpi001 VLv3 HumAb747-52 huEpi001-VHv2 huEpi001 VLv3 HumAb747-53 huEpi001-VHv3 huEpi001 VLv3 HumAb747-54 huEpi001-VHv4 huEpi001 VLv3 HumAb747-55 huEpi001-VHv1 huEpi001 VLv4 HumAb747-56 huEpi001-VHv2 huEpi001 VLv4 HumAb747-57 huEpi001-VHv3 huEpi001 VLv4 HumAb747-58 huEpi001-VHv4 huEpi001 VLv4 HumAb747-59 huEpi001-VHv5 huEpi001 VLv3 HumAb747-60 huEpi001-VHv6 huEpi001 VLv3

All 18 humanized antibodies (Table 34) and a chimeric antibody having parental mouse VH and VL domains (mAb747c) were ranked by dissociation rate constant (k _off ). Briefly, antibodies were characterized by affinity and binding kinetics by biolayer interferometry performed on an Octet® RED96 device (Pall FortéBio LLC). Antibodies were captured with Anti-HIgG Fc Capture (AHC) (Pall) biosensors at a concentration of 100 nM for 30 seconds. The sensors were then immersed in running buffer (1X pH 7.2 PBS, 0.05% Tween 20, 0.1% BSA) for 60 seconds to check baseline. Binding was measured by immersing the sensors in one concentration of recombinant human protein LAG-3-his (Novoprotein). Dissociation was obtained by immersing the sensors in the working buffer for 1200 seconds. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using FortéBio Data Analysis software (Pall). The results are shown in Table 35. In each test group, antibody dissociation rates could be compared with mAb747c dissociation rates. Dissociation rate ratios were calculated by the ratio of antibody dissociation rate to mAb747c dissociation rate per group, and all values were compared. The lower the ratio, the higher the affinity of the antibody.

Table 35: Ranking of Humanized Anti-LAG-3 Antibodies by Dissociation Rate

test group antibody Dissociation rate (k _off )
(1/s) Ratio of dissociation rate to dissociation rate of mAb747c one HumAb747-43 9.25×10 ^-3 682% HumAb747-44 1.10×10 ^-2 809% HumAb747-45 1.08×10 ^-2 797% HumAb747-46 1.09×10 ^-2 801% HumAb747-47 1.44×10 ^-2 1062% HumAb747-48 8.03×10 ^-3 592% mAb747c 1.36×10 ^-3 100% 2 HumAb747-49 7.73×10 ^-3 575% HumAb747-50 7.19×10 ^-3 534% HumAb747-51 1.19×10 ^-2 888% HumAb747-52 4.36×10 ^-3 324% HumAb747-53 4.30×10 ^-3 319% HumAb747-54 4.23×10 ^-3 314% mAb747c 1.35×10 ^-3 100% 3 HumAb747-55 1.19×10 ^-2 972% HumAb747-56 4.31×10 ^-3 352% HumAb747-57 4.11×10 ^-3 335% HumAb747-58 4.05×10 ^-3 331% mAb747c 1.23×10 ^-3 100% four HumAb747-59 1.20×10 ^-3 226% HumAb747-60 8.10×10 ^-4 153% mAb747c 5.30×10 ^-4 100%

HumAb747-60 showed a dissociation rate constant that exceeded the dissociation rate of the parental variable domain control chimeric antibody by only 1.5 times.

To further test the function of anti-LAG-3 antibodies in human PBMCs, a bacterial toxin stimulation assay was performed using superantigenic Staphylococcus aureus enterotoxin B (SEB). IL-2 production was measured using a PerkinElmer IL-2 assay kit (PerkinElmer; cat. no. TRF1221M). HumAb747-60 was able to enhance IL-2 secretion of SEB stimulated PBMCs by blocking the LAG-3 signaling pathway. The results are shown in FIG. 9. Thus, HumAb747-60 proved to be functional and was selected for further development.

Example 14 Affinity Maturation of HumAb747-60

Example 14.1: Creation and screening of an affinity maturation library

Although HumAb747-60 showed a dissociation rate constant that was only 1.5-fold higher than the chimeric control mAb747c, the PBMC-SEB assay showed that HumAb747-60 had slightly lower functional activity. To further improve affinity, CDR residues (according to the ABM numbering system) were optimized by affinity maturation based on HumAb747-39 (see tables 24, 25). Two phage libraries were designed and created. One was designed to generate mutated CDR-L1, CDR-L3 and CDR-H3 (ABM numbering), each with one randomly mutated residue. Another was designed to produce mutated CDR-L2, CDR-H1 and CDR-H2 (ABM numbering), each with one randomly mutated residue.

Phage display libraries were generated using the method described in Journal of Immunological Methods, 201:35-55 (1997). Briefly, VH-CH1 and VL-CL were amplified with degenerate primers that introduced mutations and then cloned sequentially into two multiple cloning sites (MCS) of the phagemid vector. The phagemid vectors were then electrotransformed into TG1 (Cat. No. 60502-1, Lucigen) resulting in libraries of approximately 1.2×10 ⁸ clones, respectively, showing high sequence diversity. Libraries were rescued with helper phage M13K07 (Cat. No. N0315S) at a ratio of approximately 1:20 (cell per phage). Phage display library selection was performed using recombinant human LAG-3 protein followed by washing steps. The HumAb747-39 Fab fragment was also constructed with the phagemid vector and used as a positive control. Selected phages were used to infect host cells. The binding capacity of Fab supernatants from individual clones was screened by ELISA. Briefly, Fab supernatants of individual clones were obtained by culturing overnight at 30° C. with 1 mM IPTG. 2 μg/ml of human LAG-3 protein in 100 μl of phosphate buffered saline (PBS) was applied directly to each well of 96-well plates. HRP-linked anti-c-myc antibody (Cat. No. Ab1261, Abcam) and TMB reagent were used to detect and develop an ELISA signal that was read using a plate reader (SpectraMax® Plus 384 Plate Reader, Molecular Devices) at 450 nm .

Positive clones in the screening ELISA were selected for sequencing. Given the sequence redundancy and signal strength in the screening ELISA, the following 7 clones were selected for further evaluation. The VH/VL sequences are shown in Table 36. (Mutated amino acid residues identified by affinity maturation are double underlined; CDRs are underlined according to the Kabat numbering system).

Table 36: VH/VL amino acid sequences of 7 mutated affinity matured antibodies

Affinity matured clones domain SEQID NO. Protein sequences
1234567890123456789012345678901234567890 B2-53 vh 119 EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYMHWVRQAPGQGLEWIGWIVPENGNTVYASKFQGRVTITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSS VL 120 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIK B3-21 vh 121 EVQLVQSGAEVKKPGASVKVSCKASDFNIKDDYMHWVRQAPGQGLEWIGWIVPENGNTEYASKFQGRVTITADTSINTAYMELSRLRSDDTAVYYCTVYGDVWGQGTTVTVSS VL 122 DIQMTQSPSSLSASVGDRVTINCRAMQEISGYLSWLQQKPEGTIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYAYYPLTFGGGTKVEIK B3-43 vh 123 EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYMHWVCQAPGQGLEWIGWIVPENGNTEYASKFQGRVTITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSS VL 124 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYAASHLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIK B3-46 vh 125 EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYMHWVRQAPGQGLEWIGWIVPENGLTEYASKFQGRVTITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSS VL 126 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYATSTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIK B3-48 vh 127 EVQLVQSGAEVKKPGASVKVSCKASDFSIKDDYMHWVRQAPGQGLEWIGWIVPENGKTEYASKFQGRVTITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSS VL 128 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYAAMTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIK B3-69 vh 129 EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYMHWVRQAPGQGLEWIGWIVPENGNTHYASKFQGRVTITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSS VL 130 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYEASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIK D1-70 vh 131 EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYMHWVRQAPGQGLEWIGWIVPRNGNTMYASKFQGRVTITADTSINTAYMELSRLRSDDTAVYYCTVYGDYWGQGTTVTVSS VL 132 DIQMTQSPSSLSASVGDRVTINCRASQEISGYLSWLQQKPEGTIKRLIYAASTLDLGVPSRFSGSRSGSDYTLTISSLQPEDFATYYCLQYASYPLTFGGGTKVEIK

Example 14.2: IgG Conversion and Characterization of Positive Clones

Seven Fab clones were converted to complete IgG proteins. Briefly, the VH and VL genes were generated synthetically and then cloned into vectors containing coding sequences for human IgG1 and human kappa chain constant domains, respectively. The heavy chain and cognate light chain plasmids were co-transfected into HEK 293E cells separately. Approximately six days of cell culture after transfection, supernatants were collected and subjected to protein A affinity chromatography. Purified antibodies were ranked by affinity values using biolayer interferometry on an Octet® RED96 device (see example 9.1 above). The results are shown in table 37.

Table 37: Ranking of dissociation rate of anti-LAG-3 antibodies after affinity maturation

test group Full size antibody Dissociation rate (k _off ) (1/s) Ratio of dissociation rate to dissociation rate of mAb747c one B3-21-IgG 1.98×10 ^-3 132% B3-43-IgG 5.88×10 ^-3 392% B3-46-IgG 2.48×10 ^-3 165% B3-48-IgG 2.85×10 ^-3 190% B3-69-IgG 3.59×10 ^-3 239% B2-53-IgG 1.80×10 ^-3 120% mAb747c 1.50×10 ^-3 100% 2 D1-70-IgG 2.60×10 ^-3 160% HumAb747-42 4.28×10 ^-3 263% HumAb747-39 3.91×10 ^-3 240% mAb747c 1.63×10 ^-3 100%

D1-70-IgG and B2-53-IgG showed a minimally increased dissociation rate constant compared to that of HumAb747-39, reflecting the largest increase in affinity after mutations. Therefore, D1-70-IgG and B2-53-IgG containing mutations introduced in the HumAb747-60 sequence were the best candidates after humanization.

Example 14.3: Generation and characterization of antibodies engineered further

Mutations in D1-70 identified during affinity maturation were D26G, E53R and E58M in the VH domain and S56L in the VL domain (residue position determined by the Kabat numbering system). Mutations in B2-53 identified during affinity maturation were D26G and E58V in the VH domain and T53A in the VL domain (residue position determined by the Kabat numbering system). These mutations were included in the VH/VL sequences of HumAb747-60, alone or in combination.

CDR-H2 of HumAb747-60 had an NG pattern that could lead to heterogeneity during antibody generation from deamination reactions, so NG to NA mutation was also evaluated. According to the calculations, the G55A mutation in the VH domain would not lead to disruption of HumAb747-60 activity if the NG pattern was disrupted. Amino acid sequences for antibody variants, including the mutations discussed above, are shown in Table 38. (CDRs are defined according to Kabat numbering.)

Table 38: Developed VH/VL design for HumAb747-60

Designed VH/VL ID SEQID NO. Amino acid sequences
1234567890123456789012345678901234567890 huEpi001-VHv6(G55A) 133 EVQLVQSGAEVKKPGATVKLSCTASDFNIKDDYMHWVKQRPEQGLDWIGWIVPENANTEYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001-VHv6.1 134 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNGNTMYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001-VHv6.2 135 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPENGNTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001-VHv6.3 136 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNGNTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSS huEpi001-VLv3.4 137 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELK huEpi001-VLv3.5 138 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELK huEpi001-VLv3.6 139 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELK

The engineered VH and VK genes were obtained synthetically and then respectively cloned into vectors containing the constant domains of human IgG1 and human kappa chain. Pairing of human VH and human VK resulted in 13 engineered anti-LAG-3 antibodies, named HumAb747V-61 - HumAb747V-73 (Table 25). A chimeric antibody with parental mouse VH/VL and human constant sequences (mAb747c) was also generated as a positive control for affinity comparison.

Table 39: List of engineered anti-LAG-3 antibodies obtained

Antibody identifier VH region in the heavy chain VL region in the κ light chain HumAb747V-61 huEpi001-VHv6(G55A) huEpi001 VLv3 HumAb747V-62 huEpi001-VHv6.1 huEpi001VLv3.4 HumAb747V-63 huEpi001-VHv6.1 huEpi001 VLv3.5 HumAb747V-64 huEpi001-VHv6.1 huEpi001 VLv3.6 HumAb747V-65 huEpi001-VHv6.1 huEpi001 VLv3 HumAb747V-66 huEpi001-VHv6.2 huEpi001VLv3.4 HumAb747V-67 huEpi001-VHv6.2 huEpi001 VLv3.5 HumAb747V-68 huEpi001-VHv6.2 huEpi001 VLv3.6 HumAb747V-69 huEpi001-VHv6.2 huEpi001 VLv3 HumAb747V-70 huEpi001-VHv6.3 huEpi001VLv3.4 HumAb747V-71 huEpi001-VHv6.3 huEpi001 VLv3.5 HumAb747V-72 huEpi001-VHv6.3 huEpi001 VLv3.6 HumAb747V-73 huEpi001-VHv6.3 huEpi001 VLv3

All 13 humanized antibodies (Table 39) and the chimeric anti-LAG-3 antibody mAb747c having parental mouse VH and VL domains were ranked by dissociation rate constant (k _off ) in the same manner as described in Example 9.1 above. The results are shown in Table 40. In each test group, antibody dissociation rates could be compared with mAb747c dissociation rates. Dissociation rate ratios were calculated by ratio of antibody dissociation rate to mAb747c dissociation rate per group, and all values were compared. The lower the ratio, the higher the affinity of the antibody.

Table 40: Ranking of anti-LAG-3 antibodies with further development

test group Full size antibody Dissociation rate (k _off ) (1/s) Ratio of dissociation rate to dissociation rate of mAb747c one HumAb747V-61 6.86 × 10 ^-4 112% HumAb747V-62 4.80× ^10-4 78% HumAb747V-63 5.22 × 10 ^-4 85% HumAb747V-64 4.50 × ^10-4 73% HumAb747V-65 2.92 × 10 ^-4 47% HumAb747V-66 5.84 × 10 ^-4 95% mAb747c 6.15 × 10 ^-4 100% 2 HumAb747V-67 3.19 × 10 ^-4 47% HumAb747V-68 2.95 × 10 ^-4 44% HumAb747V-69 3.57 × 10 ^-4 53% HumAb747V-70 2.73 × 10 ^-4 40% HumAb747V-71 2.92 × 10 ^-4 43% HumAb747V-72 2.47 × 10 ^-4 37% mAb747c 6.76 × 10 ^-4 100% 3 HumAb747V-73 3.21 × 10 ^-4 51% mAb747c 6.31 × 10 ^-4 100%

Most of the HumAb747-60 based antibodies containing mutations in the CDRs adopted after affinity maturation showed an improved dissociation rate compared to that of mAb747c, predicting an improvement in affinity. HumAb747V-61 showed a similar dissociation rate to mAb747, indicating that the VH G55A amino acid substitution does not impair affinity. Antibodies with ratios of dissociation rates below 60% relative to the dissociation rate of mAb747c within the test group were further evaluated using cell functional analysis.

Anti-LAG-3 activity was tested in a PBMC activation assay using staphylococcal enterotoxin B (SEB) as superantigen. Briefly, PBMC were seeded in a 96-well plate at 2×10 ⁵ cells/well. Test proteins (monoclonal anti-LAG-3 antibodies) were added to the plates and incubated with PBMC at 37°C for 30 min. The SEB solution was added at a final concentration of 10 ng/mL. Plates were incubated for 96 hours, then 100 μl of cell culture supernatant was collected and IL-2 production was measured using a PerkinElmer IL-2 Detection Kit (PerkinElmer; cat. no. TRF1221M). The results are shown in FIG. 10. The results show that engineered antibody variants can enhance IL-2 production from SEB stimulated PBMCs by blocking LAG-3 mediated signaling.

Based on the PBMC-SEB assay, HumAb747V-67, HumAb747V-72, and HumAb747V-73 showed superior LAG-3 blocking activity; therefore, these three antibodies were used to generate FIT-Ig binding proteins targeting LAG-3 as well as PD-1.

Example 15 Generation of PD-1/LAG-3 FIT-Ig Using New Anti-LAG-3 Antibody Sequences

The anti-LAG-3 antibodies HumAb747V-67, HumAb747V-72 and HumAb747V-73 prepared as described above and two anti-PD-1 antibodies, HumAb709-8 (see Tables 5 and 7 above) and HumAb713-7 ( see Tables 9 and 10 above) were used to generate FIT-Ig binding proteins following the procedures described in Example 10 above. The G55A mutation was included in the sequence structure of all VH domains of the ani-LAG-3 Fab fragments.

Example 15.1: Construction of PD-1/LAG-3 FIT-Ig Binding Protein FIT107-1-6a-1

PD-1/LAG-3 FIT-Ig, designated FIT107-1-6a-1, was constructed using coding sequences for immunoglobulin domains from mAb709-8 parent antibodies (humanized anti-PD-1, see tables 5 and 6 above) and HumAb747V-67 (humanized anti-LAG-3, see tables 38 and 39 above). FIT-Ig FIT107-1-6a-1 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb709-8 VL-CL fused directly to HumAb747V-67 VH-CH1 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region (see Table 6 above);

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb709-8; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb747V-676.

The amino acid sequences of the three FIT107-1-6a-1 expressed polypeptide chains are shown in Table 41 below.

Table 41: Amino acid chain sequences of FIT107-1-6a components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig FIT107-1-6a-1 140 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPENANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb709-8
(VL underlined) 141 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VH-CH1 mAb
HumAb747V-67
(VH underlined) 142 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPENANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKVSCK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig FIT107-1-6a-1 143 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYY
PDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb709-8
(VH underlined) 144 EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGPSVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKKNVSC Polypeptide chain #3 FIT-Ig FIT107-1-6a-1 145 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb747V-67
(VL underlined) 146 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKTHNKSSVY

Example 15.2: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-6b-1

Another bispecific Fabs-in-Tandem immunoglobulin was constructed that recognizes both human PD-1 and human LAG-3. This PD-1/LAG-3 FIT-Ig is designated FIT107-1-6b-1. The FIT107-1-6b-1 binding protein was constructed using the immunoglobulin domain coding sequences from the parent antibodies HumAb747V-67 (anti-LAG-3) and HumAb709-8 (anti-PD-1). This FIT-Ig construct had a LAG-3 binding domain at the N-terminal (outer) position and a PD-1 binding domain at the inner position fused at the C-terminus to the VL-CL domains of the LAG-3 binding region. FIT-Ig FIT107-1-6b-1 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb747-67 VL-CL fused directly to HumAb709-8 VH-CH1 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region (see Table 6 above);

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb747V-67; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) of HumAb709-8.

The amino acid sequences of the three expressed FIT107-1-6b-1 polypeptide chains are shown in Table 42 below:

Table 42: Amino acid chain sequences of FIT107-1-6b-1 components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-6b-1 147 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb747V-67 (VL underlined) 148 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKTHNKSSVY VH-CH1 mAb
HumAb709-8
(VH underlined) 149 EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGPSVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKKNVSC hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-6b-1 150 MEFGLSWLFLVAILKGVQCEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPENANTVY
ASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb747V-67
(VH underlined) 151 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPENANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKVSCK Polypeptide chain #3 FIT-Ig
FIT107-1-6b-1 152 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb709-8
(VL underlined) 153 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

Example 15.3: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-6a-2

PD-1/LAG-3 FIT-Ig, designated FIT107-1-6a-2, was constructed using the immunoglobulin domain coding sequences from the parent antibodies mAb709-8 (humanized anti-PD-1) and HumAb747V-72 (humanized anti-LAG -3). FIT-Ig FIT107-1-6a-2 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: VL-CL HumAb709-8 fused directly to VH-CH1 HumAb747V-72 fused directly to the hinge-CH2-CH3 mutant human IgG1 constant region (see Table 6 above);

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb709-8; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb747V-72.

The amino acid sequences of the three FIT107-1-6a-2 expressed polypeptide chains are shown in Table 43 below.

Table 43: Amino acid chain sequences of FIT107-1-6a components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-6a-2 154 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb709-8
(VL underlined) 155 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VH-CH1 mAb
HumAb747V-72
(VH underlined) 156 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-6a-2 157 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYY
PDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb709-8
(VH underlined) 158 EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGPSVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKKNVSC Polypeptide chain #3 FIT-Ig
FIT107-1-6a-2 159 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb747V-72 (VL underlined) 160 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

Example 15.4: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-6b-2

PD-1/LAG-3 FIT-Ig, designated FIT107-1-6b-2, was constructed using the immunoglobulin domain coding sequences from the parent antibodies HumAb709-8 (humanized anti-PD-1) and HumAb747V-72 (humanized anti-LAG -3). FIT-Ig FIT107-1-6b-2 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb747V-72 VL-CL fused directly to HumAb709-8 VH-CH1 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region;

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb747V-72; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) of HumAb709-8.

The amino acid sequences of the three FIT107-1-6b-2 expressed polypeptide chains are shown in Table 44 below.

Table 44: Amino acid chain sequences of FIT107-1-6b-2 components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-6b-2 161 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb747V-72 (VL underlined) 162 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VH-CH1 mAb
HumAb709-8 (VH underlined) 163 EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGPSVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKKNVSC hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-6b-2 164 MEFGLSWLFLVAILKGVQCEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVY
ASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb747V-72
(VH underlined) 165 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK Polypeptide chain #3 FIT-Ig
FIT107-1-6b-2 166 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb709-8
(VL underlined) 167 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

Example 15.5: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-6a-3

PD-1/LAG-3 FIT-Ig, designated FIT107-1-6a-3, was constructed using the immunoglobulin domain coding sequences from the parent antibodies mAb709-8 (humanized anti-PD-1) and HumAb747V-73 (humanized anti-LAG -3). FIT-Ig FIT107-1-6a-3 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb709-8 VL-CL fused directly to HumAb747V-73 VH-CH1 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region;

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb709-8; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb747V-73.

The amino acid sequences of the three FIT107-1-6a-3 expressed polypeptide chains are shown in Table 45 below.

Table 45: Amino acid chain sequences of FIT107-1-6a components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-6a-3 168 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb709-8
(VL underlined) 169 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VH-CH1 mAb
HumAb747V-73
(VH underlined) 170 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-6a-3 171 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYY
PDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb709-8
(VH underlined) 172 EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGPSVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKKNVSC Polypeptide chain #3 FIT-Ig
FIT107-1-6a-3 173 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb747V-73
(VL underlined) 174 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSVY

Example 15.6: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-6b-3

PD-1/LAG-3 FIT-Ig, designated FIT107-1-6b-3, was constructed using the immunoglobulin domain coding sequences from the parent antibodies HumAb709-8 (humanized anti-PD-1) and HumAb747V-73 (humanized anti-LAG -3). FIT-Ig FIT107-1-6b-3 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: VL-CL HumAb747V-73 fused directly to VH-CH1 HumAb709-8 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region;

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb747V-73; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) of HumAb709-8.

The amino acid sequences of the three FIT107-1-6b-3 expressed polypeptide chains are shown in Table 46 below.

Table 46: Amino acid chain sequences of FIT107-1-6b-3 components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-6b-3 175 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb747V-73 (VL underlined) 176 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSVY VH-CH1 mAb
HumAb709-8 (VH underlined) 177 EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMSWVRQAPGKGLEWVATISGGGRDTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGQGGNYLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGPSVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKKNVSC hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-6b-3 178 MEFGLSWLFLVAILKGVQCEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVY
ASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb747V-73
(VH underlined) 179 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK Polypeptide chain #3 FIT-Ig
FIT107-1-6b-3 180 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb709-8
(VL underlined) 181 DIVMTQSPSSLSASVGDRVTITCKASQDVNTVVAWYQQKPGKAPKVLISWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYTTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

Example 15.7: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-7a-1

The PD-1/LAG-3 FIT-Ig, designated FIT107-1-7a-1, was constructed using the immunoglobulin domain coding sequences from the parent antibodies HumAb713-7 (humanized anti-PD-1, see tables 9 and 10 above) and HumAb747V-67 (humanized anti-LAG-3). FIT-Ig FIT107-1-7a-1 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb713-7 VL-CL fused directly to HumAb747V-67 VH-CH1 fused directly to the hinge-CH2-CH3 region of a human IgG1 mutant constant region;

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb713-7; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb747V-67.

The amino acid sequences of the three FIT107-1-7a-1 expressed polypeptide chains are shown in Table 47 below.

Table 47: Amino acid chain sequences of FIT107-1-7a components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-7a-1 182 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPENANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb713-7
(VL underlined) 183 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VH-CH1 mAb
HumAb747V-67
(VH underlined) 184 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPENANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKVSCK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-7a-1 185 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYY
ADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb713-7
(VH underlined) 186 EVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYNHPSKVVTVPSSVSLGTQTYKTYK Polypeptide chain #3 FIT-Ig
FIT107-1-7a-1 187 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb747V-67
(VL underlined) 188 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKTHNKSSVY

Example 15.8: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-7b-1

PD-1/LAG-3 FIT-Ig, designated FIT107-1-7b-1, was constructed using the immunoglobulin domain coding sequences from the parent antibodies HumAb713-7 (humanized anti-PD-1) and HumAb747V-67 (humanized anti-LAG -3). FIT-Ig FIT107-1-7b-1 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb747V-67 VL-CL fused directly to HumAb713-7 VH-CH1 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region;

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb747V-67; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb713-7.

The amino acid sequences of the three FIT107-1-7b-1 expressed polypeptide chains are shown in Table 48 below.

Table 48: Amino acid chain sequences of FIT107-1-7b-1 components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-7b-1 189 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb747V-67 (VL underlined) 190 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKTHNKSSVY VH-CH1 mAb
HumAb713-7 (VH underlined) 191 EVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYNHPSKVVTVPSSVSLGTQTYKTYK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-7b-1 192 MEFGLSWLFLVAILKGVQCEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPENANTVY
ASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb747V-67
(VH underlined) 193 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPENANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKVSCK Polypeptide chain #3 FIT-Ig
FIT107-1-7b-1 194 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb713-7
(VL underlined) 195 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

Example 15.9: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-7a-2

PD-1/LAG-3 FIT-Ig, designated FIT107-1-7a-2, was constructed using the immunoglobulin domain coding sequences from the parent antibodies HumAb713-7 (humanized anti-PD-1) and HumAb747V-72 (humanized anti-LAG -3). FIT-Ig FIT107-1-7a-2 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb713-7 VL-CL fused directly to HumAb747V-72 VH-CH1 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region (see Table 6 above);

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb713-7; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb747V-72.

The amino acid sequences of the three FIT107-1-7a-2 expressed polypeptide chains are shown in Table 49 below.

Table 49: Amino acid chain sequences of FIT107-1-7a components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-7a-2 196 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb713-7
(VL underlined) 197 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VH-CH1 mAb
HumAb747V-72
(VH underlined) 198 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-7a-2 199 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb713-7
(VH underlined) 200 EVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYNHPSKVVTVPSSVSLGTQTYKTYK Polypeptide chain #3 FIT-Ig
FIT107-1-7a-2 201 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb747V-72
(VL underlined) 202 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

Example 15.10: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-7b-2

PD-1/LAG-3 FIT-Ig, designated FIT107-1-7b-2, was constructed using the immunoglobulin domain coding sequences from the parent antibodies HumAb713-7 (humanized anti-PD-1) and HumAb747V-72 (humanized anti-LAG -3). FIT-Ig FIT107-1-7b-2 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: VL-CL HumAb747V-72 fused directly to VH-CH1 HumAb713-7 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region;

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb747V-72; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb713-7.

The amino acid sequences of the three FIT107-1-7b-2 expressed polypeptide chains are shown in Table 50 below.

Table 50: Amino acid chain sequences of FIT107-1-7b-2 components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-7b-2 203 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb747V-72 (VL underlined) 204 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASALDLGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VH-CH1 mAb
HumAb713-7
(VH underlined) 205 EVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYNHPSKVVTVPSSVSLGTQTYKTYK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-7b-2 206 MEFGLSWLFLVAILKGVQCEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVY
ASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb747V-72
(VH underlined) 207 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK Polypeptide chain #3 FIT-Ig
FIT107-1-7b-2 208 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb713-7
(VL underlined) 209 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

Example 15.11: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-7a-3

PD-1/LAG-3 FIT-Ig, designated FIT107-1-7a-3, was constructed using the immunoglobulin domain coding sequences from the parent antibodies HumAb713-7 (humanized anti-PD-1) and HumAb747V-73 (humanized anti-LAG -3). FIT-Ig FIT107-1-7a-3 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: HumAb713-7 VL-CL fused directly to HumAb747V-73 VH-CH1 fused directly to the hinge-CH2-CH3 region of a human IgG1 mutant constant region;

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb713-7; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb747V-73.

The amino acid sequences of the three FIT107-1-7a-3 expressed polypeptide chains are shown in Table 51 below.

Table 51: Amino acid chain sequences of FIT107-1-7a-3 components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-7a-3 210 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb713-7
(VL underlined) 211 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VH-CH1 mAb
HumAb747V-73
(VH underlined) 212 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-7a-3 213 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYY
ADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb713-7
(VH underlined) 214 EVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYNHPSKVVTVPSSVSLGTQTYKTYK Polypeptide chain #3 FIT-Ig
FIT107-1-7a-3 215 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb747V-73
(VL underlined) 216 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSVY

Example 15.12: Preparation of PD-1/LAG-3 binding FIT-Ig protein FIT107-1-7b-3

PD-1/LAG-3 FIT-Ig, designated FIT107-1-7b-3, was constructed using the immunoglobulin domain coding sequences from the parent antibodies HumAb713-7 (humanized anti-PD-1) and HumAb747V-73 (humanized anti-LAG -3). FIT-Ig FIT107-1-7b-3 is a hexamer consisting of three-component polypeptide chains:

Polypeptide chain #1 has the domain formula: VL-CL HumAb747V-73 fused directly to VH-CH1 HumAb713-7 fused directly to the hinge-CH2-CH3 region of the human IgG1 mutant constant region;

Polypeptide chain #2 has the domain formula: VH-CH1 HumAb747V-73; and

Polypeptide chain #3 has the domain formula: light chain (VL-CL) HumAb713-7.

The amino acid sequences of the three FIT107-1-7b-3 expressed polypeptide chains are shown in Table 52 below.

Table 52: Amino acid chain sequences of FIT107-1-7b-3 components

Polypeptide SEQID NO: Amino acid sequence
1234567890123456789012345678901234567890 Polypeptide chain #1 FIT-Ig
FIT107-1-7b-3 217 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL mAb
HumAb747V-73 (VL underlined) 218 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLSWLQQKPGGAIKRLIYAASTLDSGVPSRFSGSRSGSDYTLTISSLQPEDFADYYCLQYASYPLTFGQGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKSSVY VH-CH1 mAb
HumAb713-7 (VH underlined) 219 EVQLVESGGGLVQPGGSLRLSCAASGFTSSDYGMHWVRQAPGKGLEWVSYISSGSYTIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAKRGGSSHVNVMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYNHPSKVVTVPSSVSLGTQTYKTYK hinge-CH2-CH3
human IgG1 82 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTVMTPVLDSDGSHYFFLYSKLTVLSDSDNCSWQQ Polypeptide chain #2 FIT-Ig
FIT107-1-7b-3 220 MEFGLSWLFLVAILKGVQCEVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVY
ASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Signal sequence 84 MEFGLSWLFLVAILKGVQC VH-CH1
HumAb747V-73
(VH underlined) 221 EVQLVQSGAEVKKPGATVKLSCTASGFNIKDDYMHWVKQRPEQGLDWIGWIVPRNANTVYASKFQGKATITADTSTNTAYLELSSLRSEDTAVYYCTVYGDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYKNVSCK Polypeptide chain #3 FIT-Ig
FIT107-1-7b-3 222 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Signal sequence 79 MDMRVPAQLLGLLLLWFPGSRC VL-CL
HumAb713-7
(VL underlined) 223 DIQMTQSPSSLSASVGDRVTITCKASDHINNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQYWSPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

Example 16: Characterization of new FIT-Ig proteins

Example 16.1: Expression and analysis of SEC

The 12 FIT-Ig binding proteins described above (Tables 41-52) were expressed in the same manner as described in Example 10.5 above and purified by protein A chromatography. The composition and purity of the purified FIT-Igs were analyzed by size exclusion chromatography ( SEC). Purified FIT-Ig in PBS was applied to a TSKgel SuperSW3000, 300×4.6 mm (TOSOH) column. A DIONEX™ UltiMate 3000 HPLC instrument (Thermo Scientific) was used to perform SEC using UV detection at 280 nm and 214 nm. See table 53 below.

Table 53: Expression and SEC analysis of PD-1/LAG-3 FIT-Ig binding proteins

FIT-Ig protein Molar ratio of DNA: strands #1:#2:#3 Expression level (mg/l) % Peak Monomer Fraction by SEC FIT107-1-6a-1 1:2:1.5 6.21 78.9% FIT107-1-6b-1 1:2:1.5 8.58 55.4% FIT107-1-6a-2 1:2:1.5 6.11 90.9% FIT107-1-6b-2 1:2:1.5 15.86 43.2% FIT107-1-6a-3 1:2:1.5 11.37 43.0% FIT107-1-6b-3 1:2:1.5 17.22 44.0% FIT107-1-7a-1 1:2:1.5 14.47 80.0% FIT107-1-7b-1 1:2:1.5 17.96 94.8% FIT107-1-7a-2 1:2:1.5 19.25 88.3% FIT107-1-7b-2 1:2:1.5 22.12 98.5% FIT107-1-7a-3 1:2:1.5 14.31 80.9% FIT107-1-7b-3 1:2:1.5 29.20 98.7%

FIT-Ig proteins that had a lower monomer fraction (<80%) were excluded from further protein characterization studies.

Example 16.2: Functional analyzes

PD-1/LAG-3 FIT-Ig activity was tested in a PBMC activation assay using staphylococcal enterotoxin B (SEB) as superantigen as described in Example 12. The results are shown in FIG. 11. The results showed that all tested variants of FIT-Ig can increase the secretion of IL-2 from PBMC stimulated with SEB. This increase was for some reason inverted at the highest doses of FIT107-1-7b-2 and FIT107-1-7b-3, so these two FIT-Ig proteins were not favored as leader molecules.

Example 16.3 Binding Activity

FIT-Ig binding kinetics to PD-1 and LAG-3 targets were determined by biolayer interferometry using the Octet® RED96 system (Pall FortéBio LLC). Binding affinities for both PD-1 and LAG-3 target antigens are shown in Table 54 below. All FIT-Ig proteins retained affinity for both huPD-1 and huLAG-3. All FIT-Ig proteins that were active against cynomolgus macaque antigens also showed cross-reactivity with cynomolgus macaque antigens.

Table 54: Binding Affinity of PD-1/LAG-3 FIT-Ig Binding Proteins

FIT-Ig captured on the sensor chip analyte k _on (1/ms) k _off (1/s) K _D (M) FIT107-1-7b-1 PD-1-His human 8.96×10 ⁴ 9.35×10 ^-4 1.04×10 ^-8 PD-1-his cynomolgus macaque 2.15×10 ⁵ 7.75×10 ^-4 3.61×10 ^-9 LAG-3-His human 2.00×10 ⁵ 2.43×10 ^-4 1.22×10 ^-9 LAG-3-His cynomolgus macaque 5.19×10 ⁵ 6.36×10 ^-5 1.23×10 ^-10 FIT107-1-7b-3 PD-1-His human 1.16×10 ⁵ 1.03×10 ^-3 8.86×10 ^-9 PD-1-His cynomolgus macaque 2.48×10 ⁵ 9.19×10 ^-4 3.71×10 ^-9 LAG-3-His human 1.44×10 ⁵ 3.28×10 ^-4 2.28×10 ^-9 LAG-3-His cynomolgus macaque 2.81×10 ⁵ 4.70×10 ^-5 1.67×10 ^-10 FIT107-1-6a-2 PD-1-His human 1.87×10 ⁵ 2.39×10 ^-4 1.28×10 ^-9 PD-1-His cynomolgus macaque 3.06×10 ⁵ 1.07×10 ^-3 3.49×10 ^-9 LAG-3-His human 1.15×10 ⁵ 1.17×10 ^-4 1.02×10 ^-9 LAG-3-His cynomolgus macaque 1.91×10 ⁵ 8.17×10 ^-5 4.28×10 ^-10 FIT107-1-7a-1 PD-1-His human 1.77×10 ⁵ 5.38×10 ^-4 3.04×10 ^-9 LAG-3-His human 1.69×10 ⁵ 1.24×10 ^-4 7.32×10 ^-10 FIT107-1-7a-2 PD-1-His human 1.66×10 ⁵ 5.26×10 ^-4 3.17×10 ^-9 LAG-3-His human 1.05×10 ⁵ 1.28×10 ^-4 1.22×10 ^-9 FIT107-1-7a-3 PD-1-His human 2.08×10 ⁵ 6.28×10 ^-4 3.01×10 ^-9 LAG-3-His human 9.10×10 ⁴ 1.34×10 ^-4 1.47×10 ^-9 FIT107-1-7b-2 PD-1-His human 1.05×10 ⁵ 8.27×10 ^-4 7.90×10 ^-9 LAG-3-His human 1.74×10 ⁵ 2.46×10 ^-4 1.41×10 ^-9

Example 16.4: Pharmacokinetic data obtained in rats

Based on purity after one-step purification, transient transfection expression titer, retained binding affinity, and functional activity in the PBMC-SEB assay, FIT107-1-7b-1 was selected as the lead molecule. The pharmacokinetic properties of FIT107-1-7b-1 were evaluated in male Sprague-Dawley (SD) rats. FIT-Ig protein was administered to male SD rats once intravenously at a dose of 5 mg/kg. Serum samples were collected at different time points for 28 days: after 0, 5, 15 and 30 minutes; 1, 2, 4, 8 and 24 hours; and 2, 4, 7, 10, 14, 21 and 28 days of serial tail vein blood sampling and analyzed by total ELISA. Briefly, ELISA plates were coated with goat anti-human IgG Fc (Rockland cat# 609-101-017) at 125 ng/well at 4° C. overnight, blocked with 1X PBS/1% BSA/0.05% Tween-20/0.05% ProClin ^TM 300. All serum samples were first diluted 20-fold with blocking buffer. An additional dilution was performed using 5% pooled rat serum and incubated on the plate for 60 minutes at 37°C. Detection was performed with peroxidase-conjugated goat anti-human IgG Fab (Sigma-Aldrich; Cat. No. A0293) and concentrations were determined from standard curves by fitting using a four-parameter logistic model. Pharmacokinetic parameter values were determined using a non-compartmental model using WinNonlin software (Pharsight Corporation, Mountain View, CA). According to the results shown in Table 55, FIT107-1-7b-1 showed stable properties in vivo .

Table 55: Pharmacokinetic properties of FIT107-1-7b-1

PC parameters CL Vss beta t _1/2 AUC MRT antibody ml/day/kg ml/kg day day*mcg/ml day FIT107-1-7b-1 9.17 114 8.82 436 12.4

Example 16.5: FGL1 Receptor Blocking Assay (RBA)

According to recently published reports, fibrinogen-like protein 1 (FGL1) is the main functional LAG-3 ligand independent of MHC Class II (Wang J. et al., Cell, 176 (1): 334-47 (2019)). Blockade of FGL1/LAG-3 interaction by antibodies stimulates tumor immunity. To assess the blocking activity of an anti-LAG-3 antibody or FIT-Ig, FGL1 (Wuhan USCN, cat. no. RPD022Hu01) was diluted to 5 µg/ml with saline using Dulbecco's phosphate buffer and added 100 µl to a 96-well plate and incubated at 4°C overnight. The plate was washed three times with 300 μl/well PBS+Tween 20 (PBST). HumAb747V-67, FIT107-1-7b-1, hIgG (working concentration: 100 nM, 10 nM, 1 nM, and 0.1 nM) and 1 μg/ml biotinylated LAG-3 (AcroBiosystem cat. no. H82E5) and incubated at room temperature for 2 hours. The plate was washed three times with 300 μl/well PBST, then read with a VARIOSKAN ^™ LUX microplate reader (Thermo Scientific) using the ELISA-Endpoint-TMB/HRP protocol. The results are shown in FIG. 12. The results showed that both the FIT-Ig FIT107-1-7b-1 and its parent anti-LAG-3 antibody HumAb747V-67 can block the binding of human LAG-3 to the FGL1 protein.

Example 16.6: Binding activity of cellular protein FIT107-1-7b-1 in primary cells.

The above assays have demonstrated that PD-1/LAG-3 FIT-Ig proteins can bind recombinant antigenic proteins. To further evaluate the binding capacity of FIT107-1-7b-1 to cell surfaces, the parent antibodies HumAb713-7, HumAb747V-67, and the FIT107-1-7b-1 bispecific FIT-Ig were biotinylated using biotin as a reagent (Sigma, Cat no. S3259 ). Unstimulated PBMCs were resuspended at 5×10 ⁶ cells/ml. For testing anti-PD-1 antibody (HumAb713-7) or anti-LAG-3 antibody (HumAb747V-67), 100 μg/ml antibody was added and the reaction mixture was incubated at 37°C for 40 minutes separately followed by two washes with buffer FACS. Then, 100 μl containing 5×10 ⁵ PBMC/well (untreated group, anti-PD-1 antibody treated group, and anti-LAG-3 antibody treated group) were added to the wells of a 96-well plate. Biotinylated HumAb713-7, HumAb747V-67 and FIT107-1-7b-1 were added and incubated at 37°C for 40 minutes (final working concentration starting at 100 nM with 3-fold serial dilutions) followed by a two-fold wash with FACS buffer . FITC-streptavidin and BV421 anti-human CD3 antibody were added and the assay plate was incubated at 4° C. for 30 minutes followed by washing twice with FACS buffer. The plate was analyzed using a Beckman Coulter CytoFlex flow cytometer. PBMC stimulated groups were stimulated using anti-CD3 plus anti-CD28 antibodies for 72 hours to induce PD-1 and LAG-3 expression on T cells. Binding of HumAb713-7, HumAb747V-67, and FIT107-1-7b-1 was tested on stimulated PBMCs using a grouping strategy (untreated, treated with anti-PD-1 antibody, and treated with anti-LAG-3 antibody) similar to that used in the experiment with unstimulated PBMC. The binding of test antibodies was examined on a subpopulation of CD3-T cells by FACS. The results are shown in FIG. 13. The results showed that FIT107-7b-1 exhibits a unique binding pattern indicating binding to both the PD-1 target and the LAG-3 target on T cells.

The contents of all references (including literary sources, patents, patent applications and websites) that are given in this application, are expressly incorporated in this description in its entirety by reference. For the practical implementation of the invention are used, unless otherwise indicated, the usual methods of immunology, molecular biology and cell biology, which are well known in this field.

The invention may be embodied in other specific forms without departing from the essential features of the invention described above. Therefore, the foregoing embodiments should be considered as illustrative and not limiting of the invention disclosed in the present description. The scope of the invention is defined by the appended claims.

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SEQUENCE LISTS

<110> Shanghai EpimAb Biotherapeutics Co., Ltd.

XU Xuan

GONG, Shiyong

WU Chengbin

<120> HIGH AFFINITY ANTI-PD-1 AND ANTI-LAG-3 ANTIBODIES AND DERIVED FROM

THEM BI-SPECIFIC BINDING PROTEINS

<130> EPM-107.1PCT (PF190265PCT)

<140> PCT/CN2019/085164

<141> 2019-04-30

<150> PCT/CN2018/085468

<151> 2018-05-03

<160> 223

<170>PatentIn version 3.5

<210> 1

<211> 380

<212> Protein

<213> Artificial sequence

<220>

<223> Human PD-1/Fc fusion protein

<400> 1

Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala

1 5 10 15

Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe

20 25 30

Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro

35 40 45

Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln

50 55 60

Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg

65 70 75 80

Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr

85 90 95

Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu

100 105 110

Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro

115 120 125

Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Ile

130 135 140

Glu Gly Arg Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

145 150 155 160

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

165 170 175

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

180 185 190

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

195 200 205

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

210 215 220

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

225 230 235 240

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

245 250 255

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

260 265 270

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

275 280 285

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

290 295 300

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

305 310 315 320

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

325 330 335

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

340 345 350

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

355 360 365

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375 380

<210> 2

<211> 380

<212> Protein

<213> Artificial sequence

<220>

<223> Cynomolgus monkey PD-1/Fc fusion protein

<400> 2

Leu Glu Ser Pro Asp Arg Pro Trp Asn Ala Pro Thr Phe Ser Pro Ala

1 5 10 15

Leu Leu Leu Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe

20 25 30

Ser Asn Ala Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro

35 40 45

Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln

50 55 60

Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Arg Leu Pro Asn Gly Arg

65 70 75 80

Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr

85 90 95

Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu

100 105 110

Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro

115 120 125

Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Ile

130 135 140

Glu Gly Arg Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

145 150 155 160

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

165 170 175

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

180 185 190

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

195 200 205

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

210 215 220

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

225 230 235 240

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

245 250 255

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

260 265 270

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

275 280 285

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

290 295 300

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

305 310 315 320

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

325 330 335

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

340 345 350

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

355 360 365

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375 380

<210> 3

<211> 380

<212> Protein

<213> Artificial sequence

<220>

<223> Mouse PD-1 /Fc fusion protein

<400> 3

Leu Glu Val Pro Asn Gly Pro Trp Arg Ser Leu Thr Phe Tyr Pro Ala

1 5 10 15

Trp Leu Thr Val Ser Glu Gly Ala Asn Ala Thr Phe Thr Cys Ser Leu

20 25 30

Ser Asn Trp Ser Glu Asp Leu Met Leu Asn Trp Asn Arg Leu Ser Pro

35 40 45

Ser Asn Gln Thr Glu Lys Gln Ala Ala Phe Cys Asn Gly Leu Ser Gln

50 55 60

Pro Val Gln Asp Ala Arg Phe Gln Ile Ile Gln Leu Pro Asn Arg His

65 70 75 80

Asp Phe His Met Asn Ile Leu Asp Thr Arg Arg Asn Asp Ser Gly Ile

85 90 95

Tyr Leu Cys Gly Ala Ile Ser Leu His Pro Lys Ala Lys Ile Glu Glu

100 105 110

Ser Pro Gly Ala Glu Leu Val Val Thr Glu Arg Ile Leu Glu Thr Ser

115 120 125

Thr Arg Tyr Pro Ser Pro Ser Pro Lys Pro Glu Gly Arg Phe Gln Ile

130 135 140

Glu Gly Arg Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

145 150 155 160

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

165 170 175

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

180 185 190

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

195 200 205

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

210 215 220

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

225 230 235 240

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

245 250 255

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

260 265 270

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

275 280 285

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

290 295 300

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

305 310 315 320

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

325 330 335

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

340 345 350

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

355 360 365

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375 380

<210> 4

<211> 117

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb701

<400> 4

Glu Val Leu Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Ile Arg Gln Thr Pro Glu Arg Arg Leu Glu Trp Val

35 40 45

Ala Ser Met Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 5

<211> 107

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb701

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly

1 5 10 15

Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 6

<211> 121

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb703

<400> 6

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Thr Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Met Ser Tyr Asp Gly Asn Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Leu

65 70 75 80

Leu Arg Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Asp Arg Gly Thr Thr Ile Leu Gly Gly Thr Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser

115 120

<210> 7

<211> 107

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb703

<400> 7

Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Phe Val Ser Ala Gly

1 5 10 15

Asp Arg Val Thr Ile Ala Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Phe Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 8

<211> 118

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb709

<400> 8

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu His Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 9

<211> 107

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb709

<400> 9

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Leu Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ala Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Gln Leu Glu Ile Lys

100 105

<210> 10

<211> 121

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb713

<400> 10

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Asp Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 11

<211> 107

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb713

<400> 11

Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly

1 5 10 15

Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 117

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb714

<400> 12

Glu Val His Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Phe Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn

20 25 30

Asn Val Glu Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Asp Thr Leu Tyr Ser Gln Tyr Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Gly Leu Tyr Tyr Cys

85 90 95

Ala Arg Gly Lys Ser Asp Gln Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 13

<211> 107

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb714

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly

1 5 10 15

Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu Gln Gly

65 70 75 80

Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 14

<211> 118

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb715

<400> 14

Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Leu Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Thr Tyr Leu Phe Ala Ser Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 15

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL mAb715

<400> 15

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 16

<211> 120

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb718

<400> 16

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Ala Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Glu Pro Glu Ser Gly Gly Thr Val Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Lys Leu Thr Ala Asp Lys Ser Ser Arg Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Phe Asn Ser Asp His Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 17

<211> 112

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb718

<400> 17

Asp Val Leu 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 Asn Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Phe 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 Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 18

<211> 116

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb719

<400> 18

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Ser Phe Ser Ser His

20 25 30

Leu Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ala Ile Ser Gly Gly Gly Ala Asp Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Arg Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Thr Arg Gln Ile Leu Ala Phe Asp Ser Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 19

<211> 107

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb719

<400> 19

Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Val Ser Leu Gly

1 5 10 15

Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Gly Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 20

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb709 VH.1

<400> 20

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Gly Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 21

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb709 VH.1A

<400> 21

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 22

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb709 VH.1B

<400> 22

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 23

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb709 VK.1A

<400> 23

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 Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 24

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb709 VK.1B

<400> 24

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 Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb709 VK.1C

<400> 25

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb709 VK.1D

<400> 26

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 27

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb709 VK.1E

<400> 27

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Leu Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 28

<211> 330

<212> Protein

<213> Homo sapiens

<400> 28

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 29

<211> 107

<212> Protein

<213> Homo sapiens

<400> 29

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 30

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb713 VH.1

<400> 30

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 Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 31

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb713 VH.1A

<400> 31

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 Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 32

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb713 VH.1B

<400> 32

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 Ser Ser Asp Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 33

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb713 VH.1C

<400> 33

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 Ser Ser Asp Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 34

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb713 VK.1

<400> 34

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 35

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb713 VK.1A

<400> 35

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 36

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb713 VK.1B

<400> 36

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 37

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb713 VK.1C

<400> 37

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 38

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb703 VH.1A

<400> 38

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Thr Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Met Ser Tyr Asp Gly Asn Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Thr Thr Ile Leu Gly Gly Thr Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 39

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb703 VH.1B

<400> 39

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Thr Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Met Ser Tyr Asp Gly Asn Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Thr Thr Ile Leu Gly Gly Thr Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 40

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb703 VH.1C

<400> 40

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Thr Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Tyr Met Ser Tyr Asp Gly Asn Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Thr Thr Ile Leu Gly Gly Thr Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 41

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb703 VH.1D

<400> 41

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Tyr Met Ser Tyr Asp Gly Asn Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Thr Thr Ile Leu Gly Gly Thr Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 42

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb703 VH.1E

<400> 42

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp

35 40 45

Met Gly Tyr Met Ser Tyr Asp Gly Asn Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asp Arg Gly Thr Thr Ile Leu Gly Gly Thr Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 43

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb703 VK.1

<400> 43

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 Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Phe Tyr Arg Tyr Thr 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 Asp Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 44

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb703 VK.1A

<400> 44

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 Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Phe Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Tyr 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 Asp Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb703 VK.1B

<400> 45

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 Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Phe Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr 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 Asp Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 46

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb703 VK.1C

<400> 46

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 Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Phe Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb703 VK.1D

<400> 47

Ser Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Phe Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 48

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb719 VH.1

<400> 48

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 His

20 25 30

Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Gly Gly Ala Asp Thr Tyr Tyr Pro 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 Lys Gln Ile Leu Ala Phe Asp Ser Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 49

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb719 VH.1A

<400> 49

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 His

20 25 30

Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Gly Gly Ala Asp Thr Tyr Tyr Pro 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

Thr Arg Gln Ile Leu Ala Phe Asp Ser Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 50

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb719 VH.1B

<400> 50

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 Ser Phe Ser Ser His

20 25 30

Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Gly Gly Ala Asp Thr Tyr Tyr Pro 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

Thr Arg Gln Ile Leu Ala Phe Asp Ser Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 51

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb719 VH.1C

<400> 51

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 Ser Phe Ser Ser His

20 25 30

Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ala Ala Ile Ser Gly Gly Gly Ala Asp Thr Tyr Tyr Pro 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

Thr Arg Gln Ile Leu Ala Phe Asp Ser Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 52

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb719 VH.1D

<400> 52

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 Ser Phe Ser Ser His

20 25 30

Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Ala Ile Ser Gly Gly Gly Ala Asp Thr Tyr Tyr Pro 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

Thr Arg Gln Ile Leu Ala Phe Asp Ser Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 53

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb719 VH.1E

<400> 53

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 Ser Phe Ser Ser His

20 25 30

Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ala Ala Ile Ser Gly Gly Gly Ala Asp Thr Tyr Tyr Pro Ala 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

Thr Arg Gln Ile Leu Ala Phe Asp Ala Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 54

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb719 VH.1F

<400> 54

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 Ser Phe Ser Ser His

20 25 30

Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Ala Ile Ser Gly Gly Gly Ala Asp Thr Tyr Tyr Pro Ala 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

Thr Arg Gln Ile Leu Ala Phe Asp Ala Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 55

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb719 VK.1

<400> 55

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 His Ala Ser Gln Asn Ile Tyr Val Trp

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 56

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb719 VK.1A

<400> 56

Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 57

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb719 VK.1B

<400> 57

Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ile Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 58

<211> 665

<212> Protein

<213> Artificial sequence

<220>

<223> Human LAG-3 /Fc fusion protein

<400> 58

Leu Gln Pro Gly Ala Glu Val Pro Val Val Trp Ala Gln Glu Gly Ala

1 5 10 15

Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser

20 25 30

Leu Leu Arg Arg Ala Gly Val Thr Trp Gln His Gln Pro Asp Ser Gly

35 40 45

Pro Pro Ala Ala Ala Pro Gly His Pro Leu Ala Pro Gly Pro His Pro

50 55 60

Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu

65 70 75 80

Ser Val Gly Pro Gly Gly Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro

85 90 95

Arg Val Gln Leu Asp Glu Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu

100 105 110

Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala

115 120 125

Val His Leu Arg Asp Arg Ala Leu Ser Cys Arg Leu Arg Leu Arg Leu

130 135 140

Gly Gln Ala Ser Met Thr Ala Ser Pro Pro Gly Ser Leu Arg Ala Ser

145 150 155 160

Asp Trp Val Ile Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala

165 170 175

Ser Val His Trp Phe Arg Asn Arg Gly Gln Gly Arg Val Pro Val Arg

180 185 190

Glu Ser Pro His His His Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln

195 200 205

Val Ser Pro Met Asp Ser Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg

210 215 220

Asp Gly Phe Asn Val Ser Ile Met Tyr Asn Leu Thr Val Leu Gly Leu

225 230 235 240

Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala Gly Ala Gly Ser Arg Val

245 250 255

Gly Leu Pro Cys Arg Leu Pro Ala Gly Val Gly Thr Arg Ser Phe Leu

260 265 270

Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Asp Leu Leu Val Thr

275 280 285

Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu Glu Asp Val Ser Gln Ala

290 295 300

Gln Ala Gly Thr Tyr Thr Cys His Ile His Leu Gln Glu Gln Gln Leu

305 310 315 320

Asn Ala Thr Val Thr Leu Ala Ile Ile Thr Val Thr Pro Lys Ser Phe

325 330 335

Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu Cys Glu Val Thr Pro Val

340 345 350

Ser Gly Gln Glu Arg Phe Val Trp Ser Ser Leu Asp Thr Pro Ser Gln

355 360 365

Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala Gln Glu Ala Gln Leu Leu

370 375 380

Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln Gly Glu Arg Leu Leu Gly

385 390 395 400

Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser Pro Gly Ala Gln Arg Ser

405 410 415

Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly His Leu Ile Glu Gly Arg

420 425 430

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

435 440 445

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

450 455 460

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

465 470 475 480

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

485 490 495

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

500 505 510

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

515 520 525

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

530 535 540

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

545 550 555 560

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

565 570 575

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

580 585 590

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

595 600 605

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

610 615 620

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

625 630 635 640

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

645 650 655

Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665

<210> 59

<211> 665

<212> Protein

<213> Artificial sequence

<220>

<223> Cynomolgus LAG-3/Fc fusion protein

<220>

<221> misc_feature

<222> (52)..(52)

<223> Xaa can be any naturally occurring

amino acid

<400> 59

Pro Gln Pro Gly Ala Glu Ile Ser Val Val Trp Ala Gln Glu Gly Ala

1 5 10 15

Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser

20 25 30

Leu Leu Arg Arg Ala Gly Val Thr Trp Gln His Gln Pro Asp Ser Gly

35 40 45

Pro Pro Ala Xaa Ala Pro Gly His Pro Pro Val Pro Gly His Arg Pro

50 55 60

Ala Ala Pro Tyr Ser Trp Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu

65 70 75 80

Ser Val Gly Pro Gly Gly Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro

85 90 95

Arg Val Gln Leu Asp Glu Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu

100 105 110

Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala Gly Glu Tyr Arg Ala Thr

115 120 125

Val His Leu Arg Asp Arg Ala Leu Ser Cys Arg Leu Arg Leu Arg Val

130 135 140

Gly Gln Ala Ser Met Thr Ala Ser Pro Pro Gly Ser Leu Arg Thr Ser

145 150 155 160

Asp Trp Val Ile Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala

165 170 175

Ser Val His Trp Phe Arg Ser Arg Gly Gln Gly Arg Val Pro Val Gln

180 185 190

Gly Ser Pro His His His Leu Ala Glu Ser Phe Leu Phe Leu Pro His

195 200 205

Val Gly Pro Met Asp Ser Gly Leu Trp Gly Cys Ile Leu Thr Tyr Arg

210 215 220

Asp Gly Phe Asn Val Ser Ile Met Tyr Asn Leu Thr Val Leu Gly Leu

225 230 235 240

Glu Pro Ala Thr Pro Leu Thr Val Tyr Ala Gly Ala Gly Ser Arg Val

245 250 255

Glu Leu Pro Cys Arg Leu Pro Pro Ala Val Gly Thr Gln Ser Phe Leu

260 265 270

Thr Ala Lys Trp Ala Pro Pro Gly Gly Gly Pro Asp Leu Leu Val Ala

275 280 285

Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu Glu Asp Val Ser Gln Ala

290 295 300

Gln Ala Gly Thr Tyr Ile Cys His Ile Arg Leu Gln Gly Gln Gln Leu

305 310 315 320

Asn Ala Thr Val Thr Leu Ala Ile Ile Thr Val Thr Pro Lys Ser Phe

325 330 335

Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu Cys Glu Val Thr Pro Ala

340 345 350

Ser Gly Gln Glu His Phe Val Trp Ser Pro Leu Asn Thr Pro Ser Gln

355 360 365

Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala Gln Glu Ala Gln Leu Leu

370 375 380

Ser Gln Pro Trp Gln Cys Gln Leu His Gln Gly Glu Arg Leu Leu Gly

385 390 395 400

Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser Pro Gly Ala Gln Arg Ser

405 410 415

Gly Arg Ala Pro Gly Ala Leu Arg Ala Gly His Leu Ile Glu Gly Arg

420 425 430

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

435 440 445

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

450 455 460

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

465 470 475 480

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

485 490 495

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

500 505 510

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

515 520 525

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

530 535 540

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

545 550 555 560

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

565 570 575

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

580 585 590

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

595 600 605

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

610 615 620

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

625 630 635 640

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

645 650 655

Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665

<210> 60

<211> 113

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb746 and VH mAb747

<400> 60

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

100 105 110

Ser

<210> 61

<211> 107

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb746

<400> 61

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Ser Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 62

<211> 107

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb747

<400> 62

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Ala Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 63

<211> 116

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb742

<400> 63

Gln Gly Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ile Arg Trp Gly Ser Thr Val Phe Pro Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser

115

<210> 64

<211> 112

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb742

<400> 64

Asp Gly Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Ile Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn Ser

20 25 30

Asp Gly Phe Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu 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 Tyr Cys Phe Gln Ser

85 90 95

Asn Tyr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 65

<211> 116

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb744

<400> 65

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Met Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Ala Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Asp Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ile Arg Trp Gly Thr Thr Val Phe Pro Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser

115

<210> 66

<211> 112

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb744

<400> 66

Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Ile Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn Ser

20 25 30

Asp Gly Phe Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu 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 Tyr Cys Phe Gln Ser

85 90 95

Asn Tyr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 67

<211> 112

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb748

<400> 67

Glu Val Gln Met Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Val Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

100 105 110

<210> 68

<211> 112

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb748 and VL mAb750

<400> 68

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr 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 Tyr Cys Trp Gln Gly

85 90 95

Ser His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 69

<211> 116

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb749

<400> 69

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Val His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ser Thr Trp Asp Ala Glu Glu Asn Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Ser Val Ser Ser

115

<210> 70

<211> 112

<212> Protein

<213> Mus musculus

<220>

<223> VL mAb749

<400> 70

Asp Ile Val Leu Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly

1 5 10 15

Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 71

<211> 116

<212> Protein

<213> Mus musculus

<220>

<223> VH mAb750

<400> 71

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Pro Ser Gly Leu Asn Ile Lys Asp Asp

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Cys Thr Ala Asp Tyr Arg Asn Trp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 72

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb747 VH.2A

<400> 72

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 73

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb747 VH.2B

<400> 73

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 74

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH mAb747 VH.1G

<400> 74

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 75

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb747 VK.1E

<400> 75

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Lys Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 76

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VK mAb747 VK.2A

<400> 76

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Lys Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 77

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Vk mAb747 VK.2B

<400> 77

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 78

<211> 679

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-2a

Polypeptide chain #1 FIT-Ig

<400> 78

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser His Lys Phe

20 25 30

Met Ser Thr Ser Val Gly Asp Ser Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Ser Leu Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ala Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Gln Leu Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu

245 250 255

Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp Tyr Met His Trp

260 265 270

Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile Gly Trp Ile Val

275 280 285

Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe Gln Gly Lys Ala

290 295 300

Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser

305 310 315 320

Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Val Tyr Gly

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly Lys

675

<210> 79

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> signal sequence

<400> 79

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys

twenty

<210> 80

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL mAb709

<400> 80

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Leu Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ala Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Gln Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 81

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 mAb746

<400> 81

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu 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 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

210 215

<210> 82

<211> 227

<212> Protein

<213> Artificial sequence

<220>

<223> hinge-CH2-CH3 human IgG1

<400> 82

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 83

<211> 240

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-2a

Polypeptide chain #2 FIT-Ig

<400> 83

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Phe Tyr Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu

50 55 60

Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Thr Leu Tyr Leu His Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu

100 105 110

Tyr Tyr Cys Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

<210> 84

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> signal sequence

<400> 84

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys

<210> 85

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223>VH-CH1 mouse mAb709

<400> 85

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu His Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 86

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-2a

Polypeptide chain #3 FIT-Ig

<400> 86

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Asn Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Ser Asp Gly

50 55 60

Thr Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 80

Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 87

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL mouse mAb746

<400> 87

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Ser Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 88

<211> 684

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-2b

Polypeptide chain #1 FIT-Ig

<400> 88

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Asn Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Ser Asp Gly

50 55 60

Thr Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 80

Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Lys Leu

225 230 235 240

Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr Thr Met Ser Trp

260 265 270

Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Ile Ser

275 280 285

Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu His Met Ser

305 310 315 320

Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Gly Gln Gly

325 330 335

Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

340 345 350

Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

355 360 365

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

370 375 380

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

385 390 395 400

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

405 410 415

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

420 425 430

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

435 440 445

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

450 455 460

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

465 470 475 480

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

485 490 495

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

500 505 510

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

515 520 525

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

530 535 540

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

545 550 555 560

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

565 570 575

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

580 585 590

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

595 600 605

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

610 615 620

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

625 630 635 640

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

645 650 655

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

660 665 670

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680

<210> 89

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL mAb746

<400> 89

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Ser Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 90

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 mAb709

<400> 90

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu His Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 91

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-2b

Polypeptide chain #2 FIT-Ig

<400> 91

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Asp Phe Asn Ile

35 40 45

Lys Asp Asp Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60

Asp Trp Ile Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala

65 70 75 80

Ser Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn

85 90 95

Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235

<210> 92

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 mAb746

<400> 92

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu 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 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

210 215

<210> 93

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-2b

Polypeptide chain #3 FIT-Ig

<400> 93

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser His Lys Phe

20 25 30

Met Ser Thr Ser Val Gly Asp Ser Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Ser Leu Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ala Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Gln Leu Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 94

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL mAb709

<400> 94

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Leu Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ala Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Gln Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 95

<211> 679

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-5a

Polypeptide chain #1 FIT-Ig

<400> 95

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val

245 250 255

Ser Cys Lys Ala Ser Asp Phe Asn Ile Lys Asp Asp Tyr Met His Trp

260 265 270

Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Trp Ile Val

275 280 285

Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe Gln Gly Lys Ala

290 295 300

Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr Met Glu Leu Ser

305 310 315 320

Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Thr Val Tyr Gly

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly Lys

675

<210> 96

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb709-8

<400> 96

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

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 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> 97

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 mAb

HumAb747-42

<400> 97

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 98

<211> 240

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-5a

Polypeptide chain #2 FIT-Ig

<400> 98

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Phe Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

<210> 99

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb709-8

<400> 99

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 100

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-5a

Polypeptide chain #3 FIT-Ig

<400> 100

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Asn Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly

50 55 60

Thr Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 101

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747-42

<400> 101

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

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 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> 102

<211> 684

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-5b

Polypeptide chain #1 FIT-Ig

<400> 102

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Asn Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly

50 55 60

Thr Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr Thr Met Ser Trp

260 265 270

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser

275 280 285

Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn

305 310 315 320

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Gly Gln Gly

325 330 335

Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

340 345 350

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

355 360 365

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

370 375 380

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

385 390 395 400

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

405 410 415

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

420 425 430

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

435 440 445

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

450 455 460

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

465 470 475 480

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

485 490 495

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

500 505 510

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

515 520 525

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

530 535 540

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

545 550 555 560

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

565 570 575

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

580 585 590

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

595 600 605

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

610 615 620

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

625 630 635 640

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

645 650 655

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

660 665 670

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680

<210> 103

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747-42

<400> 103

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

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 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> 104

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb709-8

<400> 104

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 105

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-5b

Polypeptide chain #2 FIT-Ig

<400> 105

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Asn Ile

35 40 45

Lys Asp Asp Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala

65 70 75 80

Ser Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ile Asn

85 90 95

Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235

<210> 106

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747-42

<400> 106

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 107

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-5b

Polypeptide chain #3 FIT-Ig

<400> 107

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 108

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL mAb HuMAb709-8

<400> 108

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

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 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> 109

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv1

<400> 109

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 110

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv2

<400> 110

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Lys Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 111

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv3

<400> 111

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 112

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv4

<400> 112

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 113

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv5

<400> 113

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 114

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv6

<400> 114

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 115

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for huEpi001 VLv1

<400> 115

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg 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 Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 116

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for huEpi001 VLv2

<400> 116

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg 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 Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 117

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for huEpi001 VLv3

<400> 117

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 118

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for huEpi001 VLv4

<400> 118

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 119

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for antibody B2-53

<400> 119

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 120

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for antibody B2-53

<400> 120

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 121

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for antibody B3-21

<400> 121

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Val Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 122

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for antibody B3-21

<400> 122

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 Asn Cys Arg Ala Met Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Tyr Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 123

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for antibody B3-43

<400> 123

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Cys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 124

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for antibody B3-43

<400> 124

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser His Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 125

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for antibody B3-46

<400> 125

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Leu Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 126

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for antibody B3-46

<400> 126

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 127

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for antibody B3-48

<400> 127

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Phe Ser Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Lys Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 128

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for antibody B3-48

<400> 128

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Met Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 129

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for antibody B3-69

<400> 129

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr His Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 130

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for antibody B3-69

<400> 130

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Glu Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 131

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for antibody D1-70

<400> 131

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Gly Asn Thr Met Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 132

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for antibody D1-70

<400> 132

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 Asn Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 133

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv6(G55A)

<400> 133

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Asp Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Ala Asn Thr Glu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 134

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv6.1

<400> 134

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Gly Asn Thr Met Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 135

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv6.2

<400> 135

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Gly Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 136

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH for huEpi001-VHv6.3

<400> 136

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Gly Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 137

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for huEpi001-VLv3.4

<400> 137

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 138

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for huEpi001-VLv3.5

<400> 138

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 139

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL for huEpi001-VLv3.6

<400> 139

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 140

<211> 679

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6a-1

Polypeptide chain #1 FIT-Ig

<400> 140

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Leu

245 250 255

Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp Tyr Met His Trp

260 265 270

Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile Gly Trp Ile Val

275 280 285

Pro Glu Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe Gln Gly Lys Ala

290 295 300

Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr Leu Glu Leu Ser

305 310 315 320

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Val Tyr Gly

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly Lys

675

<210> 141

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb709-8

<400> 141

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

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 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> 142

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-67

<400> 142

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 143

<211> 240

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6a-1

Polypeptide chain #2 FIT-Ig

<400> 143

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Phe Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

<210> 144

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb709-8

<400> 144

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 145

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6a-1

Polypeptide chain #3 FIT-Ig

<400> 145

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 146

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-67

<400> 146

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 147

<211> 684

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6b-1

Polypeptide chain #1 FIT-Ig

<400> 147

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr Thr Met Ser Trp

260 265 270

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser

275 280 285

Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn

305 310 315 320

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Gly Gln Gly

325 330 335

Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

340 345 350

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

355 360 365

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

370 375 380

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

385 390 395 400

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

405 410 415

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

420 425 430

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

435 440 445

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

450 455 460

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

465 470 475 480

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

485 490 495

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

500 505 510

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

515 520 525

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

530 535 540

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

545 550 555 560

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

565 570 575

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

580 585 590

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

595 600 605

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

610 615 620

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

625 630 635 640

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

645 650 655

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

660 665 670

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680

<210> 148

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-67

<400> 148

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 149

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb709-8

<400> 149

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 150

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6b-1

Polypeptide chain #2 FIT-Ig

<400> 150

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile

35 40 45

Lys Asp Asp Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60

Asp Trp Ile Gly Trp Ile Val Pro Glu Asn Ala Asn Thr Val Tyr Ala

65 70 75 80

Ser Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn

85 90 95

Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235

<210> 151

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-67

<400> 151

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 152

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6b-1

Polypeptide chain #3 FIT-Ig

<400> 152

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 153

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb709-8

<400> 153

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

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 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> 154

<211> 679

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6a-2

Polypeptide chain #1 FIT-Ig

<400> 154

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Leu

245 250 255

Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp Tyr Met His Trp

260 265 270

Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile Gly Trp Ile Val

275 280 285

Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe Gln Gly Lys Ala

290 295 300

Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr Leu Glu Leu Ser

305 310 315 320

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Val Tyr Gly

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly Lys

675

<210> 155

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb709-8

<400> 155

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

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 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> 156

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-72

<400> 156

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 157

<211> 240

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6a-2

Polypeptide chain #2 FIT-Ig

<400> 157

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Phe Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

<210> 158

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb709-8

<400> 158

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 159

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6a-2

Polypeptide chain #3 FIT-Ig

<400> 159

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Ala Leu Asp Leu Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 160

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-72

<400> 160

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 161

<211> 684

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6b-2

Polypeptide chain #1 FIT-Ig

<400> 161

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Ala Leu Asp Leu Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr Thr Met Ser Trp

260 265 270

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser

275 280 285

Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn

305 310 315 320

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Gly Gln Gly

325 330 335

Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

340 345 350

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

355 360 365

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

370 375 380

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

385 390 395 400

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

405 410 415

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

420 425 430

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

435 440 445

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

450 455 460

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

465 470 475 480

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

485 490 495

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

500 505 510

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

515 520 525

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

530 535 540

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

545 550 555 560

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

565 570 575

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

580 585 590

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

595 600 605

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

610 615 620

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

625 630 635 640

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

645 650 655

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

660 665 670

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680

<210> 162

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-72

<400> 162

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 163

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb709-8

<400> 163

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 164

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6b-2

Polypeptide chain #2 FIT-Ig

<400> 164

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile

35 40 45

Lys Asp Asp Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60

Asp Trp Ile Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala

65 70 75 80

Ser Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn

85 90 95

Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235

<210> 165

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-72

<400> 165

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 166

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6b-2

Polypeptide chain #3 FIT-Ig

<400> 166

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 167

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb709-8

<400> 167

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

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 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> 168

<211> 679

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6a-3

Polypeptide chain #1 FIT-Ig

<400> 168

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Leu

245 250 255

Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp Tyr Met His Trp

260 265 270

Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile Gly Trp Ile Val

275 280 285

Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe Gln Gly Lys Ala

290 295 300

Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr Leu Glu Leu Ser

305 310 315 320

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Val Tyr Gly

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly Lys

675

<210> 169

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb709-8

<400> 169

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

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 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> 170

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-73

<400> 170

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 171

<211> 240

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6a-3

Polypeptide chain #2 FIT-Ig

<400> 171

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Phe Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

<210> 172

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb709-8

<400> 172

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 173

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6a-3

Polypeptide chain #3 FIT-Ig

<400> 173

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 174

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-73

<400> 174

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 175

<211> 684

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6b-3

Polypeptide chain #1 FIT-Ig

<400> 175

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr Thr Met Ser Trp

260 265 270

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile Ser

275 280 285

Gly Gly Gly Arg Asp Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn

305 310 315 320

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Gly Gln Gly

325 330 335

Gly Asn Tyr Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

340 345 350

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

355 360 365

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

370 375 380

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

385 390 395 400

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

405 410 415

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

420 425 430

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

435 440 445

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

450 455 460

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

465 470 475 480

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

485 490 495

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

500 505 510

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

515 520 525

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

530 535 540

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

545 550 555 560

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

565 570 575

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

580 585 590

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

595 600 605

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

610 615 620

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

625 630 635 640

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

645 650 655

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

660 665 670

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680

<210> 176

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-73

<400> 176

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 177

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb709-8

<400> 177

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 Phe Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Pro 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gln Gly Gly Asn Tyr Leu Phe Ala 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

210 215 220

<210> 178

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6b-3

Polypeptide chain #2 FIT-Ig

<400> 178

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile

35 40 45

Lys Asp Asp Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60

Asp Trp Ile Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala

65 70 75 80

Ser Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn

85 90 95

Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235

<210> 179

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-73

<400> 179

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 180

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-6b-3

Polypeptide chain #3 FIT-Ig

<400> 180

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asp Val Asn Thr Val Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Val Leu Ile Ser Trp Ala Ser Thr Arg His Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

His Tyr Thr Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 181

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb709-8

<400> 181

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr

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 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> 182

<211> 679

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7a-1

Polypeptide chain #1 FIT-Ig

<400> 182

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Asp His Ile Asn Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Trp Ser Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Leu

245 250 255

Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp Tyr Met His Trp

260 265 270

Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile Gly Trp Ile Val

275 280 285

Pro Glu Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe Gln Gly Lys Ala

290 295 300

Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr Leu Glu Leu Ser

305 310 315 320

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Val Tyr Gly

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly Lys

675

<210> 183

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL mAb HumAb713-7

<400> 183

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

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 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> 184

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-67

<400> 184

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 185

<211> 243

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7a-1

Polypeptide chain #2 FIT-Ig

<400> 185

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser

35 40 45

Ser Asp Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala

65 70 75 80

Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

225 230 235 240

Lys Ser Cys

<210> 186

<211> 224

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb713-7

<400> 186

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 Ser Ser Asp Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr 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

<210> 187

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7a-1

Polypeptide chain #3 FIT-Ig

<400> 187

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 188

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-67

<400> 188

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 189

<211> 687

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7b-1

Polypeptide chain #1 FIT-Ig

<400> 189

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Asp Tyr Gly Met His Trp

260 265 270

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser

275 280 285

Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn

305 310 315 320

Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Arg Gly

325 330 335

Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly Gln Gly Thr Thr

340 345 350

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

355 360 365

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

370 375 380

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

385 390 395 400

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

405 410 415

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

420 425 430

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

435 440 445

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

450 455 460

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

465 470 475 480

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

485 490 495

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

500 505 510

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

515 520 525

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

530 535 540

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

545 550 555 560

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

565 570 575

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

580 585 590

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

595 600 605

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

610 615 620

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

625 630 635 640

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

645 650 655

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

660 665 670

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680 685

<210> 190

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-67

<400> 190

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 191

<211> 224

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb713-7

<400> 191

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 Ser Ser Asp Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr 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

<210> 192

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7b-1

Polypeptide chain #2 FIT-Ig

<400> 192

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile

35 40 45

Lys Asp Asp Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60

Asp Trp Ile Gly Trp Ile Val Pro Glu Asn Ala Asn Thr Val Tyr Ala

65 70 75 80

Ser Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn

85 90 95

Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235

<210> 193

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-67

<400> 193

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 194

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7b-1

Polypeptide chain #3 FIT-Ig

<400> 194

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Asp His Ile Asn Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Trp Ser Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 195

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb713-7

<400> 195

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

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 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> 196

<211> 679

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7a-2

Polypeptide chain #1 FIT-Ig

<400> 196

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Asp His Ile Asn Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Trp Ser Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Leu

245 250 255

Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp Tyr Met His Trp

260 265 270

Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile Gly Trp Ile Val

275 280 285

Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe Gln Gly Lys Ala

290 295 300

Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr Leu Glu Leu Ser

305 310 315 320

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Val Tyr Gly

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly Lys

675

<210> 197

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb713-7

<400> 197

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

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 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> 198

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-72

<400> 198

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 199

<211> 243

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7a-2

Polypeptide chain #2 FIT-Ig

<400> 199

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser

35 40 45

Ser Asp Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala

65 70 75 80

Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

225 230 235 240

Lys Ser Cys

<210> 200

<211> 224

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb713-7

<400> 200

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 Ser Ser Asp Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr 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

<210> 201

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7a-2

Polypeptide chain #3 FIT-Ig

<400> 201

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Ala Leu Asp Leu Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 202

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-72

<400> 202

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 203

<211> 687

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7b-2

Polypeptide chain #1 FIT-Ig

<400> 203

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Ala Leu Asp Leu Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Asp Tyr Gly Met His Trp

260 265 270

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser

275 280 285

Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn

305 310 315 320

Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Arg Gly

325 330 335

Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly Gln Gly Thr Thr

340 345 350

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

355 360 365

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

370 375 380

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

385 390 395 400

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

405 410 415

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

420 425 430

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

435 440 445

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

450 455 460

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

465 470 475 480

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

485 490 495

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

500 505 510

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

515 520 525

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

530 535 540

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

545 550 555 560

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

565 570 575

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

580 585 590

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

595 600 605

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

610 615 620

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

625 630 635 640

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

645 650 655

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

660 665 670

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680 685

<210> 204

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL mAb HumAb747V-72

<400> 204

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ala Leu Asp Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 205

<211> 224

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb713-7

<400> 205

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 Ser Ser Asp Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr 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

<210> 206

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7b-2

Polypeptide chain #2 FIT-Ig

<400> 206

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile

35 40 45

Lys Asp Asp Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60

Asp Trp Ile Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala

65 70 75 80

Ser Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn

85 90 95

Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235

<210> 207

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-72

<400> 207

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 208

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7b-2

Polypeptide chain #3 FIT-Ig

<400> 208

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Asp His Ile Asn Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Trp Ser Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 209

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb713-7

<400> 209

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

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 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> 210

<211> 679

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7a-3

Polypeptide chain #1 FIT-Ig

<400> 210

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Asp His Ile Asn Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Trp Ser Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Leu

245 250 255

Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp Tyr Met His Trp

260 265 270

Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile Gly Trp Ile Val

275 280 285

Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe Gln Gly Lys Ala

290 295 300

Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr Leu Glu Leu Ser

305 310 315 320

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Val Tyr Gly

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly Lys

675

<210> 211

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb713-7

<400> 211

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

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 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> 212

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-73

<400> 212

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 213

<211> 243

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7a-3

Polypeptide chain #2 FIT-Ig

<400> 213

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser

35 40 45

Ser Asp Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala

65 70 75 80

Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

225 230 235 240

Lys Ser Cys

<210> 214

<211> 224

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb713-7

<400> 214

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 Ser Ser Asp Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr 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

<210> 215

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7a-3

Polypeptide chain #3 FIT-Ig

<400> 215

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 216

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-73

<400> 216

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 217

<211> 687

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7b-3

Polypeptide chain #1 FIT-Ig

<400> 217

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly

50 55 60

Ala Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Val Gln Leu

225 230 235 240

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

245 250 255

Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Asp Tyr Gly Met His Trp

260 265 270

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser

275 280 285

Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn

305 310 315 320

Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Arg Gly

325 330 335

Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly Gln Gly Thr Thr

340 345 350

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

355 360 365

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

370 375 380

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

385 390 395 400

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

405 410 415

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

420 425 430

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

435 440 445

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

450 455 460

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

465 470 475 480

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

485 490 495

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

500 505 510

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

515 520 525

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

530 535 540

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

545 550 555 560

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

565 570 575

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

580 585 590

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

595 600 605

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

610 615 620

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

625 630 635 640

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

645 650 655

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

660 665 670

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

675 680 685

<210> 218

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb747V-73

<400> 218

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 Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 219

<211> 224

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb713-7

<400> 219

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 Ser Ser Asp Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Gly Ser Tyr Thr Ile Tyr Tyr Ala Asp Thr 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 Lys Arg Gly Gly Ser Ser His Val Asn Val Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr 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

<210> 220

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7b-3

Polypeptide chain #2 FIT-Ig

<400> 220

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile

35 40 45

Lys Asp Asp Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60

Asp Trp Ile Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala

65 70 75 80

Ser Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn

85 90 95

Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235

<210> 221

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 HumAb747V-73

<400> 221

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr 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 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

210 215

<210> 222

<211> 236

<212> Protein

<213> Artificial sequence

<220>

<223> FIT107-1-7b-3

Polypeptide chain #3 FIT-Ig

<400> 222

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Asp His Ile Asn Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Trp Ser Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 223

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> VL-CL HumAb713-7

<400> 223

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 Lys Ala Ser Asp His Ile Asn Asn Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Pro Pro Tyr

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 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> 224

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR-H2 from anti-LAG-3 antibody HumAb747V-67 with G55A substitution at

numbering Kabat

<400> 224

Trp Ile Val Pro Glu Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe Gln

1 5 10 15

gly

<210> 225

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR-H2 from anti-LAG-3 antibody HumAb747V-72 or HumAb747V-73

with the replacement of G55A by numbering Kabat

<400> 225

Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe Gln

1 5 10 15

gly

<210> 226

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH with G55A replacement by Kabat numbering

<400> 226

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Glu Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 227

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> VH with G55A replacement by Kabat numbering

<400> 227

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Asp Trp Ile

35 40 45

Gly Trp Ile Val Pro Arg Asn Ala Asn Thr Val Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Val Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<---

Claims (71)

1. Bispecific polyvalent binding protein containing the first, second and third polypeptide chains, and said first polypeptide chain contains, in the direction from the amino terminus to the carboxyl terminus, (i) VL _A -CL-VH _B -CH1-Fc, where CL is fused directly to VH _B , or (ii) VH _B -CH1-VL _A -CL -Fc, where CH1 is fused directly to VL _A ; the specified second polypeptide chain contains, in the direction from the amino end to the carboxyl end, VH _A -CH1; and the specified third polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _B -CL; wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, A is a PD-1 or LAG epitope -3 and B is an epitope of PD-1 or LAG-3, provided that A and B are different, and said binding protein is capable of binding both PD-1 and LAG-3; where VH _A and VL _A respectively represent VH _LAG-3 and VL _LAG-3 and VL _B and VH _B respectively represent VL _PD-1 and VH _PD-1 if A is a LAG-3 epitope and B is a PD-1 epitope , or VL_B and VH_B are respectively VL_LAG-3 and VH_LAG-3 and VL_A and VH_Aare respectively VL_PD-1 and VH_PD-1, when B is a LAG-3 epitope and A is a PD-1 epitope, and said VH _LAG-3 and VL _LAG-3 respectively contain CDRs, namely CDR-H1, CDR-H2, CDR-H3 and CDR-L1, CDR-L2, CDR-L3 selected from the group consisting of the following CDR sets: CDR-H1 containing DDYMH (residues 31-35 of SEQ ID NO: 135), CDR-H2 containing WIVPENANTVYASKFQG (SEQ ID NO: 224), CDR-H3 containing YGDY (residues 99-102 of SEQ ID NO: 135), CDR-L1 containing RASQEISGYLS (residues 24-34 of SEQ ID NO: 138), CDR-L2 containing AASALDS (residues 50-56 of SEQ ID NO: 138), and CDR-L3 containing LQYASYPLT (residues 89-97 of SEQ ID NO: 138); CDR-H1 containing DDYMH (residues 31-35 of SEQ ID NO: 136), CDR-H2 containing WIVPRNANTVYASKFQG (SEQ ID NO: 225), CDR-H3 containing YGDY (residues 99-102 of SEQ ID NO: 136), CDR-L1 containing RASQEISGYLS (residues 24-34 of SEQ ID NO: 139), CDR-L2 containing AASALDL (residues 50-56 of SEQ ID NO: 139), and CDR-L3 containing LQYASYPLT (residues 89-97 of SEQ ID NO: : 139); or CDR-H1 containing DDYMH (residues 31-35 of SEQ ID NO: 136), CDR-H2 containing WIVPRNANTVYASKFQG (SEQ ID NO: 225), CDR-H3 containing YGDY (residues 99-102 of SEQ ID NO: 136), CDR-L1 containing RASQEISGYLS (residues 24-34 of SEQ ID NO: 117), CDR-L2 containing AASTLDS (residues 50-56 of SEQ ID NO: 117), and CDR-L3 containing LQYASYPLT (residues 89-97 of SEQ ID NO: 117); and where said VH _PD-1 and VL _PD-1 respectively contain CDRs, namely CDR-H1, CDR-H2, CDR-H3 and CDR-L1, CDR-L2, CDR-L3, which are respectively as follows: CDR-H1 containing DYGMH (residues 31-35 of SEQ ID NO: 10), CDR-H2 containing YISSGSYTIYYADTVKG (residues 50-66 of SEQ ID NO: 10), CDR-H3 containing RGGSSHVNVMDY (residues 99-110 of SEQ ID NO : 10), CDR-L1 containing KASDHINNWLA (residues 24-34 of SEQ ID NO: 11), CDR-L2 containing GATSLET (residues 50-56 of SEQ ID NO: 11), and CDR-L3 containing QQYWSPPYT (residues 89 -97 SEQ ID NO: 11). 2. Bispecific polyvalent binding protein containing the first, second and third polypeptide chains, and said first polypeptide chain contains, in the direction from the amino terminus to the carboxyl terminus, (i) VL _A -CL-VH _B -CH1-Fc, where CL is fused directly to VH _B , or (ii) VH _B -CH1-VL _A -CL -Fc, where CH1 is fused directly to VL _A ; the specified second polypeptide chain contains, in the direction from the amino end to the carboxyl end, VH _A -CH1; and the specified third polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _B -CL; wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, A is a PD-1 or LAG epitope -3 and B is an epitope of PD-1 or LAG-3, provided that A and B are different, and said binding protein is capable of binding both PD-1 and LAG-3; where VH _A and VL _A respectively represent VH _LAG-3 and VL _LAG-3 and VL _B and VH _B respectively represent VL _PD-1 and VH _PD-1 if A is a LAG-3 epitope and B is a PD-1 epitope , or VL_B and VH_B are respectively VL_LAG-3 and VH_LAG-3 and VL_A and VH_Aare respectively VL_PD-1 and VH_PD-1, when B is a LAG-3 epitope and A is a PD-1 epitope, and where said VH _LAG-3 and VL _LAG-3 , respectively, contain amino acid sequences selected from the group consisting of: SEQ ID NO: 135 and SEQ ID NO: 138, SEQ ID NO: 136 and SEQ ID NO: 139, SEQ ID NO: 136 and SEQ ID NO: 117, SEQ ID NO: 226 and SEQ ID NO: 138, SEQ ID NO: 227 and SEQ ID NO: 139 and SEQ ID NO: 227 and SEQ ID NO: 117; and wherein said PD-1 VH _{and PD-1} VL _, respectively, contain the amino acid sequences as shown in SEQ ID NO: 32 and SEQ ID NO: 35. 3. The binding protein of claim 1, wherein the VL _A -CL and VH _A -CH1 domains are derived from a parent antibody that binds to one of the target PD-1 or LAG-3 antigens, and the VL _B -CL and VH _B domains are CH1 are derived from a different parental antibody that binds to a different target PD-1 or LAG-3 antigen. 4. The binding protein according to claim 3, containing the first, second and third polypeptide chains, and the specified first polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _PD-1 -CL-VH _LAG-3 -CH1-Fc, where CL merged directly with VH _LAG-3 , moreover, the specified second polypeptide chain contains, in the direction from the amino end to the carboxyl end, VH _PD-1 -CH1; and moreover, the specified third polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _LAG-3 -CL; where VL _PD-1 is the light chain variable domain of an anti-PD-1 antibody, CL is the light chain constant domain, VH _PD-1 is the heavy chain variable domain of an anti-PD-1 antibody, CH1 is the heavy chain constant domain , VL _LAG-3 is the light chain variable domain of an anti-LAG-3 antibody, VH _LAG-3 is the heavy chain variable domain of an anti-LAG-3 antibody, and Fc is the Fc region of an immunoglobulin. 5. The binding protein of claim 4, wherein in the first polypeptide chain, the VL domains _{of PD-1} -CL are the same as the light chain of the parental anti-PD-1 antibody, the VH domains _{of PD-1} -CH1 are the same as the variable the heavy chain domain and heavy chain constant domain of the parental anti-PD-1 antibody, the VL domains _{of LAG-3} -CL are the same as the light chain of the parental anti-LAG-3 antibody, and the VH domains _{of LAG-3} -CH1 are the same as as the heavy chain variable domain and the heavy chain constant domain of the parental anti-LAG-3 antibody. 6. The binding protein according to claim 3, containing the first, second and third polypeptide chains, and the specified first polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _LAG-3 -CL-VH _PD-1 -CH1-Fc, where CL merged directly with VH _PD-1 , moreover, the specified second polypeptide chain contains, in the direction from the amino end to the carboxyl end, VH _LAG-3 -CH1; and moreover, the specified third polypeptide chain contains, in the direction from the amino end to the carboxyl end, VL _PD-1 -CL; where VL _PD-1 is the light chain variable domain of an anti-PD-1 antibody, CL is the light chain constant domain, VH _PD-1 is the heavy chain variable domain of an anti-PD-1 antibody, CH1 is the heavy chain constant domain , VL _LAG-3 is the light chain variable domain of an anti-LAG-3 antibody, VH _LAG-3 is the heavy chain variable domain of an anti-LAG-3 antibody, and Fc is the Fc region of an immunoglobulin. 7. The binding protein of claim 6, wherein in the first polypeptide chain, the VL domains _{of LAG-3} -CL are the same as the light chain of the parent anti-LAG-3 antibody, the VH domains _{of LAG-3} -CH1 are the same as the variable the heavy chain domain and heavy chain constant domain of the parent anti-LAG-3 antibody, the VL domains _{of PD-1} -CL are the same as the light chain of the parent anti-PD-1 antibody, and the VH domains _{of PD-1} -CH1 are the same as the heavy chain variable domain and the heavy chain constant domain of the parental anti-PD-1 antibody. 8. The binding protein of claim 1, wherein said first polypeptide chain contains the amino acid sequence 23-679 of SEQ ID NO: 182, said second polypeptide chain contains the amino acid sequence of SEQ ID NO: 186, and said third polypeptide chain contains the amino acid sequence of SEQ ID NO: : 188. 9. The binding protein of claim 1, wherein said first polypeptide chain contains the amino acid sequence 23-687 of SEQ ID NO: 189, said second polypeptide chain contains the amino acid sequence of SEQ ID NO: 193, and said third polypeptide chain contains the amino acid sequence of SEQ ID NO: : 195. 10. The binding protein of claim 1, wherein said first polypeptide chain contains the amino acid sequence 23-679 of SEQ ID NO: 196, said second polypeptide chain contains the amino acid sequence of SEQ ID NO: 200, and said third polypeptide chain contains the amino acid sequence of SEQ ID NO: : 202. 11. The binding protein of claim 1, wherein said first polypeptide chain contains the amino acid sequence 23-687 of SEQ ID NO: 203, said second polypeptide chain contains the amino acid sequence of SEQ ID NO: 207, and said third polypeptide chain contains the amino acid sequence of SEQ ID NO: :209. 12. The binding protein of claim 1, wherein said first polypeptide chain contains the amino acid sequence 23-679 of SEQ ID NO: 210, said second polypeptide chain contains the amino acid sequence of SEQ ID NO: 214, and said third polypeptide chain contains the amino acid sequence of SEQ ID NO: : 216. 13. The binding protein of claim 1, wherein said first polypeptide chain contains the amino acid sequence 23-687 of SEQ ID NO: 217, said second polypeptide chain contains the amino acid sequence of SEQ ID NO: 221, and said third polypeptide chain contains the amino acid sequence of SEQ ID NO: :223. 14. An anti-LAG-3 antibody or antigen-binding portion thereof capable of binding human LAG-3, wherein the antibody or antigen-binding portion of the antibody comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR- L2 and CDR-L3 selected from the group consisting of the following CDR sets: CDR-H1 consisting of DDYMH (residues 31-35 of SEQ ID NO: 135), CDR-H2 consisting of WIVPENANTVYASKFQG (SEQ ID NO: 224), CDR-H3 consisting of YGDY (residues 99-102 of SEQ ID NO: 135), a CDR-L1 consisting of RASQEISGYLS (residues 24-34 of SEQ ID NO: 138), a CDR-L2 consisting of AASALDS (residues 50-56 of SEQ ID NO: 138), and a CDR-L3 consisting of LQYASYPLT ( residues 89-97 SEQ ID NO: 138); CDR-H1 consisting of DDYMH (residues 31-35 of SEQ ID NO: 136), CDR-H2 containing WIVPRNANTVYASKFQG (SEQ ID NO: 225), CDR-H3 consisting of YGDY (residues 99-102 of SEQ ID NO: 136 ), a CDR-L1 consisting of RASQEISGYLS (residues 24-34 of SEQ ID NO: 139), a CDR-L2 consisting of AASALDL (residues 50-56 of SEQ ID NO: 139), and a CDR-L3 consisting of LQYASYPLT (residues 89-97 SEQ ID NO: 139); or CDR-H1 consisting of DDYMH (residues 31-35 of SEQ ID NO: 136), CDR-H2 consisting of WIVPRNANTVYASKFQG (SEQ ID NO: 225), CDR-H3 consisting of YGDY (residues 99-102 of SEQ ID NO: 136), a CDR-L1 consisting of RASQEISGYLS (residues 24-34 of SEQ ID NO: 117), a CDR-L2 consisting of AASTLDS (residues 50-56 of SEQ ID NO: 117), and a CDR-L3 consisting of LQYASYPLT ( residues 89-97 of SEQ ID NO: 117). 15. An anti-LAG-3 antibody or antigen-binding portion thereof capable of binding human LAG-3, wherein the antibody or antigen-binding portion of the antibody comprises a pair of VH and VL domains, where the pair of VH and VL domains contains amino acid sequences selected from the group consisting of: SEQ ID NO: 135 and SEQ ID NO: 138, SEQ ID NO: 136 and SEQ ID NO: 139, SEQ ID NO: 136 and SEQ ID NO: 117, SEQ ID NO: 226 and SEQ ID NO: 138, SEQ ID NO: 227 and SEQ ID NO: 139 and SEQ ID NO: 227 and SEQ ID NO: 117. 16. An antibody according to any one of paragraphs. 14, 15 or a binding protein according to any one of paragraphs. 1-13 containing the Fc region containing SEQ ID NO: 28. 17. A pharmaceutical composition comprising at least one anti-LAG-3 antibody or antigen-binding fragment thereof according to claim 14 in an effective amount and a pharmaceutically acceptable carrier, for the treatment of a disease or disorder in which LAG-3 mediated activity is detrimental. 18. A pharmaceutical composition containing at least one bispecific binding protein according to claim 1 in an effective amount and a pharmaceutically acceptable carrier, for the treatment of a disease or disorder in which PD-1 mediated activity and/or LAG-3 mediated activity, is harmful. 19. The use of a bespecifically binding protein according to claim 1 for the treatment of a disease or disorder in which PD-1 mediated activity and/or LAG-3 mediated activity is detrimental. 20. Use according to claim 19, wherein said disease is cancer. 21. Use according to claim 20, wherein the cancer is melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cancer cervical, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, or primary bone cancer. 22. Use according to claim 21, wherein the melanoma is a metastatic malignant melanoma, the renal cancer is a clear cell carcinoma, the prostate cancer is a hormone-resistant adenocarcinoma of the prostate, the lung cancer is a non-small cell lung cancer, or the primary bone cancer is an osteosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma and chondrosarcoma. 23. A method of manufacturing a drug for the treatment of a disease or disorder in which PD-1 mediated activity and/or LAG-3 mediated activity is detrimental, comprising preparing at least one bispecific binding protein according to claim 1 with a pharmaceutically acceptable carrier. 24. The method of claim 23, wherein said disease is cancer. 25. The method of claim 24 wherein the cancer is melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cancer cervical, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, or primary bone cancer. 26. The method of claim 25, wherein the melanoma is metastatic malignant melanoma, the renal cancer is clear cell carcinoma, the prostate cancer is hormone resistant prostate adenocarcinoma, the lung cancer is non-small cell lung cancer, or the primary bone cancer is osteosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma and chondrosarcoma. 27. A method of treating a disorder in which PD-1 and/or LAG-3 mediated activity is detrimental, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to claim 17 or 18, or a combination thereof. 28. The method of claim 27, wherein said disease is cancer. 29. The method of claim 28, wherein the cancer is melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cancer cervical, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, or primary bone cancer. 30. The method of claim 29, wherein the melanoma is metastatic malignant melanoma, the renal cancer is clear cell carcinoma, the prostate cancer is hormone resistant prostate adenocarcinoma, the lung cancer is non-small cell lung cancer, or the primary bone cancer is osteosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma and chondrosarcoma. 31. The method of any one of claims 27-30, wherein the subject is a human.

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