Competitive antigen-binding proteins

Abstract

To provide antagonistic antigen binding proteins.SOLUTION: In one aspect, provided is an antagonistic antigen binding protein that specifically binds to PD-1, the antigen binding protein competing for binding to PD-1 with an antibody comprising a heavy chain variable region of amino acid sequences in a particular sequence and a light chain variable region of amino acid sequences in another particular sequence. Also, provided are a nucleic acid encoding an antagonistic antigen binding protein; a recombinant expression vector comprising the nucleic acid molecule; a host cell containing the vector; a method for producing the above-mentioned antagonistic antigen-binding protein; an antagonistic binding protein produced by the method; and a pharmaceutical composition comprising the antagonistic binding protein, the nucleic acid or the vector. Furthermore, provided are a kit comprising the pharmaceutical composition, and use of the antagonistic binding proteins in the treatment of cancer and / or chronic infection.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to competitive antigen-binding proteins, nucleic acids encoding the competitive binding proteins, recombinant expression vectors comprising the nucleic acid molecules, host cells comprising the vectors, methods for making the competitive antigen-binding proteins, competitive binding proteins produced by the methods, and pharmaceutical compositions comprising the competitive binding proteins, nucleic acids, or vectors. The present invention further relates to kits comprising the pharmaceutical compositions, and the use of the competitive binding proteins in the treatment of cancer and / or chronic infections. [Background technology]

[0002] The activation and proliferation of immune cells involved in anti-tumor responses is controlled by multiple stimulatory and inhibitory pathways that can be targeted by monoclonal antibodies (mAbs) that inhibit immunosuppressive receptors or activate costimulatory modulators (collectively known as immune checkpoints) expressed on the surface of T and B lymphocytes or NK cells, enhancing their tumor-specific responses.

[0003] Translating this concept into the clinic has led to the development of novel and effective immunotherapies. To date, three classes of human or humanized monoclonal antibodies targeting immunosuppressive receptors, such as CTLA4 (ipilimumab), PD-1 (nivolumab and pembrolizumab), and PD-L1 (atezolizumab, durvalumab, and avelumab), have been approved for the treatment of several tumors, including melanoma, NSCLC, RCC, head and neck squamous cell carcinoma, Hodgkin lymphoma, urothelial carcinoma, MSI CRC, and Merkel cell carcinoma.

[0004] Spurred by the remarkable success of these immunotherapies, many more antibodies against other immune regulatory receptors are being brought into the clinic in the hope of finding even more effective treatments by targeting novel immune pathways. In fact, despite their success, currently approved antibodies against immune checkpoints (hence collectively referred to as checkpoint inhibitors (CIs)) are effective in only approximately 20%–30% of patients. Among these new targets are co-inhibitory receptors such as lymphocyte-activation gene 3 (LAG3), an immunoinhibitory receptor expressed on activated T lymphocytes and T regulatory lymphocytes; T cell immunoglobulin 3 (TIM-3); and T cell immunoglobulin and ITIM domain (TIGIT), expressed on exhausted CD8+ T cells in cancer. In addition to improving CD8+ T cell function, blockade of Lag-3, TIM-3, and TIGIT is predicted to affect Treg cells and IL-10-producing Tr1 cells in tumor tissue. B and T lymphocyte attenuator (BTLA) is another co-inhibitory receptor whose expression is induced during T cell activation and leads to the inhibition of human CD8+ cancer-specific T cells. BTLA interacts with the B7 homolog B7H4, but unlike PD-1 and CTLA-4, BTLA exerts T cell inhibition not only through interactions with the B7 family of cell surface receptors but also with tumor necrosis factor receptors (TNF-R).

[0005] Similarly, agonistic antibodies that recognize costimulatory receptors such as OX40, a secondary costimulatory immune checkpoint molecule that prevents premature death of activated lymphocytes, and 41BB, expressed on activated CD4 and CD8 T lymphocytes, have reached the clinical stage. Other costimulatory proteins considered excellent targets for antibody-mediated immunotherapy are inducible T-cell costimulator (ICOS), an immune checkpoint protein belonging to the CD28 superfamily, and CD27, a member of the tumor necrosis factor receptor superfamily.

[0006] Cancer immunotherapy based on immune-modulating antibodies is becoming a core component of modern oncology. Checkpoint inhibitors (i.e., anti-PD-1, anti-PD-L1, and anti-CTLA4) have been shown to achieve long-lasting clinical benefits with unprecedented efficacy. Perhaps most importantly, these antibodies have a very broad range of applications, with currently approved antibodies being used to treat several tumors. Despite this, therapeutic benefit with CIs remains limited to 20%–30% of the population and has not been demonstrated in common cancer types such as colorectal, breast, and prostate cancer. However, the recent expansion of the library of monoclonal antibodies against novel targets with agonistic or antagonistic activity against costimulatory or inhibitory receptors, respectively, has made it possible to design novel combination therapies that enhance the potential of immune-based cancer therapy.

[0007] Combining antibodies against different immune checkpoint receptors may also achieve additive or synergistic activity, potentially leading to greater efficacy. Proof of concept for this approach was provided by the finding that the efficacy of ipilimumab and nivolumab combination therapy was increased versus monotherapy in the treatment of metastatic melanoma.

[0008] Currently, there are over 1000 clinical trials underway using approved or novel antibodies against immunomodulatory receptors, used as monotherapy or in combination with other biologics or small molecules. Because a large set of antibodies against various immune checkpoints is not available in a single laboratory, most of these trials are conducted without preclinical head-to-head comparisons that would allow prediction of the most effective treatment.

[0009] Using a strategy for rapid parallel screening of phage-displayed antibody libraries, we generated a large repertoire of fully human monoclonal antibodies against several immune regulatory checkpoints by directly panning against activated human lymphocytes. The monoclonal antibodies thus selected were demonstrated to have high binding affinity for their targets and improved functional activity on lymphocytes. Summary of the Invention

[0010] In a first aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein exhibits, for binding to PD-1: (i) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 3; or (ii) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12; The present invention provides an antagonistic antigen-binding protein that competes with the antibody.

[0011] In a second aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein exhibits, for binding to PD-L1: (i) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 20 and a light chain variable region of the amino acid sequence of SEQ ID NO: 21; or (ii) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 29 and a light chain variable region of the amino acid sequence of SEQ ID NO: 30; or (iii) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 83 and a light chain variable region of the amino acid sequence of SEQ ID NO: 84; or (iv) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 92 and a light chain variable region of the amino acid sequence of SEQ ID NO: 93; or (v) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 101 and a light chain variable region of the amino acid sequence of SEQ ID NO: 102; It relates to an antagonistic antigen-binding protein that competes with

[0012] In a third aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein exhibits, for binding to LAG-3: (i) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 38 and a light chain variable region of the amino acid sequence of SEQ ID NO: 39; (ii) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 47 and a light chain variable region of the amino acid sequence of SEQ ID NO: 48; (iii) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 56 and a light chain variable region of the amino acid sequence of SEQ ID NO: 57; (iv) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 65 and a light chain variable region of the amino acid sequence of SEQ ID NO: 66; or (v) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 74 and a light chain variable region of the amino acid sequence of SEQ ID NO: 75; It relates to an antagonistic antigen-binding protein that competes with

[0013] In a fourth aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to PD-1, the antigen binding protein comprising: (i) A combination of: a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 3 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 2; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 12 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 11; a combination of a light chain variable domain and a heavy chain variable domain selected from the group (ii) A combination of: a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 18; a combination of heavy chain complementarity determining region 3 (CDRH3) and light chain complementarity determining region 3 (CDRL3) selected from the group consisting of: The present invention relates to an antagonistic antigen-binding protein comprising any one of:

[0014] In a fifth aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to PD-L1, the antigen binding protein comprising: (i) A combination of: a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 21 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 20; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 30 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 29; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 84 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 83; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 93 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 92; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 102 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 101; a combination of a light chain variable domain and a heavy chain variable domain selected from the group (ii) A combination of: a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 89 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 90; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 99; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 107 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 108; a combination of heavy chain complementarity determining region 3 (CDRH3) and light chain complementarity determining region 3 (CDRL3) selected from the group consisting of: The present invention relates to an antagonistic antigen-binding protein comprising any one of:

[0015] In a sixth aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to LAG-3, the antigen binding protein comprising: (i) A combination of: a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 39 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 38; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 48 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 47; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 57 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 56; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 66 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 65; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 75 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 74; a combination of a light chain variable domain and a heavy chain variable domain selected from the group (ii) A combination of: a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 63; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 72; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 80 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 81; a combination of heavy chain complementarity determining region 3 (CDRH3) and light chain complementarity determining region 3 (CDRL3) selected from the group consisting of: The present invention relates to an antagonistic antigen-binding protein comprising any one of:

[0016] In a seventh aspect, the present invention relates to a nucleic acid encoding the antagonistic antigen-binding protein of any one of aspects 1 to 6 of the present invention.

[0017] In an eighth aspect, the present invention relates to a recombinant expression vector comprising a nucleic acid molecule according to the seventh aspect.

[0018] In a ninth aspect, the present invention relates to a host cell comprising the vector of the eighth aspect of the invention.

[0019] In a tenth aspect, the present invention relates to a method of producing an antagonistic antigen-binding protein of any one of aspects 1 to 6 of the present invention, the method comprising the step of preparing said antigen-binding protein from a host cell that expresses said antigen-binding protein.

[0020] In an eleventh aspect, the present invention relates to an antagonistic antigen-binding protein produced by expression of recombinant DNA in a host cell of the ninth aspect of the invention.

[0021] In a twelfth aspect, the present invention relates to a pharmaceutical composition comprising at least one antagonistic antigen-binding protein according to any one of aspects 1 to 6 of the invention, a nucleic acid according to the seventh aspect of the invention, or a vector according to the eighth aspect of the invention, and a pharmaceutically acceptable carrier.

[0022] In a thirteenth aspect, the present invention relates to a kit comprising a pharmaceutical composition according to the twelfth aspect of the invention and, optionally, at least one further active agent.

[0023] In a fourteenth aspect, the present invention relates to an antagonistic antigen-binding protein according to any one of aspects 1 to 6 of the invention, a nucleic acid according to the seventh aspect of the invention, a vector according to the eighth aspect of the invention or a pharmaceutical composition according to the twelfth aspect of the invention for use in the treatment of cancer and / or chronic infections.

[0024] The contents of the drawings contained in this specification are described below, and in this connection reference is also made to the detailed description of the invention set forth above and / or below. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1. Immunomic screening. The screening procedure started with a universal cycle common to the various targets (inner circle) performed by panning the unselected library against activated PBMCs. Each diverging sector represents the enrichment profile of the first to ten scFv clones for the indicated target, scored according to counts per million. The lines within each sector connect the individual enrichments obtained after cycle 2 (small circle) and cycle 3 (large circle). Both cycles 2 and 3 were performed on recombinant proteins. [Figure 2-1] Figure 1 shows the binding affinity of selected antibodies to lymphocytes. ELISA assays were performed on activated hPBMCs (black curve) or naive hPBMCs (gray curve) using increasing concentrations of monoclonal antibodies (7.5 nM, 25 nM, 50 nM, 100 nM). Antibodies were also tested at increasing concentrations against each recombinant protein / Fc by ELISA assay, and Kd values ​​are reported in the table. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 3] Figure 1 shows proliferation of hPBMCs following stimulation with 2.5 μg / ml PHA in the absence or presence of immunomodulatory antibodies. The fold increase in CD3- T cell proliferation, as determined by selected antibodies shown, was measured for activation of hPBMCs with 2.5 μg / ml PHA in the absence of antibody or in the presence of irrelevant IgG4. [Figure 4]Figure 1 shows the effect of anti-PD-1 and anti-PD-L1 antibodies on tumor cell-induced lymphocyte proliferation. Fold-increase in hPBMC proliferation as determined by normalized absorbance values ​​obtained by ELISA using anti-BrdU antibody in hPBMC samples co-cultured with MDA-MB-231 (A) or MCF-7 (B) tumor cells for 72 hours at 37°C in the absence of antibody (white bars) or in the presence of increasing concentrations (50 nM and 200 nM) of PD-L1_A (light gray bars) or PD-1_A (gray bars) antibodies. In both experiments, nivolumab was used as a positive control (black bars). [Figure 5] Figure 1 shows the effect of novel immunomodulatory antibodies on cytokine secretion by stimulated T cells. IL-2 and IFNγ values ​​obtained by ELISA assay on supernatants of hPBMCs stimulated with PHA (2.5 μg / mL) or SEB (50 ng / mL) for 18 to 66 hours at 37°C in the absence of antibody or in the presence of antibodies LAG-3_A, PD-1_A, PD-1_B, PD-L1_A, and PD-L1_B. Nivolumab and an irrelevant antibody were used as positive and negative controls, respectively. [Figure 6] Figure 1 shows the in vivo anti-tumor activity of PD-1_A and PD-L1_A antibodies. Tumor growth in mice inoculated with CT26 cells on day 0 and treated with PD-1_A, PD-L1_A, or positive control antibodies reactive against mouse PD-1 and PD-L1 (α-mPD-1, α-mPD-L1) on days 3, 6, and 10. Tumor volumes 21 days after tumor challenge are shown. [Figure 7]Figure 7 shows the binding of anti-PD-L1 antibodies to lymphocytes. To evaluate the ability of antibodies PD-L1_C, PD-L1_D, and PD-L1_E to bind to PD-L1 protein compared to PD-L1_A, binding curves were generated for all of these monoclonal antibodies on human activated lymphocytes. Human PBMCs isolated from healthy donors were activated with anti-CD3 / CD28 beads to stimulate PD-L1 expression. PD-L1_A, PD-L1_C, PD-L1_D, and PD-L1_E antibodies were then added at a range of concentrations and analyzed by flow cytometry (CytoFLEX flow cytometer, Beckman Coulter). The mean fluorescence intensity of lymphocytes binding PD-L1 was plotted against antibody concentration (Figures 7A and 7B). [Figure 8] Figure 1 shows the effect of anti-PD-L1 antibodies on cytokine secretion by stimulated T cells. IL-2 and IFNγ levels obtained by ELISA assay on supernatants of hPBMCs stimulated with PHA (2.5 μg / mL) or SEB (50 ng / mL) for 18 to 66 hours at 37°C in the absence of antibody or in the presence of antibodies PD-L1_A, PD-L1_C, and PD-L1_D. Nivolumab and an irrelevant antibody were used as positive and negative controls, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0026] List of sequences SEQ ID NO: 1: Amino acid sequence of PD-1_A SEQ ID NO: 2 PD-1_A; amino acid sequence of VH SEQ ID NO: 3: Amino acid sequence of PD-1_A;VL SEQ ID NO: 4: PD-1_A; amino acid sequence of CDRH1 SEQ ID NO: 5 PD-1_A; amino acid sequence of CDRL1 SEQ ID NO: 6: PD-1_A; amino acid sequence of CDRH2 SEQ ID NO: 7: PD-1_A; amino acid sequence of CDRL2 SEQ ID NO: 8: PD-1_A; amino acid sequence of CDRH3 SEQ ID NO: 9: PD-1_A; amino acid sequence of CDRL3 SEQ ID NO: 10: Amino acid sequence of PD-1_B SEQ ID NO: 11: PD-1_B; amino acid sequence of VH SEQ ID NO: 12: Amino acid sequence of PD-1_B;VL SEQ ID NO: 13: PD-1_B; amino acid sequence of CDRH1 SEQ ID NO: 14: PD-1_B; amino acid sequence of CDRL1 SEQ ID NO: 15: PD-1_B; amino acid sequence of CDRH2 SEQ ID NO: 16: PD-1_B; amino acid sequence of CDRL2 SEQ ID NO: 17: PD-1_B; amino acid sequence of CDRH3 SEQ ID NO: 18: PD-1_B; amino acid sequence of CDRL3 SEQ ID NO: 19: Amino acid sequence of PD-L1_A SEQ ID NO: 20: PD-L1_A; amino acid sequence of VH SEQ ID NO: 21: PD-L1_A; amino acid sequence of VL SEQ ID NO: 22: PD-L1_A; amino acid sequence of CDRH1 SEQ ID NO: 23 PD-L1_A; amino acid sequence of CDRL1 SEQ ID NO: 24: Amino acid sequence of PD-L1_A; CDRH2 SEQ ID NO: 25: PD-L1_A; amino acid sequence of CDRL2 SEQ ID NO: 26: Amino acid sequence of PD-L1_A; CDRH3 SEQ ID NO: 27: PD-L1_A; amino acid sequence of CDRL3 SEQ ID NO: 28: Amino acid sequence of PD-L1_B SEQ ID NO: 29: PD-L1_B; amino acid sequence of VH SEQ ID NO: 30: PD-L1_B; amino acid sequence of VL SEQ ID NO: 31: PD-L1_B; amino acid sequence of CDRH1 SEQ ID NO: 32: PD-L1_B; amino acid sequence of CDRL1 SEQ ID NO: 33: Amino acid sequence of PD-L1_B; CDRH2 SEQ ID NO: 34: Amino acid sequence of PD-L1_B; CDRL2 SEQ ID NO: 35: Amino acid sequence of PD-L1_B; CDRH3 SEQ ID NO: 36: PD-L1_B; amino acid sequence of CDRL3 SEQ ID NO: 37 Amino acid sequence of LAG-3_A SEQ ID NO: 38 LAG-3_A; amino acid sequence of VH SEQ ID NO: 39 LAG-3_A; amino acid sequence of VL SEQ ID NO: 40 LAG-3_A; amino acid sequence of CDRH1 SEQ ID NO: 41 LAG-3_A; amino acid sequence of CDRL1 SEQ ID NO: 42 LAG-3_A; amino acid sequence of CDRH2 SEQ ID NO: 43 LAG-3_A; amino acid sequence of CDRL2 SEQ ID NO: 44 LAG-3_A; amino acid sequence of CDRH3 SEQ ID NO: 45 LAG-3_A; amino acid sequence of CDRL3 SEQ ID NO: 46: Amino acid sequence of LAG-3_C SEQ ID NO: 47 LAG-3_C; amino acid sequence of VH SEQ ID NO: 48 LAG-3_C; amino acid sequence of VL SEQ ID NO: 49 LAG-3_C; amino acid sequence of CDRH1 SEQ ID NO: 50 LAG-3_C; amino acid sequence of CDRL1 SEQ ID NO: 51 LAG-3_C; amino acid sequence of CDRH2 SEQ ID NO: 52 LAG-3_C; amino acid sequence of CDRL2 SEQ ID NO: 53 LAG-3_C; amino acid sequence of CDRH3 SEQ ID NO: 54 LAG-3_C; amino acid sequence of CDRL3 SEQ ID NO: 55 Amino acid sequence of LAG-3_D SEQ ID NO: 56 LAG-3_D; amino acid sequence of VH SEQ ID NO: 57 LAG-3_D; amino acid sequence of VL SEQ ID NO: 58 LAG-3_D; amino acid sequence of CDRH1 SEQ ID NO: 59 LAG-3_D; amino acid sequence of CDRL1 SEQ ID NO: 60 LAG-3_D; amino acid sequence of CDRH2 SEQ ID NO: 61 LAG-3_D; amino acid sequence of CDRL2 SEQ ID NO: 62 LAG-3_D; amino acid sequence of CDRH3 SEQ ID NO: 63 LAG-3_D; amino acid sequence of CDRL3 SEQ ID NO: 64 Amino acid sequence of LAG-3_B SEQ ID NO: 65 LAG-3_B; amino acid sequence of VH SEQ ID NO: 66: LAG-3_B; amino acid sequence of VL SEQ ID NO: 67 LAG-3_B; amino acid sequence of CDRH1 SEQ ID NO: 68 LAG-3_B; amino acid sequence of CDRL1 SEQ ID NO: 69 LAG-3_B; amino acid sequence of CDRH2 SEQ ID NO: 70 LAG-3_B; amino acid sequence of CDRL2 SEQ ID NO: 71 LAG-3_B; amino acid sequence of CDRH3 SEQ ID NO: 72 LAG-3_B; amino acid sequence of CDRL3 SEQ ID NO: 73 Amino acid sequence of LAG-3_E SEQ ID NO: 74 LAG-3_E; amino acid sequence of VH SEQ ID NO: 75 LAG-3_E; amino acid sequence of VL SEQ ID NO: 76 LAG-3_E; amino acid sequence of CDRH1 SEQ ID NO: 77 LAG-3_E; amino acid sequence of CDRL1 SEQ ID NO: 78 LAG-3_E; amino acid sequence of CDRH2 SEQ ID NO: 79 LAG-3_E; amino acid sequence of CDRL2 SEQ ID NO: 80 LAG-3_E; amino acid sequence of CDRH3 SEQ ID NO: 81 LAG-3_E; amino acid sequence of CDRL3 SEQ ID NO: 82: Amino acid sequence of PD-L1_C SEQ ID NO: 83: PD-L1_C; amino acid sequence of VH SEQ ID NO: 84: Amino acid sequence of PD-L1_C;VL SEQ ID NO: 85: PD-L1_C; amino acid sequence of CDRH1 SEQ ID NO: 86 PD-L1_C; amino acid sequence of CDRL1 SEQ ID NO: 87: Amino acid sequence of PD-L1_C;CDRH2 SEQ ID NO: 88: PD-L1_C; amino acid sequence of CDRL2 SEQ ID NO: 89: Amino acid sequence of PD-L1_C; CDRH3 SEQ ID NO: 90: PD-L1_C; amino acid sequence of CDRL3 SEQ ID NO: 91: Amino acid sequence of PD-L1_D SEQ ID NO: 92: PD-L1_D; amino acid sequence of VH SEQ ID NO: 93: Amino acid sequence of PD-L1_D;VL SEQ ID NO: 94: PD-L1_D; amino acid sequence of CDRH1 SEQ ID NO: 95 PD-L1_D; amino acid sequence of CDRL1 SEQ ID NO: 96: Amino acid sequence of PD-L1_D;CDRH2 SEQ ID NO: 97: PD-L1_D; amino acid sequence of CDRL2 SEQ ID NO: 98: PD-L1_D; amino acid sequence of CDRH3 SEQ ID NO: 99: PD-L1_D; amino acid sequence of CDRL3 SEQ ID NO: 100: Amino acid sequence of PD-L1_E SEQ ID NO: 101 PD-L1_E; amino acid sequence of VH SEQ ID NO: 102: PD-L1_E; amino acid sequence of VL SEQ ID NO: 103 PD-L1_E; amino acid sequence of CDRH1 SEQ ID NO: 104 PD-L1_E; amino acid sequence of CDRL1 SEQ ID NO: 105: PD-L1_E; amino acid sequence of CDRH2 SEQ ID NO: 106: PD-L1_E; amino acid sequence of CDRL2 SEQ ID NO: 107: PD-L1_E; amino acid sequence of CDRH3 SEQ ID NO: 108 PD-L1_E; amino acid sequence of CDRL3

[0027] Before describing the present invention in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0028] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0029] Throughout the remainder of this specification and the claims, unless the context otherwise requires, the terms "comprise" and variants such as "comprises" and "comprising" shall be interpreted as meaning the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. The following sections define various aspects of the invention in more detail. Each aspect thus defined may be combined with any other one or more aspects, unless expressly indicated otherwise. In particular, any feature indicated as optional, preferred, or advantageous may be combined with any other feature or features indicated as optional, preferred, or advantageous.

[0030] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as "incorporated herein by reference." In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching recited herein, the text of this specification shall control.

[0031] The elements of the present invention are described below. While these elements are listed with specific embodiments, it is understood that these elements can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments are not to be construed as limiting the invention to only the illustratively described embodiments. The specification should be understood to support and encompass embodiments combining the illustratively described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, all permutations and combinations of all elements described in this application are deemed to be disclosed by the specification of this application.

[0032] definition Below are definitions of some of the terms frequently used herein, which will have their defined and preferred meanings in the remainder of the specification, wherever they are used.

[0033] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0034] As used herein, the term "antagonistic" refers to any molecule or portion of a molecule that blocks or attenuates a biological response by binding to and blocking a second molecule that normally provides the biological response. The term "antagonistic" includes "competitive antagonists," "non-competitive antagonists," "uncompetitive antagonists," "partial agonists," and "inverse agonists."

[0035] As used herein, the term "PD-1" refers to programmed cell death protein 1, also known as CD279.

[0036] As used herein, the term "PD-L1" refers to programmed death-ligand 1, also known as CD274 or B7 homolog 1 (B7-H1).

[0037] As used herein, the term "LAG-3" refers to lymphocyte activation gene 3, also known as CD223.

[0038] The term "antigen-binding protein," as used herein, refers to any molecule or portion of a molecule that can specifically bind to a target molecule or target epitope. Preferred binding proteins in the context of this application are (a) an antibody or antigen-binding fragment thereof, (b) an oligonucleotide, (c) an antibody-like protein, or (d) a peptidomimetic.

[0039] As used herein, a first compound (e.g., an antibody) has a dissociation constant Kd for a second compound (e.g., an antigen such as a target protein) of 1 mM or less, preferably 100 μM or less, preferably 50 μM or less, preferably 30 μM or less, preferably 20 μM or less, preferably 10 μM or less, preferably 5 μM or less, more preferably 1 μM or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, more preferably 200 nM or less, even more preferably 100 nM or less, even more preferably 90 nM or less, even more preferably 80 nM or less, even more preferably 70 nM or less, even more preferably 60 nM or less, even more preferably 50 nM or less, even more preferably 40 nM or less, even more preferably 30 nM or less, even more preferably 20 nM or less, even more preferably 10 A compound is considered to "bind" to said second compound when its binding is at or below nM.

[0040] Typically, the first compound (e.g., an antibody) has a dissociation constant Kd for the second compound (e.g., an antigen such as a target protein) of between 10 nM and 1 mM, between 10 nM and 100 μM, between 10 nM and 50 μM, between 10 nM and 1 μM, preferably between 10 nM and 900 nM, between 10 nM and 800 nM, between 10 nM and 700 nM, between 10 nM and 600 nM, more preferably between 10 nM and 500 nM, between 10 nM and 400 nM, between 10 nM and 300 nM, between 10 nM and 200 nM, between 10 nM and 100 nM, for example, between 10 nM and 90 nM, between 10 nM and 80 nM, between 10 nM and 70 nM, between 10 nM and 60 nM, between 10 nM and 50 nM, between 10 nM and 50 nM, A compound is considered to "bind" to the second compound when its binding affinity is between 10 nM and 40 nM, between 10 nM and 30 nM, or between 10 nM and 20 nM. The term "binding" according to the present invention preferably relates to specific binding. "Specific binding" means that a binding protein (e.g., an antibody) binds more strongly to a specific target, such as an epitope, than to another target. A binding protein binds to a first target more strongly than a second target if the binding protein binds to the first target with a dissociation constant (Kd) lower than the dissociation constant for the second target. Preferably, the dissociation constant (Kd) for a target to which the binding protein specifically binds is more than 10-fold, preferably more than 20-fold, more preferably more than 50-fold, even more preferably more than 100-fold, more than 200-fold, more than 500-fold, or more than 1000-fold lower than the dissociation constant (Kd) for a target to which the binding protein does not specifically bind.

[0041] For example, the dissociation constant (Kd) for a target to which the binding protein specifically binds is 10 to 1000 times lower than the dissociation constant (Kd) for a target to which the binding protein does not specifically bind, e.g., 20 to 1000 times, 50 to 1000 times, 100 to 1000 times, 200 to 1000 times, 300 to 1000 times, 400 to 1000 times, or 500 to 1000 times lower than the dissociation constant (Kd) for a target to which the binding protein does not specifically bind.

[0042] As used herein, the term "Kd" (measured in "mol / L" (sometimes abbreviated as "M")) is intended to refer to the dissociation equilibrium constant of a specific interaction between a binding protein (e.g., an antibody or fragment thereof) and a target molecule (e.g., an antigen or epitope thereof). Methods for determining the binding affinity of a compound, i.e., determining the dissociation constant Kd, are known to those skilled in the art and may be selected, for example, from the following methods known in the art: surface plasmon resonance (SPR)-based techniques, biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), flow cytometry, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). In the context of the present application, the "Kd" value is determined by surface plasmon resonance spectroscopy (Biacore™) at room temperature (25°C). For example, surface plasmon resonance analysis is typically performed at 25°C on a Biacore X100 instrument (GE Healthcare) equipped with, for example, a CM5 sensor chip (GE Healthcare), using, for example, HBS-EP buffer (10 mM Hepes, 0.15 M NaCl, 3 mM EDTA, and 0.05% surfactant P20, pH 7.4) as the running buffer (GE Healthcare).

[0043] The "IC50" value refers to the half-maximal inhibitory concentration of a substance, which is a measure of the effectiveness of the substance in inhibiting a specific biological or biochemical function. This value is usually expressed as a molar concentration. The IC50 of a drug can be determined by constructing a dose-response curve in a functional antagonism assay and examining the inhibitory effect of the test substance at various concentrations. Alternatively, a competitive binding assay can be performed to determine the IC50 value. Typically, the inhibitory antibodies of the present invention exhibit IC50 values ​​between 50 nM and 1 pM, more preferably between 10 nM and 10 pM, and even more preferably between 1 nM and 50 pM, i.e., 50 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, or 1 pM.

[0044] Cell proliferation is recognized as a reliable means of monitoring T lymphocyte activation. T lymphocyte proliferation is effectively monitored using CFSE carboxyfluorescein succinimidyl ester as described in Nat. Prot. 2007; 2(9):2049-56.

[0045] Secretion of IL-2 or IFN-γ is another reliable means of monitoring T lymphocyte activation. Detection of secreted cytokine proteins is the most widely used type of analysis to date. Because secreted proteins are biologically relevant, their detection most closely reflects the target to which the cells are responding. Secreted proteins are typically measured by ELISA. Herein, hPBMCs (1 × 10 cells) were cultured and stimulated with 2.5 μg / mL PHA-L or 50 ng / mL Staphylococcal Enterotoxin B (SEB) for 18, 42, and 66 hours in the absence of antibody or in the presence of selected anti-LAG-3 monoclonal antibodies, anti-PD-L1 monoclonal antibodies, and anti-PD-1 monoclonal antibodies (20 μg / mL), or an isotype control antibody used as a negative control. Nivolumab was tested as a positive control in parallel assays under the same conditions. The concentrations of IL-2 or IFNγ in cell culture supernatants were determined by ELISA assay (DuoSet ELISA, R&D Systems) by comparison with a standard curve according to the manufacturer's recommendations. Concentration values ​​were reported as the mean of at least three determinations (standard deviation ≤ 5%).

[0046] In animal studies, tumor size is used to assess response to anticancer therapy. The current standard technique for determining the volume of subcutaneous xenograft tumors in vivo is by external caliper, where tumor volume is calculated using the modified ellipsoid formula 1 / 2 (length × width 2).

[0047] The term "compete," when used in the context of antigen binding proteins competing for the same epitope, refers to competition between the antigen binding proteins as determined by an assay in which the antigen binding protein being tested (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., PD-1, PD-L1 and / or LAG-3 or a fragment thereof). There are many types of competitive binding assays, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct label assay, solid-phase direct label sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct label RIA using I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15), solid-phase direct biotin-avidin EIA (see, e.g., Cheung,

[0010] Competitive inhibition can be measured by direct labeling RIA (see, e.g., Moldenhauer et al., 1990, Virology 176:546-552) and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82) to determine whether one antigen-binding protein competes with another. Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen-binding protein, and a labeled reference antigen-binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess.Antigen-binding proteins identified by competition assays (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein and antigen-binding proteins that bind to an adjacent epitope that is sufficiently proximal to the epitope bound by the reference antigen-binding protein to sterically hinder it. Typically, when the competing antigen-binding protein is present in excess, the competing antigen-binding protein inhibits (e.g., reduces) specific binding of the reference antigen-binding protein to PD-1, PD-L1, and / or LAG-3 or extracellular fragments thereof by at least about 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, or 70% to 75%, e.g., about 75% or more. In some examples, binding is inhibited by at least about 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 97%, e.g., about 97% or more.

[0048] An "epitope," also known as an antigenic determinant, is a portion of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or T cells. As used herein, an "epitope" is a portion of a macromolecule that is capable of binding to a binding protein (e.g., an antibody or antigen-binding fragment thereof) as described herein. In this context, the term "binding" preferably relates to specific binding. Epitopes usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding of conformational epitopes, but not nonconformational epitopes, is lost in the presence of denaturing solvents.

[0049] As used herein, a "conformational epitope" refers to an epitope of a linear polymer (e.g., a polypeptide), which epitope is formed by the three-dimensional structure of said polymer. In the context of the present application, a "conformational epitope" is a "discontinuous epitope," i.e., a conformational epitope in a polymer (e.g., a polypeptide) that is formed from at least two separate regions in the primary sequence of the polymer (e.g., the amino acid sequence of the polypeptide). In other words, an epitope is considered to be a "conformational epitope" in the context of the present invention if it consists of at least two separate regions in the primary sequence to which a binding protein of the invention (e.g., an antibody or antigen-binding fragment thereof) simultaneously binds (these at least two separate regions are interrupted by one or more regions in the primary sequence to which a binding protein of the invention does not bind). Preferably, such a "conformational epitope" is present on a polypeptide, and two separate regions in the primary sequence are two separate amino acid sequences to which a binding protein of the invention (e.g., an antibody or antigen-binding fragment thereof) binds (these at least two separate amino acid sequences are interrupted by one or more amino acid sequences in the primary sequence to which the binding protein of the invention does not bind). Preferably, the interrupting amino acid sequences are contiguous amino acid sequences comprising two or more amino acids to which the binding protein of the invention does not bind. The at least two separate amino acid sequences to which the binding protein of the invention binds are not particularly limited in length. Such separate amino acid sequences may consist of only one amino acid, provided that the total number of amino acids in the at least two separate amino acid sequences is large enough to achieve specific binding between the binding protein and the conformational epitope.

[0050] A "paratope" is the portion of an antibody that recognizes an epitope. In the context of the present invention, a "paratope" is the portion of a binding protein as described herein (e.g., an antibody or antigen-binding fragment thereof) that recognizes an epitope.

[0051] The term "antibody" typically refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, inter-connected by disulfide bonds. The term "antibody" also includes all recombinant forms of antibodies, particularly the antibodies described herein, such as antibodies expressed in prokaryotes, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives described below. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability (termed complementarity-determining regions (CDRs)) separated by more conserved regions (termed framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0052] The term "antigen-binding fragment" (or simply "binding portion") of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) an F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH domains; (iv) an Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a combination of two or more isolated CDRs, which can optionally be linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker, allowing them to be produced using recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. A further example is a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region.Binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies are screened. A further example of an "antigen-binding fragment" is a so-called microantibody derived from a single CDR. For example, Heap et al., 2005, described a 17-amino acid residue microantibody derived from the heavy chain CDR3 of an antibody against the gp120 envelope glycoprotein of HIV-1. Other examples include miniantibody mimetics comprising two or more CDR regions fused together, preferably by cognate framework regions. Such a miniantibody mimic comprises the VH CDR1 and VL CDR3 linked by the cognate VH FR2, as described by Qiu et al., 2007.

[0053] The immunoglobulin molecules of the present invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, preferably IgG2a and IgG2b, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0054] Antibodies and antigen-binding fragments thereof that can be used in the present invention can be from any animal origin, including birds and mammals. Preferably, the antibodies or fragments are from human, chimpanzee, rodent (e.g., mouse, rat, guinea pig, or rabbit), chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog origin. It is particularly preferred that the antibodies are of human or murine origin. Antibodies of the present invention also include chimeric molecules that combine an antibody constant region from one species, preferably human, with an antigen-binding site from another species, such as mouse. Furthermore, antibodies of the present invention include humanized molecules that combine the antigen-binding site of an antibody from a non-human species (e.g., mouse) with constant and framework regions of human origin.

[0055] As exemplified herein, the antibodies of the present invention can be obtained directly from hybridomas expressing the antibodies, or can be cloned and expressed recombinantly in host cells (e.g., CHO cells or lymphocytic cells). Further examples of host cells are microorganisms, such as E. coli, and fungi, such as yeast. Alternatively, the antibodies can be produced recombinantly in transgenic non-human animals or plants.

[0056] The term "chimeric antibody" refers to an antibody in which a portion of each of the heavy and light chain amino acid sequences is homologous to corresponding sequences in antibodies from a particular species or belonging to a particular class, while the remaining segments of the chains are homologous to corresponding sequences in antibodies from another species or class. Typically, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from one mammalian species, while the constant regions are homologous to sequences in antibodies from another species. One obvious advantage of such chimeric forms is that the variable regions can be conveniently obtained from currently known sources, for example, using readily available B cells or hybridomas from non-human host organisms, in combination with constant regions from human cell preparations. While the variable regions have the advantage of being easily prepared and their specificity is unaffected by the source, constant regions from a human species are less likely to elicit an immune response from a human subject upon injection of the antibody than constant regions from non-human sources. However, the definition is not limited to this specific example.

[0057] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin of a non-human species, with the remaining immunoglobulin structure of the molecule being based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either complete variable domains fused onto constant domains, or only complementarity-determining regions (CDRs) grafted onto appropriate framework regions in the variable domains. The antigen-binding site may be wild-type or may be modified by one or more amino acid substitutions, e.g., to more closely resemble human immunoglobulins. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). Other forms have one or more CDRs altered relative to the original antibody.

[0058] Various methods for humanizing antibodies are known to those skilled in the art, as reviewed in Almagro & Fransson, 2008, the entire contents of which are incorporated herein by reference. The review article by Almagro and Fransson is briefly summarized below. Almagro and Fransson distinguish between rational and empirical approaches. Rational approaches are characterized by generating several variants of an engineered antibody and evaluating their binding or other properties of interest. If the designed variant does not produce the expected results, a new cycle of design and binding evaluation is initiated. Rational approaches include CDR grafting, resurfacing, superhumanization, and human string content optimization. In contrast, empirical approaches are based on generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high-throughput screening. Therefore, empirical approaches rely on reliable selection and / or screening systems that can explore a vast range of antibody variants. In vitro display techniques such as phage display and ribosome display meet these requirements and are well known to those skilled in the art. Empirical approaches include FR libraries, guided selection methods, framework-shuffling, and humaneering.

[0059] CDR grafting CDR grafting protocols typically involve three decision points: (1) defining the specificity-determining region of the donor antibody, i.e., the target of grafting; (2) identifying the source of human sequence to be used as the FR donor; and (3) selecting residues outside the specificity-determining region, i.e., determining the amino acid positions that will be targets for backmutation to restore or improve the affinity of the humanized antibody.

[0060] (1) Region that determines antibody specificity The experimental structure of a non-human antibody complexed with an antigen provides a detailed map of the residues that contact the antigen and thus determine its specificity. Supplementing structural information with alanine scanning mutagenesis and / or combinatorial mutagenesis can identify the residues that contribute most to the binding energy or functional paratope. Because the functional paratope is a subset of contact residues, grafting only the functional paratope reduces the number of non-human residues in the humanized product. However, experimental structures of antigen-antibody complexes and / or functional paratopes are rarely available at the start of a humanization protocol. In the absence of precise definition of the residues responsible for a given antibody specificity, CDRs are often used as specificity-defining regions. It is also possible to use a combination of CDRs and HV loops as targets for grafting. To reduce the number of residues to be grafted onto human FRs, SDR grafting, i.e., grafting of specificity-determining residues (SDRs), has been described.

[0061] (2) Source of human FR The second step in a typical CDR-grafting protocol is to identify a human FR donor. Early studies utilized FRs of human antibodies of known structure, regardless of their homology to the nonhuman antibody. This approach is known as the "fixed FR" method. Later studies used human sequences with the highest homology to the nonhuman antibody. This approach is called "best fit." While the "best fit" strategy tends to yield antibodies with higher affinity, other parameters, such as low immunogenicity and production yield, must also be taken into account when selecting FRs for humanization. Therefore, a combination of "best fit" and "fixed FR" is also possible. For example, the VL portion can be humanized according to the fixed FR method, and the VH portion can be humanized according to the best fit method, or vice versa.

[0062] Two sources of human sequences are utilized: mature sequences and germline sequences. Mature sequences, which are the product of immune responses, have somatic mutations generated by random processes and are not under species selection, resulting in potentially immunogenic residues. Therefore, to avoid immunogenic residues, human germline genes are increasingly being used as sources of FR donors. The nucleotide sequences of human germline FRs are disclosed, for example, in Appendix A and Appendix B of the paper by Dall'Acqua et al., 2005. Furthermore, antibodies based on germline genes tend to be more flexible than mature antibodies. This greater flexibility is thought to better accommodate diverse CDRs with little or no back mutations to the FRs to restore the affinity of humanized antibodies.

[0063] (3) Back mutations to restore or enhance affinity Generally, affinity decreases after CDR-grafting as a result of incompatibility between non-human CDRs and human FRs. Therefore, the third step in a typical CDR-grafting protocol is to define mutations that restore or prevent the affinity loss. Back mutations need to be carefully designed and experimentally tested based on the structure or model of the humanized antibody. A website for automated antibody modeling, called WAM, can be found at the URL: http: / / antibody.bath.ac.uk. Software for protein structure modeling can be downloaded from the sites http: / / salilab.org / modeller / modeller.html (Modeller) and http: / / spdbv.vital-it.ch (Swiss PdbViewer).

[0064] Resurfacing Resurfacing is similar to CDR grafting and shares the first two decision points: in contrast to CDR grafting, resurfacing preserves non-exposed residues of the non-human antibody; only surface residues in the non-human antibody are exchanged for human residues.

[0065] Super Humanization While CDR grafting relies on the comparison of FRs between non-human and human sequences, superhumanization is based on the comparison of CDRs, so the homology of FRs is irrelevant. This approach involves comparing non-human sequences with a repertoire of functional human germline genes. Then, select those genes that encode the same or closely related canonical structure as the mouse sequence. Next, within the range of genes that share canonical structures with non-human antibodies, select the gene with the highest homology within the CDR as the FR donor. Finally, non-human CDRs are grafted onto these FRs.

[0066] Human String Content Optimization This approach is based on a measure of the "humanity" of an antibody called human string content (HSC). Briefly, this approach compares the mouse sequence to a repertoire of human germline genes. Differences are scored as HSC. Target sequences are humanized by maximizing their HSC rather than using a measure of overall identity, generating a large number of diverse humanized variants.

[0067] Framework Library (abbreviated as FR Library) In the FR library approach, a collection of residue variants is introduced into specific positions in the FR, followed by panning of the library to select the FR that best supports the grafted CDR. Thus, this approach resembles CDR grafting, but instead of generating a few backmutations in the FR, a combinatorial library of typically over 100 mutants is constructed.

[0068] Guide Selection This approach involves combining the VH or VL domain of a given non-human antibody specific for a particular antigen with a library of human VH and VL domains. Subsequently, human V domains specific to the antigen of interest are selected. For example, a non-human antibody can be humanized by first combining the non-human VH with a library of human light chains. The library is then selected against the target antigen by phage display, and the selected VL is cloned into a library of human VH chains and selected against the target antigen. It is also possible to start by combining the non-human VL with a library of human heavy chains. This library is then selected against the target antigen by phage display, and the selected VH is cloned into a library of human VL chains and selected against the target antigen. As a result, fully human antibodies with affinities similar to those of the non-human antibody can be isolated. To avoid epitope drift, a so-called inhibition ELISA can be performed, which allows the selection of clones that recognize the same epitope as the parent antibody. Alternatively, CDR retention can be applied to avoid epitope drift. In CDR retention, one or more non-human CDRs are retained, preferably heavy chain CDR3 since it is at the center of the antigen binding site.

[0069] Framework Shuffling (abbreviated as FR Shuffling) In the FR shuffling approach, entire FRs are combined with non-human CDRs. Using FR shuffling, Dall'Acqua and coworkers humanized a mouse antibody. All six CDRs of a mouse antibody were cloned into a library containing FRs from all human germline genes (Dall'Acqua et al., 2005). The library was screened for binding using a two-step selection method, first humanizing the VL and then the VH. Subsequent studies have successfully used a one-step FR shuffling method (Damschroder et al., 2007). Oligonucleotide sequences encoding all known human germline light chain (κ) frameworks are disclosed in Appendix A of Dall'Acqua et al., 2005. Oligonucleotide sequences encoding all known human germline heavy chain frameworks are disclosed in Appendix B of Dall'Acqua et al., 2005.

[0070] Humane Ring Humaneering allows the isolation of antibodies that are 91% to 96% homologous to human germline antibodies. This method is based on experimental identification of essential minimal specificity determinants (MSDs) and sequential replacement of non-human fragments with a library of human FRs and evaluation of binding. This method begins with the CDR3 regions of the non-human VH and VL chains and gradually replaces other regions of the non-human antibody, including CDR1 and CDR2 of both VH and VL, with human FRs.

[0071] The above-described methods for humanizing antibodies are preferred for generating humanized antibodies that specifically bind to the conformational epitopes described herein. Nevertheless, the present invention is not limited to the above-described methods for humanizing antibodies.

[0072] Some of the humanization methods mentioned above, namely the "fixed FR method" (a variation of CDR grafting), superhumanization, framework shuffling, and humaneering, can be performed without information about the FR sequences in the donor antibody. Variations of the "fixed FR method" were successfully performed by Qin et al., 2007 and Chang et al., 2007. In particular, Qin et al. constructed an antibody fragment containing a human heavy chain variable region in which three CDR regions were replaced by antigenic peptides derived from the CDR sequences of a mouse antibody. Chang et al. continued these experiments and constructed an scFv fragment in which all CDRs from the VH portion and CDR3 from the VL portion were replaced by antigenic peptides derived from the CDR sequences of a mouse antibody.

[0073] As used herein, "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Human antibodies of the invention include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, e.g., as described in U.S. Patent No. 5,939,598 by Kucherlapati & Jakobovits.

[0074] The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. In one embodiment, a monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a non-human animal (e.g., a mouse) fused with an immortalized cell.

[0075] The term "recombinant antibody," as used herein, includes all antibodies prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for immunoglobulin genes, or hybridomas prepared therefrom; (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express the antibody; (c) antibodies isolated from recombinant combinatorial antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means, including splicing of immunoglobulin gene sequences into other DNA sequences.

[0076] The term "transfectoma", as used herein, includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells, or fungal, including yeast cells.

[0077] As used herein, a "heterologous antibody" is defined in relation to the transgenic organism producing such an antibody. The term generally refers to an antibody from a species other than the transgenic organism, having an amino acid sequence or encoding nucleic acid sequence that corresponds to that found in an organism not consisting of the transgenic organism.

[0078] As used herein, a "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain combined with a murine light chain is a heterohybrid antibody.

[0079] Accordingly, "antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monovalent antibodies, bispecific antibodies, heteroconjugate antibodies, multispecific antibodies, recombinant antibodies, xenoantibodies, heterohybrid antibodies, chimeric antibodies, humanized antibodies (particularly CDR-grafted antibodies), deimmunized or human antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, fragments produced by a Fab expression library, Fd, Fv, disulfide-linked Fv (dsFv), single-chain antibodies (e.g., scFv), diabodies or tetrabodies (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90(14), 6444-6448), nanobodies (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against an antibody of the invention), and epitope-binding fragments of any of the above.

[0080] The antibodies described herein are preferably isolated. "Isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities.

[0081] The term "naturally occurring," as used herein as applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally occurring.

[0082] As used herein, the term "nucleic acid aptamer" refers to a nucleic acid molecule that has been engineered to bind to a target molecule through repeated in vitro selection or SELEX (sequential evolution of molecules) (for a review, see Brody EN and Gold L. (2000), Aptamers as therapeutic and diagnostic agents. J. Biotechnol. 74 (1):5-13). Nucleic acid aptamers can be DNA or RNA molecules. Aptamers can contain modifications, such as modified nucleotides, e.g., 2'-fluorine substituted pyrimidines.

[0083] As used herein, the term "antibody-like protein" refers to a protein that has been engineered (e.g., by loop mutation) to specifically bind to a target molecule. Typically, such antibody-like proteins contain at least one variable peptide loop attached at both ends to a protein scaffold. This dual structural constraint significantly increases the binding affinity of antibody-like proteins to a level comparable to that of antibodies. The length of the variable peptide loop typically consists of 10 to 20 amino acids. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include, but are not limited to, affibodies, anticalins, and engineered ankyrin repeat proteins (for a review, see Binz HK et al. (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23(10):1257-1268). Antibody-like proteins can be derived from large libraries of mutants, for example, by panning from large phage display libraries, and isolated in the same way as regular antibodies. Antibody-like binding proteins can also be obtained by combinatorial mutagenesis of surface-exposed residues of globular proteins. Antibody-like proteins are sometimes called "peptide aptamers."

[0084] As used herein, a "peptidomimetic" is a small protein-like chain designed to mimic a peptide. Peptidomimetics typically arise from the modification of existing peptides to change the properties of the molecule. For example, peptidomimetics can arise from modifications to change the stability or biological activity of the molecule. This can have a role in the development of drug-like compounds derived from existing peptides. These modifications include changes to peptides that do not occur in nature (e.g., backbone changes and incorporation of unnatural amino acids).

[0085] "Percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity.

[0086] The term "identical" in the context of two or more nucleic acid or polypeptide sequences is used herein to refer to two or more sequences or subsequences that are the same, i.e., contain the same sequence of nucleotides or amino acids. Sequences are "substantially identical" to one another if the sequences have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity over a specified region) when compared and aligned for maximum correspondence over a comparison window, or designated region, as assessed using one of the following sequence comparison algorithms or by manual alignment and visual inspection. These definitions also apply to the complement of a test sequence. Thus, the term "at least 80% sequence identity" is used throughout this specification in reference to polypeptide sequence comparisons and polynucleotide sequence comparisons. This phrase preferably refers to at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the respective reference polypeptide or respective reference polynucleotide.

[0087] The term "sequence comparison" is used herein to refer to a method in which one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, once the necessary subsequence coordinates have been designated and sequence algorithm program parameters have been designated, the test and reference sequences are input into a computer. Default program parameters are generally used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity or similarity for the test sequence relative to the reference sequence based on the program parameters. When two sequences are compared and the reference sequence to which the percent sequence identity is calculated is not specified, the sequence identity should be calculated with reference to the longer of the two sequences to be compared, unless otherwise specified. When a reference sequence is provided, the sequence identity is determined based on the entire length of the reference sequence, as indicated by the SEQ ID NO:, unless otherwise specified.

[0088] In sequence alignment, the term "comparison window" refers to a stretch of contiguous positions of a sequence that is compared to a reference stretch of contiguous positions of a sequence having the same number of positions. The number of contiguous positions selected can range from 4 to 1000 contiguous positions, i.e., 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 contiguous positions. Typically, the number of consecutive positions ranges from about 20 to about 800 consecutive positions, from about 20 to about 600 consecutive positions, from about 50 to about 400 consecutive positions, from about 50 to about 200 consecutive positions, or from about 100 to about 150 consecutive positions.

[0089] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 Supplement)). Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in a query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when the cumulative alignment score falls by an amount X from its maximum achieved value, when the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) alignment (B) of 50, an expectation of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.

[0090] "Conservative substitutions" may be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. Amino acids can be classified into six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile, (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe. As used herein, a "conservative substitution" is defined as the replacement of an amino acid with another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the replacement of Asp with Glu results in the retention of one negative charge in the modified polypeptide. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices. Some preferred conservative substitutions within the above six groups are within the following subgroups: (i) Ala, Val, Leu, and Ile; (ii) Ser and Thr; (iii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe. Given the known genetic code and recombinant and synthetic DNA techniques, skilled scientists can easily construct DNA encoding conservative amino acid variants.

[0091] As used herein, a "non-conservative substitution" or "non-conservative amino acid exchange" is defined as the replacement of one amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[0092] The terms "nucleic acid" and "nucleic acid molecule" are used synonymously herein and are understood to refer to single- or double-stranded oligomers or polymers of deoxyribonucleotides or ribonucleotide bases, or both. Nucleotide monomers consist of a nucleobase, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Nucleic acids are typically formed via phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules, but also synthetic forms of nucleic acids containing other linkages (e.g., peptide nucleic acids as described by Nielsen et al. (Science 254:1497-1500, 1991)). Typically, nucleic acids are single- or double-stranded molecules and are composed of naturally occurring nucleotides. A description of a single strand of a nucleic acid also defines (at least in part) the sequence of the complementary strand. Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. Exemplary double-stranded nucleic acid molecules can have 3' or 5' overhangs, and are not required or expected to be completely double-stranded throughout their entire length. Nucleic acids can be obtained by any method known in the art, including biological, biochemical, or chemical synthesis, or by amplification and reverse transcription of RNA. The term nucleic acid includes chromosomes or chromosomal segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, primers, probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA (miRNA), or small interfering RNA (siRNA). Nucleic acids can be, for example, single-stranded, double-stranded, or triple-stranded and are not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence includes or encodes complementary sequences in addition to any sequence explicitly indicated.

[0093] Nucleic acids can be degraded by endonucleases or exonucleases, particularly DNases and RNases found within cells. Therefore, it may be advantageous to ensure that high concentrations of nucleic acids are maintained within cells for extended periods of time by modifying the nucleic acids of the present invention to stabilize them against degradation. Typically, such stabilization can be achieved by introducing one or more internucleotide phosphorus-based groups or one or more internucleotide non-phosphorus-based groups. Thus, nucleic acids can be composed of non-naturally occurring nucleotides and / or modifications to naturally occurring nucleotides and / or modifications to the backbone of the molecule. Modified internucleotide phosphate groups and / or non-phosphorus-based crosslinks in nucleic acids include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate, phosphorodithioate, and / or phosphate, while non-phosphorus-based internucleotide analogs include, but are not limited to, siloxane crosslinks, carbonate crosslinks, carboxymethyl ester, acetamidate crosslinks, and / or thioether crosslinks. Further examples of nucleotide modifications include, but are not limited to, phosphorylation of the 5' or 3' nucleotide to allow for prevention of ligation or exonuclease degradation / polymerase extension, respectively, amino modifications for covalent and near-covalent attachment, thiol modifications, alkyne modifications or biotinyl modifications, fluorophores and quenchers, deoxyinosine (dI), 5-bromo-deoxyuridine (5-bromo-dU), deoxyuridine, 2-aminopurine, 2,6-di ... Included are aminopurine, reverse dT, reverse dideoxy-T, dideoxycytidine (ddC), 5-methyldeoxycytidine (5-methyl dC), locked nucleic acid (LNA), 5-nitroindole, Iso-dC and Iso-dG bases, 2'-O-methyl RNA bases, hydroxymethyl dC, 5-hydroxybutyl-2'-deoxyuridine, 8-aza-7-deazaguanosine, and modified bases such as fluorine-modified bases.Thus, the nucleic acid may also be an artificial nucleic acid, including, but not limited to, polyamide nucleic acid or peptide nucleic acid (PNA), morpholino nucleic acid and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA).

[0094] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0095] The term "polynucleotide", as used in the context of the present invention, refers to a nucleic acid of greater than about 50 nucleotides in length, for example, 51 or more nucleotides in length.

[0096] Polypeptides of the present invention are prepared by any suitable method, including, but not limited to, isolation of existing or naturally occurring sequences, DNA replication or amplification, reverse transcription, cloning of appropriate sequences and restriction digestion, or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99, 1979), the phosphodiester method of Brown et al. (Meth. Enzymol. 68:109-151, 1979), the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22:1859-1862, 1981), the triester method of Matteucci et al. (J. Am. Chem. Soc. 103:3185-3191, 1981), automated synthesis, or the solid support method of U.S. Pat. No. 4,458,066, or other methods known to those skilled in the art.

[0097] As used herein, the term "vector" refers to a protein or polynucleotide, or mixture thereof, that can be introduced into a cell or that can introduce proteins and / or nucleic acids contained therein into a cell. Examples of vectors include, but are not limited to, plasmids, cosmids, phages, viruses, or artificial chromosomes. In particular, vectors are used to transport a gene product of interest, such as foreign or heterologous DNA, into a suitable host cell. A vector may contain a "replicon" polynucleotide sequence that facilitates autonomous replication of the vector within the host cell. Foreign DNA is defined as DNA not naturally found in the host cell, e.g., heterologous DNA that replicates the vector molecule, encodes a selectable or screenable marker, or encodes a transgene. Once inside the host cell, the vector may replicate independently of or simultaneously with the host chromosomal DNA, generating several copies of the vector and its inserted DNA. Furthermore, a vector may also contain necessary elements that enable transcription of the inserted DNA into an mRNA molecule or otherwise cause replication of the inserted DNA into multiple RNA copies. A vector may further contain an "expression control sequence" that regulates expression of the gene of interest. Typically, expression control sequences are polypeptides or polynucleotides, such as, but not limited to, promoters, enhancers, silencers, insulators, or repressors. In vectors containing two or more polynucleotides encoding one or more gene products of interest, expression can be controlled jointly or separately by one or more expression control sequences. More specifically, each polynucleotide contained on a vector can be controlled by a separate expression control sequence, or all polynucleotides contained on a vector can be controlled by a single expression control sequence. Polynucleotides contained on a single vector controlled by a single expression control sequence can form an open reading frame. Some expression vectors further contain sequence elements flanking the inserted DNA that increase the half-life of the expressed mRNA and / or enable translation of the mRNA into a protein molecule.Many molecules of mRNA and the polypeptide encoded by the inserted DNA can therefore be rapidly synthesized.

[0098] The term "host cell" refers to a cell that harbors a vector (e.g., a plasmid or virus). Such host cells can be either prokaryotic (e.g., bacterial) or eukaryotic (e.g., fungal, plant, or animal) cells. Host cells include both unicellular prokaryotes and eukaryotes (e.g., bacteria, yeast, and actinomycetes), as well as single cells from higher plants or animals when grown in cell culture. As used herein, a "recombinant host cell" refers to a host cell that contains a polynucleotide encoding a polypeptide fragment of interest, i.e., a fragment of a viral PA subunit or variant thereof, according to the present invention. This polynucleotide can be found within the host cell (i) as is, freely dispersed; (ii) incorporated into a recombinant vector; or (iii) integrated into the host cell genome or mitochondrial DNA. Recombinant cells can be used for expression of a polynucleotide of interest or for amplification of a polynucleotide or recombinant vector of the invention. The term "recombinant host cell" includes the progeny of an original cell transformed, transfected, or infected with a polynucleotide or recombinant vector of the invention. Recombinant host cells can be bacterial cells such as E. coli cells, yeast cells such as Saccharomyces cerevisiae or Pichia pastoris, plant cells, insect cells such as SF9 cells or High Five cells, or mammalian cells. Preferred examples of mammalian cells include Chinese hamster ovary (CHO) cells, African green monkey kidney (COS) cells, human embryonic kidney (HEK293) cells, HELA cells, etc.

[0099] The term "active agent" as used herein relates to any therapeutic activity that an agent may exhibit.

[0100] "Pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, or more specifically in humans.

[0101] The term "carrier," as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and saline solutions in oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions are preferred carriers when pharmaceutical compositions are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice flour, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. If desired, the composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Compositions can also be formulated as suppositories, using traditional binders and carriers such as triglycerides. The compounds of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions contain a therapeutically effective amount of the compound, preferably in purified form, together with an appropriate amount of carrier to provide the form for proper administration to the patient. The formulation should be compatible with the mode of administration.

[0102] Commonly known and practiced methods in the fields of molecular biology, cell biology, protein chemistry and antibody techniques are fully described in the following publications, which are continually updated: "Molecular Cloning: A Laboratory Manual" (Sambrook et al., Cold Spring Harbor), Current Protocols in Molecular Biology (F.M. Ausubel et al. Eds., Wiley & Sons), Current Protocols in Protein Science (J.E. Colligan et al. eds., Wiley & Sons), Current Protocols in Cell Biology (J.S. Bonifacino et al., Wiley & Sons), and Current Protocols in Immunology (J.E. Colligan et al., Eds., Wiley & Sons). Known techniques for cell culture and media are described in "Large Scale Mammalian Cell Culture" (Hu et al., Curr. Opin., Biotechnol. 8: 148, 1997), "Serum-free Media" (K. Kitano, Biotechnol. 17:73, 1991), and "Suspension Culture of Mammalian Cells" (Birch et al., Bioprocess Technol. 19: 251, 1990).

[0103] Embodiment Various aspects of the invention are defined in more detail below. Each aspect so defined may be combined with any other aspect or aspects, unless expressly stated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0104] In a first aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein exhibits, for binding to PD-1: (i) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 3; or (ii) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12; The present invention provides an antagonistic antigen-binding protein that competes with the antibody.

[0105] Preferably, the present invention provides antagonistic antigen-binding proteins that specifically bind to PD-1 and compete for binding to PD-1 with an antibody comprising a heavy chain variable region of the amino acid sequence in SEQ ID NO:2 and a light chain variable region of the amino acid sequence in SEQ ID NO:3.

[0106] In a preferred embodiment of the first aspect, the antagonistic antigen-binding protein that specifically binds to PD-1 is an antibody, antibody-like protein, or fragment thereof. Preferably, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a single-chain variable fragment antibody, a diabody, a Fab fragment, a F(ab)2 fragment, an antibody mimetic, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0107] In further preferred embodiments of the first aspect, the antagonistic antigen-binding protein that specifically binds to PD-1 binds to PD-1 with a Kd of less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 100 pM, less than 10 pM, or less than 5 pM. For example, the antagonistic antigen-binding protein that specifically binds to PD-1 binds to PD-1 with a Kd of between 5 pM and 100 nM, between 5 pM and 50 nM, between 5 pM and 10 nM, between 5 pM and 1 nM, between 5 pM and 100 pM, e.g., between 5 pM and 10 pM.

[0108] In further preferred embodiments of the first aspect, an antagonistic antigen-binding protein that specifically binds to PD-1 may block binding of a ligand to PD-1 with an IC50 of less than 10 nM, less than 1 nM, or less than 200 pM. For example, an antagonistic antigen-binding protein that specifically binds to PD-1 may block binding of a ligand to PD-1 with an IC50 of between 200 pM and 10 nM, e.g., between 200 pM and 1 nM.

[0109] In a further preferred embodiment of the first aspect, the antagonistic antigen binding protein that specifically binds to PD-1 stimulates T cell proliferation.

[0110] In a further preferred embodiment of the first aspect, the antagonistic antigen binding protein that specifically binds to PD-1 induces lymphocyte proliferation.

[0111] In a further preferred embodiment of the first aspect, the antagonistic antigen binding protein that specifically binds to PD-1 induces the secretion of IL-2 and / or IFNγ.

[0112] In further preferred embodiments of the first aspect, the antagonistic antigen binding protein that specifically binds to PD-1 reduces tumor volume by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to baseline values.

[0113] In a second aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein exhibits, for binding to PD-L1: (i) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 20 and a light chain variable region of the amino acid sequence of SEQ ID NO: 21; or (ii) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 29 and a light chain variable region of the amino acid sequence of SEQ ID NO: 30; or (iii) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 83 and a light chain variable region of the amino acid sequence of SEQ ID NO: 84; or (iv) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 92 and a light chain variable region of the amino acid sequence of SEQ ID NO: 93; or (v) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 101 and a light chain variable region of the amino acid sequence of SEQ ID NO: 102; The present invention provides an antagonistic antigen-binding protein that competes with the antibody.

[0114] Preferably, the invention provides antagonistic antigen binding proteins that specifically bind to PD-L1 and compete for binding to PD-L1 with an antibody comprising a heavy chain variable region of the amino acid sequence in SEQ ID NO:29 and a light chain variable region of the amino acid sequence in SEQ ID NO:30.

[0115] In a preferred embodiment of the second aspect, the antagonistic antigen-binding protein that specifically binds to PD-L1 is an antibody, antibody-like protein, or fragment thereof. Preferably, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a single-chain variable fragment antibody, a diabody, a Fab fragment, a F(ab)2 fragment, an antibody mimetic, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0116] In further preferred embodiments of the second aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 binds to PD-L1 with a Kd of less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 100 pM, less than 10 pM, or less than 5 pM. For example, the antagonistic antigen binding protein that specifically binds to PD-L1 binds to PD-L1 with a Kd of between 5 pM and 100 nM, between 5 pM and 50 nM, between 5 pM and 10 nM, between 5 pM and 1 nM, between 5 pM and 100 pM, for example, between 5 pM and 10 pM.

[0117] In further preferred embodiments of the second aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 is capable of blocking binding of a ligand to PD-L1 with an IC50 of less than 10 nM, less than 1 nM, or less than 200 pM.

[0118] For example, an antagonistic antigen binding protein that specifically binds to PD-L1 may block binding of a ligand to PD-L1 with an IC50 of between 200 pM and 10 nM, such as between 200 pM and 1 nM.

[0119] In a further preferred embodiment of the second aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 stimulates T cell proliferation.

[0120] In a further preferred embodiment of the second aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 induces lymphocyte proliferation.

[0121] In a further preferred embodiment of the second aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 induces the secretion of IL-2 and / or IFNγ.

[0122] In further preferred embodiments of the second aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 reduces tumor volume by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to baseline values.

[0123] In a third aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein exhibits, for binding to LAG-3: (i) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 38 and a light chain variable region of the amino acid sequence of SEQ ID NO: 39; (ii) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 47 and a light chain variable region of the amino acid sequence of SEQ ID NO: 48; (iii) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 56 and a light chain variable region of the amino acid sequence of SEQ ID NO: 57; (iv) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 65 and a light chain variable region of the amino acid sequence of SEQ ID NO: 66; or (v) an antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 74 and a light chain variable region of the amino acid sequence of SEQ ID NO: 75; It relates to an antagonistic antigen-binding protein that competes with

[0124] Preferably, the present invention provides an antagonistic antigen-binding protein that specifically binds to LAG-3 and competes for binding to LAG-3 with an antibody comprising a heavy chain variable region of the amino acid sequence in SEQ ID NO: 38 and a light chain variable region of the amino acid sequence in SEQ ID NO: 39.

[0125] In another preferred embodiment, the present invention provides an antagonistic antigen binding protein that specifically binds to LAG-3 and competes for binding to LAG-3 with an antibody comprising a heavy chain variable region of the amino acid sequence in SEQ ID NO: 47 and a light chain variable region of the amino acid sequence in SEQ ID NO: 48.

[0126] In a preferred embodiment of the third aspect, the competitive antigen-binding protein that specifically binds to LAG-3 is an antibody, antibody-like protein, or fragment thereof. Preferably, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a single-chain variable fragment antibody, a diabody, a Fab fragment, a F(ab)2 fragment, an antibody mimetic, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0127] In further preferred embodiments of the third aspect, an antagonistic antigen binding protein that specifically binds to LAG-3 binds to LAG-3 with a Kd of less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 100 pM, less than 10 pM, or less than 5 pM. For example, an antagonistic antigen binding protein that specifically binds to LAG-3 binds to LAG-3 with a Kd of between 5 pM and 100 nM, between 5 pM and 50 nM, between 5 pM and 10 nM, between 5 pM and 1 nM, between 5 pM and 100 pM, e.g., between 5 pM and 10 pM.

[0128] In further preferred embodiments of the third aspect, a competitive antigen binding protein that specifically binds to LAG-3 may block ligand binding to LAG-3 with an IC50 of less than 10 nM, less than 1 nM, or less than 200 pM. For example, a competitive antigen binding protein that specifically binds to LAG-3 may block ligand binding to LAG-3 with an IC50 of between 200 pM and 10 nM, e.g., between 200 pM and 1 nM.

[0129] In a further preferred embodiment of the third aspect, the antagonistic antigen binding protein that specifically binds to LAG-3 stimulates T cell proliferation.

[0130] In a further preferred embodiment of the third aspect, the antagonistic antigen binding protein that specifically binds to LAG-3 induces lymphocyte proliferation.

[0131] In a further preferred embodiment of the third aspect, the antagonistic antigen binding protein that specifically binds to LAG-3 induces the secretion of IL-2 and / or IFNγ.

[0132] In a further preferred embodiment of the third aspect, the antagonistic antigen binding protein that specifically binds to LAG-3 reduces tumor volume by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to baseline values.

[0133] In a fourth aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to PD-1, the antigen binding protein comprising: (i) A combination of: a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 3 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 2; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 12 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 11; a combination of a light chain variable domain and a heavy chain variable domain selected from the group (ii) A combination of: a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 18; a combination of heavy chain complementarity determining region 3 (CDRH3) and light chain complementarity determining region 3 (CDRL3) selected from the group consisting of: The present invention relates to an antagonistic antigen-binding protein comprising any one of:

[0134] In a preferred embodiment, the invention relates to an antagonistic antigen binding protein that specifically binds to PD-1, comprising a combination of a light chain variable domain having a sequence at least 90% identical to SEQ ID NO:3 and a heavy chain variable domain having a sequence at least 90% identical to SEQ ID NO:2.

[0135] In a further preferred embodiment, the invention relates to an antagonistic antigen binding protein that specifically binds to PD-1, comprising a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO:8 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0136] In a preferred embodiment of the fourth aspect of the invention, the antagonistic antigen binding protein specifically binds to PD-1, wherein said antigen binding protein is a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 3 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 2; a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 12 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 11; The combination of a light chain variable domain and a heavy chain variable domain is selected from the group:

[0137] In a further preferred embodiment, the invention relates to an antagonistic antigen binding protein that specifically binds to PD-1, comprising a combination of a light chain variable domain having a sequence at least 95% identical to SEQ ID NO:3 and a heavy chain variable domain having a sequence at least 95% identical to SEQ ID NO:2.

[0138] In a further preferred embodiment of the fourth aspect of the invention, the antagonistic antigen binding protein specifically binds to PD-1, wherein the antigen binding protein is a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 3 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 2; a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 12 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 11; The combination of a light chain variable domain and a heavy chain variable domain is selected from the group:

[0139] In a further preferred embodiment, the invention relates to an antagonistic antigen binding protein that specifically binds to PD-1, comprising a combination of a light chain variable domain having a sequence at least 99% identical to SEQ ID NO:3 and a heavy chain variable domain having a sequence at least 99% identical to SEQ ID NO:2.

[0140] In a further preferred embodiment of the fourth aspect of the invention, the antagonistic antigen binding protein specifically binds to PD-1, wherein the antigen binding protein is a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 18; The heavy chain complementarity determining region 3 (CDRH3) and light chain complementarity determining region 3 (CDRL3) are selected from the group consisting of:

[0141] In a further preferred embodiment, the invention relates to an antagonistic antigen-binding protein that specifically binds to PD-1, comprising a combination of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9. Preferably, the antagonistic antigen-binding protein specifically binds to PD-1, wherein said antigen-binding protein further comprises one or more selected from the group consisting of: a CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 13; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 15; a CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 14; and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 16.

[0142] For example, an antagonistic antigen binding protein specifically binds to PD-1, wherein the antigen binding protein: a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 5; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 6 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0143] For example, an antagonistic antigen binding protein specifically binds to PD-1, wherein the antigen binding protein: a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 18; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 13 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 14; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 16; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0144] In a further preferred embodiment of the fourth aspect, the antagonistic antigen-binding protein that specifically binds to PD-1 is an antibody, antibody-like protein, or fragment thereof. Preferably, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a single-chain variable fragment antibody, a diabody, a Fab fragment, a F(ab)2 fragment, an antibody mimetic, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0145] In further preferred embodiments of the fourth aspect, the antagonistic antigen binding protein that specifically binds to PD-1 binds to PD-1 with a Kd of less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 100 pM, less than 10 pM, or less than 5 pM. For example, the antagonistic antigen binding protein that specifically binds to PD-1 binds to PD-1 with a Kd of between 5 pM and 100 nM, between 5 pM and 50 nM, between 5 pM and 10 nM, between 5 pM and 1 nM, between 5 pM and 100 pM, e.g., between 5 pM and 10 pM.

[0146] In further preferred embodiments of the fourth aspect, an antagonistic antigen binding protein that specifically binds to PD-1 may block binding of a ligand to PD-1 with an IC50 of less than 10 nM, less than 1 nM, or less than 200 pM. For example, an antagonistic antigen binding protein that specifically binds to PD-1 may block binding of a ligand to PD-1 with an IC50 of between 200 pM and 10 nM, e.g., between 200 pM and 1 nM.

[0147] In a further preferred embodiment of the fourth aspect, the antagonistic antigen binding protein that specifically binds to PD-1 stimulates T cell proliferation.

[0148] In a further preferred embodiment of the fourth aspect, the antagonistic antigen binding protein that specifically binds to PD-1 induces lymphocyte proliferation.

[0149] In a further preferred embodiment of the fourth aspect, the antagonistic antigen binding protein that specifically binds to PD-1 induces the secretion of IL-2 and / or IFNγ.

[0150] In further preferred embodiments of the fourth aspect, the antagonistic antigen binding protein that specifically binds to PD-1 reduces tumor volume by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to baseline values.

[0151] In a fifth aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to PD-L1, the antigen binding protein comprising: (i) A combination of: a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 21 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 20; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 30 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 29; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 84 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 83; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 93 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 92; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 102 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 101; a combination of a light chain variable domain and a heavy chain variable domain selected from the group (ii) A combination of: a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 89 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 90; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 99; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 107 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 108; a combination of heavy chain complementarity determining region 3 (CDRH3) and light chain complementarity determining region 3 (CDRL3) selected from the group consisting of: The present invention relates to an antagonistic antigen-binding protein comprising any one of:

[0152] In a preferred embodiment, the invention relates to an antagonistic antigen binding protein that specifically binds to PD-L1, comprising a combination of a light chain variable domain having a sequence at least 90% identical to SEQ ID NO: 30 and a heavy chain variable domain having a sequence at least 90% identical to SEQ ID NO: 29.

[0153] In a further preferred embodiment, the invention relates to an antagonistic antigen binding protein that specifically binds to PD-L1, comprising a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35, and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36.

[0154] In a preferred embodiment of the fifth aspect of the invention, the antagonistic antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein is a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 21 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 20; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 30 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 29; a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 84 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 83; a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 93 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 92; a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 102 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 101; The combination of a light chain variable domain and a heavy chain variable domain is selected from the group:

[0155] In a further preferred embodiment, the invention relates to an antagonistic antigen binding protein that specifically binds to PD-L1, comprising a combination of a light chain variable domain having a sequence at least 95% identical to SEQ ID NO: 30, and a heavy chain variable domain having a sequence at least 95% identical to SEQ ID NO: 29.

[0156] In a further preferred embodiment of the fifth aspect of the invention, the antagonistic antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein is a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 21 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 20; a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 30 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 29; a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 84 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 83; a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 93 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 92; a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 102 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 101; The combination of a light chain variable domain and a heavy chain variable domain is selected from the group:

[0157] In a preferred embodiment, the invention relates to an antagonistic antigen binding protein that specifically binds to PD-L1, comprising a combination of a light chain variable domain having a sequence at least 99% identical to SEQ ID NO: 30 and a heavy chain variable domain having a sequence at least 99% identical to SEQ ID NO: 29.

[0158] In a further preferred embodiment of the fifth aspect of the invention, the antagonistic antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein is a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 89 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 90; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 99; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 107 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 108; The heavy chain complementarity determining region 3 (CDRH3) and light chain complementarity determining region 3 (CDRL3) are selected from the group consisting of:

[0159] In a further preferred embodiment, the invention relates to an antagonistic antigen binding protein that specifically binds to PD-L1, comprising a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35, and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36.

[0160] Preferably, the antagonistic antigen-binding protein specifically binds to PD-L1, wherein the antigen-binding protein further comprises one or more selected from the group consisting of: CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 31; CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 33; CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 32; and CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 34.

[0161] For example, an antagonistic antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein: a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 25; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0162] For example, an antagonistic antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein: a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 31 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 32; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 33 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 34; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0163] For example, an antagonistic antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein: a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 89 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 90; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 85 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 86; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 87 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 88; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0164] For example, an antagonistic antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein: a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 99; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 94 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 95; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 96 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 97; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0165] For example, an antagonistic antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein: a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 107 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 108; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 103 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 104; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 105 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 106; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0166] In a further preferred embodiment of the fifth aspect, the antagonistic antigen-binding protein that specifically binds to PD-L1 is an antibody, antibody-like protein, or fragment thereof. Preferably, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a single-chain variable fragment antibody, a diabody, a Fab fragment, a F(ab)2 fragment, an antibody mimetic, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0167] In further preferred embodiments of the fifth aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 binds to PD-L1 with a Kd of less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 100 pM, less than 10 pM, or less than 5 pM. For example, the antagonistic antigen binding protein that specifically binds to PD-L1 binds to PD-L1 with a Kd of between 5 pM and 100 nM, between 5 pM and 50 nM, between 5 pM and 10 nM, between 5 pM and 1 nM, between 5 pM and 100 pM, for example, between 5 pM and 10 pM.

[0168] In further preferred embodiments of the fifth aspect, an antagonistic antigen binding protein that specifically binds to PD-L1 may block the binding of a ligand to PD-L1 with an IC50 of less than 10 nM, less than 1 nM, or less than 200 pM. For example, an antagonistic antigen binding protein that specifically binds to PD-L1 may block the binding of a ligand to PD-L1 with an IC50 of between 200 pM and 10 nM, for example between 200 pM and 1 nM.

[0169] In a further preferred embodiment of the fifth aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 stimulates T cell proliferation.

[0170] In a further preferred embodiment of the fifth aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 induces lymphocyte proliferation.

[0171] In a further preferred embodiment of the fifth aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 induces the secretion of IL-2 and / or IFNγ.

[0172] In a further preferred embodiment of the fifth aspect, the antagonistic antigen binding protein that specifically binds to PD-L1 reduces tumor volume by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to baseline values.

[0173] In a sixth aspect, the present invention provides an antagonistic antigen binding protein that specifically binds to LAG-3, the antigen binding protein comprising: (i) A combination of: a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 39 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 38; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 48 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 47; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 57 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 56; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 66 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 65; a combination consisting of a light chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 75 and a heavy chain variable domain having a sequence that has at least 90% identity to SEQ ID NO: 74; a combination of a light chain variable domain and a heavy chain variable domain selected from the group (ii) A combination of: a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 63; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 72; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 80 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 81; a combination of heavy chain complementarity determining region 3 (CDRH3) and light chain complementarity determining region 3 (CDRL3) selected from the group consisting of: The present invention relates to an antagonistic antigen-binding protein comprising any one of:

[0174] In a preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a light chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 39 and a heavy chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 38.

[0175] In a further preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45.

[0176] In a further preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a light chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 48 and a heavy chain variable domain having a sequence with at least 90% identity to SEQ ID NO: 47.

[0177] In a further preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54.

[0178] In a preferred embodiment of the sixth aspect of the invention, the antagonistic antigen binding protein specifically binds to LAG-3, wherein said antigen binding protein comprises: a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 39 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 38; a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 48 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 47; a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 57 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 56; a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 66 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 65; a combination consisting of a light chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 75 and a heavy chain variable domain having a sequence that has at least 95% identity to SEQ ID NO: 74; The combination of a light chain variable domain and a heavy chain variable domain is selected from the group:

[0179] In a further preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a light chain variable domain having a sequence with at least 95% identity to SEQ ID NO: 39 and a heavy chain variable domain having a sequence with at least 95% identity to SEQ ID NO: 38.

[0180] In a further preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a light chain variable domain having a sequence with at least 95% identity to SEQ ID NO: 48 and a heavy chain variable domain having a sequence with at least 95% identity to SEQ ID NO: 47.

[0181] In a further preferred embodiment of the sixth aspect of the invention, the antagonistic antigen binding protein specifically binds to LAG-3, wherein said antigen binding protein comprises: a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 39 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 38; a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 48 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 47; a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 57 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 56; a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 66 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 65; a combination consisting of a light chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 75 and a heavy chain variable domain having a sequence that has at least 99% identity to SEQ ID NO: 74; The combination of a light chain variable domain and a heavy chain variable domain is selected from the group:

[0182] In a further preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a light chain variable domain having a sequence with at least 99% identity to SEQ ID NO: 39 and a heavy chain variable domain having a sequence with at least 99% identity to SEQ ID NO: 38.

[0183] In a further preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a light chain variable domain having a sequence with at least 99% identity to SEQ ID NO: 48 and a heavy chain variable domain having a sequence with at least 99% identity to SEQ ID NO: 47.

[0184] In a further preferred embodiment of the sixth aspect of the invention, the antagonistic antigen binding protein specifically binds to LAG-3, wherein said antigen binding protein is a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 63; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 72; a combination consisting of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 80 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 81; The heavy chain complementarity determining region 3 (CDRH3) and light chain complementarity determining region 3 (CDRL3) are selected from the group consisting of:

[0185] In a further preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45.

[0186] In a further preferred embodiment, the present invention relates to an antagonistic antigen-binding protein that specifically binds to LAG-3, comprising a combination of a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54.

[0187] Preferably, the antagonistic antigen binding protein specifically binds to LAG-3, wherein said antigen binding protein further comprises one or more selected from the group consisting of: CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:58, SEQ ID NO:67 or SEQ ID NO:76; CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO:42, SEQ ID NO:51, SEQ ID NO:60, SEQ ID NO:69 or SEQ ID NO:78; CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO:41, SEQ ID NO:50, SEQ ID NO:59, SEQ ID NO:68 or SEQ ID NO:77; and CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:61, SEQ ID NO:70 or SEQ ID NO:79.

[0188] For example, an antagonistic antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein is a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 40 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 41; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 42 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 43; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0189] For example, an antagonistic antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein is a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54; a CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 49 and a CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 50; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 51 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 52; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0190] For example, an antagonistic antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein is a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 63; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 58 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 59; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 60 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 61; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0191] For example, an antagonistic antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein is a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 72; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 67 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 68; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 69 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 70; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0192] For example, an antagonistic antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein is a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 80 and a CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 81; CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 76 and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 77; a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 78 and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 79; Also within the scope of the present invention are the above-identified competitive antigen binding proteins which preferably comprise one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2.

[0193] In a further preferred embodiment of the sixth aspect, the antagonistic antigen-binding protein that specifically binds to LAG-3 is an antibody, antibody-like protein, or fragment thereof. Preferably, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a single-chain variable fragment antibody, a diabody, a Fab fragment, a F(ab)2 fragment, an antibody mimetic, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0194] In a further preferred embodiment of the sixth aspect, an antagonistic antigen binding protein that specifically binds to LAG-3 binds to LAG-3 with a Kd of less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 100 pM, less than 10 pM, or less than 5 pM. For example, an antagonistic antigen binding protein that specifically binds to LAG-3 binds to LAG-3 with a Kd of between 5 pM and 100 nM, between 5 pM and 50 nM, between 5 pM and 10 nM, between 5 pM and 1 nM, between 5 pM and 100 pM, e.g., between 5 pM and 10 pM.

[0195] In further preferred embodiments of the sixth aspect, a competitive antigen binding protein that specifically binds to LAG-3 may block ligand binding to LAG-3 with an IC50 of less than 10 nM, less than 1 nM, or less than 200 pM. For example, a competitive antigen binding protein that specifically binds to LAG-3 may block ligand binding to LAG-3 with an IC50 of between 200 pM and 10 nM, e.g., between 200 pM and 1 nM.

[0196] In a further preferred embodiment of the sixth aspect, the antagonistic antigen binding protein that specifically binds to LAG-3 stimulates T cell proliferation.

[0197] In a further preferred embodiment of the sixth aspect, the antagonistic antigen binding protein that specifically binds to LAG-3 induces lymphocyte proliferation.

[0198] In a further preferred embodiment of the sixth aspect, the antagonistic antigen binding protein that specifically binds to LAG-3 induces the secretion of IL-2 and / or IFNγ.

[0199] In a further preferred embodiment of the sixth aspect, the antagonistic antigen binding protein that specifically binds to LAG-3 reduces tumor volume by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to baseline values.

[0200] In a seventh aspect, the present invention relates to a nucleic acid encoding the antagonistic antigen-binding protein of any one of aspects 1 to 6 of the present invention.

[0201] In an eighth aspect, the present invention relates to a recombinant expression vector comprising the nucleic acid molecule of the seventh aspect.

[0202] In a ninth aspect, the present invention relates to a host cell comprising the vector of the eighth aspect of the invention.

[0203] In a tenth aspect, the present invention relates to a method of producing an antagonistic antigen-binding protein according to any one of Aspects 1 to 6 of the present invention, comprising the step of preparing said antigen-binding protein from a host cell that expresses said antigen-binding protein.

[0204] In an eleventh aspect, the present invention relates to an antagonistic antigen-binding protein produced by expression of recombinant DNA in a host cell of the ninth aspect of the invention.

[0205] In a twelfth aspect, the present invention relates to a pharmaceutical composition comprising at least one antagonistic antigen-binding protein according to any one of aspects 1 to 6 of the invention, a nucleic acid according to the seventh aspect of the invention, or a vector according to the eighth aspect of the invention, and a pharmaceutically acceptable carrier.

[0206] In a preferred embodiment of the twelfth aspect, the pharmaceutical composition is adapted for parenteral administration. Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the composition substantially isotonic with the blood of the intended recipient. Other components that may be present in such compositions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. Compositions adapted for parenteral administration may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, such as sterile physiological saline solution for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0207] In a preferred embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the injection site. Generally, these ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by injection, the composition can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile saline can be provided so that the ingredients can be mixed prior to administration.

[0208] In a preferred embodiment of the twelfth aspect of the invention, the pharmaceutical composition comprises at least one further active agent. Preferably, the at least one further active agent is selected from the group consisting of another competitive antigen-binding protein according to any one of aspects 1 to 6 of the invention, a checkpoint inhibitor, a chemotherapeutic agent, a radiotherapeutic agent, an angiogenesis inhibitor, a cancer vaccine, and an oncolytic virus. For example, the at least one further active agent is selected from the group consisting of carboplatin-paclitaxel, trastuzumab, pertuzumab, erlotinib, lapatinib, imatinib, vemurafenib, dabrafenib, trametinib, bevacizumab, sunitinib, and pazopanib.

[0209] In a thirteenth aspect, the present invention relates to a kit comprising a pharmaceutical composition according to the twelfth aspect of the invention and, optionally, at least one further active agent. Preferably, the at least one further active agent is selected from the group consisting of another competitive antigen-binding protein according to any one of aspects 1 to 6 of the invention, a checkpoint inhibitor, a chemotherapeutic agent, a radiotherapeutic agent, an angiogenesis inhibitor, a cancer vaccine, and an oncolytic virus. For example, the at least one further active agent is selected from the group consisting of carboplatin-paclitaxel, trastuzumab, pertuzumab, erlotinib, lapatinib, imatinib, vemurafenib, dabrafenib, trametinib, bevacizumab, sunitinib, and pazopanib.

[0210] In a fourteenth aspect, the present invention relates to an antagonistic antigen-binding protein according to any one of aspects 1 to 6 of the invention, a nucleic acid according to the seventh aspect of the invention, a vector according to the eighth aspect of the invention or a pharmaceutical composition according to the twelfth aspect of the invention for use in the treatment of cancer and / or chronic infections.

[0211] In a preferred embodiment of the fourteenth aspect of the invention, the cancer is (a) malignant neoplasms of the lips, oral cavity and pharynx; and / or (b) malignant neoplasms of the digestive tract, and / or (c) malignant neoplasms of the respiratory and intrathoracic organs, and / or (d) malignant neoplasms of bone and articular cartilage, and / or (e) melanoma and other malignant neoplasms of the skin, and / or (f) malignant neoplasms of the mesothelium and soft tissues, and / or (g) malignant neoplasm of the breast, and / or (h) Malignant neoplasms of the female genital tract, and / or (i) malignant neoplasms of the male genital tract, and / or (j) malignant neoplasms of the urinary tract, and / or (k) malignant neoplasms of the eye, brain, and other parts of the central nervous system; and / or (l) malignant neoplasms of the thyroid gland and other endocrine glands, and / or (m) Malignant neoplasms of the lymphatic system, hematopoietic system and related tissues; is selected from the group consisting of:

[0212] Preferably, the cancer is selected from the group consisting of NSCLC (non-small cell lung cancer), melanoma, MSI (microsatellite instability associated cancer), bladder cancer, kidney cancer, head and neck cancer, Hodgkin's lymphoma, hepatocellular carcinoma (HCC) and gastric cancer.

[0213] In a further preferred embodiment of the fourteenth aspect of the invention, the chronic infection is selected from the group consisting of HBV, HCV, HIV, HSV, HPV, EBV, CMV and chlamydia. Preferably, the chronic infection is caused by HBV, HCV, HIV and HPV.

[0214] In a further preferred embodiment of the fourteenth aspect of the invention, at least one further active agent is administered to a subject in need thereof. Preferably, the at least one further active agent and the competitive binding protein of any of aspects 1 to 6 of the invention, the nucleic acid of the seventh aspect of the invention, the vector of the eighth aspect of the invention or the pharmaceutical composition of the twelfth aspect of the invention are administered simultaneously, sequentially or in a combination thereof.

[0215] The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention as defined by the appended claims. [Example]

[0216] Materials and Methods Antibodies and Human Recombinant Proteins The following antibodies were used: horseradish peroxidase (HRP)-conjugated anti-His mouse monoclonal antibody (Qiagen, Hilden, Germany), (HRP)-conjugated anti-M13 mouse monoclonal antibody (GE Healthcare, Chalfont St. Giles, UK), anti-human LAG-3 mouse monoclonal antibody (R&D Systems, Minneapolis, USA), anti-human PD-1 human monoclonal antibody nivolumab (Opdivo™, Bristol-Myers Squibb, Princeton, NJ, USA), anti-human PD-L1 human monoclonal antibody (G&P Biosciences, Santa Clara, CA, USA), (HRP)-conjugated anti-human IgG (Promega, Madison, WI, USA), PE / anti-human CD2 mouse monoclonal antibody (BioLegend Inc., San Diego, CA, USA), APC / anti-human CD3 mouse antibody, PE / anti-human CD4 mouse monoclonal antibody, PerCP / anti-human CD8 mouse monoclonal antibody (all from BD Biosciences, San Jose, CA, USA), (HRP)-conjugated anti-human IgG (Fab')2 goat monoclonal antibody (Abcam, Cambridge, UK), APC / anti-human IgG Fc mouse antibody, APC / Cy7 anti-human CD366 (TIM3) mouse antibody, APC / anti-human CD272 (BTLA) mouse antibody, APC / anti-human CD137 (4-1BB) mouse antibody, PE / anti-human CD134 (OX40) mouse antibody, Brilliant Violet 510™ / anti-mouse / rat / human CD27 hamster antibody, APC / anti-human / mouse / rat CD278 (ICOS) hamster antibody (all from BioLegend), FITC / anti-human TIGIT mouse antibody (Thermo Fisher Scientific, Waltham, MA, USA). The following recombinant chimeric proteins were used: human LAG-3 / Fc, human PD-1 / Fc, human PD-L1 / Fc, TIM3 / Fc, BTLA / Fc, TIGIT / Fc, OX40 / Fc, 4-1BB / Fc, CD27 / Fc, and ICOS / Fc, and human IgG1-Fc (all from R&D Systems).

[0217] cell culture MDA-MB-231 cells were cultured in Dulbecco's modified Eagle's medium (Gibco™ DMEM, Thermo Fisher Scientific). MCF7 cells were cultured in modified Eagle's medium (Gibco™ MEM, Thermo Fisher Scientific). The medium was supplemented with 10% (vol / vol) heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 50 IU ml-1 penicillin, 50 μg ml-1 streptomycin, and 2 nM L-glutamine (all Gibco™, Thermo Fisher Scientific). Cell lines were purchased from the American Type Culture Collection (ATCC) and cultured at 37°C in a humidified atmosphere containing 5% CO2.

[0218] Isolation of human peripheral blood mononuclear cells (hPBMCs) Human peripheral blood mononuclear cells (hPBMCs) were isolated from healthy donor blood using ACCUSPIN™ System-Histopaque™-1077 (Sigma-Aldrich) according to the manufacturer's instructions and frozen in a solution containing 90% FBS and 10% DMSO until use. Cryopreserved cell vials were gently thawed using RPMI 1640 medium (Gibco™, Thermo Fisher Scientific) supplemented with 1% L-glutamine, 1% CTL-Wash™ (Cellular Technology Limited, Cleveland, OH, USA), and 100 U / ml benzonase (Merck Millipore, Billerica, MA, USA). The collected hPBMCs were then washed by centrifugation, plated, and incubated overnight at 37°C in R10 medium consisting of RPMI 1640 (Gibco™, Thermo Fisher Scientific) supplemented with 10% inactivated fetal bovine serum (FBS, Sigma-Aldrich), 1% L-glutamine, 50 U ml-1 penicillin, 50 μg ml-1 streptomycin, and 1% HEPES (all Gibco™, Thermo Fisher Scientific). After overnight incubation, the hPBMCs were collected in PBS, counted using a Muse™ cell analyzer (Merck Millipore), and resuspended at a density of 1 x 106 cells / ml.

[0219] FACS analysis of immune checkpoint expression levels in hPBMCs Rescued and counted human peripheral blood mononuclear cells (hPBMCs) were activated with Dynabeads™ Human T-Activator CD3 / CD28 at a concentration of 1 x 103 beads / ml (Gibco™, Thermo Fisher Scientific). 24 to 96 hours after activation, cells were seeded into round-bottom 96-well plates (1 x 106 cells / well) and then centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed. Unlabeled anti-LAG-3 primary antibody, anti-PD-1 primary antibody (nivolumab), or anti-PD-L1 primary antibody was added to each well at a concentration of 10 μg / ml and incubated at room temperature for 90 minutes with gentle shaking. After thorough washing, cells were stained with 100 μl of APC / anti-human IgG Fc antibody and 10 μg / ml of PE / anti-human CD2 antibody (BioLegend) in FACS buffer (PBS, 1% FBS) for 45 minutes at room temperature by gentle shaking. Labeled antibodies APC / Cy7 anti-human CD366 (TIM3), APC / anti-human CD272 (BTLA), APC / anti-human CD137 (4-1BB), PE / anti-human CD134 (OX40), Brilliant Violet 510™ / anti-mouse / rat / human CD27, APC / anti-human / mouse / rat CD278 (ICOS), and FITC / anti-human TIGIT antibodies were added to each well at a concentration of 10 μg / ml and incubated for 90 minutes at room temperature by gentle shaking. After washing twice with FACS buffer, cells were resuspended in PBS, transferred to 5 ml polystyrene round-bottom tubes (BD Biosciences), and analyzed on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA).

[0220] Flow cytometry binding assay of monoclonal antibodies to hPBMC Human peripheral blood mononuclear cells (hPBMCs) isolated from healthy donors were thawed in RPMI 1640 medium (Gibco) supplemented with 2 mM L-glutamine, 10% (v / v) CTL wash supplement (CTL, Shaker Heights, OH, USA), and 100 U / ml benzonase (Merck Millipore, Burlington, MA, USA). After centrifugation at 1200 rpm for 10 min, they were resuspended in complete RPMI medium supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS, Sigma), 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin (Gibco), and 10 mM HEPES (Gibco). After 16 hours at 37°C in a humidified atmosphere containing 5% CO2, hPBMCs were counted, resuspended in complete RPMI medium (as described above) at a concentration of 106 viable cells / ml, and activated with Dynabeads Human T-Activator CD3 / CD28 (Gibco) at 25 μl beads / 106 viable cells. 24 hours after activation, cells were centrifuged and plated at 4 × 105 cells / well in PBS in a round-bottom 96-well plate. After one wash in PBS, they were incubated with 50 μl LIVE / DEAD™ Fixable Violet Dead Cell Stain (Invitrogen) for 30 minutes at +4°C and washed again. Monoclonal antibodies diluted in 100 μl PBS at concentrations of 48 pM and 9.6 pM were added to the cells and incubated for 1 hour and 30 minutes at room temperature in the dark with gentle shaking. Cells were washed twice as described above and then incubated with 5 μl of PE-conjugated anti-human CD2 (BD Biosciences) and APC-conjugated anti-human IgG, Fcγ-specific antibody (Jackson Immunoresearch) diluted 1:2000 in 100 μl of FACS buffer (PBS (1×) 1% (v / v) FBS)).After 45 min of incubation under the same conditions as above, cells were washed twice with FACS buffer and resuspended in 150 μl of PBS (1×) for acquisition on a CytoFLEX flow cytometer (Beckman Coulter).

[0221] Selection of scFv-phage clones Phagemid particles were recovered from the library using M13-K07 helper phage (Invitrogen, Thermo Fisher Scientific) as previously described (De Lorenzo C, Clinical Cancer Research, 2002). For each selection round, phage (10 cfu) were blocked with 5% (wt / vol) skim milk powder (Fluka Analytical, Sigma-Aldrich) in PBS. In the first selection round, blocked phage were subjected to a single round of selection by incubating with activated lymphocytes (1 × 10 cells) overnight at 4°C with rotation. After extensive washing with PBS, bound phage were eluted from the activated lymphocytes with 76 mM citric acid in PBS (pH 2.5) for 5 min and then neutralized with 1 M Tris·HCl (pH 8.0). The recovered phage were amplified by infecting E. coli TG1 cells to prepare phage for the next selection round against the purified chimeric protein. For this purpose, Nunc™ polypropylene tubes (Fisher Scientific, Thermo Fisher Scientific) were coated with the selected recombinant chimeric protein at a concentration of 20 μg / ml in 0.05 M NaHCO3 solution for 72 hours at 4°C. Blocked phage were subjected to two subsequent rounds of negative selection by rotating and incubating the tubes coated with rhIgG1-Fc protein for 2 hours at 4°C. Unbound phage recovered in the supernatant were then incubated overnight at 4°C in the coated tubes prepared as described above for positive selection and eluted as described above. Alternatively, trypsin was used for elution. Briefly, after extensive washing with PBS, bound phage were incubated with 50 mM Tris·HCl (pH 8.0) buffer containing 1 mM CaCl2 for 1 hour at 4°C with rotation, followed by elution from the chimeric protein with trypsin (1 μg / ml) by gentle shaking for an additional 15 minutes at 25°C.The reaction was then blocked by using protease inhibitors (cOmplete™ EDTA-free protease inhibitor cocktail, Sigma-Aldrich), after which the phages were collected and stored at 4°C until use.

[0222] DNA fragment preparation and high-throughput sequencing For each sublibrary, scFv-containing phagemid double-stranded DNA was purified from superinfected E. coli TG1 cell cultures using the Endo-free Plasmid Maxi Kit (Qiagen). Full-length scFvs were excised with the restriction enzymes BamHI and HindIII (New England Biolabs) and purified from a 1.2% agarose gel using the Wizard™ SV Gel and PCR Clean-Up System (Promega). A second enzymatic excision with NcoI and XhoI (New England Biolabs) was performed to isolate VHs from the previously purified material, followed by extraction from a 1.4% agarose gel. Library preparation for NGS, sequencing, and preliminary bioinformatics analysis of the data were performed at the Center for Translational Genomics and Bioinformatics, San Raffaele Hospital, Milan, Italy. VHs extracted from the sublibraries were barcoded using the TruSeq ChIP Sample Preparation Kit (Illumina). To achieve deep sequencing of the VH mixture of several subcycles, a complementary barcoding scheme was implemented. Subcycles 2 and 3 of each target were mixed in a dedicated run. To compensate for the greater complexity, the first universal cycle 1 was sequenced separately. The barcoded samples were diluted to a final concentration of 10 pM and sequenced on an Illumina MiSeq instrument using a 2 × 300 nt SBS Kit v3.

[0223] Recovery of scFv Clones of interest were isolated from the sublibrary at cycle 3 of the corresponding target. High-ranking clones were cloned using overlapping primers designed within the corresponding HCDR3 region using the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) as previously described (26). Briefly, the extension reaction consisted of: 50 ng–250 ng template, 2 / 5 μL of QuickSolution reagent, 1 μL of Pfu Ultra High Fidelity DNA Polymerase (2.5 U / μL), 5 μL of 10× reaction buffer, 1 μL of dNTP mix, 125 ng of forward primer, 125 ng of reverse primer, and HO to a final volume of 50 μL. Template DNA was removed by restriction with 1 μL of DpnI enzyme as suggested by the kit supplier. An appropriate amount of the reaction was used to transform XL10-GOLD ultracompetent cells (Agilent Technologies) and then plated on LB / agar containing 100 μg / ml ampicillin. Several colonies were picked and evaluated by double digestion and sequencing. For low-ranking clones, DNA samples were isolated from cycle 3 by overlapping PCR. Briefly, two PCR reactions were performed using primers designed within the corresponding HCDR3 regions and the constant regions of the plasmid upstream and downstream of the VH and VL fragments using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) to obtain the VH and VL fragments separately. In a second step, the PCR fragments corresponding to each clone were mixed and extended to obtain the complete scFv.The reaction was composed as follows: 150 ng of template for amplification of the VH and VL fragments and 10 ng of template for amplification of the complete scFv (VH and VL fragments), 0.5 μL of Phusion DNA polymerase (0.02 U / μL), 10 μL of 5× Phusion HF buffer, 1 μL of dNTP mix, 0.5 μM forward primer, 0.5 μM reverse primer, 1.5 μL of DMSO, and HO to a final volume of 50 μL.

[0224] Antibody production and purification The scFvs of interest were converted to whole IgG4 antibodies by cloning the corresponding VH and VL cDNAs into pEU vectors 8.2VH and 4.2VL (Paciello R, J Gen Virol., 2016), which express the antibody's constant heavy and light chains, respectively. Briefly, VH and VL were amplified using specific primers with CloneAmp HiFi PCR Premix under standard conditions and purified from a 1.3% agarose gel using the Wizard™ SV Gel and PCR Clean-Up System (Promega). Using the In-Fusion HD Cloning Kit (Clontech Laboratories, Mountain View, CA, USA), VH was cloned into the pEU8.2VH vector linearized with BamHI and BssHII (New England Biolabs), and VL was cloned into the pEU4.2VL vector linearized with ApaLI and AvrII (New England Biolabs). Stellar competent cells (Clontech Laboratories, Inc., Mountain View, CA, USA) were transformed with the obtained vector, and colonies were screened by digestion and sequence analysis. The correct preparations were co-transfected into HEK293-EBNA using Lipofectamine transfection reagent (Life Technologies, Inc.) and grown in chemically defined CD CHO medium (Gibco, Life Technologies, Inc.) supplemented with 5 ml of L-glutamine (200 mM) (Gibco, Life Technologies) and 5 ml of penicillin-streptomycin (10,000 U / mL-10 mg / mL) (Sigma-Aldirch) at 37°C for approximately 10 days in 6-well plates or 150 mm Corning™ tissue culture-treated dishes.Conditioned media was collected and antibodies were purified using Protein A HP SpinTrap (GE Healthcare Life Sciences, NY, USA). The purity of the final product was assessed by SDS-PAGE NuPAGE™ 4%-12% Bis-Tris protein gel, 1.0 mm, 10-well (Thermo Fisher Scientific), followed by staining with Coomassie Blue solution (Biorad) for 20 min and destaining with 7% CH3COOH and 20% Et-OH.

[0225] Enzyme-linked immunosorbent assay (ELISA) To confirm the binding specificity of the purified monoclonal antibodies, ELISA assays were performed on the chimeric proteins (coated at 5 μg / ml), tumor cells (PD-L1-positive breast cancer MDA-MB-231 cells or PD-L1-negative breast cancer MCF7 cells), and naive or activated hPBMCs. Nunc™ flat-bottom 96-well plates (Fisher Scientific, Thermo Fisher Scientific) were coated with 5 μg / ml of rhPD-1 recombinant protein, rhPD-1 recombinant protein, and rhLAG-3 recombinant protein in 0.05 M NaHCO3 solution at 37°C for 72 hours. After blocking the coated 96-well plates with 5% nonfat dry milk in PBS at 37°C for 1 hour, purified monoclonal antibodies were added to the plates at increasing concentrations (10 nM to 200 nM) in 2.5% nonfat dry milk in PBS and incubated at room temperature for 2 hours with gentle shaking. After extensive washing with PBS, the plates were incubated with (HRP)-conjugated anti-human IgG (Fab')2 goat monoclonal antibody (Abcam, Cambridge, UK) for 1 hour, washed again, and incubated with TMB reagent for 10 minutes before quenching with an equal volume of 1 N HCl.

[0226] Cell ELISA assays were performed by plating cells in round-bottom 96-well plates (2 × 10 cells or 2 × 10 lymphocytes per well) and incubating them with increasing concentrations (0.5 nM to 200 nM) of monoclonal antibodies in 2.5% nonfat dry milk for 2 hours at room temperature with gentle agitation. The plates were then centrifuged, and the cell pellets were washed with PBS and incubated with HRP-conjugated anti-human IgG goat polyclonal antibodies for 1 hour at room temperature. Following further washing with PBS (1X), TMB reagent was added for 10 minutes before quenching with an equal volume of 1N HCl. Absorbance at 450 nm was measured using an Envision plate reader (Perkin Elmer, Waltham, MA, USA).

[0227] Competitive ELISA assay To determine whether the novel anti-PD-1 monoclonal antibodies recognize epitopes distinct from those of nivolumab, a competitive ELISA assay was performed on the coated PD-1 / Fc chimeric protein (5 μg / ml). After blocking with 5% nonfat dry milk in PBS, the coated 96-well plates were preincubated with saturating concentrations of each unlabeled monoclonal antibody (400 nM) in 2.5% nonfat dry milk in PBS for 2 hours at room temperature with agitation. After extensive washing with PBS, increasing concentrations of biotinylated nivolumab monoclonal antibody were added. To detect binding, the plates were incubated with HRP-conjugated streptavidin (Biorad) for 30 minutes at room temperature with agitation, washed again, and analyzed as described above.

[0228] Analysis of antibody binding to hPBMCs by FACS Human peripheral blood mononuclear cells (hPBMCs) were rescued the day after thawing, counted, and then activated with Dynabeads™ Human T-Activator CD3 / CD28 at a concentration of 1 x 103 beads / ml (Gibco™, Thermo Fisher Scientific). 24 to 96 hours after activation, cells were seeded into round-bottom 96-well plates (4 x 105 cells / well) and then centrifuged at 1200 rpm / min for 5 minutes, and the supernatant was removed. Anti-PD-L1 monoclonal antibodies PD-L1_A and PD-L1_C were added at a concentration of 10 μg / ml and incubated at room temperature for 90 minutes with gentle shaking. After thorough washing, cells were stained with 100 μl of APC / anti-human IgG Fc antibody and 10 μg / ml of PE / anti-human CD2 antibody (BioLegend) in FACS buffer (PBS, 1% FBS) for 45 minutes at room temperature by gentle shaking. After two washes with FACS buffer, cells were resuspended in PBS, transferred to polystyrene round-bottom tubes (BD Biosciences), and analyzed on a CytoFLEX flow cytometer (Beckman Coulter).

[0229] Lymphocyte proliferation assay Lymphocyte proliferation assays were performed using hPBMCs, counted as described above, and resuspended at a density of 2 × 10 cells / ml in prewarmed 0.1% BSA / PBS solution. For staining, hPBMCs were resuspended at a density of 1 × 10 cells / ml using prewarmed 0.1% BSA / PBS solution containing 10 μM CFDA-SE (Vybrant™ Cell Tracer Kit, Invitrogen™, Thermo Fisher Scientific) and incubated at 37°C for 10 minutes. The cells were then permeabilized using ice-cold R10 medium by incubating on ice for an additional 5 minutes. Next, the cells were washed three times with PBS. Between the second and third washes, the cells were incubated at 37°C for 5 minutes to completely remove excess CFDA-SE. After the final wash and centrifugation at 1200 rpm for 10 minutes, cells were resuspended in R10 at a density of 1 × 10 cells / ml and plated into 48-well plates (1 × 10 cells / well). Lymphocyte proliferation assays were performed by incubating plated lymphocytes with 2.5 μg / ml phytohemagglutinin-L (PHA-L, Roche) in the absence or presence of selected anti-LAG-3 monoclonal antibodies (10 μg / ml). The plates were then incubated at 37°C for 5 days. After treatment, each sample was collected, resuspended in 100 μl of PBS, and transferred to a round-bottom 96-well plate. Cells were first incubated with violet LIVE / DEAD solution (Thermo Fisher Scientific) for 30 minutes at 4°C. After several washes, the samples were further incubated with anti-human CD3 (APC), anti-CD4 (PE), and anti-CD8 (PerCP) antibodies (10 μl / sample) for 1 hour at 4° C. Finally, the cells were thoroughly washed, resuspended in 200 μl of PBS, and transferred to a 5 ml polystyrene round-bottom tube (BD Biosciences) for cytometry acquisition using a CytoFLEX flow cytometer (Beckman Coulter).

[0230] Example 1: Selection of scFvs on activated human T lymphocytes Our goal was to generate a human antibody repertoire against immune checkpoints (ICs). Because many ICs are expressed on the surface of T lymphocytes and their expression increases upon exposure of T cells to antigen-dependent or antigen-independent stimuli, we attempted to screen a library of human single-chain antibody fragments (scFvs) using unfractionated human peripheral blood mononuclear cells (hPBMCs). To optimize the selection process, we first evaluated the kinetics and expression levels of 10 different ICs (listed in Table 1) after in vitro activation with anti-CD3 / CD28 beads. As shown in Table 1, peak expression measured by flow cytometry analysis varied among various immune modulators, but most of them reached maximum levels of expression after 96 hours of stimulation (see Table 1). After 96 hours of stimulation, all ICs were well expressed in more than 50% of the gated population, except for BTLA, which maintained the same level of surface expression between naive and activated hPBMCs, and TIGIT, which slightly increased its expression level. Based on this analysis, 96 hours was chosen as the time point of lymphocyte activation to be used for selection of the human scFv library.

[0231] Table 1 - Percentage of expression of each target in human lymphocytes untreated or activated for various time intervals [Table 1]

[0232] Approximately 1 million phage particles were selected by panning the library against activated hPBMCs (selection cycle 1) as described in the Experimental Procedures section. This pool of phage potentially represents a large collection of binders to many different ICs and is hereafter referred to as the "immunome library." To facilitate the identification of binders to specific ICs, 10 different parallel selection rounds of the immunome library were performed using Fc-fusion recombinant proteins in successive pannings, resulting in IC-specific repertoires.

[0233] Example 2: Identification of scFv binders by next generation sequencing To identify individual phage clones selected by the combined ex vivo / in vitro approach, we sequenced the VH regions of IC-specific repertoires by massively parallel sequencing on the MiSeq Illumina platform (see the Materials and Methods section for details). Starting with the immunome library (cycle 1), two subsequent cycles of selection against Fc-fusion recombinant proteins (cycles 2 and 3) were performed to efficiently enrich for target-specific phages. Sequence analysis of the entire selection set revealed that enrichment had already occurred after cycle 2, but significantly higher levels of enrichment (i.e., at least 10-fold) were obtained after the third cycle (Figure 1). Analysis of the parallel sequencing data allowed us to remove VH sequences common to all selections, likely due to enrichment of Fc binders common to the 10 biochemical baits. Similarly, nonspecific biologically enriched clones with stop codons within the scFv-encoding sequence were removed from the list of potential binders. Finally, a threshold of 85 counts per million was set for the various selection cycle 3 lists to obtain the most relevant and specific clones for each target. In this way, a detailed snapshot of the best potential binders for 9 out of 10 targets was obtained (shown in Figure 1). The TIGIT selection was actually excluded from the analysis, since it showed little sequence enrichment, likely due to its low expression in activated hPBMCs (see Table 1).

[0234] Binders specific for three targets, namely LAG-3, PD-1, and PD-L1, were selected for further study, along with antibodies specific for them, such as the anti-PD-1 antibody nivolumab, which have previously been developed and widely used in clinical settings with demonstrated therapeutic efficacy, so they can be used for comparison in biological assays. The best scFvs were rescued from the IC-specific repertoires for LAG-3, PD-1, and PD-L1, with the aim of identifying at least five antibodies active against each of the three targets for further characterization and conversion to fully human IgG4.

[0235] Example 3: Human IgG generated from selected binders exhibits high binding affinity and receptor / ligand competition activity Human IgG4 antibodies generated from the top-ranked LAG-3, PD-1, and PD-L1 binders were confirmed to recognize activated PBMCs and recombinant proteins with low or subnanomolar affinity (Figure 2 and the table therein). Notably, several anti-PD-1 monoclonal antibodies (i.e., PD-1_A and PD-1_B) showed comparable or better apparent affinity compared to the clinically validated nivolumab. Most anti-PD-1 and anti-PD-L1 antibodies, including nivolumab, showed stronger binding to recombinant proteins compared to activated hPBMCs, whereas the opposite was true for anti-LAG3 antibodies. Because anti-PD-L1 antibodies have been shown to provide clinical benefit by blocking the interaction between PD-L1 expressed on cancer cells and PD-1 presented by T cells, we further tested whether anti-PD-L1 antibodies recognize their targets on the surface of cancer cells. All anti-PD-L1 antibodies also showed high affinity for PD-L1-expressing tumor cells, such as breast MDA-MB-231 tumor cells, although the hierarchy of tumor cell binding activity was different from that observed with activated hPBMCs (Table in Figure 2). Furthermore, monoclonal antibodies PD-L1_A and PD-L1_B were shown to be able to block the recognition of PD-L1 by its two receptors, PD-1 and B7.1, in a competitive ELISA assay.

[0236] Additionally, selected antibodies were tested for binding to their mouse orthologues. Unlike nivolumab, one of the anti-PD-1 antibodies was found to be cross-reactive with mouse PD-L1. Therefore, PD-1_A likely recognizes a different epitope than nivolumab, as supported by a competitive ELISA assay performed by measuring the binding of biotinylated nivolumab to PD-1 in the absence or presence of a saturating concentration of unlabeled PD-1_A monoclonal antibody. Simultaneous binding of PD-1_A and nivolumab revealed that the two monoclonal antibodies do not interfere with their receptor interactions.

[0237] Example 4: High-affinity IgG against immune checkpoint molecules exhibits T cell immunostimulatory activity and effector function Previous reports have shown that CD3+ primary resting cells in unfractionated human PBMCs can be induced to proliferate in vitro using staphylococcal enterotoxin B (SEB) or phytohemagglutinin (PHA), and that this activity is regulated by CI (Macon-Lemaitre L, Immunology, 2005; Wang C, Cancer Immunol Res., 2014; Selby MJ, Plos One, 2016). Therefore, using the lymphocyte proliferation assay described above, we tested whether selected antibodies against LAG-3, PD-1, or PD-L1 could increase cell division. CFDA-SE stained lymphocytes were stimulated with PHA and incubated in the absence or presence of antibodies to induce antigen-specific T cell proliferation. In this assay, nivolumab consistently produced a 50% increase in proliferative activity, and the anti-PD-1 antibodies PD-1_A and PD-1_B could also effectively stimulate T cell proliferation, with PD-1_A showing greater activity than the clinically active nivolumab (Figure 3). Similarly, all three antibodies against PD-L1 and one of the three LAG-3 antibodies induced varying degrees of proliferation. The ability to stimulate proliferation did not always correlate with avidity, suggesting that different antibodies may have different modes of interaction with their targets.

[0238] Interestingly, the antibodies PD-1_A and PD-L1_A, which were most active against human lymphocytes, also confirmed this ability against mouse lymphocytes, suggesting that they are cross-reactive with mouse PD-1 and PD-L1. We utilized MDA-MB-231 breast tumor cells, which express high levels of PD-L1 on their surface, to test the ability of anti-PD-L1 and anti-PD-1 antibodies to suppress the inhibitory effect of PD-1 / PD-L1 interaction on lymphocyte proliferation when the two cell types were co-cultured. As shown in Figure 4, the monoclonal antibodies PD-L1_A and PD-1_A induced lymphocyte proliferation in a dose-dependent manner, whereas only a minimal effect was detected in lymphocytes treated with the monoclonal antibodies PD-L1_B and PD-1_B. Again, monoclonal antibodies PD-L1_A and PD-1_A showed greater activity than nivolumab in this assay, whereas no effect on lymphocyte proliferation was observed when PD-L1-negative MCF7 tumor cells were used. Interestingly, in some cases, such as in the case of monoclonal antibodies LAG3_A and LAG3_C (Figure 2 and Table of Figure 2), the ability to stimulate proliferation did not correlate with the highest apparent affinity.

[0239] Three additional anti-PD-L1 antibodies were identified and characterized against PD-L1_A. The three antibodies, PD-L1_C, PD-L1_D, and PD-L1_E, were evaluated for their binding to human PBMCs that had been pre-activated to induce PD-L1 surface expression. A higher percentage of lymphocytes were stained by the three antibodies against PD-L1_A (see Figure 7).

[0240] Example 5: Effect of novel antibodies on cytokine production by stimulated hPBMCs hPBMCs (1 x 10 cells) were cultured and stimulated with 2.5 μg / mL PHA-L or 50 ng / mL Staphylococcal enterotoxin B (SEB, Sigma-Aldrich) for 18, 42, and 66 hours in the absence of antibody or in the presence of selected anti-LAG-3, anti-PD-L1, and anti-PD-1 monoclonal antibodies (20 μg / mL) or an isotype control antibody used as a negative control. Nivolumab was tested as a positive control in parallel assays under the same conditions. IL-2 or IFNγ levels in cell culture supernatants were measured by ELISA assay (DuoSet ELISA, R&D Systems) according to the manufacturer's recommendations.

[0241] Immunomodulatory antibodies such as nivolumab and ipilimumab have been shown to improve T cell effector function, potentially enhancing their clinical benefit (Macon-Lemaitre L, Immunology, 2005; Wang C, Cancer Immunol Res., 2014; Selby MJ, Plos One, 2016). Therefore, five antibodies (LAG-3_A, PD-1_A, PD-1_B, PD-L1_A, and PD-L1_B) with the highest ability to induce T cell proliferation were tested in a previously reported cytokine secretion assay (Wang C, Cancer Immunol Res., 2014) to characterize nivolumab. As shown in Figure 5, all tested antibodies were able to increase both IL-2 and IFNγ secretion by hPBMCs stimulated with either PHA or SEB. Cytokine secretion increased over time when various antibodies were added to the cell culture mixture. The PD-1_1 monoclonal antibody appeared to be consistently more potent than all other tested antibodies in its ability to stimulate secretion of both cytokines. The newly identified antibodies also performed comparable to nivolumab in this assay, further supporting the conclusion that our generated immunome library is enriched with binders with great potential for clinical development.

[0242] Two additional anti-PD-L1 antibodies (PD-L1_C and PD-L1_D) that were identified and characterized against PD-L1_A were also evaluated in comparison to nivolumab in the described cytokine secretion assay. As shown in Figure 8, all antibodies tested were able to increase the secretion of both IL-2 and IFNγ by hPBMCs stimulated with either PHA or SEB.

[0243] Example 6: In vivo antitumor activity Six-week-old female BalBC mice (Envigo) were used for in vivo studies. Mice were implanted subcutaneously with 2 × 10 cells in the right flank (day 0) and treated intraperitoneally with 200 μg of α-mPD-L1 (BioXcell, clone 10F.9G2), α-mPD-1 (BioXcell, clone RMP114), PD-1_A, or PD-L1_A on days 3, 6, and 10. Tumor growth was measured every 3 to 4 days using a caliper using the formula L × W2 / 2 (L is the maximum diameter of the tumor, and W is the minimum diameter of the tumor). Animals were sacrificed immediately upon signs of distress or tumor volume exceeding 2000 mm3.

[0244] Two antibodies, PD-1_A and PD-L1_A, were also tested in vivo in a CT26 colon cancer model, given their cross-reactivity with mouse PD-1 and PD-L1. Mice were implanted with CT-26 cells (day 0) and subsequently treated with PD-1_A and PD-L1 antibodies (days 3, 6, and 10). Two commercially available antibodies reactive with mouse PD-1 and PD-L1 (α-mPD-1 and α-mPD-L1) and a previously in vivo validated antibody (reference) were used as positive controls. Tumor growth in untreated mice was very rapid and uncontrollable, with the majority of tumors reaching a size of over 650 mm3 by day 21, whereas mice treated with PD-1_A showed a reduction in tumor volume (p=0.03). The activity of the cross-reactive anti-PD-1 antibodies was comparable to that of commercially available antibodies against mouse PD-1. Comparable activity trends were also observed with the two anti-PD-L1 antibodies (Figure 6). Treated mice consistently exhibited a dichotomy in response to PD-1 and PD-L1 blockade, with two distinct treatment outcomes: responder and non-responder mice, which are well described for this and other cancer models. Thus, the biological and functional activities of the two novel monoclonal antibodies were also confirmed in relevant in vivo models.

[0245] Summary of the experiment We adapted phage display technology to enable the rapid identification of a large set of antibodies recognizing several different ICs. To this end, we chose live, activated hPBMCs as selectors for the generation of an unbiased library of IC binders, i.e., an immunome library, from which scFvs specifically recognizing a given receptor can be elicited through subsequent affinity selection cycles using recombinant proteins or peptides or other target-specific baits. The rationale for using hPBMCs as the starting point for the selection process was also to ensure the selection of antibodies capable of recognizing native receptors with the correct post-translational modifications displayed by live human cells. Consistent with this working hypothesis, human IgG4 generated using the selected scFvs was able to recognize cell-presented receptors with apparent affinities in the low nanomolar range, down to 0.1 nM. This binding affinity is comparable to or exceeds that exhibited by clinically effective checkpoint-specific antibodies and is superior to that of nivolumab, an anti-PD-1 monoclonal antibody approved for the treatment of many types of cancer (Wang C, Cancer Immunol Res., 2014). To improve the efficiency of selection in hPBMCs, the expression levels of target ICs were increased by anti-CD3 cross-linking of T cells.

[0246] To facilitate the identification of binders that recognize specific receptors, the immunome library was further affinity-selected using a panel of 10 recombinant proteins. Aiming to develop a universal protocol for the selection of antibodies against cell surface-displayed proteins, two subsequent cycles of selection were performed against recombinant Fc-fusion proteins. This strategy represented a compromise between the efficiency of selection and the generation of a highly diverse IC-specific repertoire, thus enabling the identification of antibodies that bind to different regions of the target IC with distinct biological activities.

[0247] All selections achieved enrichment levels of 100- to 1000-fold, with four of them (PD-1, 4-1BB, CD27, and OX40) reaching even higher enrichments. Comparative sequence analysis of the different selection cycles confirmed that most selections (i.e., 7 out of 9) showed a significant improvement (i.e., at least 10-fold) in clonal enrichment after the second cycle, whereas selections on LAG3 and OX40 did not appear to improve beyond the second cycle.

[0248] Our results support the conclusion that the combined ex vivo / in vitro selection approach used in the context of this invention is more rapid and efficient than our previous approach of combining live cell selection with direct screening against selected targets to identify specific binders (Monaci P, Plos One, 2008). A total of 16 IgGs were confirmed to bind (5 anti-LAG3, 5 anti-PD-L1, and 6 anti-PD-1), with 11 exhibiting low nanomolar apparent affinity for their cognate receptors expressed in hPBMCs, and three of these (PD-L1_2, PD-1_1, and PD-1_2) possessing subnanomolar apparent affinity comparable to that of nivolumab. Notably, at least two to three of the five converted antibodies against each target were found to be highly specific for both recombinant purified targets and activated lymphocytes, without significant binding to naive lymphocytes or Fc, and were therefore selected for further biological and functional assays.

[0249] When the antibodies were tested for their ability to stimulate T cell proliferation, six of the eight antibodies (LAG-3_A, PD-L1_A, PD-L1_B, PD-1_A, and PD-1_B) demonstrated improved biological activity compared to nivolumab. PD-1_A also demonstrated significant activity in promoting T cell proliferation in coculture with the PD-L1-highly expressing tumor cell line MDA-MB-231. In this assay, the PD-L1_A monoclonal antibody also nearly tripled T cell proliferation, consistent with its ability to disrupt the PD-L1 / PD-1 interaction. The most active antibody did not necessarily correspond to an antibody with stronger binding affinity; for example, the monoclonal antibody LAG-3_A bound to hPBMCs with lower apparent affinity than the monoclonal antibody LAG-3_C, yet LAG-3_A was able to stimulate T cell proliferation, whereas LAG-3_C was not. These results also indirectly suggest that the selected antibodies recognize different epitopes on the target protein. We were able to demonstrate that some of the antibodies identified in this study can stimulate T cells to secrete IL2 and IFNγ. PD-1_1 monoclonal antibodies consistently showed the highest activity in stimulating cytokine secretion, extending the observation that some of the antibodies identified in this study exhibit similar binding affinity and biological properties to clinically validated nivolumab. Indeed, when tested in cell-based assays using cocultures of lymphocytes with APCs or tumor cells, some of the new antibodies demonstrated even greater activity compared to nivolumab. These assays may better represent in vivo conditions because they recapitulate not only the adverse effects of the interaction between the receptor and its ligand (i.e., PD-1 / PD-L1) but also other potential biological effects that the new antibodies may antagonize. This may explain why the novel anti-PD-1 and anti-PD-L1 antibodies exhibit stronger effects on stimulating T cell proliferation and cytokine secretion than those exerted by nivolumab, whose activity may primarily depend on blocking PD-1 and PD-L1 interactions. This hypothesis is also supported by the intriguing observation that the novel anti-PD-1 antibodies recognize different epitopes than nivolumab and therefore may act via a different mechanism of action.Indeed, PD-1_A was able to cross-react with mouse lymphocytes in a manner similar to PD-L1_A, which distinguishes PD-1_A from nivolumab. Because of this property, these two novel antibodies were also tested in vivo in mice bearing CT26 colon carcinoma and were found to effectively suppress tumor growth.

[0250] Overall, the data presented in this study support the conclusion that the selection procedure used makes it possible to generate antibodies that recognize target receptors specifically in their native conformation and with high affinity without the need for further affinity maturation, resulting in antibodies that specifically bind checkpoint inhibitors with superior properties compared to prior art antibodies. [Explanation of symbols]

[0251] Drawing translation Figure 1 Immunome universal cycle 1 Immunome universal cycle 1 Cycle 2 Cycle 3 Figure 2 Absorbance Activated hPBMCs Activated hPBMCs Untreated hPBMCs MDA-MB-231 cells MDA-MB-231 cells Nivolumab Figure 3 Fold increase of CD3-T cell proliferation hIgG4 control hIgG4 control Nivolumab Figure 4 Fold increase rate Nivolumab Figure 5 hIgG4 control hIgG4 control Nivolumab Time (hours) Figure 6 Tumor volume (mm3) untreated Figure 7 Binding to activated hPBMC IgG concentration Figure 8 hIgG4 control hIgG4 control Nivolumab Time (hours)

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to PD-L1, The antibody or antigen-binding fragment thereof （i） CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, and CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 25; a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26, and CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; Or, (ii) CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 31, and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 32; CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 33, and CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 34; a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35, and CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36; Or, (iii) CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, and CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 25; a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 89, and CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; Or, (iv) CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23; CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, and CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 25; a CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 98, and CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27; Including, An antibody or antigen-binding fragment thereof.

2. 2. The antibody or antigen-binding fragment thereof of claim 1, The antibody or antigen-binding fragment thereof (i) a light chain variable domain having a sequence having at least 90% identity to SEQ ID NO: 21 and a heavy chain variable domain having a sequence having at least 90% identity to SEQ ID NO: 20; (ii) a light chain variable domain having a sequence having at least 90% identity to SEQ ID NO: 30 and a heavy chain variable domain having a sequence having at least 90% identity to SEQ ID NO: 29; (iii) a light chain variable domain having a sequence having at least 90% identity to SEQ ID NO: 21 and a heavy chain variable domain having a sequence having at least 90% identity to SEQ ID NO: 83; (iv) a light chain variable domain having a sequence having at least 90% identity to SEQ ID NO: 21 and a heavy chain variable domain having a sequence having at least 90% identity to SEQ ID NO: 92; Including, The antibody or antigen-binding fragment thereof according to claim 1.

3. 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, (a) the antibody is selected from the group consisting of a human antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a recombinant antibody, an antibody mimetic, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody; or the antigen-binding fragment is selected from the group consisting of a single-chain antibody, a single-chain variable fragment antibody, a diabody, a Fab fragment, and a F(ab)2 fragment; and / or (b) the antibody or antigen-binding fragment thereof has the following characteristics: (i) binds to PD-L1 with a Kd of less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 100 pM, less than 10 pM, or less than 5 pM as assessed by ELISA; (ii) an IC of less than 10 nM, less than 1 nM, or less than 200 pM as assessed by ELISA 50 the ability to block ligand binding to PD-L1; (iii) the property of stimulating T cell proliferation; (iv) the property of inducing lymphocyte proliferation; (v) the ability to induce the secretion of IL-2 and / or IFNγ, and / or (vi) reducing tumor volume by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to baseline; having at least one of The antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A recombinant expression vector comprising the nucleic acid of claim 4.

6. A host cell comprising the vector of claim 5.

7. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising preparing the antibody or antigen-binding fragment thereof from a host cell that expresses the antibody or antigen-binding fragment thereof.

8. A pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, a nucleic acid according to claim 4, or a vector according to claim 5, and a pharmaceutically acceptable carrier.

9. 10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises at least one additional active agent.

10. 10. A kit comprising the pharmaceutical composition of claim 8 or 9 and, optionally, at least one further active agent.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid according to claim 4, the vector according to claim 5, or the pharmaceutical composition according to claim 8 or 9, for use in the treatment of cancer and / or chronic infections.

12. 12. A medicament comprising an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, or a pharmaceutical composition according to claim 11 for use in the treatment of cancer and / or chronic infections, (i) the cancer is (a) malignant neoplasms of the lips, oral cavity and pharynx; and / or (b) malignant neoplasms of the digestive tract, and / or (c) malignant neoplasms of the respiratory and intrathoracic organs, and / or (d) malignant neoplasms of bone and articular cartilage; and / or (e) melanoma and other malignant neoplasms of the skin; and / or (f) malignant neoplasms of the mesothelial and soft tissues; and / or (g) malignant neoplasm of the breast, and / or (h) Malignant neoplasms of the female genital tract, and / or (i) malignant neoplasms of the male genital tract, and / or (j) malignant neoplasms of the urinary tract, and / or (k) Malignant neoplasms of the eye, brain, and other parts of the central nervous system; and / or (l) malignant neoplasms of the thyroid gland and other endocrine glands, and / or (m) Malignant neoplasms of the lymphatic system, hematopoietic system and related tissues; and / or selected from the group consisting of (ii) the chronic infection is selected from the group consisting of HBV, HCV, HIV, HSV, HPV, EBV, CMV and chlamydia; and / or (iii) at least one additional active agent is administered to a subject in need thereof; or At least one further active agent is administered to a subject in need thereof, and the at least one further active agent and the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid according to claim 4, the vector according to claim 5, or the pharmaceutical composition according to claim 8 or 9 are administered simultaneously or sequentially, or in combination thereof. A medicament comprising an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, or a pharmaceutical composition according to claim 11, for use in the treatment of cancer and / or chronic infections.

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