EA043373B1 - BISPECIFIC MOLECULES WITH IMMUNOREACTIVITY AGAINST PD-1 AND CTLA-4, AND METHODS OF THEIR APPLICATION - Google Patents

Description

Cross reference to related applications

This application is a continuation in part of, and claims priority to, U.S. Patent Application Serial No. 62/266,944 (filed December 14, 2015, pending), which is incorporated herein in its entirety.

Link to sequence list

This application includes one or more sequence listings in accordance with the Code of Federal Regulations of the United States (C.F.R., 37, 1.821 et seq.), which are presented on machine-readable media (file name: 1301_0134PCT_ST25.txt, created December 4, 2016, size - 186,040 bytes); said file is incorporated herein by reference in its entirety.

Field of technology

The present invention provides bispecific molecules (e.g., diabodies, bispecific antibodies, trivalent binding molecules, etc.) that contain at least one epitope binding site that is immunospecific for a PD-1 epitope and at least one binding site an epitope that is immunospecific for the CTLA-4 epitope (i.e., molecules bispecific for PD-1 x CTLA-4). The present invention provides such molecules, bispecific for PD-1 x CTLA-4, containing two epitope binding sites having immune specificity for one (or two) PD-1 epitope(s), and two epitope binding sites having immune specificity for against one (or two) CTLA-4 epitope(s). The present invention also provides such PD-1 x CTLA-4 bispecific molecules that additionally contain an immunoglobulin Fc moiety. The PD-1 x CTLA-4 bispecific molecules of the present invention are capable of simultaneously binding to PD-1 and CTLA-4, in particular when said molecules are located on the surface of human cells. The invention relates to pharmaceutical compositions that contain said PD-1 x CTLA-4 bispecific molecules, and methods including the use of said bispecific molecules in the treatment of cancer and other diseases and conditions. The present invention also provides methods including the use of such PD-1 x CTLA-4 bispecific molecules to stimulate an immune response.

State of the art

I. Immune system response to cancer.

The mammalian immune system has the natural ability to recognize and destroy cancer cells (Topalian, S.L. et al. (2015) Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy Cancer Cell 27:450-461). The immune system of healthy subjects is quiescent and its activity is suppressed by a combination of various inhibitory receptors and ligands. Such immune checkpoint pathways are important in maintaining autotolerance (ie, preventing a subject's immune system from attacking his/her own autoimmune cells) and limiting collateral tissue damage during antimicrobial or antiallergic immune responses. Following recognition of a cancer antigen, detection of a microbial pathogen, or the presence of an allergen, a series of activating receptors and ligands cause activation of the immune system. This activation results in the activation of macrophages, natural killer (NK) cells, and antigen-specific cytotoxic T cells and stimulates the release of various cytokines, which act to combat the detected threat to the subject's health (Dong, C. et al. (2003) ) Immune Regulation by Novel Costimulatory Molecules, Immunolog. Res. 28(1):39-48; Viglietta, V. et al. (2007) Modulating Co-Stimulation Neurotherapeutics 4:666-675; Korman, A. J. et al. (2007) ) Checkpoint Blockade in Cancer Immunotherapy Adv. Immunol. 90:297-339). The immune system is able to return to its normal resting state when compensatory inhibitory immune signals prevail over activating immune signals.

Thus, the pathological condition in cancer (and, in fact, the pathological condition in infectious diseases) can be considered to reflect an inability to adequately activate the immune system of the subject. Such failure may reflect inadequate presentation of activating immune signals or inadequate ability to attenuate inhibitory immune signals in the subject. Based on some examples, researchers have determined that cancer cells can evade detection by the immune system (Topalian, S.L. et al. (2015) Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy Cancer Cell 27:450-461).

Of particular importance is the binding between ligands B7-1 (CD80) and B7-2 (CD86), which are expressed on the surface of the antigen-presenting cell, and the CD28 and CTLA-4 receptors of the CD4 ⁺ T lymphocyte (Sharpe, AH et al. (2002) The B7-CD28 Superfamily Nature Rev. Immunol. 2:116-126; Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules Immunolog. Res. 28(1):39-48; Lindley, PS et al. (2009) The Clinical Utility of Inhibiting CD28-Mediated Costimulation Immunol Rev 229:307-321). Binding of B7-1 or B7-2 to CD28 stimulates T cell activation; binding of B7-1 or B7-2 to CTLA-4 inhibits T cell activation (Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules Immunolog. Res. 28(1):39-48; Lindley, P.S. et al. (2009) The Clinical Utility Of Inhibition- 1 043373 ing CD28-Mediated Costimulation Immunol. Rev. 229:307-321; Greenwald, R. J. et al. (2005) The B7 Family Revisited, Ann. Rev. Immunol. 23 :515-548). CD28 is constitutively expressed on the surface of T cells (Gross, J., et al. (1992) Identification And Distribution Of The Costimulatory Receptor CD28 In The Mouse J. Immunol. 149:380-388), and CTLA-4 expression is rapidly increased after T activation -cells (Linsley, P. et al. (1996) Intracellular Trafficking Of CTLA4 And Focal Localization Towards Sites Of TCR Engagement Immunity 4:535-543). Because the CTLA-4 receptor has higher affinity (Sharpe, A.H. et al. (2002) The B7-CD28 Superfamily Nature Rev. Immunol. 2:116-126; Topalian, S.L. et al. (2015) Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy Cancer Cell 27:450-461), ligand binding first leads to T cell proliferation (via CD28) and then its inhibition (as a result of the onset of CTLA-4 expression), thus causing a suppressive effect, when proliferation is no longer required.

II. CTLA-4.

Cytotoxic T-lymphocyte associated protein-4 (CTLA-4; CD152) is a type I membrane protein with a single transmembrane domain that forms a disulfide-linked homodimer (Schwartz J.C., et al. (2001) Structural Basis For Co-Stimulation By The Human CTLA-4/B72 Complex Nature 410:604–608). Alternative splice variants have been characterized encoding various isoforms, including a soluble isoform that functions as a monomer (Magistrelli G., et al. (1999) A Soluble Form Of CtLA-4 Generated By Alternative Splicing Is Expressed By Nonstimulated Human T Cells Eur. J Immunol 29:3596-3602 Oaks MK et al (2000) A Native Soluble Form of CTLA-4, Cell Immunol 201:144-153).

CTLA-4 is primarily an intracellular antigen, the expression of which on the cell surface is strictly regulated by limited migration to the cell surface and rapid internalization (Alegre M-L, et al., (1996) Regulation Of Surface And Intracellular Expression Of CTLA4 OnMouse T Cells J. Immunol 157:4762-4770 Linsley, P. S. et al (1996) Intracellular Trafficking Of CTLA-4 And Focal Localization Towards Sites Of TCR Engagement Immunity 4:535-543). CTLA-4 is expressed at low levels on the surface of naive effector T cells (Alegre, M.L., et al. (1996) Regulation Of Surface And Intracellular Expression Of CTLA4 On Mouse T Cells J Immunol 157:4762-70) and is constitutively expressed on regulatory T cells (Wang, X.B., et al. (2002) Expression Of CTLA-4 By Human Monocytes Scand. J. Immunol. 55:53-60).

The extracellular region of CTLA-4 contains a single extracellular Ig(V) domain, followed by a transmembrane region (TM) and a small intracellular cytoplasmic tail (37 amino acids). The intracellular tail contains two tyrosine motifs that interact with several intracellular proteins, including the lipid kinase phosphatidylinositol 3-kinase (PI3K), the phosphatases SHP-2 and PP2A, and the clathrin adapter proteins AP-1 and AP-2 (Rudd, C.E. et al. ( 2003) Unifying Concepts In CD28, ICOS And CTLA4 Co-Receptor Signaling, Nat Rev Immunol. 3:544-56). CTLA-4 is close to CD28, with approximately 29% amino acid identity between the two proteins (Harper, K. (1991) CTLA-4 And CD28 Activated Lymphocyte Molecules Are Closely Related In Mouse And Human As To Sequence, Message Expression, Gene Structure, And Chromosomal Location J Immunol 147:1037–1044).

When a naive effector T cell is activated through its T cell receptors (TCRs), CTLA-4 is recruited to the cell surface (Linsley, P.S., et al. (1996) Intracellular Trafficking Of CTLA-4 And Focal Localization Towards Sites Of TCR Engagement Immunity 4 :535-43). Once CTLA-4 begins to be expressed on the surface of T cells, it competes with CD28 (constitutively expressed on T cells) for binding to CD80/CD86, thereby turning off further TCR-mediated signaling and thus inhibiting any further development of T -cellular response via the TCR signaling pathway (Carreno, B.M., et al. (2000) CTLA-4 (CD152) Can Inhibit T Cell Activation By Two Different Mechanisms Depending On Its Level Of Cell Surface Expression, J Immunol 165:1352-6; Chuang , E., et al (1999) Regulation Of Cytotoxic T Lymphocyte-Associated Molecule-4 By Src Kinases, J Immunol 162:1270-7). Thus, CTLA-4 acts as a negative regulator of effector T cell activation, which reduces effector function and determines the efficiency and duration of the T cell response (Linsley, P.S., et al. (1996) Intracellular Trafficking Of CTLA-4 And Focal Localization Towards Sites Of TCR Engagement Immunity 4:535-43).

In addition, CTLA-4 may play a role in enhancing the negative influence of regulatory T cells in the immune response to cancer (Tai, Y.T., et al., (2012) Potent in vitro And in vivo Activity Of An Fc-Engineered Humanized Anti-HM1 24 Antibody Against Multiple Myeloma via Augmented Effector Function, Blood 119:2074-82). CTLA-4 has a much higher affinity for B7 family proteins compared to CD28 and thus its expression on a T cell determines the predominance of negative regulation of that T cell (Allison, J.P., et al. (1995) Manipulation Of Costimulatory Signals To Enhance Antitumor T-Cell Responses, Curr Opin Immunol 7:682-6). The mechanism by which CTLA-4 participates in the suppressor function of regulatory T cells is not fully understood, but expression of CTLA on non-regulatory T cells enhances the suppressor function of these cells (Tai, Y.T., et al., (2012) Potent in vitro And in vivo Activity Of An Fc-Engineered Humanized Anti-HM1.24 Antibody Against Multiple Myeloma via Aug

- 2 043373 mented Effector Function; Blood 119:2074-82).

Blocking CTLA-4 has been reported to enhance T cell responses in vitro (Walunas, T.L., et al. (1994) CTLA-4 Can Function As A Negative Regulator Of T Cell Activation, Immunity 1:405-413) and in vivo ( Kearney, E.R., et al (1995) Antigen-Dependent Clonal Expansion Of A Trace Population Of Antigen-Specific CD4+ T Cells in Vivo Is Dependent On CD28 Costimulation And Inhibited By CTLA-4, J. Immunol. 155:10321036).

It has therefore been suggested that blocking CTLA-4 using anti-CTLA-4 antibodies could provide new treatments for diseases, particularly human diseases where stimulation of the immune system would have a beneficial effect, such as cancer and infectious diseases. diseases (see Leach, D.R. et al. (1996) Enhancement Of Antitumor Immunity By CTLA-4 Blockade Science 271:1734-1736 and international publications according to PCT No. WO 01/14424; WO 00/37504). The development of CTLA-4 function blockers has focused on the use of monoclonal antibodies such as ipilimumab (see, for example, Hodi, F.S., et al., (2003) Biologic Activity Of Cytotoxic T Lymphocyte-Associated Antigen 4 Antibody Blockade In Previously Vaccinated Metastatic Melanoma And Ovarian Carcinoma Patients Proc. Natl. Acad. Sci. (USA) 100:4717-4717) and tremelimumab (Ribas, A. et al. (2005) Antitumor Activity In Melanoma And Anti-Self Responses In A Phase I Trial With The Anti -Cytotoxic T Lymphocyte-Associated Antigen 4 Monoclonal Antibody CP-675,206 Oncologist 12: 873883).

III. Programmed death receptor 1 (PD-1).

Programmed death receptor 1 (PD-1, also known as CD279), is a type I membrane protein of the large CD28/CTLA-4 family of T cell regulators that negatively regulate a wide range of immune responses (Ishida, Y. et al. ( 1992) Induced Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death, EMBO J. 11:3887-3895; US Patent Application Publications No. 2007/0202100; 2008/0311117; 2009/00110667; US Patents No. 6,808,710; 7,101,550; 7,488,802; 7,635,757; 7,722,868; PCT International Publication No. WO 01/14557).

The receptor-ligand interactions of the PD-1 system appear to be even more complex than those of the CD28/CTLA-4 system. PD-1 is expressed on the cell surface of activated T cells, B cells and monocytes (Agata, Y. et al. (1996) Expression Of The PD-1 Antigen On The Surface Of Stimulated Mouse T And B Lymphocytes Int. Immunol. 8 (5):765-772; Yamazaki, T. et al. (2002) Expression Of Programmed Death 1 Ligands By Murine T-Cells And APC J. Immunol. 169:5538-5545), and is also expressed at low levels in T -natural killer (NK) cells (Nishimura, H. et al. (2000) Facilitation Of Beta Selection And Modification Of Positive Selection In The Thymus Of PD-1Deficient Mice, J. Exp. Med. 191:891-898 Martin-Orozco, N. et al (2007) Inhibitory Costimulation And Anti-Tumor Immunity Semin. Cancer Biol. 17(4):288-298).

The extracellular region of PD-1 consists of a single immunoglobulin (Ig)V domain, 23% identical to the equivalent domain of CTLA-4 (Martin-Orozco, N. et al. (2007) Inhibitory Costimulation And Anti-Tumor Immunity Semin. Cancer Biol. 17 (4):288-298). The extracellular domain of IgV is followed by a transmembrane domain and an intracellular tail. The intracellular tail contains two phosphorylation sites located at the immunoreceptor tyrosine-based inhibitory motif and the immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively regulates TCR signaling (Ishida, Y. et al. (1992) Induced Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death EMBO J. 11:3887-3895 Blank, C. et al (2006) Contribution Of The PD-L1/PD-1 Pathway To T -CeII Exhaustion: An Update On Implications For Chronic Infections And Tumor Evasion Cancer, Immunol. Immunother. 56(5):739-745).

PD-1 exerts immune system inhibition by binding to B7-H1 and B7-DC (also known as PD-L1 and PD-L2, Flies, D.B. et al. (2007) The New B7s: Playing a Pivotal Role in Tumor Immunity, J. Immunother 30(3):251-260; US Patent Nos. 6,803,192; 7,794,710; US Patent Application Publications No. 2005/0059051; 2009/0055944; 2009/0274666; 2009/0313687; International PCT Publications No. WO 01/39722; WO 02/086083).

B7-H1 and B7-DC are widely expressed on the surface of many types of human and mouse tissues, such as heart, placenta, muscle, fetal liver, spleen, lymph nodes and thymus, as well as liver, lung and kidney, pancreatic islet cells and mouse small intestine (Martin-Orozco, N. et al. (2007) Inhibitory Costimulation And Anti-Tumor Immunity, Semin. Cancer Biol. 17(4):288-298). In humans, B7-H1 protein expression has been found in endothelial cells (Chen, Y. et al. (2005) Expression of B7-H1 in Inflammatory Renal Tubular Epithelial Cells Nephron. Exp. Nephrol. 102:e81-e92; de Haij, S et al (2005) Renal Tubular Epithelial Cells Modulate T-Cell Responses Via ICOS-L And B7-H1 Kidney Int 68:2091-2102 Mazanet, M. M. et al (2002) B7-H1 Is Expressed By Human Endothelial Cells And Suppresses T-Cell Cytokine Synthesis J. Immunol. 169:3581-3588), myocardium (Brown, J.A. et al. (2003) Blockade Of Programmed Death-1 Ligands On Dendritic Cells Enhances T-Cell Activation And Cytokine Production J. Immunol. 170:1257-1266), syncytial layer of trophoblast (Petroff, M.G. et al. (2002) B7 Family Molecules: Novel Immunomodulators At The Maternal-Fetal Interface Placenta 23:S95-S101).

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These molecules are also expressed by resident macrophages of some tissues, macrophages activated by interferon (IFN)-γ or tumor necrosis factor (TNF)-α (Latchman, Y. et al.

(2001) PD-L2 Is a Second Ligand For PD-1 And Inhibits T-Cell Activation, Nat. Immunol 2:261-268), and in tumors (Dong, H. (2003) B7-H1 Pathway And Its Role In The Evasion Of Tumor Immunity J. Mol. Med.

81:281-287).

It was found that the interaction between B7-H1 and PD-1 leads to the transmission of a critical costimulatory negative signal to T and B cells (Martin-Orozco, N. et al. (2007) Inhibitory Costimulation And Anti-Tumor Immunity Semin. Cancer Biol. 17(4):288-298) and acts as an inducer of cell death (Ishida, Y. et al. (1992) Induced Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death, EMBO J. 11:3887-3895; Subudhi, SK et al. (2005) The Balance Of Immune Responses: Costimulation Verse Coinhibition, J. Molec. Med. 83:193-202). More specifically, the interaction between the PD-1 receptor and the B7-H1 ligand at low concentrations was found to result in the transmission of an inhibitory signal that strongly suppresses the proliferation of antigen-specific CD8 ⁺ T cells; at higher concentrations, interaction with PD-1 does not inhibit T cell proliferation but significantly reduces the production of multiple cytokines (Sharpe, AH et al. (2002) The B7-CD28 Superfamily Nature Rev. Immunol. 2:116-126). Proliferation and cytokine production in both resting and previously activated CD4 and CD8 T cells, and even naïve T cells from umbilical cord blood, have been found to be suppressed by soluble B7H1-Fc fusion proteins (Freeman, G. J. et al. (2000) Engagement Of The PD-1 Immunoinhibitory Receptor By A Novel B7 Family Member Leads To Negative Regulation Of Lymphocyte Activation J Exp Med 192:1-9 Latchman, Y et al (2001) PD-L2 Is A Second Ligand For PD-1 And Inhibits T-Cell Activation, Nature Immunol.2:261268;Carter, L. et al. (2002) PD-LPD-L Inhibitory Pathway Affects Both CD4(+) and CD8(+) T-cells And Is Overcome By IL-2, Eur J Immunol 32(3):634-643; Sharpe, AH et al (2002) The B7-CD28 Superfamily, Nature Rev Immunol 2:116-126).

The involvement of B7-H1 and PD-1 in suppressing T cell activation and proliferation indicates that these biomolecules may serve as therapeutic targets for the treatment of inflammation and cancer. In this regard, the use of anti-PD-1 antibodies has been proposed for the treatment of infections and tumors and for the positive regulation of the acquired immune response (see US patent application publications No. 2010/0040614; 2010/0028330; 2004/0241745; 2008/0311117; 2009 /0217401; US patents No. 7,521,051; 7,563,869; 7,595,048; PCT international publications No. WO 2004/056875; WO 2008/083174). Antibodies that can specifically bind to PD-1 have been described (Agata, T. et al. (1996) Expression Of The PD-1 Antigen On The Surface Of Stimulated Mouse T And B Lymphocytes Int. Immunol. 8(5):765- 772; and Berger, R. et al. (2008) Phase I Safety And Pharmacokinetic Study Of CT-011, A Humanized Antibody Interacting With PD-1, In Patients With Advanced Hematologic Malignancies Clin. Cancer Res. 14(10):30443051 ; see also US Patent Nos. 8,008,449 and 8,552,154; US Patent Publications No. 2007/0166281; 2012/0114648; 2012/0114649; 2013/0017199; 2013/0230514 and 2014/0044738 and international patents publications according to PCT No. WO 2003/099196; WO 2004/004771; WO 2004/056875; WO 2004/072286; WO 2006/121168; WO 2007/005874; WO 2008/083174; WO 2009/014708; WO 2 009/073533; WO 2012/135408, WO 2012/145549 and WO 2013/014668).

However, despite all such advances in the art, there remains a need for improved compositions capable of more actively targeting the body's immune system against cancer cells or pathogen-infected cells, particularly at lower therapeutic concentrations and/or with reduced side effects. Although adaptive immunity can be a powerful defense mechanism against cancer and disease, its action is often hampered by immunosuppressive/immune evasion mechanisms in the tumor microenvironment, such as PD-1 and CTLA-4 expression. Moreover, co-inhibitory molecules expressed by tumor cells, immune cells, and stromal cells in the tumor environment can significantly dampen T cell responses against cancer cells. In addition, the use of anti-CTLA-4 antibodies causes well-known side effects called immune-mediated adverse events (iAEs). Immune-mediated adverse events (iAEs) include colitis/diarrhea, dermatitis, hepatitis, endocrinopathies and inflammatory myopathy. These isolated side effects have been described to occur due to a breakdown of immune tolerance when blocking CTLA-4 (Di Giacomo, A.M., et al. (2010) The Emerging Toxicity Profiles Of Anti-CTLA-4 Antibodies Across Clinical Indications Semin Oncol. 37 :499-507). Thus, therapeutic treatments that avoid these limitations would be of great advantage.

As described in detail below, the present invention solves the described problem by providing PD-1 x CTLA-4 bispecific molecules. Such bispecific molecules are capable of binding to PD-1 and CTLA-4 molecules that are present on the surface of exhausted and tolerant tumor-infiltrating lymphocytes and other cell types, and thus impairing the ability of such cell surface molecules to respond to the corresponding ligands. Thus, the PD-1 x CTLA-4 bispecific molecules of the present invention act by blocking PD-1 and CTLA-4 mediated suppression of the immune system to promote activation or

- 4 043373 continued activation of the immune system. These properties enable the use of such bispecific molecules in stimulating the immune system and, in particular, in the treatment of cancer and pathogen-related diseases and conditions. The present invention is aimed at achieving these and other objectives.

Brief description of the invention

The present invention is directed to bispecific molecules (e.g., diabodies, bispecific antibodies, trivalent binding molecules, etc.) containing at least one binding site having immunospecificity for a PD-1 epitope and at least one binding site having immunospecificity for the CTLA-4 epitope (i.e. bispecific for PD-1 x CTLA-4 molecules). The present invention provides such PD-1 x CTLA-4 bispecific molecules that contain two epitope binding sites that are immunospecific for one (or two) PD-1 epitope(s) and two epitope binding sites that are immune specific. against one (or two) CTLA-4 epitope(s). The present invention is also directed to such PD-1 x CTLA-4 bispecific molecules that further comprise an immunoglobulin Fc moiety. The PD-1 x CTLA-4 bispecific molecules of the present invention are capable of simultaneously binding to PD-1 and CTLA-4, particularly as these molecules are distributed on the surface of human cells. The invention is directed to pharmaceutical compositions that contain such PD-1 x CTLA-4 bispecific molecules, and methods including the use of such bispecific molecules for the treatment of cancer and other diseases and conditions. The present invention also provides methods including the use of such PD-1 x CTLA-4 bispecific molecules to stimulate an immune response.

In more detail, the invention provides a bispecific molecule comprising one or more epitope binding sites capable of immunospecifically binding to PD-1 epitope(s), and one or more epitope binding sites capable of immunospecifically binding to CTLA-4 epitope(s), wherein said molecule contains:

(A) a heavy chain variable domain and a light chain variable domain of an antibody that binds to PD-1; and (B) a heavy chain variable domain and a light chain variable domain of an antibody that binds CTLA-4;

wherein said bispecific binding molecule is:

(i) a diabody, wherein said diabody is a covalently linked complex that contains two, three, four or five polypeptide chains; or (ii) a trivalent binding molecule, wherein said trivalent binding molecule is a covalently linked complex that contains three, four, five or more polypeptide chains.

The invention relates to an embodiment of such bispecific molecules, wherein said bispecific binding molecule exhibits activity greater than that of two monospecific molecules, one of which contains a heavy chain variable domain and a light chain variable domain of an antibody that binds PD-1, and the other of which contains a heavy chain variable domain and a light chain variable domain of an antibody that binds to CTLA-4.

The invention provides an embodiment of all such bispecific molecules, wherein the molecule causes fewer immune-mediated adverse events (iAEs) when administered to a subject requiring said administration compared to iAEs caused by administration of a monospecific antibody that binds to CTLA-4, such as ipilimumab .

The invention further relates to an embodiment of such bispecific molecules, wherein said molecule contains an Fc moiety. The invention further relates to an embodiment of such bispecific molecules, wherein said Fc fragment is a variant Fc fragment that contains:

(A) one or more amino acid modifications that reduce the affinity of the Fc fragment variant for FcyR, and/or (B) one or more amino acid modifications that increase the serum half-life of the Fc fragment variant.

The invention further relates to an embodiment of such bispecific molecules, where modifications leading to a decrease in the affinity of the specified Fc fragment variant for FcyR include substitution of L234A; L235A or L234A and L235A, where the numbering corresponds to the EC index according to Kabat.

The invention further relates to an embodiment of such bispecific molecules, where modifications resulting in an increase in the serum half-life of the Fc fragment variant include substitution of M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E or K288D and H435K, where the numbering corresponds to the Kabat EC index.

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The invention further provides an embodiment of all such bispecific molecules, wherein the molecule is a diabody and contains two epitope binding sites capable of immunospecifically binding to a PD-1 epitope and two epitope binding sites capable of immunospecifically binding to a CTLA-4 epitope.

The invention further provides an embodiment of all such bispecific molecules, wherein the molecule is a trivalent binding molecule and contains two epitope binding sites capable of immunospecifically binding to a PD-1 epitope and one epitope binding site capable of immunospecifically binding to a CTLA-4 epitope.

The invention further relates to an embodiment of all such bispecific molecules, wherein the molecule is capable of binding to PD-1 and CTLA-4 molecules present on the cell surface.

The invention further relates to an embodiment of all such bispecific molecules, wherein the molecule is capable of simultaneously binding to PD-1 and CTLA-4.

The invention further relates to an embodiment of all such bispecific molecules, wherein said molecule is capable of stimulating cells of the immune system, in particular wherein said stimulation results in:

(A) proliferation of immune cells, and/or (B) production and/or release by immune cells of at least one cytokine, and/or (C) production and/or release by immune cells of at least one lytic molecule, and/or ( D) expression by immune cells of at least one activation marker.

The invention further relates to an embodiment of all such bispecific molecules wherein the immune system cell is a T lymphocyte or a natural killer cell (NK cell).

The invention further provides an embodiment of all such bispecific molecules, wherein the epitope binding sites capable of immunospecifically binding to the PD-1 epitope comprise:

(A) VH domain of PD-1 mAb 1 (SEQ ID NO: 47) and VL domain of PD-1 mAb 1 (SEQ ID NO: 48); or (b) the VH domain of PD-1 mAb 2 (SEQ ID NO: 49) and the VL domain of PD-1 mAb 2 (SEQ ID NO: 50); or (C) the VH domain of PD-1 mAb 3 (SEQ ID NO: 51) and the VL domain of PD-1 mAb 3 (SEQ ID NO: 52); or (D) the VH domain of PD-1 mAb 4 (SEQ ID NO: 53) and the VL domain of PD-1 mAb 4 (SEQ ID NO: 54); or (E) VH domain of PD-1 mAb 5 (SEQ ID NO: 55) and VL domain of PD-1 mAb 5 (SEQ ID NO: 56); or (F) VH domain of PD-1 mAb 6 (SEQ ID NO: 57) and VL domain of PD-1 mAb 6 (SEQ ID NO: 58); or (G) the VH domain of PD-1 mAb 6-I VH (SEQ ID NO: 86) and the VL domain of PD-1 mAb 6-SQ VL (SEQ ID NO: 87); or (H) VH domain of PD-1 mAb 7 (SEQ ID NO: 59) and VL domain of PD-1 mAb 7 (SEQ ID NO: 60); or (I) VH domain anti-PD-1 mAb 8 (SEQ ID NO: 61) and VL domain anti-PD-1 mAb 8 (SEQ ID NO: 62)

The invention further provides an embodiment of all such bispecific molecules, wherein the epitope binding site(s) capable of immunospecifically binding to a CTLA-4 epitope comprises:

(A) VH domain CTLA-4 mAb 1 (SEQ ID NO: 76) and VL domain anti-CTLA-4 mAb (SEQ ID NO: 77); or (B) the VH domain of CTLA-4 mAb 2 (SEQ ID NO: 78) and the VL domain of CTLA-4 mAb 2 (SEQ ID NO: 79); or (C) the VH domain of CTLA-4 mAb 3 (SEQ ID NO: 90) and the VL domain of CTLA-4 mAb 3 (SEQ ID NO: 91).

The invention further relates to such bispecific molecules, where:

(A) Epitope binding sites capable of immunospecifically binding to the PD-1 epitope contain the VH domain of PD-1 mAb 6-I VH (SEQ ID NO: 86) and the VL domain of PD-1 mAb 6-SQ (SEQ ID NO : 87) and (B) the epitope binding site(s) capable of immunospecifically binding to the CTLA-4 epitope comprise(s) the VH domain of CTLA-4 mAb 3 (SEQ ID NO: 90) and the VL domain of CTLA-4 mAb 3 (SEQ ID NO: 91).

The invention further relates to an embodiment of all of these bispecific molecules, wherein said molecule contains:

(A) two polypeptide chains having the sequence SEQ ID NO: 95, and two polypeptide chains having the sequence SEQ ID NO: 96; or (B) two polypeptide chains having the sequence SEQ ID NO: 97 and two polypeptide chains having the sequence SEQ ID NO: 98; or (C) two polypeptide chains having the sequence SEQ ID NO: 99 and two polypeptide chains having the sequence SEQ ID NO: 100; or (D) two polypeptide chains having the sequence SEQ ID NO: 102 and two polypeptide chains having the sequence SEQ ID NO: 103; or (E) two polypeptide chains having the sequence SEQ ID NO: 101 and two polypeptide chains having the sequence SEQ ID NO: 100; or

- 6 043373 (F) one polypeptide chain having the sequence SEQ ID NO: 104, one polypeptide chain having the sequence SEQ ID NO: 105, one polypeptide chain having the sequence SEQ ID NO: 106, and one polypeptide chain having the sequence SEQ ID NO:

107; or (G) one polypeptide chain having the sequence SEQ ID NO: 108, one polypeptide chain having the sequence SEQ ID NO: 105, one polypeptide chain having the sequence SEQ ID NO: 109, and one polypeptide chain having the sequence SEQ ID NO: : 107.

The invention further relates to such bispecific molecules, wherein said molecule comprises an albumin binding domain, in particular a deimmunized albumin binding domain.

The invention further relates to a pharmaceutical composition that contains an effective amount of any of such bispecific molecules and a pharmaceutically acceptable carrier.

The invention further relates to the use of such a pharmaceutical composition or any of the bispecific molecules described above for stimulating an immune response in a subject in need thereof, in particular wherein said molecule is capable of stimulating cells of the immune system and, in particular, stimulating natural killer cells (NK cells). ) and/or T lymphocytes. The invention, in particular, relates to embodiments where said stimulation leads to proliferation of immune cells, production and/or release of cytokines by immune cells (for example, IFNy, IL-2, TNFa, etc.), production and/or release by immune cells cells lytic molecules (eg granzyme, perforin, etc.) and/or immune cell expression of activation markers (eg CD69, CD25, CD107a, etc.). The invention further provides methods for treating cancer or other diseases, comprising the use or administration of any of the PD-1 x CTLA-4 bispecific molecules described above to stimulate an immune system-mediated response. The invention particularly relates to embodiments in which the immune system stimulating activity of any of the PD-1 x CTLA-4 bispecific molecules described above exceeds the activity provided by co- or combined administration of a separate anti-PD-1 antibody and a separate anti-CTLA antibody. -4 (particularly where such antibodies are monospecific for said molecules). The invention also relates to embodiments in which increased proliferation, altered production and/or release of cytokines (e.g., IFNy, IL-2, TNFa, etc.), altered production and/or release of lytic molecules, and/or altered expression of activation markers compared to such cells stimulated with co- or combined administration of a single antibodies against PD-1 and a separate antibody against CTLA-4. The invention also provides embodiments in which the PD-1 x CTLA-4 bispecific molecules described above are less likely to cause immune-mediated adverse events (iAEs). The invention further provides embodiments in which any of the PD-1 x CTLA-4 bispecific molecules described above are used to treat a disease or condition associated with immune suppression, particularly cancer or infection.

The invention further relates to such use for the treatment of a disease or condition associated with suppression of the immune system, or use in the treatment of the specified disease or condition. The invention particularly relates to such use in the treatment of a disease or condition associated with suppression of the immune system, or to such use where the disease or condition is cancer or infection (in particular, an infection characterized by the presence of a bacterial, fungal, viral or protozoan pathogen).

The invention relates in particular to applications where:

(A) said use is the treatment of cancer, and said cancer is characterized by the presence of a cancer cell selected from the group consisting of a cell: adrenal tumor, AIDS-associated cancer, alveolar soft tissue sarcoma, astrocytic tumor, bladder cancer, bone cancer, cancer brain and spinal cord, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's sarcoma, extraskeletal mucoid chondrosarcoma, osseous fibrogenesis imperfecta, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germinal cell tumor, head and neck cancer, hepatocellular carcinoma, tumor pancreatic islet cells, Kaposi's sarcoma, kidney cancer, leukemia, lipoma/benign lipomatous tumor, liposarcoma/malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, me-

- 7 043373 lanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyrocarcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer gland, posterior uveal melanoma, rare hematological disorder, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymus cancer, thymoma, metastatic thyroid cancer and uterine cancer; or (B) the specified use is a use in the treatment of an infection and the specified infection is a chronic viral, bacterial, fungal and parasitic infection characterized by the presence of Epstein-Barr virus, hepatitis A virus (HAV); hepatitis B virus (HBV); hepatitis C virus (HCV); herpes viruses (e.g. HSV-1, HSV-2, HHV-6, CMV), human immunodeficiency virus (HIV), vesicular stomatitis virus (VSV), bacteria of the Bacilli class, bacteria of the Citrobacter genus, Vibrio cholerae, diphthyria bacilli, enterobacteria, gonococci , bacteria of the Helicobacter pylori species, bacteria of the genus Klebsiella and Legionella, meningococci, mycobacteria, pseudomonas, pneumococci, rickettsia, salmonella, bacteria of the genus Serratia, staphylococci, streptococci, tetanus bacillus, fungi of the genus Aspergillus (A.fumigatus, A. niger, etc. .), fungi of the genus Blastomyces dermatitidis, fungi of the genus Candida (C. albicans, C. krusei, C. glabrata, C. tropicalis, etc.), fungi of the genus Cryptococcus neoformans, fungi of the genus Mucorales (mucor, absidia, rhizopus), fungi of the species Sporothrix schenkii, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum, bacteria that cause leptospirosis, bacteria of the species Borrelia burgdorferi, helminths (nematodes, tapeworms, trematodes, flatworms (for example, schistosomes), intestinal Giardia, worms of the genus Trichinella, amoebae sp. Dientamoeba Fragilis, trypanosomes of the species Trypanosoma brucei and Trypanosoma cruzi or protists of the species Leishmania donovani.

The invention particularly relates to such use in the treatment of cancer, wherein said cancer is colorectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma, sarcoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphoblastic leukemia (CLL), hairy cell leukemia (BCL), blastoid plasmacytoid dendritic cell neoplasia (BPDN), non-Hodgkin's lymphomas (NHL), including mantle cell leukemia (MCL) and small cell lymphocytic lymphoma (SCLL), Hodgkin's lymphoma, systemic mastocytosis or Burkitt's lymphoma.

Brief description of graphic materials

In fig. 1 is a schematic representation of a typical covalently bound diabody containing two epitope binding sites and consisting of two polypeptide chains, each containing a heterodimer-promoting E-coil or K-coil domain (alternative heterodimer-promoting domains are shown below). A cysteine residue may be present in the linker and/or heterodimer-promoting domain, as shown in FIG. 3B. VL and VH domains that recognize the same epitope are shown using the same pattern or color.

In fig. 2 is a schematic representation of a typical covalently linked diabody molecule containing two epitope binding sites and consisting of two polypeptide chains, each containing a CH2 and CH3 domain such that the linked chains form an entire Fc fragment or part of an Fc fragment. VL and VH domains that recognize the same epitope are shown using the same pattern or color.

In fig. 3A-3C are schematic views showing typical covalently linked tetravalent diabodies containing four epitope binding sites and consisting of two pairs of polypeptide chains (ie, a total of four polypeptide chains). One polypeptide of each pair contains a CH2 and a CH3 domain such that the linked chains form an entire Fc fragment or part of an Fc fragment. VL and VH domains that recognize the same epitope are shown using the same pattern or color. Two pairs of polypeptide chains can be identical. In such embodiments, where the two pairs of polypeptide chains are the same and the VL and VH domains recognize different epitopes (as shown in FIGS. 3A-3B), the resulting molecule contains four epitope binding sites and is bispecific and bivalent for each associated epitope. In such embodiments, where the VL and VH domains recognize the same epitope (e.g., the same VL domain CDRs and the same VH domain CDRs are used on both strands), the resulting molecule contains four epitope binding sites and is monospecific and tetravalent for a single epitope. Alternatively, the two polypeptide pairs may be different. In such embodiments, where two pairs of polypeptide chains are different and the VL and VH domains of each pair of polypeptides recognize different epitopes (as indicated by the different pattern and color in Fig. 3C), the resulting molecule contains four epitope binding sites and is tetraspe

- 8 043373 digital and monovalent in relation to each associated epitope. In fig. 3A shows an Fc fragment-containing diabody that contains a peptide heterodimer-promoting domain containing a cysteine residue. In fig. 3B shows an Fc moiety-containing diabody that contains heterodimer-promoting E-coil and K-coil domains containing a cysteine residue and a linker (optionally with a cysteine residue). In fig. 3C shows an Fc fragment-containing diabody that contains the CH1 and CL antibody domains.

In fig. 4A and 4B are schematic representations of a typical covalently linked diabody molecule containing two epitope binding sites and consisting of three polypeptide chains. Two of the polypeptide chains contain a CH2 and CH3 domain such that the linked chains form an entire Fc fragment or part thereof. Polypeptide chains containing the VL and VH domains additionally contain a domain that stimulates heterodimer formation. VL and VH domains that recognize the same epitope are shown using the same pattern or color.

In fig. 5 is a schematic representation of a typical covalently linked diabody molecule containing four epitope binding sites and consisting of five polypeptide chains. Two of these polypeptide chains contain CH2 and CH3 domains such that the linked chains form an Fc fragment that contains all or part of the Fc fragment. Polypeptide chains containing linked VL and VH domains additionally contain a domain that stimulates heterodimer formation. VL and VH domains that recognize the same epitope are shown using the same pattern or color.

In fig. 6A-6F are schematic representations of typical Fc-containing trivalent binding molecules containing three epitope binding sites. In fig. 6A and 6B schematically show the domains of trivalent binding molecules containing two diabody binding domains and a Fab binding domain, with different domain distributions, where the diabody binding domain is located on the N-terminal side or the C-terminal side in relation to the Fc fragment, respectively. Molecules in Fig. 6A and 6B contain four circuits. In fig. 6C and 6D schematically depict the domains of trivalent binding molecules containing two diabody binding domains located N-terminal to the Fc moiety and an attached Fab binding domain or scFv binding domain, respectively. In fig. 6E and 6F schematically show the domains of trivalent binding molecules containing two diabody-type binding domains located C-terminal to the Fc moiety, and a Fab-type binding domain, where the light chain and the heavy chain are connected through a polypeptide spacer or domain scFv type binding, respectively. The trivalent binding molecules in FIG. 6C-6F contain three chains. VL and VH domains that recognize the same epitope are shown using the same pattern and color.

Fig. 7 illustrates the principles of the present invention, demonstrating that an example bispecific molecule (a PD-1 x LAG-3 bispecific molecule, designated DART A) is capable of stimulating cytokine production to levels greater than those observed with co- or combined administration of the parent anti-PD antibodies. -1 and LAG-3. IFNy secretion profiles are shown for peripheral blood mononuclear cells (PBMCs) from a representative donor that were stimulated with SEB (0.5 ng/ml) and treated with an example bispecific molecule (PD-1 x LAG-3 bispecific DART A molecule) or anti-PD antibodies. -1 and LAG-3 separately or in combination.

In fig. 8A-8D show the results of an ELISA study based on measuring the binding of serial dilutions of binding molecules to human CTLA-4 and PD-1. In fig. 8A-8B show the binding curves of CTLA-4 mAb 3 G4P, DART D, TRIDENT A or DART B to soluble hCTLA-4-Avi-His (1 μg/ml) (Fig. 8A) or hPD-1-His (1 μg /ml) (Fig. 8B) applied to the substrate tablets. Goat Fc-specific HRP-conjugated anti-human immunoglobulin antibody (1:10,000) was used as a secondary detection molecule to detect binding. In fig. 8C-8D show the results of a study on the effect of changing binding domains and their distribution on binding. PD-1 x CTLA-4 bispecific molecules containing the CTLA-4 binding domains of the antibodies CTLA-4 mAb 1 (for example, DART B) and CTLA-4 mAb 3 (for example, DART C and DART D) were incubated in the presence of soluble human PD-1 (Fig. 8C) or soluble human CTLA-4-Avi-His (Fig. 8D) coated on a plate support. Goat Fcγ-specific HRP-conjugated anti-human immunoglobulin antibodies were used as a secondary detection molecule to detect binding using the chemiluminescent substrate PICO.

In fig. 9A-9E show the results of evaluating the ability of DART D, TRIDENT A, PD-1 mAb 6 G4P and CTLA-4 mAb 3 G4P and the control molecule TRIDENT (having two RSV binding sites and one CTLA-4 binding site) to block ligand binding to PD- 1 and CTLA-1, alone and in combination. Blocking of PD-L1 binding to PD-1 was assessed by ELISA in the presence of an equal amount of irrelevant antigen (Fig. 9A) and in the presence of an equal amount of CTLA-4

- 9 043373 (Fig. 9B), and blocking B7-1 binding to CTLA-4 was assessed in the presence of an equal amount of irrelevant antigen (Fig. 9C) and in the presence of an equal amount of PD-1 (Fig. 9D), as well as in the presence of fourfold PD-1 concentrations (Fig. 9E).

In fig. 10A-10B show the ability of DART B, DART D, TRIDENT A, anti-CTLA-4 antibodies CTLA-4 mAb 1, CTLA-4 mAb 3 G4P and control antibody hIgG to bind to CHO cells expressing cynomolgus CTLA-4 (Fig. 10A) or human CTLA-4 (Fig. 10B). Binding was detected using Fc-specific secondary antibodies against human immunoglobulin.

In fig. 11A-11B show the results of evaluating the ability of DART C, DART D, DART E, TRIDENT A, anti-CTLA-4 antibodies CTLA-4 mAb 1, CTLA-4 mAb 3 G1AA and anti-PD-1 antibodies PD-1 mAb 6 G4P to bind to Jurkat cells (which express huCTLA-4 on their surface but do not express PD-1). Binding of DART and TRIDENT molecules to human CTLA-4 was detected using Fc-specific secondary antibodies against human immunoglobulin (fluorescence-activated cell sorting, FACS). In fig. 11A shows the results of evaluation of DART C, DART D, DART E, CTLA-4 mAb 1, CTLA-4 mAb 3 G1AA and PD-1 mAb 6 G4P. In fig. 11B shows the evaluation results of CTLA-4 mAb 1, CTLA-4 mAb 3 G1AA, PD-1 mAb 6 G4P, and TRIDENT A.

In fig. 12A-12B show the results of evaluating the ability of DART D, TRIDENT A and anti-CTLA-4 antibodies CTLA-4 mAb 1, CTLA-4 mAb 3 G1AA to block CTLA-4 ligands B7-1 and B7-2 in a cell-based assay. Histidine (His)-tagged derivatives of B7-1 and B7-2 were incubated in the presence of Jurkat cells and artificial antigen-presenting cells (Promega). The binding of His-B7-1 and His-B7-2 was detected using an anti-His antibody. The evaluation results are shown in Fig. 12A (His-B7-1) and FIG. 12B (His-B7-2)

In fig. 13 shows the ability of DART C, DART D, TRIDENT A, CTLA-4 mAb 3 G1AA and PD-1 mAb 6 G4P to reverse the CTLA-4 immune checkpoint inhibitory signal as demonstrated in a reporter gene assay on IL-2 cells. Luc-Jurkat-CTLA-4 based on increased luciferase expression. IL-2/Luc-Jurkat-CTLA-4 cells were incubated in the presence of the indicated binding molecules (R:S = 1:0.3) for 30 min at 37°C, after which artificial antigen-presenting Raji cells were added and continued incubate for 6 hours. Reversal of the CTLA-4 immune checkpoint inhibitory signal was determined using a luciferase assay.

In fig. 14 shows the ability of DART D, TRIDENT A, PD-1 mAb 6 G4P and CTLA-4 mAb 3 G1AA to bind to NSO cells expressing PD-1 but not CTLA-4. The binding molecules were incubated in the presence of cells and the average fluorescence of the cells was measured.

In fig. 15A-15B show the results of evaluating the ability of DART D, TRIDENT A, PD-1 mAb 6 G4P and CTLA-4 mAb 3 G1AA to block binding between PD-1 and its ligands PD-L1 and PD-L2 in a cell-based assay. PD-L1-PE or PD-L2-PE were incubated in the presence of the indicated binding molecules and their ability to bind to NSO-PD-1 cells was assessed by fluorescence-activated cell sorting (FACS). Fig. 15A (PD-L1); fig. 15B (PD-L2).

In fig. 16 shows the results of an evaluation of the ability of DART D, TRIDENT A, CTLA-4 mAb 3 G1AA and PD-1 mAb 6 G4P to block the inhibition of the immune response resulting from the PD-1/PD-L1 interaction. Binding molecules were incubated in the presence of PD-L1+ CHO cells and Jurkat effector cells and assessed for their ability to block inhibition of the immune response (by blocking the PD-1/PD-L1 interaction) based on the extent of CD3-mediated activation observed (as indicated by increased luciferase expression in an assay on NFAT-luc/PD1 Jurkat cells; Promega).

In fig. 17 shows the results of evaluating the ability of DART D, TRIDENT A, and the negative control antibody to bind simultaneously to PD-1 and CTLA-4 in the DiscoverX protein fragment complementation enzyme assay. Aliquots of a suspension of the U2OS CTLA-4(1195)-PK PD-1(1-199)-EA cell line, clone #9, were plated in quadruplicate at a concentration of 5,000 cells/well in DiscoverX CP5 plate culture medium on 384-well plates . Cells were allowed to attach for 4 hours at 37°C/5% CO ₂ . Each of the binding molecules in serial dilutions of 1:3 (11 points) were then added to PD-1-CTLA-4 cells and DART D and TRIDENT A samples were added to PD-1-LAG-3 cells. The plates were incubated overnight (16 hours) at 37°C/5% CO ₂ . PathHunter detection reagent was added to the wells and the plate was then incubated for 1 hour at room temperature in the dark and read on an Envision luminometer.

In fig. Figure 18 shows the results of assessing the ability of DART D, TRIDENT A, CTLA-4 mAb 3 G1AA, PD-1 mAb 6 G4P and the combination CTLA-4 mAb 3 G1AA/PD-1 mAb 6 G4P (Ab Combo 1) to enhance the response in a mixed culture reaction lymphocytes (LCL). Monocyte-derived dendritic cells were generated by treating CD14+ monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) administered on day 1 of the incubation period) and IL-4 (administered on day 7 of the incubation period). On day 8 of the incubation period, the SCL reaction was initiated by incubating CD4+ T cells with monocytic-derived dendritic cells (provided on day 8 of the incubation period) and binding molecules against CTLA-4 and PD-1 (provided on day 8 of the incubation period). . IFN-γ release is graphed in FIG. 18. Both bispecific DART D and TRIDENT molecules were found to enhance the response in the SCL response to the same extent or slightly more than the combination of individual parent antibodies. The data shown includes seven groups of columns (each representing a different binding molecule: hIgG4 control; PD-1 mAb 6 G4P; CTLA-4 mAb 3 G1AA; CTLA4 mAb 3 G1AA/PD-1 mAb 6 G4P combination (Ab Combo 1) ; DART D; TRIDENT A and control hIgG1, from left to right, respectively); each group consists of six columns (each representing a different concentration of the molecule represented: 0.016, 0.08, 0.4, 2, 10, or 50 nM, from left to right, respectively).

In fig. 19A-19D show the effect of administration of DART D, TRIDENT A, CTLA-4 mAb 3 G1AA, PD-1 mAb 6 G4P, and the combination of CTLA-4 mAb 1/PD-1 mAb 1 (Ab Combo 1) on T cell responses by Staphylococcus aureus enterotoxin type B (SEB) repeat stimulation assay. In fig. 19A-19B show dot plots of fluorescence-activated cell sorting (FACS) data reflecting the expression of PD-1 by such PBMCs in a CTLA-1-dependent manner in the absence (FIG. 19A) or presence (FIG. 19B) of SEB stimulation. In fig. 19C shows the effect of SEB stimulation on IFN-γ secretion. PBMCs were stimulated with Staphylococcus aureus enterotoxin type B (SEB) at a concentration of 0.5 ng/ml for 48 hours. Cells were then harvested, washed, and replated into 96-well plates with various concentrations of antibodies in the presence of fresh SEB for an additional 48 hours. The supernatant was then collected and IFN-γ production was analyzed by flow cytometry/ELISA. Both bispecific DART and TRIDENT protein showed increased IFN-γ production responses that were consistent with the net response observed with the combination of individual parent mAbs. Similar results were observed in the SEB stimulation assay, in which PBMCs were cultured with a high concentration (500 ng/ml) of SEB for 72 hours. Six groups of columns are presented, each representing a different binding molecule. Each group consists of seven columns that reflect the result obtained using the binding molecule at a concentration of 25 nM, 6.25 nM, 1.56 nM, 0.39 nM, 0.09 nM, 0.02 nM or 0.006 nM (left to the right respectively). In fig. 19D shows IL-2 release for a typical donor. PBMCs were stimulated with 0.5 ng/ml SEB for 48 hours, collected, washed and replated into 96-well plates with fresh SEB in the presence of either DART D, TRIDENT A, CTLA-4 mAb 3 G1AA, PD-1 mAb 6 G4P , or the combination CTLA-4 mAb 3 G1AA/PD-1 mAb 6 G4P (Ab Combo 1) for an additional 48 hours and the released IL-2 was measured. Seven groups of columns are presented, each representing a different binding molecule or state. Each group consists of three columns representing the result obtained using the binding molecule at a concentration of 0.5 nM, 5 nM or 50 nM, respectively (from left to right). When using antibodies in combination, each antibody was added at the indicated concentration such that the total concentration of antibody added was doubled.

In fig. 20A-20B show the activity of PD-1 x CTLA-4 bispecific molecules in a graft-versus-host disease (GVHD) model of NOG mice transplanted with PBMCs. CD3+ T cell enumeration was performed by fluorescence activated cell sorting (FACS) on the day of the study (Fig. 20A) in mice that received DART D at a dose of 50 mg/kg or 500 mg/kg (Fig. 20A). Survival was monitored throughout the study; the data are plotted as percentage survival in FIG. 20B.

In fig. 21A-21C show serum concentration versus time plots for cynomolgus monkeys (identified by a 6-digit alphanumeric code) that received DART D at a concentration of 50 mg/kg on days 1, 8, and 15 of the study (FIG. 21A). , DART D at a concentration of 75 mg/kg on days 1, 8 and 15 of the study (Fig. 21B) or Trident A at a concentration of 5 mg/kg on day 1 of the study (Fig. 21C).

In fig. 22A-22B show the effect of DART D administration on absolute lymphocyte count (ALC) in treated cynomolgus monkeys. In fig. 22A shows ACL in thousands of cells/μL (thousand cells/μL). In fig. 22B shows the percentage change in ASL normalized to the day 1 value (D1).

In fig. 23A-23B show CD4+ T cell proliferation and PD-1 receptor occupancy on T cells in cynomolgus monkeys administered DART D at a concentration of 50 mg/kg (FIG. 23A) or DART D at a concentration of 75 mg/kg (FIG. 23B)

In fig. 24A-24B show the effect of DART D administration on CD4+ T cell proliferation in cynomolgus monkeys administered DART D at a concentration of 50 mg/kg (FIG. 24A) or DART D at a concentration of 75 mg/kg (FIG. 24B).

Detailed Description of the Invention

The present invention is directed to bispecific molecules (e.g., diabodies, bispecific antibodies, trivalent binding molecules, etc.) containing at least one binding site having immunospecificity for a PD-1 epitope and at least one binding site having immunospecificity for the CTLA-4 epitope (i.e. bispecies

- 11 043373 molecules specific to PD-1 x CTLA-4). The present invention provides such PD-1 x CTLA-4 bispecific molecules that contain two epitope binding sites that are immunospecific for one (or two) PD-1 epitope(s), and two epitope binding sites that are immune specific for against one (or two) CTLA-4 epitope(s). The present invention is also directed to such PD-1 x CTLA-4 bispecific molecules that further comprise an immunoglobulin Fc moiety. The PD-1 x CTLA-4 bispecific molecules of the present invention are capable of simultaneously binding to PD-1 and CTLA-4, in particular in the form in which these molecules are distributed on the surface of human cells. The invention is directed to pharmaceutical compositions that contain such PD-1 x CTLA-4 bispecific molecules, and methods including the use of such bispecific molecules in the treatment of cancer and other diseases and conditions. The present invention also provides methods including the use of such PD-1CTLA-4 bispecific molecules to stimulate an immune response.

T cell activation requires two separate signals (Viglietta, V. et al. (2007) Modulating CoStimulation Neurotherapeutics 4:666-675; Korman, A.J. et al. (2007) Checkpoint Blockade in Cancer Immunotherapy Adv. Immunol. 90:297 -339). The first signal is provided by a T cell receptor (TCR) molecule expressed on the surface of a T cell that has recognized a peptide antigen bound to a human leukocyte antigen (HLA) expressed on the surface of an antigen presenting cell (APC). The second signal is provided by the interaction of cognate pairs of costimulatory ligands B7-1 and B7-2, expressed on APCs, and their corresponding receptors CD28 and CTLA-4, expressed on T cells.

Binding of B7-1 and B7-2 molecules to CD28 stimulates T cell proliferation and further induces increased expression of CTLA-4. CTLA-4 is a negative regulator that competes with B7-1 and B7-2 for binding to CD28. Thus, the response to disease involves two phases: the initial phase involves stimulation of T cell proliferation; the subsequent phase nullifies the immune response and returns the subject's immune system to a dormant state. Antibodies that bind to CD28 can mimic the binding of B7-1 or B7-2 and thus induce or enhance T cell effector functions and the generation of an immune response aimed at eliminating the tumor; such antibodies are costimulatory. In contrast, antibodies that block CTLA-4 binding to B7-1 and B7-2 can prevent T cells from returning to a resting state; thus maintaining the continuous proliferation of these T cells, which can lead to an autoimmune reaction and the development of immune-mediated adverse events (iAEs) (Wang, L. et al. (March 7, 2011) VISTA, A Novel Mouse Ig Superfamily Ligand That Negatively Regulates T-Cell Responses J. Exp. Med. 10.1084/jem.20100619:l-16; Lepenies, B. et al. (2008) The Role Of Negative Costimulators During Parasitic Infections Endocrine, Metabolic & Immune Disorders - Drug Targets 8: 279-288). Of particular importance is the binding between the B7-1 (CD80) and B7-2 (CD86) ligands of the antigen-presenting cell and the CD28 and CTLA-4 receptors of the CD4+ T lymphocyte (Sharpe, A.H. et al. (2002) The B7-CD28 Superfamily , Nature Rev. Immunol. 2:116-126; Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules Immunolog. Res. 28(1):39-48; Lindley, P. S. et al. (2009) The Clinical Utility of Inhibiting CD28Mediated Costimulation, Immunol Rev 229:307–321). Binding of B7-1 or B7-2 to CD28 stimulates T cell activation; binding of B7-1 or B7-2 to CTLA-4 inhibits this activation (Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules, Immunolog. Res. 28(1):39-48; Lindley, P.S. et al. (2009) The Clinical Utility Of Inhibiting CD28-Mediated Costimulation Immunol. Rev. 229:307-321; Greenwald, R. J. et al. (2005) The B7 Family Revisited, Ann. Rev. Immunol. 23:515-548) . CD28 is constitutively expressed on the surface of T cells (Gross, J., et al. (1992) Identification And Distribution Of The Costimulatory Receptor CD28 In The Mouse, J. Immunol. 149:380-388), whereas CTLA-4 expression is stimulated immediately after T cell activation (Linsley, P. et al. (1996) Intracellular Trafficking Of CTLA4 And Focal Localization Towards Sites Of TCR Engagement Immunity 4:535-543). Because the CTLA-4 receptor has higher affinity (Sharpe, A.H. et al. (2002) The B7-CD28 Superfamily Nature Rev. Immunol. 2:116-126), ligand binding first induces T cell proliferation (via CD28) and it is then inhibited (as a result of the onset of CTLA-4 expression), thereby suppressing the effect when proliferation is no longer required.

Along with the interactions described above, a second set of receptors and binding ligands functions as an inhibitor of the immune system, thus acting as a brake to slow the CD28/B7-1/B7-2 mediated enhancement of the immune response. This additional response involves binding of the programmed cell death protein-1 (PD-1) receptor, expressed on the surface of T cells, to the corresponding ligands: PD-L1, expressed on antigen presenting cells (APCs), and PD-L2, expressed on epithelial cells (Chen L. et al. (2013) Molecular Mechanisms Of T-Cell Co-Stimulation And Co-Inhibition Nature Reviews Immunology 13(4):227-242). Unlike agonist antibodies, which bind to CD28, allowing direct stimulation of T cell responses, antibodies that bind to PD-1 or PD-L1 act as antagonists or block PD-1/PD-L1 recruitment and thus , support T activation

- 12 043373 cells by preventing the transmission of a negative signal to T cells. in such a case, antibodies that bind to PD-1 or PD-L1 enhance or support T cell proliferation, cytotoxicity, and/or cytokine secretion. Taken together, agonist antibodies, such as anti-CD28 antibodies, target positive signaling pathways and are thus co-stimulatory, whereas antagonist antibodies, such as anti-CTLA-4 antibodies and anti-PD-1 antibodies, target negative signaling pathways and are called immune checkpoint inhibitors.

As described above, CTLA-4 and PD-1 are canonical immune checkpoint inhibitors that exhibit distinct inhibitory effects on T cell activation. The PD-1 x CTLA-4 bispecific molecules of the present invention are capable of binding to the PD-1 and CTLA-4 cell surface molecules that are present on the surface of lymphocytes and thereby impairing the ability of these cell surface molecules to respond to their respective receptors. Without being limited to any theory or mechanism, we believe that PD-1 binding may result in reversal of T cell inhibition (eg, at tumor sites and/or as a result of infection) and that CTLA-1 binding may stimulate polyclonal activation and stimulation. In such a case, the PD-1 x CTLA-4 bispecific molecules of the present invention are able to attenuate PD-1 and CTLA-4 mediated inhibition of the immune system and provide sustained activation of the immune system. In the present application, it has been shown that bispecific molecules that target two immune modulatory pathways are more active than a combination of separate antibodies. The present invention also provides PD1 x CTLA-4 bispecific molecules with PD-1:CTLA-4 binding ratios of 1:1, 1:2, 2:2 and 2:1, which allow complete blocking of both PD-1, and CTLA-4, as well as a block that extends to CTLA-4 when it is co-expressed with PD-1. Thus, the PD-1 x CTLA-4 bispecific molecules of the present invention provide an unexpected advantage over the combination of individual anti-PD-1 and anti-CTLA-4 antibodies. Additionally, the PD-1 x CTLA-4 bispecific molecules of the present invention may provide stimulation of the immune response with a reduced risk of immune-mediated adverse events (iAEs).

I. Antibodies and their binding domains.

The antibodies of the present invention are immunoglobulin molecules capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable domain of the immunoglobulin molecule. As used herein, the terms antibody and antibodies refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, hybrid antibodies, polyclonal antibodies, camelized antibodies, single chain Fv fragments (scFv), single chain antibodies, Fab fragments , F(ab') fragments, disulfide-linked bispecific Fv fragments (sdFv), intrabodies and epitope-binding fragments of any of the above molecules. In particular, the term antibody includes immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e. molecules that contain an epitope binding site. 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. As used herein, an Fc fragment is considered to belong to a particular IgG isotype, class or subclass if its amino acid sequence has the greatest homology with that isotype compared to other IgG isotypes. In addition to the known uses of antibodies in diagnostics, antibodies have been shown to be useful as therapeutic agents. Antibodies are capable of immunospecifically binding to a polypeptide or protein or non-protein molecule due to the presence of a specific domain or fragment or structure (epitope) in such a molecule. The epitope-containing molecule may have immunogenic activity, causing an antibody response in the animal; such molecules are called antigens. The past few decades have seen renewed interest in the therapeutic potential of antibodies, and antibodies have become one of the leading classes of biotechnologically derived drugs (Chan, C.E. et al. (2009) The Use Of Antibodies In The Treatment Of Infectious Diseases Singapore Med. J. 50( 7):663-666). More than 200 antibody-based drugs have been approved for use or are in development.

The term monoclonal antibody refers to a homogeneous population of antibodies, wherein said monoclonal antibody is composed of amino acids (natural and non-natural) that are involved in selective antigen binding. Monoclonal antibodies are highly specific and directed against a single epitope (or antigenic site). The term monoclonal antibody includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2 Fv), single chain antibodies (scFv), their mutants, fusion proteins containing part of the antibody , humanized monoclonal antibodies, chimeric monoclonal antibodies and any other modified configuration of the immunoglobulin molecule having an antigen recognition site, the necessary specificity and binding ability

- 13 043373 with antigen. It is not intended that the present invention limit the source of the antibody or the method of its production (eg, production using hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins, as well as fragments, etc., described above, within the scope of the definition of antibody. Methods for producing monoclonal antibodies are known in the art. One method that can be used is the Kohler method (Kohler, G. et al. (1975) Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity', Nature 256:495-497) or a modification thereof. Monoclonal antibodies are usually produced in mice, rats or rabbits. Antibodies are produced by immunizing an animal with an immunogenic number of cells, cell extracts, or protein preparations that contain the desired epitope. The immunogen may be, but is not limited to, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissue. The cells used for immunization can be cultured for a period of time (eg, at least 24 hours) before being used as an immunogen. Cells can be used as immunogens alone or in combination with a non-denaturing adjuvant such as Ribi (see, for example, Jennings, V.M. (\995) Review of Selected Adjuvants Used in Antibody Production, ILAR J. 37(3):119-125) . In general, cells should be kept intact and preferably viable when used as immunogens. Intact cells may allow better detection of antigens in an immunized animal compared to disrupted cells. The use of denaturing or harsh adjuvants, such as Freund's adjuvant, may lead to cell rupture and is therefore not recommended. The immunogen can be administered repeatedly at periodic intervals, for example, twice a week or once a week, or can be administered in a manner that maintains viability in the animal (eg, in a tissue recombinant). Alternatively, existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for the desired pathogenic epitope can be sequenced and recombinantly produced using any methods known in the art. In one embodiment, the antibody is sequenced and the polynucleotide sequence is then cloned into an expression or amplification vector. The coding sequence for the antibody of interest can be maintained in a vector in a host cell, and the host cell can then be expanded and frozen for future use. The polynucleotide sequence of such an antibody can be used for genetic manipulation to create monospecific or multispecific (eg, bispecific, trispecific and tetraspecific) molecules of the invention, as well as affinity optimized molecules, chimeric antibodies, humanized antibodies and/or canine antibodies to improve affinity or other characteristics of the antibody. The general principle in humanizing an antibody involves maintaining the core sequence of the antigen-binding portion of the antibody while replacing the remainder of the non-human antibody with human antibody sequences.

Natural antibodies (such as IgG antibodies) consist of two light chains combined with two heavy chains. Each light chain contains a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH), three constant domains (CH1, CH2 and CH3) and a hinge region located between the CH1 and CH2 domains. The basic structural unit of natural immunoglobulins (eg, IgG) is therefore a tetramer containing two light chains and two heavy chains, which are typically expressed as a glycoprotein with a molecular weight of approximately 150,000 Da. The amino-terminal (N-terminal) part of each chain contains a variable domain consisting of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (C-terminal) portion of each chain defines a constant region, with light chains having a single constant domain and heavy chains typically containing three constant domains and a hinge region. Thus, the structure of the light chains of the IgG molecule is n-VL-CL-c, and the structure of the heavy chains of IgG is n-VH-CH1H-CH2-CH3-C (where H is the hinge region and n and c are N-terminus and C-terminus of the polypeptide, respectively). The variable domains of an IgG molecule consist of hypervariable regions (CDRs), which contain epitope-contacting residues, and non-CDR segments called framework regions (FRs), which generally support the structure and determine the orientation of the CDR loops to allow the specified contact (although specific framework residues may also contact the antigen). Thus, the VL and VH domains have the structure n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c. Polypeptides that are or can serve as the first, second and third CDRs of an antibody light chain are referred to herein as the CDRL1 domain, CDRL2 domain and CDRL3 domain, respectively. Likewise, polypeptides that are or can serve as the first, second, and third CDRs of an antibody heavy chain are referred to herein as the CDR _H 1 domain, the CDR _H 2 domain, and the CDRH3 domain, respectively. Thus, the terms CDRL1 domain, CDRL2 domain, CDRL3 domain, CDR _H 1 domain, CDR _H 2 domain, and CDR _H 3 domain refer to polypeptides that, when incorporated into a protein, give it the ability to bind to a specific epitope, whether or not with

- 14 043373 specified protein antibody containing light and heavy chains, diabody or single chain binding molecule (eg scFv, a BiTe, etc.) or other type of protein. Accordingly, as used herein, the term epitope binding fragment refers to an antibody fragment capable of immunospecifically binding to an epitope, and the term epitope binding site refers to the portion of the molecule containing the epitope binding fragment. The epitope-binding fragment may contain 1, 2, 3, 4, 5 or all 6 of the CDR domains of the specified antibody and, despite the ability to immunospecifically bind to the specified epitope, may exhibit immunospecificity, affinity or selectivity for such an epitope that is different from the epitope of the specified antibodies. However, preferably the epitope binding fragment contains all 6 of the CDR domains of the specified antibody. The epitope-binding antibody fragment may be a single polypeptide chain (e.g., scFv) or may contain two or more polypeptide chains, each containing an amino terminus and a carboxy terminus (e.g., diabody, Fab fragment, Fab ₂ fragment, etc.) . Unless specifically stated, the domains of the protein molecules described in this application are located in the direction from the N-terminus to the C-terminus.

The invention particularly includes bispecific PD-1 x CTLA-4 binding molecules comprising one, two or more than two single chain variable domain fragments (scFv) of an anti-PD-1 antibody and one, two or more than two single chain variable domain fragments of an antibody against CTLA-4. Single chain variable domain fragments are produced by linking the light and heavy chain variable domains using a short linker peptide. Linkers can be modified to provide additional functionality, such as the ability to attach drugs or attach to solid supports. Single chain variants can be produced either recombinantly or synthetically. An automatic synthesizer can be used to synthesize scFv. To recombinantly produce scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell - eukaryotic, such as a yeast, plant, insect or mammalian cell, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be obtained using standard procedures such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification methods known in the art.

The invention in particular also includes PD-1 x CTLA-4 bispecific molecules comprising humanized anti-PD-1 and anti-CTLA-4 antibodies. The term humanized antibody refers to a hybrid molecule generally produced by recombinant methods and containing an immunoglobulin antigen binding site from a species of non-human origin with a retained structure of the immunoglobulin molecule based on the structure and/or sequence of human immunoglobulin. The polynucleotide sequence of the variable domains of such an antibody can be used for genetic manipulation to create such derivatives and to improve the affinity or other characteristics of the antibody. The general principle in humanizing an antibody involves maintaining the core sequence of the antigen-binding portion of the antibody while replacing the remainder of the non-human antibody with human antibody sequences. There are four general steps in humanizing a monoclonal antibody: (1) determining the nucleotide and predicted amino acid sequence of the light and heavy chain variable domains of the parent antibody; (2) designing a humanized antibody or a canine antibody, i.e. deciding which antibody framework will be used during the humanization or caninization process; (3) actual implementation of humanization or caninization methods/technologies; and (4) transfection and expression of the humanized antibody. See, for example, US Pat. No. 4,816,567; 5 807 715; 5,866,692 and 6,331,415.

The antigen binding site may comprise a full-length variable domain fused to a constant domain, or only the hypervariable regions (CDRs) of the variable domain grafted onto suitable framework regions. Antigen binding sites may be wild-type sites or sites modified by one or more amino acid substitutions. This eliminates the constant region that is an immunogen in human subjects, but the potential for an immune response to the foreign variable domain remains (LoBuglio, A.F. et al. (1989) Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response Proc Natl Acad Sci (USA) 86:4220-4224). Another approach focuses not only on providing constant regions of human origin, but also on modifying variable domains and reconstructing them as closely as possible to the form of human origin. It is known that the variable domains of both heavy and light chains contain three hypervariable regions (CDRs), which differ depending on the specific antigen and determine the binding ability. These domains are flanked by four framework regions (FRs) that are relatively conserved in the species and are thought to provide support for the CDR. When creating non-human antibodies directed against a specific antigen, the variable domains can be reconstructed or humanized by

- 15 043373 grafting CDRs derived from an antibody of non-human origin to FRs present in the human antibody to be modified. The application of this approach to various antibodies has been described by Sato, K. et al. (1993) Cancer Res 53:851–856. Riechmann, L. et al. (1988) Reshaping Human Antibodies for Therapy Nature 332:323-327; Verhoeyen, M. et al. (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity Science 239:1534-1536; Kettleborough, C. A. et al. (1991) Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation Protein Engineering 4:773-3783; Maeda, H. et al. (1991) Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity Human Antibodies Hybridoma 2:124-134; Gorman, S. D. et al. (1991) Reshaping A Therapeutic CD4 Antibody Proc. Natl. Acad. Sci. (USA) 88:4181-4185; Tempest, P.R. et al. (1991) Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in Vivo Bio/Technology 9:266-271; Co, M. S. et al. (1991) Humanized Antibodies For Antiviral Therapy Proc. Natl. Acad. Sci. (USA) 88:2869-2873; Carter, P. et al. (1992) Humanization Of An Antipl85her2 Antibody For Human Cancer Therapy Proc. Natl. Acad. Sci. (USA) 89:4285-4289; Co, M.S. et al. (1992) Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen J. Immunol. 148:11491154. In some embodiments, humanized antibodies retain all CDR sequences (eg, a humanized mouse antibody that contains all six CDRs from mouse antibodies). In other embodiments, humanized antibodies comprise one or more CDRs (one, two, three, four, five, or six) that differ in sequence from the parent antibody.

A number of humanized antibody molecules containing an antigen binding site derived from a non-human immunoglobulin have been described, including hybrid antibodies containing a rodent variable domain and associated CDRs or a modified variable domain and associated rodent CDRs. fused to human immunoglobulin constant domains (see, for example, Winter et al. (1991) Man-made Antibodies Nature 349:293299; Lobuglio et al. (1989) Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response Proc. Natl Acad Sci (USA) 86:4220-4224 (1989), Shaw et al (1987) Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor-Associated Antigen J. Immunol 138:4534-4538, Brown et al (1987) Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody Cancer Res 47:3577-3583). Other references describe rodent CDRs grafted onto a human framework region (FR) prior to fusion with a suitable human antibody constant domain (see, for example, Riechmann, L. et al. (1988) Reshaping Human Antibodies for Therapy Nature 332:323-327; Verhoeyen, M. et al (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity Science 239:1534-1536 and Jones et al (1986) Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse Nature 321 :522-525). Another reference describes rodent CDRs that are supported by recombinantly venated rodent scaffold regions. See, for example, European Patent Publication No. 519,596. These humanized molecules are designed to minimize unwanted immune response to rodent antibody molecules directed against human immunoglobulin, limiting the duration and effectiveness of therapeutic use of these fragments in human recipients. Other methods for humanizing antibodies that can also be used are described in Daugherty et al. (1991) Polymerase Chain Reaction Facilitates The Cloning, CDRGrafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins Nucl. Acids Res. 19:2471-2476 and US Pat. No. 6,180,377; 6,054,297; 5,997,867 and 5,866,692.

II. Fcy receptors (FcyRs).

The CH2 and CH3 domains of the two heavy chains interact to form the Fc moiety, which is a domain recognized by cellular Fc receptors, including but not limited to Fc receptor gamma (FcyRs). In this application, the term Fc fragment is used to define the C-terminal region of the IgG heavy chain. The amino acid sequence of the CH2-CH3 domain of an example human IgG1 is (SEQ ID NO: 1) 231 240 250 260 270 280

APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA

340 350 360 370 380 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE

390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

440 447 ALHNHYTQKS LSLSPGX when numbered according to the Kabat EU index, where X represents lysine (K) or is absent.

The amino acid sequence of the CH2-CH3 domain of an example human IgG2 is (SEQ ID NO: 2)

- 16 043373

231 240 250 260 270 280 APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVQFNWYVD

290 300 310 320 330 GVEVHNAKTK PREEQFNSTF RWSVLTWH QDWLNGKEYK CKVSNKGLPA

340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE

390 400 410 420 430

WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

440 447 ALHNHYTQKS LSLSPGX when numbered according to the Kabat EU index, where X represents lysine (K) or is absent.

The amino acid sequence of the CH2-CH3 domain of an example human IgG3 is (SEQ ID NO: 3)

231 240 250 260 270 280

APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVQFKWYVD

290 300 310 320 330 GVEVHNAKTK PREEQYNSTF RWSVLTVLH QDWLNGKEYK CKVSNKALPA

340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE

390 400 410 420 430 WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE

440 447 ALHNRFTQKS LSLSPGX when numbered according to the Kabat EU index, where X represents lysine (K) or is absent.

The amino acid sequence of the CH2-CH3 domain of an example human IgG4 is (SEQ ID NO: 4)

231 240 250 260 270 280

APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD

290 300 310 320 330 GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS

340 350 360 370 380 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE

390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE

440 447 ALHNHYTQKS LSLSLGX when numbered according to the Kabat EU index, where X represents lysine (K) or is absent.

Throughout this application, the numbering of residues in the constant region of the IgG heavy chain is the numbering according to the EC index according to Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, ^5th Ed. Public Health Service, NH1, MD (1991) ( Kabat, wherein said reference is specifically incorporated herein by reference. The term Kabat EC index refers to the numbering of human IgG1 according to the European Antibody Numbering System. Amino acids from the variable domains of the mature heavy and light chains of immunoglobulins are designated by the number of the position of the amino acid in the chain. Kabat described multiple antibody amino acid sequences, identified consensus amino acid sequences for each subgroup, and assigned each amino acid a residue number. CDRs identified as determined by Kabat (it should be understood that CDRH1 starts five residues earlier according to Chothia, C. & Lesk numbering , AM ((1987) Canonical structures for the hypervariable regions of immunoglobulins J. Mol. Biol. 196:901-917). The Kabat numbering scheme extends to antibodies not included in its listed antibodies by aligning the antibody of interest to one of the Kabat consensus sequences with reference to conserved amino acids. This method of assigning residue numbers has become standard in the art and allows amino acids at equivalent positions to be easily identified in various antibodies, including hybrid or humanized variants. For example, the amino acid at position 50 of the light chain of a human antibody occupies a position equivalent to the amino acid at position 50 of the light chain of a mouse antibody.

Polymorphisms have been found at several different positions within the antibody constant regions (eg, positions in the Fc region, including but not limited to positions 270, 272, 312, 315, 356 and 358 when numbered according to the Kabat EC index) and, thus, there may be slight differences between the presented sequence and the sequences according to the prior art. Polymorphic forms of human immunoglobulins have been well characterized. Currently, 18 Gm allotypes are known: G1m (1, 2, 3, 17) or G1m (a,

- 17 043373 x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5) (Lefranc, et al., The Human IgG Subclasses: Molecular Analysis Of Structure, Function And Regulation:' Pergamon , Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199211). It is specifically contemplated that the antibodies of the present invention may include any allotype, isoallotype or haplotype of any immunoglobulin gene and are not limited to the allotype, isoallotype or haplotype of the sequences presented herein. Moreover, in some expression systems, the C-terminal amino acid residue (shown in bold above) of the CH3 domain may be removed post-translationally. Thus, the C-terminal residue of the CH3 domain is an optional amino acid residue in the PD-1 x CTLA-4 bispecific molecules of the invention. The present invention specifically includes PD-1 x CTLA-4 bispecific molecules lacking the C-terminal residue of the CH3 domain. The present invention also specifically includes those constructs that contain the C-terminal lysine residue of the CH3 domain.

As stated above, the Fc fragment of natural IgG antibodies is capable of binding to cellular Fcgamma receptors (FcyR). This binding results in the transmission of activating or inhibitory signals to the immune system. The ability of such binding to stimulate diametrically opposed functions reflects structural differences between different FcyRs and, in particular, indicates whether the bound FcyR contains an immunoreceptor tyrosine-based activating motif (ITAM) or an immunoreceptor tyrosine-based inhibitory motif (ITIM). The recruitment of various cytoplasmic enzymes to these structures determines the outcome of FcyR-mediated cellular responses. ITAM-containing FcyRs include FcyRI, FcyRIIA, FcyRIIIA and activate the immune system upon binding to the Fc fragment. FcyRIIB is the only naturally occurring ITIM-containing FcyR known to date; it acts to weaken or suppress the immune system when bound to the Fc fragment. Human neutrophils express the FcyRIIA gene. Clustering of FcyRIIA by immune complex formation or cross-linking of specific antibodies serves to aggregate ITAMs to receptor-associated kinases that facilitate ITAM phosphorylation. Phosphorylated ITAM serves as a docking site for Syk kinase, the activation of which leads to the activation of downstream substrates (e.g., PI3K). Cellular activation leads to the release of proinflammatory mediators. The FcyRIIB gene is expressed in B lymphocytes; its extracellular domain is 96% identical to FcyRIIA and binds indistinguishably to IgG complexes. The presence of ITIM in the cytoplasmic domain of FcyRIIB defines this inhibitory subclass of FcyR. The molecular basis of the described inhibition has recently been elucidated. When co-ligated with the activating FcyR, the ITIM motif of the FcyRIIB receptor is phosphorylated and recruits the SH2 domain of inositol polyphosphate 5'-phosphatase (SHIP), which hydrolyzes phosphoinositol messengers released as a result of tyrosine kinase activation mediated by the ITAM-containing FcyR, resulting in the prevention of intracellular Ca entry. Thus, FcyRIIB cross-linking extinguishes the activating response to FcyR ligation and suppresses cellular responsiveness. Thus, the activation and proliferation of B cells and their secretion of antibodies stops.

Bispecific antibodies, multispecific diabodies and DART® diabodies.

The ability of an antibody to bind to an antigen epitope depends on the presence and amino acid sequence of the VL and VH domains of the antibody. As a result of the interaction of the light and heavy chains of an antibody and, in particular, the interaction of its VL and VH domains, one of two binding sites for a natural antibody epitope, such as IgG, is formed. Natural antibodies are capable of binding to only one type of epitope (i.e., they are monospecific), despite the fact that they can bind to multiple copies of epitopes of a given type (i.e., they exhibit bivalence or multivalency).

The antibody binding domains and PD-1 x CTLA-4 bispecific molecules of the present invention bind to epitopes in an immune-specific manner. As used herein, an antibody, diabody, or other epitope-binding molecule will immunospecifically bind to a region of another molecule (i.e., an epitope) if it reacts or binds more frequently, faster, with greater duration, and/or with greater affinity to said epitope. compared to alternative epitopes. For example, an antibody that immunospecifically binds to a viral epitope is an antibody that binds to that viral epitope with greater affinity, avidity, speed, and/or duration compared to immunospecific binding to other viral epitopes or nonviral epitopes. When encountering this definition, it should also be understood that, for example, an antibody (or fragment or epitope) that immunospecifically binds to a first target may or may not specifically or preferentially bind to a second target. In this regard, immune-specific binding does not necessarily require exclusive binding (although it may involve exclusive binding). In general, reference to binding refers to (but not necessarily) immune-specific binding. It is indicated that two molecules are able to bind to each other with

- 18 043373 physiological specificity if such binding is characterized by the specificity with which the receptors bind to their respective ligands.

One aspect of the present invention is based on the knowledge that the functional activity of antibodies can be enhanced by creating multispecific antibody molecules that can simultaneously bind to one or more PD-1 epitopes as well as one or more CTLA epitopes. -4. For molecules containing more than one epitope binding site that is immune specific for a PD-1 epitope, the epitopes may be identical to each other, overlap, or be separate from each other; binding to one such epitope may or may not compete with binding to other such epitopes. Likewise, for molecules containing more than one epitope binding site that is immune specific for a CTLA-4 epitope, such epitopes may be identical to each other, overlap, or be separate from each other; binding to one such epitope may or may not compete with binding to a second such epitope. It is specifically contemplated that these characteristics can be independently varied to produce PD-1 x CTLA-4 bispecific molecules which, for example, have:

(1) the ability to bind to two identical epitopes of PD-1 and to:

(a) two identical CTLA-4 epitopes; or (b) two overlapping CTLA-4 epitopes; or (c) two separate CTLA-4 epitopes; or (2) the ability to bind to two overlapping epitopes of PD-1 and to:

(a) two identical CTLA-4 epitopes; or (b) two overlapping CTLA-4 epitopes; or (c) two separate CTLA-4 epitopes; or (3) the ability to bind to two distinct PD-1 epitopes and to:

(a) two identical CTLA-4 epitopes; or (b) two overlapping CTLA-4 epitopes; or (c) two separate CTLA-4 epitopes.

To obtain molecules with a higher binding capacity compared to natural antibodies, a large number of different recombinant bispecific antibodies have been developed (see, for example, international publications according to PCT No. WO 2008/003116, WO 2009/132876, WO 2008/003103, WO 2007 /146968, WO 2009/018386, WO 2012/009544, WO 2013/070565), most of which use linker peptides to fuse an additional epitope-binding moiety (e.g. scFv, VL, VH, etc.) to the antibody or to fuse within its framework structure (IgA, IgD, IgE, IgG or IgM) or for the fusion of several epitope-binding fragments (for example, two Fab fragments or scFv). Alternative forms use linker peptides to fuse an epitope-binding moiety (eg scFv, VL, VH, etc.) with a dimerization domain such as CH2-CH3 domains or alternative polypeptides (WO 2005/070966, WO 2006/107786A WO 2006/ 107617A, WO 2007/046893). PCT Publications No. WO 2013/174873, WO 2011/133886 and WO 2010/136172 describe a trispecific antibody in which the CL and CH1 domains have been moved from their respective natural positions and the VL and VH domains have been altered (WO 2008/027236; WO 2010 /108127) so as to allow said antibody to bind to more than one antigen. PCT Publications No. WO 2013/163427 and WO 2013/119903 describe modification of the CH2 domain so that it contains a fusion protein adduct having a binding domain. PCT Publications No. WO 2010/028797, WO2010028796 and WO 2010/028795 describe recombinant antibodies whose Fc fragments have been replaced by additional VL and VH domains so as to form trivalent binding molecules. Publications according to PCT No. WO 2003/025018 and WO2003012069 describe recombinant diabodies whose individual chains contain scFv domains. International publication according to PCT No. WO 2013/006544 describes multivalent Fab molecules synthesized as a single polypeptide chain and then subjected to proteolysis to obtain heterodimeric structures. Publications according to PCT No. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2008/024188, WO 2007/024715, WO 2007/07 5270, WO 1998/002463, WO 1992/022583 and WO 1991/003493 describe the addition of additional binding domains or functional groups to an antibody or part of an antibody (for example, adding a diabody to the light chain of an antibody, or adding additional VL and VH domains to the light or heavy chains of an antibody, or adding a heterologous hybrid protein, or the coupling of several Fab domains to each other).

The prior art also describes the ability to produce diabodies that differ from such natural antibodies by the ability to bind to two or more different epitopes (i.e., exhibiting bispecificity or multispecificity in addition to bivalence or multivalency) (see, for example, Holliger et al. (1993) 'Diabodies': Small Bivalent And Bispecific Antibody Fragments, Proc.

- 19 043373

Natl. Acad. Sci. (USA) 90:6444-6448; US 2004/0058400 (Hollinger et al.); US 2004/0220388 / WO 02/02781 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J. Biol. Chem. 280(20): 19665-19672; WO 02/02781 (Mertens et al); Olafsen, T. et al. (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications Protein Eng. Des. Sel. 17(1):21-27; Wu, A. et al. (2001) Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange Protein Engineering 14(2): 1025-1033; Asano et al. (2004) A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain Abstract 3P683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13(8):583-588; Baeuerle, P. A. et al. (2009) Bispecific T-Cell Engaging Antibodies For Cancer Therapy Cancer Res. 69(12):4941-4944).

The basis of the diabody design is an antibody derivative known as a single chain fragment variable domain (scFv). Such molecules are prepared by linking the light and/or heavy chain variable domain with a short binding peptide. Bird et al. (1988) (Single-Chain Antigen-Binding Proteins Science 242:423-426, describes an example of binding peptides that provide an approximately 3.5 nm bond between the carboxy terminus of one variable domain and the amino terminus of another variable domain. Linkers with other sequences have been used (Bird et al. (1988) (Single-Chain Antigen-Binding Proteins Science 242:423-426). Linkers, in turn, can be modified to provide additional functions, such as attaching drugs or attaching to solid supports. Single-stranded variants can be produced either recombinantly or synthetically. An automated synthesizer can be used to produce scFv synthetically. To produce scFv recombinantly, a suitable plasmid containing the polynucleotide that encodes the scFv can be introduced into a suitable eukaryotic host cell, such as yeast, plant, insect or mammalian cells, or prokaryotic such as E. coli. Polynucleotides encoding the target scFv can be obtained using standard procedures such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification methods known in the art.

The production of bispecific binding molecules (e.g., non-monospecific diabodies) provides significant advantages over antibodies, including trans-binding ability sufficient to co-ligate and/or provide colocalization of different cells that express different epitopes, and/or the ability to cis-linking sufficient to coligate and/or provide colocalization of different molecules expressed by the same cell, but not limited to the above. Thus, there are various uses of bispecific binding molecules (eg, non-monospecific diabodies), including therapy and immunodiagnostics. Bispecificity provides highly flexible capabilities in the design and construction of diabodies for a variety of applications, providing increased avidity to multimeric antigens, cross-linking of different antigens, and targeting of specific cell types based on the presence of both target antigens. Due to their increased valency, low dissociation rate, and rapid absorption from the bloodstream (for small diabodies, ~50 kDa or less), diabody molecules known in the art have also shown particular utility in the field of tumor imaging (Fitzgerald et al. (1997) ) Improved Tumor Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris, Protein Eng. 10:1221).

The ability to create bispecific diabodies has led to their use in co-ligating two cells (trans-linking), for example by co-ligating receptors present on the surface of different cells (eg, cross-linking cytotoxic T cells to tumor cells) (Staerz et al (1985) Hybrid Antibodies Can Target Sites For Attack By T Cells, Nature 314:628-631, and Holliger et al (1996) Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, Protein Eng. 9 :299-305; Marvin et al (2005) Recombinant Approaches To IgG-Like Bispecific Antibodies Acta Pharmacol Sin 26:649-658). Alternatively or additionally, bispecific diabodies can be used to co-ligate molecules, such as receptors, etc., that are present on the surface of the same cell (cis-linking). Co-ligation of different cells and/or receptors can be used to modulate effector functions and/or cell signaling of the immune system. However, the price for these benefits is high. The formation of such non-monospecific diabodies requires the successful combination of two or more separate and distinct polypeptides (i.e., the formation of these non-monospecific diabodies requires that they be formed by heterodimerization of polypeptide chains of different species), as opposed to monospecific diabodies, which are formed by homodimerization of identical polypeptides chains. Because it is necessary to provide at least two different polypeptides (i.e., two kinds of polypeptides) to form a non-monospecific diabody, and because homodimerization of such polypeptides

- 20 043373 leads to the production of inactive molecules (Takemura, S. et al. (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,' Protein Eng. 13(8):583-588), obtain such polypeptides are needed in such a way as to prevent the formation of covalent bonds between polypeptides of the same species (i.e., to prevent homodimerization) (Takemura, S. et al. (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng 13(8):583–588). Accordingly, non-covalent linking of such polypeptides has been proposed in the art (see, for example, Olafsen et al. (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications, Prot. Engr. Des. Sel 17:21-27; Asano et al. (2004) A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng 13(8):583588; Lu, D. et al (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity J Biol Chem 280(20): 19665-19672).

However, work in the art has confirmed that bispecific diabodies, consisting of non-covalently linked polypeptides, are unstable and readily dissociate to form non-functional monomers (see, for example, Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J Biol Chem 280(20): 19665-19672).

In addressing this challenge, the art has achieved success in the development of stable covalently linked heterodimeric non-monospecific diabodies called DART® (Dual Affinity Re-Targeting Reagents) diabodies; see, for example, US Patent Publication No. 2013-0295121; 2010-0174053 and 2009-0060910; European Patent Publications No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and international publications according to PCT No. WO 2012/162068; WO 2012/018687; WO 2010/080538 and Sloan, D.D. et al. (2015) Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells, PLoS Pathog. 11(11):e1005233. doi: 10.1371/journal.ppat. 1005233; Al Hussaini, M. et al. (2015) Targeting CD 123 In AML Using A T-Cell Directed Dual-Affinity Re-Targeting (DART®) Platform, Blood pii: blood-2014-05-575704; Chichili, G.R. et al. (2015) A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates Sci. Transl. Med. 7(289):289ra82; Moore, P.A. et al. (2011) Application Of Dual Affinity Retargeting Molecules To Achieve Optimally Redirected T-Cell Killing Of B-Cell Lymphoma, Blood 117(17):45424551; Veri, M.C. et al. (2010) Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold Arthritis Rheum. 62(7): 19331943; Johnson, S. et al. (2010) Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And In Vivo B-Cell Depletion J. Mol. Biol. 399(3):436-449). Such diabodies contain two or more covalently linked polypeptides and involve the insertion into each of the polypeptide species used of one or more cysteine residues allowing the formation of disulfide bonds and thereby covalently linking one or more pairs of such polypeptide chains to each other. For example, it has been shown that the addition of a cysteine residue to the C-terminus of such a construct allows the formation of a disulfide bond between the involved polypeptide chains, which stabilizes the resulting diabody without affecting the binding characteristics of the diabody.

Many variants of such molecules have been described (see, for example, US patent publications No. 2015/0175697; 2014/0255407; 2014/0099318; 2013/0295121; 2010/0174053; 2009/0060910; 2007-0004909; European patent publications No. EP 2 714079;EP 2601216; EP 2376109; EP 2158221; EP 1868650 and international publications according to PCT No. WO 2012/162068; WO 2012/018687; WO 2010/080538; WO 2006/113665) and proposed in the present application.

Alternative designs are known in the art for use in cases where a tetravalent molecule is desired but an Fc moiety is not required, including, without limitation, tetravalent tandem antibodies, also referred to as TandAbs (see, for example, US Patent Publication No. 2005-0079170, 2007 -0031436, 2010-0099853, 2011-020667 2013-0189263; European patent publications No. EP 1078004, EP 2371866, EP 2361936 and EP 1293514; publications according to PCT No. WO 1999/057150, WO 2 003/025018 and WO 2013/013700), which are formed by homodimerization of two identical chains, each containing a VH1, VL2, VH2 and VL2 domain.

IV. Preferred PD-1 x CTLA-4 bispecific molecules.

One embodiment of the present invention provides PD-1 x CTLA-4 bispecific molecules that are capable of binding to a first epitope and a second epitope, wherein said epitopes are not identical to each other. Such bispecific molecules contain VLr7VH1 domains, which are capable of binding to the first epitope, and VL2/VH2 domains, which are capable of binding to the second epitope. The designations VL1 and VH1 refer, respectively, to va

- 21 043373 to the light chain variable domain and the heavy chain variable domain of such bispecific molecules, wherein said domains bind to a first epitope. Likewise, the designations VL2 and VH2 refer, respectively, to the light chain variable domain and the heavy chain variable domain of such bispecific molecules, wherein said domains bind a second epitope. It does not matter whether a particular epitope is designated as the first or second epitope; such designations are important only from the point of view of the presence and orientation of the polypeptide chain domains of the binding molecule of the present invention. In one embodiment, one such epitope is a human PD-1 epitope and the other such epitope is a CTLA4 epitope. In certain embodiments, the bispecific molecule contains more than two epitope binding sites. Such bispecific molecules bind to at least one PD-1 epitope and at least one CTLA-4 epitope and may further bind to additional PD-1 epitopes and/or additional CTLA-4 epitopes.

The present invention particularly relates to PD-1 x CTLA-4 bispecific molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) that contain epitope-binding antibody fragments that provide their ability to coordinately bind to at least one PD-1 epitope and at least one CTLA-4 epitope. The choice of VL and VH domains for the polypeptide domains of such molecules is coordinated so that the VL domain and VH domain of the same polypeptide chain cannot form an epitope binding site capable of binding to PD-1 or CTLA-4. The selection of said domains is further coordinated such that the assembly of the polypeptide chains that form said PD-1 x CTLA-4 bispecific molecules occurs to form at least one functional antigen binding site specific for at least one PD-1 epitope. 1, and at least one functional antigen binding site specific for at least one CTLA-4 epitope.

The present invention particularly relates to such molecules, bispecific for PD-1 x CTLA-4, which exhibit activity greater than that of two monospecific molecules, one of which contains a heavy chain variable domain and a light chain variable domain of the antibody that binds with PD-1, and the other of which contains a heavy chain variable domain and a light chain variable domain of an antibody that binds to CTLA-4. Examples of such activity include attenuation of PD-1 activity, attenuation of CTLA-4 activity, enhancement of immune system activation, enhancement of effector function, and enhancement of antitumor activity. As used herein, said reduction in activity refers to a reduction of 10% or more, a reduction of 20% or more, a reduction of 50% or more, a reduction of 80% or more, or a reduction of 90% or more of PD-1 inhibitory activity and/or CTLA-4 or complete elimination of such inhibitory activity of PD-1 and/or CTLA-4. As used herein, said enhancement of activity refers to an enhancement of 10% or more, an enhancement of 20% or more, an enhancement of 50% or more, an enhancement of 80% or more, or an enhancement of 90% or more of immune system stimulating, mediated or dependent activity. from the expression or presence of PD-1 and/or CTLA-4, compared with the activity exhibited by two monospecific molecules, one containing the heavy chain variable domain and the light chain variable domain of an antibody that binds PD-1, and the other from which contains a heavy chain variable domain and a light chain variable domain of an antibody that binds to CTLA-4. Examples of activities leading to activation of the immune system include, but are not limited to, immune cell proliferation (eg, T lymphocytes, natural killer (NK) cells), production and/or release of cytokines by immune cells, production and/or or the release of lytic molecules (eg, granzyme, perforin, etc.) by immune cells and/or the expression of activation markers by immune cells. Cytokines that are released upon activation of the immune system are known in the art and include, but are not limited to: IFNy, IL-2 and TNFa, (see for example, Janeway, C.A. et al. 2011) Immunobiology 8th ed. Garland Science Publishing, NY; Banyer, J.L. (2000) Cytokines in innate and adaptive immunity Rev Immunogenet. 2:359-373). Activation markers expressed by immune cells are known in the art and include, but are not limited to: CD69, CD25 and CD107a (see, for example, Janeway, C.A. et al. (2011) IMMUNOBIOLOGY 8th ed. Garland Science Publishing, NY; Shipkova , M. and Wieland, E. (2012) Surface markers of lymphocyte activation and markers of cell proliferation, Clin Chim Acta 413:1338-1349).

A. PD-1 x CTLA-4 bispecific antibodies.

The present invention includes bispecific antibodies capable of simultaneously binding to PD-1 and CTLA-4. In some embodiments, a bispecific antibody capable of simultaneously binding to PD-1 and CTLA-4 is produced by any of the methods described in international publications according to PCT No. WO 1998/002463, WO 2005/070966, WO 2006/107786 WO 2007 /024715, WO 2007/075270, WO 2006/107617, WO 2007/046893, WO 2007/146968, WO 2008/003103, WO 2008/003116, WO 2008/027236, WO 2008/0 24188, WO 2009/132876, WO 2009 /018386, WO 2010/028797, WO2010028796, WO 2010/028795, WO 2010/108127, WO 2010/136172,

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WO 2011/086091, WO 2011/133886, WO 2012/009544, WO 2013/003652, WO 2013/070565, WO

2012/162583, WO 2012/156430, WO 2013/174873 and WO 2014/022540, each of which is hereby incorporated by reference in its entirety.

B. PD-1 x CTLA-4 bispecific diabodies without Fc fragments.

One embodiment of the present invention relates to bispecific diabodies that contain, and most preferably consist of, a first polypeptide chain and a second polypeptide chain, the sequences of which allow the polypeptide chains to covalently link to each other to form a covalently linked diabody that is capable of simultaneously binding to a PD -1 and CTLA-4.

The first polypeptide chain of this embodiment of bispecific diabodies contains (in the direction from N-terminus to C-terminus) the N-terminus, VL domain of a monoclonal antibody capable of binding to PD-1 or CTLA-4 (i.e., VL _PD-1 or VL _ctla-4 ), first intermediate spacer peptide (linker 1), a VH domain of a monoclonal antibody capable of binding to CTLA-4 (if said first polypeptide chain contains VL _PD-1 ) or PD-1 (if said first polypeptide chain the chain contains VL _ctla-4 ), a second intermediate spacer peptide (linker 2), optionally containing a cysteine residue, a heterodimer-promoting domain, and a C terminus (Fig. 1).

The second polypeptide chain of the specified embodiment of the bispecific diabodies contains (in the direction from the N-terminus to the C-terminus) the N-terminus, VL domain of a monoclonal antibody capable of binding to PD-1 or CTLA-4 (i.e., VL _PD-1 or VL _ctla-4 , where the VL domain is not included in the first polypeptide chain of the diabody), intermediate spacer peptide (linker 1), VH domain of a monoclonal antibody capable of binding to CTLA-4 (if said second polypeptide chain contains VL _PD-1 ) or with PD-1 (if said second polypeptide chain contains VL _ctla-4 ), a second intermediate spacer peptide (linker 2), optionally containing a cysteine residue, a heterodimer-promoting domain and a C-terminus (Fig. 1).

The VL domain of the first polypeptide chain interacts with the VH domain of the second polypeptide chain to form a first functional antigen binding site specific for the first antigen (ie, PD-1 or CTLA-4). Likewise, the VL domain of the second polypeptide chain interacts with the VH domain of the first polypeptide chain to form a second functional antigen binding site specific for the second antigen (ie, CTLA-4 or PD-1). Thus, the selection of the VL and VH domains of the first and second polypeptide chains is coordinated so that the two polypeptide chains of the diabody together contain VL and VH domains capable of binding to both the PD-1 epitope and the CTLA-4 epitope ( i.e. they contained VL _PD-1 /VH _PD-1 and VL _ctla-4 /VH _ctla-4 together).

The length of the intermediate linker peptide (linker 1, which separates said VL and VH domains) is most preferably selected to substantially or completely prevent the VL and VH domains of the polypeptide chain from binding to each other (e.g., the length of the intermediate linker is between 0. 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid residues). Thus, the VL and VH domains of the first polypeptide chain are essentially or completely unable to bind to each other. Likewise, the VL and VH domains of the second polypeptide chain are substantially or completely incapable of binding to each other. A preferred intermediate spacer peptide (linker 1) has the sequence (SEQ ID NO: 5): GGGSGGGG.

The length and composition of the second intermediate spacer peptide (linker 2) is selected based on the selection of one or more polypeptide domains that promote said dimerization (ie, heterodimer-promoting domains). Typically the second intermediate spacer peptide (linker 2) contains 3-20 amino acid residues. In particular, when the heterodimer formation-promoting domain(s) used contains/does not contain a cysteine residue, a second cysteine-containing intermediate spacer peptide (linker 2) is used. The second cysteine-containing intermediate spacer peptide (linker 2) contains 1, 2, 3 or more cysteines. A preferred cysteine-containing spacer peptide (linker 2) has the sequence SEQ ID NO: 6: GGCGGG. Alternatively, Linker 2 does not contain a cysteine (e.g., GGG, GGGS (SEQ ID NO: 7), LGGGSG (SEQ ID NO: 8), GGGSGGGSGGG (SEQ ID NO: 9), ASTKG (SEQ ID NO: 10), LEPKSS (SEQ ID NO: 11), APSSS (SEQ ID NO: 12), etc.), and a cysteine-containing heterodimer-promoting domain is used, as described below. Optionally, both a cysteine-containing linker 2 and a cysteine-containing heterodimer-promoting domain are used.

The heterodimer-promoting domains may be GVEPKSC (SEQ ID NO: 13) or VEPKSC (SEQ ID NO: 14) or AEPKSC (SEQ ID NO: 15) on one polypeptide chain and GFNRGEC (SEQ ID NO: 16) or FNRGEC (SEQ ID NO: 17) on another polypeptide chain (US2007/0004909).

In a preferred embodiment, the heterodimer-promoting domains comprise tandemly repeating helical domains of opposite charge, for example E-coil helical domains (SEQ ID NO:18: evaalek-evaalek-evaalek-evaalek), the glutamate residues of which will have a negative charge at pH 7, and K-helical domains (SEQ ID NO: 19: kvaalke-kvaalke-kvaalke-kvaalke), the lysine residues of which will have a positive charge at pH 7.

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The presence of such charged domains promotes binding between the first and second polypeptides and thus promotes heterodimer formation. Heterodimer-promoting domains can be used that contain modifications of the E-coil and K-coil sequences described above to include one or more cysteine residues. The presence of such cysteine residues allows the covalent binding of a helical domain present on one polypeptide chain to a complementary helical domain present on another polypeptide chain, thereby ensuring covalent binding of the polypeptide chains to each other and increasing the stability of the diabody. Examples of such preferred heterodimer formation promoting domains specifically include a modified E-coil domain having the amino acid sequence evaacek-evaalek-evaalek-evaalek (SEQ ID NO: 20) and a modified K-coil domain having the amino acid sequence kvaacke-kvaalke- kvaalke-kvaalke (SEQ ID NO: 21)

As described in International Patent Application Publication WO 2012/018687, to improve the pharmacokinetic properties of the diabody in vivo, the diabody can be modified to contain a polypeptide moiety of a serum binding protein at one or more ends of the diabody. In the most preferred embodiment, such a polypeptide portion of the serum binding protein is added at the C-terminus of the diabody. Albumin is the most abundant protein in blood plasma, with a half-life of 19 days in humans. Albumin contains several small molecule binding sites that allow it to bind non-covalently to other proteins and thus increase their serum half-life. Albumin-binding domain 3 (ABD3) of the G protein of Streptococcus strain G148 consists of 46 amino acid residues forming a stable three-stranded strand and has high specificity for binding to albumin (Johansson, M.U. et al. (2002) Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Modules J Biol Chem 277(10):8114–8120). Thus, a particularly preferred polypeptide moiety of the serum binding protein for improving the pharmacokinetic properties of the diabody in vivo is the albumin binding domain (ABD) of the Streptococcus G protein, and more preferably the albumin binding domain 3 (ABD3) of the Streptococcus G protein G148 (SEQ ID NO: 22): LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

As described in International Patent Application Publication WO 2012/162068 (which is incorporated herein by reference), the deimmunized variants of SEQ ID NO: 22 are capable of reducing or preventing the binding of MHC class II. Based on the results of combining mutations, the following combinations of substitutions are considered preferable for the formation of such a deimmunized ABD: 66D/70S +71A; 66S/70S +71A; 66S/70S +79A; 64A/65A/71A; 64A/65A/71A+66S; 64A/65A/71A+66D; 64A/65A/71A+66E; 64A/65A/79A+66S; 64A/65A/79A+66D; 64A/65A/79A+66E. ABD variants with modifications L64A, I65A and D79A, or modifications N66S, T70S and D79A. Deimmunized ABD variant having amino acid sequence

LAEAKVLANR ELDKYGVSDY YKNLIDggNAKSvo A71EGVKALIDE ILAALP (SEQ

ID NO:23), or amino acid sequence

LAEAKVLANR ELDKYGVSDY YKNA ₆₄ A ₆ 5NNAKT VEGVKALIA79E ILAALP (SEQ

ID NO:24), or amino acid sequence

LAEAKVLANR ELDKYGVSDY YKNLIS66NAKS70 VEGVKALIA79E ILAALP (SEQ ID NO:25) are particularly preferred for use as such deimmunized ABD and substantially exhibit binding consistent with that of the wild type molecule while providing attenuation of binding of MHC class II molecules. Thus, the first polypeptide chain of such an ABD-containing diabody contains a third linker (linker 3), preferably located C-terminal to the E-coil (or K-coil) domain of such polypeptide chain such that it is located between the domain E-coil (or K-coil) and ABD (which is preferably deimmunized ABD). The preferred sequence for such linker 3 is SEQ ID NO: 7: GGGS.

C. PD-1 x CTLA-4 bispecific diabodies containing Fc fragments.

One embodiment of the present invention provides bispecific Fc fragment-containing diabodies capable of simultaneously binding to PD-1 and CTLA-4. Addition of an IgG CH2CH3 domain to one or both of the polypeptide chains of a diabody such that the association of chains of said diabody results in the formation of an Fc moiety, increases the biological half-life and/or changes the valence of the diabody. The inclusion of the CH2-CH3 domains of IgG in both polypeptide chains of the specified diabody allows the formation of a double-chain bispecific Fc-fragment-containing diabody (Fig. 2).

Alternatively, inclusion of the CH2-CH3 domains of an IgG into only one of the polypeptide chains of the diabody allows the formation of a more complex four-chain bispecific Fc moiety-containing diabody (Figures 3A-3C). Figure 3C shows a typical four-chain diabody

- 24 043373 containing a light chain constant domain (CL) and a heavy chain constant domain CH1, however, as an alternative, fragments of such domains, as well as other polypeptides, can be used (see, for example, (Fig. 3A and 3B, US patent publication No. 2013- 0295121; 2010-0174053 and 2009-0060910; European patent publications No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and international publications according to PCT No. WO 2012/162068; WO 2012/018687; WO 2010/080538) Thus, for example, instead of the CH1 domain, a peptide having the amino acid sequence GVEPKSC (SEQ ID NO: 13), VEPKSC (SEQ ID NO: 14) or AEPKSC (SEQ ID NO: 15) derived from the hinge region of human IgG, and instead of the CL domain, can be used the C-terminal 6 amino acids of the human immunoglobulin kappa light chain, GFNRGEC (SEQ ID NO: 16) or FNRGEC (SEQ ID NO: 17) may be used. A typical peptide-containing four-chain diabody is shown in Fig. 3A. Alternatively or additionally, use a peptide containing tandem helical domains of opposite charge, such as E-coil helical domains (SEQ ID NO: 18: EVAALEK-EVAALEK-EVAALEK-EVAALEK OR SEQ ID NO: 19: EVAACEK-EVAALEK-EVAALEK-EVAALEK) and K domains -COil (SEQ ID NO: 20: kvaalke-kvaalke-kvaalke-kvaalke or SEQ ID NO: 21 : kvaacke kvaalke-kvaalke-kvaalke). An example of a helical domain-containing four-chain diabody is shown in FIG. 3B.

The bispecific Fc fragment-containing molecules of the present invention may contain additional intermediate spacer peptides (linkers). Such linkers are typically inserted between the heterodimer-promoting domain of the peptide (eg, E-coil or K-coil) and the CH2-CH3 domains and/or between the CH2-CH3 domains and the variable domain (ie, VH or VL). Typically, additional linkers contain 3-20 amino acid residues. Linkers that can be used in the bispecific Fc-containing diabody molecules of the present invention include: GGGS (SEQ ID NO: 7), LGGGSG (SEQ ID NO: 8), GGGSGGGSGGG (SEQ ID NO: 9), ASTKG (SEQ ID NO: : 10), DKTHTCPPCP (SEQ ID NO: 26), EPKSCDKTHTCPPCP (SEQ ID NO: 27), LEPKSS (SEQ ID NO: 11), APSSS (SEQ ID NO:28) and APSSSPME (SEQ ID NO: 29), LEPKSADKTHTCPPC SEQ ID NO: 30), GGC and GGG. SEQ ID NO: 11 can be used in place of GGG or GGC to facilitate cloning. In addition, the amino acids GGG or SEQ ID NO: 11 may be immediately followed by the sequence SEQ ID NO: 26 to form alternative linkers: GGGDKTHTCPPCP (SEQ ID NO: 31) and LEPKSSDKTHTCPPCP (SEQ ID NO: 32). The bispecific Fc moiety-containing molecules of the present invention may include an IgG hinge region in addition to or instead of a linker. Examples of hinge regions include: EPKSCDKTHTCPPCP (SEQ ID NO: 33) from IgG1, ERKCCVECPPCP (SEQ ID NO: 34) from IgG2, ESKYGPPCPSCP (SEQ ID NO: 35) from IgG4, and eskygppcppcp (SEQ ID NO: 36) a variant of the IgG4 hinge region , containing a stabilizing replacement S228P (when numbered according to the Kabat EC index) to reduce the rate of circuit exchange.

As shown in FIG. 3A-3C, the bispecific Fc moiety-containing diabodies of the invention may contain four different chains. The first and third polypeptide chains of such a diabody contain three domains: (i) a VL1-containing domain, (ii) a VH2-containing domain, (iii) a domain that stimulates heterodimer formation, and (iv) a domain containing the CH2-CH3 sequence. The second and fourth polypeptide chains comprise: (i) a VL2-containing domain, (ii) a VH1-containing domain, and (iii) a heterodimer-promoting domain, wherein said heterodimer-promoting domains promote dimerization of the first/third polypeptide chains with the second/fourth polypeptide chains . The VL and/or VH domains of the third and fourth polypeptide chains and the VL and/or VH domains of the first and second polypeptide chains may be the same or may differ so as to allow tetravalent binding that is monospecific, bispecific or tetraspecific. The designations VL3 and VH3 refer, respectively, to the light chain variable domain and the heavy chain variable domain of the diabody that bind to the third epitope. Likewise, the designations VL4 and VH4 refer, respectively, to the light chain variable domain and the heavy chain variable domain of the diabody that bind to the fourth epitope. The general structure of the polypeptide chains of typical four-chain bispecific Fc-fragment containing diabodies according to the invention is presented in table. 1.

Table 1 Bispecific molecule 2nd chain NH2-VL2-VH1-HPD-COOH 1st chain NH ₂ -VL 1 - VH2-HPD-CH2-CH3 -COOH 1st chain NH ₂ -VL 1 - VH2-HPD-CH2-CH3 -COOH 2nd chain NH ₂ -VL2-VH1-HPD-COOH Tetraspecific molecule 2nd chain NH ₂ -VL2-VH1-HPD-COOH 1st chain NH ₂ -VL 1 - VH2-HPD-CH2-CH3 -COOH 3rd chain NH ₂ -VL3 - VH4-HPD-CH2-CH3 -COOH 4th chain NH ₂ -VL4-VH3-HPD-COOH

HPD = heterodimer formation promoting domain.

In a specific embodiment, the diabodies of the present invention are bispecific tetravalent (i.e., having four epitope binding sites) Fc-containing diabodies that are composed of four (total) polypeptide chains

- 25 043373 (Fig. 3A-3C). The bispecific tetravalent Fc-containing diabodies of the invention contain two epitope binding sites that are immunospecific for PD-1 (which may be capable of binding to the same or different epitopes of PD-1) and two epitope binding sites that are immunospecific for CTLA-1. 4 (which may be able to bind to the same or different CTLA-4 epitopes).

According to another embodiment of the invention, bispecific Fc-containing diabodies may contain three polypeptide chains. The first polypeptide of such a diabody contains three domains: (i) a VL1-containing domain, (ii) a VH2-containing domain, and (iii) a domain containing the CH2-CH3 sequence. The second polypeptide of such a diabody contains: (i) a VL2-containing domain, (ii) a VH1-containing domain, and (iii) a domain that promotes heterodimerization and the formation of a covalent bond with the first polypeptide chain of the specified diabody. The third polypeptide of such a diabody contains the sequence CH2-CH3. Thus, the first and second polypeptide chains of such a diabody are joined together to form a VL1/VH1 binding site that is capable of binding to the first epitope (i.e., PD-1 or CTLA-4), as well as a VL2/VH2 binding site that is capable of bind to the second epitope (ie, CTLA-4 or PD-1). The first and second polypeptides are linked to each other through a disulfide bond involving cysteine residues in the respective third domains. It should be noted that the first and third polypeptide chains combine with each other to form the Fc fragment, which is stabilized through a disulfide bond. Such bispecific diabodies have increased activity. In fig. 4A and 4B show the structures of such diabodies. Such bispecific diabodies containing the Fc fragment can have any of two orientations (Table 2).

table 2 First orientation 3rd chain NH2-CH2-CH3-COOH 1st chain NH2-VLI-VH2-HPD-CH2-CH3-COOH 2nd chain NH ₂ -VL2-VH1-HPD-COOH Second orientation 3rd chain NH2-CH2-CH3-COOH 1st chain NH2-CH2-CH3 -VL 1-VH2-HPD-COOH 2nd chain NH ₂ -VL2-VH1-HPD-COOH

HPD = heterodimer formation promoting domain.

In a specific embodiment, the diabodies of the present invention are bispecific bivalent (i.e., containing two epitope binding sites) Fc-containing diabodies that generally consist of three polypeptide chains ((FIGS. 4A-4B). Bispecific bivalent Fc- containing diabodies of the invention contain one epitope binding site with immunospecificity for PD-1 and one epitope binding site with specificity for CTLA-4.

In another embodiment, the bispecific Fc moiety-containing diabodies may comprise a total of five polypeptide chains. According to a specific embodiment of the invention, two of the five polypeptide chains have the same amino acid sequence. The first polypeptide chain of such a diabody contains: (i) a VH1-containing domain, (ii) a CH1-containing domain, and (iii) a domain containing the CH2-CH3 sequence. The first polypeptide chain may be an antibody heavy chain that contains VH1 and a heavy chain constant region. The second and fifth polypeptide chains of such a diabody contain: (i) a VL1-containing domain and (ii) a CL-containing domain. The second and/or fifth polypeptide chains of such a diabody may be antibody light chains that contain a VL1 complementary to the VH1 of the first/third polypeptide chain. The first, second and/or fifth polypeptide chains can be isolated from a natural antibody. Alternatively, these chains can be constructed recombinantly. The third polypeptide chain of such a diabody contains: (i) a VH1-containing domain, (ii) a CH1-containing domain, (iii) a domain containing the sequence CH2-CH3, (iv) a VL2-containing domain, (v) a VH3-containing domain, and (vi ) a heterodimer formation promoting domain, wherein said heterodimer formation promoting domains promote dimerization of the third strand with the fourth strand. The fourth polypeptide of such diabodies contains: (i) a VL3-containing domain, (ii) a VH2-containing domain, and (iii) a heterodimerization domain and forms a covalent bond with the third polypeptide chain of the diabody.

Thus, the first, second, third and fifth polypeptide chains of such diabodies are joined together to form two VL1/VH1 binding sites capable of binding to the first epitope. The third and fourth polypeptide chains of such diabodies bind together to form a VL2/VH2 binding site that is capable of binding to a second epitope, as well as a VL3/VH3 binding site that is capable of binding to a third epitope. The first and third polypeptides are linked to each other through a disulfide bond involving cysteine residues in the corresponding constant regions. It should be noted that the first and third polypeptide chains combine with each other to form the Fc fragment. Such bispecific diabodies have increased activity. In fig. Figure 5 shows the structure of such diabodies. It should be understood that the VL1/VH1, VL2/VH2 and VL3/VH3 domains may

- 26 043373 be the same or may differ in such a way as to allow binding that is monospecific, bispecific or trispecific. However, as proposed herein, these domains are preferably selected to provide binding to PD-1 and

CTLA-4.

The VL and VH domains of the polypeptide chains are selected to form a VL/VH binding site specific for the desired epitope. The VL/VH binding sites formed by joining the polypeptide chains may be the same or different so as to allow tetravalent binding that is monospecific, bispecific, trispecific or tetraspecific. In particular, the VL and VH domains may be selected such that the bispecific diabody may contain two binding sites for a first epitope and two binding sites for a second epitope, or three binding sites for a first epitope and one binding site for a second epitope, or two sites binding site for the first epitope, one binding site for the second epitope, and one binding site for the third epitope (as depicted in Fig. 5). The general structure of the polypeptide chains of typical five-chain Fc-fragment containing diabodies according to the invention is presented in table. 3. _____________________________________________________

Table 3 Bispecific molecules (2x2) 2nd chain NH2-VLI-CL-COOH 1st chain NH2-VH1-CH1-CH2-CH3-COOH 3rd chain NH2-VHI-CHI-CH2-CH3-VL2-VH2-HPD-COOH 5th chain NH2-VLI-CL-COOH 4th chain NH2-VL2-VH2-HPD-COOH Bispecific molecules (3x1) 2nd chain NH2-VLI-CL-COOH 1st chain NH2-VH1-CH1-CH2-CH3-COOH 3rd chain NH2-VHI -CHI -СН2-СНЗ -VL 1 - VH2-HPD-COOH 5th chain NH2-VLI-CL-COOH 4th chain NH ₂ -VL2-VH1-HPD-COOH Trispecific molecules (2x1x1) 2nd chain NH2-VLI-CL-COOH 1st chain NH2-VH1-CH1-CH2-CH3-COOH 3rd chain NH2-VH 1 -CH 1-CH2-CHZ - VL2-VH3 -HPD-COOH 5th chain NH ₂ -VL1-CL-COOH 4th chain NH ₂ -VL3 - VH2-HPD-COOH

HPD = heterodimer formation promoting domain.

In a specific embodiment, the diabodies of the present invention are bispecific tetravalent (i.e., containing four epitope binding sites) Fc-containing diabodies that consist of five (total) polypeptide chains containing two epitope binding sites that are immunospecific for PD-1 (which may be able to bind to one or different epitopes of PD-1) and two CTLA-4-specific epitope binding sites (which may be able to bind to one or different epitopes of CTLA-4). In another embodiment, the bispecific tetravalent Fc-containing diabodies of the invention comprise three epitope binding sites that are immunospecific for PD-1 (which may be capable of binding to one or two or three different PD-1 epitopes) and one epitope binding site , specific for CTLA-4. In another embodiment, the bispecific tetravalent Fc-containing diabodies of the invention contain one epitope binding site that is immunospecific for PD-1 and three epitope binding sites that are specific for CTLA-4 (which may be capable of binding to one or two or three different CTLA-4 epitopes).

D. PD-1 x CTLA-4 bispecific trivalent binding molecules containing Fc fragments.

An additional embodiment of the present invention provides bispecific trivalent binding molecules containing an Fc moiety capable of simultaneously binding to an epitope of PD-1 and an epitope present on CTLA-4. Such bispecific trivalent binding molecules contain three epitope binding sites, two of which are diabody-type binding domains that provide binding site A and binding site B, and one of which is a Fab-type binding domain (or binding site B). scFv) that provides the C binding site (see, for example, (Fig. 6A-6F and PCT Application Nos. PCT/US15/33081 and PCT/US15/33076). Such bispecific trivalent molecules thus contain VL1/VH1 domains, that are capable of binding to the first epitope, VL2/VH2 domains that are capable of binding to the second epitope, and VL3 and VH3 domains that are capable of binding to the third epitope of such a trivalent molecule. A diabody binding domain is a type of epitope binding site present on a diabody and, in particular, Diatel DART®, as described above.Each of the Fab-binding domain and the scFv-binding domain is an epitope binding site that is formed by interaction

- 27 043373

VL domain of immunoglobulin light chain and complementary VH domain of immunoglobulin heavy chain. Fab binding domains differ from a diabody binding domain in that the two polypeptide chains that form a Fab binding domain contain only one epitope binding site, whereas the two polypeptide chains that form a diabody binding domain contain at least one epitope binding site. two epitope binding sites. Likewise, scFv-type binding domains also differ from diabody-type binding domains in that they contain only one epitope binding site. Thus, as used herein, the Fab-type and scFv-type binding domains are different from the diabody-type binding domain.

Typically, the trivalent binding molecules of the present invention contain four different polypeptide chains (see (Fig. 6A-6B), however, the molecules may contain fewer or more polypeptide chains, for example, as a result of the fusion of such polypeptide chains with each other (for example, through a peptide bond) or by separating such polypeptide chains to form additional polypeptide chains or by joining fewer or additional polypeptide chains through disulfide bonds (FIGS. 6C-6F illustrate this aspect of the present invention by schematic representation of such molecules containing three polypeptide chains As illustrated in Figures 6A-6F, the trivalent binding molecules of the present invention may have alternative orientations in which the diabody binding domain is located at the N-terminus (Figures 6A, 6C and 6D) or the C-terminus (Figures 6A, 6C and 6D). (Fig. 6B, 6E and 6F) in relation to the Fc fragment.

In specific embodiments, the first polypeptide chain of such trivalent binding molecules of the present invention comprises: (i) a VL1-containing domain, (ii) a VH2-containing domain, (iii) a heterodimer-promoting domain, and (iv) a domain containing a CH2-CH3 sequence. The VL1 and VL2 domains are located at the N-terminal side or the C-terminal side relative to the CH2-CH3-containing domain, as shown in Table. 4 (see also (FIGS. 6A and 6B). The second polypeptide chain of such embodiments of trivalent binding molecules contains: (i) a VL2-containing domain, (ii) a VHl-containing domain, and (iii) a heterodimer-stimulating domain. The third polypeptide chain the chain of such embodiments of trivalent binding molecules comprises: (i) a VH3-containing domain, (ii) a CHI-containing domain, and (iii) a domain containing the sequence CH2-CH3. The third polypeptide chain may be an antibody heavy chain that contains VH3 and a heavy chain constant region, or a polypeptide that contains these domains. The fourth polypeptide of such embodiments contains: (i) a VL3-containing domain and (ii) a CL-containing domain. The fourth polypeptide chains may be an antibody light chain that contains VL3 , complementary to VH3 of the third polypeptide chain, or a polypeptide that contains these domains.The third or fourth polypeptide chain can be isolated from natural antibodies. Alternatively, these polypeptide chains may be generated recombinantly, synthetically, or by other means.

The light chain variable domain of the first and second polypeptide chains is separated from the heavy chain variable domains of said polypeptide chains by an intermediate spacer peptide that is too short in length to allow the VL1/VH2 domains (or VL2/VH1 domains) of said chains to combine to form an epitope binding site , capable of binding to either the first or second epitope. The preferred intermediate spacer peptide (linker 1) used for this purpose has the sequence (SEQ ID NO: 5): GGGSGGGG. Other domains of trivalent binding molecules may be separated by one or more intermediate spacer peptides (linkers), optionally containing a cysteine residue. In particular, as presented above, such linkers are typically inserted between variable domains (i.e., VH or VL) and heterodimer-promoting peptide domains (e.g., E-coil or K-coil) and between such heterodimer-promoting peptides domains (for example, E-coil or K-coil) and CH2-CH3 domains. Examples of linkers useful for creating trivalent linking molecules are presented above and also described in PCT Application Nos: PCT/US15/33081 and PCT/US15/33076. Thus, the first and second polypeptide chains of such a trivalent binding molecule bind together to form a VL1/VH1 binding site capable of binding to the first epitope, as well as a VL2/VH2 binding site that is capable of binding to the second epitope. The third and fourth polypeptide chains of such trivalent binding molecules bind together to form a VL3/VH3 binding site that is capable of binding to the third epitope.

As described above, the trivalent binding molecules of the present invention may comprise three polypeptides. Trivalent binding molecules containing three polypeptide chains can be prepared by linking domains of a fourth polypeptide located N-terminal to the VH3-containing domain of a third polypeptide (eg, using an intermediate spacer peptide (linker 4)). Alternatively, a third polypeptide chain of the trivalent binding molecule of the invention is used, containing the following domains: (i) a VL3-containing domain, (ii) a VH3-containing domain, and (iii) a domain containing the sequence CH2-CH3, where VL3 and VH3 are separated from each other by an intermediate spacer peptide, length

- 28 043373 which is sufficient (consists of at least 9 or more amino acid residues) to allow the specified domains to join to form an epitope binding site. One preferred intermediate spacer peptide that can be used for this purpose has the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 37).

It should be understood that the VL1/VH1, VL2/VH2 and VL3/VH3 domains of such trivalent binding molecules may be different, allowing for binding that is bispecific or trispecific. However, as proposed herein, these domains are selected to provide a trivalent binding molecule capable of binding to PD-1 and CTLA4.

In particular, the VL and VH domains can be selected such that the trivalent binding molecule contains two PD-1 binding sites (which may be capable of binding to the same PD-1 epitope or to different PD-1 epitopes) and one binding site CTLA-4, or one PD-1 binding site and two CTLA-4 binding sites (which may be able to bind to one CTLA-4 epitope or to different CTLA-4 epitopes), or one binding site for PD-1, one site binding site for CTLA-4 and one binding site for a third antigen that is not PD-1 or CTLA-4. The general structure of the polypeptide chains of typical trivalent binding molecules according to the invention is shown in FIG. 6A-6F and in table. 4

Table 4

Four-chain molecule 1st orientation option 2nd chain NH ₂ -VL2-VH1-HPD-COOH 1st chain NH2-VL 1 - VH2-HPD-CH2-CH3 -COOH 3rd chain NH ₂ -VH3 -CH 1-CH2-CHZ -COOH 2nd chain NH ₂ -VL3-CL-COOH Four-chain molecule 2nd orientation option 2nd chain NH ₂ -VL2-VH1 -hpd-cooh 1st chain NH ₂ -CH2-CH3 - VL 1 - VH2-HPD-COOH 3rd chain NH ₂ -VH3 -CH 1-CH2-CH3 -COOH 2nd chain NH ₂ -VL3-CL-COOH Three-chain molecule 1st orientation option 2nd chain NH ₂ -VL2-VH1-HPD-COOH 1st chain NH ₂ -VL 1 - VH2-HPD-CH2-CH3 -COOH 3rd chain NH ₂ -VL3 - VH3 -HPD-CH2-CH3 -COOH Three-chain molecule 2nd orientation option 2nd chain NH ₂ -VL2-VH1-HPD-COOH 1st chain NH ₂ -CH2-CH3 -VL 1-VH2-HPD-COOH 3rd chain NH ₂ -VL3 - VH3 -HPD-CH2-CH3 -COOH

HPD = heterodimer formation promoting domain.

One embodiment of the present invention provides bispecific trivalent binding molecules that contain two epitope binding sites for PD-1 and one epitope binding site for CTLA-4.

The two epitope binding sites for PD-1 can bind to the same epitope or to different epitopes. Another embodiment of the present invention provides bispecific trivalent binding molecules that contain one epitope binding site for PD-1 and two epitope binding sites for CTLA-4. The two epitope binding sites for CTLA-4 can bind to the same epitope or to different epitopes of CTLA-4. As described above, such bispecific trivalent binding molecules may contain three, four, five or more polypeptide chains.

V. Constant domains and Fc fragments.

The present application provides antibody constant domains useful for creating PD-1 x CTLA-4 bispecific molecules (eg, antibodies, diabodies, trivalent binding molecules, etc.) according to the invention.

A preferred CL domain is the human IgG kappa CL domain. The amino acid sequence of an example human kappa CL domain is (SEQ ID NO: 38)

RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

Alternatively, an example of a CL domain is a human IgG lambda CL domain. The amino acid sequence of an example human lambda CL domain is (SEQ ID NO: 39)

QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA

GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS

As proposed herein, the PD-1 x CTLA-4 bispecific molecules of the invention may contain an Fc moiety. The Fc portion of such molecules according to the invention may be of any isotype (eg IgG1, IgG2, IgG3 or IgG4). The PD-1 x CTLA-4 bispecific molecules of the invention may further comprise a CH1 domain and/or a hinge region. The CH1 domain and/or hinge region, if present, may be of any isotype (e.g. IgG1, IgG2, IgG3 or IgG4) and are preferably of the same isotype as

- 29 043373 desired Fc fragment.

An example of a CHI domain is the CH domain of human IgG1. The amino acid sequence of an example CH1 domain of human IgG1 is (SEQ ID NO: 40)

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV

HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKRV

An example CH1 domain is the CH domain of human IgG2. The amino acid sequence of an example human IgG2 CH1 domain is (SEQ ID NO: 41)

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTV

An example of a CH1 domain is the CH1 domain of human IgG4. The amino acid sequence of an example human IgG4 CH1 domain is (SEQ ID NO: 42)

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRV

One example of a hinge region is the human IgG1 hinge region. The amino acid sequence of an example human IgG1 hinge region is (SEQ ID NO: 33): EPKSCDKTHTCPPCP.

Another example of a hinge region is the human IgG2 hinge region. The amino acid sequence of an example human IgG2 hinge region is (SEQ ID NO: 34): ERKCCVECPPCP.

Another example of a hinge region is the human IgG4 hinge region. The amino acid sequence of an example human IgG4 hinge region is (SEQ ID NO: 35): ESKYGPPCPSCP. As described herein, the IgG4 hinge region may contain a stabilizing mutation, such as the S228P substitution. An example amino acid sequence of a stabilized IgG4 hinge region is (SEQ ID NO: 36): ESKYGPPCPPCP.

The Fc fragment of Fc fragment containing molecules (eg, antibodies, diabodies, trivalent molecules, etc.) according to the present invention may be a complete Fc fragment (eg, a complete IgG Fc fragment) or only a portion of the Fc fragment. Optionally, the Fc portion of the Fc portion-containing molecule of the present invention does not contain a C-terminal amino acid residue that is lysine.

In classical immune responses, the interaction of antigen-antibody complexes with immune cells results in a wide range of responses ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation and phagocytosis to immunomodulatory signals such as those regulating lymphocyte proliferation and antibody secretion . All of these interactions are initiated through the binding of the Fc fragment of an antibody or immune complexes to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses elicited by antibodies and immune complexes results from the structural heterogeneity of the three Fc receptors: FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16). FcyRI (CD64), FcyRIIA (CD32A), and FcyRIII (CD16) are activating receptors (i.e., they stimulate the immune system); FcyRIIB (CD32B) is an inhibitory receptor (ie it suppresses the immune system). In addition, interaction with the neonatal Fc receptor (FcRn) mediates the recycling of IgG molecules from endosomes to the cell surface and their release into the blood. Examples of the amino acid sequence of wild type IgG1 (SEQ ID NO: 1), IgG2 (SEQ ID NO: 2), IgG3 (SEQ ID NO: 3) and IgG4 (SEQ ID NO: 4) are shown above.

Modification of the Fc fragment may result in a change in phenotype, such as changes in serum half-life, changes in stability, changes in sensitivity to cellular enzymes, or changes in effector function. Thus, it may be desirable to modify the Fc moiety-containing PD-1 x CTLA-4 bispecific molecule of the present invention to alter its effector function, for example, to enhance the effectiveness of such a molecule in the treatment of cancer. Reducing or eliminating effector function is desirable in certain cases, for example, in the case of antibodies, the mechanism of action of which involves blocking or antagonizing, but not killing, cells containing the target antigen. Increasing effector function is generally desirable when targeting unwanted cells, such as tumor and foreign cells where FcyRs are expressed at low levels, such as tumor-specific B cells with low levels of FcyRIIB (eg, non-Hodgkin's lymphoma, CLL and lymphoma cells Burkitt). Molecules of the invention having such acquired or altered effector functional activity are useful for the treatment and/or prevention of a disease, disorder or infection in which increased efficiency of the effector functional activity of said molecules is desired.

Thus, in certain embodiments, the Fc moiety of the Fc moiety-containing molecules of the present invention may be an engineered version of the Fc moiety. Although the Fc moiety of the bispecific Fc moiety containing molecules of the present invention may have the ability to bind to one or more Fc receptors

- 30 043373 (for example, FcyR)), more preferably, such a variant of the Fc fragment has altered binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (compared to binding that the wild-type Fc fragment exhibits), for example, has an increased ability to bind to the activating receptor and/or has a significantly reduced ability or no ability to bind to the inhibitory receptor(s). Thus, the Fc fragment of the Fc fragment containing molecules of the present invention may contain some or all of the CH2 domains and/or some or all of the CH3 domains of the full-length Fc fragment, or may contain a CH2 sequence variant and/or a CH3 sequence variant (which may contain, for example, one or more insertions and/or one or more deletions relative to the CH2 or CH3 domains of the full-length Fc fragment). Such Fc fragments may contain non-Fc polypeptide portions or portions of unnatural full-length Fc fragments, or may have an unnatural orientation of the CH2 and/or CH3 domains (for example, two CH2 domains or two CH3 domains or a CH3 domain linked to a CH2 domain in the direction from N-terminus to C-terminus, etc.

Fc fragment modifications identified as altering effector functions are known in the art, including modifications that increase binding to activating receptors (eg, FcyRIIA (CD16A)) and reduce binding to inhibitory receptors (eg, FcyRIIB (CD32B) (see e.g. , Stavenhagen, J.B. et al (2007) Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors Cancer Res. 57(18):8882-8890). Table 5 lists examples of one, two, three, four and five substitutions (as compared to the amino acid sequence of SEQ ID NO: 1) examples of modifications that increase binding to activating receptors and/or decrease binding to inhibitory receptors._______________________

Table 5 Options for preferred activating substitutions of Fc fragments Options with one replacement F243L R292G D270E R292P Y300L P396L Options with two replacements F243L and R292P F243L and Y300L D270E and P396L R292P and V305I R292P and РЗ 96L Y3 00L and РЗ 96L K392T and P396L Options with three substitutions F243L, P247LhN421K F243L, R292P and Y300L F243L, R292P and V305I F243L, R292P and P396L F243L, Y300L and P396L V284M, R292LhK370N Options with four substitutions L234F, F243L, R292P and Y300L L234F, F243L, R292P and Y300L L235I, F243L, R292P and Y300L L235Q, F243L, R292P and Y300L F243L and РЗ96L R292P and Y300L P396L and Q419H P247L and N421K R255LhP396L R292PhP305I P247L, D270EhN421K R255L, D270E and P396L D270E, G316DhR416G D270E, K392TiR396b D270E, P396LhQ419H R292P, Y300L and P396L F243L, P247L, D270E and N421K F243L, R255L, D270E and P396L F243L, D270E, G316D hR416G F243L, D270E, K392T and P396L P247L, D270E, Y300L and N421K F243L, R292P, Y300L and P396L R255L, D270E, R292G and P396L F243L, R292P, V305I and P396L R255L, D270E, Y300L and P396L F243L, D270E, P396L and Q419H D270E, G316D, P396L hR416G Options with five substitutions L235V, F243L, R292P, Y300L and P396L F243L, R292P, V305I, Y300L and P396L L235P, F243L, R292P, Y300L and P396L

Examples of human IgG1 Fc variants with decreased binding to CD32B and/or increased binding to CD16A include the F243L, R292P, Y300L, V305I, or P296L substitutions. These amino acid substitutions may be present in the Fc fragment of human IgG1 in any combination. In one embodiment, the human IgG1 Fc fragment variant contains the substitutions F243L, R292P, and Y300L. According to another embodiment of the invention, the human IgG1 Fc fragment variant contains the substitution F243L, R292P, Y300L, V305I and P296L.

In specific embodiments, the Fc portions of the PD-1 x CTLA-4 bispecific molecules of the present invention preferably have a reduced (or substantially no) ability to bind to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (compared to the binding exhibited by wild-type IgG1 Fc fragment (SEQ ID NO: 1). In a particular embodiment, the PD-1 x CTLA-4 bispecific molecules of the present invention comprise an IgG Fc fragment , which has reduced effector function associated with antibody-dependent cellular cytotoxicity (ADCC).In a preferred embodiment, the CH2-CH3 domains of such PD-1 x CTLA-4 bispecific molecules contain any 1, 2, 3 or 4 of the substitutions: L234A , L235A, D265A, N297Q, and N297G. In another embodiment, the CH2-CH3 domains contain the N297Q substitution, the N297G substitution, the L234A and L235A substitutions, or the D265A substitution because these mutations prevent FcR binding. As an alternative, the CH2-CH3 domain of the natural Fc fragment is used, which itself has a reduced (by

- 31 043373 essentially lacks) FcyRIIIA (CD16a) binding ability and/or reduced effector function (compared to wild-type IgG1 Fc fragment binding and effector function (SEQ ID NO: 1)). In a specific embodiment, the PD-1 x CTLA-4 bispecific molecules of the present invention comprise an IgG2 Fc fragment (SEQ ID NO: 2) or an IgG4 Fc fragment (SEQ ID:NO:4). When using the Fc fragment of IgG4, the present invention also includes the introduction of a stabilizing mutation, such as the S228P substitution in the hinge region described above (see, for example, SEQ ID NO: 36). Because the N297G, N297Q, L234A, L235A, and D265A substitutions prevent effector function, these substitutions are preferably not used under conditions in which effector function is desired.

The preferred IgG1 sequence for the CH2 and CH3 domains of Fc-containing molecules of the present invention with reduced or eliminated effector function will contain the L234A/L235A substitutions (SEQ ID NO: 43)

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSK L TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGX where, X represents lysine (K) or is absent.

The serum half-life of proteins containing Fc fragments can be increased by increasing the binding affinity of the Fc fragment for FcRn. The term half-life as used herein refers to a pharmacokinetic property of a molecule that is a measure of the average lifetime of the molecules after administration. Half-life can be expressed as the time required for fifty percent (50%) of a known amount of a molecule to be cleared from the body of a subject (e.g., a human or other mammalian patient) or from a specific compartment thereof, e.g., when measured in serum, i.e. . as half-life in the circulatory system or in other tissues. In general, an increase in half-life results in an increase in the mean retention time (MRT) of the administered molecule in the bloodstream.

In some embodiments, the PD-1 x CTLA-4 bispecific molecules of the present invention comprise a variant Fc fragment, wherein the variant Fc fragment contains at least one amino acid modification relative to the wild type Fc fragment that provides the molecule with increased half-life (compared to a molecule containing the wild-type Fc fragment). In some embodiments, the PD-1 x CTLA-4 bispecific molecules of the present invention comprise a variant Fc fragment of an IgG, wherein the variant Fc fragment comprises a half-life-increasing amino acid substitution at one or more positions selected from the group consisting of 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 3 78, 380, 382, 413, 424, 428, 433, 434, 435, and 436. A variety of mutations capable of increasing the half-life of an Fc moiety-containing molecule are known in the art and include, for example, M252Y, S254T, T256E, and combinations thereof. See, for example, the mutations described in US patents No. 6,277,375, 7,083,784; 7,217,797, 8,088,376; US Patent Publications No. 2002/0147311; 2007/0148164 and international publications No. WO 98/23289; WO 2009/058492 and WO 2010/033279, which are incorporated herein by reference in their entirety. PD1 x CTLA-4 bispecific molecules with extended half-life also include molecules containing Fc fragment variants with substitutions of two or more of residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378 , 428, 433, 434, 435 and 436. Specifically, two or more replacements are selected from T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K and Y436I.

In a specific embodiment, the PD-1 x CTLA-4 bispecific molecules comprise an IgG Fc variant containing the following substitutions:

(A) M252Y, S254T and T256E;

(B) M252Y and S254T;

(C) M252Y and T256E;

(D) T250Q and M428L;

(E) T307Q and N434A;

(F) A378V and N434A;

(G) N434A and Y436I;

(H) V308P and N434A or (I) K288D and H435K.

In a preferred embodiment, the PD-1 x CTLA-4 bispecific molecules comprise an IgG Fc variant containing any 1, 2 or 3 of M252Y, S254T and T256E substitutions. The invention further includes PD-1 x CTLA-4 bispecific molecules containing a variant Fc fragment comprising:

(A) one or more mutations that alter effector functions and/or FcyR; and (B) one or more mutations that increase serum half-life.

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The preferred IgG1 sequence for the CH2 and CH3 domains of Fc-containing molecules of the present invention having an increased serum half-life contains substitutions M252Y, S254T and T256E (SEQ ID NO: 80) APEAAGGPSV FLFPPKPKDT LYITREPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTV LH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGX where X represents lysine (K) or is absent.

It should be noted that the CH2-CH3 domains of SEQ ID NO: 80 contain substitutions of amino acids at positions 234 and 235 with alanine, thus the resulting Fc fragment binds less (or essentially does not bind) to FcyRIA (CD64), FcyRIIA (CD32A ), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (compared to the indicated binding for the wild type Fc fragment (SEQ ID NO: 1). The invention also includes those IgG1 CH2-CH3 domains that contain alanine residues wild type, alternative and/or additional substitutions that modify effector function and/or Fc fragment binding activity to FyR.

Preferred IgG4 sequence for the CH2 and CH3 domains of the Fc-containing molecules of the present invention with increased serum half-life contain substitutions M252Y, S254T and T256E (SEQ ID NO: 81)

APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFF LYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGX where, X represents lysine (K) or is absent.

In the case of particular antibodies, diabodies and trivalent binding molecules in which the first and third polypeptide chains containing the Fc moiety are not identical, it is desirable to reduce or prevent the occurrence of homodimerization between the CH2-CH3 domains of the first two polypeptide chains or between the CH2-CH3 domains of two third polypeptide chains. The CH2 and/or CH3 domains of such polypeptide chains need not be identical in sequence and are preferably modified to facilitate the joining of the two polypeptide chains. For example, an amino acid substitution (preferably an amino acid substitution containing a bulky side group forming a knob, such as tryptophan) can be introduced into the CH2 or CH3 domain such that the steric effect prevents interaction with the similarly modified domain and forces the mutant domain to pair with the domain , in which a complementary or adaptive mutation was created, i.e. depression (for example, replacement with glycine). Such sets of mutations can be inserted into any pair of CH2-CH3 domain-containing polypeptides that form the Fc moiety to promote heterodimerization. Protein engineering techniques that promote heterodimerization over homodimerization are well known in the art, particularly with respect to the engineering of immunoglobulin-like molecules, and are included herein (see, for example, Ridgway et al. (1996) 'Knobs-Into -Holes' Engineering Of Antibody CHS Domains For Heavy Chain Heterodimerization, Protein Engr 9:617–621, Atwell et al (1997) Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library, J. Mol. Biol 270: 26-35, and Xie et al (2005) A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis, J Immunol Methods 296:95-101, each of which therefore incorporated herein by reference in its entirety).

Preferably, the protrusion is created by modifying the Fc portion of the IgG so that it contains the T366W modification. Preferably, the cavity is created by modifying the Fc portion of the IgG so that it contains the modification T366S, L368A and Y407V. To facilitate purification of the homodimer containing the dimpled third polypeptide chain from the final bispecific heterodimeric Fc fragment-containing molecule, the protein A binding site of the dimpled CH2 and CH3 domains of the third polypeptide chain is preferably modified by introducing an amino acid substitution at position 435 (H435R). Thus, a homodimer of the dimpled third polypeptide chain will not bind to protein A, whereas a bispecific heterodimer will retain its ability to bind to protein A through the protein A binding site on the first polypeptide chain. According to an alternative embodiment of the invention, the cavity-containing third polypeptide chain may contain amino acid substitutions at positions 434 and 435 (N434A/N435K).

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The preferred IgG1 amino acid sequence for the CH2 and CH3 domains of the first polypeptide chain of the Fc moiety of the present invention will comprise a knob sequence (SEQ ID NO: 44)

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSK L TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGX where X is lysine (K) or absent.

The preferred IgG1 amino acid sequence for the CH2 and CH3 domains of the second polypeptide chain of a two-polypeptide chain Fc-containing molecule of the present invention (or the third polypeptide chain of a three, four or five polypeptide chain Fc-containing molecule) will be cavity containing sequence (SEQ ID NO: 45)

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD

GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGX where X represents lysine (K) or absent.

It should be noted that the CH2-CH3 domains of SEQ ID NO: 44 and SEQ ID NO: 45 include substitutions of amino acids at positions 234 and 235 with alanine and, thus, the resulting Fc fragment binds weaker (essentially does not bind) to FcyRIA (CD64) , FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (compared to binding for the wild type Fc fragment (SEQ ID NO: 1)). The invention also includes those IgG1 CH2-CH3 domains that contain wild-type alanine residues, alternative and/or additional substitutions that modify the effector function and/or FyR binding activity of the Fc fragment. The invention also includes such CH2-CH3 domains that further contain one or more half-life increasing amino acid substitutions. In particular, as described above, the invention includes such pit-containing and such protrusion-containing CH2-CH3 domains, which further comprise M252Y/S254T/T256E.

The preferred IgG1 amino acid sequence for the additional M252Y/S254T/T256E containing CH2 and CH3 domains of the first polypeptide chain of the Fc moiety of the present invention will be a knob-containing sequence (SEQ ID NO: 82)

APEAAGGPSV FLFPPKPKDT LYITREPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSK L TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGX where X is lysine (K) or absent.

Preferred IgG1 amino acid sequence for the additional M252Y/S254T/T256E CH2 and CH3 domains of a second polypeptide chain of an Fc-containing molecule of the present invention that contains two polypeptide chains (or a third polypeptide chain of an Fc-containing molecule that contains three, four, or five polypeptide chains) will have a cavity-containing sequence (SEQ ID NO: 83)

APEAAGGPSV FLFPPKPKDT LYITREPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSK L TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGX where X is lysine (K) or absent.

The preferred IgG4 amino acid sequence for the M252Y/S254T/T256E containing CH2 and CH3 domains of the first polypeptide chain containing an Fc moiety of the present invention will have a knob sequence (SEQ ID NO: 84)

APEFLGGPSV FLFPPKPKDT LYITREPEVT CWVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFF LYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGX where X is lysine (K) or absent.

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Preferred IgG4 amino acid sequence for the M252Y/S254T/T256E-containing CH2 and CH3 domains of a second polypeptide chain of an Fc-containing molecule of the present invention that contains two polypeptide chains (or a third polypeptide chain of an Fc-containing molecule that contains three, four or five polypeptide chains) will have a cavity-containing sequence (SEQ ID NO: 85)

APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFF LVSRL TVDKSRWQEG NVFSCSVMHE ALHNRYTQKS LSLSLGX where X is lysine (K) or absent.

It should be noted that the CH2-CH3 domains of SEQ ID NO: 84 and SEQ ID NO: 85 contain substitutions M252Y/S254T/T256E and thus form the Fc fragment of IgG4, providing an increased half-life in blood serum. The invention also includes the CH2-CH3 domains of IgG4, which contain wild-type M252/S254/T256 residues.

The CH2-CH3 domains of the first polypeptide chain preferably have a knob-containing sequence, such as the sequence of SEQ ID NO: 44. However, it should be understood that in the first polypeptide chain, CH2-CH3 trench-containing domains (for example, SEQ ID NO: 45) can be used, in If so, CH2-CH3 overhang domains (e.g., SEQ ID NO: 44) will be used in a second polypeptide chain of an Fc moiety of the present invention that contains two polypeptide chains (or a third polypeptide chain of an Fc moiety that contains three , four or five polypeptide chains).

In other embodiments, the invention includes PD-1 x CTLA-4 bispecific molecules containing CH2 and/or CH3 domains that have been engineered to promote heterodimerization as opposed to homodimerization, using mutations known in the art techniques such as mutations described in international publications according to PCT No. WO 2007/110205; WO 2011/143545; WO 2012/058768; WO 2013/06867, all of which are incorporated herein by reference in their entirety.

VI. PD-1 binding ability.

Antibodies are known that are immunospecific for PD-1 (see, for example, US patent applications No. 62/198,867; 62/239,559; 62/255,140, US patent No. 8,008,449; 8,552,154; international patent publications according to PCT WO 2012/135408; WO 2012/145549 and WO 2013/014668). A preferred PD-1 binding ability useful for creating PD-1 x CTLA-4 bispecific molecules of the present invention is the ability to bind to a continuous or discontinuous (e.g., conformational) portion (epitope) of human PD-1 (CD279) and preferably also the ability to bind to the PD-1 molecule of one or more non-human species, in particular primate species (especially primate species such as the cynomolgus macaque). Additional desired antibodies can be obtained by isolating hybridomas with antibody secretion caused by the use of PD-1 or a peptide fragment thereof. A representative human PD-1 polypeptide (sequence in NCBI NP_005009.2; including the 20 amino acid residue signal sequence shown underlined) and the 268 amino acid residues of the mature protein have the amino acid sequence (SEQ ID NO: 46)

MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLWTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGWGGLLGS LVLLVWV LAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL

Preferred PD-1 binding molecules (e.g., antibodies) useful for generating PD-1 x CTLA-4 bispecific molecules of the present invention comprise the VL and/or VH domains of the anti-human PD-1 monoclonal antibody PD-1 mAb 1 (nivolumab, registration number CAS:946414-94-4, also known as 5C4, BMS-936558, ONO-4538, MDX-1106 and commercially available as OPDIVO® from Bristol-Myers Squibb); PD-1 mAb 2 (pembrolizumab, (formerly known as lambrolizumab), CAS registration number: 1374853-91-4, also known as MK-3475, SCH-900475 and marketed as KEYTRUDA® from Merck); PD-1 mAb 3 (EH12.2H7; Dana Farber), PD-1 mAb 4 (pidilizumab, CAS registration number: 1036730-42-3, also known as CT-011, CureTech) or any of the anti-PD-1 antibodies from table 6 and, more preferably, contain 1, 2 or all 3 of the CDRL VL domain and/or 1, 2 or all 3 of the CDRH VH domain of such anti-PD-1 monoclonal antibody. Additional anti-PD-1 antibodies having unique binding properties useful in the methods and compositions of the present invention have recently been identified (see US Patent Application Nos. 62/198,867; 62/239,559; 62/255,140). Particularly preferred are PD-1 binding molecules that contain a humanized VH and/or VL domain of the anti-PD-1 antibody PD-1 mAb 5 (hPD-1 mAb 2,

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MacroGenics); PD-1 mAb 6 (hPD-1 mAb 7, MacroGenics); PD-1 mAb 7 (hPD-1 mAb 9, MacroGenics) or

PD-1 mAb 8 (hPD-1 mAb 15, MacroGenics) and more preferably contain 1, 2 or all 3 of the sites

CDRL of the VL domain and/or 1, 2 or all 3 of the CDRH regions of the VH domain of said humanized anti-PD-1 monoclonal antibody.

A. PD-1 mAb 1.

The amino acid sequence of the VH domain of PD-1 mAb 1 (SEQ ID NO: 47) is shown below (os- CDRH tags are underlined). QVQLVESGGG WQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSS Amino acid sequence of the VL domain of PD-1 mAb 1 (SEQ ID NO: 48) shown below (os- CDRL pads are underlined). EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIK B. PD-1 mAb 2. Amino acid sequence of the VH domain of PD-1 mAb 2 (SEQ ID NO: 49) shown below (os- CDRH tags are underlined). QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS Amino acid sequence of the VL domain of PD-1 mAb 2 (SEQ ID NO: 50) shown below (os- CDRL pads are underlined). EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES GVPARFSSGG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEIK C. PD-1 mAb 3. Amino acid sequence of the VH domain of PD-1 mAb 3 (SEQ ID NO: 51) shown below (os- CDRH tags are underlined). QVQLQQSGAE LAKPGASVQM SCKASGYSFT SSWIHWVKQR PGQGLEWIGY IYPSTGFTEY NQKFKDKATL TADKSSSTAY MQLSSLTSED SAVYYCARWR DSSGYHAMDY WGQGTSVTVSS Amino acid sequence of the VL domain of PD-1 mAb 3 (SEQ ID NO: 52) shown below (os- CDRL pads are underlined). DIVLTQSPAS LTVSLGQRAT ISCRASQSVS TSGYSYMHWY QQKPGQPPKL LIKFGSNLES GIPARFSGSG SGTDFTLNIH PVEEEDTATY YCQHSWEIPY TFGGGTKLEI K D. PD-1 mAb 4. Amino acid sequence of the VH domain of PD-1 mAb 4 (SEQ ID NO: 53) shown below (os- CDRH tags are underlined). QVQLVQSGSE LKKPGASVKI SCKASGYTFT NYGMNWVRQA PGQGLQWMGW INTDSGESTY AEEFKGRFVF SLDTSVNTAY LQITSLTAED TGMYFCVRVG YDALDYWGQG TLVTVSS Amino acid sequence of the VL domain of PD-1 mAb 4 (SEQ ID NO: 54) shown below (os-

CDRL pads are underlined).

EIVLTQSPSS LSASVGDRVT ITCSARSSVS YMHWFQQKPG KAPKLWIYRT

SNLASGVPSR FSGSGSGTSY CLTINSLQPE DFATYYCQQR SSFPLTFGGG

TKLEIK

E. PD-1 mAb 5.

The amino acid sequence of the VH domain of PD-1 mAb 5 (SEQ ID NO: 55) is shown below (CDRH residues are underlined).

EVQLVESGGG LVQPGGSLRL SCAASGFVFS SFGMHWVRQA PGKGLEWVAY ISSGSMSISY ADTVKGRFTI SRDNAKNTLY LQMNSLRTED TALYYCASLS DYFDYWGQGT TVTVSS

The amino acid sequence of the VL domain of PD-1 mAb 5 (SEQ ID NO: 56) is shown below (CDRL residues are underlined).

DWMTQSPLS LPVTLGQPAS ISCRSSQSLV HSTGNTYLHW YLQKPGQSPQ LLIYRVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQTTHVP WTFGQGTKLE IK F. PD-1 mAb 6.

The amino acid sequence of the VH domain of PD-1 mAb 6 (SEQ ID NO: 57) is shown below (CDRH residues are underlined).

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWXiGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSS where Xi represents I or A.

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The amino acid sequence of the VL domain of PD-1 mAb 6 (SEQ ID NO: 58) is shown below (CDRL residues are underlined).

EIVLTQSPAT LSLSPGERAT LSCRAXiESVD NYGMSFMNWF QQKPGQPPKL LIHAASNX ₂ GS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI K where: Xi represents N or S and X ₂ represents Q or R; or

Xi represents N and Xr represents Q; or Xi represents S and X ₂ represents Q; or Xi is S and X ₂ is R.

Specifically, embodiments of the amino acid sequence of PD-1 mAb 6 include:

(a) SEQ ID NO: 57, where X1 is I, and SEQ ID NO: 58, where X1 is N and X ₂ is Q; or (b) SEQ ID NO: 57, where X1 is I, and SEQ ID NO: 58, where X1 is S and X ₂ is Q.

An example of an anti-PD-1 VH domain antibody, designated PD-1 mAb 6-I VH, contains SEQ ID NO: 57, where X1 is I, and has the amino acid sequence (SEQ ID NO: 86) QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSS

An example anti-PD-1 VL domain, designated PD-1 mAb 6-SQ VL, contains SEQ ID NO: 58, where X1 is S and X ₂ is Q, and has the amino acid sequence (SEQ ID NO: 87)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI K

An example of an anti-PD-1 antibody that contains the VH domain of PD-1 mAb 6-I and the VL domain of PD-1 mAb 6-SQ is designated PD-1 mAb 6-ISQ.

G. PD-1 mAb 7.

The amino acid sequence of the VH domain of PD-1 mAb 7 (SEQ ID NO: 59) is shown below (CDR _H residues are underlined).

EVQLVESGGG LXiRPGGSLKL SCAASGFTFS SYLVX ₂ WVRQA PGKGLEWX3AT ISGGGGNTYY SDSVKGRFTI SRDNAKNSLY LQMNSX4RAED TATYYCARYG FDGAWFAYWG QGTLVTVSS where: Xi represents V or A; X ₂ represents S or G; Xs represents V or T; X ₄ represents L or A; or

Xi represents V, X ₂ represents S, X3 represents V and X ₄ represents L; or

Xi represents A, X ₂ represents G, X3 represents T and X ₄ represents A.

The amino acid sequence of the VL domain of PD-1 mAb 7 (SEQ ID NO: 60) is shown below (CDRL residues are underlined).

where: Xi represents S or N and X ₂ represents N or D; or Xi represents S and X ₂ represents N; or

Xi represents N and X ₂ represents D

Specifically, embodiments of PD-1 mAb 7 include:

(a) SEQ ID NO: 59 where X ₁ is V, X2 is S, X ₃ is V and X4 is L, and SEQ ID NO: 60 where X1 is S and X2 is N; or (b) SEQ ID NO: 59 wherein X1 is A, X2 is G, _X3 is T and X4 is A, and SEQ ID NO: 60 wherein X1 is N and X2 is D.

H. mAb against PD-1 8.

The amino acid sequence of the VH domain of anti-PD-1 mAb 8 (SEQ ID NO: 61) is shown below (CDRH residues are underlined).

EVQLVESGGG LVRPGGSLRL SCAASGFTFS SYLISWVRQA PGKGLEWVAA ISGGGADTYY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TATYYCARRG TYAMDYWGQG TLVTVSS

The amino acid sequence of the VL domain of anti-PD-1 mAb 8 (SEQ ID NO: 62) is shown below (CDRL residues are underlined).

DIQMTQSPSS LSASVGDRVT ITCRASENIY NYLAWYQQKP GKAPKLLIYD AKTLAAGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQH HYAVPWTFGQ GTKLEIK

I. Additional anti-PD-1 antibodies.

Additional anti-PD-1 antibodies that can be used to create PD-1 x CTLA-4 bispecific molecules according to the present invention are presented in table. 6.

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Table 6: Additional Anti-PD-1 Antibodies Antibodies against PD-1 Link/Source PD1-17; PD1-28; PD1-33; PD1-35 and PD1-F2 US Patents No. 7,488,802; 7,521,051 and 8,088,905; international patent publication according to PCT WO 2004/056875 17D8; 2D3; 4H1; 5S4; 4A11; 7D3 and 5F4 US Patents No. 8,008,449; 8,779,105 and 9,084,776; international patent publication according to PCTWO 2006/121168 hPD-1.08A; hPD-1.09A; 109A; K09A; 409A; h409All; h409A16; h409A17; codon-optimized version 109A and codon-optimized version 409A US Patents No. 8,354,509; 8,900,587 and 5,952,136; international patent publication according to PCTWO 2008/156712 1EZ; 1E8 and 1NZ US Patent Publication 2014/0044738; international patent publication according to PCTWO 2012/145493 9A2; 10B11; 6E9; ARE1922; ARE1923; ARE1924; ARE1950; ARE1963 and ARE2058 International patent publication according to PCT WO 2014/179664 GAI; GA2; GB1; GB6; GH1; A2; C7; H7; SH-A4; SH-A9; RG1H10; RG1H11; RG2H7; RG2H10; RG3E12; RG4A6; RG5D9; RG1H10-H2A-22-1S; RG1H10-H2A-27-2S; RG1H10-3C; RG1H10-16C; RG1H10-17C; RG1H10-19C; RG1H10-21C and RG1H10-23C2 US Patent Publication 2014/0356363; international patent publication according to PCTWO 2014/194302 H1M7789N; H1M7799N; H1M7800N; H2M7780N; H2M7788N; H2M7790N; H2M7791N; H2M7794N; H2M7795N; H2M7796N; H2M7798N; N4N9019R; Н4хН9034Р2; Н4хН9035Р2; Н4хН9037Р2; Н4хН9045Р2; Н4хН9048Р2; N4N9057R2; N4N9068R2; Н4хН9119Р2; Н4хН9120Р2; H4Xh9128p2; H4Xh9135p2; H4Xh9145p2; H4Xh8992p; H4Xh8999p and H4Xh9008p; US Patent Publication 2015/0203579; international patent publication according to PCTWO 2015/112800 PD-1 mAb 1; PD-1 mAb 2; mAb against hPD-1 2; PD-1 mAb 3; PD-1 mAb 4; PD-1 mAb 5; PD-1 mAb 6; PD-1 mAb 7; mAb against hPD-1 7; mAb against PD-1 8; mAb against PD-1 9; mAb against hPD-1 9; PD-1 mAb 10; PD-1 mAb 11; PD-1 mAb 12; PD-1 mAb 13; PD-1 mAb 14; PD-1 mAb 15 and anti-hPD-1 mAb 15 US Patent Applications no. 62/198.867 and 62/239.559

J. Example of anti-PD-1 antibody.

An example anti-PD-1 antibody, designated PD-1 mAb 6 G4P, contains: heavy chain containing VH domain PD-1 mAb 6I (SEQ ID NO: 86), CH1 domain IgG4 (SEQ ID NO: 42), stabilized hinge region of IgG 4 (SEQ ID NO: 36) and CH2-CH3 domains of IgG4 without C-terminal lysine (SEQ ID NO: 4), light chain containing VL domain of PD-1 mAb 6SQ (SEQ ID NO: 87) and kappa-CL (SEQ ID NO: 38).

The amino acid sequence of the entire heavy chain of PD-1 mAb 6 G4P (SEQ ID NO: 88) is shown below.

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSWTV PSSSLGTKTY TCNVDHKPSN TKVDKRVESK YGPPCPPCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VWDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR WSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG QPREPQVYTL P PSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLG

The amino acid sequence of the entire light chain of PD-1 mAb 6 G4P (SEQ ID NO: 89) is shown below.

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL

LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY

TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV

THQGLSSPVT KSFNRGEC

VI I. Binding ability to CTLA-4.

Antibodies are known that are immunospecific for CTLA-4 (see, for example, US patents No. 6,984,720; 6,682,736; 7,034,121; 7,109,003; 7,132,281; 7,411,057; 7,605,238; 7,807,797; 7,82 4,679; 8,017,114; 8,143,379; 8,318,916; 8,491,895; 8,784,815 and 8,883,984; patent publications US 2009/0123477; 2009/0252741 and 2014/0105914; international patent publications according to PCT No. WO 00/37504; WO 01/14424; WO 01/54732; WO 2006/029219; WO 2006/066568 and WO 20 12/120125 and table 7). Preferably, the CTLA-4 binding ability useful for creating the PD-1 x CTLA-4 bispecific molecules of the present invention is the ability to bind to a continuous or discontinuous (eg, conformational) portion (epitope) of human CTLA-4 and preferably also includes the ability to bind to CTLA-4 molecules of one or more species of non-human origin, in particular primate species (and, in particular, primate species such as cynomolgus macaques). Additional desired antibodies can be generated by isolating antibody-secreting hybridomas stimulated with CTLA-4 or its peptide

- 38 043373 fragments. A typical human CTLA-4 polypeptide (NCBI sequence NP_005205.2; including the 35 amino acid residue signal sequence (shown underlined) and the 188 amino acid residues of the mature protein) has the amino acid sequence (SEQ ID NO: 75)

MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQ PAWL ASS RGIASFVCEY ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAV SSGLFFYSFL LTAVSLSKML K KRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN

Preferred CTLA-4 binding molecules (e.g., antibodies) useful for generating PD-1 x CTLA-4 bispecific molecules of the present invention comprise the VL and/or VH domains of the anti-human CTLA-4 monoclonal antibody mAb CTLA- 4 1 (ipilimumab, CAS registration number: 477202-00-9, also known as MDX010 and marketed as YERVOY® from Bristol-Myers Squibb); mAb CTLA-4 2 (tremelimumab, CAS registration number: 745013-59-6, also known as CP-675206); mAb CTLA-4 3 (4B6, as presented in Table 7) or any of the other anti-CTLA-4 antibodies from Table. 7 and, more preferably, contain 1, 2 or all 3 of the CDRL VL domain and/or 1, 2 or all 3 of the CDRH VH domain of such anti-CTLA-4 monoclonal antibodies.

A. CTLA-4 mAb 1.

The amino acid sequence of the anti-VH domain antibody CTLA-4 mAb 1 (SEQ ID NO: 76) is shown below (CDRH residues are underlined).

QVQLVESGGG WQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGNNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAIYYCARTG WLGPFDYWGQ GTLVTVSS

The amino acid sequence of the VL domain mAb against CTLA-4 (SEQ ID NO: 77) is shown below (CDR _L residues are underlined).

EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY

GAFSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIK

B. CTLA-4 mAb 2.

The amino acid sequence of the VH domain of anti-CTLA-4 mAb2 (SEQ ID NO: 78) is shown below (CDRH residues are underlined).

QVQLVESGGG WQPGRSLRL SCAASGFTFS SYGMHWVRQA PGKGLEWVAV IWYDGSNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP RGATLYYYYY GMDVWGQGTT VTVSS

The amino acid sequence of the VL domain of anti-CTLA-4 mAb2 (SEQ ID NO: 79) is shown below (CDR _L residues are underlined).

DIQMTQSPSS LSASVGDRVT ITCRASQSIN SYLDWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYSTPFTFGP GTKVEIK

C. CTLA-4 mAb 3.

The amino acid sequence of the VH domain of anti-CTLA-4 mAb3 (SEQ ID NO: 90) is shown below (CDRH residues are underlined).

QVQLVESGGG WQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGSNKHY ADSVKGRFTV SRDNSKNTLY LQMNSLRAED TAIYYCARTG WLGPFDYWGQ GTLVTVSS

The amino acid sequence of the VL domain of anti-CTLA-4 mAb3 (SEQ ID NO: 91) is shown below (CDR _L residues are underlined).

EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSFLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIK

D. Additional anti-CTLA-4 antibodies.

Additional anti-CTLA-4 antibodies that can be used to create PD-1 x CTLA-4 bispecific molecules according to the present invention are presented in table. 7.

Table 7: Additional Anti-CTLA-4 Antibodies Antibodies against CTLA-4 Link/Source mAb 26 US Patent No. 7,034,121; international patent publication according to PCT WO 01/54732 10D1; 1E2i4B6 US Patents No. 6,984,720; 7 605 238; 8 017 114; 8,318,916 and 8,784,815; international patent publication according to PCT WO 01/14424 2.1.3; 3.1.1; 4.1.1; 4.8.1; 4.9.1; 4.10.2; 4.13.1; 4.14.3; 6.1.1; 11.2.1; 11.6.1; 11.7.1; 12.2.1; 12.3.1; 12.3.1.1; 12.9.1 and 12.9.1.1 US Patents No. 6,682,736; 7 109 003; 7 132 281; 7 411 057; 7 807 797; 7,824,679; 8 143 379; 8,491,895 and 8,883,984; international patent publication according to PCT WO 00/37504 ZV10; 8H5; 8Н5-1В1; 3B10-4F7; 7V9-1AZ; 2C7-1G10; ZV10-6EZ and 8N5-1A1 US Patent Publication 2014/0105914; international patent publication according to PCT WO 2012/120125 3.7F10A2; 4.3F6B5; 4.4A7F4; 4.6С1ЭЗ; 4.7A8N8; 4.7E11F1; 4.8Н10Н5; TGN2122 and TGN2422 US Patent Publication 2009/0123477; international patent publication according to PCT WO 2006/066568 L3D10; L1B11; K4G4; KM10 and YL2 US Patent Publication 2009/0252741; international patent publication according to PCT WO 2006/029219

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E. Examples of anti-CTLA-4 antibodies.

An example anti-CTLA-4 antibody designated CTLA-4 mAb 3 G1AA contains a heavy chain containing the VH domain of CTLA-4 mAb 3 (SEQ ID NO: 90), CH1 domain of IgG1 (SEQ ID NO: 40), hinge region IgG1 (SEQ ID NO: 33) and CH2-CH3 domains of IgG1 with substitutions L234A/L235A (SEQ ID NO:

43).

The amino acid sequence of the entire heavy chain of CTLA-4 mAb 3 G1AA (SEQ ID NO: 92) is shown below.

QVQLVESGGG WQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGSNKHY ADSVKGRFTV SRDNSKNTLY LQMNSLRAED TAIYYCARTG WLGPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTC LV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

An example of an alternative anti-CTLA-4 antibody, designated CTLA-4 mAb 3 G4P, contains a heavy chain containing the VH domain of CTLA-4 mAb 3 (SEQ ID NO: 90), CH1 domain of IgG4 (SEQ ID NO: 42), stabilized hinge region of IgG4 (SEQ ID NO: 36) and CH2-CH3 domains of IgG4 without C-terminal lysine (SEQ ID NO: 4). The amino acid sequence of the entire heavy chain of CTLA-4 mAb 3 G4P is shown below (SEQ ID NO: 93).

QVQLVESGGG WQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGSNKHY ADSVKGRFTV SRDNSKNTLY LQMNSLRAED TAIYYCARTG WLGPFDYWGQ GTLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SS SLGTKTYT CNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLM ISRTPEVTCV WDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLV KGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLG

Amino acid sequence of the entire light chain of CTLA-4 mAb 3 G1AA and CTLA-4 mAb 3

G4P (SEQ ID NO: 94) is shown below.

EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSFLAWYQQK PGQAPRLLIY

GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG

QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASWCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

Both of the example anti-CTLA-4 antibodies, CTLA-4 mAb 3 G1AA and CTLA-4 mAb 3 G4P, contain a light chain containing the VL domain of CTLA-4 mAb 3 (SEQ ID NO: 91) and CL kappa (SEQ ID NO : 38).

VIII. Examples of PD-1 x CTLA-4 bispecific molecules.

A. Examples of four-chain, Fc-containing diabodies having E/K-coil domains.

Three examples of PD-1 x CTLA-4 bispecific four-chain Fc-containing diabodies having E/K-coil domains promoting heterodimer formation (designated DART B, DART C, and DART D) were generated. The structure of these Fc moiety containing diabodies is described in detail below. These examples of PD-1 x CTLA-4 bispecific diabodies are intended to illustrate the present invention and are not intended to limit its scope in any way.

1. DART B.

DART B is a bispecific four-chain Fc-containing diabody containing two PD-1-specific binding sites, two CTLA-4-specific binding sites, an IgG4 Fc variant engineered to increase half-life, and E domains /K-coil, promoting the formation of a heterodimer. The first and third polypeptide chains of DART B contain (in the direction from the N-terminus to the C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to CTLA-4 (VL _CTLA - ₄ CTLA-4 mAb 1 VL) ( SEQ ID NO: 77); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to PD-1 (VH _PD -1 PD-1 mAb 6-I VH) (SEQ ID NO: 86); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing domain promoting heterodimer formation (E-coil) (EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 20)); stabilized IgG4 hinge region (SEQ ID NO: 36); a variant of the CH2-CH3 domain of IgG4 containing the substitutions M252Y/S254T/T256E and not containing the C-terminal residue (SEQ ID NO: 81), and the C-terminus.

- 40 043373

The amino acid sequence of the first and third polypeptide chains of DART B is (SEQ ID NO: 95)

EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY GAFSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIKGG GSGGGGQVQL VQSGAEVKKP GASVKVSCKA SGYSFTSYWM NWVRQAPGQG LEWIGVIHPS D SETWLDQKF KDRVTITVDK STSTAYMELS SLRSEDTAVY YCAREHYGTS PFAYWGQGTL VTVSSGGCGG GEVAACEKEV AALEKEVAAL EKEVAALEKE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLYITREPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST Y RWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLG

The second and fourth polypeptide chains of DART B contain (in the direction from N-terminus to C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to PD-1 (VL _PD-1 PD-1 mAb 6-SQ VL ) (SEQ ID NO: 87); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to CTLA-4 (VHCTLA-4 CTLA-4 mAb 1 VH) (SEQ ID NO: 76); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing heterodimer-promoting domain (K-coil) (KVAACKEKVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 21) and C-terminus.

The amino acid sequence of the second and fourth polypeptide chains of DART B is (SEQ ID NO: 96)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS YTMHWVRQAP GKGLEWVTFI SYDGNNKYYA DSVK GRFTIS RDNSKNTLYL QMNSLRAEDT AIYYCARTGW LGPFDYWGQG TLVTVSSGGC GGGKVAACKE KVAALKEKVA ALKEKVAALK E

2. DART C.

DART C is a bispecific four-chain Fc-containing diabody that contains two PD-1-specific binding sites, two CTLA-4-specific binding sites, an IgG4 Fc variant engineered to increase half-life, and domains E/K-coils promoting heterodimer formation. The first and third polypeptide chains of DART C contain (in the direction from N-terminus to C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to CTLA-4 (VLCTLA-4 CTLA-4 mAb 3 VL) (SEQ ID NO: 91); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to PD-1 (VH _PD-1 PD-1 mAb 6-I VH) (SEQ ID NO: 86); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing domain promoting heterodimer formation (E-coil) (EVAACEK-EVAALEK-EVAALEKEVAALEK (SEQ ID NO: 20)); stabilized IgG4 hinge region (SEQ ID NO: 36); an IgG4 CH2-CH3 domain variant containing the M252Y/S254T/T256E substitutions and lacking the C-terminal residue (SEQ ID NO: 81); and C-terminus.

The amino acid sequence of the first and third polypeptide chains of DART C is (SEQ ID NO: 97)

EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSFLAWYQQK PGQAPRLLIY

GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG

QGTKVEIKGG GSGGGGQVQL VQSGAEVKKP GASVKVSCKA SGYSFTSYWM

NWVRQAPGQG LEWIGVIHPS DSETWLDQKF KDRVTTITVDK STSTAYMELS

SLRSEDTAVY YCAREHYGTS PFAYWGQGTL VTVSSGGCGG GEVAACEKEV

AALEKEVAAL EKEVAALEKE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD

TLYITREPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST

YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY

TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLG

The second and fourth polypeptide chains of DART C contain (in the direction from N-terminus to C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to PD-1 (VL _PD-1 PD-1 mAb 6-SQ VL ) (SEQ ID NO: 87); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding CTLA-4 (VHCTLA-4 CTLA-4 mAb 3 VH) (SEQ ID NO: 90); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing heterodimer-promoting domain (K-coil) (KVAACKEKVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 21) and C-terminus.

- 41 043373

The amino acid sequence of the second and fourth polypeptide chains of DART C is (SEQ ID NO: 98)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS YTMHWVRQAP GKGLEWVTFI SYDGSNKHYA DSVKG RFTVS RDNSKNTLYL QMNSLRAEDT AIYYCARTGW LGPFDYWGQG TLVTVSSGGC GGGKVAACKE KVAALKEKVA ALKEKVAALK E

3. DART D.

DART D is a bispecific four-chain Fc-containing diabody that contains two PD-1-specific binding sites, two CTLA-4-specific binding sites, an IgG4 Fc variant engineered to increase half-life, and domains E/K-coils promoting heterodimer formation. The first and third polypeptide chains of DART D contain (in the direction from N-terminus to C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to PD-1 (VLPD-1 PD-1 mAb 6-SQ VL) (SEQ ID NO: 87); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding CTLA-4 (VH _CTLA - ₄ CTLA-4 mAb 3 VH) (SEQ ID NO: 90); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing domain promoting heterodimer formation (E-coil) (EVAACEK-EVAALEK-EVAALEKEVAALEK (SEQ ID NO: 20)); stabilized IgG4 hinge region (SEQ ID NO: 36); an IgG4 CH2-CH3 domain variant containing the M252Y/S254T/T256E substitutions and lacking the C-terminal residue (SEQ ID NO: 81); and C-terminus.

The amino acid sequence of the first and third polypeptide chains of DART D is (SEQ ID NO: 99)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS YTMHWVRQAP GKGLEWVTFI SYDGSNKHYA DSVKG RFTVS RDNSKNTLYL QMNSLRAEDT AIYYCARTGW LGPFDYWGQG TLVTVSSGGC GGGEVAACEK EVAALEKEVA ALEKEVAALE KESKYGPPCP PCPAPEFLGG PSVFLFPPKP KDTLYITREP EVTCVWDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRWSVLT VLHQDWL NGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLG

The second and fourth polypeptide chains of DART D contain (in the direction from N-terminus to C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to CTLA-4 (VLCTLA-4 CTLA-4 mAb 3 VL) (SEQ ID NO: 91); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to PD-1 (VHPD-1 PD-1 mAb 6-I VH) (SEQ ID NO: 86); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing heterodimer-promoting domain (K-coil) (KVAACKEKVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 21) and C-terminus.

The amino acid sequence of the second and fourth polypeptide chains of DART D is (SEQ ID NO: 100)

EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSFLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIKGG GSGGGGQVQL VQSGAEVKKP GASVKVSCKA SGYSFTSYWM NWVRQAPGQG LEWIGVIHPS DSE TWLDQKF KDRVTITVDK STSTAYMELS SLRSEDTAVY YCAREHYGTS PFAYWGQGTL VTVSSGGCGG GKVAACKEKV AALKEKVAAL KEKVAALKE

4. DART F.

DART F is a bispecific four-chain Fc-containing diabody that contains two PD-1-specific binding sites, two CTLA-4-specific binding sites, an IgG1 Fc variant engineered to reduce/eliminate effector function and to increase half-life, and E/K-coil domains to promote heterodimer formation. The first and third polypeptide chains of DART F contain (in the direction from N-terminus to C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to PD-1 (VLPD-1 PD-1 mAb 6-SQ VL) (SEQ ID NO: 87); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding CTLA-4 (VH _CTLA - ₄ CTLA-4 mAb 3 VH) (SEQ ID NO: 90); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing domain promoting heterodimer formation (E-coil) (EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 20)); linker (SEQ ID NO: 30); a variant of the CH2-CH3 domain of IgG1 containing the substitutions L235A/L235A/M252Y/S254T/T256E and not containing the C-terminal residue (SEQ ID NO: 80); and C-terminus.

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Amino acid sequence of the first and third polypeptide chains of DART F (SEQ ID NO:

101) represents

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS YTMHWVRQAP GKGLEWVTFI SYDGSNKHYA DSVKG RFTVS RDNSKNTLYL QMNSLRAEDT AIYYCARTGW LGPFDYWGQG TLVTVSSGGC GGGEVAACEK EVAALEKEVA ALEKEVAALE KLEPKSADKT HTCPPCPAPE AAGGPSVFLF PPKPKDTLYI TREPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLH QDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPG

The second and fourth polypeptide chains of DART F contain (in the direction from N-terminus to C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to CTLA-4 (VLCTLA-4 CTLA-4 mAb 3 VL) (SEQ ID NO: 91); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to PD-1 (VH _PD-1 PD-1 mAb 6-I VH) (SEQ ID NO: 86); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing heterodimer-promoting domain (K-coil) (KVAACKEKVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 21) and C-terminus.

The amino acid sequence of the second and fourth polypeptide chains of DART F is the same as the amino acid sequence of the second and fourth polypeptide chains of DART D (SEQ ID NO: 100).

B. Examples of four-chain Fc-containing diabodies that contain CL/CH1 domains: DART E.

An example of a PD-1 x CTLA-4 bispecific four-chain Fc-containing diabody that contains CL/CH1 domains was generated and designated DART E. The structure of these Fc-containing diabodies is described in detail below. This example of a PD-1 x CTLA-4 bispecific diabody is intended to illustrate the present invention and is not intended to limit its scope in any way.

DART E is a bispecific four-chain Fc-containing diabody containing two PD-1-specific binding sites, two CTLA-4-specific binding sites, CL/CH1 domains, and an IgG4 Fc variant engineered to increase half-life time. The first and third polypeptide chains of DART E contain (in the direction from N-terminus to C-terminus): N-terminus; VL domain of a monoclonal antibody capable of binding to CTLA-4 (VL _CTLA - ₄ CTLA-4 mAb 3 VL) (SEQ ID NO: 91); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to PD-1 (VHPD-1 PD-1 mAb 6-I VH) (SEQ ID NO: 86); intermediate linker peptide (linker 2: LGGGSG (SEQ ID NO: 8)); CH1 domain of IgG4 (SEQ ID NO: 42); stabilized IgG4 hinge region (SEQ ID NO: 36); a variant of the CH2-CH3 domain of IgG4 containing the substitutions M252Y/S254T/T256E and not containing the C-terminal residue (SEQ ID NO: 81), and the C-terminus.

The amino acid sequence of the first and third polypeptide chains of DART E is (SEQ ID NO: 102)

EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSFLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIKGG GSGGGGQVQL VQSGAEVKKP GASVKVSCKA SGYSFTSYWM NWVRQAPGQG LEWIGVIHPS DSE TWLDQKF KDRVTITVDK STSTAYMELS SLRSEDTAVY YCAREHYGTS PFAYWGQGTL VTVSSLGGGS GASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS V FLFPPKPKD TLYITREPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLG

The second and fourth polypeptide chains of DART E contain (in the direction from N-terminus to C-terminus): N-terminus; VL domain of a monoclonal antibody capable of binding to PD-1 (VL _PD -1 PD-1 mAb 6-SQ VL) (SEQ ID NO: 87); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding CTLA-4 (VH _CTLA - ₄ CTLA-4 mAb 3 VH) (SEQ ID NO: 90); intermediate linker peptide (linker 2: LGGGSG (SEQ ID NO: 8)); Kappa CL domain (SEQ ID NO: 38) and C terminus.

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The amino acid sequence of the second and fourth polypeptide chains of DART E is (SEQ ID NO: 103)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVESGGGV VQPGRSLRLS CAASGFTFSS YTMHWVRQAP GKGLEWVTFI SYDGSNKHYA DSVKG RFTVS RDNSKNTLYL QMNSLRAEDT AIYYCARTGW LGPFDYWGQG TLVTVSSLGG GSGRTVAAPS VFIFPPSDEQ LKSGTASWC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

C. Examples of trivalent binding molecules containing Fc fragments.

Two examples of PD-1 x CTLA-4 bispecific four-chain Fc-containing trivalent binding molecules (designated TRIDENT A and TRIDENT B) were generated. The structure of these Fc-containing trivalent binding molecules is described in detail below. Also shown below is a three-stranded variant, designated TRIDENT C, that can be created. These examples of PD-1 x CTLA-4 bispecific trivalent binding molecules are provided to illustrate the present invention and are not intended to limit its scope in any way.

1. TRIDENT A (TRIDENT A).

TRIDENT A is a bispecific, four-chain, Fc-containing trivalent binding molecule containing two PD-1-specific binding sites, one CTLA-4-specific binding site, a knob/valve variant of the IgG4 Fc fragment, designed to increase half-life, E/K-coil domains that promote heterodimer formation, and CL/CH1 domains. The first polypeptide chain of TRIDENT A contains (from N-terminus to C-terminus): the VL domain of a monoclonal antibody capable of binding to PD-1 (VLPD-1 PD-1 mAb 6-SQ VL) (SEQ ID NO: 87 ); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to PD-1 (VH _PD -1 PD-1 mAb 6-I VH) (SEQ ID NO: 86); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing domain promoting heterodimer formation (E-coil) (EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 20)); stabilized IgG4 hinge region (SEQ ID NO: 36); the IgG4 CH2-CH3 domain containing the overhang and the M252Y/S254T/T256E substitutions and lacking the C-terminal residue (SEQ ID NO: 84), and the C-terminus.

The amino acid sequence of the first polypeptide chain of TRIDENT A is (SEQ ID NO: 104)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL

LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY

TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYSFTS

YWMNWVRQAP GQGLEWIGVI HPSDSETWLD QKFKDRVTIT VDKSTSTAYM

ELSSLRSEDT AVYYCAREHY GTSPFAYWGQ GTLVTVSSGG CGGGEVAACE

KEVAALEKEV AALEKEVAAL EKESKYGPPC PPCPAPEFLG GPSVFLFPPK

PKDTLYITRE PEVTCVWDV SQEDPEVQFN WYVDGVEVHN AKTKPREEQF

NSTYRWSVL TVLHQDWLNG KEYKCKVSNK GLPSSIEKTI SKAKGQPREP

QVYTLPPSQE EMTKNQVSLW CLVKGFYPSD IAVEWESNGQ PENNYKTTPP

VLDSDGSFFL YSRLTVDKSR WQEGNVFSCS VMHEALHNHY TQKSLSLSLG

The second polypeptide chain of TRIDENT A contains (in the direction from N-terminus to C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to PD-1 (VLPD-1 PD-1 mAb 6-SQ VL) (SEQ ID NO: 87); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to PD-1 (VH _PD -1 PD-1 mAb 6-I VH) (SEQ ID NO: 86); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); a cysteine-containing heterodimer-promoting domain (K-coil) (KVAACKE-KVAALKEKVAALKE-KVAALKE (SEQ ID NO: 21)) and a C-terminus.

The amino acid sequence of the second polypeptide chain of TRIDENT A is (SEQ ID NO: 105)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYSFTS YWMNWVRQAP GQGLEWIGVI HPSDSETWLD QKFK DRVTIT VDKSTSTAYM ELSSLRSEDT AVYYCAREHY GTSPFAYWGQ GTLVTVSSGG CGGGKVAACK EKVAALKEKV AALKEKVAAL KE

The third polypeptide chain of TRIDENT A contains (in the direction from the N-terminus to the C-terminus): N-terminus; VH domain of a monoclonal antibody capable of binding CTLA-4 (VH _CTLA - ₄ CTLA-4 mAb 3 VH) (SEQ ID NO: 90); CH1 domain of IgG4 (SEQ ID NO: 42); stabilized IgG4 hinge region (SEQ ID NO: 36); the CH2-CH3 domain of IgG4 containing the cavity and substitutions M252Y/S254T/T256E and not containing the C-terminal residue (SEQ ID NO: 81), and the C-terminus.

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The amino acid sequence of the third polypeptide chain of TRIDENT A is (SEQ ID NO: 106)

QVQLVESGGG WQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGSNKHY ADSVKGRFTV SRDNSKNTLY LQMNSLRAED TAIYYCARTG WLGPFDYWGQ GTLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SS SLGTKTYT CNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLY ITREPEVTCV WDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLSCAV KGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSRLTV DKSRWQEGNV FSCSVMHEAL HNRYTQKSLS LSLG

The fourth polypeptide chain of TRIDENT A contains (in the direction from N-terminus to C-terminus): Nterminus; VL domain of a monoclonal antibody capable of binding to CTLA-4 (VL _ctla-4 CTLA-4 mAb 3 VL) (SEQ ID NO: 91); Kappa CL domain (SEQ ID NO: 38) and C terminus.

The amino acid sequence of the fourth polypeptide chain of TRIDENT A is (SEQ ID NO: 107)

EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSFLAWYQQK PGQAPRLLIY

GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG

QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASWCLLNNF YPREAKVQWK

VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ

GLSSPVTKSF NRGEC

2. TRIDENT B (TRIDENT B).

TRIDENT B is a bispecific four-chain Fc-containing trivalent binding molecule that contains two PD-1-specific binding sites, one CTLA-4-specific binding site, an IgG1 Fc-containing knob/valve variant engineered to reduce/eliminate effector function and to increase half-life, E/K-coil domains promoting heterodimer formation and CL/CH1 domains. The first polypeptide chain of TRIDENT B contains (from N-terminus to C-terminus): the VL domain of a monoclonal antibody capable of binding to PD-1 (VL _PD-1 PD-1 mAb 6-SQ VL) (SEQ ID NO: 87) ; intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to PD-1 (VH _PD-1 PD-1 mAb 6-I VH) (SEQ ID NO: 86); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); cysteine-containing domain promoting heterodimer formation (E-coil) (EVAACEKEVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 20)); linker (SEQ ID NO: 31); the CH2-CH3 domain of IgG1 containing the overhang and substitutions L234A/L235A/M252Y/S254T/T256E and not containing the C-terminal residue (SEQ ID NO: 82), and the C-terminus.

The amino acid sequence of the first polypeptide chain of TRIDENT B is (SEQ ID NO: 108)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYSFTS YWMNWVRQAP GQGLEWIGVI HPSDSETWLD QKFK DRVTIT VDKSTSTAYM ELSSLRSEDT AVYYCAREHY GTSPFAYWGQ GTLVTVSSGG CGGGEVAACE KEVAALEKEV AALEKEVAAL EKGGGDKTHT CPPCPAPEAA GGPSVFLFPP KPKDTLYITR EPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDW LN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL WCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

The second polypeptide chain of TRIDENT B contains (in the direction from N-terminus to C-terminus): N-terminus, VL domain of a monoclonal antibody capable of binding to PD-1 (VL _PD-1 PD-1 mAb 6-SQ VL) ( SEQ ID NO: 87); intermediate linker peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); VH domain of a monoclonal antibody capable of binding to PD-1 (VH _PD-1 PD-1 mAb 6-I VH) (SEQ ID NO: 86); cysteine-containing intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); a cysteine-containing heterodimer-promoting domain (K-coil) (KVAACKE-KVAALKEKVAALKE-KVAALKE (SEQ ID NO: 21)) and a C-terminus.

The amino acid sequence of the second polypeptide chain of TRIDENT B is the same as the amino acid sequence of the second polypeptide chain of TRIDENT A (SEQ ID NO: 105):

The third polypeptide chain of TRIDENT B contains (in the direction from N-terminus to C-terminus): N-terminus; VH domain of a monoclonal antibody capable of binding CTLA-4 (VH _CTLA-4 CTLA-4 mAb 3 VH) (SEQ ID NO: 90); CHI-domain IgG1 (SEQ ID NO: 40); IgG1 hinge region (SEQ ID NO: 33); the CH2-CH3 domain of IgG1 containing the cavity and substitutions L234A/L235A/M252Y/S254T/T256E and not containing the C-terminal residue (SEQ ID NO: 83), and the C-terminus.

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The amino acid sequence of the third polypeptide chain of TRIDENT B is (SEQ ID NO: 109)

QVQLVESGGG WQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGSNKHY ADSVKGRFTV SRDNSKNTLY LQMNSLRAED TAIYYCARTG IWLGPFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSWTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLYITREPEV TCVWDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSL SCAV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK

The fourth polypeptide chain of TRIDENT B contains (in the direction from N-terminus to C-terminus): Nterminus; VL domain of a monoclonal antibody capable of binding to CTLA-4 (VLCTLA-4 CTLA-4 mAb 3 VL) (SEQ ID NO: 91); Kappa CL domain (SEQ ID NO: 38) and C terminus.

The amino acid sequence of the fourth polypeptide chain of TRIDENT B is the same as the amino acid sequence of the second polypeptide chain of TRIDENT A (SEQ ID NO: 107).

3. TRIDENT C (TRIDENT C).

As proposed herein, trivalent binding molecules containing three polypeptide chains can be created by combining the binding domains of two separate polypeptide chains into a single chain (eg, by hybridizing coding polynucleotides, etc.). One bispecific three-chain Fc-containing trivalent binding molecule that can be designed contains two PD-1-specific binding sites, one CTLA-4-specific binding site, a knob/valve variant of the IgG4 Fc fragment, engineered to increase half-life, and E/K-coil domains promoting heterodimer formation (TRIDENT C). The first and second polypeptide chains of TRIDENT C may be identical to the first and second polypeptide chains of TRIDENT A presented above.

If the first and second chains are identical to the first and second chains of TRIDENT A, the third polypeptide chain of TRIDENT C may contain (in the direction from N-terminus to C-terminus): N-terminus; VL domain of a monoclonal antibody capable of binding to CTLA-4 (VLCTLA-4 CTLA-4 mAb 3 VL) (SEQ ID NO: 91); intermediate spacer peptide (GGGGSGGGGSGGGGS (SEQ ID NO: 37)); VH domain of a monoclonal antibody capable of binding to CTLA-4 (VHCTLA-4 CTLA-4 mAb 3 VH) (SEQ ID NO: 90); stabilized IgG4 hinge region (SEQ ID NO: 36); the CH2-CH3 domain of IgG4 containing the cavity and substitutions M252Y/S254T/T256E and not containing the C-terminal residue (SEQ ID NO: 85), and the C-terminal.

Thus, the amino acid sequence of the third polypeptide chain of TRIDENT C is (SEQ ID NO: 110)

EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSFLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIKGG GGSGGGGSGG GGSQVQLVES GGGWQPGRS LRLSCAASGF TFSSYTMHWV RQAPGKGLEW VTFISYDGSN K HYADSVKGR FTVSRDNSKN TLYLQMNSLR AEDTAIYYCA RTGWLGPFDY WGQGTLVTVS SESKYGPPCP PCPAPEFLGG PSVFLFPPKP KDTLYITREP EVTCWVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRWSVLT VLHQDWLNGK EYKCKVSNKG LPSS IEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLSC AVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLV SRLTVDKSRW QEGNVFSCSV MHEALHNRYT QKSLSLSLG

IX. Methods of obtaining.

The PD-1 x CTLA-4 bispecific molecules of the present invention are most preferably prepared by recombinant expression of nucleic acid molecules encoding these polypeptides, as is well known in the art.

The polypeptides of the invention can be successfully prepared using solid-phase peptide synthesis (Merrifield, B. (1986) Solid-Phase Synthesis Science 232(4748):341-347; Houghten, R.A. (1985) General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Ganesan, A. (2006) Solid-Phase Synthesis In The Twenty -First Century Mini Rev Med Chem 6(1):3-10).

Alternatively, antibodies can be produced recombinantly and expressed using a method known in the art. Antibodies can be produced recombinantly by first isolating an antibody obtained from a host animal, determining its gene sequence, and using said gene sequence to recombinantly express the antibody in host cells (eg, CHO cells). Another method that can be used is to express the antibody sequence in plants (eg tobacco) or the milk of transgenic animals. Suitable methods for recombinant expression of antibodies in plants or milk have been described (see, for example, Peeters et al. (2001) Production Of Antibodies And Antibody Fragments In Plants Vaccine 19:2756; Lonberg, N. et al. (1995) Human Antibodies From Transgenic Mice Int. Rev. Immunol

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13:65-93; and Pollock et al. (1999) Transgenic Milk As A Method For The Production Of Recombinant Antibodies J. Immunol Methods 231:147-157). Suitable methods for creating antibody derivatives, eg, humanized antibodies, single chain antibodies, etc., are known in the art and have been described above. In another alternative, antibodies can be produced recombinantly using phage display technology (see, for example, US Pat. Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150; and Winter, G. et al. (1994) Making Antibodies By Phage Display Technology Annu. Rev. Immunol. 12.433-455).

Vectors containing polynucleotides of interest (e.g., polynucleotides encoding the polypeptide chains of the PD-1 x CTLA-4 bispecific molecules of the present invention) can be introduced into a host cell by any of a number of suitable methods, including electroporation, calcium chloride transfection , rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microparticle bombardment; lipofection and infection (eg, where the vector is an infectious agent such as vaccinia virus). The choice of vectors or polynucleotides to be introduced often depends on the characteristics of the host cell.

Any host cell capable of expressing heterologous DNA at elevated levels can be used to express the polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa and CHO cells.

The invention includes polypeptides containing the amino acid sequence of the PD-1 x CTLA-4 bispecific molecules of the present invention. The polypeptides of the present invention can be prepared using procedures known in the art. Polypeptides can be produced by proteolytic or other degradation of antibodies, by recombinant methods (ie, as single or hybrid polypeptides) as described above, or by chemical synthesis. Antibody polypeptides, particularly short polypeptides of up to about 50 amino acids in size, are conveniently produced by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available.

The invention includes variants of PD-1 x CTLA-4 bispecific molecules containing functionally equivalent polypeptides that do not significantly affect the properties of these molecules, as well as variants that have increased or decreased activity. Modification of polypeptides is standard practice in the art and does not need to be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, with one or more deletions or additions of amino acids that do not significantly adversely affect functional activity, or using chemical analogues. Amino acid residues that can be substituted for each other as conservative substitutions include, but are not limited to: glycine/alanine; serine/threonine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; lysine/arginine and phenylalanine/tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with various sugars, acetylation and phosphorylation. Preferably, the amino acid substitutions are conservative, i.e. the replacement amino acid has chemical properties similar to those of the original amino acid. Such conservative substitutions are known in the art and examples have been provided above. Amino acid modifications can range from changing or modifying one or more amino acids to completely remodeling a region, such as a variable domain. Changes in the variable domain may result in changes in binding affinity and/or specificity. Other modification methods include the use of coupling methods known in the art, including, but not limited to, enzymatic methods, oxidative displacement and chelation. The modifications can be used, for example, to attach labels for immunoassays, such as attaching radioactive moieties for radioimmunoassays. Modified polypeptides can be prepared using methods conventional in the art and screened using standard assays known in the art.

The invention includes fusion proteins containing one or more of the polypeptides or antibodies of the present invention. According to one embodiment of the invention, a hybrid polypeptide is provided that contains a light chain, a heavy chain, or a light and a heavy chain. According to another embodiment of the invention, the hybrid polypeptide contains a heterologous immunoglobulin constant domain. In another embodiment, the hybrid polypeptide comprises a light chain variable domain and a heavy chain variable domain of an antibody obtained from a publicly available deposited hybridoma. For purposes of the present invention, the antibody fusion protein contains one or more polypeptide domains that specifically bind PD-1 and/or CTLA-4, and another amino acid sequence to which it is not attached in the native molecule, for example, a heterologous sequence or a homologous sequence from another area.

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X. Methods of using the PD-1 x CTLA-4 bispecific molecules of the present invention.

The present invention includes compositions, including pharmaceutical compositions, containing PD-1 x CTLA-4 bispecific molecules of the present invention (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.), polypeptides derived from such molecules, polynucleotides containing sequences encoding such molecules or polypeptides, and other agents described in this application.

As discussed above, PD-1 and CTLA-4 play important roles in the negative regulation of immune responses (eg, immune cell proliferation, function, and homeostasis). The PD-1 x CTLA-4 bispecific molecules of the present invention are capable of inhibiting PD-1 function and thereby reversing PD-1-mediated suppression of the immune system. In addition, the PD-1 x CTLA-4 bispecific molecules of the present invention are capable of inhibiting CTLA-4 function and thereby stimulating the immune system by blocking PD-1 and CTLA-4 mediated suppression of the immune system. The PD-1 x CTLA-4 bispecific molecules of the present invention also allow complete blocking of PD-1 and CTLA-4, as well as preferential blocking of CTLA-4 when co-expressed with PD-1. Thus, the PD-1 x CTLA-4 bispecific molecules of the invention are useful for attenuating T cell exhaustion and/or enhancing an immune response (e.g., T cell and/or natural killer cell (NK) cell-mediated immune response cells)) of the subject. In particular, the PD-1 x CTLA-4 bispecific molecules of the invention can be used to treat any disease or condition associated with unwanted suppression of the immune system, including cancer and diseases that are associated with the presence of a pathogen (e.g., bacterial, fungal, viral or protozoal infection).

Cancers that can be treated with the PD-1 x CTLA-4 bispecific molecules of the present invention include cancers characterized by the presence of a cancer cell selected from the group consisting of: adrenal tumor, AIDS-associated cancer, alveolar sarcoma soft tissue, astrocytic tumor, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, cancer colon, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's sarcoma, extraskeletal mucoid chondrosarcoma, osseous fibrogenesis imperfecta, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germinal cell tumor, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, lipoma/benign lipomatous tumor, liposarcoma/malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma , meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyrocarcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal cancer melanoma, rare hematological disorder, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymus cancer, thymoma, metastatic thyroid cancer and uterine cancer.

In particular, the PD-1 x CTLA-4 bispecific molecules of the present invention can be used in the treatment of colorectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma; sarcoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer and colorectal cancer.

Infections that can be treated with the PD-1 x CTLA-4 bispecific molecules of the present invention include chronic viral, bacterial, fungal and parasitic infections. Chronic infections that can be treated with the PD-1 x CTLA-4 bispecific molecules of the present invention include infections caused by Epstein-Barr virus, hepatitis A virus (HAV); hepatitis B virus (HBV); hepatitis C virus (HCV); herpes viruses (e.g. HSV-1, HSV-2, HHV-6, CMV), human immunodeficiency viruses (HIV), vesicular stomatitis virus (VSV), bacteria of the Bacilli class, bacteria of the Citrobacter genus, Vibrio cholerae, diphthyria bacilli, enterobacteria, gonococci , bacteria of the Helicobacter pylori species, bacteria of the genera Klebsiella and Legionella, meningococci, mycobacteria, bacteria of the genus Pseudomonas, pneumococci, rickettsia, salmonella, bacteria of the genus Serratia, staphylococci, streptococci, tetanus bacillus, fungi of the genus Aspergillus (A. fumigatus, A. niger, etc. .d.), fungi of the species Blastomyces dermatitidis, fungi of the genus Candida (C. albicans, C. krusei, C. glabrata, C.

- 48 043373 tropicalis, etc.), fungi of the species Cryptococcus neoformans, fungi of the genus Mucorales (mucor, absidia, rhizopus), fungi of the species Sporothrix schenkii, Paracoccidioides brasiliensis, Coccidioides immitis, Histoplasma capsulatum, bacteria that cause leptospirosis, bacteria of the species Borrelia burgdorferi , helminths (nematodes, tapeworms, trematodes, flatworms (for example, schistosomes), intestinal giardia, worms of the genus Trichinella, amoebae of the species Dientamoeba Fragilis, trypanosomes of the species Trypanosoma brucei and Trypanosoma cruzi and protists of the species Leishmania donovani.

XI. Pharmaceutical compositions.

The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (eg, crude or non-sterile compositions), and pharmaceutical compositions (ie, compositions suitable for administration to a subject or patient) which can be used in the preparation of a unit dosage form. Such compositions contain a prophylactically or therapeutically effective amount of the PD-1 x CTLA-4 bispecific molecules of the present invention or a combination of such agents and a pharmaceutically acceptable carrier. Preferably, the compositions of the invention contain a prophylactically or therapeutically effective amount of the PD-1 x CTLA-4 bispecific molecules of the present invention and a pharmaceutically acceptable carrier. The invention also includes such pharmaceutical compositions that further comprise a second therapeutic antibody (eg, a tumor-specific monoclonal antibody) that is specific for a particular cancer antigen, and a pharmaceutically acceptable carrier.

In a specific embodiment, the term pharmaceutically acceptable means that the substance is approved by a regulatory agency of the federal or state government of the United States or is listed in the United States Pharmacopoeia or other generally accepted pharmacopoeias for use in animals and, more specifically, in humans. The term carrier refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete), excipient or excipient with which the therapeutic agent is administered. In general, the ingredients of the composition according to the invention are provided either separately or mixed in a unit dosage form, for example , in the form of a dry lyophilized powder or an anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the amount of active agent.When the composition is to be administered by infusion, it can be dosed using an infusion bottle containing sterile water or saline solution of pharmaceutical grade. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

The invention also provides a pharmaceutical kit or kit containing one or more containers filled with the PD-1 x CTLA-4 bispecific molecules of the present invention, alone or with said pharmaceutically acceptable carrier. In addition, one or more other prophylactic or therapeutic agents useful for treating the disease may also be included in the pharmaceutical kit or kit. The invention also provides a pharmaceutical kit or kit containing one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. Optionally, the specified container(s) may be accompanied by a legend in the form recommended by the government agency regulating the manufacture, use or sale of pharmaceuticals or biological products, where the specified legend reflects the approval by the specified authority of the manufacture, use or sale of the specified device for administration to humans.

The present invention provides kits that can be used in the above methods. The kit may contain any of the PD-1 x CTLA-4 bispecific molecules of the present invention. The kit may further contain one or more other prophylactic and/or therapeutic agents useful for treating cancer in one or more containers.

XII. Methods of administration.

The compositions of the present invention may be provided for the treatment, prevention, and amelioration of one or more symptoms associated with a disease, disorder, or infection by administering to a subject an effective amount of a fusion protein or conjugated molecule of the invention or a pharmaceutical composition comprising a fusion protein or conjugated molecule of the invention. In a preferred aspect, such compositions are substantially purified (ie, substantially free of substances that limit the effectiveness of the compositions or cause undesirable side effects). In a specific embodiment, the subject is an animal, preferably a mammal, such as a non-primate mammal (eg, bovine, pig, horse, dog, rodent, etc.) or a primate (eg, a monkey such as a Javan monkey). macaques, humans, etc.). In a preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer the compositions of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing an antibody or receptor-mediated fusion protein

- 49 043373 endocytosis (see, for example, Wu et al. (1987) Receptor-Mediated In Vitro Gene Transformation By A Soluble

DNA Carrier System, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.

Methods of administration of the molecule of the invention include, but are not limited to, parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and transmucosal administration (eg, intranasal and oral routes of administration). In a specific embodiment, the PD-1 x CTLA-4 bispecific molecules of the present invention are administered intramuscularly, intravenously, or subcutaneously. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelium or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration may be systemic or local. In addition, pulmonary administration can also be used, for example, by using an inhaler or nebulizer and an aerosol agent dosage form. See, for example, US Patent No. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540 and 4,880,078 and international publications according to PCT No. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346 and WO 99/66903, each of which is incorporated herein by reference in its entirety.

The invention also provides that formulations of the PD-1 x CTLA-4 bispecific molecules of the present invention are packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the number of molecules. In one embodiment, such molecules are provided as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be reconstituted, for example, with water or saline to a suitable concentration for administration to a subject. Preferably, the PD-1 x CTLA-4 bispecific molecules of the present invention are presented as a dry, sterile, lyophilized powder in a hermetically sealed container.

Lyophilized preparations of the PD-1 x CTLA-4 bispecific molecules of the present invention should be stored at a temperature between 2°C and 8°C in their original container, and the molecules should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours or within 1 hour after they are restored. According to an alternative embodiment of the invention, such molecules are presented in liquid form in a hermetically sealed container indicating the amount and concentration of the specified molecule, fusion protein or conjugated molecule. If such PD-1 x CTLA-4 bispecific molecules are provided in liquid form, they are preferably provided in a hermetically sealed container.

The amount of the compositions of the invention that will be effective in treating, preventing or alleviating one or more symptoms associated with the disorder can be determined using standard clinical techniques. The exact dose to be used in the dosage form also depends on the route of administration and the severity of the condition and should be decided in accordance with the judgment of the practitioner and the conditions for each patient. Effective doses can be extrapolated from dose response curves obtained from in vitro test systems or animal model test systems.

As used herein, an effective amount of a pharmaceutical composition according to one embodiment of the invention is an amount sufficient to achieve beneficial or desired results, including, but not limited to, clinical results such as reduction of symptoms resulting from a disease, reduction of a symptom of an infection (eg, viral stress, fever, pain, sepsis, etc.) or a symptom of cancer (eg, proliferation of cancer cells, presence of tumor, tumor metastasis, etc.), thus providing an increase in the quality of life of diseased subjects, reducing the dose of others drugs required to treat a disease, enhancing the effect of another drug, for example, by targeting and/or internalization, delaying the progression of the disease and/or increasing the survival time of subjects.

An effective amount may be administered over one or more administrations. For purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to kill and/or reduce proliferation of cancer cells and/or to eliminate, reduce and/or delay the development of metastasis from the primary site of a cancer tumor or reduce proliferation ( or effect) of an infectious pathogen and reducing and/or delaying the development of pathogen-mediated disease, directly or indirectly. In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an effective amount may be considered in the context of administering one or more chemotherapeutic agents, and one agent may be considered to be provided in an effective amount if said agent, together with one or more other agents, can or is capable of achieving the desired result. Despite individual differences in needs, determination of the optimal ranges of effective amounts of each component is within the skill of the person skilled in the art.

For the PD-1 x CTLA-4 bispecific molecules included according to the invention, the dose administered to the patient is preferably determined based on the body weight (kg) of the recipient subject. For the PD-1 x CTLA-4 bispecific molecules included according to the invention, the dose administered to a patient is typically from about 0.01 μg/kg to about 150 mg/kg or more of the subject's body weight.

The dose and frequency of administration of the PD-1 x CTLA-4 bispecific molecules of the present invention can be reduced or modified by enhancing the uptake of the molecule and its penetration into tissues through modifications such as, for example, lipidation.

The dose of the PD-1 x CTLA-4 bispecific molecule of the invention administered to a patient for use as monotherapy can be calculated. Alternatively, the molecule can be used in combination with other therapeutic compositions, and the dose administered to the patient will be lower compared to using the molecule as monotherapy.

The pharmaceutical compositions of the invention may be administered topically to the area requiring treatment, which may be achieved, for example (without limitation), by local infusion, injection, or by means of an implant, wherein said implant consists of a porous, non-porous or gel-like material, including membranes such as Silastic membranes or fibers. Preferably, when administering the molecule according to the invention, care should be taken to ensure that materials are used to which the molecule is not absorbed.

The composition according to the invention can be delivered in a vesicle, in particular a liposome (see Langer (1990) New Methods Of Drug Delivery, Science 249:1527-1533); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein, and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 3 17-327).

When the composition of the invention is a nucleic acid encoding a PD-1 x CTLA-4 bispecific molecule of the present invention, the nucleic acid can be administered in vivo to cause expression of the PD-1 x CTLA-4 bispecific molecule encoded by insertion said nucleic acid as part of a suitable nucleic acid expression vector and introducing said vector in such a way as to make it intracellular, for example, by use of a retroviral vector (see US Pat. No. 4,980,286) or by direct injection or by microparticle bombardment (eg, gene gun; Biolistic, Dupont) or by coating with lipids or cell surface receptors or transfection agents or by introducing it in conjunction with a homeobox-like peptide known to enter the nucleus (see, for example, Joliot et al. ( 1991) Antennapedia Homeobox Peptide Regulates Neural Morphogenesis, Proc. Natl. Acad. Sci. (USA) 88:18641868), etc. Alternatively, the nucleic acid can be introduced into the cell and inserted into the DNA of the host cell for expression by homologous recombination.

Treatment of a subject with a therapeutically or prophylactically effective amount of the PD-1 x CTLA-4 bispecific molecules of the present invention may comprise a single course of treatment or, preferably, may comprise a series of courses of treatment. In a preferred example, the subject is treated with said diabody once a week for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably for about 4, 5 or 6 weeks. The pharmaceutical compositions of the invention can be administered once a day, twice a day or three times a day. Alternatively, the pharmaceutical compositions may be administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, or once in year. It should also be borne in mind that the effective dose of the molecules used for treatment can be increased or decreased during a particular treatment.

XIII. Examples of implementation options.

The invention relates in particular to embodiments E1-E26.

E1. A bispecific molecule comprising one or more epitope binding sites capable of immunospecifically binding to PD-1 epitope(s), and one or more epitope binding sites capable of immunospecifically binding to CTLA-4 epitope(s), wherein said molecule comprises:

(A) a heavy chain variable domain and a light chain variable domain of an antibody that binds to PD-1; and (B) a heavy chain variable domain and a light chain variable domain of an antibody that binds CTLA-4;

where the specified molecule is:

(i) a diabody, wherein said diabody is a covalently linked complex that contains two, three, four or five polypeptide chains; or

- 51 043373 (ii) a trivalent binding molecule, wherein said trivalent binding molecule is a covalently linked complex that contains three, four, five or more polypeptide chains.

E2. The bispecific molecule of Embodiment E1, wherein said molecule exhibits an activity greater than said activity exhibited by two monospecific molecules, one of which comprises said heavy chain variable domain and said light chain variable domain of an antibody that binds PD-1, and the other of which comprises said heavy chain variable domain and said light chain variable domain of an antibody that binds to CTLA-4.

E3. The bispecific molecule of embodiment E1 or E2, wherein the molecule causes fewer immune-mediated adverse events (iAEs) when administered to a subject requiring said administration compared to those iAEs caused by administration of a monospecific antibody that binds to CTLA-4.

E4. The bispecific molecule of embodiment E3, wherein said monospecific antibody that binds to CTLA-4 is ipilimumab.

E5. The bispecific molecule according to any one of embodiments E1-E4, wherein the molecule contains an Fc moiety.

E6. A bispecific molecule according to embodiment E5, wherein said Fc fragment is a variant Fc fragment that contains:

(A) one or more amino acid modifications that reduce the affinity of the Fc fragment variant for FcyR, and/or (B) one or more amino acid modifications that increase the serum half-life of the Fc fragment variant.

E7. The bispecific molecule of embodiment E6, wherein said modifications that reduce the affinity of the Fc fragment variant for FcyR include the L234A substitution; L235A, or L234A and L235A, where the indicated numbering corresponds to the EC index according to Kabat.

E8. The bispecific molecule of embodiment E6 or E7, wherein said modifications that increase the serum half-life of the Fc fragment variant include an M252Y substitution; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, where the indicated numbering corresponds to the EC index according to Kabat.

E9. The bispecific molecule according to any one of embodiments E1-E8, wherein the molecule is said diabody and contains two epitope binding sites capable of immunospecifically binding to a PD-1 epitope and two epitope binding sites capable of immunospecifically binding to a CTLA-4 epitope.

E10. The bispecific molecule according to any one of embodiments E1-E8, wherein said molecule is said trivalent binding molecule and comprises two epitope binding sites capable of immunospecifically binding to a PD-1 epitope and one epitope binding site capable of immunospecifically binding to a CTLA-4 epitope .

E11. The bispecific molecule according to any one of embodiments E1-E10, wherein the molecule is capable of binding to PD-1 and CTLA-4 molecules present on the cell surface.

E12. The bispecific molecule according to any one of embodiments E1-E11, wherein the molecule is capable of simultaneously binding to PD-1 and CTLA-4.

E13. The bispecific molecule according to any one of embodiments E1-E12, wherein said molecule is capable of stimulating cells of the immune system.

E14. A bispecific molecule according to embodiment E13, wherein said stimulation of immune cells results in:

(A) proliferation of immune cells and/or (B) production and/or release by immune cells of at least one cytokine and/or (C) production and/or release by immune cells of at least one lytic molecule and/or (D) expression immune cells of at least one activation marker.

E15. The bispecific molecule of embodiment E13 or E14, wherein said immune system cell is a T lymphocyte or a natural killer (NK) cell.

E16. The bispecific molecule according to any one of embodiments E1-E15, wherein said epitope binding sites capable of immunospecifically binding to a PD-1 epitope comprise:

(A) VH domain of PD-1 mAb 1 (SEQ ID NO: 47) and VL domain of PD-1 mAb 1 (SEQ ID NO: 48); or (B) the VH domain of PD-1 mAb 2 (SEQ ID NO: 49) and the VL domain of PD-1 mAb 2 (SEQ ID NO: 50); or (c) the VH domain of PD-1 mAb 3 (SEQ ID NO: 51) and the VL domain of PD-1 mAb 3 (SEQ ID NO: 52); or (d) the VH domain of PD-1 mAb 4 (SEQ ID NO: 53) and the VL domain of PD-1 mAb 4 (SEQ ID NO: 54); or

- 52 043373 (E) VH domain of PD-1 mAb 5 (SEQ ID NO: 55) and VL domain of PD-1 mAb 5 (SEQ ID NO: 56); or (F) VH domain of PD-1 mAb 6 (SEQ ID NO: 57) and VL domain of PD-1 mAb 6 (SEQ ID NO: 58); or (G) the VH domain of PD-1 mAb 6-I VH (SEQ ID NO: 86) and the VL domain of PD-1 mAb 6-SQ VL (SEQ ID NO: 87);

or (H) VH domain of PD-1 mAb 7 (SEQ ID NO: 59) and VL domain of PD-1 mAb 7 (SEQ ID NO: 60); or (I) the VH domain of the anti-PD-1 mAb 8 (SEQ ID NO: 61) and the VL domain of the anti-PD-1 mAb 8 (SEQ ID NO: 62).

E17. The bispecific molecule according to any one of embodiments E1-E16, wherein said epitope binding site(s) capable of immunospecifically binding to a CTLA-4 epitope comprises:

(A) VH domain CTLA-4 mAb 1 (SEQ ID NO: 76) and VL domain anti-CTLA-4 mAb (SEQ ID NO: 77); or (B) the VH domain of CTLA-4 mAb 2 (SEQ ID NO: 78) and the VL domain of CTLA-4 mAb 2 (SEQ ID NO: 79); or (C) VH domain CTLA-4 mAb 3 (SEQ ID NO: 90) and VL domain CTLA-4 mAb 3 (SEQ ID NO: 91)

E18. The bispecific molecule of Embodiment 17, wherein:

(A) The indicated epitope binding sites capable of immunospecifically binding to the PD-1 epitope comprise the VH domain of PD-1 mAb 6-I VH (SEQ ID NO: 86) and the VL domain of PD-1 mAb 6-SQ (SEQ ID NO: 87) and (B) said epitope binding site(s) capable of immunospecifically binding to the CTLA-4 epitope comprise(s) the VH domain of CTLA-4 mAb 3 (SEQ ID NO: 90) and the VL domain of CTLA- 4 mAb 3 (SEQ ID NO: 91).

E19. A bispecific molecule according to any one of embodiments E1-E18, wherein said molecule comprises:

(A) two polypeptide chains having the sequence SEQ ID NO: 95, and two polypeptide chains having the sequence SEQ ID NO: 96; or (B) two polypeptide chains having the sequence SEQ ID NO: 97 and two polypeptide chains having the sequence SEQ ID NO: 98; or (C) two polypeptide chains having the sequence SEQ ID NO: 99 and two polypeptide chains having the sequence SEQ ID NO: 100; or (D) two polypeptide chains having the sequence SEQ ID NO: 102 and two polypeptide chains having the sequence SEQ ID NO: 103; or (E) two polypeptide chains having the sequence SEQ ID NO: 101 and two polypeptide chains having the sequence SEQ ID NO: 100; or (F) one polypeptide chain having the sequence SEQ ID NO: 104, one polypeptide chain having the sequence SEQ ID NO: 105, one polypeptide chain having the sequence SEQ ID NO: 106, and one polypeptide chain having the sequence SEQ ID NO: : 107; or (G) one polypeptide chain having the sequence SEQ ID NO: 108, one polypeptide chain having the sequence SEQ ID NO: 105, one polypeptide chain having the sequence SEQ ID NO: 109, and one polypeptide chain having the sequence SEQ ID NO: : 107.

E20. A pharmaceutical composition comprising an effective amount of a bispecific molecule according to any one of embodiments E1-E19 and a pharmaceutically acceptable carrier.

E21. The bispecific molecule according to any one of embodiments E1-E19, wherein the molecule is used to activate the stimulation of an immune system-mediated response in a subject in need thereof.

E22. The bispecific molecule according to any one of embodiments E1-E19, wherein the molecule is used to treat a disease or condition associated with suppression of the immune system.

E23. The bispecific molecule of embodiment E22, wherein said disease or condition is cancer or infection.

E24. A bispecific molecule according to embodiment E23, wherein said cancer is characterized by the presence of a cancer cell selected from the group consisting of: adrenal tumor, AIDS-associated cancer, alveolar soft tissue sarcoma, astrocytic tumor, bladder cancer, bone cancer, brain and spinal cancer brain, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor , ependymomas, Ewing's sarcoma, extraskeletal mucoid chondrosarcoma, osseous fibrogenesis imperfecta, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germinal cell tumor, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumor

- 53 043373 glands, Kaposi's sarcoma, kidney cancer, leukemia, lipoma/benign lipomatous tumor, liposarcoma/malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma astomy , neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyrocarcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disorder, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymus cancer, thymoma, metastatic thyroid cancer and uterine cancer.

E25. A bispecific molecule according to embodiment E24, wherein said infection is characterized by the presence of a bacterial, fungal, viral or protozoal pathogen.

E26. A bispecific molecule according to embodiment E25, wherein said infection is characterized by the presence of Epstein-Barr virus, hepatitis A virus (HAV); hepatitis B virus (HBV); hepatitis C virus (HCV); herpes viruses (for example, HSV-1, HSV-2, HHV-6, CMV), human immunodeficiency virus (HIV), vesicular stomatitis virus (VSV), bacteria of the Bacilli class, bacteria of the Citrobacter genus, Vibrio cholerae, diphthyria bacilli, enterobacteria, gonococci, bacteria of the Helicobacter pylori species, bacteria of the genera Klebsiella and Legionella, meningococci, mycobacteria, pseudomonas, pneumococci, rickettsia, salmonella, bacteria of the genus Serratia, staphylococci, streptococci, tetanus bacillus, fungi of the genus Aspergillus (A. fumigatus, A. niger, etc.) etc.), fungi of the Blastomyces dermatitidis species, fungi of the genus Candida (C. albicans, C. krusei, C. glabrata, C. tropicalis, etc.), fungi of the species Cryptococcus neoformans, fungi of the genus Mucorales (mucor, absidia, rhizopus) , fungi of the species Sporothrix schenkii, Paracoccidioides brasiliensis, Coccidioides immitis, Histoplasma capsulatum, bacteria that cause leptospirosis, bacteria of the species Borrelia burgdorferi, helminths (nematodes, tapeworms, trematodes, flatworms (for example, schistosomes), intestinal lamblia, worms of the genus Trichinella, amoebas species Dientamoeba Fragilis, trypanosomes species Trypanosoma brucei and Trypanosoma cruzi and protists species Leishmania donovani.

Examples

The invention described as a whole will be more accessible to understanding after reading the examples below. The following examples illustrate various ways of using the compositions in the diagnostic or treatment methods of the invention. The examples are provided by way of illustration and are not intended to limit the scope of the present invention in any way.

Example 1: Bispecific molecules provide increased stimulation of immune responses.

A bispecific molecule with specificity for individual cell surface proteins regulating two immune modulatory pathways, PD-1 and LAG-3, was created and designated DART A.

DART A is a bispecific four-chain Fc-containing diabody that contains two PD-1-specific binding sites, two LAG-3-specific binding sites, an IgG4 Fc variant engineered to increase half-life, and cysteine-containing E/K-coil domains promoting heterodimer formation. As described in more detail below, DART A has the binding specificity (ie, VH and VL domains) of a humanized anti-PD-1 antibody (hPD-1 mAb 6) and a humanized anti-LAG-3 antibody (hLAG-3 mAb 1). The amino acid sequence of the first and third polypeptide chains of DART A is (SEQ ID NO: 63)

DIQMTQSPSS LSASVGDRVT ITCRASQDVS SWAWYQQKP GKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYSTPWTFGG GTKLEIKGGG SGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYSFTSYWMN WVRQAPGQGL EWIGVIHPSD SETWLDQK FK DRVTITVDKS TSTAYMELSSS LRSEDTAVYY CAREHYGTSP FAYWGQGTLV TVSSGGCGGG EVAACEKEVA ALEKEVAALE KEVAALEKES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH Q DWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG

In SEQ ID NO: 63, amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of a humanized monoclonal antibody capable of binding to LAG-3 (hLAG-3 mAb 1); residues 108-115 correspond to the intermediate spacer peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); residues 116-234 correspond to the VH domain of a monoclonal antibody capable of binding to PD-1 (hPD-1 mAb 6, SEQ ID NO: 57, where X1 is I); residues 235-240 correspond to the intermediate spacer peptide (linker 2: GGCGGG (SEQ ID NO: 6)); residues 241-268 correspond to the cysteine-containing domain promoting heterodimer formation (E-coil) (EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 20)); residues 269-280 correspond to the stabilized hinge region of IgG4 (SEQ ID NO: 36); residues up to 281-496 correspond to the variant CH2-CH3 domain of IgG4, containing the M252Y/S254T/T256E substitutions and not containing the C-terminal residue.

The amino acid sequence of the second and fourth polypeptide chains of DART A is (SEQ ID NO: 64)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTD YNMDWVRQAP GQGLEWMGDI NPDNGVTIYN QK FEGRVTMT TDTSTSTAYM ELRSLRSDDT AVYYCAREAD YFYFDYWGQG TTLTVSSGGC GGGKVAACKE KVAALKEKVA ALKEKVAALK E

In SEQ ID NO: 64, amino acid residues 1-111 correspond to the amino acid sequence of the VL domain of a monoclonal antibody capable of binding to PD-1 (hPD-1 mAb 6, SEQ ID NO: 58, where X ₁ represents S and X2 represents Q); residues 112-119 correspond to the intermediate spacer peptide (linker 1: GGGSGGGG (SEQ ID NO: 5)); residues 120237 correspond to the VH domain of a humanized monoclonal antibody capable of binding to LAG-3 (hLAG-3 mAb 1); residues 238-243 correspond to a cysteine-containing spacer linker peptide (linker 2: GGCGGG (SEQ ID NO: 6)); residues 244-271 correspond to the cysteine-containing heterodimer-promoting (K-helical) domain (KVAACKE-KVAALKEKVAALKE-KVAALKE (SEQ ID NO: 21)).

The ability of DART A to stimulate T cells was assessed in an assay using Staphylococcus aureus enterotoxin type B (SEB). SEB is a microbial superantigen capable of activating a significant proportion of T cells (5-30%) in SEB-sensitive donors. SEB binds to an MHC II molecule outside the peptide-binding loop and thus the SEB signal is dependent on, but not limited to, MHC II and is mediated by the TCR. SEB stimulation of T cells results in oligoclonal T cell proliferation and cytokine production (although donor-dependent variability may be observed). Within 48 hours after SEB stimulation, positive regulation of PD-1 and LAG-3 is observed in PBMCs, while on day 5 (after secondary stimulation by cultivation in a 96-well plate in the presence of SEB), additional stimulation of these proteins is observed. Up-regulation of immune checkpoint proteins PD-1 and LAG-3 following SEB stimulation of PBMCs limits cytokine release upon SEB re-stimulation. The ability of DART A to enhance cytokine release by immune checkpoint inhibition was assessed and compared with the activity of parent antibodies against PD-1 and LAG-3 alone and in combination.

Briefly, PBMCs were purified from whole blood obtained from healthy donors with informed consent (Biological Specialty Corporation) using the Ficoll-Paque Plus density gradient centrifugation method (GE Healthcare) according to the manufacturer's instructions and then purified T cells using a kit to obtain Dynabeads® human T cells (Life Technologies) according to the manufacturer's instructions. Purified PBMCs were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin in T-25 volume flasks for 2-3 days alone or together with SEB (Sigma-Aldrich) at a concentration of 0.5 ng/ml (primary stimulation). At the end of the first cycle of SEB stimulation, PBMCs were washed twice with phosphate-buffered saline (PBS) and immediately seeded onto 96-well tissue culture plates at a concentration of 1-5 x 105 cells per well in just medium, in medium containing control antibody, in medium containing SEB at a concentration of 0.5 ng/ml (secondary stimulation) in the absence of antibody, or medium containing SEB and DART A, control IgG or anti-PD-1 antibody +/- anti-LAG-3 mAb and cultured for an additional 2-3 days. At the end of the second stimulation, supernatants were collected for measurement of cytokine secretion using DuoSet human cell ELISA kits for IFNy, TNFa, IL-10, and IL-4 (R&D Systems) according to the manufacturer's instructions.

In these assays, DART A (PD-1 x LAG-3 bispecific molecule) and anti-PD-1 and LAG-3 antibodies were used at concentrations of 0.0061, 0.024, 0.09, 0.39, 1.56, 6 .25 or 25 nM. For those studies in which a combination of antibodies was used, each antibody was provided at the indicated concentration such that the total antibody concentration was twice the concentration used for each antibody (i.e., 0.0122, 0.048, 0.18, 0 .78, 3.12, 12.5 or 50 nM). In fig. Figure 7 shows IFNy secretion profiles for SEB-stimulated PBMCs from a typical donor (D: 56041). Similar results were observed for PD-1 x LAG-3 bispecific molecules containing VH/VL domains from alternative antibodies against PD-1 and LAG-3, and for PD-1 x LAG-3 bispecific molecules having alternative structures (see, for example, Fig. 3C), in several donors.

The results of these studies demonstrate that PD-1 x LAG-3 bispecific molecules significantly enhance IFNy production in SEB-stimulated PBMCs upon repeated stimulation. These results indicate that bispecific molecules targeting two immune modulatory pathways are more active than a combination of individual ones.

- 55 043373 antibodies targeting the same pathways.

Example 2: PD-1 x CTLA-4 Bispecific Molecules.

Bispecific molecules having specificity for PD-1 and CTLA-4 can be created using methods proposed herein and known in the art. The general structure of the polypeptide chains of several molecules bispecific for PD-1 x CTLA-4 is presented in Table. 8. In particular, it is possible to obtain bispecific bivalent diabody molecules containing two polypeptide chains having one binding site for PD-1 and one binding site for CTLA-4, wherein said polypeptide chains have the general structure of variant I (also see, for example, Fig. 1). Bispecific bivalent diabody molecules can be created containing three polypeptide chains having one PD-1 binding site, one CTLA-4 binding site and an Fc moiety, wherein said polypeptide chains have the general structure of variant II (see also, for example, FIG. 4A). Bispecific tetravalent diabody molecules can be created containing four polypeptide chains having two identical PD-1 binding sites, two identical CTLA-4 binding sites and an Fc moiety, wherein said polypeptide chains have the general structure of variants III or IV (also see, for example, (Fig. 3A-3C) In addition, bispecific trivalent molecules can be created containing four polypeptide chains having two PD-1 binding sites and one CTLA-4 binding site (or two CTLA-4 binding sites and one PD-1 binding) and Fc fragment, wherein said polypeptide chains have the general structure of variant V (also see, e.g., Fig. 6A).Additionally, bispecific bivalent antibody molecules can be generated containing four polypeptide chains having a single site PD-1 binding site, one CTLA-4 binding site, and an Fc moiety wherein said polypeptide chains have the general structure of variant VI (also see, for example, US Pat. No. 7,695,936 and PCT International Patent Publication WO 2011/143545). ___________________________________________________

Table 8 Option Polypeptide chain Domains I First (VL 1) - (linker 1) - (VH2) - (linker 2) - (HPD) Second (VL2) - (linker 1) - (VH1) - (linker 2) - (HPD) II First (VL1) - (linker 1) - (VH2) - (linker 2) - (HPD) - (linker 3) - (modified CH2-CH3 domain) Second (VL2) - (linker 1) - (VH1) - (linker 2) - (HPD) Third (linker3) - (modified CH2-CH3 domain) III First and Third (VL1) - (linker 1) - (VH2) - (linker 2) - (HPD) (linker 3) - (CH2-CH3 domain) Second and Fourth (VL2) - (linker 1) - (VH1) - (linker 2) - (HPD) IV First and Third (VL1) - (linker 1) - (VH2) - (linker 2) - (CH1) - (Hinge) - (CH2-CH3 domain) Second and Fourth (VL2) - (linker 1) - (VH1) - (linker 2) - (CL) V First (VL1) - (linker 1) - (VH2) - (linker 2) - (HPD) (linker 3) - (modified CH2-CH3 domain) Second (VL2) - (linker 1) - (VH1) - (linker 2) - (HPD) Third (VH3) - (CH1) - (hinge) - (modified CH2-CH3 domain) Fourth (VL3)—(CL) VI First (VL1) - (linker 1) - (VH2) - (linker 2) - (HPD) - (linker 3) - (modified CH2-CH3 domain) Second (VL2) - (linker 1) - (VH1) - (linker 2) - (HPD) Third (VL3) - (linker 4) - (VH3) - (CH1) - (Hinge) - (modified CH2-CH3 domain) VII First (VH1) - (CH1) - (hinge) - (modified CH2-CH3 domain) Second (VLl)-(CL) Third (VH2) - (CH1) - (hinge) - (modified CH2-CH3 domain) Fourth (VL2)—(CL)

HPD = heterodimer formation promoting domain.

For each variant of bispecific molecules presented in table. 8:

(a) VL1 and VH1 are variable domains of an anti-PD-1 antibody, and VL2 and VH2 are variable domains of an anti-CTLA-4 antibody; or (b) VL1 and VH1 are variable domains of an anti-CTLA-4 antibody, and VL2 and VH2 are variable domains of an anti-PD-1 antibody.

For variants V and VI: VL3 and VH3 are the variable domains of an anti-PD-1 antibody or are the variable domains of an anti-CTLA-4 antibody.

Linkers, heterodimer formation domains and constant regions (eg, CH1, hinge region, CH2-CH3 domains) useful in creating such bispecific molecules are presented above. In particular, as described in detail herein, the CH2-CH3 domains of molecules whose first and third polypeptide chains are not identical are modified in such a way that the modifications promote heterodimerization and reduce or prevent homodimerization, for example, by modifying the CH2-CH3 domains of one chain so that they contain a depression, and modify the CH2-CH3 domains on the other chain so that they contain a protrusion.

As described in detail above, the hinge region and/or CH2-CH3 domains may contain amino acid substitutions that stabilize the bispecific molecules and/or alter their effector functions and/or increase their serum half-life.

Example 3. Universal bispecific adapter molecules (UBA).

Alternatively, a bispecific molecule (e.g., bispecific antibody, bispecific diabody, trivalent binding molecule, etc.) can be constructed containing one epitope binding site that specifically binds PD-1 (or CTLA-4) and a second an epitope binding site that specifically binds to a hapten, such as fluorescein isothiocyanate (also known as fluoroisothiocyanate, or FITC). Such a bispecific molecule serves as a universal bispecific adapter (UBA) molecule capable of coligating a PD-1 (or CTLA-4)-specific binding domain with fluorescein-conjugated binding molecules (e.g., antibody, scFv, etc. .) specific for CTLA-4 (or PD-1). For example, the FITC-reactive moiety of such a universal bispecific adapter molecule can be used to bind to a FITC-labeled antibody that binds CTLA-4 (or PD1), thereby creating a universal bispecific adapter molecule that is tailored to bind PD-1 and CTLA-4. 4. Such universal bispecific adapter molecules are useful for rapid evaluation of bispecific molecules.

Anti-fluorescein antibody, 4-4-20 (mAb 4-4-20), can be used as a source of FITC-specific binding domains (Gruber, M. et al. (1994) Efficient Tumor Cell Lysis Mediated By A Bispecific Single Chain Antibody Expressed in Escherichia coli, J. Immunol. 152(11): 5368-5374).

Amino acid sequence of mAb 4-4-20 heavy chain variable domain (SEQ ID NO: 65) (CDR _H residues are underlined)

EVKLDETGGG LVQPGRPMKL SCVASGFTFS DYWMNWVRQS PEKGLEWVAQ IRNKPYNYET YYSDSVKGRF TISRDDSKSS VYLQMNNLRV EDMGIYYCTG SYYGMDYWGQ GTSVTVSS

Amino acid sequence of mAb 4-4-20 light chain variable domain (SEQ ID NO: 66) (CDR _l residues are underlined)

DWMTQTPFS LPVSLGDQAS ISCRSSQSLV HSNGNTYLRW YLQKPGQSPK VLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP WTFGGGTKLE IK

Any bispecific structures proposed in this application can be used (see, for example, tables 1, 2, 3 and 4). Preferred bispecific molecules contain only one hapten (eg, fluorescein) binding site and bind to a single hapten-labeled antibody, thereby providing a 1:1 universal adapter bispecific molecule:hapten-labeled antibody ratio in the resulting complexes. Such universal bispecific adapter molecules can be created using, for example, the VL and VH domains of an anti-PD-1 antibody and an anti-fluorescein antibody. Preferably, such a universal bispecific adapter molecule is a covalently linked diabody or trivalent linking molecule containing two, three, four, five or more polypeptide chains. Typical universal bispecific adapter molecules that can be created are presented below.

A.UBA1.

One of the versatile bispecific adapter molecules that can be created is a covalently linked diabody consisting of two polypeptide chains and containing one PD-1 epitope binding site and one fluorescein binding site (UBA 1).

The first polypeptide chain of UBA 1 contains (in the direction from N-terminus to C-terminus) the N-terminus, VL domain of mAb 4-4-20 (SEQ ID NO: 66), intermediate spacer peptide (linker 1, GGGSGGGG (SEQ ID NO: 5)), VH domain of PD-1 mAb 6 (SEQ ID NO: 57, where X ₁ is I)), intermediate spacer peptide (linker 2, GGCGGG (SEQ ID NO: 6)), promoting the formation of E- heterodimer coil domain: EVAALEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 18)) and C-terminus.

Thus, the amino acid sequence of the first polypeptide chain of UBA 1 is (SEQ ID NO: 67)

DWMTQTPFS LPVSLGDQAS ISCRSSQSLV HSNGNTYLRW YLQKPGQSPK

VLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP

WTFGGGTKLE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT

SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY

MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSG GCGGGEVAAL

EKEVAALEKE VAALEKEVAA LEK

The second polypeptide chain of UBA 1 contains (in the direction from N-terminus to C-terminus) the N-terminus, VL domain of PD-1 mAb 6 (SEQ ID NO: 58, where X1 is S and X2 is Q)), an intermediate spacer peptide (linker 1, GGGSGGGG (SEQ ID NO: 5)), VH domain of mAb 4-4-20 (SEQ ID NO: 65)), intermediate spacer peptide (linker 2, GGCGGG (SEQ ID NO: 6)), K-coil heterodimer formation stimulating domain: KVAALKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 19)) and C-57 043373 end.

Thus, the amino acid sequence of the second polypeptide chain of UBA 1 is (SEQ ID NO: 68)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGGSGGGG EVKLDETGGG LVQPGRPMKL SCVASGFTFS DYWMNWVRQS PEKGLEWVAQ IRNKPYNYET YY SDSVKGRF TISRDDSKSS VYLQMNNLRV EDMGIYYCTG SYYGMDYWGQ GTSVTVSSGG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE

B.UBA2.

As suggested above, insertion of the CH2-CH3 domains of an IgG into a single polypeptide chain of a diabody such as UBA 1 may allow the formation of a more complex four-chain bispecific Fc fragment-containing diabody. Thus, the second universal bispecific adapter molecule that can be created is a covalently linked diabody consisting of four polypeptide chains containing two PD-1 epitope binding sites, two fluorescein binding sites and an Fc fragment (UBA 2). It should be noted that UBA 2 can bind to two fluorescein-labeled molecules through two fluorescein binding sites.

The first and third polypeptide chains of UBA 2 contain (from N-terminus to C-terminus) Nterminus, VL domain of mAb 4-4-20 (SEQ ID NO: 66), intermediate spacer peptide (linker 1, GGGSGGGG (SEQ ID NO: 5)), VH domain of PD-1 mAb 6 (SEQ ID NO: 57, where Xi is I)), intermediate spacer peptide (linker 2, GGCGGG (SEQ ID NO: 7)), heterodimer formation promoting domain E-coil: EVAALEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 18)), intermediate spacer peptide (linker 3, GGGDKTHTCPPCP (SEQ ID NO: 31)), IgG1 Fc fragment containing substitutions L234A/L235A (SEQ ID NO: 43), where X represents K), and the C-terminus.

Thus, the amino acid sequence of the first and third polypeptide chains of UBA 2 is (SEQ ID NO: 69)

DWMTQTPFS LPVSLGDQAS ISCRSSQSLV HSNGNTYLRW YLQKPGQSPK VLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP WTFGGGTKLE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSD SETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSG GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGGDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The second and fourth polypeptide chains of UBA 2 are identical to the second polypeptide chain of UBA 1. Thus, each of the second and fourth polypeptide chains of UBA 2 has the amino acid sequence of SEQ ID NO: 68.

C.UBA3.

The third universal bispecific adapter molecule that can be created is a covalently linked diabody consisting of three polypeptide chains and contains one PD-1 epitope binding site, one fluorescein binding site and an Fc moiety (UBA 3).

The first polypeptide chain of UBA 3 contains (in the direction from N-terminus to C-terminus), the N-terminus, the VL domain of mAb 4-4-20 (SEQ ID NO: 66), the intermediate spacer peptide (linker 1, GGGSGGGG (SEQ ID NO : 5)), VH domain of PD-1 mAb 6 (SEQ ID NO: 57, where X ₁ is I)), intermediate spacer peptide (linker 2, GGCGGG (SEQ ID NO: 6)), heterodimer formation promoting domain E-coil: EVAALEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 18)), intermediate spacer peptide (linker 3, GGGDKTHTCPPCP (SEQ ID NO: 31)), containing an overhang IgG1 Fc fragment containing L234A/L235A substitutions ( SEQ ID NO: 44 where X represents K)), and the C terminus.

Thus, the amino acid sequence of the first polypeptide chain of UBA 3 is (SEQ ID NO: 70)

DWMTQTPFS LPVSLGDQAS ISCRSSQSLV HSNGNTYLRW YLQKPGQSPK VLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP WTFGGGTKLE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSD SETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSG GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGGDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The second polypeptide chain of UBA 3 may be identical to the second polypeptide chain of UBA 1. Thus, the second polypeptide chain of UBA 3 has the amino acid sequence SEQ ID NO:

68.

The third polypeptide chain UBA 3 contains (in the direction from N-terminal to C-terminal), N-terminal spacer peptide (linker 3, DKTHTCPPCP (SEQ ID NO: 26)), containing the cavity Fc fragment of IgG1 with substitutions L234A/L235A (SEQ ID NO: 45, where X represents K), and the C-terminus.

Thus, the amino acid sequence of the third polypeptide chain of UBA 3 is (SEQ ID NO: 71)

DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK

D. UBA 4.

The fourth universal bispecific adapter molecule that can be created is a covalently linked trivalent binding molecule consisting of four polypeptide chains and containing two PD-1 epitope binding sites, one fluorescein binding site and an Fc moiety (UBA 4).

The first polypeptide chain of UBA 4 is identical to the first polypeptide chain of UBA 3. Thus, the first polypeptide chain of UBA 4 has the amino acid sequence of SEQ ID NO: 70.

The second polypeptide chain of UBA 4 is identical to the second polypeptide chain of UBA 1. Thus, the second polypeptide chain of UBA 4 has the amino acid sequence of SEQ ID NO: 68.

The third polypeptide chain of UBA 4 contains (from N-terminus to C-terminus) the VH domain of PD-1 mAb 6 (SEQ ID NO: 57, where X1 is I), the CH1 domain of IgG1 (SEQ ID NO: 40) , the hinge region of IgG1 (SEQ ID NO: 33), containing the cavity Fc fragment of IgG1 with substitutions L234A/L235A (SEQ ID NO: 45, where X is K) and the C-terminus.

Thus, the amino acid sequence of the third polypeptide chain of UBA 4 is (SEQ ID NO: 72)

QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSWTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV Y TLPPSREEM TKNQVSLSCA VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLVS KLTVDKSRWQ QGNVFSCSVM HEALHNRYTQ KSLSLSPGK

The fourth polypeptide chain UBA 4 contains (from N-terminus to C-terminus) the VL domain of PD-1 mAb 6 (SEQ ID NO: 58, where X1 is S and X ₂ is Q), the CL domain ( for example, the kappa domain of IgG (SEQ ID NO: 38)) and the C-terminus.

Thus, the amino acid sequence of the fourth polypeptide chain of UBA 4 is (SEQ ID NO: 73)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL

LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY

TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV

QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV

THQGLSSPVT KSFNRGEC

E. UBA 5.

The fifth universal bispecific adapter molecule that can be created is a covalently linked trivalent binding molecule consisting of three polypeptide chains and containing two PD-1 epitope binding sites, one fluorescein binding site and an Fc moiety (UBA 4, see e.g. , Fig. 6C-6D).

The first polypeptide chain of UBA 5 is identical to the first polypeptide chain of UBA 3. Thus, the first polypeptide chain of UBA 5 has the amino acid sequence of SEQ ID NO: 70.

The second polypeptide chain of UBA 5 is identical to the second polypeptide chain of UBA 1. Thus, the second polypeptide chain of UBA 5 has the amino acid sequence of SEQ ID NO: 68.

The third polypeptide chain of UBA 5 contains (from N-terminus to C-terminus) the VL domain of PD-1 mAb 6 (SEQ ID NO: 58, where X ₁ is S and X ₂ is Q), an intermediate spacer peptide (linker 4, GGGGSGGGGSGGGGS (SEQ ID NO: 37)), VH domain of PD-1 mAb 6 (SEQ ID NO: 57, where X1 is I), CH1 domain of IgG1 (SEQ ID NO: 40), hinge region of IgG1 (SEQ ID NO: 33) containing a cavity Fc fragment of IgG1 with substitutions L234A/L235A (SEQ ID NO: 45, where X represents the K and C terminus.

- 59 043373

Thus, the amino acid sequence of the third polypeptide chain of UBA 5 is (SEQ ID NO: 74)

EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGGSGGGG SGGGGSQVQL VQSGAEVKKP GASVKVSCKA SGYSFTSYWM NWVRQAPGQG LEWIGVI HPS DSETWLDQKF KDRVTITVDK STSTAYMELS SLRSEDTAVY YCAREHYGTS PFAYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSWTVPSSS LGTQTYICNV NHKPSNTKVD KRVEPKSCDK THTCPPCPAP EA AGGPSVFL FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLSCAVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV FSCSVMHEAL HNRYTQKSLS LSPGK

Antibodies against CTLA-4 can be labeled with fluorescein using standard methods. When such a tagged molecule is incubated in the presence of the universal bispecific adapter molecule presented above having an epitope binding site that binds to PD-1 and an epitope binding site that binds to fluorescein, PD-1 x CTLA-4 bispecific molecules are formed, which can be assessed as described below.

Given the principles proposed herein, it is apparent that various VH domains, VL domains, linkers, heterodimer formation domains and/or constant domains of IgG can be used to create alternative universal bispecific adapter molecules. For example, the VH and VL domains of an anti-CTLA-4 antibody and/or another anti-PD-1 antibody can be used in place of the VH and VL domains of a used anti-PD-1 antibody to create alternative or equivalent universal bispecific adapter molecules. Alternatively, the VH and VL domains of the anti-CTLA-4 antibody can be used in place of the VH and VL domains of the anti-fluorescein antibody to create PD-1 x CTLA-4 bispecific molecules having the general structure of options I, II, III , V and VI proposed above. Such PD-1 x CTLA-4 bispecific molecules can be used directly in the assays described below.

Example 4. Analyzes.

The PD-1 x CTLA-4 bispecific molecules of the present invention can be characterized using any of various methods. In particular, the ability of the PD-1 x CTLA-4 bispecific molecules of the invention to bind immunospecifically to PD-1 and CTLA-4 molecules (e.g. as present on the cell surface, etc.) can be analyzed. , and/or the binding kinetics of interaction with the antigen can be determined. When bispecific molecules contain an Fc moiety (or part thereof), their ability to mediate Fc-FcyR interactions, such as specific binding of the Fc moiety (or part thereof) to FcyR, mediate effector function, signal transduction, etc., can be analyzed. The in vivo immunomodulatory activity and/or antitumor efficacy of the PD-1 x CTLA4 bispecific molecules of the invention can be assessed using in vitro and in vivo assays known in the art.

A. Preparation of immune cells and cells expressing PD-1 and/or CTLA-4.

1. Isolation of PBMCs and Immune Cell Subsets from Human Whole Blood PBMCs were isolated from the whole blood of healthy human donors using, for example, Ficoll gradient centrifugation. Briefly, whole blood was diluted 1:1 in sterile phosphate-buffered saline (PBS). Diluted blood (35 ml) was layered onto 15 ml of FicollPaque™ Plus solution in a 50 ml tube and the tubes were centrifuged at 400 x g (1320 rpm) for 30 minutes with braking. The buffy coat between the two phases was collected in 50 ml tubes and centrifuged at 600 x g (1620 rpm) for 5 minutes. The supernatant was discarded and the cell pellet was washed 3 times with PBS (eg, by centrifuging the tubes at 600 x g (1620 rpm) for 5 minutes). The number of viable cells was determined using trypan blue dye. PBMCs were resuspended in complete growth medium (eg, RPMI 1640, 10% FBS, 1% penicillin/streptomycin) and incubated at 37°C in the presence of 5% CO ₂ overnight or further processed to isolate the desired subset of immune cells, such as T -cells (eg, regulatory T cells, CD8 cells, CD4 cells), NK cells, dendritic cells and monocytes, as described below.

Specific immune cell subpopulations are rapidly isolated from PBMCs using a commercial subset recovery kit (eg, Untouched™ Human T Cell Isolation Kit for T Cells, CD4 T Cells, CD8 T Cells, Monocytes, Dendritic Cells (Life Technologies/ThermoFisher Scientific); DYNABEADS® Regulatory CD4+/CD35+ T Cell Isolation Kit (ThermoFisher), etc.), according to the manufacturer's instructions. Once isolated, a subpopulation of immune cells (eg T cells)

- 60 043373 resuspended in a suitable complete culture medium (e.g. RPMI 1640, 10% FBS, 1% penicillin/streptomycin) to which cytokines (e.g. IL-2, GM-CF, IL-4, TNF-α and etc.) and incubated at 37°C in the presence of 5% CO ₂ overnight. As proposed herein, such purified subpopulations are useful for assessing cell surface expression of PD-1 and/or CTLA-4 and for assessing the immune system-stimulating activity of the PD-1 x CTLA-4 bispecific molecules of the invention.

2. Isolation of PBMCs from whole blood of cynomolgus or rhesus monkeys.

Cynomolgus or rhesus monkey PBMC were isolated from whole blood, for example, using Ficoll gradient centrifugation. Briefly, whole blood was diluted 1:3 in sterile PBS. Diluted blood (35 ml) was layered onto 15 ml of 90% Ficoll-Paque™ Plus solution (90 ml Ficoll + 10 ml PBS) in a 50 ml polypropylene centrifuge tube and centrifuged at 931 x g (2000 rpm) for 30 minutes at room temperature with braking. The buffy coat between the two phases was collected and transferred to a clean 50 ml tube and washed with 45 ml PBS by centrifuging the tubes at 600 x g (1620 rpm) for 5 minutes. The supernatant was discarded and the sediment was washed 3x with PBS. PBMCs from cynomolgus or rhesus monkeys were then resuspended in 30 ml of complete culture medium and the number of viable cells was determined by trypan blue dye exclusion.

Specific subsets of immune cells can be easily isolated from non-human primate PBMC using a commercial subset preparation kit (e.g. pan T cell, CD4+ T cell and CD4+/CD25+ regulatory T cell isolation kits (Miltenyl Biotech) ), in accordance with the manufacturer's instructions. Alternatively, flow cytometry-based sorting can be used using non-human primate specific mAbs or cross-reactive mAbs for sorting.

3. Generation of immature or mature human myeloid-derived dendritic cells (mDC) from isolated human monocytes.

Human monocytes were isolated from donor-derived purified PBMCs using a commercial PBMC collection kit, such as the Untouched™ Human Monocyte Collection Kit (Life Technologies/ThermoFisher Scientific) according to the manufacturer's instructions. Isolated human monocytes were differentiated into immature human mDCs by culturing them (e.g., in alpha minimal essential medium with nucleosides (aMEM) supplemented with 2% AB negative human serum and 1% penicillin/streptomycin) for 5-7 days in the presence of recombinant granulocyte-derived cells. human macrophage colony-stimulating factor (eg, hGM-CSF; Peprotech, 100 ng/ml) and recombinant human interleukin-4 (hIL-4; Peprotech, 40 ng/ml). Immature mDCs were collected and washed with PBS by centrifuging the tubes at 600 x g (1620 rpm) for 5 minutes for use as stimulating cells in an allogeneic mixed culture lymphocyte reaction (allo-SCL) assay, such as the assays described below.

In specific experiments using allo-SCL, differentiation of immature mDCs was induced by adding TNFa or a cocktail of additional cytokines (IFNy, IL-1e) and mitogens (LPS) for two additional days of culture (see, for example, Han, T. (2009) Evaluation of 3 Clinical Dendritic Cell Maturation Protocols Containing LPS and IFN-gamma, J Immunother 32:399). The purity, maturation and activation of mDCs can be assessed by flow cytometry using one or more of the following antibodies: anti-CD14, anti-CD80, anti-CD83, anti-CD86, anti-HLA-DR and suitable isotype controls. Flow cytometry data from this assessment can be acquired on a FACSCalibur/Fortessa instrument (Becton Dickinson/BD Biosciences) and analyzed using FlowJo software (TreeStar).

4. Expression of PD-1 and CTLA-4.

Cells expressing PD-1 and/or CTLA-4 can be generated using methods known in the art. For example, cells (such as NSO, Jurkat, CHO, etc.) can be generated to express PD-1 and/or CTLA-4 using retroviral vectors containing a suitable gene (for example, the PD-1 gene 1). Alternatively, cells of the immune system can be stimulated to induce or increase the expression of PD-1 and/or CTLA-4. Briefly, purified immune cells (eg, PBMCs, T cells, dendritic cells, etc.) were isolated as described above, cultured for 2-6 days in the presence or absence of mitogen, and PD-1 and/or expression assessed. or CTLA-4 in untreated (naive) and stimulated cells, for example, using flow cytometry. Commercial anti-PD-1 antibodies and anti-CTLA-4 antibodies can be used to preliminarily assess expression patterns in naïve cells and in response to mitogen stimulation. Additionally or optionally, PD-1 x CTLA-4 bispecific molecules according to the invention can be used.

Mitogens that can be used for such studies are well known in the art and include, but are not limited to: CD3/CD28 beads, lipopolysaccharides (LPS), Staphylococcus aureus enterotoxin types A-E (e.g., SEB), phorbol myristate acetate (PMA) ,

- 61 043373 phytohemagglutinin (PHA), concanavalin A (conA), milkweed mitogen (PWM), etc. The mitogen(s) identified to induce/increase the expression of PD-1 and/or CTLA-4 can be used in functional assays to evaluate the stimulatory activity of bispecifics against

PD-1 x CTLA-4 molecules according to the present invention. See, for example, the SEB and SCL assays described herein.

B. Binding Assays.

Immunoassays that can be used to analyze immune-specific binding to PD-1 or CTLA-4 molecules, binding cross-reactivity, or Fc-FcyR interactions include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blotting , radioimunological tests, solid phase ELISA (solid -phase immunological analysis), immunological analysis of the type of sandwich, immun -proportion tests, precipitation reactions, diffuse precipitation reactions in gel, immunochromatographic analyzes, immuno -diffuseal analyzes, agglutination reactions, analysis of complement fixation, radio impulses, fluor Enterbic immunological tests and immunoassays based on protein A, etc. (See for example Ausubel et al., 2008, Current Protocols in Molecular Biology). Binding affinity to a target antigen is typically measured or determined using standard antibody-antigen assays such as Biacore competition assays, saturation assays, or immunoassays such as ELISA or RIA. Fluorescence activated cell sorting (FACS) using methods known to one skilled in the art was used for immunological or functional assays to characterize the PD-1 x CTLA-4 bispecific molecules of the invention.

For example, PBMC can be obtained as described above. When the desired immune cell subtypes (e.g., T-regulatory, T-helper, APC, etc.) can be isolated from the purified PBMCs, the isolated cells are then assessed for PD-1 and CTLA-4 expression for the various cell subtypes (eg, T-regulator, T-helper, APC, etc.) by co-staining and fluorescence-activated cell sorting (FACS) analysis as described below.

1. Cell surface binding (saturation assay).

The ability of PD-1 x CTLA-4 bispecific molecules to bind to PD-1 and/or CTLA-4 expressed on the cell surface can be measured in saturation/dilution assays using a cell that expresses PD-1 and/or CTLA-4 (target cells). Such cells may be immune cells stimulated to express PD-1 and/or CTLA-4, or a cell line (eg, NSO cells) generated to stably express PD-1 molecules at elevated levels. 1 and/or CTLA-4. Briefly, cultured target cells (eg, PD1+ expressing NSO cells) were collected and resuspended (eg, at a concentration of approximately 5 x 10 ⁶ cells/ml) in blocking buffer (eg, FACS buffer supplemented with 10% AB human serum). A PD-1 x CTLA-4 bispecific molecule, an anti-PD-1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody were prepared for dilution in a separate microtiter plate, starting at equal molar concentrations ( eg 20 nM in a total volume of 200 µl) and then serial dilutions (eg 1:4, 1:5, 1:10, etc.) were made 5-12 times to obtain a 5-12 point curve. The maximum initial concentration in all experiments was determined experimentally. An equal volume (eg, 50 μl) of each dilution was added to a new microtiter plate and target cells were added to each well (eg, at a concentration of 0.25 x 10 ⁶ cells per well) and incubated (eg, at 4-25°C for 30 -120 minutes). Cells were washed 1-3 times (eg, microtiter plate spun at 600 x g (1620 rpm) for 5 minutes and then washed in blocking buffer and spun again) and resuspended in blocking buffer. For secondary staining, a suitable secondary reagent was selected, e.g. goat anti-human immunoglobulin Fc-specific IgG conjugated to APC can be used to detect human primary antibodies, while goat anti-mouse immunoglobulin Fc-specific IgG conjugated to Alexa Fluor 647 used to detect mouse primary antibodies. The selected secondary reagent was diluted in blocking buffer and, based on the concentration of each individual secondary reagent, a stock solution and an equal volume of secondary reagent mixture per well were prepared, aliquoted into separate wells and incubated (eg, 4-25°C for 30-120 minutes). Cells were washed as described above and resuspended in blocking buffer. Stained cells were analyzed by flow cytometry. Flow cytometry data can be acquired on a FACSCalibur/Fortessa instrument (Becton Dickinson/Fortessa), analyzed as mean fluorescence intensity using FlowJo software (TreeStar), and plotted and normalized using the log(agonist)-effect-variable slope function ( four parameters) in Prism6 (Graphpad).

2. Receptor/ligand binding and signaling pathway assays.

Assays that can be used to evaluate the ability of the PD-1 x CTLA-4 bispecific molecules of the invention to modulate (eg, block, inhibit, stimulate, etc.) ligand binding and signaling pathways are described in more detail below.

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A. PD-1 receptor/ligand binding.

The ability of PD-1 x CTLA-4 bispecific molecules to inhibit the binding of PD-1 to PD-L1 and/or PD-L2 can be assessed using cells that express PD-1 (target cells). Such cells may be immune cells stimulated to express PD-1 or a cell line engineered to express PD-1 molecules, such as NSO cells retrovirally transfected with the PD-1 gene person. Briefly, PD-1 expressing cells (eg, NSO/PDCD1 (NSO-PD1+)) were collected and resuspended (eg, at a concentration of approximately 1.5 x 10 ⁶ cells/ml) in blocking buffer (eg, FACS buffer supplemented with 10% serum AB human) and seeded onto a microtiter plate (eg at a concentration of 0.25x10 ⁶ cells per well). A PD-1 x CTLA-4 bispecific molecule, an anti-PD-1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody were prepared for dilution in a separate microtiter plate, starting at the same molar concentration ( eg 20 nM in a total volume of 200 µl) and serial dilutions (eg 1:4, 1:5, 1:10, etc.) were made 5-12 times to obtain a 5-12 point curve. The maximum initial concentration in all experiments was determined empirically. An equal volume (eg, 50 μl) of each dilution was added to each well of a microtiter plate containing the target cells. To assess inhibition of PD-L1 binding, a soluble PD-L1 fusion protein (e.g., hPD-L1(B7H1)TEV-hIgG1-Fcbiotin (Ancell)) was added to each well, except for unstained negative control wells, and incubated (e.g., 4 -25°C for 30-120 minutes). To assess inhibition of PD-L2 binding, a soluble PD-L2 fusion block (e.g., CD273 (PD-L2) muIgG/biotin (Ancell)) was added to each well, excluding unstained negative control wells, and incubated (e.g., 4-25 °C for 30-120 minutes). Cells were washed 1-3 times (eg, microtiter plate spun at 600 x g (1620 rpm) for 5 minutes and then washed with blocking buffer and spun again). Cells were resuspended in blocking buffer. Appropriate secondary reagent (excluding unstained negative control wells) to detect PD-L1 or PD-L2 fusion protein (eg, streptavidin-PE labeled secondary reagent (eBiosciences)) was added and incubated (eg, 4-25°C for 15 -120 minutes). Cells were washed as described above and resuspended in blocking buffer. Stained cells can be analyzed using flow cytometry. Flow cytometry data can be obtained on a FACSCalibur/Fortessa instrument (Becton Dickinson/Fortessa) and analyzed for the decrease in mean fluorescence intensity of labeled sPD-L1 or sPD-L2 in the presence of PD-1 x CTLA-4 bispecific molecules, anti-PD-1 antibodies, anti-CTLA-4 antibodies or a combination of anti-PD-1 antibody and anti-CTLA-4 antibody using FlowJo software (TreeStar) and plotted and normalized using the log(agonist) - effect - variable slope (four parameters) function in the program Prism6 (Graphpad).

b. CTLA-4 receptor/ligand binding.

The ability of PD-1 x CTLA-4 bispecific molecules to inhibit the binding of CTLA-4 to CD80 and/or CD86 can be assessed using cells that express CTLA-4 (target cells). Such cells may be immune cells stimulated to express CTLA-4 or a cell line generated to express CTLA-4, such as NSO cells retrovirally transfected with the human CTLA-4 gene . Briefly, CTLA-4-expressing cells were collected and resuspended in blocking buffer (e.g., FACS buffer supplemented with 10% human AB serum) and plated on a microtiter plate (e.g., at a concentration of 0.25 x 10 ⁶ -1.0 x 10 ⁶ cells per well) . A PD-1 x CTLA-4 bispecific molecule, an anti-PD-1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody were prepared for dilution in a separate microtiter plate, starting at the same molar concentration ( eg 20 nM in a total volume of 200 µl) and serial dilutions (eg 1:4, 1:5, 1:10, etc.) were made 5-12 times to obtain a 5-12 point curve. The maximum initial concentration in all experiments was determined empirically. Each dilution in the same volume (eg, 50 μl) was added to each well of the microtiter plate containing the target cells. To assess inhibition of CD80 binding, a soluble CD80 fusion protein (e.g., hCD80-muIg-biotin (ADIPOGEN®)) was added to each well, except unstained negative control wells, and incubated (e.g., at 4-25°C for 30-120 minutes). To assess inhibition of CD86 binding, a soluble CD86 fusion protein (e.g., hCD86-muIg-biotin (ADIPOGEN®)) was added to each well, except unstained negative control wells, and incubated (e.g., at 4-25°C for 30-120 minutes). Cells were washed 1-3 times (eg microtiter plate spun at 600xg (1620 rpm) for 5 minutes and then washed with blocking buffer and spun again). Cells were resuspended in blocking buffer. Add a suitable secondary reagent to detect CD80 or CD86 fusion protein (e.g. streptavidin-PE labeled

- 63 043373 secondary (eBiosciences)), excluding unstained negative control wells, and incubated (eg at 4-25°C for 15-120 minutes). Cells were washed as described above and resuspended in blocking buffer. Stained cells can be analyzed using flow cytometry. Flow cytometry data can be obtained using the FACSCalibur/Fortessa system (Becton Dickinson/Fortessa) and analyze the decrease in mean fluorescence intensity of labeled CD86 or CD80 in the presence of PD-1 x CTLA-4 bispecific molecules, anti-PD-1 antibody, anti-CTLA antibody -4 or combinations of anti-PD-1 and anti-CTLA-4 using FlowJo software (TreeStar) and plotted and normalized using the log(agonist) - effect - variable slope (four parameters) function in Prism6 software ( Graphpad).

C. Reporter assays.

The functional activity of PD-1 x CTLA-4 bispecific molecules in blocking the interaction of PD-1 with PD-L1 can be assessed using a commercially available reporter system developed by Promega, according to the manufacturer's recommendations. Briefly, two cell lines engineered to function as either a stimulatory line or a reporter cell line were used. The stimulatory line was generated from the original CHO line so that it expresses PD-L1 molecules and T cell activator, which is a membrane-bound agonist anti-CD3 mAb [CHO/PDL1 cells]. A reporter cell line was generated from a parental CD3+ Jurkat cell line such that the line expressed a luciferase reporter construct under the control of nuclear factor of activated T cells (NFAT) transcription [NFATluc2/PD-1 Jurkat cells]. When co-cultured together, the anti-CD3 agonist expressed on the CHO-PDL1 cell line triggers luciferase expression through the NFAT signaling pathway, mediated by the engagement of the TCR/CD3 signaling complex present in the Jurkat-NFAT-luc/PD-1 cell line. In the absence of antibodies against PD-1 or PD-L1, luciferase is expressed at levels driven by the activity of the TCR/CD3 signaling pathway, but is suppressed or inhibited in the presence of the PD-1/PD-L1 pathway, which acts as a brake. In the presence of molecules that inhibit the PD-1/PD-L1 signaling pathway (eg, anti-PD-1 or PD-L1 antibodies), this inhibitory pathway, or brake, is turned off, allowing an increase in luciferase expression that can be measured. Accordingly, the PD-1 inhibitory activity of PD-1 x CTLA-4 bispecific molecules can be assessed by culturing CHO/PDL1 cells with NFAT-luc2/PD1 Jurkat (3H-D5). Briefly, CHO-PDL1 cells were seeded onto a microtiter plate (eg, at a concentration of 4.0 x 10 ⁴ cells per well) and cultured overnight (eg, in RPMI medium containing 10% FBS and 100 μg/ml hygromycin B and 500 μg/ml ml G418). The next day, prepare assay buffer (e.g., RPMI medium supplemented with 2% FBS) and serial dilutions of the PD-1 x CTLA-4 bispecific molecule or anti-PD-1 antibody in assay buffer, including 5-12 spots, with the highest dilution at the same molar equivalent (eg, 100-200 nM) and 5-12 serial dilutions were made (eg, 1:4, 1:5, 1:10, etc.). In the next step, a portion of the culture medium was removed from the microtiter plate containing adherent CHO/PDL1 cells, and aliquots of each dilution were added to the indicated CHO/PDL1 cells. Cultured NFAT-luc2/PD-1 Jurkat cells were collected and resuspended in assay buffer and added (eg, at a concentration of 5.0 x 10 ⁴ cells per well in a volume of 40 μl per well) to CHO/PDL1 cells. The cultures were co-incubated (eg, 6 hours at 37°C). At the end of incubation, Bio-Glo substrate (Promega) was reconstituted and added to the microtiter plate at room temperature. After incubation (e.g., 5-10 minutes), the absorbance of each well was read on a VICTOR™ X4 Multi-Channel Plate Reader (Perkin Elmer #2030-0040) at 450 nm to obtain relative luminescence units (RLU) values as output data. The data can then be plotted and normalized using the log(agonist) - effect - variable slope (four parameters) function using Prism6 (Graphpad).

Similar reporter assays are available for the CTLA-4 signaling pathway (e.g., CTLA-4 Blockade Bioassay Kit (Promega)) and/or can be easily constructed to assess the functional activity of PD-1 x CTLA-4 bispecific molecules in blocking CTLA-4 interactions. 4 using the appropriate ligand(s).

D. Immunomodulatory Activity Assays.

Assays that can be used to evaluate the immunomodulatory activity of the PD-1 x CTLA-4 bispecific molecules of the invention include mitogenic stimulation assays, such as the SEB assay described above, and mixed culture lymphocyte (MSC) response assays, such as analyzes described in more detail below. The ability of the PD-1 x CTLA-4 bispecific molecules of the invention to modulate both PD-1 and CTLA-4 inhibitory pathways is expected to result in enhanced stimulation in assays compared to anti-PD1 antibody and anti-CTLA-4 antibody separately or in combination.

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PBMCs or T cells were isolated from the blood of healthy donor(s) - from the blood of the same patient (autologous cells) or from unrelated patients (allogeneic cells) - by Ficoll-Paque™ gradient centrifugation as described above and resuspended in complete nutrient medium. For allo-SCL assays using mDCs, monocytes were purified and differentiated as described above. For one-way (unidirectional) analysis using the allo-SCL reaction, responder cells (eg, PBMCs) were cocultured with stimulating cells in a microtiter plate. Depending on the context, the stimulating cells were dendritic cells (DCs), autologous PBMCs (for the auto-SCL reaction, i.e., negative control) or allogeneic PBMCs (for the allo-SCL reaction, i.e., positive control). The ratio of responder cells:stimulator cells is usually 1:1 or 2:1, but can vary. Cocultures were performed in the presence of equal molar amounts of a PD-1 x CTLA-4 bispecific molecule, an anti-PD-1 antibody, an anti-CTLA-4 antibody, a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody, or appropriate isotype mAbs in serial dilutions (for example, 1:4 1:5, 1:10, etc.). Serial dilutions of the antibody can be prepared as described above. In addition, control populations of cells of the same type, stimulated with or without the addition of anti-CD3 +/- CD28 mAb, can be used as controls in such experiments. The stimulating cells (stimulators) were pre-irradiated (eg, at 45 Gray [Gy] (4500 rad) using a Gammacell® 3000 Elan Blood/Cell Irradiator (Theratronics)) to prevent proliferation of the stimulating cells and to allow the proliferation of only the responder cell to be measured. (responder). After 5-7 days (time adjusted to ensure PD-1 and CTLA-4 expression during assay), [ ^3H ]-thymidine (eg, 1 μCi/well (Perkin Elmer)) was added for an additional 18-48 hours. The radioactivity of the tag incorporated into the DNA was measured (for example, using a TOPCount NXT β-scintillation counter (Perkin Elmer)). Results were expressed as either average counts per minute (cpm) or stimulation ratio (SI), allowing comparison of results from different donors. SI was calculated as follows: mean counts per minute (cpm) for stimulated cells divided by mean cpm for unstimulated cells. Responses in the SCL were considered positive when SI was >3 for PBMC-evoked stimulation and SI >6 for DC-evoked stimulation. Alternatively, proliferation can be measured using a CEFSE-based proliferation assay (Boks, M.A., et al. (2010) An optimized CFSE based T-cell suppression assay to evaluate the suppressive capacity of regulatory T-cells induced by human tolerogenic dendritic cells ,' Scand J Immunol 72:158–168).

Additional SCR assays that can be used to evaluate the immune system stimulating activity of the PD-1 x CTLA-4 bispecific molecules of the invention are known in the art. See, for example, Davies, J.K. et al. (2011) Induction of alloantigen-specific anergy in human peripheral blood mononuclear cells by alloantigen stimulation with costimulatory signal blockade Journal of Visualized Experiments : JoVE, (49), 2673; Kruisbeek, A. M., et al. (2004) Proliferative Assays for T cell Function, CURRENT PROTOCOLS IN IMMUNOLOGY, 60:111:3.12.1-3.12.20; Wallgren, A.C. et al. (2006) The Direct Pathway Of Human T-Cell Allorecognition Is Not Tolerized By Stimulation With Allogeneic Peripheral Blood Mononuclear Cells Irradiates With High-Dose Ultraviolet Ba. Scand J of Immunol 63:90–96; Levitsky, J. et al. (2009) The Human 'Treg MLR' Immune Monitoring for Foxp3+ T regulatory cell generation, Transplantation 88:1303-11.

E. In vivo antitumor assays.

The antitumor activity of the PD-1 x CTLA-4 bispecific molecules of the invention can be assessed in various animal models known in the art. Treatment with the PD-1 x CTLA-4 bispecific molecules of the invention is expected to inhibit tumor formation and/or tumor growth to a greater extent compared to treatment with an anti-PD1 antibody and an anti-CTLA-4 antibody alone or a combination of such antibodies.

In particular, murine tumor xenograft models can be used. Briefly, mice are implanted with a cancer cell line or tumor cells of interest and treated with (i) PD-1 x CTLA-4 bispecific molecules, (ii) anti-PD-1 antibodies, (iii) anti-CTLA-4 antibodies, (iv ) a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody and (vi) a no-treatment control, which may be a vehicle and/or irrelevant antibody alone. Treatment can begin before implantation (for example, 1 day (i.e. -1 day)); on the day of implantation (i.e., day 0) or after tumor formation (i.e., day 7). Animals may receive one course of treatment or multiple courses of treatment (eg, every week after implantation). Animals are observed over an extended period of time to determine the in vivo effect of these molecules on tumor formation and/or growth. The growth of tumors can be monitored by measuring them and determining their volume (height x width x length). Treated animals that exhibit complete tumor regression can be used to assess the tumor-specific immune response by restimulation using the same or tumor cells and irrelevant tumor cells as controls. In addition, these models can be modified to provide a treatment method in combination with standard treatments such as chemotherapy, radiation therapy, etc.

A variety of cancer cell lines that can be transplanted and used in such xenograft models are known in the art. These lines include, but are not limited to: MDST8, SW480 and SW620 colon and rectal cancer cells; AGS gastric cancer cells; UACC-62, A2058 and LOX IMVI melanoma cells; prostate cancer cells 22rv; pancreatic cancer cells AsPC-1 and BxPc-3; kidney cancer cells Caki-1, A498 and 786-0; HT-1197 bladder cancer cells; 4T1 breast cancer cells, MDA-MB-231; lung cancer cells A549, WX322; fibrosarcoma cells HT1080; human mantle cell lymphoma HBL-2 cells; Raji Burkitt lymphoma cells. In particular, patient-derived xenograft (PDX) models are preferred. Such cancer cell lines or patient-derived tumors are transplanted into immunodeficient strain mice (eg, Nude mice, Scid mice, NOD mice, Rag 1 null mice, etc. (see, for example, Belizario, J.E., (2009) Immunodeficient Mouse Models: An Overview Bentham Open 1874-2262/09) or humanized mice, such as human HLA-A2 transgenic mice (see, for example, Shultz, L.D., et al. (2012) Humanized mice for immune system investigation: progress, promise and challenges Nature Rev Immunol 12:786-798) as described above. Additionally, to evaluate molecules that modulate immune checkpoints, components of the human immune system can be transplanted into immunodeficient mice (e.g., immune reconstitution with PBMCs, stem cells , human progenitor immune cells, etc.) prior to or simultaneously with implantation of the desired tumor cells and treatment as detailed above.

Example 5: Binding studies of PD-1 x CTLA-4 bispecific molecules.

Several PD-1 x CTLA-4 bispecific molecules have been generated, including Fc-containing diabodies and Fc-containing trivalent molecules having four polypeptide chains. Three diabodies having four polypeptide chains and containing E/K-coil domains promoting heterodimer formation were created and designated DART B, DART C and DART D. One diabody having four chains and containing CH1/CL domains was created and designated it as DART E. Two trivalent binding molecules were created, having four chains and containing E/K-coil domains promoting heterodimer formation, and CH1/CL domains, and designated these molecules as TRIDENT A and TRIDENT B.

In addition, several antibodies have been generated that are specific for PD-1 or CTLA-4. One antibody specific for PD-1 was generated and designated PD-1 mAb 6 G4P. Three antibodies specific for CTLA-4 were generated and designated CTLA-4 mAb 1, CTLA-4 mAb 3 G1AA, and CTLA-4 mAb 3 G4P.

The structure and amino acid sequences of these bispecific PD-1 x CTLA-4 molecules, anti-PD-1 antibodies, anti-CTLA-4 antibodies are presented above and summarized in table. 9 below.

Table 9 Name Variable regions Fc* chains SEQ ID NO: Other components DART B CTLA-4 mAb 1 PD-1 mAb 6-ISQ IgG4 (YTE) 1 2 3 4 95 96 95 96 E/Kcoil Domains; see Figure ZV DART C CTLA-4 mAb 3 PD-1 mAb 6-ISQ IgG4 1 2 3 4 97 98 97 98 E/Kcoil Domains; see Figure ZV DARTD PD-1 mAb 6-ISQ CTLA-4 mAb 3 IgG4 (YTE) 1 2 3 4 99 100 99 100 E/Kcoil Domains; see Figure ZV DARTE CTLA-4 mAb 3 PD-1 mAb 6-ISQ IgG4 (YTE) 1 2 3 4 102 103 102 103 CL/CH1; see Figure ZS DARTF PD-1 mAb 6-ISQ CTLA-4 mAb 3 IgGl (AA/YTE) 1 2 3 4 101 100 101 100 E/Kcoil Domains; see Figure ZV

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TRIDENT A PD-1 mAb 6-ISQ CTLA-4 mAb 3 IgG4 (YTE) 1 2 3 4 104 105 106 107 E/K-coil domains hCL/CHI; cm. Figure 6A 1 108 TRIDENT B PD-1 mAb 6-ISQ IgGl 2 105 Domains E/K-coil hCL/CHL cm CTLA-4 mAb 3 (AA/YTE) 3 4 109 107 Figure 6A 1 88 PD-1 mAb 6 2 89 natural G4P PD-1 mAb 6-ISQ IgG4 3 88 structure antibodies 4 89 CTLA-4 CTLA-4 mAb 1 natural mAb 1 (copy IgGl 4 ** structure ipilimumab) antibodies CTLA-4 1 2 92 94 natural mAb 3 G1AA CTLA-4 mAb 3 IgGl (AA) 3 4 92 94 antibody structure 1 93 CTLA-4 2 94 natural mAb 3 G4P CTLA-4 mAb 3 IgG4 3 93 structure antibodies 4 94

J Molecules containing IgG4 Fc fragments also include a stabilized IgG4 hinge region.

** Same amino acid sequence as ipilimumab (see e.g. IMGT 3D and 2D structure database, accession numbers 8568_H and 8568_L).

Additional PD-1 x CTLA-4 bispecific molecules containing alternative PD-1 and/or CTLA-4 epitope binding sites can be easily generated by incorporating different VH and VL domains. Likewise, molecules can be created containing alternative linkers, Fc moieties and/or having alternative structures, as proposed herein (see, for example, Table 8).

A. Binding studies by ELISA.

ELISA studies were performed to measure the binding of serial dilutions of binding molecules (CTLA-4 mAb 3 G4P, DART D, TRIDENT A, or DARTB antibody) to soluble hCTLA-4-Avi-His (1 μg/ml) or hPD-1 -His (1 μg/ml), which was applied to the substrate plates. Goat anti-human immunoglobulin Fc-specific antibody conjugated to HRP (1:10,000) was used as a secondary detection molecule to detect binding. The results of such a study are shown in table. 10 and fig. 8A-8B. Data show that binding to PD-1 and CTLA-4 of PD-1 x CTLA-4 bispecific molecules containing two binding sites for PD-1 and CTLA-4 (e.g., DART D and DART B) was similar to binding of their respective parent anti-PD-1 and anti-CTLA-4 antibodies. Binding to PD-1 of PD-1 x CTLA-4 bispecific molecules containing two PD-1 and one CTLA-4 binding sites (e.g., TRIDENT A) was similar to the binding of the parent anti-PD-1 antibody , and binding to CTLA-4 was reduced compared to that of the parent antibody due to the reduced avidity of the trivalent molecule containing only one binding site for CTLA-4. Similar binding results were observed for DART F and TRIDENT B containing CH1 IgG1 and/or (AA/YTE) IgG1 Fc fragments.

Table 10 Construct ECso for CTLA-binding 4 (nM) ECso for PD1 binding (nM) CTLA-4 mAb 3 G4P 0.4 N/A PD-1 mAb 6 G4P N/A o, s DARTD 0.4 0.3 TRIDENT A L0 0.4 DART B 0.4 0.4

The effect of changing domain distribution and connectivity on binding was examined by incubating PD-1 x CTLA-4 bispecific molecules containing the CTLA-4 binding domains of CTLA-4 mAb 1 (e.g., DART B) and CTLA-4 mAb 3 (e.g., DART C and DART D) in the presence of soluble human PD-1 (Fig. 8C) or soluble human CTLA-4-Avi-His (Fig. 8D), which were applied to the support plates. Goat Fcy-specific anti-human immunoglobulin antibodies conjugated to HRP were used as secondary detection molecules to detect binding using the chemiluminescent substrate PICO. The results indicate that PD-1 x CTLA-4 bispecific molecules containing the CTLA-4 binding domains of CTLA-4 mAb 1 (e.g., DART B) and CTLA-4 mAb 3 (e.g., DART C and DART D) bound to CTLA-4 in a similar manner. It was found that binding to PD-1 or CTLA-4 did not vary significantly depending on the distribution of binding domains, i.e. their location on the first or second chain (for comparison, the binding of DART C and DART D).

B. ELISA blocking studies.

A series of ELISAs were performed to evaluate the ability of the bispecific molecules of the invention to block ligand binding to PD-1 and CTLA-1, alone and in combination. Blokirova

- 67 043373 The binding of PD-L1 to PD-1 was assessed in the presence of an equal amount of irrelevant antigen and an equal amount of CTLA-4. The plates were coated with a 1:1 mixture of histidine (His)-tagged soluble human PD-1 (shPD-1) and His-tagged irrelevant antigen (irrAg) (2 μg/ml each) or a mixture of shPD-1 and His-tagged soluble CTLA-4 human (shCTLA-4), 1:1, (2 μg/ml each). PD1 mAb 6 G4P, DART D, TRIDENT A or TRIDENT control molecule (containing two binding sites for RSV and one binding site for CTLA-4) at the indicated concentration was premixed for 5 min with biotin-labeled PD-L1 at a concentration of 6 μg/ ml and added to the plates. PD-L1 binding was detected using HRP-conjugated streptavidin (1:3,000). The evaluation results are presented in Fig. 9A-9B. All of the PD-1 binding molecules tested were found to be able to inhibit PD-L1 binding to cPD-1.

Blocking B7-1 binding to CTLA-4 was assessed in the presence of an equal amount of irrelevant antigen and an equal or fourfold greater amount of PD-1. The plates were coated with a 1:1 mixture of shCTLA-4 and irrAg (2 μg/ml each), a 1:1 mixture of shCTLA-4 shPD-1 (2 μg/ml each), or a mixture of shCTLA-4 (0.8 μg/ml). ml) and shPD-1 (3.2 μg/ml), 1:4. PD-1 mAb 6 G4P, DART D, TRIDENT A, CTLA-4 mAb 3 G4P or control molecule at the indicated concentration was pre-mixed for 5 min with labeled biotin B7-1 at a concentration of 0.2 μg/ml and added to the plates. B7-1 binding was detected using HRP-conjugated streptavidin (1:3,000). The evaluation results are presented in Fig. 9C-9E. It was found that all of the CTLA-4 binding molecules tested were able to inhibit the binding of B7-1 to CTLA-4. It was found that the blocking of B7-1 binding by TRIDENT A was enhanced by the interaction of its PD-1 binding moiety with immobilized PD-1 (compared to the control TRIDENT molecule, which does not bind PD-1) (Fig. 9D). Moreover, it was found that at a CTLA-4:PD-1 ratio of 1:4, where these conditions better mimic the relative expression level observed in stimulated cells (see FIG. 19A), TRIDENT blocked B7-1 binding A was further enhanced (i.e., the TRIDENT A curve was further shifted relative to the curve of the control molecule TRIDENT, which does not bind PD-1) (Fig. 9E).

The results of these ELISA studies demonstrate that all of the PD-1 binding molecules tested were able to inhibit the binding of PD-L1 to PD-1 (Fig. 9A-9B). All of these molecules are bivalent for PD-1 and exhibited Similar Inhibition Profiles All of the CTLA-4 binding molecules tested were able to inhibit the binding of B7-1 to immobilized CTLA-4 (Figures 9C-9E), with molecules containing two PD-1 binding sites and one CTLA-binding site 4 caused stronger inhibition in the presence of PD-1 (Figures 9D-9E) Thus, trivalent molecules containing a single CTLA-4 binding site caused PD-1-mediated blocking of CTLA-4 ligands, demonstrating that the CTLA-4 interaction 4 can be adapted by adjusting the valence.

C. BIACORE® Research.

The binding affinities of DART A, TRIDENT A and CTLA-4 mAb 1 to human and cynomolgus CTLA-4 were examined using the BIACORE® assay. Briefly, His-tagged soluble CTLA-4 (the extracellular portion of human or cynomolgus CTLA-4 fused with a histidine-containing peptide) was captured onto immobilized anti-pentahistidine (penta-His) molecules and then CTLA-4 binding molecules at various concentrations (12.5- 200 nM) was passed along the immobilized CTLA-4 proteins. Binding kinetics were determined using the BIACORE® assay (1:1 Langmuir binding affinity (simultaneous ka/kd fit); or avidity using separate ka/kd curve fit, 1:1). The calculated k _a , k _d and K _D from these studies are presented in table. eleven

Table 11 Molecule CTLA-4 people CTLA-4 cynomolgus macaque H (xlO ⁵ ) N4 (xlO' ⁴ ) KD (nM) y (χιό ⁵ ) ^to % (χίο' ³ ) KD (nM) CTLA-4 mAb G 6.6 8.9 1.4 10 1.3 1.3 DART D* 2.3 7.1 3.1 3.5 1.7 4.9 TRIDENT At 1.2 32 26.7 2.5 65 260

* Avidity by separate approximation of ka/kd curves, 1:1.

$ Affinity according to Langmuir binding model, 1:1.

DART D is bivalent for CTLA-4 and exhibits binding affinity for human and cynomolgus CTLA4 within approximately 2 to 4 times the affinity of CTLA-4 mAb 1. TRIDENT A, which is monovalent for CTLA-4, has a lower affinity for both human and cynomolgus CTLA-4, which is expected given its reduced avidity.

The binding affinities of DART A, TRIDENT A, PD-1 mAb 6 G4P and CTLA-4 mAb 3 G1AA to human PD-1 were examined using the BIACORE® assay. Binding molecules were captured on immobilized F(ab) ₂ goat Fc-specific anti-human immunoglobulin antibodies and then His-tagged soluble human PD-1 at various concentrations (6.25-100 nM) was passed along the immobilized binding molecules and binding kinetics determined by analysis

BIACORE® (binding curve fitting using Langmuir equation, 1:1). The calculated k _a , kd and KD from these studies are presented in table. 12 (nd - not detectable).

Table 12 Molecule human PD-1 k (X10 ⁵ ) ^k d (χίο' ⁴ ) KD (nM) CTLA-4 mAb 3 G1AA n.d. n.d. n.d. PD-1 mAb 6 G4P 6.2 6.7 1.1 DARTD 4.8 8.1 1.7 TRIDEM A 5.2 6.8 1.3

DART A, TRIDENT A, PD-1 mAb 6 G4P are all bivalent for PD-1 and have comparable binding affinities. As expected, CTLA-4 mAb 3 G1AA did not exhibit any detectable binding to human PD-1.

D. CTLA-4 Cellular Assays.

The binding of DART B, DART D, TRIDENT A, anti-CTLA-4 CTLA-4 mAb 1, CTLA-4 mAb 3 G4P and control antibody hIgG to CHO cells expressing cynoCTLA-4 (cynoCTLA-4) or CTLA- was assessed. 4 people (huCTLA-4). The results of this evaluation are shown in FIG. 10A-10B. The binding molecules were incubated in the presence of CHO cells that expressed either cynomolgus CTLA-4 (Fig. 10A) or human CTLA-4 (Fig. 10B). Binding to these cells was detected using secondary antibodies against human immunoglobulin Fc. The results show that all molecules tested were able to bind to human and cynomolgus CTLA-4 expressed on the surface of CHO cells. Anti-CTLA-4 antibodies showed similar binding profiles to huCTLA-4; the bivalent bispecific molecules DART B and DART D exhibited slightly weaker binding, and the trivalent binding molecule TRIDENT A, which is monovalent for CTLA-4, exhibited weaker binding compared to higher valency molecules for CTLA-4. The control antibody did not bind. Similar results were shown for binding to cynoCTLA-4.

The binding of DART C, DART D, DART E, TRIDENT A, anti-CTLA-4 antibodies CTLA-4 mAb 1, CTLA-4 mAb 3 G1AA and anti-PD-1 antibodies PD-1 mAb 6 G4P to Jurkat cells that express huCTLA-4 on their surface but do not express PD-1. The binding of DART and TRIDENT molecules to human CTLA-4 was detected using secondary Abs against human immunoglobulin Fc (FACS analysis). The evaluation results are shown in table. 13 and fig. 11A (DART C, DART D, DART E, CTLA-4 mAb 1, CTLA-4 mAb 3 G1AA and PD-1 mAb 6 G4P) and FIG. 11B (CTLA-4 mAb 1, CTLA-4 mAb 3 G1AA, PD-1 mAb 6 G4P and TRIDENT A). As shown in FIG. 11A-11B, PD-1 antibody did not bind to CTLA-4, but all CTLA-4 binding molecules tested were able to bind to huCTLA-4 expressed on the surface of Jurkat cells. Anti-CTLA-4 antibodies showed similar binding profiles; the bivalent bispecific molecules DART C, DART D and DART E showed weaker binding to Jurkat cells, and the trivalent binding molecule TRIDENT A, which is monovalent for CTLA-4, showed weaker binding compared to higher valency molecules in regarding CTLA-4._____

Table 13 Molecule EC50 (nM) CTLA-4 mAb 1 0.4215 PD-1 mAb 6 G4P 6,557 CTLA-4 mAb 3 G1AA 0.3728 DARTE 1,269 DART C 0.7575 DARTD 0.8829 TRIDENT A 4.638

The ability of DART D, TRIDENT A and anti-CTLA-4 antibodies CTLA-4 mAb 1, CTLA-4 mAb 3 G1AA to block CTLA-4 ligands B7-1 and B7-2 was assessed. His-tagged derivatives of B7-1 and B7-2 were incubated in the presence of CTLA-4-Jurkat cells. The binding of His-B7-1 and His-B7-2 was detected using anti-His antibodies. The results of this evaluation are shown in FIG. 12A (His-B7-1) and FIG. 12B (His-B7-2). It was found that all molecules tested were able to inhibit the binding of B7-1 and B7-2 to CTLA-4 expressed on the surface of Jurkat cells. Anti-CTLA-4 antibodies showed similar inhibition profiles; the bivalent bispecific molecule DART D was a slightly less active inhibitor, and the trivalent binding molecule TRIDENT A, which is monovalent on CTLA-4, was less active compared to either of the higher valency molecules on CTLA-4. The control antibody caused no inhibition at all. The results of the ELISA studies described above suggest that TRIDENT A and similar molecules containing two PD-1 binding sites and one CTLA-4 binding site might be more active inhibitors in the presence of PD-1.

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A reporter gene assay on IL-2/Luc Jurkat-CTLA-4 cells was used to evaluate the ability of DART C, DART D, TRIDENT A, CTLA-4 mAb 3 G1AA, and PD-1 mAb 6 G4P to reverse the CTLA- immune checkpoint inhibitory signal. 4, as indicated by increased luciferase expression. IL-2/Luc-Jurkat-CTLA-4 cells were thus incubated in the presence of such molecules (R:S = 1:0.3) for 30 min at 37°C, after which artificial antigen-presenting Raji cells were added and continued to incubate for 6 hours. Artificial antigen-presenting cells activate the TCR/CD3 complex on Jurkat reporter cells. The evaluation results are shown in Fig. 13. All of the CTLA-4 binding molecules tested were able to reverse the CTLA-4 immune checkpoint inhibitory signal as determined by the luciferase assay. The TRIDENT A molecule, which is monovalent for CTLA-4, was less active in this assay compared to either of the higher valent molecules for CTLA-4. The control antibody caused no inhibition at all. The results of the ELISA study described above suggest that TRIDENT A and similar molecules containing two PD-1 binding sites and one CTLA-4 binding site may be more active in the presence of PD-1.

E. Cellular PD-1 assays.

The ability of DART D, TRIDENT A, PD-1 mAb 6 G4P, and CTLA-4 mAb 3 G1AA to bind to NSO cells expressing PD-1 but not CTLA-4 was assessed. The binding molecules were incubated in the presence of cells and the average fluorescence of the cells was measured. The results of this evaluation are presented in Fig. 14. As expected, the CTLA-4 antibody did not bind. It was found that all bispecific binding molecules were able to bind to PD-1 expressed on the surface of NSO cells. All bispecific molecules are bivalent for PD-1 and bound to NSO cells in a similar manner.

The ability of DART D, TRIDENT A, PD-1 mAb 6 G4P and CTLA-4 mAb 3 G1AA to block the binding between cell surface expressed PD-1 and its ligands PD-L1 and PDL2 was assessed. PD-L1-PE or PD-L2-PE was incubated in the presence of the indicated binding molecule and its ability to bind to NSO-PD-1 cells was assessed by fluorescence-activated cell sorting (FACS). The results of this evaluation are presented in Fig. 15A (PD-L1) and FIG. 15B (PD-L2). As expected, the CTLA-4 antibody did not cause inhibition, and all of the PD-1 binding molecules tested were able to inhibit the binding of both PD-L1 (Fig. 15A) and PD-L2 (Fig. 15B) to PD-1 , expressed on the surface of NSO cells. All PD-1 binding molecules are bivalent for PD-1 and had similar inhibition profiles.

DART D, TRIDENT A, CTLA-4 mAb 3 G1AA and PD-1 mAb 6 G4P were also evaluated in the PD-1 blocking reporter assay. These binding molecules were incubated in the presence of PDL1 ⁺ CHO cells and Jurkat effector cells, and the ability of the binding molecules to block inhibition of the immune response (by blocking the PD-1/PD-L1 interaction) was assessed by observing the extent of CD3-mediated activation (as indicated by increased luciferase expression in analysis on NFAT-luc/PD-1 Jurkat cells; Promega). The results of this evaluation are presented in Fig. 16. All of the PD-1 binding molecules tested were able to reverse the PD-1 immune checkpoint inhibitory signal, as indicated by increased luciferase expression. All PD-1 binding molecules are bivalent for PD-1 and had a similar ability to inhibit PD-1-mediated blockade of T cell signaling. The CTLA-4 antibody did not inhibit this system at all.

F. CTLA-4/PD-1 Cellular Assays.

The ability of DART D, TRIDENT A, and a negative control antibody to simultaneously bind to PD-1 and CTLA-4 was tested in a protein fragment complementation enzymatic assay, DiscoverX. Briefly, U2OS CTLA-4(1-195)-PK PD-1(1199)-EA #9 cells were aliquoted in quadruplicate at a concentration of 5,000 cells/well in DiscoverX CP5 seeding medium in 384-well plates. Cells were allowed to attach for 4 hours at 37°C/5% CO ₂ . Each binding molecule was then added to PD-1 - CTLA-4 cells in serial dilutions of 1:3 (11 spots). The plates were incubated overnight (16 hours) at 37°C/5% CO ₂ . PathHunter detection reagent was added to the wells and then incubated for 1 hour at room temperature in the dark before the plate was read on an Envision luminometer. The results of this assessment are presented in table. 14 and fig. 17 (cell line U2OS CTLA-4 (1-195)-PK PD-1(1-199)-EA #9). Both bispecific molecules DART D and TRIDENT A showed comparable simultaneous engagement of PD-1 and CTLA-4 molecules on cells in which both receptors were co-expressed, as shown by enzymatic protein fragment complementation assays, indicating that the bispecific The molecules of the invention are capable of simultaneously binding to PD-1 and CTLA-4, and further indicates that binding via PD-1 compensates for the reduced avidity of the TRIDENT A molecule for CTLA-4 when both target receptors are expressed. this finding is consistent with the results of the ELISA inhibition studies described above. The negative control did not cause a significant increase in signal in the PD1-70 043373 cell line

CTLA4. Incubation in the presence of a higher concentration of TRIDENT A resulted in a strong increase in signal in the U2OS cell line with PD1-CTLA4 dimerization (S:B=12.7). Reply to DART

D in the dose response study on the PD-1 - CTLA-4 cell line was smaller in magnitude (S:B=9.2), but EC50 values were similar for both molecules (EC50=20 pM).

Table 14 Negative control TRIDENT A DARTD Slope of the curve -15.99 1.103 0.8095 EC50 (nM) -6.883 x 10' ¹⁰ 2.123 x 10' ¹¹ 2,090 x 10' ¹¹

We assessed the ability of DART D, TRIDENT A, CTLA-4 mAb 3 G1AA, PD-1 mAb 6 G4P, and the combination CTLA-4 mAb 3 G1AA/PD-1 mAb 6 G4P (Ab Combo 1) to enhance the response in a mixed lymphocyte culture reaction (MLC). ). Monocyte-derived dendritic cells were generated by treating CD14+ monocytes (isolated from PBMCs using a Miltenyi positive selection kit) with GMCSF (100 ng/ml) and IL-4 (10 ng/ml) and then culturing the cells for 7 days. On day 7, cells were harvested and seeded into 96-well plates and cultured for 24 hours. On day 8, CD4+ T cells (isolated by negative selection using the Myltenyi kit) were seeded at a concentration of 200,000 cells per well, test products were added and cultured in within 3 days. IFN-γ levels in culture supernatants were then measured using DuoSet human cell ELISA kits for IFN-γ (R&D Systems) according to the manufacturer's instructions. When using antibodies in combination, each of these antibodies was added at the indicated concentration such that the total concentration of the added antibody was doubled. IFN-γ release is graphed in FIG. 18. Both bispecific DART D and TRIDENT A molecules were found to enhance the response in SCL to the same extent or slightly more than the combination of individual parent antibodies.

The ability of DART D, TRIDENT A, CTLA-4 mAb 3 G1AA, PD-1 mAb 6 G4P, and the CTLA-4 mAb 1/PD-1 mAb 1 combination (Ab Combo 1) to enhance cytokine release through checkpoint inhibition was also assessed in a replicate assay. stimulation by enterotoxin type B Staphylococcus aureus (SEB). In general, PBMCs were purified from whole blood of healthy donors (eg, using the Ficoll-Paque Plus density gradient centrifugation method (GE Healthcare) according to the manufacturer's instructions). Purified PBMCs were cultured in RPMI medium supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin in T-25 volumetric vials for 2-3 days in the absence or presence of SEB (eg, Sigma-Aldrich) at a concentration of 0.5 ng/ ml (primary stimulation). At the end of the first cycle of SEB stimulation, PBMCs were washed twice with PBS and immediately seeded onto 96-well tissue culture plates at a concentration of 1-5 x 10 ⁵ cells per well in medium alone, in medium with control or test product, in medium with SEB at a concentration of 0.5 ng/ml (secondary stimulation) and without antibody or in medium with SEB and control IgG or test article and cultured for an additional 2-3 days. At the end of secondary stimulation, supernatants were collected to measure cytokine secretion (eg, using DuoSet human cell ELISA kits for IFNy, IL-2, TNFa, IL-10, and IL-4 (R&D Systems) according to the manufacturer's instructions).

In fig. 19A-19B show fluorescent activated cell sorter (FACS) dot plots of PD-1 expression versus CTLA-1 expression by such PBMCs in the absence (FIG. 19A) or presence (FIG. 19B) of SEB- stimulation. In fig. 19C shows the effect of SEB stimulation on IFN-γ secretion. PBMCs were stimulated with Staphylococcus aureus enterotoxin type B (SEB) at a concentration of 0.5 ng/ml for 48 hours. Cells were then harvested, washed, and replated into 96-well plates containing various concentrations of antibodies with fresh SEB for an additional 48 hours. The supernatant was then collected and IFN-γ production was analyzed by flow cytometry-ELISA. Both bispecific DART and TRIDENT protein showed increased IFN-γ production responses that were consistent with the net response observed with the combination of individual parent mAbs. Similar results were observed in the SEB stimulation assay, in which PBMCs were cultured in the presence of a high concentration (500 ng/ml) of SEB for 72 hours. To further investigate the effect of the PD1 x CTLA-4 bispecific molecule on T cell response, PBMCs were stimulated with SEB at a concentration of 0.5 ng/ml for 48 hours, collected, washed and replated into 96-well plates in the presence of fresh SEB and either DART D, TRIDENT A, CTLA-4 mAb 3 G1AA, PD-1 mAb 6 G4P, or a combination of CTLA-4 mAb 3 G1AA / PD-1 mAb 6 G4P (Ab Combo 1) for an additional 48 hours and measured the released IL -2 (Fig. 19D). In fig. 19A-19D show that administration of PD1 x CTLA-4 bispecific molecules resulted in significantly enhanced T cell responses. When using antibodies in combination, each antibody was added at the indicated concentration such that the total concentration of antibody added was doubled.

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Example 6: In vivo studies.

A. Activity of PD-1 x CTLA-4 bispecific molecules in a mouse model of GVHD.

Activity of a typical PD1 x CTLA-4 bispecific molecule, DART

D, was evaluated in a graft-versus-host disease (GvHD) model of NOG mice transplanted with PBMCs. The research plan is presented in table. 15.________________

Table 15 Group N/gender Treatment Dose (mcg/kg) Method/mode of administration Cell graft(s) 1. 7/F DARTD 500 IV/Q7D x 7 MKPK (IP, 1E7) 2. 7/F DARTD 50 IV/Q7D x 7 MKPK (IP, 1E7) 3. 7/F DARTD 5 IV/Q7D x 7 MKPK (IP, 1E7) 4. 7/F Filler 0 IV/Q7D x 7 MKPK (IP, 1E7)

The number of CD3+ T cells was counted by fluorescence activated cell sorting (FACS) on day 14 of the study and is graphed in FIG. 20A. Survival was monitored throughout the study and is presented as percentage survival in the graph in FIG. 20B. The animal treated with DART D at 500 μg/kg exhibited increased T cell growth and increased GVHD, consistent with an increased T cell immune response.

B. Toxicological and pharmacokinetic study of PD-1 x CTLA-4 bispecific molecules.

The safety profile of a typical PD1 x CTLA-4 bispecific bivalent molecule, DART D, and a typical PD1 x CTLA-4 bispecific trivalent molecule, TRIDENT A, were evaluated in a dose study in cynomolgus monkeys (non-GLP compliant). laboratory research). Additionally, some markers of pharmacodynamic activity were checked.

The present study assessed the potential toxicity of PD-1 x CTLA-4 bispecific molecules when administered by multiple intravenous infusions. The research plan is presented in table. 16. ____________________________________________________

Table 16 Group Product under test Dose (mg/kg) Dose days Number of animals 1 Control 0 1, 8, 15 1 male, 1 female 2 DARTD 50 1, 8, 15 3 males, 3 females 3 DARTD 75 15,22, 29 3 males, 3 females 4 TRIDENTA 5 1 2 males, 1 female

Thus, a 2-week interval was allowed between the administration of the 50 mg/kg dose and the dose increase to 75 mg/kg. The following parameters and endpoints were assessed in this study: clinical signs, body weight, food intake, body temperature, clinical pathological parameters (coagulation, clinical chemistry and hematological data before dosing and 23 hours after dosing for groups 1-3; up to 22 days for group 4), bioanalytical and toxicokinetic parameters, flow cytometry data (before dosing and 23 hours after dosing), cytokines (2, 6, 22 hours after dosing). Anti-drug antibodies were assessed only for group 4 at days 8, 15 and 22. Necropsy was performed 48 hours after the 3rd dose for groups 1-3 only. The binding and in vivo activity of the PD-1 x CTLA-4 bispecific molecules were also assessed as described below.

All animals survived until scheduled euthanasia. No adverse clinical events were observed in animals receiving 3 doses up to 75 mg/kg/week. In particular, no diarrhea was observed. Histopathological changes were also insignificant. There was an increase in globulin levels in the treatment groups and an increase in the organ mass of the spleen and thymus in groups 2-3 (see Table 17, no necropsies were performed on animals from group 4), which was expected when the immune system was stimulated. Serum concentration versus time plots for each treatment group are shown in FIG. 21A-21C and are consistent with data for molecules containing human Fc fragments in cynomolgus monkeys.

Table 17 Group Product under test Dose (mg/kg) Spleen weight: body weight Thymus weight: body weight 1 Control 0 0.080 (average, n=2) 0.035 (average, n=2) 2 DARTD 50 0.239 (average, n=6) 0.088 (average, n=6) 3 DARTD 75 0.225 (average, n=6) 0.084 (average, n=6)

It has been reported that increases in absolute lymphocyte count (ALC) following treatment with the anti-CTLA-4 antibody ipilimumab appear to correlate with clinical benefit and overall survival (see, for example, Ku, G.Y., et al. (2010) Single-Institution Experience With Ipilimumab

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In Advanced Melanoma Patients In The Compassionate Use Setting: Lymphocyte Count After 2 Doses Correlates With Survival' Cancer 116(7): 1767–1775), indicating that ASL may be a useful pharmacodynamic (PD) endpoint. ASL was checked in each of the groups described above before treatment and after treatment on days 2, 8, 9, 15 and 16. DART D or TRIDENT A binding site occupancy on PD-1+ T cells was determined by measuring the mean detected fluorescence intensity (MDI) of anti-human immunoglobulin IgG4 antibodies coupled to Alexa 488+ in the CD4+/PD-1+ and CD8+/PD populations -1+ T cells under two conditions for each monkey blood sample. In the first condition, SIF values obtained in the presence of excess DART D or TRIDENT A were used to determine the maximum intensity of DART D or TRIDENT A binding on PD-1+ cells within each cell population. In the second condition, SIF values obtained in the presence of excess negative controls were used to determine the intensity of PD-1+ cell binding within each cell population in a DART D- or TRIDENT A-treated animal at the time of sample collection. The difference between the two conditions was used to calculate the % occupancy of DART D or TRIDENT A binding sites on PD-1+ T cell subtypes in DART D or TRIDENT A treated animals, as follows:

% Occupancy of DART D or TRIDENT A binding sites on a subpopulation of PD-1+ T cells

SIF detected by Anti-HuIgG4+ in the presence of excess AEX1367

SIF detected Anti-HuIgG4+ in the presence of excess DART D or TRIDENT A

The absolute content and percentage change normalized to 1 day are plotted in FIG. 22A (in thousands of cells/μL (thousand/μL)) and FIG. 22B (percentage change in ASL normalized to 1 day (D1)). In each of the treatment groups receiving DART D, there was an initial decrease in ASL immediately after treatment, followed by an increase in ASL to a level significantly above baseline. A similar trend was observed in the TRIDENT A treatment group, which received only one lower dose.

In addition, CD4+ T cell proliferation and PD-1 occupancy on T cells were tested for groups 1–3 described above. Briefly, CD3+/PD-1+ T cells were analyzed by FACS to assess the percentage of cells bound by DART D. Forty microliters of negative control molecules (respiratory syncytial virus (RSV) x fluoresceinIgG4/KFc) were added to a 96-well deep well plate DART) or the investigational device (DART D or TRIDENT A) at a concentration of 35 µg/ml. 100 microliters of well-mixed anticoagulated whole blood was then added to each well, mixed thoroughly using a pipette, and incubated in the dark for 45-75 minutes at ambient temperature. 100 microliters of 1x BD FACS lysis solution was then added to each well and mixed using a pipette; the plate was then incubated in the dark for an additional 10–20 minutes at ambient temperature. The plate was then centrifuged at 400 x g for 5 minutes and the supernatant was discarded. 100 microliters of FACS buffer was added to each well and mixed to complete the wash step. The plate was then centrifuged at 400 x g for 5 minutes and the supernatant was discarded. The cell pellet was resuspended in twenty microliters of panel 1 antibody mixture and incubated for 30-60 minutes at ambient temperature. The plate was washed as in the previous washing steps. At the end of the incubation, the plate was washed again and the cell pellet was finally resuspended in 300 microliters of FACS buffer and samples were analyzed using a BD FACSCanto II cell analyzer. The results of the analysis are shown in Fig. 23A-23B.

As shown in FIG. 23A (for DART D administered at 50 mg/kg) and FIG. 23B (for DART D administered at 75 mg/kg), PD-1 occupancy (i.e., DART D binding) was maximal throughout treatment for groups 2 and 3. CD4+ T cell proliferation was assessed using FACS-based co-expression of Ki-67 (cellular proliferation marker).

microliters of antibody mixture A (containing antibodies that bind to cell surface markers: CD45, CD3, CD4 and CD8) was added to a 96-well deep well plate. Fifty microliters of well-mixed anticoagulated whole blood was then added to each well, mixed thoroughly using a pipette, and incubated in the dark for 15 to 45 minutes at ambient temperature. 500 microliters of 1x BD FACS L lysis solution was then added to each well and mixed using a pipette; the plate was then incubated in the dark for an additional 10–20 minutes at ambient temperature. The plate was centrifuged at 1200 rpm for 5 minutes and the supernatant was discarded. 500 microliters of FACS buffer was then added to each well and mixed to complete the washing step. The plate was then centrifuged at 1200 rpm for 5 minutes and the supernatant was discarded. The cell pellet was resuspended in antibody mixture B (containing antibodies that bind to the intracellular marker Ki-67) or resuspended in antibody isotype composition (containing isotype control as an intracellular marker) and incubated in the dark for 15 to 45 minutes. After washing, the cell pellet was resuspended in 300 microliters of FACS buffer and samples were analyzed using a BD FACSCanto II Cell Analyzer.

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Based on analysis using a panel of intracellular T-cell staining, the percentage of CD4+ and CD8+ cells was determined as a fraction of total leukocytes stained for CD45+. The number of Ki 67+ cells in the fraction stained for CD4+ cells was counted, and the percentage of CD4+/Ki 67+ T cells (proliferative CD4 T cells) was determined as a fraction of total CD4+ cells. Similarly, the percentage of CD8+/Ki 67+ T cells (proliferative CD8 T cells) was determined as a fraction of total CD8+ cells. The results of the analysis are shown in Fig. 24A-24B.

As shown in FIG. 24A-24B, the proliferation of CD4+ T cells was significantly increased by treatment in treatment groups 2 and 3 throughout treatment. The results of the present study indicate that administration of a PD1 x CTLA-4 bispecific molecule to cynomolgus monkeys is well tolerated at concentrations up to 75 mg/kg, which is significantly higher than the 5 mg/kg dose at which adverse events have been reported in treated cynomolgus monkeys. ipilimumab. The molecules had a favorable pharmacokinetic profile, and a number of markers of pharmacodynamic activity were observed, including increased lymphocyte counts, increased globulin levels, increased splenic and thymic organ weights, increased T cell proliferation (both T cell number and Ki-67 expression), and maximum PD-1 occupancy on T cells.

All publications and patents mentioned in this application are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and separately stated to be incorporated by reference in its entirety. It should be understood that while the present invention has been described in connection with specific embodiments, further modifications are possible. It is intended that any variations, uses or adaptations according to the invention that are generally consistent with the teachings of the present invention are included in this application. This application includes such departures from the invention as being within the limits of known or common practice in the field of technology to which the present invention relates and as being applicable to the essential features set forth above in this application.

SEQUENCE LIST <110> MacroGenics, Inc.

Johnson, Leslie S.

Chichili, Gurunadh Reddy

Shah, Kalpana

La Motte-Mohs, Ross Moore, Paul A.

Bonvini, Ezio

Koenig, Scott <120> Bispecific molecules immunoreactive to PD-1 and CTLA-4 and their uses <130> 1301.0134PCT <150> US62/266,944 <151> 2015-12-14 <160>110 <170 > PatentIn, version 3.5 <210>1 <211>217 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1).. (217) <223> Example human IgG1 CH2-CH3 domain <220>

- 74 043373 <221> MISC_FEATURE <222> (217) ..(217) <223> Xaa is lysine (K) or missing <400>1

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

5 1015

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 2530

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 4045

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 5560

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 7580

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 9095

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

100 105110

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

115 120125

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

130 135140

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

145 150 155160

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

165 170175

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

180 185190

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

195 200205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210215

- 75 043373 <210> 2 <211> 216 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1)..(216) < 223> Example human IgG2 CH2-CH3 domain <220>

< 221> MISC_FEATURE < 222> (216)..(216) <223> Xaa is lysine (K) or missing <400>2

Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 1015

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

2530

Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 4045

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 5560

Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 65 7075

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 9095

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

100 105110

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

115 120125

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

130 135140

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

145 150155

Pro

Val

Val

Gln

Gln 80

Gly

Pro

Thr

Ser

Tyr 160

- 76 043373

Lys Thr Thr Pro Pro 165 Met Leu Asp Ser Asp 170 Gly Ser Phe Phe Leu 175 Tyr Ser Lys Leu Thr 180 Val Asp Lys Ser Arg 185 Trp Gln Gln Gly Asn 190 Val Phe Ser Cys Ser 195 Val Met His Glu Ala 200 Leu His Asn His Tyr 205 Thr Gln Lys Ser Leu 210 Ser Leu Ser Pro Gly 215 Xaa

<210> 3 <211> 217 <212> PROTEIN <213> Homo sapiens <220>

<221> MISC_FEATURE <222> (1).. (217) <223> Human IgG3 example CH2-CH3 domain <220>

<221> MISC_FEATURE <222> (217)..(217) <223> Xaa is lysine (K) or missing <400> 3

Ala 1 Pro Glu Leu Leu 5 Gly Gly Pro Ser Val 10 Phe Leu Phe Pro Pro 15 Lys Pro Lys Asp Thr 20 Leu Met Ile Ser Arg 25 Thr Pro Glu Val Thr 30 Cys Val Val Val Asp 35 Val Ser His Glu Asp 40 Pro Glu Val Gln Phe 45 Lys Trp Tyr Val Asp 50 Gly Val Glu Val His 55 Asn Ala Lys Thr Lys 60 Pro Arg Glu Glu Gln 65 Tyr Asn Ser Thr Phe 70 Arg Val Val Ser Val 75 Leu Thr Val Leu His 80 Gln Asp Trp Leu Asn 85 Gly Lys Glu Tyr Lys 90 Cys Lys Val Ser Asn 95 Lys Ala Leu Pro Ala 100 Pro Ile Glu Lys Thr 105 Ile Ser Lys Thr Lys 110 Gly Gln

- 77 043373

Pro Arg Glu Pro Gln Val Tyr 115

Thr Leu Pro Pro Ser Arg Glu Glu

120 125

Thr Lys Asn Gln Val Ser Leu

130 135

Thr Cys Leu Val Lys Gly Phe Tyr

140

Ser Asp Ile Ala Val Glu Trp

145 150

Glu Ser Ser Gly Gln Pro Glu Asn

155

Tyr Asn Thr Thr Pro Pro Met 165

Leu Asp Ser Asp Gly Ser Phe Phe

170 175

Tyr Ser Lys Leu Thr Val Asp

180

Lys Ser Arg Trp Gln Gln Gly Asn

185 190

Phe Ser Cys Ser Val Met His 195

Glu Ala Leu His Asn Arg Phe Thr

200 205

Met

Pro

Asn 160

Leu

Ile

Gln

Lys Ser Leu Ser Leu Ser Pro

210215

Gly Xaa <210>4 <211>217 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1).. (217) < 223> Human IgG4 example CH2-CH3 domain <220>

< 221> MISC_FEATURE < 222> (217)..(217) < 223> Xaa is lysine (K) or missing < 400>4

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 1 5 1015

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

2530

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 35 4045

Lys

Val

Tyr

- 78 043373

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 65 70 75

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

90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155

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

165 170 175

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

180 185 190

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

195 200 205

Glu

His 80

Lys

Gln

Met

Pro

Asn 160

Leu

Val

Gln

Lys Ser Leu Ser Leu Ser Leu Gly Xaa

210 215 <210> 5 <211> 8 <212> PROTEIN <213> Artificial sequence <220>

<223> Intermediate spacer peptide (linker 1) <400> 5

Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 6 <211> 5

- 79 043373 <212> PROTEIN <213> Artificial sequence <220>

<223> Cysteine-containing spacer peptide (linker 2) <400>6

Gly Gly Cys Gly Gly <210>7 <211>4 <212> PROTEIN <213> Artificial sequence <220>

<223>Alternative spacer peptide (linker 2) <400>7

Gly Gly Gly Ser < 210>8 < 211>6 < 212> PROTEIN < 213> Artificial sequence <220>

<223>Alternative spacer peptide (linker 2) <400>8

Leu Gly Gly Gly Ser Gly <210>9 <211>11 <212> PROTEIN <213> Artificial sequence <220>

<223> Alternative spacer peptide (linker 2) <400>9

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

510 <210>10 <211>5 <212> PROTEIN <213> Artificial sequence <220>

- 80 043373 <223> Alternative spacer peptide (linker 2) <400>10

Ala Ser Thr Lys Gly 15 < 210>11 < 211>6 < 212> PROTEIN < 213> Artificial sequence <220>

<223>Alternative spacer peptide (linker 2) <400>11

Leu Glu Pro Lys Ser Ser <210>12 <211>5 <212> PROTEIN <213> Artificial sequence <220>

<223>Alternative spacer peptide (linker 2) <400>12

Ala Pro Ser Ser Ser <210>13 <211>7 <212> PROTEIN <213> Artificial sequence <220>

<223>Heterodimer-promoting domain <400>13

Gly Val Glu Pro Lys Ser Cys <210>14 <211>6 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE <222> (1)..(6) < 223> Heterodimer-promoting hinge region domain

- 81 043373

Human IgG <400>14

Val Glu Pro Lys Ser Cys <210>15 <211>6 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1)..(6) < 223> Human IgG hinge region heterodimer-promoting domain < 400>15

Ala Glu Pro Lys Ser Cys <210>16 <211>6 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1)..(6) < 223> Human immunoglobulin kappa light chain heterodimer-promoting domain < 400>16

Gly Phe Asn Gly Glu Cys <210>17 <211>6 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1)..(6) < 223> Human immunoglobulin kappa light chain heterodimer-promoting domain < 400> 17

Phe Asn Arg Gly Glu Cys

5

- 82 043373 <210> 18 <211> 28 <212> PROTEIN <213> Artificial sequence <220>

<223>Heterodimer-promoting E-coil domain <400>18

Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val 1 5 1015

Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

2025 < 210>19 < 211>28 < 212> PROTEIN < 213> Artificial sequence <220>

<223>Heterodimer-promoting K-coil domain <400>19

Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val 1 5 1015

Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu

2025 < 210>20 < 211>28 < 212> PROTEIN < 213> Artificial sequence <220>

< 223> Cysteine-containing heterodimer-promoting domain

E-coil <400>20

Glu Val Ala Ala Cys Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val

5 1015

Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

2025 <210>21 <211>28 <212> PROTEIN

- 83 043373 <213> Artificial sequence <220>

< 223> Cysteine-containing heterodimer-promoting domain

K-coil <400>21

Lys Val Ala Ala Cys Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val

5 1015

Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu 2025 <210>22 <211>46 <212> PROTEIN <213> Streptococcus dysgalactiae <220>

<221> MISC_FEATURE <222> (1).. (46)

<223> Albumin-binding G148 domain 3 (ABD3) squirrel G strain Streptococcus <400> 22 Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly 1 5 10 15 Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asp Asn Ala Lys Ser Ala Glu 20 25 thirty Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro 35 40 45

< 210>23 < 211>46 < 212> PROTEIN < 213> Artificial sequence <220>

<223> Deimmunized variant of albumin binding domain 3 (ABD3) protein

G strain Streptococcus G148 <400>23

Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly

5 1015

Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asp Asn Ala Lys Ser Ala Glu

2530

- 84 043373

Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro

4045 <210>24 <211>46 <212> PROTEIN <213> Artificial sequence <220>

<223> Deimmunized variant of albumin binding domain 3 protein

G strain Streptococcus G148 <400>24

Leu 1 Ala Glu Ala Lys 5 Val Leu Ala Asn Arg 10 Glu Leu Asp Lys Tyr 15 Gly Val Ser Asp Tyr 20 Tyr Lys Asn Ala Ala 25 Asn Asn Ala Lys Thr 30 Val Glu Gly Val Lys 35 Ala Leu Ile Ala Glu 40 Ile Leu Ala Ala Leu 45 Pro

<210>25 <211>46 <212> PROTEIN <213> Artificial sequence <220>

<223> Deimmunized variant of albumin binding domain 3 protein

G strain Streptococcus G148 <400> 25

Leu Ala Glu Ala 1

Lys Val 5

Val Ser Asp Tyr 20

Tyr Lys

Gly Val Lys Ala 35

Leu Ile

Leu Ala Asn Arg Glu Leu Asp 10

Asn Leu Ile Ser Asn Ala Lys 25

Ala Glu Ile Leu Ala Ala Leu 40 45

Lys Tyr Gly 15

Ser Val Glu 30

Pro (ABD3) (ABD3) <210> 26 <211> 10 <212> PROTEIN <213> Artificial sequence <220>

- 85 043373 <223> Intermediate spacer linker <400>26

Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 510 < 210>27 < 211>15 < 212> PROTEIN < 213> Artificial sequence < 220>

<223> Intermediate spacer linker <400>27

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

5 1015 < 210>28 < 211>5 < 212> PROTEIN < 213> Artificial sequence <220>

<223> Intermediate spacer linker <400>28

Ala Pro Ser Ser Ser <210>29 <211>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Intermediate spacer linker <400>29

Ala Pro Ser Ser Ser Pro Met Glu < 210>30 < 211>15 < 212> PROTEIN < 213> Artificial sequence <220>

< 223 > Intermediate spacer linker < 400 > 30

Leu Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys

- 86 043373 <210> 31 <211> 13 <212> PROTEIN <213> Artificial sequence <220>

<223> Intermediate spacer linker <400>31

Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 510 < 210>32 < 211>16 < 212> PROTEIN < 213> Artificial sequence <220>

<223> Intermediate spacer linker <400>32

Leu Glu Pro Lys Ser Ser Asp Lys Thr His 1 510

Thr Cys Pro Pro Cys Pro 15 <210>33 <211>15 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1).. (15) < 223> Example of an IgG1 hinge region <400>33

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 1015 <210>34 <211>12 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1)..(12) <223> Example of an IgG2 hinge region <400> 34

- 87 043373

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro <210> 35 <211> 12 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1)..(12) <223> Example of an IgG4 hinge region <400>35

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 510 <210>36 <211>12 <212> PROTEIN <213> Artificial sequence <220>

<223> IgG4 hinge region variant containing stabilizing substitution S228P <400>36

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

510 <210>37 <211>15 <212> PROTEIN <213> Artificial sequence <220>

<223> Intermediate spacer peptide <400>37

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 1015 <210>38 <211>107 <212> PROTEIN <213> Homo sapiens <220>

<221> MISC_FEATURE <222> (1)..(107)

- 88 043373 <223> Example of a human IgG kappa CL domain <400>38

Arg 1 Thr Val Ala Ala 5 Pro Ser Val Phe Ile 10 Phe Pro Pro Ser Asp 15 Glu Gln Leu Lys Ser 20 Gly Thr Ala Ser Val 25 Val Cys Leu Leu Asn 30 Asn Phe Tyr Pro Arg 35 Glu Ala Lys Val Gln 40 Trp Lys Val Asp Asn 45 Ala Leu Gln Ser Gly 50 Asn Ser Gln Glu Ser 55 Val Thr Glu Gln Asp 60 Ser Lys Asp Ser Thr 65 Tyr Ser Leu Ser Ser 70 Thr Leu Thr Leu Ser 75 Lys Ala Asp Tyr Glu 80 Lys His Lys Val Tyr 85 Ala Cys Glu Val Thr 90 His Gln Gly Leu Ser 95 Ser Pro Val Thr Lys 100 Ser Phe Asn Arg Gly 105 Glu Cys

<210>39 <211>104 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1).. (104) <223> Example of a human IgG lambda CL domain <400>39

Gln Pro Lys Ala Ala Pro Ser Val 15

Thr Leu Phe Pro Pro Ser Ser Glu

1015

Glu Leu Gln Ala Asn Lys Ala Thr 20

Leu Val Cys Leu Ile Ser Asp Phe 25 30

Tyr Pro Gly Ala Val Thr Val Ala

40

Trp Lys Ala Asp Ser Ser Pro Val 45

Lys Ala Gly Val Glu Thr Thr Pro

55

Ser Lys Gln Ser Asn Asn Lys Tyr 60

- 89 043373

Ala 65 Ala Ser Ser Tyr Leu 70 Ser Leu Thr Pro Glu 75 Gln Trp Lys Ser His 80 Arg Ser Tyr Ser Cys 85 Gln Val Thr His Glu 90 Gly Ser Thr Val Glu 95 Lys Thr Val Ala Pro 100 Thr Glu Cys Ser

<210> 40 <211> 98 <212> PROTEIN <213> Homo sapiens <220>

<221> MISC_FEATURE <222> (1)..(98) <223> Human IgG1 CH1 domain <400> 40

Ala 1 Ser Thr Lys Gly 5 Pro Ser Val Phe Pro 10 Leu Ala Pro Ser Ser 15 Lys Ser Thr Ser Gly 20 Gly Thr Ala Ala Leu 25 Gly Cys Leu Val Lys 30 Asp Tyr Phe Pro Glu 35 Pro Val Thr Val Ser 40 Trp Asn Ser Gly Ala 45 Leu Thr Ser Gly Val 50 His Thr Phe Pro Ala 55 Val Leu Gln Ser Ser 60 Gly Leu Tyr Ser Leu 65 Ser Ser Val Val Thr 70 Val Pro Ser Ser Ser 75 Leu Gly Thr Gln Thr 80 Tyr Ile Cys Asn Val 85 Asn His Lys Pro Ser 90 Asn Thr Lys Val Asp 95 Lys

Arg Val <210> 41 <211> 98 <212> PROTEIN <213> Homo sapiens

- 90 043373

Leu Ala Pro Cys Ser Arg <220>

<221> MISC_FEATURE <222> (1)..(98) <223> Human IgG2 CH1 domain <400> 41

Ala Ser Thr Lys Gly Pro Ser Val Phe 1 5

Ser Thr Ser Glu Ser Thr Ala Ala Leu

25

Phe Pro Glu Pro Val Thr Val Ser Trp

40

Gly Val His Thr Phe Pro Ala Val Leu

55

Leu Ser Ser Val Val Thr Val Pro Ser 65 70

Tyr Thr Cys Asn Val Asp His Lys Pro 85

Thr Val <210> 42 <211> 98 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1)..(98) < 223> Human IgG4 CH1 domain < 400> 42

Ala Ser Thr Lys Gly Pro Ser Val Phe 1 5

Ser Thr Ser Glu Ser Thr Ala Ala Leu

25

Phe Pro Glu Pro Val Thr Val Ser Trp

40

Cys Leu Val Lys Asp Tyr 30

Ser Gly Ala Leu Thr Ser 45

Ser Ser Gly Leu Tyr Ser 60

Asn Phe Gly Thr Gln Thr 75 80

Asn Thr Lys Val Asp Lys 95

Leu Ala Pro Cys Ser Arg 15

Cys Leu Val Lys Asp Tyr 30

Ser Gly Ala Leu Thr Ser 45

- 91 043373

Gly Val His Thr Phe Pro Ala Val

55

Leu Ser Ser Val Val Thr Val Pro 65 70

Tyr Thr Cys Asn Val Asp His Lys 85

Leu Gln Ser Ser 60 Gly Leu Tyr Ser Ser Ser Ser 75 Leu Gly Thr Lys Thr 80 Pro Ser 90 Asn Thr Lys Val Asp 95 Lys

Arg Val <210> 43 <211> 217 <212> PROTEIN <213> Artificial sequence <220>

<223> CH2-CH3 domains of IgG1 containing substitutions L234A and L235A (according to Kabat) <220>

<221> MISC_FEATURE <222> (217)..(217) <223> Xaa is lysine (K) or missing <400>43

Ala Pro Glu Ala Ala Gly 1 5

Pro Lys Asp Thr Leu Met 20

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1015

Ile Ser Arg Thr Pro Glu Val Thr Cys Val

2530

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 4045

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 5560

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 7580

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 9095

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

100 105110

- 92 043373

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

115 120

Ser Arg Glu Glu Met

125

Thr Lys Asn Gln Val Ser Leu

130 135

Thr Cys Leu Val Lys Gly Phe Tyr Pro

140

Ser Asp Ile Ala Val Glu Trp

145 150

Glu Ser Asn Gly Gln Pro Glu Asn Asn

155 160

Tyr Lys Thr Thr Pro Pro Val

165

Leu Asp Ser Asp Gly Ser Phe Phe Leu

170 175

Tyr Ser Lys Leu Thr Val Asp 180

Lys Ser Arg Trp Gln Gln Gly Asn Val

185 190

Phe Ser Cys Ser Val Met His 195

Glu Ala Leu His Asn His Tyr Thr Gln

200 205

Lys

Ser Leu Ser Leu Ser Pro Gly Xaa

210215 <210>44 <211>217 <212> PROTEIN <213> Artificial sequence <220>

<223> IgG1 CH2-CH3 knob-containing domains <220>

< 221> MISC_FEATURE < 222> (217) ..(217) <223> Xaa is lysine (K) or missing <400> 44

Ala 1 Pro Glu Ala Ala 5 Gly Gly Pro Ser Val 10 Phe Leu Phe Pro Pro 15 Lys Pro Lys Asp Thr 20 Leu Met Ile Ser Arg 25 Thr Pro Glu Val Thr 30 Cys Val Val Val Asp 35 Val Ser His Glu Asp 40 Pro Glu Val Lys Phe 45 Asn Trp Tyr Val Asp 50 Gly Val Glu Val His 55 Asn Ala Lys Thr Lys 60 Pro Arg Glu Glu

- 93 043373

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 65 70 75

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

90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155

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

165 170 175

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

180 185 190

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

195 200 205

His 80

Lys

Gln

Met

Pro

Asn 160

Leu

Val

Gln

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210215 <210>45 <211>217 <212> PROTEIN <213> Artificial sequence <220>

<223> IgG1 CH2-CH3 cavity-containing domains <220>

< 221> MISC_FEATURE < 222> (217) ..(217) < 223> Xaa is lysine (K) or missing <400> 45

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 1 5 10 15

Lys

- 94 043373

Pro Lys Asp Thr Leu 20

Met Ile Ser Arg Thr 25

Pro Glu Val Thr Cys 30

Val Val Asp Val Ser 35

His Glu Asp Pro Glu 40

Val Lys Phe Asn Trp 45

Val Asp Gly Val Glu 50

Val His Asn Ala Lys 55

Thr Lys Pro Arg Glu 60

Gln Tyr Asn Ser Thr 65

Tyr Arg Val Val Ser 70

Val Leu Thr Val Leu 75

Gln Asp Trp Leu Asn 85

Gly Lys Glu Tyr Lys

Cys Lys Val Ser Asn

Ala Leu Pro Ala Pro

100

Ile Glu Lys Thr Ile

105

Ser Lys Ala Lys Gly

110

Pro Arg Glu Pro Gln 115

Val Tyr Thr Leu Pro

120

Pro Ser Arg Glu Glu 125

Thr Lys Asn Gln Val

130

Ser Leu Ser Cys Ala

135

Val Lys Gly Phe Tyr

140

Ser Asp Ile Ala Val 145

Glu Trp Glu Ser Asn 150

Gly Gln Pro Glu Asn 155

Tyr Lys Thr Thr Pro

165

Pro Val Leu Asp Ser

170

Asp Gly Ser Phe Phe

175

Val Ser Lys Leu Thr

180

Val Asp Lys Ser Arg

185

Trp Gln Gln Gly Asn

190

Phe Ser Cys Ser Val 195

Met His Glu Ala Leu

200

His Asn Arg Tyr Thr

205

Val

Tyr

Glu

His 80

Lys

Gln

Met

Pro

Asn 160

Leu

Val

Gln

Lys Ser Leu Ser Leu 210

Ser Pro Gly Xaa

215 <210> 46 <211> 288 <212> PROTEIN <213> Homo sapiens <220>

- 95 043373 <221> MISC_FEATURE <222> (1).. (288) <223> Human PD-1, including signal sequence in NCBI (sequence NP_005009.2) <220>

< 221> SIGNAL < 222> (1)..(20) < 223> Signal sequence < 400> 46

Met Gln

Ile Pro Gln Ala 5

Leu Gly

Trp Arg Pro Gly 20

Asn Pro

Pro Thr Phe Ser 35

Pro Trp Pro Val Val Trp Ala Val Leu 1015

Trp Phe Leu Asp Ser Pro Asp Arg Pro 2530

Pro Ala Leu Leu Val Val Thr Glu Gly 4045

Asn Ala Thr Phe Thr Cys Ser

55

Phe Ser Asn Thr Ser Glu Ser Phe 60

Leu Asn Trp Tyr Arg Met Ser 65 70

Pro Ser Asn Gln Thr Asp Lys Leu 75

Ala Phe Pro Glu Asp Arg Ser 85

Gln Pro Gly Gln Asp Cys Arg Phe

95

Val Thr Gln Leu Pro Asn Gly

100

Arg Asp Phe His Met Ser Val Val

105 110

Ala Arg Arg Asn Asp Ser Gly

115

Thr Tyr Leu Cys Gly Ala Ile Ser

120 125

Ala Pro Lys Ala Gln Ile Lys

130 135

Glu Ser Leu Arg Ala Glu Leu Arg

140

Thr Glu Arg Arg Ala Glu Val

145 150

Pro Thr Ala His Pro Ser Pro Ser

155

Arg Pro Ala Gly Gln Phe Gln 165

Thr Leu Val Val Gly Val Val Gly

170 175

Leu Leu Gly Ser Leu Val Leu

180

Leu Val Trp Val Leu Ala Val Ile

185 190

Gln

Trp

Asp

Val

Ala 80

Arg

Arg

Leu

Val

Pro 160

Gly

Cys

- 96 043373

Ser Arg Ala Ala Arg Gly Thr Ile

195 200

Ala Arg Arg Thr Gly Gln Pro

205

Leu Lys Glu Asp Pro Ser Ala Val

210 215

Val Phe Ser Val Asp Tyr Gly

220

Glu Leu Asp Phe Gln Trp Arg Glu

225 230

Thr Pro Glu Pro Pro Val Pro

235 240

Cys Val Pro Glu Gln Thr Glu Tyr

245

Thr Ile Val Phe Pro Ser Gly

250 255

Met Gly Thr Ser Ser Pro Ala Arg

260

Gly Ser Ala Asp Gly Pro Arg

270

Ser Ala Gln Pro Leu Arg Pro Glu

275 280

Gly His Cys Ser Trp Pro Leu

285 <210> 47 <211> 113 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1).. (113) <223> Antibody VL domain against <400> 47

Gln Val Gln Leu Val Glu Ser Gly 1 5 human PD-1 mAb 1 (nivolumab)

Gly Val Val Gln Pro Gly Arg

15

Ser Leu Arg Leu Asp Cys Lys Ala 20

Gly Ile Thr Phe Ser Asn Ser 30

Gly Met His Trp Val Arg Gln Ala

40

Gly Lys Gly Leu Glu Trp Val 45

Ala Val Ile Trp Tyr Asp Gly Ser

55

Arg Tyr Tyr Ala Asp Ser Val 60

Lys Gly Arg Phe Thr Ile Ser Arg 65 70

Asn Ser Lys Asn Thr Leu Phe

80

Leu Gln Met Asn Ser Leu Arg Ala 85

Asp Thr Ala Val Tyr Tyr Cys

95

- 97 043373

Ala Thr Asn Asp Asp Tyr Trp Gly

100

Gln Gly Thr Leu Val Thr Val Ser

105 110

Ser <210> 48 <211> 107 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1).. (107) < 223> VL domain of anti-human PD-1 antibody PD-1 mAb 1 (nivolumab) < 400>48

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

5 1015

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 2530

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 4045

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 7580

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 9095

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100105 <210>49 <211>120 <212> PROTEIN <213> Artificial sequence <220>

<223> VL domain of humanized anti-human PD-1 antibody PD-1 mAb 2 (pembrolizumab)

- 98 043373 <400>49

Gln Val Gln Leu Val Gln Ser Gly 15

Val Glu Val Lys Lys Pro Gly Ala

1015

Ser Val Lys Val Ser Cys Lys Ala 20

Ser Gly Tyr Thr Phe Thr Asn Tyr 25 30

Tyr Met Tyr Trp Val Arg Gln Ala

40

Pro Gly Gln Gly Leu Glu Trp Met 45

Gly Gly Ile Asn Pro Ser Asn Gly

55

Gly Thr Asn Phe Asn Glu Lys Phe 60

Lys Asn Arg Val Thr Leu Thr Thr 65 70

Asp Ser Ser Thr Thr Thr Ala Tyr

80

Met Glu Leu Lys Ser Leu Gln Phe 85

Asp Asp Thr Ala Val Tyr Tyr Cys

95

Ala Arg Arg Asp Tyr Arg Phe Asp

100

Met Gly Phe Asp Tyr Trp Gly Gln

105 110

Gly Thr Thr Val Thr Val Ser Ser

115120 <210>50 <211>111 <212> PROTEIN <213> Artificial sequence <220>

<223> VL domain of humanized anti-human PD-1 antibody PD mAb 2 (pembrolizumab) <400> 50

Glu 1 Ile Val Leu Thr 5 Gln Ser Pro Ala Thr 10 Leu Ser Leu Ser Pro 15 Gly Glu Arg Ala Thr 20 Leu Ser Cys Arg Ala 25 Ser Lys Gly Val Ser 30 Thr Ser Gly Tyr Ser 35 Tyr Leu His Trp Tyr 40 Gln Gln Lys Pro Gly 45 Gln Ala Pro Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

- 99 043373

55 60

Arg Phe Ser Gly 65

Ser Leu Glu Pro

Ser Gly 70

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

80

Glu Asp 85

Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

95

Asp Leu Pro Leu

100

Thr Phe

Gly Gly Gly Thr Lys Val Glu Ile Lys

105110 <210>51 <211>121 <212> PROTEIN <213> Mus musculus <220>

< 221> MISC_FEATURE < 222> (1).. (121) < 223> VH domain of mouse anti-human PD-1 antibody PD-1 mAb 3 (EH12.2H7) < 400>51

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

5 1015

Ser Val Gln Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Ser 20 2530

Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 4045

Gly Tyr Ile Tyr Pro Ser Thr Gly Phe Thr Glu Tyr Asn Gln Lys Phe 50 5560

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 7580

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

9095

Ala Arg Trp Arg Asp Ser Ser Gly Tyr His Ala Met Asp Tyr Trp Gly

100 105110

Gln Gly Thr Ser Val Thr Val Ser Ser

115120

- 100 043373 <210> 52 <211> 111 <212> PROTEIN <213> Mus musculus <220>

< 221> MISC_FEATURE < 222> (1).. (111) < 223> VH domain of mouse anti-human PD-1 antibody PD-1 mAb 3 (EH12.2H7) < 400>52

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

5 1015

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 2530

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 4045

Lys Leu Leu Ile Lys Phe Gly Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 5560

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 7580

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

9095

Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105110 < 210>53 < 211>117 < 212> PROTEIN < 213> Artificial sequence <220>

<223> VH domain of humanized anti-human PD-1 antibody PD-1 mAb 4 (pidilizumab) <400> 53

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15

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

- 101 043373

Gly Gln Gly Leu Gln Trp Met

Gly Met Asn Trp Val Arg Gln Ala Pro

3540

Gly Trp Ile Asn Thr Asp Ser Gly Glu Ser

5055

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr 6570

Leu Gln Ile Thr Ser Leu Thr Ala Glu Asp

8590

Val Arg Val Gly Tyr Asp Ala Leu Asp Tyr

100105

Thr Tyr Ala Glu Glu Phe 60

Ser Val Asn Thr Ala Tyr 75 80

Thr Gly Met Tyr Phe Cys

Trp Gly Gln Gly Thr Leu

110

Val Thr Val Ser Ser

115 <210>54 <211>106 <212> PROTEIN <213> Artificial sequence <220>

<223> VH domain of humanized anti-human PD-1 antibody PD mAb 4 (pidilizumab) <400> 54

Glu 1 Ile Val Leu Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Gly Asp Arg Val Thr 20 Ile Thr Cys Ser Ala 25 Arg Ser Ser Val Ser 30 Tyr Met His Trp Phe 35 Gln Gln Lys Pro Gly 40 Lys Ala Pro Lys Leu 45 Trp Ile Tyr Arg Thr 50 Ser Asn Leu Ala Ser 55 Gly Val Pro Ser Arg 60 Phe Ser Gly Ser Gly 65 Ser Gly Thr Ser Tyr 70 Cys Leu Thr Ile Asn 75 Ser Leu Gln Pro Glu 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Phe Pro Leu Thr

- 102 043373

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100

105 <210> 55 <211> 116 <212> PROTEIN <213> Artificial sequence <220>

<223> VH domain of humanized anti-human PD-1 antibody (PD-1 mAb 5) <400>55

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Phe 20 2530

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Tyr Ile Ser Ser Gly Ser Met Ser Ile Ser Tyr Ala Asp Thr Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 7580

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

9095

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

100 105110

Thr Val Ser Ser

115 <210>56 <211>112 <212> PROTEIN <213> Artificial sequence <220>

< 223 > VL domain of humanized anti-human PD-1 antibody (PD-1 mAb 5) < 400 > 56

- 103 043373

Asp Val Val Met Thr Gln Ser Pro Leu Ser 1 5 10

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser

25

Leu Pro Val Thr Leu Gly 15

Gln Ser Leu Val His Ser 30

Thr Gly Asn Thr Tyr Leu His 35

Trp Tyr Leu Gln Lys Pro Gly Gln Ser 40 45

Pro Gln Leu Leu Ile 50

Tyr Arg Val 55

Ser Asn Arg Phe Ser Gly Val Pro 60

Asp Arg Phe Ser Gly 65

Ser Gly Ser 70

Gly Thr Asp Phe Thr Leu Lys Ile

80

Ser Arg Val Glu Ala 85

Glu Asp Val

Gly Val Tyr Tyr Cys Ser Gln Thr

95

Thr His Val Pro Trp

100

Thr Phe Gly

Gln Gly Thr Lys Leu Glu Ile Lys

105 110 <210> 57 <211> 119 <212> PROTEIN <213> Artificial sequence <220>

<223>VH domain of humanized anti-human PD-1 antibody (PD-1 mAb 6) <220>

< 221> MISC_FEATURE < 222> (48)..(48) < 223> Xaa is isoleucine (I) or alanine (A) < 400> 57

Gln Val Gln Leu Val Gln Ser Gly 1 5

Ala Glu Val Lys Lys Pro Gly Ala

15

Ser Val Lys Val Ser Cys Lys Ala 20

Ser Gly Tyr Ser Phe Thr Ser Tyr 25 30

Trp Met Asn Trp Val Arg Gln Ala

40

Pro Gly Gln Gly Leu Glu Trp Xaa 45

Gly Val Ile His Pro Ser Asp Ser

55

Glu Thr Trp Leu Asp Gln Lys Phe 60

- 104 043373

Lys Asp Arg Val 65

Met Glu Leu Ser

Thr Ile Thr Val 70

Asp Lys Ser Thr Ser Thr Ala Tyr

80

Ser Leu Arg Ser 85

Glu Asp Thr Ala Val Tyr Tyr Cys

95

Ala Arg Glu His

100

Tyr Gly Thr Ser

Pro Phe Ala Tyr Trp Gly Gln Gly

105110

Thr Leu Val Thr Val Ser Ser

115 <210>58 <211>111 <212> PROTEIN <213> Artificial sequence <220>

<223> VL domain of humanized anti-human PD-1 antibody (PD mAb 6) <220>

< 221> MISC_FEATURE < 222> (26)..(26) < 223> Xaa is asparagine (N) or serine (S) <220>

< 221> MISC_FEATURE < 222> (58)..(58) <223> Xaa is glutamine (Q) or arginine (R) <400> 58

Glu 1 Ile Val Leu Thr 5 Gln Ser Pro Ala Thr 10 Leu Ser Leu Ser Pro 15 Gly Glu Arg Ala Thr 20 Leu Ser Cys Arg Ala 25 Xaa Glu Ser Val Asp 30 Asn Tyr Gly Met Ser 35 Phe Met Asn Trp Phe 40 Gln Gln Lys Pro Gly 45 Gln Pro Pro Lys Leu 50 Leu Ile His Ala Ala 55 Ser Asn Xaa Gly Ser 60 Gly Val Pro Ser Arg 65 Phe Ser Gly Ser Gly 70 Ser Gly Thr Asp Phe 75 Thr Leu Thr Ile Ser 80

- 105 043373

Ser Leu Glu Pro

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Lys 85 9095

Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105110 <210>59 <211>119 <212> PROTEIN <213> Artificial sequence <220>

<223>VH domain of humanized anti-human PD-1 antibody (PD-1 mAb 7) <220>

< 221> MISC_FEATURE < 222> (12)..(12) < 223> Xaa is valine (V) or alanine (A) < 220>

< 221> MISC_FEATURE < 222> (35)..(35) < 223> Xaa is serine (S) or glycine (G) < 220>

< 221> MISC_FEATURE < 222> (48).. (48) < 223> Xaa is valine (V) or threonine (T) < 220>

< 221> MISC_FEATURE < 222> (86).. (86) < 223> Xaa is leucine (L) or alanine (A) < 400>59

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

5 1015

Ser Leu Lys

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 2530

Leu Val Xaa Trp Val Arg Gln Ala Pro Gly Lys

3540

Ala Thr Ile Ser Gly Gly Gly Gly Asn Thr Tyr

5055

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 65 7075

Gly Leu Glu Trp Xaa 45

Tyr Ser Asp Ser Val 60

Lys Asn Ser Leu Tyr 80

- 106 043373

Leu Gln Met Asn Ser 85 Xaa Arg Ala Glu Asp 90 Thr Ala Thr Tyr Tyr 95 Cys Ala Arg Tyr Gly 100 Phe Asp Gly Ala Trp 105 Phe Ala Tyr Trp Gly 110 Gln Gly Thr Leu Val 115 Thr Val Ser Ser

<210> 60 <211> 107 <212> PROTEIN <213> Artificial sequence <220>

<223> VL domain of humanized anti-human PD-1 antibody (PD-1 mAb 7) <220>

<221> MISC_FEATURE <222> (31) ..(31)

<223> Xaa is is serine (S) or asparagine (N) <220> <221> <222> <223> MISC (50) Xaa _FEATURE ..(50) represents is asparagine (N) or aspartate (D) <400> 60 Asp Ile 1 Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Gly Asp Arg Val Thr 20 Ile Thr Cys Arg Ala 25 Ser Glu Asn Ile Tyr 30 Xaa Tyr Leu Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Lys Ala Pro Lys 45 Leu Leu Ile Tyr Xaa 50 Ala Lys Thr Leu Ala 55 Ala Gly Val Pro Ser 60 Arg Phe Ser Gly Ser Gly 65 Ser Gly Thr Asp 70 Phe Thr Leu Thr Ile 75 Ser Ser Leu Gln Pro 80 Glu Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 His Tyr Ala Val Pro 95 Trp Thr Phe Gly Gln 100 Gly Thr Lys Leu Glu 105 Ile Lys

- 107 043373 <210> 61 <211> 117 <212> PROTEIN <213> Artificial sequence <220>

<223>VH domain of humanized anti-human PD-1 antibody (PD-1 mAb 8) <400>61

Glu Val Gln Leu Val Glu Ser Gly Gly 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser 2025

Leu Ile Ser Trp Val Arg Gln Ala Pro

3540

Gly Leu Val Arg Pro Gly Gly

15

Gly Phe Thr Phe Ser Ser Tyr 30

Gly Lys Gly Leu Glu Trp Val 45

Ala Ala Ile Ser 50

Lys Gly Arg Phe 65

Leu Gln Met Asn

Ala Arg Arg Gly

100

Gly Gly Gly Ala Asp Thr Tyr Tyr Ala Asp

5560

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

7075

Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr 8590

Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

105110

Ser Val

Leu Tyr 80

Tyr Cys 95

Thr Leu

Val Thr Val Ser Ser

115 < 210 > 62 < 211 > 107 < 212 > PROTEIN < 213 > Artificial sequence < 220 >

< 223 > VL domain of humanized anti-human PD-1 antibody (PD-1 mAb 8) < 400 > 62

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

- 108 043373

Asp Arg Val

Thr Ile Thr Cys Arg Ala Ser 20 25

Glu Asn Ile Tyr Asn Tyr 30

Leu Ala Trp Tyr 35

Gln Gln Lys Pro 40

Tyr Asp Ala Lys 50

Thr Leu Ala Ala

Ser Gly Ser Gly 65

Thr Asp Phe Thr 70

Glu Asp Phe Ala

Thr Tyr Tyr Cys 85

Gly Lys Ala Pro Lys Leu Leu Ile 45

Gly Val Pro Ser Arg Phe Ser Gly 60

Leu Thr Ile Ser Ser Leu Gln Pro 75 80

Gln His His Tyr Ala Val Pro Trp 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100105 <210>63 <211>496 <212> PROTEIN <213> Artificial sequence <220>

<223> First or third polypeptide chain PD-1 x LAG-3 DART A <400>63

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 1015

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Ser Val 20 2530

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 4045

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 7580

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 9095

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Ser Gly

100 105110

- 109 043373

Gly Gly Gly

115

Gln Val Gln

Leu Val Gln

120

Ser Gly Ala

Glu Val Lys 125

Pro Gly Ala

130

Ser Val Lys

Val Ser Cys 135

Lys Ala Ser

140

Gly Tyr Ser

Thr Ser Tyr 145

Trp Met Asn

150

Trp Val Arg

Gln Ala Pro

155

Gly Gln Gly

Glu Trp Ile

Gly Val Ile 165

His Pro Ser

Asp Ser Glu 170

Thr Trp Leu

175

Gln Lys Phe

Lys Asp Arg 180

Val Thr Ile

185

Thr Val Asp

Lys Ser Thr 190

Thr Ala Tyr

195

Met Glu Leu

Ser Ser Leu

200

Arg Ser Glu

Asp Thr Ala 205

Tyr Tyr Cys

210

Ala Arg Glu

His Tyr Gly 215

Thr Ser Pro

220

Phe Ala Tyr

Gly Gln Gly 225

Thr Leu Val

230

Thr Val Ser

Ser Gly Gly

235

Cys Gly Gly

Glu Val Ala

Ala Cys Glu 245

Lys Glu Val

Ala Ala Leu 250

Glu Lys Glu

255

Ala Ala Leu

Glu Lys Glu 260

Val Ala Ala

265

Leu Glu Lys

Glu Ser Lys

270

Gly Pro Pro

275

Cys Pro Pro

Cys Pro Ala

280

Pro Glu Phe

Leu Gly Gly 285

Ser Val Phe

290

Leu Phe Pro

Pro Lys Pro 295

Lys Asp Thr

300

Leu Tyr Ile

Arg Glu Pro 305

Glu Val Thr

310

Cys Val Val

Val Asp Val

315

Ser Gln Glu

Pro Glu Val

Gln Phe Asn

325

Trp Tyr Val

Asp Gly Val 330

Glu Val His

335

Ala Lys Thr

Lys Pro Arg 340

Glu Glu Gln

345

Phe Asn Ser

Thr Tyr Arg 350

Lys

Phe

Leu 160

Asp

Ser

Val

Trp

Gly 240

Val

Tyr

Pro

Thr

Asp 320

Asn

Val

- 110 043373

Val Ser Val Leu Thr Val Leu His

355 360

Gln Asp Trp Leu Asn Gly Lys Glu 365

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

370 375380

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

385 390 395400

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

405 410415

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

420 425430

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

435 440445

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

450 455460

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

465 470 475480

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

485 490495 <210>64 <211>271 <212> PROTEIN <213> Artificial sequence <220>

<223> Second or fourth polypeptide chain PD-1 x LAG-3 DART A <400> 64

Glu Ile Val Leu Thr Gln

5

Ser Pro Ala Thr

Glu Arg Ala Thr Leu Ser 20

Gly Met Ser Phe Met Asn 35

Cys Arg Ala Ser 25

Trp Phe Gln Gln 40

Leu Ser Leu Ser Pro Gly 15

Glu Ser Val Asp Asn Tyr 30

Lys Pro Gly Gln Pro Pro 45

- 111 043373

Lys Leu Leu Ile His Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 5560

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 7580

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

9095

Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

100 105110

Gly Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala

115 120125

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

130 135140

Gly Tyr Thr Phe Thr Asp Tyr Asn Met Asp Trp Val Arg Gln Ala Pro

145 150 155160

Gly Gln Gly Leu Glu Trp Met Gly Asp Ile Asn Pro Asp Asn Gly Val

165 170175

Thr Ile Tyr Asn Gln Lys Phe Glu Gly Arg Val Thr Met Thr Thr Asp

180 185190

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

195 200205

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Ala Asp Tyr Phe Tyr Phe

210 215220

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Cys

225 230 235240

Gly Gly Gly Lys Val Ala Ala Cys Lys Glu Lys Val Ala Ala Leu Lys

245 250255

Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu

260 265270 <210>65 <211>118 <212> PROTEIN <213> Mus musculus

- 112 043373

Gly Leu Val Gln Pro Gly Arg

15 <220>

< 221> MISC_FEATURE < 222> (1).. (118) < 223> VH domain mAb 4-4-20 < 400> 65

Glu Val Lys Leu Asp Glu Thr Gly 1 5

Pro Met Lys Leu Ser Cys Val Ala 20

Gly Phe Thr Phe Ser Asp Tyr 30

Trp Met Asn Trp Val Arg Gln Ser

40

Glu Lys Gly Leu Glu Trp Val 45

Ala Gln Ile Arg Asn Lys Pro Tyr

55

Tyr Glu Thr Tyr Tyr Ser Asp 60

Ser Val Lys Gly Arg Phe Thr Ile 65 70

Arg Asp Asp Ser Lys Ser Ser

80

Val Tyr Leu Gln Met Asn Asn Leu 85

Val Glu Asp Met Gly Ile Tyr

95

Tyr Cys Thr Gly Ser Tyr Tyr Gly 100

Asp Tyr Trp Gly Gln Gly Thr

110

Ser Val Thr Val Ser Ser

115 <210> 66 <211> 112 <212> PROTEIN <213> Mus musculus <220>

< 221> MISC_FEATURE < 222> (1)..(112) <223> VL domain mAb 4-4-20 <400> 66

Asp Val Val Met Thr Gln Thr Pro 1 5

Ser Leu Pro Val Ser Leu Gly

15

Asp Gln Ala Ser Ile Ser Cys Arg 20

Ser Gln Ser Leu Val His Ser

- 113 043373

Asn Gly Asn Thr Tyr Leu Arg Trp

40

Tyr Leu

Gln Lys

Pro Gly Gln Ser 45

Pro Lys Val Leu Ile Tyr Lys Val

55

Ser Asn Arg Phe Ser Gly Val Pro 60

Asp Arg Phe Ser Gly Ser Gly Ser 65 70

Gly Thr Asp Phe Thr Leu Lys Ile

80

Ser Arg Val Glu Ala Glu Asp Leu 85

Gly Val Tyr Phe Cys Ser Gln Ser

95

Thr His Val Pro Trp Thr Phe Gly

100

Gly Gly Thr Lys Leu Glu Ile Lys

105 110 <210> 67 <211> 273 <212> PROTEIN <213> Artificial sequence <220>

< 223> The first polypeptide chain of a universal bispecific adapter molecule

UBA-1 <400> 67

Asp 1 Val Val Met Thr 5 Gln Thr Pro Phe Ser 10 Leu Pro Val Ser Leu 15 Gly Asp Gln Ala Ser 20 Ile Ser Cys Arg Ser 25 Ser Gln Ser Leu Val 30 His Ser Asn Gly Asn 35 Thr Tyr Leu Arg Trp 40 Tyr Leu Gln Lys Pro 45 Gly Gln Ser Pro Lys 50 Val Leu Ile Tyr Lys 55 Val Ser Asn Arg Phe 60 Ser Gly Val Pro Asp 65 Arg Phe Ser Gly Ser 70 Gly Ser Gly Thr Asp 75 Phe Thr Leu Lys Ile 80 Ser Arg Val Glu Ala 85 Glu Asp Leu Gly Val 90 Tyr Phe Cys Ser Gln 95 Ser Thr His Val Pro 100 Trp Thr Phe Gly Gly 105 Gly Thr Lys Leu Glu 110 Ile Lys

- 114 043373

Gly Gly Gly Ser Gly Gly Gly Gly

115 120

Gln Val Gln Leu Val Gln Ser Gly

125

Ala Glu Val Lys Lys Pro Gly Ala

130135

Ser Gly Tyr Ser Phe Thr Ser Tyr

145150

Ser Val Lys Val Ser Cys Lys Ala 140

Trp Met Asn Trp Val Arg Gln Ala 155160

Pro Gly Gln Gly Leu

165

Glu Trp

Ile Gly Val Ile His Pro Ser Asp Ser

170 175

Glu Thr Trp Leu Asp

180

Gln Lys

Phe Lys Asp Arg Val Thr Ile Thr Val

185 190

Asp Lys Ser Thr Ser 195

Thr Ala

Tyr Met Glu Leu Ser Ser Leu Arg Ser

200 205

Glu Asp Thr Ala Val 210

Tyr Tyr

215

Cys Ala Arg Glu His Tyr Gly Thr Ser

220

Pro Phe Ala Tyr Trp 225

Gly Gln 230

Gly Thr Leu Val Thr Val Ser Ser Gly

235 240

Gly Cys

Leu Glu

Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala

245 250 255

Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu

260 265 270

Lys <210> 68 <211> 272 <212> PROTEIN <213> Artificial sequence <220>

<223> Second polypeptide chain of a universal bispecific adapter molecule

UBA-1 <400> 68

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

5 10 15

- 115 043373

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 2530

Gly Met Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 4045

Lys Leu Leu Ile His Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 5560

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 7580

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Lys 85 9095

Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

100 105110

Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Lys Leu Asp Glu Thr Gly

115 120125

Gly Gly Leu Val Gln Pro Gly Arg Pro Met Lys Leu Ser Cys Val Ala

130 135140

Ser Gly Phe Thr Phe Ser Asp Tyr Trp Met Asn Trp Val Arg Gln Ser

145 150 155160

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

165 170175

Asn Tyr Glu Thr Tyr Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile

180 185190

Ser Arg Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu

195 200205

Arg Val Glu Asp Met Gly Ile Tyr Tyr Cys Thr Gly Ser Tyr Tyr Gly

210 215220

Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly

225 230 235240

Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu

245 250255

- 116 043373

Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu

260 265270 < 210>69 < 211>503 < 212> PROTEIN < 213> Artificial sequence <220>

<223> First and third polypeptide chain of a universal bispecific adapter molecule

UBA-2 <400> 69

Asp 1 Val Val Met Thr 5 Gln Thr Pro Phe Ser 10 Leu Pro Val Ser Leu 15 Gly Asp Gln Ala Ser 20 Ile Ser Cys Arg Ser 25 Ser Gln Ser Leu Val 30 His Ser Asn Gly Asn 35 Thr Tyr Leu Arg Trp 40 Tyr Leu Gln Lys Pro 45 Gly Gln Ser Pro Lys 50 Val Leu Ile Tyr Lys 55 Val Ser Asn Arg Phe 60 Ser Gly Val Pro Asp 65 Arg Phe Ser Gly Ser 70 Gly Ser Gly Thr Asp 75 Phe Thr Leu Lys Ile 80 Ser Arg Val Glu Ala 85 Glu Asp Leu Gly Val 90 Tyr Phe Cys Ser Gln 95 Ser Thr His Val Pro 100 Trp Thr Phe Gly Gly 105 Gly Thr Lys Leu Glu 110 Ile Lys Gly Gly Gly 115 Ser Gly Gly Gly Gly 120 Gln Val Gln Leu Val 125 Gln Ser Gly Ala Glu 130 Val Lys Lys Pro Gly 135 Ala Ser Val Lys Val 140 Ser Cys Lys Ala Ser 145 Gly Tyr Ser Phe Thr 150 Ser Tyr Trp Met Asn 155 Trp Val Arg Gln Ala 160 Pro Gly Gln Gly Leu Glu Trp Ile Gly Val Ile His Pro Ser Asp Ser

165 170 175

- 117 043373

Glu

Asp

Glu

Pro 225

Gly

Leu

Lys

Glu

Asp 305

Asp

Gly

Asn

T rp

Pro 385

Glu

Thr Trp Leu

180

Lys Ser Thr

195

Asp Thr Ala 210

Phe Ala Tyr

Cys Gly Gly

Glu Lys Glu

260

Gly Gly Gly

275

Ala Ala Gly 290

Thr Leu Met

Val Ser His

Val Glu Val

340

Ser Thr Tyr

355

Leu Asn Gly 370

Ala Pro Ile

Pro Gln Val

Asp Gln Lys

Ser Thr Ala

Val Tyr Tyr

215

Trp Gly Gln

230

Gly Glu Val 245

Val Ala Ala

Asp Lys Thr

Gly Pro Ser

295

Ile Ser Arg

310

Glu Asp Pro 325

His Asn Ala

Arg Val Val

Lys Glu Tyr

375

Glu Lys Thr 390

Tyr Thr Leu 405

Phe Lys Asp

185

Tyr Met Glu 200

Cys Ala Arg

Gly Thr Leu

Ala Ala Leu

250

Leu Glu Lys

265

His Thr Cys 280

Val Phe Leu

Thr Pro Glu

Glu Val Lys

330

Lys Thr Lys

345

Ser Val Leu 360

Lys Cys Lys

Ile Ser Lys

Pro Pro Ser

410

Arg Val Thr

Leu Ser Ser

205

Glu His Tyr

220

Val Thr Val 235

Glu Lys Glu

Glu Val Ala

Pro Pro Cys

285

Phe Pro Pro

300

Val Thr Cys 315

Phe Asn Trp

Pro Arg Glu

Thr Val Leu

365

Val Ser Asn

380

Ala Lys Gly 395

Arg Glu Glu

Ile Thr Val 190

Leu Arg Ser

Gly Thr Ser

Ser Ser Gly

240

Val Ala Ala

255

Ala Leu Glu 270

Pro Ala Pro

Lys Pro Lys

Val Val Val

320

Tyr Val Asp

335

Glu Gln Tyr 350

His Gln Asp

Lys Ala Leu

Gln Pro Arg

400

Met Thr Lys

415

- 118 043373

Asn

Gln

Val

Ser

420

Leu

Thr

Cys

Leu

Val

425

Lys

Gly

Phe

Tyr

Pro

430

Ser

Asp

Ile

Ala

Val

435

Glu

Trp

Glu

Ser

Asn

440

Gly

Gln

Pro

Glu

Asn

445

Asn

Tyr

Lys

Thr

Thr

450

Pro

Pro

Val

Leu

Asp 455

Ser

Asp

Gly

Ser

Phe

460

Phe

Leu

Tyr

Ser

Lys

465

Leu

Thr

Val

Asp

Lys 470

Ser

Arg

Trp

Gln

Gln

475

Gly

Asn

Val

Phe

Ser

480

Cys

Ser

Val

Met

His

485

Glu

Ala

Leu

His

Asn

490

His

Tyr

Thr

Gln

Lys

495

Ser

Leu

Ser

Leu

Ser

500

Pro

Gly

Lys <210> 70 <211> 503 <212> PROTEIN <213> Artificial sequence <220>

< 223> The first polypeptide chain of a universal bispecific adapter molecule

UBA-3 <400> 70

Asp Val Val Met 1

Asp Gln Ala Ser 20

Asn Gly Asn Thr 35

Thr Gln Thr Pro Phe 5

Ile Ser Cys Arg Ser

Tyr Leu Arg Trp Tyr

Ser 10 Leu Pro Val Ser Leu 15 Gly Ser Gln Ser Leu Val 30 His Ser Leu Gln Lys Pro 45 Gly Gln Ser

Pro Lys Val 50

Leu Ile

Tyr Lys Val 55

Asp Arg Phe 65

Ser Gly

Ser Gly Ser 70

Ser Arg Val

Glu Ala

Glu Asp Leu

Ser Asn Arg Phe Ser Gly Val Pro 60

Gly Thr Asp Phe Thr Leu Lys Ile 75 80

Gly Val Tyr Phe Cys Ser Gln Ser 90 95

- 119 043373

Thr His Val Pro Trp Thr Phe Gly

100

Gly Gly Gly Ser Gly Gly Gly Gly

115 120

Ala Glu Val Lys Lys Pro Gly Ala

130 135

Ser Gly Tyr Ser Phe Thr Ser Tyr

145 150

Gly 105 Gly Thr Lys Leu Glu 110 Ile Lys Gln Val Gln Leu Val 125 Gln Ser Gly Ser Val Lys Val 140 Ser Cys Lys Ala Trp Met Asn 155 Trp Val Arg Gln Ala 160

Pro Gly

Glu Thr

Asp Lys

Glu Asp

210

Gln Gly Leu

165

Trp Leu Asp

180

Ser Thr Ser 195

Thr Ala Val

Glu Trp Ile Gly Val 170 Ile His Pro Ser Asp 175 Ser Gln Lys Phe Lys 185 Asp Arg Val Thr Ile 190 Thr Val Thr Ala Tyr 200 Met Glu Leu Ser Ser 205 Leu Arg Ser Tyr Tyr 215 Cys Ala Arg Glu His 220 Tyr Gly Thr Ser

Pro Phe Ala 225

Gly Cys Gly

Tyr Trp Gly Gln Gly Thr

230

Leu Val Thr Val Ser Ser Gly

235 240

Leu Glu Lys

Lys Gly Gly

275

Gly Gly Glu Val Ala Ala 245

Glu Val Ala Ala Leu Glu

260 265

Gly Asp Lys Thr His Thr

280

Glu Ala Ala Gly Gly Pro Ser Val Phe

290 295

Asp Thr Leu Met Ile Ser Arg Thr Pro 305 310

Asp Val Ser His Glu Asp Pro Glu Val

325

Leu Glu Lys Glu Val Ala Ala

250 255

Lys Glu Val Ala Ala Leu Glu

270

Cys Pro Pro Cys Pro Ala Pro

285

Leu Phe Pro Pro Lys Pro Lys

300

Glu Val Thr Cys Val Val Val

315 320

Lys Phe Asn Trp Tyr Val Asp

330 335

- 120 043373

Gly

Val

Glu

Val

340

His

Asn

Ala

Lys

Thr

345

Lys

Pro

Arg

Glu

Glu

350

Gln

Tyr

Asn

Ser

Thr

355

Tyr

Arg

Val

Val

Ser

360

Val

Leu

Thr

Val

Leu

365

His

Gln

Asp

Trp

Leu

370

Asn

Gly

Lys

Glu

Tyr 375

Lys

Cys

Lys

Val

Ser

380

Asn

Lys

Ala

Leu

Pro

385

Ala

Pro

Ile

Glu

Lys 390

Thr

Ile

Ser

Lys

Ala

395

Lys

Gly

Gln

Pro

Arg 400

Glu

Pro

Gln

Val

Tyr 405

Thr

Leu

Pro

Pro

Ser

410

Arg

Glu

Glu

Met

Thr

415

Lys

Asn

Gln

Val

Ser

420

Leu

Trp

Cys

Leu

Val

425

Lys

Gly

Phe

Tyr

Pro

430

Ser

Asp

Ile

Ala

Val

435

Glu

Trp

Glu

Ser

Asn

440

Gly

Gln

Pro

Glu

Asn

445

Asn

Tyr

Lys

Thr

Thr

450

Pro

Pro

Val

Leu

Asp 455

Ser

Asp

Gly

Ser

Phe

460

Phe

Leu

Tyr

Ser

Lys

465

Leu

Thr

Val

Asp

Lys 470

Ser

Arg

Trp

Gln

Gln

475

Gly

Asn

Val

Phe

Ser

480

Cys

Ser

Val

Met

His

485

Glu

Ala

Leu

His

Asn

490

His

Tyr

Thr

Gln

Lys

495

Ser

Leu

Ser

Leu

Ser

500

Pro

Gly

Lys <210> 71 <211> 227 <212> PROTEIN <213> Artificial sequence <220>

<223> Third polypeptide chain of a universal bispecific adapter molecule

UBA-3 <400> 71

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

5 10 15

- 121 043373

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

2025

Pro Lys Asp Thr Leu Met 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 4045

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

5560

His Asn Ala 65

Arg Val Val

Lys Glu Tyr

Lys Thr

Ser Val

Lys Pro Arg Glu 70

Leu Thr Val Leu

Glu Gln Tyr Asn Ser Thr Tyr

80

His Gln Asp Trp Leu Asn Gly

95

Glu Lys

Tyr Thr

130

Leu Ser 145

Lys Cys 100

Thr Ile Ser 115

Leu Pro Pro

Cys Ala Val

Lys Val Ser Asn

105

Lys Ala Lys

120

Ser Arg Glu

135

Lys Gly Phe 150

Lys Ala Leu Pro Ala Pro Ile

110

Gly Gln Pro Arg Glu Pro Gln Val 125

Glu Met Thr Lys Asn Gln Val Ser 140

Tyr Pro Ser Asp Ile Ala Val Glu 155 160

Trp Glu Ser Asn Gly 165 Gln Pro Glu Asn Asn 170 Tyr Val Leu Asp Ser 180 Asp Gly Ser Phe Phe 185 Leu Val Asp Lys Ser 195 Arg Trp Gln Gln Gly 200 Asn Val Phe His Glu 210 Ala Leu His Asn Arg 215 Tyr Thr Gln Lys

Lys Thr Thr Pro Pro

175

Ser Lys Leu Thr Val

190

Ser Cys Ser Val Met

205

Ser Leu Ser Leu Ser 220

Pro Gly Lys 225 <210> 72 <211> 449

- 122 043373 <212> PROTEIN <213> Artificial sequence <220>

<223> Third polypeptide chain of a universal bispecific adapter molecule

UBA-4 <400>72

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

5 1015

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 2530

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 4045

Gly Val Ile His Pro Ser Asp Ser Glu Thr Trp Leu Asp Gln Lys Phe 50 5560

Lys Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 7580

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

9095

Ala Arg Glu His Tyr Gly Thr Ser Pro Phe Ala Tyr Trp Gly Gln Gly

100 105110

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

115 120125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170175

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

180 185190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200205

- 123 043373

Ser Asn Thr Lys Val Asp Lys Arg Val

210 215

Thr His Thr Cys Pro Pro Cys Pro Ala

225 230

Ser Val Phe Leu Phe Pro Pro Lys Pro

245

Arg Thr Pro Glu Val Thr Cys Val Val

260 265

Pro Glu Val Lys Phe Asn Trp Tyr Val

275 280

Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295

Val Ser Val Leu Thr Val Leu His Gln

305 310

Tyr Lys Cys Lys Val Ser Asn Lys Ala

325

Thr Ile Ser Lys Ala Lys Gly Gln Pro

340 345

Leu Pro Pro Ser Arg Glu Glu Met Thr

355 360

Cys Ala Val Lys Gly Phe Tyr Pro Ser

370 375

Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390

Asp Ser Asp Gly Ser Phe Phe Leu Val 405

Ser Arg Trp Gln Gln Gly Asn Val Phe

420 425

Ala Leu His Asn Arg Tyr Thr Gln Lys

435 440

Pro Lys Ser Cys Asp Lys

220

Glu Ala Ala Gly Gly Pro

235 240

Asp Thr Leu Met Ile Ser

255

Asp Val Ser His Glu Asp

270

Gly Val Glu Val His Asn

285

Asn Ser Thr Tyr Arg Val 300

Trp Leu Asn Gly Lys Glu

315 320

Pro Ala Pro Ile Glu Lys

335

Glu Pro Gln Val Tyr Thr

350

Asn Gln Val Ser Leu Ser

365

Ile Ala Val Glu Trp Glu

380

Thr Thr Pro Pro Val Leu

395 400

Lys Leu Thr Val Asp Lys

415

Cys Ser Val Met His Glu

430

Leu Ser Leu Ser Pro Gly

445

- 124 043373

Lys < 210> 73 < 211> 218 < 212> PROTEIN < 213> Artificial sequence < 220>

< 223> Fourth polypeptide chain of a universal bispecific adapter molecule

UBA-4 <400> 73

Glu Ile Val 1

Leu Thr

Gln Ser Pro Ala

Thr Leu Ser Leu Ser Pro Gly

15

Glu Arg Ala

Thr Leu 20

Ser Cys Arg Ala 25

Ser Glu Ser Val Asp Asn Tyr 30

Gly Met Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 4045

Lys Leu Leu Ile His Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 5560

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 7580

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

9095

Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105110

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

115 120125

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

130 135140

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

145 150 155160

Gly Asn Ser Gln Glu Ser Val Thr

165

Glu Gln Asp Ser Lys Asp Ser Thr

170 175

- 125 043373

Leu Ser Lys Ala Asp Tyr Glu Lys

185 190

Tyr Ser Leu Ser 180 Ser Thr Leu Thr His Lys Val Tyr Ala Cys Glu Val 195 200 Val Thr Lys Ser Phe Asn Arg Gly 210 215

Glu Cys

Thr His Gln Gly Leu Ser Ser Pro

205 <210> 74 <211> 575 <212> PROTEIN <213> Artificial sequence <220>

<223> Third polypeptide chain of a universal bispecific adapter molecule

UBA-5 <400> 74

Glu Ile Val 1

Leu Thr

Glu Arg Ala

Thr Leu 20

Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 10 15

Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 25 30

Gly Met

Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45

Lys Leu Leu 50

Arg Phe Ser 65

Ser Leu Glu

Ile His Ala Ala Ser Asn Gln 55

Gly Ser Gly Ser Gly Thr Asp 70

Pro Glu Asp Phe Ala Val Tyr 85 90

Gly Ser Gly Val Pro Ser 60

Phe Thr Leu Thr Ile Ser

80

Phe Cys Gln Gln Ser Lys

Glu Val Pro

Tyr Thr Phe Gly Gly Gly Thr

100 105

Gly Gly Gly

115

Ser Gly Gly Gly Gly Ser Gly

120

Gln Leu Val

130

Gln Ser Gly Ala Glu Val Lys 135

Lys Val Glu Ile Lys Gly 110

Gly Gly Gly Ser Gln Val 125

Lys Pro Gly Ala Ser Val 140

- 126 043373

Lys Val Ser Cys Lys Ala Ser Gly

145 150

Tyr Ser Phe Thr Ser Tyr Trp Met

155 160

Asn Trp Val Arg Gln Ala Pro Gly

165

Gln Gly Leu Glu Trp Ile Gly Val

170 175

Ile His Pro Ser Asp Ser Glu Thr

180

Trp Leu Asp Gln Lys Phe Lys Asp

185 190

Arg Val Thr Ile Thr Val Asp Lys

195 200

Ser Thr Ser Thr Ala Tyr Met Glu

205

Leu Ser Ser Leu Arg Ser

210

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

215 220

Glu His Tyr Gly Thr Ser

225 230

Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu

235 240

Val Thr Val Ser Ser Ala

245

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

250 255

Ala Pro Ser Ser Lys Ser 260

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

265 270

Leu Val Lys Asp Tyr Phe

275

Pro Glu Pro Val Thr Val Ser Trp Asn Ser

280 285

Gly Ala Leu Thr 290

Ser Gly Val

295

His Thr Phe Pro Ala Val Leu Gln Ser

300

Ser Gly Leu Tyr 305

Ser Leu Ser

310

Ser Val Val Thr Val Pro Ser Ser Ser

315 320

Leu Gly Thr Gln

Thr Tyr Ile 325

Cys Asn Val Asn His Lys Pro Ser Asn

330 335

Thr Lys Val Asp

340

Lys Arg Val

Glu Pro Lys Ser Cys Asp Lys Thr His

345 350

Thr Cys Pro

355

Pro Cys

Pro Ala

Pro Glu Ala Ala Gly Gly Pro Ser Val 360 365

Phe Leu Phe

370

Pro Pro

Lys Pro

375

Lys Asp Thr Leu Met Ile Ser Arg Thr

380

- 127 043373

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

385 390 395400

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

405 410415

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

420 425430

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

435 440445

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

450 455460

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

465 470 475480

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala

485 490495

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

500 505510

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

515 520525

Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg

530 535540

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

545 550 555560

His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570575 <210>75 <211>223 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1).. (223) < 223> Human CTLA-4, including signal sequence (sequence

- 128 043373 activity in NCBI NP_005205.2) <220>

< 221> SIGNAL < 222> (1).. (34) < 223> Signal sequence < 400> 75

Met Ala Cys Leu Gly Phe Gln Arg His 1 5

Thr Arg Thr Trp Pro Cys Thr Leu Leu 20 25

Val Phe Cys Lys Ala Met His Val Ala

40

Ser Ser Arg Gly Ile Ala Ser Phe Val

55

Lys Ala Thr Glu Val Arg Val Thr Val 65 70

Val Thr Glu Val Cys Ala Ala Thr Tyr 85

Phe Leu Asp Asp Ser Ile Cys Thr Gly

100 105

Asn Leu Thr Ile Gln Gly Leu Arg Ala

115 120

Cys Lys Val Glu Leu Met Tyr Pro Pro

130 135

Asn Gly Thr Gln Ile Tyr Val Ile Asp 145 150

Asp Phe Leu Leu Trp Ile Leu Ala Ala

165

Tyr Ser Phe Leu Leu Thr Ala Val Ser

180 185

Arg Ser Pro Leu Thr Thr Gly Val Tyr

195 200

Ala Gln Leu Asn Leu Ala

Phe Leu Leu Phe Ile Pro

Pro Ala Val Val Leu Ala

Glu Tyr Ala Ser Pro Gly 60

Arg Gln Ala Asp Ser Gln 75 80

Met Gly Asn Glu Leu Thr

Ser Ser Gly Asn Gln Val

110

Asp Thr Gly Leu Tyr Ile

125

Tyr Tyr Leu Gly Ile Gly 140

Glu Pro Cys Pro Asp Ser

155 160

Ser Ser Gly Leu Phe Phe

175

Ser Lys Met Leu Lys Lys

190

Lys Met Pro Pro Thr Glu

205

- 129 043373

Pro Glu Cys Glu Lys Gln Phe Gln

210 215

Pro Tyr Phe Ile Pro Ile Asn

220 <210> 76 <211> 118 <212> PROTEIN <213> Homo sapiens <220>

<221> MISC_FEATURE <222> (1).. (118) <223> VH domain of anti-human CTLA-4 antibody CTLA-4 mAb 1 mab) (ipilimu<400> 76

Gln 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Val Val Gln Pro Gly 15 Arg Ser Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Phe Ser 30 Ser Tyr Thr Met His 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Val Thr Phe 50 Ile Ser Tyr Asp Gly 55 Asn Asn Lys Tyr Tyr 60 Ala Asp Ser Val Lys 65 Gly Arg Phe Thr Ile 70 Ser Arg Asp Asn Ser 75 Lys Asn Thr Leu Tyr 80 Leu Gln Met Asn Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala Ile Tyr Tyr 95 Cys Ala Arg Thr Gly 100 Trp Leu Gly Pro Phe 105 Asp Tyr Trp Gly Gln 110 Gly Thr

Leu Val Thr Val Ser Ser

115 <210> 77 <211> 108 <212> PROTEIN <213> Homo sapiens <220>

<221> MISC_FEATURE

- 130 043373 antibodies against

CTLA-4 people

CTLA-4 mAb 1 (ipilimu<222>

<223> mab) (1) . . (108)

VL domain <400>

Glu 1

Ile

Val

Leu

Thr

Gln

Ser

Pro

Gly

Thr 10

Leu

Ser

Leu Ser Pro Gly 15

Glu

Arg

Ala

Thr 20

Leu

Ser

Cys

Arg

Ala 25

Ser

Gln

Ser

Val Gly Ser Ser 30

Tyr

Leu

Ala 35

Trp

Tyr

Gln

Gln

Lys 40

Pro

Gly

Gln

Ala

Pro Arg Leu Leu 45

Ile

Tyr 50

Gly

Ala

Phe

Ser

Arg 55

Ala

Thr

Gly

Ile

Pro 60

Asp Arg Phe Ser

Gly 65

Ser

Gly

Ser

Gly

Thr 70

Asp

Phe

Thr

Leu

Thr 75

Ile

Ser Arg Leu Glu 80

Pro

Glu

Asp

Phe

Ala 85

Val

Tyr

Tyr

Cys

Gln 90

Gln

Tyr

Gly Ser Ser Pro 95

Trp

Thr

Phe

Gly 100

Gln

Gly

Thr

Lys

Val

105

Glu

Ile

Lys <210> 78 <211> 125 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1)..(125) < 223> VH domain of anti-human CTLA-4 antibody

CTLA-4 mAb 2 (tremelimumab) <400> 78

Gln Val Gln Leu Val Glu 1 5

Ser Leu Arg Leu Ser Cys 20

Gly Met His Trp Val Arg 35

Ser Gly Gly Gly Val Val 10

Ala Ala Ser Gly Phe Thr 25

Gln Ala Pro Gly Lys Gly 40

Gln Pro Gly Arg

Phe Ser Ser Tyr 30

Leu Glu Trp Val 45

- 131 043373

Ala Val Ile Trp 50

Tyr Asp Gly Ser Asn Lys Tyr Tyr

60

Ala Asp Ser Val

Lys Gly Arg Phe 65

Thr Ile Ser Arg Asp Asn Ser Lys

75

Asn Thr Leu Tyr 80

Leu Gln Met Asn

Ser Leu Arg Ala Glu Asp Thr Ala 85 90

Val Tyr Tyr Cys

Ala Arg Asp Pro

100

Arg Gly Ala Thr Leu Tyr Tyr Tyr

105

Tyr Tyr Gly Met

110

Asp Val Trp Gly

115

Gln Gly Thr Thr Val Thr Val Ser

120

Ser

125 <210> 79 <211> 107 <212> PROTEIN <213> Homo sapiens <220>

< 221> MISC_FEATURE < 222> (1).. (107) < 223> VL domain of anti-human CTLA-4 antibody (tremelimumab) <400> 79

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

5 10

CTLA-4 mAb 2

Ala Ser Val Gly

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 20 25

Ile Asn Ser Tyr 30

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

40

Lys Leu Leu Ile 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

55 60

Arg Phe Ser Gly

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75

Ser Leu Gln Pro

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

90

Ser Thr Pro Phe

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

- 132 043373 <210> 80 <211> 217 <212> PROTEIN <213> Artificial sequence <220>

<223> CH2-CH3 domains of the IgG1 Fc fragment containing substitutions M252Y, S254T and T256E <220>

<221> MISC_FEATURE <222> (217) ..(217) <223> Xaa is lysine (K) or missing <400> 80

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15

Pro Lys Asp Thr Leu Tyr 20

Ile Thr Arg Glu Pro Glu Val 25

Thr Cys Val 30

Val Val Asp Val

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 4045

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 5560

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 7580

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 9095

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

100 105110

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

115 120125

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

130 135140

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

145 150 155160

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

- 133 043373

175

Trp Gln Gln Gly Asn Val

190

165

170

Tyr Ser Lys

Leu Thr Val Asp Lys Ser Arg

180 185

Phe Ser Cys

195

Ser Val Met His Glu Ala Leu

200

His Asn His Tyr Thr Gln

205

Lys Ser Leu 210

Ser Leu Ser Pro Gly Xaa

215 <210> 81 <211> 217 <212> PROTEIN <213> Artificial sequence <220>

<223> CH2-CH3 domains of the IgG4 Fc fragment containing substitutions M252Y, S254T and T256E <220>

<221> MISC_FEATURE <222> (217) ..(217) <223> Xaa is lysine (K) or missing <400> 81

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val 20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95

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

100 105 110

- 134 043373

Pro Arg Glu 115 Pro Gln Val Tyr Thr 120 Leu Pro Pro Ser Gln 125 Glu Glu Met Thr Lys 130 Asn Gln Val Ser Leu 135 Thr Cys Leu Val Lys 140 Gly Phe Tyr Pro Ser 145 Asp Ile Ala Val Glu 150 Trp Glu Ser Asn Gly 155 Gln Pro Glu Asn Asn 160 Tyr Lys Thr Thr Pro 165 Pro Val Leu Asp Ser 170 Asp Gly Ser Phe Phe 175 Leu Tyr Ser Arg Leu 180 Thr Val Asp Lys Ser 185 Arg Trp Gln Glu Gly 190 Asn Val Phe Ser Cys 195 Ser Val Met His Glu 200 Ala Leu His Asn His 205 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Xaa

210 215 <210> 82 <211> 217 <212> PROTEIN <213> Artificial sequence <220>

<223> Overhang variant of the CH2-CH3 domains of the IgG1 Fc fragment containing substitutions M252Y, S254T and T256E <220>

<221> MISC_FEATURE <222> (217) ..(217) <223> Xaa is lysine (K) or missing <400> 82

Ala 1 Pro Glu Ala Ala 5 Gly Gly Pro Ser Val 10 Phe Leu Phe Pro Pro 15 Lys Pro Lys Asp Thr 20 Leu Tyr Ile Thr Arg 25 Glu Pro Glu Val Thr 30 Cys Val Val Val Asp 35 Val Ser His Glu Asp 40 Pro Glu Val Lys Phe 45 Asn Trp Tyr Val Asp 50 Gly Val Glu Val His 55 Asn Ala Lys Thr Lys 60 Pro Arg Glu Glu

- 135 043373

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 65 70 75

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 85 90

Leu Thr Val Leu His 80

Lys Val Ser Asn Lys 95

Ala Leu Pro Ala Pro Ile 100

Pro Arg Glu Pro Gln Val 115

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

105 110

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

120 125

Thr Lys Asn Gln Val Ser Leu

130 135

Trp Cys Leu Val Lys Gly Phe Tyr Pro

140

Ser Asp Ile Ala Val Glu Trp

145 150

Glu Ser Asn Gly Gln Pro Glu Asn Asn

155 160

Tyr Lys Thr Thr Pro Pro Val 165

Leu Asp Ser Asp Gly Ser Phe Phe Leu

170 175

Tyr Ser Lys Leu Thr Val Asp

180

Lys Ser Arg Trp Gln Gln Gly Asn Val

185 190

Phe Ser Cys Ser Val Met His 195

Glu Ala Leu His Asn His Tyr Thr Gln

200 205

Lys

Ser Leu Ser Leu Ser Pro Gly Xaa

210 215 <210> 83 <211> 217 <212> PROTEIN <213> Artificial sequence <220>

<223> A dimpled variant of the CH2-CH3 domains of the IgG1 Fc fragment containing substitutions M252Y, S254T and T256E <220>

< 221> MISC_FEATURE < 222> (217) ..(217) < 223> Xaa is lysine (K) or missing <400> 83

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15

- 136 043373

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val 20 2530

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 4045

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 5560

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 7580

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 9095

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

100 105110

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

115 120125

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

130 135140

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

145 150 155160

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

165 170175

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

180 185190

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

195 200205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210215 <210>84 <211>217 <212> PROTEIN <213> Artificial sequence <220>

- 137 043373 <223> Overhang variant of the CH2-CH3 domains of the IgG4 Fc fragment containing substitutions M252Y, S254T and T256E <220>

< 221> MISC_FEATURE < 222> (217)..(217) < 223> Xaa is lysine (K) or missing < 400> 84

Ala Pro Glu Phe Leu Gly Gly Pro 1 5

Ser Val Phe Leu Phe Pro Pro Lys

15

Pro Lys Asp Thr Leu Tyr Ile Thr 20

Arg Glu Pro Glu Val Thr Cys Val 25 30

Val Val Asp Val Ser Gln Glu Asp

40

Pro Glu Val Gln Phe Asn Trp Tyr 45

Val Asp Gly Val Glu Val His Asn

55

Ala Lys Thr Lys Pro Arg Glu Glu 60

Gln Phe Asn Ser Thr Tyr Arg Val 65 70

Val Ser Val Leu Thr Val Leu His

80

Gln Asp Trp Leu Asn Gly Lys Glu 85

Tyr Lys Cys Lys Val Ser Asn Lys

95

Gly Leu Pro Ser Ser Ile Glu Lys 100

Thr Ile Ser Lys Ala Lys Gly Gln

105 110

Pro Arg Glu Pro Gln Val Tyr Thr

115 120

Leu Pro Pro Ser Gln Glu Glu Met

125

Thr Lys Asn Gln Val Ser Leu Trp

130 135

Cys Leu Val Lys Gly Phe Tyr Pro

140

Ser Asp Ile Ala Val Glu Trp Glu

145 150

Ser Asn Gly Gln Pro Glu Asn Asn

155 160

Tyr Lys Thr Thr Pro Pro Val Leu

165

Asp Ser Asp Gly Ser Phe Phe Leu

170 175

Tyr Ser Arg Leu Thr Val Asp Lys

180

Ser Arg Trp Gln Glu Gly Asn Val

185 190

Phe Ser Cys Ser Val Met His Glu

Ala Leu His Asn His Tyr Thr Gln

- 138 043373

195

200

205

Lys Ser Leu Ser Leu Ser Leu Gly Xaa

210

215 < 210> 85 < 211> 217 < 212> PROTEIN < 213> Artificial sequence < 220>

<223> A dimpled variant of the CH2-CH3 domains of the IgG4 Fc fragment containing substitutions M252Y, S254T and T256E <220>

< 221> MISC_FEATURE < 222> (217)..(217) < 223> Xaa is lysine (K) or missing <400>85

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 1015

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val 20 2530

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 4045

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 5560

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 7580

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 9095

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

100 105110

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

115 120125

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

130 135140

- 139 043373

Ser Asp Ile Ala Val 145

Glu Trp Glu Ser Asn 150

Gly Gln Pro Glu Asn Asn

155 160

Tyr Lys Thr Thr Pro

165

Pro Val Leu Asp Ser

170

Asp Gly Ser Phe Phe Leu

175

Val Ser Arg Leu Thr

180

Val Asp Lys Ser Arg

185

Trp Gln Glu Gly Asn Val

190

Phe Ser Cys Ser Val 195

Met His Glu Ala Leu

200

His Asn Arg Tyr Thr Gln

205

Lys Ser Leu Ser Leu 210

Ser Leu Gly Xaa

215 <210> 86 <211> 119 <212> PROTEIN <213> Artificial sequence <220>

< 223 > VH domain of humanized anti-human PD-1 antibody (PD-1 mAb 6 I VH) < 400 > 86

Gln 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 Tyr Ser Phe Thr Ser Tyr 20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro

40

Gly Gln Gly Leu Glu Trp Ile 45

Gly Val Ile His Pro 50

Ser Asp Ser Glu Thr Trp Leu Asp

60

Gln Lys Phe

Lys Asp Arg Val Thr 65

Ile Thr Val Asp Lys Ser Thr Ser 70 75

Thr Ala Tyr

Met Glu Leu Ser Ser

Leu Arg Ser Glu Asp Thr Ala Val 90

Tyr Tyr Cys 95

Ala Arg Glu His Tyr

100

Gly Thr Ser Pro Phe Ala Tyr Trp

105

Gly Gln Gly 110

Thr Leu Val Thr Val Ser Ser

- 140 043373

115 <210> 87 <211> 111 <212> PROTEIN <213> Artificial sequence <220>

<223> VL domain of humanized anti-human PD-1 antibody (PD mAb 6 SQ

VL) <400> 87

Glu Ile Val Leu Thr Gln Ser Pro 1 5

Ala Thr Leu Ser Leu Ser Pro Gly

15

Glu Arg Ala Thr Leu Ser Cys Arg 20

Ala Ser Glu Ser Val Asp Asn Tyr 25 30

Gly Met Ser Phe Met Asn Trp Phe

40

Gln Gln Lys Pro Gly Gln Pro Pro 45

Lys Leu Leu Ile His Ala Ala Ser

55

Asn Gln Gly Ser Gly Val Pro Ser 60

Arg Phe Ser Gly Ser Gly Ser Gly 65 70

Thr Asp Phe Thr Leu Thr Ile Ser

80

Ser Leu Glu Pro Glu Asp Phe Ala 85

Val Tyr Phe Cys Gln Gln Ser Lys

95

Glu Val Pro Tyr Thr Phe Gly Gly

100

Gly Thr Lys Val Glu Ile Lys

105 110 <210> 88 <211> 445 <212> PROTEIN <213> Artificial sequence <220>

<223>Anti-human PD-1 antibody heavy chain PD-1 mAb 6 G4P <400>88

Gln Val Gln Leu Val Gln Ser Gly Ala 15

Glu Val Lys Lys Pro Gly Ala

15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 2530

- 141 043373

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 35 40 45

Gly Val Ile His Pro Ser Asp Ser Glu Thr Trp Leu Asp Gln Lys 50 55 60

Lys Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala 65 70 75

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

90 95

Ala Arg Glu His Tyr Gly Thr Ser Pro Phe Ala Tyr Trp Gly Gln

100 105 110

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

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

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

210 215 220

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

225 230 235

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Ile

Phe

Tyr 80

Cys

Gly

Phe

Leu

Trp 160

Leu

Ser

Pro

Pro

Phe 240

Pro

Val

- 142 043373

Gln Phe Asn

275

Trp

Lys Pro Arg

290

Glu

Leu Thr Val 305

Leu

Tyr Val Asp Gly 280

Glu Gln Phe Asn 295

His Gln Asp Trp 310

Val Glu Val His Asn Ala 285

Ser Thr Tyr Arg Val Val 300

Leu Asn Gly Lys Glu Tyr

315

Lys Thr

Ser Val

Lys Cys

320

Lys Val

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

325 330 335

Lys Ala

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

340 345 350

Ser Gln

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

355 360 365

Lys Gly

370

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

375 380

Gln Pro 385

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

390 395 400

Gly Ser

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

405 410 415

Gln Glu

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

420 425 430

Asn His

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445 <210> 89 <211> 218 <212> PROTEIN <213> Artificial sequence <220>

<223> Anti-human PD-1 light chain PD-1 mAb 6 G4P <400> 89

Glu Ile Val 1

Leu Thr Gln Ser Pro Ala Thr

10

Leu Ser Leu Ser Pro Gly

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

- 143 043373

Gly

Gln

Pro

Gly

Met

Ser 35

Phe

Met

Asn

Trp

Phe 40

Gln

Gln

Lys

Pro

Lys

Leu 50

Leu

Ile

His

Ala

Ala 55

Ser

Asn

Gln

Gly

Ser 60

Gly

Val

Pro

Arg 65

Phe

Ser

Gly

Ser

Gly 70

Ser

Gly

Thr

Asp

Phe 75

Thr

Leu

Thr

Ile

Ser

Leu

Glu

Pro

Glu 85

Asp

Phe

Ala

Val

Tyr 90

Phe

Cys

Gln

Gln

Ser 95

Glu

Val

Pro

Tyr 100

Thr

Phe

Gly

Gly

Gly 105

Thr

Lys

Val

Glu

Ile

110

Lys

Thr

Val

Ala

115

Ala

Pro

Ser

Val

Phe

120

Ile

Phe

Pro

Pro

Ser

125

Asp

Glu

Leu

Lys 130

Ser

Gly

Thr

Ala

Ser

135

Val

Val

Cys

Leu

Leu

140

Asn

Asn

Phe

Pro

145

Arg

Glu

Ala

Lys

Val

150

Gln

Trp

Lys

Val

Asp 155

Asn

Ala

Leu

Gln

Gly

Asn

Ser

Gln

Glu

165

Ser

Val

Thr

Glu

Gln

170

Asp

Ser

Lys

Asp

Ser

175

Tyr

Ser

Leu

Ser

180

Ser

Thr

Leu

Thr

Leu

185

Ser

Lys

Ala

Asp

Tyr 190

Glu

Lys

Val

195

Tyr

Ala

Cys

Glu

Val

200

Thr

His

Gln

Gly

Leu

205

Ser

Ser

Pro

Ser

Ser 80

Lys

Arg

Gln

Tyr

Ser 160

Thr

Lys

Pro

Val

Thr

210

Lys

Ser

Phe

Asn

Arg 215

Gly

Glu

Cys <210>

<211>

<212>

<213>

118

PROTEIN

Mus musculus <220>

<221>

<222>

MISC_FEATURE(1) . . (118)

- 144 043373 <223> VH domain of antibody against human CTLA-4 <400> 90

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val 1 5 10

CTLA-4 mAb 3

Gln Pro Gly Arg

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 20 25

Phe Ser Ser Tyr 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly

40

Leu Glu Trp Val 45

Thr Phe Ile Ser Tyr Asp Gly Ser Asn Lys His Tyr

55 60

Ala Asp Ser Val

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys 65 70 75

Asn Thr Leu Tyr 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

90

Ile Tyr Tyr Cys 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp

100 105

Gly Gln Gly Thr

110

Leu Val Thr Val Ser Ser

115 <210> 91 <211> 108 <212> PROTEIN <213> Mus musculus <220>

< 221> MISC_FEATURE < 222> (1).. (108) < 223> VL domain of anti-human CTLA-4 antibody

CTLA-4 mAb 3 <400> 91

Glu Ile Val 1

Glu Arg Ala

Phe Leu Ala

Leu Thr Gln

Thr Leu Ser 20

Trp Tyr Gln

Ser Pro Gly

Cys Arg Ala 25

Gln Lys Pro 40

Thr Leu Ser 10

Ser Gln Ser

Gly Gln Ala

Leu Ser Pro Gly 15

Val Ser Ser Ser 30

Pro Arg Leu Leu 45

- 145 043373

Ile Tyr 50 Gly Ala Ser Ser Arg 55 Ala Thr Gly Ile Pro 60 Asp Arg Phe Ser Gly 65 Ser Gly Ser Gly Thr 70 Asp Phe Thr Leu Thr 75 Ile Ser Arg Leu Glu 80 Pro Glu Asp Phe Ala 85 Val Tyr Tyr Cys Gln 90 Gln Tyr Gly Ser Ser 95 Pro T rp Thr Phe Gly 100 Gln Gly Thr Lys Val 105 Glu Ile Lys

<210> 92 <211> 448 <212> PROTEIN <213> Mus musculus <220>

<221> MISC_FEATURE <222> (1).. (448) <223> Anti-human CTLA-4 antibody heavy chain mAb 3 G1AA <400> 92

Gln 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Val Val Gln Pro Gly 15 Arg Ser Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Phe Ser 30 Ser Tyr Thr Met His 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Val Thr Phe 50 Ile Ser Tyr Asp Gly 55 Ser Asn Lys His Tyr 60 Ala Asp Ser Val Lys 65 Gly Arg Phe Thr Val 70 Ser Arg Asp Asn Ser 75 Lys Asn Thr Leu Tyr 80 Leu Gln Met Asn Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala Ile Tyr Tyr 95 Cys Ala Arg Thr Gly 100 Trp Leu Gly Pro Phe 105 Asp Tyr Trp Gly Gln 110 Gly Thr Leu Val Thr 115 Val Ser Ser Ala Ser 120 Thr Lys Gly Pro Ser 125 Val Phe Pro

- 146 043373

Leu

Cys 145

Ser

Ser

Ser

Asn

His 225

Val

Thr

Glu

Lys

Ser 305

Lys

Ile

Pro

Ala Pro Ser 130

Leu Val Lys

Gly Ala Leu

Ser Gly Leu

180

Leu Gly Thr

195

Thr Lys Val 210

Thr Cys Pro

Phe Leu Phe

Pro Glu Val

260

Val Lys Phe 275

Thr Lys Pro 290

Val Leu Thr

Cys Lys Val

Ser Lys Ala

340

Pro Ser Arg

355

Ser Lys Ser

135

Thr Ser Gly

Gly Thr Ala

140

Ala Leu Gly

Asp Tyr Phe 150

Pro Glu Pro

Val Thr Val 155

Ser Trp Asn

160

Thr Ser Gly 165

Tyr Ser Leu

Gln Thr Tyr

Asp Lys Arg

215

Pro Cys Pro

230

Pro Pro Lys 245

Thr Cys Val

Asn Trp Tyr

Arg Glu Glu

295

Val Leu His

310

Ser Asn Lys 325

Lys Gly Gln

Glu Glu Met

Val His Thr

170

Phe Pro Ala

Val Leu Gln

175

Ser Ser Val

185

Ile Cys Asn 200

Val Glu Pro

Ala Pro Glu

Pro Lys Asp

250

Val Val Asp

265

Val Asp Gly 280

Gln Tyr Asn

Gln Asp Trp

Ala Leu Pro

330

Pro Arg Glu

345

Thr Lys Asn 360

Val Thr Val

Val Asn His

205

Lys Ser Cys

220

Ala Ala Gly 235

Thr Leu Met

Val Ser His

Val Glu Val

285

Ser Thr Tyr 300

Leu Asn Gly 315

Ala Pro Ile

Pro Gln Val

Gln Val Ser

365

Pro Ser Ser 190

Lys Pro Ser

Asp Lys Thr

Gly Pro Ser

240

Ile Ser Arg

255

Glu Asp Pro 270

His Asn Ala

Arg Val Val

Lys Glu Tyr

320

Glu Lys Thr

335

Tyr Thr Leu 350

Leu Thr Cys

- 147 043373

Leu Val

370

Lys Gly Phe Tyr Pro

375

Ser Asp Ile Ala Val Glu Trp Glu Ser 380

Asn Gly 385

Gln Pro Glu Asn Asn

390

Tyr Lys Thr Thr Pro Pro Val Leu Asp

395 400

Ser Asp

Gly Ser Phe Phe Leu 405

Tyr Ser Lys Leu Thr Val Asp Lys Ser

410 415

Arg Trp Gln Gln Gly Asn Val Phe 420

Leu His Asn His Tyr Thr Gln Lys 435 440

Ser Cys Ser Val Met His Glu Ala

425 430

Ser Leu Ser Leu Ser Pro Gly Lys 445 <210> 93 <211> 444 <212> PROTEIN <213> Mus musculus <220>

< 221> MISC_FEATURE < 222> (1).. (444) < 223> Anti-human CTLA-4 antibody heavy chain mAb 3 G4P <400>93

Gln Val Gln Leu Val Glu Ser Gly 15

Gly Gly Val Val Gln Pro Gly Arg

1015

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Ser Tyr 25 30

Thr Met His Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Thr Phe Ile Ser Tyr Asp Gly Ser

55

Asn Lys His Tyr Ala Asp Ser Val 60

Lys Gly Arg Phe Thr Val Ser Arg 65 70

Asp Asn Ser Lys Asn Thr Leu Tyr

80

Leu Gln Met Asn Ser Leu Arg Ala 85

Glu Asp Thr Ala Ile Tyr Tyr Cys

95

Ala Arg Thr Gly Trp Leu Gly Pro

Phe Asp Tyr Trp Gly Gln Gly Thr

- 148 043373

Val Phe Pro

100

105

110

Leu

Leu

Cys 145

Ser

Ser

Ser

Asn

Pro 225

Phe

Val

Phe

Pro

Thr 305

Val

Ala

Val Thr Val

115

Ala Pro Cys 130

Leu Val Lys

Gly Ala Leu

Ser Gly Leu

180

Leu Gly Thr

195

Thr Lys Val 210

Pro Cys Pro

Pro Pro Lys

Thr Cys Val

260

Asn Trp Tyr

275

Arg Glu Glu 290

Val Leu His

Ser Asn Lys

Lys Gly Gln

Ser Ser Ala

Ser Thr Lys 120

Gly Pro Ser

125

Ser Arg Ser

135

Thr Ser Glu

Ser Thr Ala

140

Ala Leu Gly

Asp Tyr Phe

150

Pro Glu Pro

Val Thr Val 155

Ser Trp Asn

160

Thr Ser Gly 165

Tyr Ser Leu

Lys Thr Tyr

Asp Lys Arg

215

Ala Pro Glu

230

Pro Lys Asp 245

Val Val Asp

Val Asp Gly

Gln Phe Asn

295

Gln Asp Trp 310

Gly Leu Pro 325

Pro Arg Glu

Val His Thr

170

Phe Pro Ala

Val Leu Gln

175

Ser Ser Val

185

Thr Cys Asn 200

Val Glu Ser

Phe Leu Gly

Thr Leu Met

250

Val Ser Gln

265

Val Glu Val 280

Ser Thr Tyr

Leu Asn Gly

Ser Ser Ile

330

Pro Gln Val

Val Thr Val

Val Asp His

205

Lys Tyr Gly

220

Gly Pro Ser 235

Ile Ser Arg

Glu Asp Pro

His Asn Ala

285

Arg Val Val 300

Lys Glu Tyr 315

Glu Lys Thr

Tyr Thr Leu

Pro Ser Ser 190

Lys Pro Ser

Pro Pro Cys

Val Phe Leu

240

Thr Pro Glu

255

Glu Val Gln 270

Lys Thr Lys

Ser Val Leu

Lys Cys Lys

320

Ile Ser Lys

335

Pro Pro Ser

- 149 043373

340

345

350

Gln Glu Glu 355 Met Thr Lys Asn Gln 360 Val Ser Leu Thr Cys 365 Leu Val Lys Gly Phe 370 Tyr Pro Ser Asp Ile 375 Ala Val Glu Trp Glu 380 Ser Asn Gly Gln Pro 385 Glu Asn Asn Tyr Lys 390 Thr Thr Pro Pro Val 395 Leu Asp Ser Asp Gly 400 Ser Phe Phe Leu Tyr 405 Ser Arg Leu Thr Val 410 Asp Lys Ser Arg Trp 415 Gln Glu Gly Asn Val 420 Phe Ser Cys Ser Val 425 Met His Glu Ala Leu 430 His Asn His Tyr Thr 435 Gln Lys Ser Leu Ser 440 Leu Ser Leu Gly

<210> 94 <211> 215 <212> PROTEIN <213> Mus musculus <220>

<221> MISC_FEATURE <222> (1).. (215) <223> Anti-human CTLA-4 light chain mAb 3 G1AA and mAb 3 G4P <400> 94

Glu 1 Ile Val Leu Thr 5 Gln Ser Pro Gly Thr 10 Leu Ser Leu Ser Pro 15 Gly Glu Arg Ala Thr 20 Leu Ser Cys Arg Ala 25 Ser Gln Ser Val Ser 30 Ser Ser Phe Leu Ala 35 Trp Tyr Gln Gln Lys 40 Pro Gly Gln Ala Pro 45 Arg Leu Leu Ile Tyr 50 Gly Ala Ser Ser Arg 55 Ala Thr Gly Ile Pro 60 Asp Arg Phe Ser Gly 65 Ser Gly Ser Gly Thr 70 Asp Phe Thr Leu Thr 75 Ile Ser Arg Leu Glu 80

- 150 043373

Pro Glu Asp Phe Ala 85

Val Tyr Tyr Cys Gln

Gln Tyr Gly Ser Ser

Trp Thr Phe Gly Gln

100

Gly Thr Lys Val Glu

105

Ile Lys Arg Thr Val

110

Ala Pro Ser Val Phe

115

Ile Phe Pro Pro Ser

120

Asp Glu Gln Leu Lys

125

Gly Thr Ala Ser Val 130

Val Cys Leu Leu Asn

135

Asn Phe Tyr Pro Arg 140

Ala Lys Val Gln Trp 145

Lys Val Asp Asn Ala 150

Leu Gln Ser Gly Asn 155

Gln Glu Ser Val Thr

165

Glu Gln Asp Ser Lys

170

Asp Ser Thr Tyr Ser

175

Ser Ser Thr Leu Thr

180

Leu Ser Lys Ala Asp

185

Tyr Glu Lys His Lys

190

Tyr Ala Cys Glu Val 195

Thr His Gln Gly Leu 200

Ser Ser Pro Val Thr

205

Pro

Ala

Ser

Glu

Ser 160

Leu

Val

Lys

Ser Phe Asn Arg Gly 210

Glu Cys

215 <210> 95 <211> 497 <212> PROTEIN <213> Artificial sequence <220>

<223>First or third polypeptide chain DART B <400>95

Glu 1 Ile Val Leu Thr 5 Gln Ser Pro Gly Thr 10 Leu Ser Leu Ser Pro 15 Glu Arg Ala Thr 20 Leu Ser Cys Arg Ala 25 Ser Gln Ser Val Gly 30 Ser Tyr Leu Ala 35 Trp Tyr Gln Gln Lys 40 Pro Gly Gln Ala Pro 45 Arg Leu Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe

Gly

Ser

Leu

Ser

- 151 043373

Gly 65

Pro

T rp

Gly

Lys

Phe 145

Leu

Asp

Ser

Val

Trp 225

Gly

Val

Tyr

Pro

Ser Gly Ser Gly Thr

Glu Asp Phe Ala Val

Thr Phe Gly Gln Gly 100

Gly Gly Gly Gln Val

115

Pro Gly Ala Ser Val 130

Thr Ser Tyr Trp Met

150

Glu Trp Ile Gly Val

165

Gln Lys Phe Lys Asp 180

Thr Ala Tyr Met Glu 195

Tyr Tyr Cys Ala Arg 210

Gly Gln Gly Thr Leu

230

Glu Val Ala Ala Cys

245

Ala Ala Leu Glu Lys 260

Gly Pro Pro Cys Pro 275

Ser Val Phe Leu Phe

Asp Phe Thr Leu Thr

Tyr Tyr Cys Gln Gln 90

Thr Lys Val Glu Ile 105

Gln Leu Val Gln Ser

120

Lys Val Ser Cys Lys 135

Asn Trp Val Arg Gln

155

Ile His Pro Ser Asp

170

Arg Val Thr Ile Thr 185

Leu Ser Ser Leu Arg 200

Glu His Tyr Gly Thr 215

Val Thr Val Ser Ser

235

Glu Lys Glu Val Ala

250

Glu Val Ala Ala Leu

265

Pro Cys Pro Ala Pro 280

Pro Pro Lys Pro Lys

Ile Ser Arg Leu Glu

Tyr Gly Ser Ser Pro

Lys Gly Gly Gly Ser

110

Gly Ala Glu Val Lys 125

Ala Ser Gly Tyr Ser 140

Ala Pro Gly Gln Gly

160

Ser Glu Thr Trp Leu

175

Val Asp Lys Ser Thr

190

Ser Glu Asp Thr Ala

205

Ser Pro Phe Ala Tyr 220

Gly Gly Cys Gly Gly

240

Ala Leu Glu Lys Glu

255

Glu Lys Glu Ser Lys 270

Glu Phe Leu Gly Gly 285

Asp Thr Leu Tyr Ile

- 152 043373

290

295

300

Thr Arg Glu 305

Pro Glu Val

310

Asp Pro Glu

Val Gln Phe

325

Asn Ala Lys

Thr Lys Pro 340

Val Val Ser

355

Val Leu Thr

Glu Tyr Lys

370

Cys Lys Val

Lys Thr Ile 385

Ser Lys Ala

390

Thr Leu Pro

Pro Ser Gln

405

Thr Cys Leu

Val Lys Gly 420

Glu Ser Asn

435

Gly Gln Pro

Leu Asp Ser

450

Asp Gly Ser

Lys Ser Arg 465

Trp Gln Glu

470

Glu Ala Leu

His Asn His

485

Thr Cys Val

Asn Trp Tyr

Arg Glu Glu 345

Val Leu His 360

Ser Asn Lys 375

Lys Gly Gln

Glu Glu Met

Phe Tyr Pro 425

Glu Asn Asn 440

Phe Phe Leu 455

Gly Asn Val

Tyr Thr Gln

Val Val Asp

315

Val Asp Gly 330

Gln Phe Asn

Gln Asp Trp

Gly Leu Pro

380

Pro Arg Glu

395

Thr Lys Asn 410

Ser Asp Ile

Tyr Lys Thr

Tyr Ser Arg

460

Phe Ser Cys 475

Lys Ser Leu 490

Gly <210> 96 <211> 271 <212> PROTEIN <213> Artificial sequence

Val Ser Gln

Val Glu Val

335

Ser Thr Tyr

350

Leu Asn Gly 365

Ser Ser Ile

Pro Gln Val

Gln Val Ser

415

Ala Val Glu

430

Thr Pro Pro 445

Leu Thr Val

Ser Val Met

Ser Leu Ser

495

Glu 320

His

Arg

Lys

Glu

Tyr 400

Leu

Trp

Val

Asp

His 480

Leu

- 153 043373 <220>

<223> Second or fourth polypeptide chain DART B <400> 96

Glu Ile Val Leu Thr

5

Gln Ser Pro Ala Thr

Leu Ser Leu Ser Pro 15

Glu Arg Ala Thr Leu

Ser Cys Arg Ala Ser 25

Glu Ser Val Asp Asn 30

Gly Met Ser Phe Met 35

Asn Trp Phe Gln Gln 40

Lys Pro Gly Gln Pro 45

Lys Leu Leu Ile His 50

Ala Ala Ser Asn Gln 55

Gly Ser Gly Val Pro 60

Arg Phe Ser Gly Ser 65

Gly Ser Gly Thr Asp 70

Phe Thr Leu Thr Ile 75

Ser Leu Glu Pro Glu

Asp Phe Ala Val Tyr 90

Phe Cys Gln Gln Ser 95

Glu Val Pro Tyr Thr

100

Phe Gly Gly Gly Thr

105

Lys Val Glu Ile Lys

110

Gly Gly Ser Gly Gly

115

Gly Gly Gln Val Gln 120

Leu Val Glu Ser Gly

125

Gly Val Val Gln Pro 130

Gly Arg Ser Leu Arg 135

Leu Ser Cys Ala Ala 140

Gly Phe Thr Phe Ser 145

Ser Tyr Thr Met His 150

Trp Val Arg Gln Ala 155

Gly Lys Gly Leu Glu

165

Trp Val Thr Phe Ile

170

Ser Tyr Asp Gly Asn

175

Lys Tyr Tyr Ala Asp

180

Ser Val Lys Gly Arg

185

Phe Thr Ile Ser Arg

190

Asn Ser Lys Asn Thr 195

Leu Tyr Leu Gln Met

200

Asn Ser Leu Arg Ala 205

Asp Thr Ala Ile Tyr

210

Tyr Cys Ala Arg Thr

215

Gly Trp Leu Gly Pro 220

Gly

Tyr

Pro

Ser

Ser 80

Lys

Gly

Gly

Ser

Pro 160

Asn

Asp

Glu

Phe

- 154 043373

Asp 225 Tyr Trp Gly Gln Gly 230 Thr Leu Val Thr Val 235 Ser Ser Gly Gly Gly Gly Gly Lys Val 245 Ala Ala Cys Lys Glu 250 Lys Val Ala Ala Leu 255 Glu Lys Val Ala 260 Ala Leu Lys Glu Lys 265 Val Ala Ala Leu Lys 270 Glu

Cys 240

Lys <210> 97 <211> 497 <212> PROTEIN <213> Artificial sequence <220>

<223>First or third polypeptide chain DART C <400>97

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser

25 30

Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe 50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 65 70 75

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser 85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly

100 105 110

Gly Gly Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

115 120 125

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr

130 135 140

Gly

Ser

Leu

Ser

Glu 80

Pro

Ser

Lys

Ser

Gly

Phe Thr Ser Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln

- 155 043373

145

Leu

Asp

Ser

Val

T rp 225

Gly

Val

Tyr

Pro

Thr 305

Asp

Asn

Val

Glu

Lys

Glu Trp Ile

Gln Lys Phe

180

Thr Ala Tyr

195

Tyr Tyr Cys 210

Gly Gln Gly

Glu Val Ala

Ala Ala Leu

260

Gly Pro Pro

275

Ser Val Phe 290

Arg Glu Pro

Pro Glu Val

Ala Lys Thr

340

Val Ser Val

355

Tyr Lys Cys 370

Thr Ile Ser

150 155 160

Gly Val Ile 165

Lys Asp Arg

Met Glu Leu

Ala Arg Glu

215

Thr Leu Val

230

Ala Cys Glu 245

Glu Lys Glu

Cys Pro Pro

Leu Phe Pro

295

Glu Val Thr

310

Gln Phe Asn 325

Lys Pro Arg

Leu Thr Val

Lys Val Ser

375

Lys Ala Lys

His Pro Ser

170

Asp Ser Glu

Thr Trp Leu

175

Val Thr Ile

185

Thr Val Asp

Lys Ser Thr 190

Ser Ser Leu 200

His Tyr Gly

Thr Val Ser

Lys Glu Val

250

Val Ala Ala

265

Cys Pro Ala 280

Pro Lys Pro

Cys Val Val

Trp Tyr Val

330

Glu Glu Gln

345

Leu His Gln 360

Asn Lys Gly

Gly Gln Pro

Arg Ser Glu

205

Asp Thr Ala

Thr Ser Pro

220

Ser Gly Gly 235

Ala Ala Leu

Leu Glu Lys

Pro Glu Phe

285

Lys Asp Thr

300

Val Asp Val 315

Asp Gly Val

Phe Asn Ser

Asp Trp Leu

365

Leu Pro Ser

380

Arg Glu Pro

Phe Ala Tyr

Cys Gly Gly

240

Glu Lys Glu

255

Glu Ser Lys 270

Leu Gly Gly

Leu Tyr Ile

Ser Gln Glu 320

Glu Val His 335

Thr Tyr Arg 350

Asn Gly Lys

Ser Ile Glu

Gln Val Tyr

- 156 043373

Lys Asn Gln Val Ser

415

385

390

395

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

405 410

Thr Cys Leu Val Lys

420

Gly Phe Tyr Pro Ser

425

Asp Ile Ala Val Glu

430

Glu Ser Asn Gly Gln 435

Pro Glu Asn Asn Tyr 440

Lys Thr Thr Pro Pro

445

Leu Asp Ser Asp Gly 450

Ser Phe Phe Leu Tyr 455

Ser Arg Leu Thr Val 460

Lys Ser Arg Trp Gln 465

Glu Gly Asn Val Phe 470

Ser Cys Ser Val Met 475

Glu Ala Leu His Asn

485

His Tyr Thr Gln Lys

490

Ser Leu Ser Leu Ser

495

400

Leu

Trp

Val

Asp

His 480

Leu

Gly <210> 98 <211> 271 <212> PROTEIN <213> Artificial sequence <220>

<223> Second or fourth polypeptide chain DART C <400> 98

Glu Ile Val Leu Thr

5

Gln Ser Pro Ala Thr

Leu Ser Leu Ser Pro 15

Glu Arg Ala Thr Leu

Ser Cys Arg Ala Ser 25

Glu Ser Val Asp Asn 30

Gly Met Ser Phe Met 35

Asn Trp Phe Gln Gln 40

Lys Pro Gly Gln Pro 45

Lys Leu Leu Ile His 50

Ala Ala Ser Asn Gln 55

Gly Ser Gly Val Pro 60

Arg Phe Ser Gly Ser 65

Gly Ser Gly Thr Asp 70

Phe Thr Leu Thr Ile 75

Gly

Tyr

Pro

Ser

Ser 80

- 157 043373

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

9095

Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

100 105110

Gly Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Glu Ser Gly Gly

115 120125

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

130 135140

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

145 150 155160

Gly Lys Gly Leu Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Ser Asn

165 170175

Lys His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp

180 185190

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

195 200205

Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe

210 215220

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys

225 230 235240

Gly Gly Gly Lys Val Ala Ala Cys Lys Glu Lys Val Ala Ala Leu Lys

245 250255

Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu

260 265270 < 210>99 < 211>499 < 212> PROTEIN < 213> Artificial sequence <220>

<223>First or third polypeptide chain DART D <400>99

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

- 158 043373

Asp

Glu

Gly

Lys

Arg 65

Ser

Glu

Gly

Gly

Gly 145

Gly

Lys

Asn

Asp

Asp 225

Gly

Arg

Met

Leu 50

Phe

Leu

Val

Gly

Val

130

Phe

Lys

His

Ser

Thr

210

Tyr

Ala

Thr 20

Leu

Ser

Cys

Arg

Ala 25

Ser

Glu

Ser

Val

Asn

Ser

Leu

Ser

Glu

Pro

Ser

115

Val

Thr

Gly

Tyr

Lys

195

Ala

Trp

Phe

Met

Asn

Trp

Phe 40

Gln

Gln

Lys

Pro

Gly 45

Gln

Pro

Ile

Gly

Pro

Tyr 100

Gly

Gln

Phe

Leu

Ala

180

Asn

Ile

Gly

His

Ala

Ala 55

Ser

Asn

Gln

Gly

Ser 60

Gly

Val

Pro

Ser

Gly 70

Ser

Gly

Thr

Asp

Phe 75

Thr

Leu

Thr

Ile

Glu 85

Thr

Gly

Pro

Ser

Glu

165

Asp

Thr

Tyr

Gln

Asp

Phe

Ala

Val

Tyr 90

Phe

Cys

Gln

Gln

Ser 95

Phe

Gly

Gly

Gly 105

Thr

Lys

Val

Glu

Ile

110

Lys

Gly

Gly

Gln

120

Val

Gln

Leu

Val

Glu

125

Ser

Gly

Gly

Ser

150

Trp

Ser

Leu

Tyr

Gly 230

Arg 135

Tyr

Val

Val

Tyr

Cys

215

Thr

Ser

Leu

Arg

Leu

Ser

140

Cys

Ala

Ala

Thr

Thr

Lys

Leu

200

Ala

Leu

Met

His

Trp 155

Val

Arg

Gln

Ala

Phe

Gly 185

Gln

Arg

Val

Ile

170

Ser

Tyr

Asp

Gly

Ser

175

Arg

Met

Thr

Thr

Phe

Thr

Val

Ser

190

Arg

Asn

Gly

Val

235

Ser

Leu

205

Arg

Ala

Trp 220

Leu

Gly

Pro

Ser

Ser

Gly

Gly

Tyr

Pro

Ser

Ser 80

Lys

Gly

Gly

Ser

Pro 160

Asn

Asp

Glu

Phe

Cys 240

Glu

Gly

Gly

Glu

Val

Ala

Ala

Cys

Glu

Lys

Glu

Val

Ala

Ala

Leu

- 159 043373

245

255

250

Lys

Ser

Gly

Tyr 305

Gln

Val

Tyr

Gly

Ile 385

Val

Ser

Glu

Pro

Val 465

Met

Glu Val

Lys Tyr

275

Gly Pro 290

Ile Thr

Glu Asp

His Asn

Arg Val

355

Lys Glu 370

Glu Lys

Tyr Thr

Leu Thr

Trp Glu

435

Val Leu 450

Asp Lys

His Glu

Ala Ala 260

Gly Pro

Ser Val

Arg Glu

Pro Glu

325

Ala Lys 340

Val Ser

Tyr Lys

Thr Ile

Leu Pro

405

Cys Leu 420

Ser Asn

Asp Ser

Ser Arg

Ala Leu

Leu Glu

Lys Glu

265

Val Ala

Ala Leu

Pro Cys

Phe Leu

295

Pro Glu 310

Val Gln

Thr Lys

Val Leu

Cys Lys

375

Ser Lys 390

Pro Ser

Val Lys

Gly Gln

Asp Gly

455

Trp Gln 470

His Asn

Pro Pro 280

Phe Pro

Val Thr

Phe Asn

Pro Arg

345

Thr Val 360

Val Ser

Ala Lys

Gln Glu

Gly Phe

425

Pro Glu 440

Ser Phe

Glu Gly

His Tyr

Cys Pro

Pro Lys

Cys Val

315

Trp Tyr 330

Glu Glu

Leu His

Asn Lys

Gly Gln

395

Glu Met 410

Tyr Pro

Asn Asn

Phe Leu

Asn Val

475

Thr Gln

Ala Pro

285

Pro Lys 300

Val Val

Val Asp

Gln Phe

Gln Asp

365

Gly Leu 380

Pro Arg

Thr Lys

Ser Asp

Tyr Lys

445

Tyr Ser 460

Phe Ser

Lys Ser

Glu Lys 270

Glu Phe

Asp Thr

Asp Val

Gly Val

335

Asn Ser 350

Trp Leu

Pro Ser

Glu Pro

Asn Gln

415

Ile Ala 430

Thr Thr

Arg Leu

Cys Ser

Leu Ser

Glu

Leu

Leu

Ser 320

Glu

Thr

Asn

Ser

Gln 400

Val

Val

Pro

Thr

Val 480

Leu

- 160 043373

485

495

490

Ser Leu Gly

<210> <211> <212> <213> 100 269 PROTEIN Artificial [ subsequence <220> <223> <400> Second or fourth 100 DART polypeptide chains D Glu 1 Ile Val Leu Thr 5 Gln Ser Pro Gly Thr 10 Leu Ser Leu Ser Pro 15 Gly Glu Arg Ala Thr 20 Leu Ser Cys Arg Ala 25 Ser Gln Ser Val Ser 30 Ser Ser Phe Leu Ala 35 Trp Tyr Gln Gln Lys 40 Pro Gly Gln Ala Pro 45 Arg Leu Leu Ile Tyr 50 Gly Ala Ser Ser Arg 55 Ala Thr Gly Ile Pro 60 Asp Arg Phe Ser Gly 65 Ser Gly Ser Gly Thr 70 Asp Phe Thr Leu Thr 75 Ile Ser Arg Leu Glu 80 Pro Glu Asp Phe Ala 85 Val Tyr Tyr Cys Gln 90 Gln Tyr Gly Ser Ser 95 Pro T rp Thr Phe Gly 100 Gln Gly Thr Lys Val 105 Glu Ile Lys Gly Gly 110 Gly Ser Gly Gly Gly 115 Gly Gln Val Gln Leu 120 Val Gln Ser Gly Ala 125 Glu Val Lys Lys Pro 130 Gly Ala Ser Val Lys 135 Val Ser Cys Lys Ala 140 Ser Gly Tyr Ser Phe 145 Thr Ser Tyr Trp Met 150 Asn Trp Val Arg Gln 155 Ala Pro Gly Gln Gly 160 Leu Glu Trp Ile Gly 165 Val Ile His Pro Ser 170 Asp Ser Glu Thr Trp 175 Leu

- 161 043373

Asp Gln Lys Phe Lys Asp Arg Val

180

Thr Ile Thr Val Asp Lys Ser

185 190

Ser Thr Ala Tyr Met Glu Leu Ser

195 200

Ser Leu Arg Ser Glu Asp Thr

205

Val Tyr Tyr Cys Ala Arg Glu His

210 215

Tyr Gly Thr Ser Pro Phe Ala 220

Trp Gly Gln Gly Thr 225

Leu Val Thr Val Ser Ser Gly Gly Cys Gly

230 235

Gly Lys Val Ala Ala

245

Cys Lys Glu Lys Val Ala Ala Leu Lys Glu

250 255

Thr

Ala

Tyr

Gly 240

Lys

Val Ala Ala Leu Lys

260

Glu Lys Val Ala Ala Leu Lys Glu

265 <210> 101 <211> 503 <212> PROTEIN <213> Artificial sequence <220>

<223> First or third polypeptide chain DART F <400> 101

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

5 10 15

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

25 30

Gly Met Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro 35 40 45

Lys Leu Leu Ile His Ala Ala Ser Asn Gln Gly Ser Gly Val Pro 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75

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

90 95

Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

Gly

Tyr

Pro

Ser

Ser 80

Lys

Gly

- 162 043373

Ser Gly Gly

100

105

110

Gly

Gly

Gly 145

Gly

Lys

Asn

Asp

Asp 225

Gly

Lys

Glu

Pro

Lys 305

Val

Asp

Gly Ser Gly

115

Gly Gly Gly

Gln Val Gln 120

Leu Val Glu

125

Val Val Gln 130

Pro Gly Arg

135

Ser Leu Arg

Leu Ser Cys

140

Ala Ala Ser

Phe Thr Phe

Lys Gly Leu

His Tyr Ala

180

Ser Lys Asn

195

Thr Ala Ile 210

Tyr Trp Gly

Gly Gly Glu

Glu Val Ala

260

Pro Lys Ser 275

Glu Ala Ala 290

Asp Thr Leu

Asp Val Ser

Gly Val Glu

Ser Ser Tyr 150

Glu Trp Val 165

Asp Ser Val

Thr Leu Tyr

Tyr Tyr Cys

215

Gln Gly Thr 230

Val Ala Ala 245

Ala Leu Glu

Ala Asp Lys

Gly Gly Pro

295

Tyr Ile Thr 310

His Glu Asp 325

Val His Asn

Thr Met His

Thr Phe Ile

170

Lys Gly Arg

185

Leu Gln Met 200

Ala Arg Thr

Leu Val Thr

Cys Glu Lys

250

Lys Glu Val

265

Thr His Thr 280

Ser Val Phe

Arg Glu Pro

Pro Glu Val

330

Ala Lys Thr

Trp Val Arg 155

Gln Ala Pro

160

Ser Tyr Asp

Gly Ser Asn

175

Phe Thr Val

Ser Arg Asp 190

Asn Ser Leu

205

Arg Ala Glu

Gly Trp Leu

220

Gly Pro Phe

Val Ser Ser 235

Gly Gly Cys

240

Glu Val Ala

Ala Leu Glu

255

Ala Ala Leu

Cys Pro Pro

285

Leu Phe Pro

300

Glu Val Thr 315

Lys Phe Asn

Lys Pro Arg

Glu Lys Leu 270

Cys Pro Ala

Pro Lys Pro

Cys Val Val

320

Trp Tyr Val

335

Glu Glu Gln

- 163 043373

Leu Thr Val Leu His

365

340

345

350

Tyr Asn Ser Thr Tyr

355

Arg Val Val Ser Val 360

Asp Trp Leu Asn Gly

370

Lys Glu Tyr Lys Cys

375

Lys Val Ser Asn Lys

380

Leu Pro Ala Pro Ile 385

Glu Lys Thr Ile Ser 390

Lys Ala Lys Gly Gln 395

Arg Glu Pro Gln Val

405

Tyr Thr Leu Pro Pro

410

Ser Arg Glu Glu Met

415

Lys Asn Gln Val Ser

420

Leu Thr Cys Leu Val

425

Lys Gly Phe Tyr Pro

430

Asp Ile Ala Val Glu 435

Trp Glu Ser Asn Gly 440

Gln Pro Glu Asn Asn

445

Lys Thr Thr Pro Pro

450

Val Leu Asp Ser Asp

455

Gly Ser Phe Phe Leu 460

Ser Lys Leu Thr Val 465

Asp Lys Ser Arg Trp 470

Gln Gln Gly Asn Val 475

Ser Cys Ser Val Met

485

His Glu Ala Leu His

490

Asn His Tyr Thr Gln

495

Gln

Ala

Pro 400

Thr

Ser

Tyr

Tyr

Phe 480

Lys

Ser Leu Ser Leu Ser

500

Pro Gly <210> 102 <211> 567 <212> PROTEIN <213> Artificial Sequence <220>

< 223> First or third polypeptide chain DART E < 400>102

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 1 5 1015

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser

2530

Gly

Ser

- 164 043373

Phe

Ile

Gly 65

Pro

T rp

Gly

Lys

Phe 145

Leu

Asp

Ser

Val

Trp 225

Gly

Arg

Leu Ala Trp Tyr Gln 35

Tyr Gly Ala Ser Ser 50

Ser Gly Ser Gly Thr

Glu Asp Phe Ala Val

Thr Phe Gly Gln Gly 100

Gly Gly Gly Gln Val

115

Pro Gly Ala Ser Val 130

Thr Ser Tyr Trp Met

150

Glu Trp Ile Gly Val

165

Gln Lys Phe Lys Asp 180

Thr Ala Tyr Met Glu

195

Tyr Tyr Cys Ala Arg 210

Gly Gln Gly Thr Leu

230

Ala Ser Thr Lys Gly

245

Ser Thr Ser Glu Ser

260

Gln Lys Pro Gly Gln 40

Arg Ala Thr Gly Ile 55

Asp Phe Thr Leu Thr

Tyr Tyr Cys Gln Gln 90

Thr Lys Val Glu Ile 105

Gln Leu Val Gln Ser

120

Lys Val Ser Cys Lys 135

Asn Trp Val Arg Gln

155

Ile His Pro Ser Asp

170

Arg Val Thr Ile Thr 185

Leu Ser Ser Leu Arg 200

Glu His Tyr Gly Thr 215

Val Thr Val Ser Ser

235

Pro Ser Val Phe Pro

250

Thr Ala Ala Leu Gly 265

Ala Pro Arg Leu Leu 45

Pro Asp Arg Phe Ser 60

Ile Ser Arg Leu Glu

Tyr Gly Ser Ser Pro

Lys Gly Gly Gly Ser

110

Gly Ala Glu Val Lys 125

Ala Ser Gly Tyr Ser 140

Ala Pro Gly Gln Gly

160

Ser Glu Thr Trp Leu

175

Val Asp Lys Ser Thr

190

Ser Glu Asp Thr Ala

205

Ser Pro Phe Ala Tyr 220

Leu Gly Gly Gly Ser

240

Leu Ala Pro Cys Ser

255

Cys Leu Val Lys Asp

270

- 165 043373

Tyr Phe Pro

275

Glu Pro Val

Thr Val Ser

280

Trp Asn Ser

Gly Ala Leu 285

Ser Gly Val

290

His Thr Phe

Pro Ala Val 295

Leu Gln Ser

300

Ser Gly Leu

Ser Leu Ser 305

Ser Val Val

310

Thr Val Pro

Ser Ser Ser

315

Leu Gly Thr

Thr Tyr Thr

Cys Asn Val

325

Asp His Lys

Pro Ser Asn 330

Thr Lys Val

335

Lys Arg Val

Glu Ser Lys 340

Tyr Gly Pro

345

Pro Cys Pro

Pro Cys Pro

350

Pro Glu Phe

355

Leu Gly Gly

Pro Ser Val

360

Phe Leu Phe

Pro Pro Lys 365

Lys Asp Thr

370

Leu Tyr Ile

Thr Arg Glu 375

Pro Glu Val

380

Thr Cys Val

Val Asp Val 385

Ser Gln Glu

390

Asp Pro Glu

Val Gln Phe

395

Asn Trp Tyr

Asp Gly Val

Glu Val His

405

Asn Ala Lys

Thr Lys Pro 410

Arg Glu Glu

415

Phe Asn Ser

Thr Tyr Arg 420

Val Val Ser

425

Val Leu Thr

Val Leu His

430

Asp Trp Leu

435

Asn Gly Lys

Glu Tyr Lys

440

Cys Lys Val

Ser Asn Lys 445

Leu Pro Ser

450

Ser Ile Glu

Lys Thr Ile 455

Ser Lys Ala

460

Lys Gly Gln

Arg Glu Pro 465

Gln Val Tyr

470

Thr Leu Pro

Pro Ser Gln

475

Glu Glu Met

Lys Asn Gln

Val Ser Leu

485

Thr Cys Leu

Val Lys Gly 490

Phe Tyr Pro

495

Asp Ile Ala

Val Glu Trp 500

Glu Ser Asn

505

Gly Gln Pro

Glu Asn Asn

510

Thr

Tyr

Lys 320

Asp

Ala

Pro

Val

Val 400

Gln

Gln

Gly

Pro

Thr 480

Ser

Tyr

- 166 043373

Lys Thr Thr 515 Pro Pro Val Leu Asp 520 Ser Asp Gly Ser Phe 525 Phe Leu Ser Arg 530 Leu Thr Val Asp Lys 535 Ser Arg Trp Gln Glu 540 Gly Asn Val Ser 545 Cys Ser Val Met His 550 Glu Ala Leu His Asn 555 His Tyr Thr Gln

Tyr

Phe

Lys 560

Ser Leu Ser Leu Ser Leu Gly

565 <210> 103 <211> 350 <212> PROTEIN <213> Artificial sequence <220>

<223> Second or fourth polypeptide chain DART E <400> 103

Glu Ile Val Leu Thr

5

Gln Ser Pro Ala Thr

Leu Ser Leu Ser Pro 15

Glu Arg Ala Thr Leu 20

Ser Cys Arg Ala Ser 25

Glu Ser Val Asp Asn 30

Gly Met Ser Phe Met 35

Asn Trp Phe Gln Gln 40

Lys Pro Gly Gln Pro 45

Lys Leu Leu Ile His 50

Ala Ala Ser Asn Gln 55

Gly Ser Gly Val Pro 60

Arg Phe Ser Gly Ser 65

Gly Ser Gly Thr Asp 70

Phe Thr Leu Thr Ile 75

Ser Leu Glu Pro Glu

Asp Phe Ala Val Tyr

Phe Cys Gln Gln Ser

Glu Val Pro Tyr Thr

100

Phe Gly Gly Gly Thr

105

Lys Val Glu Ile Lys

110

Gly Gly Ser Gly Gly

115

Gly Gly Gln Val Gln 120

Leu Val Glu Ser Gly

125

Gly

Tyr

Pro

Ser

Ser 80

Lys

Gly

Gly

Ser

Gly Val Val Gln Pro

Gly Arg Ser Leu Arg

Leu Ser Cys Ala Ala

- 167 043373

130

135

140

Gly 145

Phe

Thr

Phe

Ser

Ser

150

Tyr

Thr

Met

His

Trp 155

Val

Arg

Gln

Ala

Gly

Lys

Gly

Leu

Glu

165

Trp

Val

Thr

Phe

Ile

170

Ser

Tyr

Asp

Gly

Ser

175

Lys

His

Tyr

Ala

180

Asp

Ser

Val

Lys

Gly 185

Arg

Phe

Thr

Val

Ser

190

Arg

Asn

Ser

Lys

195

Asn

Thr

Leu

Tyr

Leu

200

Gln

Met

Asn

Ser

Leu

205

Arg

Ala

Asp

Thr

210

Ala

Ile

Tyr

Tyr

Cys

215

Ala

Arg

Thr

Gly

Trp 220

Leu

Gly

Pro

Asp

225

Tyr

Trp

Gly

Gln

Gly 230

Thr

Leu

Val

Thr

Val

235

Ser

Ser

Leu

Gly

Gly

Ser

Gly

Arg

Thr

245

Val

Ala

Ala

Pro

Ser

250

Val

Phe

Ile

Phe

Pro

255

Ser

Asp

Glu

Gln

260

Leu

Lys

Ser

Gly

Thr

265

Ala

Ser

Val

Val

Cys

270

Leu

Asn

Asn

Phe

275

Tyr

Pro

Arg

Glu

Ala

280

Lys

Val

Gln

Trp

Lys 285

Val

Asp

Ala

Leu

290

Gln

Ser

Gly

Asn

Ser 295

Gln

Glu

Ser

Val

Thr

300

Glu

Gln

Asp

Lys 305

Asp

Ser

Thr

Tyr

Ser

310

Leu

Ser

Ser

Thr

Leu

315

Thr

Leu

Ser

Lys

Asp

Tyr

Glu

Lys

His

325

Lys

Val

Tyr

Ala

Cys

330

Glu

Val

Thr

His

Gln

335

Pro 160

Asn

Asp

Glu

Phe

Gly 240

Pro

Leu

Asn

Ser

Ala 320

Gly

Leu

Ser

Ser

Pro

340

Val

Thr

Lys

Ser

Phe

345

Asn

Arg

Gly

Glu

Cys 350 <210>

<211>

<212>

<213>

104

500

PROTEIN

Artificial sequence

- 168 043373

Leu Ser Leu Ser Pro <220>

<223> First polypeptide chain TRIDENT A <400> 104

Glu Ile Val Leu Thr

5

Gln Ser Pro Ala Thr

Glu Arg Ala Thr Leu

Ser Cys Arg Ala Ser 25

Glu Ser Val Asp Asn 30

Gly Met Ser Phe Met 35

Asn Trp Phe Gln Gln 40

Lys Pro Gly Gln Pro 45

Lys Leu Leu Ile His 50

Ala Ala Ser Asn Gln 55

Gly Ser Gly Val Pro 60

Arg Phe Ser Gly Ser 65

Gly Ser Gly Thr Asp 70

Phe Thr Leu Thr Ile 75

Ser Leu Glu Pro Glu

Asp Phe Ala Val Tyr 90

Phe Cys Gln Gln Ser 95

Glu Val Pro Tyr Thr

100

Phe Gly Gly Gly Thr

105

Lys Val Glu Ile Lys

110

Gly Gly Ser Gly Gly

115

Gly Gly Gln Val Gln 120

Leu Val Gln Ser Gly

125

Glu Val Lys Lys Pro 130

Gly Ala Ser Val Lys 135

Val Ser Cys Lys Ala

140

Gly Tyr Ser Phe Thr 145

Ser Tyr Trp Met Asn 150

Trp Val Arg Gln Ala 155

Gly Gln Gly Leu Glu

165

Trp Ile Gly Val Ile

170

His Pro Ser Asp Ser

175

Thr Trp Leu Asp Gln

180

Lys Phe Lys Asp Arg

185

Val Thr Ile Thr Val

190

Lys Ser Thr Ser Thr 195

Ala Tyr Met Glu Leu 200

Ser Ser Leu Arg Ser 205

Asp Thr Ala Val Tyr

210

Tyr Cys Ala Arg Glu

215

His Tyr Gly Thr Ser 220

Gly

Tyr

Pro

Ser

Ser 80

Lys

Gly

Ala

Ser

Pro 160

Glu

Asp

Glu

Pro

- 169 043373

Phe 225

Cys

Glu

Glu

Leu

Leu 305

Ser

Glu

Thr

Asn

Ser 385

Gln

Val

Val

Pro

Ala Tyr Trp

Gly Gly Gly

Lys Glu Val

260

Ser Lys Tyr

275

Gly Gly Pro 290

Tyr Ile Thr

Gln Glu Asp

Val His Asn

340

Tyr Arg Val

355

Gly Lys Glu 370

Ile Glu Lys

Val Tyr Thr

Ser Leu Trp

420

Glu Trp Glu

435

Pro Val Leu 450

Gly Gln Gly 230

Glu Val Ala 245

Ala Ala Leu

Gly Pro Pro

Ser Val Phe

295

Arg Glu Pro 310

Pro Glu Val 325

Ala Lys Thr

Val Ser Val

Tyr Lys Cys

375

Thr Ile Ser

390

Leu Pro Pro 405

Cys Leu Val

Ser Asn Gly

Asp Ser Asp

455

Thr Leu Val

Thr Val Ser 235

Ser Gly Gly

240

Ala Cys Glu

250

Glu Lys Glu 265

Cys Pro Pro 280

Leu Phe Pro

Glu Val Thr

Gln Phe Asn

330

Lys Pro Arg

345

Leu Thr Val 360

Lys Val Ser

Lys Ala Lys

Ser Gln Glu

410

Lys Gly Phe 425

Gln Pro Glu 440

Gly Ser Phe

Lys Glu Val

Ala Ala Leu

255

Val Ala Ala

Cys Pro Ala

285

Pro Lys Pro

300

Cys Val Val 315

Trp Tyr Val

Glu Glu Gln

Leu His Gln

365

Asn Lys Gly

380

Gly Gln Pro 395

Glu Met Thr

Tyr Pro Ser

Asn Asn Tyr

445

Phe Leu Tyr

460

Leu Glu Lys 270

Pro Glu Phe

Lys Asp Thr

Val Asp Val

320

Asp Gly Val

335

Phe Asn Ser 350

Asp Trp Leu

Leu Pro Ser

Arg Glu Pro

400

Lys Asn Gln

415

Asp Ile Ala 430

Lys Thr Thr

Ser Arg Leu

- 170 043373

Thr 465 Val Asp Lys Ser Arg 470 Trp Gln Glu Gly Asn 475 Val Phe Ser Cys Val Met His Glu Ala 485 Leu His Asn His Tyr 490 Thr Gln Lys Ser Leu 495 Leu Ser Leu Gly 500

Ser 480

Ser <210> 105 <211> 272 <212> PROTEIN <213> Artificial sequence <220>

< 223> Second polypeptide chain TRIDENT A < 400> 105

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro 1 5 10 15

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

25 30

Gly Met Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro 35 40 45

Lys Leu Leu Ile His Ala Ala Ser Asn Gln Gly Ser Gly Val Pro 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75

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

90 95

Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Gln Ser Gly

115 120 125

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala

130 135 140

Gly Tyr Ser Phe Thr Ser Tyr Trp Met Asn Trp Val Arg Gln Ala

Gly

Tyr

Pro

Ser

Ser 80

Lys

Gly

Ala

Ser

Pro

- 171 043373

Ile His Pro Ser Asp Ser Glu

170 175

145

150

155

160

Gly Gln Gly Leu Glu Trp Ile Gly Val

165

Thr Trp Leu Asp

180

Gln Lys

Phe Lys Asp Arg Val Thr Ile Thr Val Asp

185 190

Lys Ser Thr Ser

195

Thr Ala

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

200 205

Asp Thr Ala Val

210

Tyr Tyr

Cys Ala Arg Glu His Tyr Gly Thr Ser Pro

215 220

Phe Ala 225

Cys Gly

Lys Glu

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

230 235240

Gly Gly Lys Val Ala Ala Cys Lys Glu Lys Val Ala Ala Leu

245 250255

Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu

260 265270 <210> 106 <211> 444 <212> PROTEIN <213> Artificial sequence <220>

< 223> Third polypeptide chain TRIDENT A < 400> 106

Gln Val Gln Leu Val 1 5

Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

15

Ser Leu Arg

Leu Ser Cys Ala Ala Ser Gly 20 25

Phe Thr Phe Ser Ser Tyr 30

Thr Met His

Thr Phe Ile

Lys Gly Arg 65

Trp Val Arg Gln Ala Pro Gly 40

Ser Tyr Asp Gly Ser Asn Lys 55

Phe Thr Val Ser Arg Asp Asn 70

Lys Gly Leu Glu Trp Val 45

His Tyr Ala Asp Ser Val 60

Ser Lys Asn Thr Leu Tyr 75 80

- 172 043373

Leu

Ala

Leu

Leu

Cys 145

Ser

Ser

Ser

Asn

Pro 225

Phe

Val

Phe

Pro

Thr 305

Gln Met Asn

Ser Leu Arg 85

Ala Glu Asp

Thr Ala Ile

Tyr Tyr Cys

Arg Thr Gly

100

Val Thr Val

115

Ala Pro Cys 130

Leu Val Lys

Gly Ala Leu

Ser Gly Leu

180

Leu Gly Thr

195

Thr Lys Val 210

Pro Cys Pro

Pro Pro Lys

Thr Cys Val

260

Asn Trp Tyr

275

Arg Glu Glu 290

Val Leu His

Trp Leu Gly

Pro Phe Asp 105

Tyr Trp Gly

Gln Gly Thr 110

Ser Ser Ala

Ser Arg Ser

135

Asp Tyr Phe 150

Thr Ser Gly 165

Tyr Ser Leu

Lys Thr Tyr

Asp Lys Arg

215

Ala Pro Glu

230

Pro Lys Asp 245

Val Val Asp

Val Asp Gly

Gln Phe Asn

295

Gln Asp Trp

310

Ser Thr Lys 120

Gly Pro Ser

125

Val Phe Pro

Thr Ser Glu

Ser Thr Ala

140

Ala Leu Gly

Pro Glu Pro

Val Thr Val 155

Ser Trp Asn

160

Val His Thr

170

Phe Pro Ala

Val Leu Gln

175

Ser Ser Val

185

Thr Cys Asn 200

Val Glu Ser

Phe Leu Gly

Thr Leu Tyr

250

Val Ser Gln

265

Val Glu Val 280

Ser Thr Tyr

Leu Asn Gly

Val Thr Val

Val Asp His

205

Lys Tyr Gly

220

Gly Pro Ser 235

Ile Thr Arg

Glu Asp Pro

His Asn Ala

285

Arg Val Val 300

Lys Glu Tyr 315

Pro Ser Ser 190

Lys Pro Ser

Pro Pro Cys

Val Phe Leu

240

Glu Pro Glu

255

Glu Val Gln 270

Lys Thr Lys

Ser Val Leu

Lys Cys Lys

320

- 173 043373

Val Ser

Ala Lys

Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330

Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345

Lys Thr Ile Ser Lys

335

Thr Leu Pro Pro Ser

350

Gln Glu

Glu Met Thr Lys Asn Gln Val Ser Leu 355 360

Ser Cys Ala Val Lys

365

Gly Phe

370

Tyr Pro Ser Asp Ile Ala Val Glu Trp

375

Glu Ser Asn Gly Gln 380

Pro Glu 385

Asn Asn Tyr Lys Thr Thr Pro Pro Val

390 395

Leu Asp Ser Asp Gly

400

Ser Phe

Phe Leu Val Ser Arg Leu Thr Val Asp

405 410

Lys Ser Arg Trp Gln

415

Glu Gly Asn Val Phe

420

Ser Cys

Ser Val Met His Glu Ala Leu His Asn

425 430

Arg Tyr Thr Gln Lys 435

Ser Leu Ser Leu Ser Leu Gly

440 <210> 107 <211> 215 <212> PROTEIN <213> Artificial sequence <220>

< 223> Fourth polypeptide chain TRIDENT A < 400> 107

Glu 1 Ile Val Leu Thr 5 Gln Ser Pro Gly Thr 10 Leu Ser Leu Ser Pro 15 Gly Glu Arg Ala Thr 20 Leu Ser Cys Arg Ala 25 Ser Gln Ser Val Ser 30 Ser Ser Phe Leu Ala 35 Trp Tyr Gln Gln Lys 40 Pro Gly Gln Ala Pro 45 Arg Leu Leu Ile Tyr 50 Gly Ala Ser Ser Arg 55 Ala Thr Gly Ile Pro 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

- 174 043373

Pro Glu Asp

Trp Thr Phe

Ala Pro Ser 115

Gly Thr Ala 130

Ala Lys Val 145

Gln Glu Ser

Ser Ser Thr

Tyr Ala Cys 195

Ser Phe Asn 210

Phe Ala Val 85

Gly Gln Gly 100

Val Phe Ile

Ser Val Val

Gln Trp Lys

150

Val Thr Glu

165

Leu Thr Leu 180

Glu Val Thr

Arg Gly Glu

Tyr Tyr Cys

Thr Lys Val

105

Phe Pro Pro

120

Cys Leu Leu 135

Val Asp Asn

Gln Asp Ser

Ser Lys Ala

185

His Gln Gly 200

Cys

215

Gln Gln Tyr 90

Glu Ile Lys

Ser Asp Glu

Asn Asn Phe

140

Ala Leu Gln 155

Lys Asp Ser 170

Asp Tyr Glu

Leu Ser Ser

Gly Ser Ser

Arg Thr Val

110

Gln Leu Lys 125

Tyr Pro Arg

Ser Gly Asn

Thr Tyr Ser

175

Lys His Lys

190

Pro Val Thr 205

Pro

Ala

Ser

Glu

Ser 160

Leu

Val

Lys <210> 108 <211> 502 <212> PROTEIN <213> Artificial sequence <220>

< 223> First polypeptide chain TRIDENT B < 400> 108

Glu 1 Ile Val Leu Thr 5 Gln Ser Pro Ala Thr 10 Leu Ser Leu Ser Pro 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Asn 20 25 thirty Gly Met Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro 35 40 45

Gly

Tyr

Pro

- 175 043373

Lys Leu Leu Ile His Ala Ala Ser Asn

55

Arg Phe Ser Gly Ser Gly Ser Gly Thr

70

Ser Leu Glu Pro Glu Asp Phe Ala Val 85

Glu Val Pro Tyr Thr Phe Gly Gly Gly

100 105

Gly Gly Ser Gly Gly Gly Gly Gln Val

115 120

Glu Val Lys Lys Pro Gly Ala Ser Val

130 135

Gly Tyr Ser Phe Thr Ser Tyr Trp Met 145 150

Gly Gln Gly Leu Glu Trp Ile Gly Val

165

Thr Trp Leu Asp Gln Lys Phe Lys Asp

180 185

Lys Ser Thr Ser Thr Ala Tyr Met Glu

195 200

Asp Thr Ala Val Tyr Tyr Cys Ala Arg

210 215

Phe Ala Tyr Trp Gly Gln Gly Thr Leu 225 230

Cys Gly Gly Gly Glu Val Ala Ala Cys

245

Glu Lys Glu Val Ala Ala Leu Glu Lys

260 265

Gly Gly Gly Asp Lys Thr His Thr Cys

275 280

Gly Ser Gly Val Pro Ser 60

Phe Thr Leu Thr Ile Ser

80

Phe Cys Gln Gln Ser Lys 95

Lys Val Glu Ile Lys Gly

110

Leu Val Gln Ser Gly Ala

125

Val Ser Cys Lys Ala Ser

140

Trp Val Arg Gln Ala Pro

155 160

His Pro Ser Asp Ser Glu

175

Val Thr Ile Thr Val Asp

190

Ser Ser Leu Arg Ser Glu

205

His Tyr Gly Thr Ser Pro

220

Thr Val Ser Ser Gly Gly

235 240

Lys Glu Val Ala Ala Leu

255

Val Ala Ala Leu Glu Lys

270

Pro Cys Pro Ala Pro Glu

285

- 176 043373

Ala

Ala

290

Gly

Gly

Pro

Ser

Val

295

Phe

Leu

Phe

Pro

Pro

300

Lys

Pro

Lys

Thr

305

Leu

Tyr

Ile

Thr

Arg 310

Glu

Pro

Glu

Val

Thr

315

Cys

Val

Val

Val

Val

Ser

His

Glu

Asp 325

Pro

Glu

Val

Lys

Phe

330

Asn

Trp

Tyr

Val

Asp 335

Val

Glu

Val

His

340

Asn

Ala

Lys

Thr

Lys 345

Pro

Arg

Glu

Glu

Gln

350

Tyr

Ser

Thr

Tyr 355

Arg

Val

Val

Ser

Val

360

Leu

Thr

Val

Leu

His

365

Gln

Asp

Leu

Asn

370

Gly

Lys

Glu

Tyr

Lys

375

Cys

Lys

Val

Ser

Asn

380

Lys

Ala

Leu

Ala

385

Pro

Ile

Glu

Lys

Thr

390

Ile

Ser

Lys

Ala

Lys 395

Gly

Gln

Pro

Arg

Pro

Gln

Val

Tyr

Thr

405

Leu

Pro

Pro

Ser

Arg 410

Glu

Glu

Met

Thr

Lys

415

Gln

Val

Ser

Leu

420

Trp

Cys

Leu

Val

Lys

425

Gly

Phe

Tyr

Pro

Ser

430

Asp

Ala

Val

Glu

435

Trp

Glu

Ser

Asn

Gly 440

Gln

Pro

Glu

Asn

Asn

445

Tyr

Lys

Thr

Pro

450

Pro

Val

Leu

Asp

Ser

455

Asp

Gly

Ser

Phe

Phe

460

Leu

Tyr

Ser

Leu

465

Thr

Val

Asp

Lys

Ser

470

Arg

Trp

Gln

Gln

Gly 475

Asn

Val

Phe

Ser

Ser

Val

Met

His

Glu

485

Ala

Leu

His

Asn

His

490

Tyr

Thr

Gln

Lys

Ser

495

Asp

Asp 320

Gly

Asn

Trp

Pro

Glu 400

Asn

Ile

Thr

Lys

Cys 480

Leu

Ser

Leu

Ser

Pro

500

Gly

Lys <210>

<211>

<212>

109

448 PROTEIN

- 177 043373

Val Val Gln Pro Gly <213> Artificial Sequence <220>

<223> Third polypeptide chain TRIDENT B <400> 109

Gln Val Gln Leu Val 1 5

Glu Ser Gly Gly Gly

Ser Leu Arg Leu Ser 20

Cys Ala Ala Ser Gly 25

Phe Thr Phe Ser Ser

Thr Met His Trp Val 35

Arg Gln Ala Pro Gly 40

Lys Gly Leu Glu Trp 45

Thr Phe Ile Ser Tyr 50

Asp Gly Ser Asn Lys 55

His Tyr Ala Asp Ser 60

Lys Gly Arg Phe Thr 65

Val Ser Arg Asp Asn 70

Ser Lys Asn Thr Leu 75

Leu Gln Met Asn Ser

Leu Arg Ala Glu Asp 90

Thr Ala Ile Tyr Tyr

Ala Arg Thr Gly Trp

100

Leu Gly Pro Phe Asp

105

Tyr Trp Gly Gln Gly

110

Leu Val Thr Val Ser

115

Ser Ala Ser Thr Lys

120

Gly Pro Ser Val Phe 125

Leu Ala Pro Ser Ser

130

Lys Ser Thr Ser Gly 135

Gly Thr Ala Ala Leu 140

Cys Leu Val Lys Asp 145

Tyr Phe Pro Glu Pro 150

Val Thr Val Ser Trp 155

Ser Gly Ala Leu Thr

165

Ser Gly Val His Thr

170

Phe Pro Ala Val Leu

175

Ser Ser Gly Leu Tyr

180

Ser Leu Ser Ser Val

185

Val Thr Val Pro Ser

190

Ser Leu Gly Thr Gln 195

Thr Tyr Ile Cys Asn 200

Val Asn His Lys Pro

205

Asn Thr Lys Val Asp

Lys Arg Val Glu Pro

Lys Ser Cys Asp Lys

Arg

Tyr

Val

Val

Tyr 80

Cys

Thr

Pro

Gly

Asn 160

Gln

Ser

Ser

Thr

- 178 043373

Ala Ala Gly Gly Pro Ser

235 240

210 215

220

His Thr Cys Pro Pro Cys Pro Ala Pro 225 230

Val Phe Leu Phe Pro Pro Lys Pro Lys

245

Glu Pro Glu Val Thr Cys Val Val Val

260 265

Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280

Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295

Ser Val Leu Thr Val Leu His Gln Asp

305 310

Lys Cys Lys Val Ser Asn Lys Ala Leu

325

Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345

Pro Pro Ser Arg Glu Glu Met Thr Lys

355 360

Ala Val Lys Gly Phe Tyr Pro Ser Asp

370 375

Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390

Ser Asp Gly Ser Phe Phe Leu Val Ser 405

Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425

Leu His Asn Arg Tyr Thr Gln Lys Ser

435 440

Thr Leu Tyr Ile Thr Arg

255

Val Ser His Glu Asp Pro

270

Val Glu Val His Asn Ala

285

Ser Thr Tyr Arg Val Val 300

Leu Asn Gly Lys Glu Tyr

315 320

Ala Pro Ile Glu Lys Thr

335

Pro Gln Val Tyr Thr Leu

350

Gln Val Ser Leu Ser Cys

365

Ala Val Glu Trp Glu Ser

380

Thr Pro Pro Val Leu Asp

395 400

Leu Thr Val Asp Lys Ser

415

Ser Val Met His Glu Ala

430

Ser Leu Ser Pro Gly Lys

445 <210> 110

- 179 043373 <211> 469 <212> PROTEIN <213> Artificial sequence <220>

<223> Third polypeptide chain TRIDENT C <400> 110

Glu Ile Val Leu

Thr Gln Ser Pro 5

Gly Thr Leu Ser 10

Leu Ser Pro Gly 15

Glu Arg Ala Thr 20

Leu Ser Cys Arg

Ala Ser Gln Ser 25

Val Ser Ser Ser 30

Phe Leu Ala Trp 35

Tyr Gln Gln Lys

Pro Gly Gln Ala

Pro Arg Leu Leu 45

Ile Tyr Gly Ala 50

Ser Ser Arg Ala 55

Thr Gly Ile Pro 60

Asp Arg Phe Ser

Gly Ser Gly Ser 65

Gly Thr Asp Phe 70

Thr Leu Thr Ile

Ser Arg Leu Glu 80

Pro Glu Asp Phe

Ala Val Tyr Tyr 85

Cys Gln Gln Tyr 90

Gly Ser Ser Pro 95

Trp Thr Phe Gly

100

Gln Gly Thr Lys

Val Glu Ile Lys 105

Gly Gly Gly Gly

110

Ser Gly Gly Gly

115

Gly Ser Gly Gly

120

Gly Gly Ser Gln

Val Gln Leu Val

125

Glu Ser Gly Gly 130

Gly Val Val Gln

135

Pro Gly Arg Ser

140

Leu Arg Leu Ser

Cys Ala Ala Ser 145

Gly Phe Thr Phe 150

Ser Ser Tyr Thr

155

Met His Trp Val

160

Arg Gln Ala Pro

Gly Lys Gly Leu 165

Glu Trp Val Thr 170

Phe Ile Ser Tyr

175

Asp Gly Ser Asn

180

Lys His Tyr Ala

Asp Ser Val Lys 185

Gly Arg Phe Thr 190

Val Ser Arg Asp

195

Asn Ser Lys Asn

200

Thr Leu Tyr Leu

Gln Met Asn Ser 205

- 180 043373

Leu

Leu 225

Ser

Phe

Thr

Val

Val 305

Ser

Leu

Ser

Pro

Gln 385

Ala

Thr

Leu

Arg Ala Glu 210

Gly Pro Phe

Glu Ser Lys

Leu Gly Gly

260

Leu Tyr Ile 275

Ser Gln Glu 290

Glu Val His

Thr Tyr Arg

Asn Gly Lys

340

Ser Ile Glu

355

Gln Val Tyr 370

Val Ser Leu

Val Glu Trp

Pro Pro Val

420

Thr Val Asp

435

Asp Thr Ala

215

Asp Tyr Trp

230

Tyr Gly Pro 245

Pro Ser Val

Thr Arg Glu

Asp Pro Glu

295

Asn Ala Lys

310

Val Val Ser 325

Glu Tyr Lys

Lys Thr Ile

Thr Leu Pro

375

Ser Cys Ala 390

Glu Ser Asn 405

Leu Asp Ser

Lys Ser Arg

Ile Tyr Tyr

Gly Gln Gly

Pro Cys Pro

250

Phe Leu Phe

265

Pro Glu Val 280

Val Gln Phe

Thr Lys Pro

Val Leu Thr

330

Cys Lys Val

345

Ser Lys Ala 360

Pro Ser Gln

Val Lys Gly

Gly Gln Pro

410

Asp Gly Ser 425

Trp Gln Glu 440

Cys Ala Arg

220

Thr Leu Val 235

Pro Cys Pro

Pro Pro Lys

Thr Cys Val

285

Asn Trp Tyr

300

Arg Glu Glu 315

Val Leu His

Ser Asn Lys

Lys Gly Gln

365

Glu Glu Met

380

Phe Tyr Pro 395

Glu Asn Asn

Phe Phe Leu

Gly Asn Val

445

Thr Gly Trp

Thr Val Ser

240

Ala Pro Glu

255

Pro Lys Asp 270

Val Val Asp

Val Asp Gly

Gln Phe Asn

320

Gln Asp Trp

335

Gly Leu Pro 350

Pro Arg Glu

Thr Lys Asn

Ser Asp Ile

400

Tyr Lys Thr

415

Val Ser Arg 430

Phe Ser Cys

- 181 -

Claims (33)

Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Leu Gly 465 CLAIM 1. Bispecific molecule containing: (A) one or more epitope binding sites capable of immunospecifically binding to a PD-1 epitope comprising the VH domain of PD-1 mAb 6-I VH (SEQ ID NO: 86) and the VL domain of PD-1 mAb 6-SQ ( SEQ ID NO: 87); and (B) one or more epitope binding sites capable of immunospecifically binding to a CTLA-4 epitope comprising the VH domain of CTLA-4 mAb 3 (SEQ ID NO: 90) and the VL domain of CTLA-4 mAb 3 (SEQ ID NO: 91). 2. A bispecific molecule according to claim 1, characterized in that said molecule contains an Fc fragment. 3. A bispecific molecule according to claim 2, characterized in that said Fc fragment is a variant of the Fc fragment which contains: one or more amino acid modifications that reduce the affinity of the Fc fragment variant for FcyR, or one or more amino acid modifications that increase the serum half-life of the Fc fragment variant, or one or more amino acid modifications that reduce the affinity of the Fc fragment variant for FcyR and one or more amino acid modifications that increase the serum half-life of the Fc fragment variant. 4. The bispecific molecule according to claim 3, characterized in that said modifications that reduce the affinity of the Fc fragment variant for FcyR include the L234A substitution; L235A or L234A and L235A, where the indicated numbering corresponds to the EC index according to Kabat. 5. The bispecific molecule according to claim 3 or 4, characterized in that said modifications, which increase the half-life of the Fc fragment variant in blood serum, include the replacement of M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E or K288D and H435K, where the indicated numbering corresponds to the Kabat EC index. 6. A bispecific molecule according to any one of claims 1 to 5, containing two polypeptide chains containing SEQ ID NO: 99, and two polypeptide chains containing SEQ ID NO: 100. 7. A bispecific molecule according to any one of claims 1 to 5, containing one polypeptide chain containing SEQ ID NO: 104, one polypeptide chain containing SEQ ID NO: 105, one polypeptide chain containing SEQ ID NO: 106, and one polypeptide chain containing SEQ ID NO: 107. 8. A bispecific molecule according to any one of claims 1 to 5, containing two polypeptide chains containing SEQ ID NO: 101, and two polypeptide chains containing SEQ ID NO: 100. 9. A bispecific molecule according to any one of claims 1 to 5, containing one polypeptide chain containing SEQ ID NO: 108, one polypeptide chain containing SEQ ID NO: 105, one polypeptide chain containing SEQ ID NO: 109, and one polypeptide chain containing SEQ ID NO: 107. 10. A pharmaceutical composition containing a bispecific molecule according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier. 11. The pharmaceutical composition according to claim 10, wherein said pharmaceutical composition contains an effective amount of said bispecific molecule. 12. Use of a pharmaceutical composition according to claim 10 or 11 in the manufacture of a medicament suitable for stimulating an immune system-mediated response. 13. Use of a pharmaceutical composition according to claim 10 or 11 in the manufacture of a medicament suitable for treating a disease or condition associated with suppression of the immune system. 14. Use of a bispecific molecule according to any one of claims 1 to 9 in the manufacture of a medicament suitable for stimulating an immune system-mediated response. 15. Use of a bispecific molecule according to any one of claims 1 to 9 in the manufacture of a medicament useful for treating a disease or condition associated with suppression of the immune system. 16. Use of a pharmaceutical composition according to claim 10 or 11 for stimulating an immune system-mediated response. 17. Use of a pharmaceutical composition according to claim 10 or 11 for the treatment of a disease or condition associated with suppression of the immune system. - 182 043373 18. Use of a bispecific molecule according to any one of claims 1 to 9 for stimulating an immune system-mediated response. 19. Use of a bispecific molecule according to any one of claims 1 to 9 for the treatment of a disease or condition associated with suppression of the immune system. 20. Use according to any one of claims 13, 15, 17 and 19, characterized in that said disease or condition is cancer or infection. 21. The use of claim 20, characterized in that said cancer is characterized by the presence of a cancer cell selected from the group consisting of an adrenal tumor cell, an AIDS-associated cancer, an alveolar soft tissue sarcoma, an astrocytic tumor, a bladder cancer, a bone cancer, a cancer brain and spinal cord, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's sarcoma, extraskeletal mucoid chondrosarcoma, osseous fibrogenesis imperfecta, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germinal cell tumor, head and neck cancer, hepatocellular carcinoma, tumor pancreatic islet cell, Kaposi's sarcoma, kidney cancer, leukemia, lipoma/benign lipomatous tumor, liposarcoma/malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma , neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyrocarcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disorder, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymus cancer, thymoma, metastatic thyroid cancer and uterine cancer. 22. Use according to claim 20, characterized in that said cancer is characterized by the presence of a cancer cell selected from the group consisting of colon and rectal cancer, lung cancer, cervical cancer, head and neck cancer, prostate cancer, sarcoma and thymomas. 23. Use according to claim 20, characterized in that said cancer is characterized by the presence of a cancer cell selected from the group consisting of colon and rectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, bladder cancer , neuroblastoma, sarcoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer and colorectal cancer. 24. Use according to claim 20, characterized in that said cancer is characterized by the presence of a cancer cell selected from the group consisting of colon and rectal cancer, gastric cancer, melanoma, prostate cancer, pancreatic cancer, kidney cancer, urinary cancer bladder, breast cancer, lung cancer, fibrosarcoma, human mantle cell lymphoma and Raji Burkitt lymphoma. 25. Use according to claim 20, characterized in that said infection is characterized by the presence of a bacterial, fungal, viral or protozoal pathogen. 26. A pharmaceutical composition containing a bispecific molecule according to any one of claims 1 to 9 and a prophylactic agent or a therapeutic agent. 27. The pharmaceutical composition according to claim 26, characterized in that said prophylactic agent contains a tumor-specific antibody. 28. A kit for stimulating an immune system-mediated response, comprising a bispecific molecule according to any one of claims 1 to 9 and a prophylactic agent or a therapeutic agent. 29. A kit for treating a disease or condition associated with suppression of the immune system in a subject, comprising a bispecific molecule according to any one of claims 1 to 9 and a prophylactic agent or therapeutic agent. 30. The kit according to claim 28 or 29, characterized in that said prophylactic agent or therapeutic agent contains a tumor-specific antibody. 31. The kit according to any one of claims 28 to 30, characterized in that said bispecific molecule is in a first container and said prophylactic agent or therapeutic agent is in a second container. 32. The kit according to any one of claims 28-30, characterized in that said bispecific molecule and said prophylactic agent or therapeutic agent are in the same container. 33. A method of treating a disease or condition associated with suppression of the immune system in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition according to claim 10. - 183 -