Antagonistic PD-1, PD-L1 and LAG-3 binding proteins

Abstract

The present invention relates to an antagonistic antigen binding protein, a nucleic acid encoding the antagonistic binding protein, a recombinant expression vector comprising the nucleic acid molecule, a host cell comprising the vector, a method for preparing the antagonistic antigen binding protein, the antagonistic binding protein prepared by the method, and a recombinant expression vector comprising the antagonistic binding protein, pharmaceutical compositions of nucleic acids or vectors. The invention also relates to a kit comprising said pharmaceutical composition and to the use of said antagonistic binding protein in the treatment of cancer and / or chronic infectious diseases.

Description

[0001] This application is a divisional application of the invention patent application with application date of March 22, 2019, application number 201980020762.9, and invention name “Antagonist PD-1, PD-L1 and LAG-3 Binding Protein”. Technical Field

[0002] The present invention relates to an antagonist antigen binding protein, a nucleic acid encoding the antagonist binding protein, a recombinant expression vector comprising the nucleic acid molecule, a host cell comprising the vector, a method for preparing the antagonist antigen binding protein, an antagonist binding protein prepared by the method, and a pharmaceutical composition comprising the antagonist binding protein, nucleic acid or vector. The present invention also relates to a kit comprising the pharmaceutical composition and the use of the antagonist binding protein in treating cancer and / or chronic infectious diseases. Background Art

[0003] The activation and proliferation of immune cells involved in antitumor responses are regulated by a variety of stimulatory and inhibitory pathways. Monoclonal antibodies (mAbs) can target a variety of stimulatory and inhibitory pathways, which can inhibit immunosuppressive receptors or activate co-stimulatory regulators (collectively known as immune checkpoints) expressed on the surface of T and B lymphocytes or NK cells to enhance their tumor-specific responses.

[0004] The application of this concept in the clinic has led to the development of new and effective immunotherapies. To date, human or humanized monoclonal antibodies targeting three types of 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 a variety of tumors including melanoma, NSCLC, RCC, head and neck squamous cell carcinoma, Hodgkin lymphoma, urothelial carcinoma, MSI CRC, and Merkell cell carcinoma.

[0005] Stimulated by the great success of these immunotherapies, more antibodies against other immunomodulatory receptors have been introduced into the clinic, hoping to find more effective treatments by targeting new immune pathways. In fact, despite their success, the currently approved antibodies against immune checkpoints (thus collectively referred to as checkpoint inhibitors; CI) are only effective in about 20% to 30% of patients. Among the new targets are co-inhibitory receptors such as lymphocyte activation gene 3 (LAG3), an immunosuppressive receptor expressed on activated T lymphocytes and T regulatory lymphocytes, T cell immunoglobulin 3 (TIM-3), T cell immunoglobulin and ITIM domain expressed on CD8+ T cells exhausted in cancer (TIGIT). In addition to improving CD8 T cell function, Lag-3, TIM-3 and TIGIT blockade are expected to affect tumor tissue regulatory T cells (Treg cells) and IL-10-producing Tr1 cells. B lymphocyte and T lymphocyte attenuation factor (BTLA) is another co-inhibitory receptor whose expression is induced during T cell activation, which 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 exhibits T cell inhibitory effects by interacting with the tumor necrosis family receptor (TNF-R) rather than just the B7 family of cell surface receptors.

[0006] Similarly, agonist antibodies that recognize co-stimulatory receptors have entered the clinical stage, such as OX40, a secondary co-stimulatory immune checkpoint molecule that prevents premature death of activated lymphocytes, and 41BB expressed on activated CD4 and CD8 T lymphocytes. Other co-stimulatory proteins that are considered good targets for antibody-mediated immunotherapy are inducible T-cell co-stimulatory protein (ICOS), an immune checkpoint protein of the CD28 superfamily, and CD27, a member of the tumor necrosis factor receptor superfamily.

[0007] Cancer immunotherapy based on immunomodulatory antibodies is becoming a mainstream in modern oncology. Checkpoint inhibitors (i.e., anti-PD-1, anti-PD-L1, and anti-CTLA4) have been shown to achieve unprecedented efficacy and have long-term clinical benefits. Perhaps most importantly, these antibodies have extremely broad applications and are currently approved for the treatment of a variety of tumors. Despite this, the therapeutic effect of CIs is still limited to 20% to 30% of the population and has not been shown to be effective in highly prevalent cancer types such as colon and rectal cancer, breast cancer, and prostate cancer. However, the recent enrichment of monoclonal antibody libraries against new targets with agonist or antagonist activity on co-stimulatory or inhibitory receptors, respectively, allows the design of new combination therapies to enhance the potential of immune-based cancer therapies.

[0008] Antibodies targeting different immune checkpoint receptors can also be combined to achieve additive or synergistic activity, potentially translating into improved efficacy. The finding that the combination of ipilimumab and nivolumab was more effective than monotherapy in treating metastatic melanoma provides proof of concept for this approach.

[0009] Currently, more than a thousand clinical trials are ongoing for approved or novel antibodies targeting immune regulatory receptors, either as monotherapy or in combination with other biologics or small molecules. Most of these trials do not have preclinical head-to-head comparisons to predict the most effective treatment, as a large number of antibodies targeting different immune checkpoints are not available in one laboratory.

[0010] The inventors have used a strategy of rapid, parallel screening of phage-displayed antibody libraries by direct panning on activated human lymphocytes to generate a large number of fully human mAbs against several immune regulatory checkpoints. The mAbs thus selected were demonstrated to have high binding affinity to their targets and improved functional activity on lymphocytes. Summary of the invention

[0011] In a first aspect, the present invention provides an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein competes with an antibody for binding to PD-1, the antibody

[0012] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3; or

[0013] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

[0014] In a second aspect, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein competes with an antibody for binding to PD-L1, the antibody

[0015] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21; or

[0016] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30;

[0017] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 83; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 84; or

[0018] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 92; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 93; or

[0019] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 101; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 102.

[0020] In a third aspect, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein competes with an antibody for binding to LAG-3, the antibody

[0021] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39;

[0022] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48;

[0023] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57;

[0024] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 66; or

[0025] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 74; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75.

[0026] In a fourth aspect, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises any one of the following:

[0027] (i) a combination of a light chain variable domain and a heavy chain variable domain, the combination being selected from:

[0028] A light chain variable domain comprising a sequence having at least 90% identity to SEQ ID NO: 3 and

[0029] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 2;

[0030] A light chain variable domain comprising a sequence having at least 90% identity to SEQ ID NO: 12 and

[0031] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 11;

[0032] (ii) a combination of a heavy chain complementarity determining region 3 (CDRH3) and a light chain complementarity determining region 3 (CDRL3), the combination being selected from the group consisting of:

[0033] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and

[0034] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9;

[0035] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17 and

[0036] A CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:18.

[0037] In a fifth aspect, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises any one of the following:

[0038] (i) a combination of a light chain variable domain and a heavy chain variable domain, the combination being selected from:

[0039] A light chain variable domain comprising a sequence having at least 90% identity to SEQ ID NO: 21 and

[0040] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 20;

[0041] A light chain variable domain comprising a sequence having at least 90% identity to SEQ ID NO: 30 and

[0042] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 29;

[0043] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 84 and

[0044] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 83;

[0045] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 93 and

[0046] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 92;

[0047] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 102 and

[0048] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 101;

[0049] (ii) a combination of a heavy chain complementarity determining region 3 (CDRH3) and a light chain complementarity determining region 3 (CDRL3), the combination being selected from the group consisting of:

[0050] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and

[0051] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27;

[0052] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and

[0053] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36;

[0054] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 89 and

[0055] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 90;

[0056] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and

[0057] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 99;

[0058] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 107 and

[0059] A CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:108.

[0060] In a sixth aspect, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises any one of the following

[0061] (i) a combination of a light chain variable domain and a heavy chain variable domain, the combination being selected from:

[0062] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 39 and

[0063] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 38;

[0064] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 48 and

[0065] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 47;

[0066] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 57 and

[0067] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO:56;

[0068] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 66 and

[0069] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 65;

[0070] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 75 and

[0071] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 74;

[0072] (ii) a combination of a heavy chain complementarity determining region 3 (CDRH3) and a light chain complementarity determining region 3 (CDRL3), the combination being selected from the group consisting of:

[0073] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and

[0074] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45;

[0075] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and

[0076] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54;

[0077] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and

[0078] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 63;

[0079] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71 and

[0080] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 72;

[0081] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 80 and

[0082] A CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:81.

[0083] In a seventh aspect, the present invention relates to a nucleic acid encoding the antagonist antigen binding protein of any one of aspects 1 to 6 of the present invention.

[0084] In an eighth aspect, the present invention relates to a recombinant expression vector comprising the nucleic acid molecule according to the seventh aspect.

[0085] In a ninth aspect, the present invention relates to a host cell comprising the vector of the eighth aspect of the present invention.

[0086] In a tenth aspect, the present invention relates to a method for preparing the antagonist antigen binding protein of any one of aspects 1 to 6 of the present invention, comprising the step of preparing the antigen binding protein from a host cell expressing the antigen binding protein.

[0087] In an eleventh aspect, the present invention relates to an antagonist antigen binding protein prepared by expressing a recombinant DNA in a host cell of the ninth aspect of the present invention.

[0088] In the twelfth aspect, the present invention relates to a pharmaceutical composition comprising at least one antagonist antigen binding protein according to any one of aspects 1 to 6 of the present invention, a nucleic acid according to the seventh aspect of the present invention, or a vector according to the eighth aspect of the present invention and a pharmaceutically acceptable carrier.

[0089] 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.

[0090] In a fourteenth aspect, the present invention relates to the use of the antagonist antigen binding protein according to any one of aspects 1 to 6 of the present invention, the nucleic acid according to the seventh aspect of the present invention, the vector according to the eighth aspect of the present invention, or the pharmaceutical composition according to the twelfth aspect of the present invention for treating cancer and / or chronic infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] In the following, the contents of the drawings included in the present description are described. In this context, reference is also made to the above and / or below detailed description of the invention.

[0092] Figure 1: Immunogroup screening. The screening procedure starts with a universal cycle, universal for different targets, by panning unselected libraries on activated PBMC (inner circle). Each different circle sector describes the enrichment profile of the ten best scFv clones indicating the target, scored according to their one-millionth count value. The lines in each sector connect the individual enrichments, which are obtained after cycle 2 (small circles) and cycle 3 (large circles). Cycles 2 and 3 are performed on the recombinant protein.

[0093] Figure 2 : Binding affinity of selected antibodies to lymphocytes. ELISA was performed with monoclonal antibodies at increasing concentrations (7.5nM, 25nM, 50nM, 100nM) on activated hPBMC (black curve) or untreated hPBMC (grey curve). Antibodies were also tested by ELISA at increasing concentrations on each recombinant protein / Fc, and Kd values ​​are reported in the table.

[0094] Figure 3 : hPBMC proliferation after 2.5 μg / ml PHA stimulation in the absence or presence of immunomodulatory antibodies. hPBMC were activated with 2.5 μg / ml PHA in the absence of antibodies or in the presence of irrelevant IgG4 and the fold increase in CD3-T cell proliferation determined by the indicated selected antibodies was measured.

[0095] Figure 4 : Effect of anti-PD-1 and anti-PD-L1 antibodies on tumor cell-induced lymphocyte proliferation. At 37°C for 72 hours, the fold increase in hPBMC proliferation was determined by the normalized absorbance values ​​obtained by ELISA with anti-BrdU antibody in hPBMC samples, which were co-cultured with MDA-MB-231 (A) or MCF-7 (B) tumor cells in the absence (white column) or in the presence of increasing concentrations (50nm and 200nm) of PD-L1_A (light gray column) or PD-1_A (gray column) antibodies. In both experiments, nivolumab was used as a positive control (black column).

[0096] Figure 5 : Effects of novel immunomodulatory antibodies on cytokine secretion by stimulated T cells. hPBMC supernatants were stimulated with PHA (2.5 μg / mL) or SEB (50 ng / mL) for 18 to 66 hours at 37°C in the absence or presence of antibodies LAG-3_A, PD-1_A, PD-1_B, PD-L1_A, PD-L1_B, and ELISA was performed to obtain IL-2 and IFNγ values. Nivolumab and an irrelevant antibody were used as positive and negative controls, respectively.

[0097] Figure 6 : In vivo antitumor 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 (α_mPD-1, α-mPD-L1) reactive with murine PD-1 and PD-L1 on days 3, 6, and 10. Tumor volumes 21 days after tumor challenge are shown.

[0098] Figure 7 : Binding of anti-PD-L1 antibodies to lymphocytes. To evaluate whether antibodies PD-L1 C, PD-L1 D and PD-L1 E are able to bind to the PD-L1 protein compared to PD-L1 A, binding curves of all these mAbs to human activated lymphocytes were generated. 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, PD-L1_E antibodies were then added at a wide range of concentrations and analyzed by flow cytometry (CytoFLEX flow cytometer, Beckman Coulter). The mean fluorescence intensity of lymphocytes binding to PD-L1 was plotted against the antibody concentration ( Figure 7 A. Figure 7 B).

[0099] Figure 8 : Effect of anti-PD-L1 antibodies on cytokine secretion by stimulated T cells. hPBMC supernatants were stimulated with PHA (2.5 μg / mL) or SEB (50 ng / mL) for 18 to 66 hours at 37°C in the absence or presence of antibodies PD-L1_A, PD-L1_C, and PD-L1_D, and ELISA was performed to obtain IL-2 and IFNγ values. Nivolumab and irrelevant antibodies were used as positive and negative controls, respectively.

[0100] Sequence Listing

[0101] SEQ ID NO: 1 Amino acid sequence of PD-1_A

[0102] SEQ ID NO: 2 Amino acid sequence of PD-1_A; VH

[0103] SEQ ID NO: 3 Amino acid sequence of PD-1_A; VL

[0104] SEQ ID NO:4 Amino acid sequence of PD-1_A; CDRH1

[0105] SEQ ID NO:5 Amino acid sequence of PD-1_A; CDRL1

[0106] SEQ ID NO:6 Amino acid sequence of PD-1_A; CDRH2

[0107] SEQ ID NO:7 Amino acid sequence of PD-1_A; CDRL2

[0108] SEQ ID NO: 8 Amino acid sequence of PD-1_A; CDRH3

[0109] SEQ ID NO: 9 Amino acid sequence of PD-1_A; CDRL3

[0110] SEQ ID NO: 10 Amino acid sequence of PD-1_B

[0111] SEQ ID NO: 11 Amino acid sequence of PD-1_B; VH

[0112] SEQ ID NO: 12 Amino acid sequence of PD-1_B; VL

[0113] SEQ ID NO: 13 Amino acid sequence of PD-1_B; CDRH1

[0114] SEQ ID NO: 14 Amino acid sequence of PD-1_B; CDRL1

[0115] SEQ ID NO: 15 Amino acid sequence of PD-1_B; CDRH2

[0116] SEQ ID NO: 16 Amino acid sequence of PD-1_B; CDRL2

[0117] SEQ ID NO: 17 Amino acid sequence of PD-1_B; CDRH3

[0118] SEQ ID NO: 18 Amino acid sequence of PD-1_B; CDRL3

[0119] SEQ ID NO: 19 Amino acid sequence of PD-L1_A

[0120] SEQ ID NO: 20 Amino acid sequence of PD-L1_A; VH

[0121] SEQ ID NO: 21 Amino acid sequence of PD-L1_A; VL

[0122] SEQ ID NO: 22 Amino acid sequence of PD-L1_A; CDRH1

[0123] SEQ ID NO: 23 Amino acid sequence of PD-L1_A; CDRL1

[0124] SEQ ID NO: 24 Amino acid sequence of PD-L1_A; CDRH2

[0125] SEQ ID NO: 25 Amino acid sequence of PD-L1_A; CDRL2

[0126] SEQ ID NO: 26 Amino acid sequence of PD-L1_A; CDRH3

[0127] SEQ ID NO: 27 Amino acid sequence of PD-L1_A; CDRL3

[0128] SEQ ID NO: 28 Amino acid sequence of PD-L1_A

[0129] SEQ ID NO: 29 Amino acid sequence of PD-L1_B; VH

[0130] SEQ ID NO: 30 Amino acid sequence of PD-L1_B; VL

[0131] SEQ ID NO:31 Amino acid sequence of PD-L1_B; CDRH1

[0132] SEQ ID NO:32 Amino acid sequence of PD-L1_B; CDRL1

[0133] SEQ ID NO: 33 Amino acid sequence of PD-L1_B; CDRH2

[0134] SEQ ID NO: 34 Amino acid sequence of PD-L1_B; CDRL2

[0135] SEQ ID NO: 35 Amino acid sequence of PD-L1_B; CDRH3

[0136] SEQ ID NO:36 Amino acid sequence of PD-L1_B; CDRL3

[0137] SEQ ID NO: 37 Amino acid sequence of LAG-3_A

[0138] SEQ ID NO:38 Amino acid sequence of LAG-3_A; VH

[0139] SEQ ID NO:39 Amino acid sequence of LAG-3_A; VL

[0140] SEQ ID NO:40 Amino acid sequence of LAG-3_A; CDRH1

[0141] SEQ ID NO:41 Amino acid sequence of LAG-3_A; CDRL1

[0142] SEQ ID NO:42 Amino acid sequence of LAG-3_A; CDRH2

[0143] SEQ ID NO:43 Amino acid sequence of LAG-3_A; CDRL2

[0144] SEQ ID NO:44 Amino acid sequence of LAG-3_A; CDRH3

[0145] SEQ ID NO:45 Amino acid sequence of LAG-3_A; CDRL3

[0146] SEQ ID NO:46 Amino acid sequence of LAG-3_C

[0147] SEQ ID NO:47 Amino acid sequence of LAG-3_C; VH

[0148] SEQ ID NO:48 Amino acid sequence of LAG-3_C; VL

[0149] SEQ ID NO:49 Amino acid sequence of LAG-3_C; CDRH1

[0150] SEQ ID NO:50 Amino acid sequence of LAG-3_C; CDRL1

[0151] SEQ ID NO:51 Amino acid sequence of LAG-3_C; CDRH2

[0152] SEQ ID NO:52 Amino acid sequence of LAG-3_C; CDRL2

[0153] SEQ ID NO:53 Amino acid sequence of LAG-3_C; CDRH3

[0154] SEQ ID NO:54 Amino acid sequence of LAG-3_C; CDRL3

[0155] SEQ ID NO:55 Amino acid sequence of LAG-3_D

[0156] SEQ ID NO:56 Amino acid sequence of LAG-3_D; VH

[0157] SEQ ID NO:57 Amino acid sequence of LAG-3_D; VL

[0158] SEQ ID NO:58 Amino acid sequence of LAG-3_D; CDRH1

[0159] SEQ ID NO:59 Amino acid sequence of LAG-3_D; CDRL1

[0160] SEQ ID NO:60 Amino acid sequence of LAG-3_D; CDRH2

[0161] SEQ ID NO:61 Amino acid sequence of LAG-3_D; CDRL2

[0162] SEQ ID NO:62 Amino acid sequence of LAG-3_D; CDRH3

[0163] SEQ ID NO:63 Amino acid sequence of LAG-3_D; CDRL3

[0164] SEQ ID NO:64 Amino acid sequence of LAG-3_B

[0165] SEQ ID NO:65 Amino acid sequence of LAG-3_B; VH

[0166] SEQ ID NO:66 Amino acid sequence of LAG-3_B; VL

[0167] SEQ ID NO:67 Amino acid sequence of LAG-3_B; CDRH1

[0168] SEQ ID NO:68 Amino acid sequence of LAG-3_B; CDRL1

[0169] SEQ ID NO:69 Amino acid sequence of LAG-3_B; CDRH2

[0170] SEQ ID NO:70 Amino acid sequence of LAG-3_B; CDRL2

[0171] SEQ ID NO:71 Amino acid sequence of LAG-3_B; CDRH3

[0172] SEQ ID NO:72 Amino acid sequence of LAG-3_B; CDRL3

[0173] SEQ ID NO:73 Amino acid sequence of LAG-3_E

[0174] SEQ ID NO:74 Amino acid sequence of LAG-3_E; VH

[0175] SEQ ID NO:75 Amino acid sequence of LAG-3_E; VL

[0176] SEQ ID NO:76 Amino acid sequence of LAG-3_E; CDRH1

[0177] SEQ ID NO:77 Amino acid sequence of LAG-3_E; CDRL1

[0178] SEQ ID NO:78 Amino acid sequence of LAG-3_E; CDRH2

[0179] SEQ ID NO:79 Amino acid sequence of LAG-3_E; CDRL2

[0180] SEQ ID NO:80 Amino acid sequence of LAG-3_E; CDRH3

[0181] SEQ ID NO:81 Amino acid sequence of LAG-3_E; CDRL3

[0182] SEQ ID NO: 82 Amino acid sequence of PD-L1_C

[0183] SEQ ID NO: 83 Amino acid sequence of PD-L1_C; VH

[0184] SEQ ID NO: 84 Amino acid sequence of PD-L1_C; VL

[0185] SEQ ID NO: 85 Amino acid sequence of PD-L1_C; CDRH1

[0186] SEQ ID NO: 86 Amino acid sequence of PD-L1_C; CDRL1

[0187] SEQ ID NO: 87 Amino acid sequence of PD-L1_C; CDRH2

[0188] SEQ ID NO: 88 Amino acid sequence of PD-L1_C; CDRL2

[0189] SEQ ID NO: 89 Amino acid sequence of PD-L1_C; CDRH3

[0190] SEQ ID NO: 90 Amino acid sequence of PD-L1_C; CDRL3

[0191] SEQ ID NO: 91 Amino acid sequence of PD-L1_D

[0192] SEQ ID NO: 92 Amino acid sequence of PD-L1_D; VH

[0193] SEQ ID NO: 93 Amino acid sequence of PD-L1_D; VL

[0194] SEQ ID NO: 94 Amino acid sequence of PD-L1_D; CDRH1

[0195] SEQ ID NO: 95 Amino acid sequence of PD-L1_D; CDRL1

[0196] SEQ ID NO: 96 Amino acid sequence of PD-L1_D; CDRH2

[0197] SEQ ID NO: 97 Amino acid sequence of PD-L1_D; CDRL2

[0198] SEQ ID NO: 98 Amino acid sequence of PD-L1_D; CDRH3

[0199] SEQ ID NO: 99 Amino acid sequence of PD-L1_D; CDRL3

[0200] SEQ ID NO: 100 Amino acid sequence of PD-L1_E

[0201] SEQ ID NO: 101 Amino acid sequence of PD-L1_E; VH

[0202] SEQ ID NO: 102 Amino acid sequence of PD-L1_E; VL

[0203] SEQ ID NO: 103 Amino acid sequence of PD-L1_E; CDRH1

[0204] SEQ ID NO: 104 Amino acid sequence of PD-L1_E; CDRL1

[0205] SEQ ID NO: 105 Amino acid sequence of PD-L1_E; CDRH2

[0206] SEQ ID NO: 106 Amino acid sequence of PD-L1_E; CDRL2

[0207] SEQ ID NO: 107 Amino acid sequence of PD-L1_E; CDRH3

[0208] SEQ ID NO: 108 Amino acid sequence of PD-L1_E; CDRL3 DETAILED DESCRIPTION

[0209] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as they may vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are 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 those generally understood by those of ordinary skill in the art.

[0210] Preferably, the terms used herein are defined as described in "Multilingual Glossary of Biotechnology Terms: (IUPAC Recommendations)" (Leuenberger, HGW, Nagel, B. and Klbl, H. eds. (1995), Helvetica ChimicaActa, CH-4010 Basel, Switzerland):.

[0211] Throughout this specification and in the claims that follow, the word "comprising" and its variants, unless the context otherwise requires, will be understood to mean the inclusion of the stated integers or steps or integers or step groups but not the exclusion of any other integers or steps or integers or step groups. In the following paragraphs, different aspects of the invention are defined in more detail. Each aspect so defined can be combined with any other one or more than one aspect, unless clearly indicated to the contrary. In particular, any feature indicated as optional, preferred or advantageous can be combined with any other one or more than one feature indicated as optional, preferred or advantageous.

[0212] Several documents are cited in the full text of this specification. Whether above or below, each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, specifications, etc.) is incorporated herein by reference in its entirety. Nothing herein should be construed as admitting that the present invention is not entitled to precede such disclosures by virtue of prior invention. Certain documents cited herein are characterized as "incorporated by reference". In the event of a conflict between the definition or teaching of such incorporated citation and the definition or teaching cited in this specification, the text of this specification shall prevail.

[0213] Hereinafter, elements of the present invention will be described. These elements are listed together with specific embodiments, but it should be understood that they can be combined in any manner and in any quantity to create additional embodiments. The different described embodiments and preferred embodiments should not be construed as limiting the present invention to only the embodiments clearly described. This description should be understood to support and encompass embodiments that combine the embodiments clearly described with any number of disclosed and / or preferred elements. In addition, unless the context otherwise indicates, any arrangement and combination of all described elements in the present application should be considered to be disclosed in the description of the present application.

[0214] definition

[0215] In the following, some definitions of terms frequently used in this specification are provided. In the remainder of the specification, these terms will have the defined meanings and preferred meanings, respectively, in each case where they are used.

[0216] As used in this specification and the appended claims, a word preceding an element includes plural referents unless the context clearly dictates otherwise.

[0217] As used herein, the term "antagonist" 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 "antagonist" includes "competitive antagonists," "noncompetitive antagonists," "uncompetitive antagonists," "partial agonists," and "inverse agonists."

[0218] As used herein, the term "PD-1" refers to programmed cell death protein 1, also known as CD279.

[0219] As used herein, the term "PD-L1" refers to programmed death-ligand 1, also known as CD274 or B7 homolog 1 (B7-H1).

[0220] As used herein, the term "LAG-3" refers to lymphocyte activation gene 3, also known as CD223.

[0221] As used herein, the term "antigen binding protein" refers to any molecule or part of a molecule that can specifically bind to a target molecule or target epitope. In the context of the present application, preferred binding proteins are (a) antibodies or antigen binding fragments thereof; (b) oligonucleotides; (c) antibody-like proteins; or (d) peptoids.

[0222] As used herein, if the dissociation constant K of a first compound (eg, an antibody) and a second compound (eg, an antigen, eg, a target protein) is dThe molecular weight of the present invention is 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 400 nM or less, more preferably 500 nM or less, more preferably 500 nM or less, more preferably 600 nM or less, more preferably 7 ... The first compound is considered to be "bound to" the second compound if the molecular weight of the first compound is 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 nM or less.

[0223] Generally, if the dissociation constant K of a first compound (eg, an antibody) and a second compound (eg, an antigen, eg, a target protein) is d The first compound is considered to "bind" to the second compound if the concentration of the first compound is 10nM to 1mM, 10nM to 100μM, 10nM to 50μM, 10nM to 1μM, preferably 10nM to 900nM, 10nM to 800nM, 10nM to 700nM, 10nM to 600nM, more preferably 10nM to 500nM, 10nM to 400nM, 10nM to 300nM, 10nM to 200nM, 10nM to 100nM, for example 10nM to 90nM, 10nM to 80nM, 10nM to 70nM, 10nM to 60nM, 10nM to 50nM, 10nM to 40nM, 10nM to 30nM, 10nM to 20nM. The term "binding" according to the invention preferably relates to specific binding. "Specific binding" means that a binding protein (e.g., an antibody) binds more strongly to a target, such as an epitope, than to another target. If a binding protein binds to a first target, the dissociation constant (K) is greater than that of a second target. d ) is lower than the dissociation constant of the second target, the binding protein binds more strongly to the first target. Preferably, the dissociation constant (K d ) is lower than the dissociation constant (K) of the target nonspecifically bound by the binding protein. d), preferably one-twentieth, more preferably one-fiftieth, even more preferably one-hundredth, one-twenty-hundredth, one-fifth, or one-thousandth of ).

[0224] For example, the dissociation constant (K) of a target specifically bound to a binding protein d ) is the dissociation constant (K) of the target nonspecifically bound to the binding protein d ), for example, 1 / 20 to 1 / 1000, 1 / 50 to 1 / 1000, 1 / 100 to 1 / 1000, 1 / 200 to 1 / 1000, 1 / 300 and 1 / 1000, 1 / 400 to 1 / 1000, 1 / 500 to 1 / 1000.

[0225] As used herein, the term "Kd" (measured in "mole / L", sometimes abbreviated as "M") refers to the dissociation equilibrium constant for 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., for determining the dissociation constant K D The methods of detecting the presence of a K are known to those of ordinary skill in the art and can be selected, for example, from the following methods known in the art: techniques based on surface plasmon resonance (SPR), 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, “K d The values ​​were determined by surface plasmon resonance spectroscopy (Biacore TM ) is determined. For example, surface plasmon resonance measurements are performed at 25° C. on a Biacore X100 instrument (GE Healthcare) typically equipped with, for example, a CM5 sensor chip (GE Healthcare), wherein, for example, HBS-EP buffer (using 10 mM Hepes, 0.15 M NaCl, 3 mM EDTA and 0.05% surfactant P20 pH 7.4) is used as running buffer (GE Healthcare).

[0226] “IC 50 The IC value refers to half the maximal inhibitory concentration of a substance and is therefore a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. The value is usually expressed as a molar concentration. The IC of a drug can be determined in a functional antagonism assay by constructing a dose-response curve and examining the inhibitory effect of the substance under examination at different concentrations. 50 Alternatively, a competition binding assay can be performed to determine the IC 50Typically, the inhibitory antibodies of the present invention exhibit an IC 50 The value is 50nM to 1pM, more preferably 10nM to 10pM, even more preferably 1nM to 50pM, i.e. 50nM, 10nM, 1nM, 900pM, 800pM, 700pM, 600pM, 500pM, 400pM, 300pM, 200pM, 100pM, 50pM or 1pM.

[0227] Cell proliferation is considered a reliable means of monitoring T lymphocyte activation. As described in Nat. Prot. 2007; 2(9):2049-56, CFSE carboxyfluorescein succinimidyl ester can effectively monitor T lymphocyte proliferation.

[0228] The secretion of IL-2 or IFN-γ is another reliable method to monitor T lymphocyte activation. Detection of secreted cytokine proteins is by far the most widely used type of analysis. Since secreted proteins are the biologically relevant fraction, their detection can most closely represent the ongoing response of the cells. Secreted proteins are usually measured by ELISA. In this article, hPBMC (1x10 6 Cells) were stimulated with 2.5 μg / mL PHA-L or 50 ng / mL Staphylococcal enterotoxin B (SEB) for 18 h, 42 h, and 66 h. Nivolumab was tested as a positive control in parallel under the same conditions. The concentration of IL-2 or IFNγ in the cell culture supernatant was determined by ELISA assay (DuoSet ELISA, R&D System) and compared with the standard curve according to the manufacturer's recommendations. Concentration values ​​are reported as the mean of at least three determinations (standard deviation ≤ 5%).

[0229] In animal studies, tumor size is used to assess response to anticancer therapy. The current standard technique for determining subcutaneous xenograft tumor volume in vivo is by external caliper measurement using a modified ellipsoid formula of 1 / 2 (length × width 2 ) to calculate tumor volume.

[0230] When used in the context of antigen binding proteins competing for the same epitope, the term "competes" refers to competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., or PD-1, PD-L1 and / or LAG-3 or a fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen binding protein competes for binding with another, for example: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:249-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using an I-125 label (see, e.g., Morel et al., 1988, Molec. J. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J Immunol. 32:77-82). Typically, such assays involve the use of purified antigen bound to a solid surface or cells carrying either of these, unlabeled test antigen binding proteins, and labeled reference antigen binding proteins. 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 bound to the reference antigen binding protein, as well as antigen binding proteins that bind to adjacent epitopes that are close enough to the epitope bound to the reference antigen binding protein to cause steric hindrance. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antigen binding protein to PD-1, PD-L1 and / or LAG-3, or an extracellular fragment 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%, such as about 75% or greater.In some cases, binding is inhibited by at least about 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 97%, such as about 97% or greater than 97%.

[0231] "Epitope", also known as antigenic determinant, is the part of a macromolecule that is recognized by the immune system, particularly antibodies, B cells or T cells. As used herein, an "epitope" is a part of a macromolecule that is capable of binding to a binding protein as described herein (e.g., an antibody or antigen binding fragment thereof). In this article, the term "binding" preferably relates to specific binding. Epitopes are generally composed of chemically active surface groups of molecules such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that the binding to the former is lost in the presence of a denaturing solvent but not the latter.

[0232] As used herein, "conformational epitope" refers to an epitope of a linear macromolecule (e.g., a polypeptide) formed by the three-dimensional structure of the macromolecule. In the context of the present application, a "conformational epitope" is a "non-continuous epitope", i.e., a conformational epitope on a macromolecule (e.g., a polypeptide) is formed by at least two separate regions in the primary sequence of the macromolecule (e.g., the amino acid sequence of the polypeptide). In other words, in the context of the present invention, if the epitope is composed of at least two separate regions in the primary sequence that simultaneously binds to the binding protein of the present invention (e.g., an antibody or its antigen-binding fragment), wherein the at least two separate regions are interrupted by another region in the primary sequence that is not bound to the binding protein of the present invention, then the epitope is considered to be a "conformational epitope". Preferably, this "conformational epitope" is present on a polypeptide, and the two separate regions in the primary sequence are two separate amino acid sequences that bind to the binding protein of the present invention (e.g., an antibody or its antigen-binding fragment), wherein the at least two separate amino acid sequences are interrupted by one or more amino acid sequences in the primary sequence that is not bound to the binding protein of the present invention. Preferably, the interrupted amino acid sequence is a continuous amino acid sequence comprising two or more amino acids that are not bound to the binding protein. The length of at least two separated amino acid sequences that bind to the binding protein of the present invention is not particularly limited. As long as the total number of amino acids in the at least two separated amino acid sequences is large enough to achieve specific binding between the binding protein and the conformational epitope, the separated amino acid sequence can be composed of only one amino acid.

[0233] A "paratope" is a portion of an antibody that recognizes an epitope. In the context of the present invention, a "paratope" is a portion of a binding protein (eg, an antibody or antigen-binding fragment thereof) as described herein that recognizes an epitope.

[0234] The term "antibody" generally refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. The term "antibody" also includes all recombinant forms of antibodies, particularly antibodies described herein, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives described below. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH or VH). H ) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL or V L ) and light chain constant regions. The VH and VL regions can also be subdivided into hypervariable regions called complementarity determining regions (CDRs), and interspersed more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins 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.

[0235] As used herein, the term "antigen-binding fragment" (or simply "binding portion") of an antibody 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 by the term "antigen-binding portion" of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of the VL, VH, CL, and CH domains; (ii) F(ab') 2Fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), consisting of a VH domain; (vi) isolated complementarity determining regions (CDRs); and (vii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be connected by a synthetic linker using recombinant methods to make them into a single protein chain in which the VL region and the VH region pair to form a monovalent molecule (called 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. Another 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 may be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are also described in US2003 / 0118592 and US2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for fragments that have the same utility as intact antibodies. Other examples of "antigen-binding fragments" are so-called miniantibodies derived from a single CDR. For example, Heap et al., 2005, described a miniantibody derived from 17 amino acid residues of the heavy chain CDR3 of an antibody against the gp120 envelope glycoprotein of HIV-1. Other examples include small antibody mimetics that contain two or more CDR regions fused to each other, preferably through homologous framework regions. Qiu et al., 2007, have described a miniantibody containing a CDR region fused to each other through homologous V H FR2 connected to V H CDR1 and V L This small antibody mimetic of CDR3.

[0236] The immunoglobulin molecules of the present invention may 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 molecules.

[0237] Antibodies and antigen-binding fragments thereof that can be used in the present invention can be from any animal source, including birds and mammals. Preferably, the antibody or fragment is from a human, chimpanzee, rodent (e.g., mouse, rat, guinea pig or rabbit), chicken, turkey, pig, sheep, goat, camel, cattle, horse, donkey, cat or dog. Particularly preferred antibodies are human or mouse-derived. The antibodies of the present invention also include chimeric molecules, in which the antibody constant region derived from a species, preferably a human, is combined with an antigen-binding site derived from another species, such as a mouse. In addition, the antibodies of the present invention include humanized molecules, in which the antigen-binding site of an antibody derived from a non-human species (e.g., derived from a mouse) is combined with a constant region and framework region of human origin.

[0238] As exemplified herein, the antibodies of the invention can be obtained directly from hybridomas expressing the antibodies, or can be cloned and recombinantly expressed in host cells (e.g., CHO cells or lymphocytes). Other examples of host cells are microorganisms, such as Escherichia coli (E. coli) and fungi, such as yeast. Alternatively, they can be recombinantly produced in transgenic non-human animals or plants.

[0239] The term "chimeric antibody" refers to those antibodies in which a portion of each amino acid sequence of the heavy and light chains is homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class, while the rest of the chain is homologous to the corresponding sequence in another species or class. Typically, the variable regions of the light and heavy chains mimic the variable regions of antibodies derived from one mammal, while the constant portion is homologous to the antibody sequence derived from another mammal. An obvious advantage of this chimeric form is that the variable region can be conveniently obtained from currently known sources by combining ready-made B cells or hybridomas from non-human host organisms with constant regions from, for example, human cell preparations. Although the variable region has the advantages of being easy to prepare and specificity not being affected by the source, when the antibody is injected, the constant region of the human is unlikely to cause an immune response from a human subject, while the constant region of non-human origin may cause an immune response from a human subject. However, this definition is not limited to this specific example.

[0240] The term "humanized antibody" refers to a molecule with an antigen binding site that is substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain or may comprise only a complementary determining region (CDR) grafted onto an appropriate framework region in the variable domain. The antigen binding site may be wild type or modified by one or more than one amino acid substitution, for example, modified to be closer to a human immunoglobulin. Some forms of humanized antibodies retain all CDR sequences (e.g., humanized mouse antibodies comprising all six CDRs from a mouse antibody). Other forms have one or more than one CDR that has been altered relative to the original antibody.

[0241] Different methods for humanizing antibodies are known to those skilled in the art, as described in the review of Almagro and Fransson, 2008, the contents of which are incorporated herein by reference in their entirety. The review article of Almagro and Fransson is briefly summarized as follows. Almagro and Fransson distinguished between rational and empirical methods. The rational method is characterized by producing a few variants of engineered antibodies and evaluating their binding or the properties of any other target. If the designed variant does not produce the expected result, a new design and combination evaluation cycle is initiated. The rational method includes CDR grafting, surface remodeling, superhumanization and human sequence content optimization. In contrast, the empirical method is based on the generation of large libraries of humanized variants and the best clone selection using enrichment technology or high-throughput screening. Therefore, the empirical method relies on reliable selection and / or screening systems that can search for a large number of antibody variants. In vitro display technologies such as phage display and ribosome display meet these requirements and are well known to technicians. The empirical method includes FR libraries, guide selection, framework mixing and human engineering.

[0242] CDR grafting

[0243] CDR grafting strategies generally involve three decision points: (1) defining the regions that determine the specificity of the donor antibody, i.e., the grafting targets, (2) determining the source of human sequences to be used as FR donors, and (3) selecting residues outside the regions that define specificity, i.e., determining the amino acid positions that serve as targets for backmutations to restore or improve the affinity of the humanized antibody.

[0244] (1) Determine the region of antibody specificity

[0245] The experimental structure of the non-human antibody compounded with the antigen provides a detailed map of the residues in contact with the antigen, and therefore provides the residues responsible for determining its specificity. Structural information can be supplemented by alanine scanning mutagenesis and / or combinatorial mutagenesis to determine the residues that contribute the most to binding energy or to the functional paratope. Since the functional paratope is a subset of the residues in contact, only grafting the functional paratope will reduce the number of non-human residues in the humanized product. However, only in rare cases can the experimental structure of antigenized antibody complexes and / or functional paratopes be used at the beginning of the humanization scheme. In the absence of an accurate definition of the residues responsible for determining the specificity of the antibody, CDR is often used as a region defining specificity. The combination of CDR and HV loops can also be used as a grafting target. In order to reduce the number of residues to be grafted on people's FR, SDR grafting has been described, that is, the grafting of specificity determining residues (SDR).

[0246] (2) Origin of human FR

[0247] The second step of a typical CDR grafting protocol is to identify the human FR donor. Initial work utilized FRs of human antibodies of known structure, regardless of their homology to non-human antibodies. This approach is called the "fixed FR method." Later work used human sequences with the highest homology to non-human antibodies. This approach is called "best match." Although the "best match" strategy tends to produce antibodies with higher affinity, other parameters such as low immunogenicity and yield must also be considered when selecting FRs for humanization. Therefore, a combination of "best match" and "fixed FR" is also possible. For example, V can be selected according to the fixed FR method L Partial humanization can be performed according to the best matching method. H Partially humanized and vice versa.

[0248] Two sources of human sequences have been used: mature sequences and germline sequences. Mature sequences are products of immune responses and carry somatic mutations generated by random processes without species selection, resulting in the possible presence of immunogenic residues. Therefore, in order to avoid immunogenic residues, human germline genes have been increasingly used as a source of FR donors. The nucleotide sequences of human germline FRs are disclosed in, for example, Appendix A and B of an article published by Dall'Acqua et al. in 2005. In addition, antibodies based on germline genes tend to be more flexible than mature antibodies. It is believed that this higher flexibility can better accommodate the entry of various CDRs with fewer or no back mutations into FRs to restore the affinity of humanized antibodies.

[0249] (3) Reversion mutations that restore or enhance affinity

[0250] Typically, due to incompatibility between non-human CDR and human FR, affinity is reduced after CDR grafting. Therefore, the third step in a typical CDR grafting scheme is to define mutations that will restore or prevent loss of affinity. Back mutations must be carefully designed based on the structure or model of the humanized antibody and tested experimentally. An automatic antibody modeling website called WAM can be found at URL http: / / antibody.bath.ac.uk. Software for protein structure modeling can be downloaded at http: / / salilab.org / modeller / modeller.html (Modeller) and http: / / spdbv.vital-it.ch (Swiss PdbViewer) sites.

[0251] Surface remodeling

[0252] Resurfacing is similar to CDR grafting and shares the first two decision points. In contrast to CDR grafting, resurfacing preserves the unexposed residues of the non-human antibody. Only surface residues in the non-human antibody are changed to human residues.

[0253] Superhumanization

[0254] While CDR grafting relies on FR comparisons between non-human sequences and human sequences, superhumanization is based on CDR comparisons, so FR homology is irrelevant. The method involves comparing non-human sequences with a functional human germline gene library. Then select those genes that encode canonical structures that are identical or closely related to the mouse sequence. Next, among the genes that share canonical structures with non-human antibodies, those with the highest homology in CDR are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs.

[0255] Human sequence content optimization

[0256] This method is based on a metric for the "humanity" of an antibody, called human sequence content (HSC). In short, this method compares the mouse sequence to all human germline genes. Differences are recorded as HSC. The target sequence is humanized by maximizing its HSC rather than using a global identity metric to generate multiple different humanized variants.

[0257] Framework library (abbreviated as: FR library)

[0258] In the FR library approach, a collection of residue variants is introduced into specific positions in the FRs, and the library is then panned to select the FRs that best support the grafted CDRs. Thus, this approach is similar to CDR grafting, but instead of creating a few back mutations in the FRs, a combinatorial library of typically more than 100 mutation variants is constructed.

[0259] Guide selection

[0260] The method comprises converting the V of a given non-human antibody specific for a particular antigen into H or V L Domain structure and human V H and V L Subsequently, a specific human V domain is selected for the target antigen. For example, a non-human V H The non-human antibody is humanized by combining it with a human light chain library. The library against the target antigen is then selected by phage display, and the selected V L Cloned into human V H The non-human V L The library is then selected against the target antigen by phage display, and the selected V H Cloned into human V L The library of heavy chains is selected for the target antigen. As a result, fully human antibodies with similar affinity to non-human antibodies can be isolated. To avoid epitope drift, a so-called inhibition ELISA can be implemented, which can select 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, because this CDR is located in the center of the antigen binding site.

[0261] Frame Mix (abbreviation: FR Mix)

[0262] In the FR shuffling method, the entire FR is combined with a non-human CDR. Dall'Acqua and colleagues humanized a mouse antibody by FR shuffling. All six CDRs of a mouse antibody were cloned into a library containing all human germline gene FRs (Dall'Acqua et al., 2005). The library was screened for binding via a two-step selection process, firstly humanized V L , then V H In a subsequent study, a one-step FR shuffling approach was used successfully (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.

[0263] Human Engineering

[0264] Human engineering allows the isolation of antibodies that are 91% to 96% homologous to human germline antibodies. The approach is based on the experimental identification of the essential minimum specificity determinant (MSD) and sequentially replacing non-human fragments into a human FR library and assessing binding. H and V L Starting from the CDR3 region of the chain, other regions of the non-human antibody are gradually replaced with human FRs, including V H and V L CDR1 and CDR2.

[0265] When preparing a humanized antibody that specifically binds to a conformational epitope described herein, the above-described methods for humanizing antibodies are preferred. However, the present invention is not limited to the above-described methods for humanizing antibodies.

[0266] Some of the above-mentioned humanization methods, namely the "fixed FR method" (a variation of CDR grafting), superhumanization, framework shuffling, and human engineering, can be performed without information about the FR sequences in the donor antibody. Qin et al., 2007 and Chang et al., 2007 successfully performed variations of the "fixed FR method." In particular, Qin et al. constructed an antibody fragment comprising 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 the CDR regions from the V H All CDRs of the part and from V L Part of CDR3 was replaced by an antigenic peptide derived from the CDR sequence of a murine antibody.

[0267] As used herein, "human antibodies" include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The 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). The human antibodies of the invention include antibodies isolated from human immunoglobulin libraries or antibodies from one or more than one transgenic human immunoglobulin animals, and do not express endogenous immunoglobulins, as described in, for example, U.S. Pat. No. 5,939,598 to Kucherlapati and Jakobovits.

[0268] 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, such as a mouse, and fused to an immortalized cell.

[0269] As used herein, the term "recombinant antibody" 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 transformed to express the antibodies, such as from transfectomas; (c) antibodies isolated from recombinant combinatorial antibody libraries; and (d) antibodies prepared, expressed, created or isolated by any other method involving splicing of immunoglobulin gene sequences to other DNA sequences.

[0270] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells, or fungi, including yeast cells, that express the antibody.

[0271] As used herein, "heterologous antibody" is defined based on the transgenic organism that produces such antibody. The term refers to an antibody having an amino acid sequence or encoding nucleic acid sequence corresponding to an amino acid sequence or encoding nucleic acid sequence found in an organism that does not consist of a transgenic organism, which antibody is generally derived from a species other than the transgenic organism.

[0272] As used herein, "hybrid antibody" refers to an antibody having light and heavy chains of different biological origins. For example, an antibody having a human heavy chain associated with a mouse light chain is a hybrid antibody.

[0273] Therefore, "antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, hybrid, chimeric, humanized (especially CDR grafted), deimmunized or human antibodies, Fab fragments, Fab' fragments, F(ab') fragments, 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 called single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies of the antibodies of the invention), and epitope-binding fragments of any of the above.

[0274] The antibodies described herein are preferably isolated. As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities.

[0275] As used herein, the term "naturally occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified by man in the laboratory is naturally occurring.

[0276] As used herein, the term "aptamer" refers to a nucleic acid molecule that has been engineered to bind to a target molecule by repeated rounds of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) (for review, see: Brody EN and Gold L. (2000), Aptamers as therapeutic and diagnostic agents, J. Biotechnol. 74 (1): 5-13). Aptamers can be DNA or RNA molecules. Aptamers can contain modifications, such as modified nucleotides, such as 2'-fluoro substituted pyrimidines.

[0277] As used herein, the term "antibody-like protein" refers to a protein that has been engineered (e.g., by mutagenic loop engineering) to specifically bind to a target molecule. Typically, such an antibody-like protein comprises at least one variable peptide loop, both ends of which are connected to a protein scaffold. This dual structural restriction greatly increases the binding affinity of the antibody-like protein, making its affinity level comparable to that of an antibody. The length of the variable peptide loop is typically 10 to 20 amino acids. The scaffold protein can be any protein with good solubility. Preferably, the scaffold protein is a globular protein. Antibody-like proteins include, but are not limited to, affibodies, anticalins, and designed ankyrin repeat proteins (for 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 mutant libraries, such as panning from large phage display libraries, and can be isolated similarly to conventional antibodies. Likewise, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins. Antibody-like proteins are sometimes referred to as "peptide aptamers".

[0278] As used herein, "peptide mimetics" are small protein-like chains designed to mimic peptides. Peptide mimetics are generally derived from modifications to existing peptides to change the properties of the molecule. For example, they may come from modifications that change the stability or biological activity of the molecule. This may play a role in drug-like compounds developed based on existing peptides. These modifications involve changes to the peptide that would not occur naturally (e.g., backbone changes and the incorporation of unnatural amino acids).

[0279] The "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence (excluding additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences to produce the number of matched positions, and 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 percentage of sequence identity.

[0280] In the context of two or more nucleic acids or peptide sequences, the term "identical" as used herein refers to two or more sequences or subsequences that are identical, i.e., comprising identical nucleotides or amino acid sequences. When using one of the following sequence comparison algorithms or measuring a specified region by manual calibration and visual inspection or comparing and comparing maximum correspondence in a comparison window, if the sequence has a specific percentage of identical nucleotides or amino acid residues (for example, in a specific region there is 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), then "substantially identical" between sequences. These definitions also relate to the complementary sequence of a test sequence. Therefore, regarding polypeptide and polynucleotide sequence comparisons, the term "at least 80% sequence identity" is used throughout the specification. This expression 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 a corresponding reference polypeptide or a corresponding reference polynucleotide.

[0281] The term "sequence comparison" as used herein refers to a method in which one sequence acts as a reference sequence, which is compared to a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters are usually used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity or similarity percentage of the test sequence relative to the reference sequence based on the program parameters. In the case of comparing two sequences and not specifying a reference sequence to be compared with a sequence identity percentage to be calculated, if not otherwise specified, the sequence identity is calculated with reference to the longer sequence in the two sequences to be compared. If not otherwise specified, if a reference sequence is specified, the sequence identity is determined based on the full length of the reference sequence indicated by SEQ ID.

[0282] In sequence alignment, the term "comparison window" refers to those consecutive position segments of a sequence that are compared with a consecutive position reference segment of a sequence having the same number of positions. The number of selected consecutive positions can be 4 to 1000, i.e., can include 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 consecutive positions. Typically, the number of consecutive positions is from about 20 to 800 consecutive positions, from about 20 to 600 consecutive positions, from about 50 to 400 consecutive positions, from about 50 to about 200 consecutive positions, from about 100 to about 150 consecutive positions.

[0283] Methods for comparing sequences are well known in the art. Optimal alignment of sequences for comparison can be performed, 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 search for similarity 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 Wisconsin genetics software package Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)). Suitable algorithms for determining percentages of sequence identity and sequence similarity are 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 analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which match or satisfy some positive threshold score T when the short word of length W is aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits are used as seeds to initiate searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score is zero or below zero due to the accumulation of one or more negatively scored residue alignments; or 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 a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and two chains as default values. For amino acid sequences, the BLASTP program uses a word length of 3, an expected value (E) of 10, a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) alignment (B) of 50, an expected value (E) of 10, M=5, N=-4, and two chains as default values. The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). A similarity measure provided by the BLAST algorithm is the minimum and probability (P(N)), which provides an indication of the probability of accidental matching between two nucleotide or amino acid sequences. 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.

[0284] "Conservative substitutions" can be made, for example, based on similarities in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. Amino acids can be divided into the following six standard amino acid groups:

[0285] (1) Hydrophobic amino acids: Met, Ala, Val, Leu, Ile;

[0286] (2) Neutral hydrophilic amino acids: Cys, Ser, Thr, Asn, Gln;

[0287] (3) Acidic amino acids: Asp, Glu;

[0288] (4) Basic amino acids: His, Lys, Arg;

[0289] (5) Residues that affect chain orientation: Gly, Pro; and

[0290] (6) Aromatic amino acids: Trp, Tyr, Phe.

[0291] As used herein, "conservative substitution" is defined as an exchange of an amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, a negative charge is retained in the polypeptide modified in this way by exchanging Asp for Glu. In addition, glycine and proline can replace each other based on their ability to disrupt α-helices. Some preferred conservative substitutions in the above six groups are exchanges in 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. In view of the known genetic code, as well as recombinant and synthetic DNA technology, skilled scientists can easily construct DNA encoding conservative amino acid variants.

[0292] As used herein, "non-conservative substitutions" or "non-conservative amino acid exchanges" are defined as exchanges 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.

[0293] The terms "nucleic acid" and "nucleic acid molecule" are used synonymously herein and are understood to be single-stranded or double-stranded oligomers or polymers of deoxyribonucleotides or ribonucleotide bases or both. Nucleotide monomers consist of a nucleobase, a pentose (such as, but not limited to, ribose or 2'-deoxyribose) and one to three phosphate groups. Typically, nucleic acids are formed by 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 includes synthetic forms of nucleic acids containing other connections (e.g., peptide nucleic acids described in Nielsen et al., Science 254: 1497-1500, 1991). Typically, nucleic acids are single-stranded or double-stranded molecules and are composed of naturally occurring nucleotides. The description of a single-stranded nucleic acid also (at least in part) defines the sequence of the complementary chain. Nucleic acids can be single-stranded or double-stranded, or can contain a portion of double-stranded and single-stranded sequences. Exemplary double-stranded nucleic acid molecules may have 3' or 5' overhangs, so it is not necessary or assumed that they are completely double-stranded over their entire length. Nucleic acids can be obtained by biological, biochemical or chemical synthesis methods or any method known in the art, including but not limited to RNA amplification and reverse transcription methods. The term nucleic acid includes chromosomes or chromosome 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 specific length. Unless otherwise indicated, in addition to any sequence explicitly indicated, a specific nucleic acid sequence also includes or encodes a complementary sequence.

[0294] Nucleic acid can be degraded by endonucleases or exonucleases, particularly by DNA enzymes and RNA enzymes found in cells. Therefore, it may be advantageous to modify the nucleic acid of the present invention so that it stably resists degradation, thereby ensuring that the nucleic acid of high concentration is maintained for a long time in the cell. Usually, this stabilization can be obtained by introducing one or more than one phosphorus-based nucleotides or by introducing one or more than one non-phosphorus nucleotides. Therefore, nucleic acid can be composed of non-naturally occurring nucleotides and / or the modification of naturally occurring nucleotides and / or the change of molecular skeleton. The modified nucleotides in nucleic acid include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates, phosphorodithioates and / or phosphates, and non-phosphorus nucleotides include, but are not limited to, siloxane bridges, carbonate bridges, carboxymethyl esters, acetamide bridges and / or thioether bridges. Other examples of nucleotide modifications include, but are not limited to: phosphorylation of the 5' or 3' nucleotide to allow ligation or prevent exonuclease degradation / polymerase extension, respectively; covalently and near-covalently attached amino, thiol, alkyne, or biotinyl modifications; fluorescent and quenching agents; and modified bases such as deoxyinosine (dI), 5-bromo-deoxyuridine (5-bromo-dU), deoxyuridine, 2-aminopurine, 2,6-diaminopurine, trans-dT, trans-dideoxy-T, dideoxycytidine (ddC 5-methyldeoxycytidine (5-methyl dC), locked nucleic acids (LNA's), 5-nitroindole, iso-dC and -dG bases, 2'-O-methyl RNA bases, hydroxymethyl dC, 5-hydroxybutyne-2'-deoxyuridine, 8-aza-7-deazaguanosine and fluorine-modified bases. Therefore, the nucleic acid can also be an artificial nucleic acid, which includes but is not limited to polyamide or peptide nucleic acids (PNA), morpholino and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA).

[0295] 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 coding sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0296] The term "polynucleotide" when used in the context of the present invention refers to a nucleic acid that is greater than about 50 nucleotides in length, such as 51 or more nucleotides in length.

[0297] The polypeptides of the present invention are prepared by any suitable method, including but not limited to isolation of existing or natural sequences, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or by direct chemical synthesis 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 methods; or the solid support method of U.S. Pat. No. 4,458,066, or other methods known to those skilled in the art.

[0298] The term "vector" as used herein refers to a protein or polynucleotide or a mixture thereof that can be introduced into or can introduce the protein and / or nucleic acid contained therein into a cell. Examples of vectors include, but are not limited to, plasmids, cosmids, bacteriophages, viruses or artificial chromosomes. In particular, vectors are used to transport target gene products, such as transporting exogenous DNA or heterologous DNA into suitable host cells. The vector may contain a "replicon" polynucleotide sequence that facilitates the autonomous replication of the vector in the host cell. Exogenous DNA is defined as heterologous DNA, which is DNA that does not naturally exist in the host cell, which, for example, replicates vector molecules, encodes selectable or screenable markers, or encodes transgenics. After entering the host cell, the vector can replicate independently of the host chromosomal DNA or simultaneously with the host chromosomal DNA, and can produce several copies of the vector and its inserted DNA. In addition, the vector may also contain essential elements that allow the inserted DNA to be transcribed into mRNA molecules or otherwise replicate the inserted DNA into multiple copies of RNA. The vector may also include an "expression control sequence" that regulates the expression of the target gene. Typically, an expression control sequence is a polypeptide or polynucleotide, such as, but not limited to, a promoter, an enhancer, a silencer, an insulator or a repressor. In a vector comprising more than one polynucleotide encoding one or more than one target gene product, expression can be controlled together or separately by one or more than one expression control sequence. More specifically, each polynucleotide contained on the vector can be controlled by a single expression control sequence or all polynucleotides contained on the vector can be controlled by a single expression control sequence. The polynucleotide contained in a single vector controlled by a single expression control sequence can form an open reading frame. Some expression vectors additionally contain sequence elements adjacent to the inserted DNA, which increase the half-life of the expressed mRNA and / or allow the mRNA to be translated into protein molecules. Therefore, many mRNAs and polypeptide molecules encoded by the inserted DNA can be quickly synthesized.

[0299] The term "host cell" refers to a cell with a vector (e.g., a plasmid or a virus). Such a host cell can be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a fungal, plant, or animal cell). Host cells include unicellular prokaryotes and eukaryotic organisms (e.g., bacteria, yeast, and actinomycetes), as well as unicellular cells from higher plants or animals when grown in cell culture. As used herein, a "recombinant host cell" refers to a host cell comprising a polynucleotide encoding a polypeptide fragment of interest, i.e., a fragment of a viral PA subunit or a variant thereof according to the present invention. The polynucleotides that can be found in the host cell are (i) freely dispersed, (ii) incorporated into a recombinant vector, or (iii) integrated into the host cell genome or mitochondrial DNA. Recombinant cells can be used to express a polynucleotide of interest or to amplify a polynucleotide or recombinant vector of the present invention. The term "recombinant host cell" includes descendants of the original cell that has been transformed, transfected, or infected by a polynucleotide or recombinant vector of the present invention. The recombinant host cell can be a bacterial cell such as an Escherichia coli cell, a yeast cell such as Saccharomyces cerevisiae or Pichia pastoris, a plant cell, an insect cell such as SF9 or HighFive cells or a mammalian cell. Preferred examples of mammalian cells are Chinese hamster ovary (CHO) cells, African green monkey kidney (COS) cells, human embryonic kidney (HEK293) cells, HELA cells, etc.

[0300] As used herein, the term "active agent" refers to any agent that can exhibit therapeutic activity.

[0301] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0302] The term "carrier" as used herein refers to a diluent, adjuvant, excipient or vehicle used together with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, saline solutions are preferred pharmaceutical carriers. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid pharmaceutical carriers, particularly for injectable solutions. Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain a small amount of wetting agent or emulsifier, or pH buffer. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, etc. The composition can be formulated into a suppository with conventional adhesives and pharmaceutical carriers such as triglycerides. The compounds of the present invention can be formulated into neutral forms or salt forms. Pharmaceutically acceptable salts include salts formed with free amino groups, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with free carboxyl groups, such as salts derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such a composition will contain a therapeutically effective amount of the compound, preferably a purified form of the compound, and an appropriate amount of carrier to provide a suitable form of administration for the patient. The preparation should be suitable for the mode of administration.

[0303] Methods commonly known and practiced in the fields of molecular biology, cell biology, protein chemistry, and antibody technology are fully described in the continually updated publications “Molecular Cloning: A Laboratory Manual”, (Sambrook et al., Cold Spring Harbor); Current Protocols in Molecular Biology (FM Ausubel et al., eds., Wiley & Sons); Current Protocols in Protein Science (JE Colligan et al., eds., Wiley & Sons); Current Protocols in Cell Biology (JS Bonifacino et al., Wiley & Sons) and Current Protocols in Immunology (JE Colligan et al., eds., Wiley & Sons). Known techniques related to cell culture and culture 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).

[0304] Implementation

[0305] In the following, different aspects of the present invention will be defined in more detail. Each aspect so defined can be combined with any other one or more than one aspect, unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous can be combined with any other one or more than one feature indicated as being preferred or advantageous.

[0306] In a first aspect, the present invention provides an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein competes with an antibody for binding to PD-1, the antibody

[0307] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3; or

[0308] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

[0309] Preferably, the present invention provides an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein competes with an antibody for binding to PD-1, the antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 2; and a light chain variable region of the amino acid sequence of SEQ ID NO: 3.

[0310] In a preferred embodiment of the first aspect, the antagonist antigen binding protein that specifically binds to PD-1 is an antibody, an antibody-like protein or a fragment thereof. Preferably, the antibody is selected from human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, single-chain variable fragment antibodies, double antibodies, Fab fragments, F(ab)2 fragments, antibody mimetics, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies and IgG4 antibodies.

[0311] In another preferred embodiment of the first aspect, the antagonist 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 antagonist antigen binding protein that specifically binds to PD-1 binds to PD-1 with a Kd of 5 pM to 100 nM, 5 pM to 50 nM, 5 pM to 10 nM, 5 pM to 1 nM, 5 pM to 100 pM, for example, 5 pM to 10 pM.

[0312] In another preferred embodiment of the first aspect, the antagonist antigen binding protein that specifically binds to PD-1 can block the binding of the ligand to PD-1 with an IC50 of less than 10 nM, less than 1 nM, or less than 200 pM. For example, the antagonist antigen binding protein that specifically binds to PD-1 can block the binding of the ligand to PD-1 with an IC50 of 200 pM to 10 nM, such as 200 pM to 1 nM.

[0313] In another preferred embodiment of the first aspect, the antagonist antigen binding protein that specifically binds to PD-1 stimulates T cell proliferation.

[0314] In another preferred embodiment of the first aspect, the antagonist antigen binding protein that specifically binds to PD-1 induces lymphocyte proliferation.

[0315] In another preferred embodiment of the first aspect, the antagonist antigen binding protein that specifically binds to PD-1 induces the secretion of IL-2 and / or IFNγ.

[0316] In another preferred embodiment of the first aspect, the antagonist 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 a baseline value.

[0317] In a second aspect, the present invention provides an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein competes with an antibody for binding to PD-L1, the antibody

[0318] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21; or

[0319] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; or

[0320] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 83; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 84; or

[0321] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 92; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 93; or

[0322] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 101; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 102.

[0323] Preferably, the present invention provides an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein competes with an antibody for binding to PD-L1, the 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.

[0324] In a preferred embodiment of the second aspect, the antagonist antigen binding protein that specifically binds to PD-L1 is an antibody, an antibody-like protein or a fragment thereof. Preferably, the antibody is selected from human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, single-chain variable fragment antibodies, double antibodies, Fab fragments, F(ab)2 fragments, antibody mimetics, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies and IgG4 antibodies.

[0325] In another preferred embodiment of the second aspect, the antagonist 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 antagonist antigen binding protein that specifically binds to PD-L1 binds to PD-L1 with a Kd of 5 pM to 100 nM, 5 pM to 50 nM, 5 pM to 10 nM, 5 pM to 1 nM, 5 pM to 100 pM, for example, 5 pM to 10 pM.

[0326] In another preferred embodiment of the second aspect, the antagonist antigen binding protein that specifically binds to PD-L1 can block the binding of the ligand to PD-L1 with an IC50 of less than 10 nM, less than 1 nM or less than 200 pM.

[0327] For example, an antagonist antigen binding protein that specifically binds to PD-L1 can block the binding of the ligand to PD-L1 with an IC50 of 200 pM to 10 nM, such as 200 pM to 1 nM.

[0328] In another preferred embodiment of the second aspect, the antagonist antigen binding protein that specifically binds to PD-L1 stimulates T cell proliferation.

[0329] In another preferred embodiment of the second aspect, the antagonist antigen binding protein that specifically binds to PD-L1 induces lymphocyte proliferation.

[0330] In another preferred embodiment of the second aspect, the antagonist antigen binding protein that specifically binds to PD-L1 induces the secretion of IL-2 and / or IFNγ.

[0331] In another preferred embodiment of the second aspect, the antagonist 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 a baseline value.

[0332] In a third aspect, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein competes with an antibody for binding to LAG-3, the antibody

[0333] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39;

[0334] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48;

[0335] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57;

[0336] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 66; or

[0337] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 74; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75.

[0338] Preferably, the present invention provides an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein competes for binding to LAG-3 with 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.

[0339] In another preferred embodiment, the present invention provides an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein competes for binding to LAG-3 with 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.

[0340] In a preferred embodiment of the third aspect, the antagonist antigen-binding protein that specifically binds to LAG-3 is an antibody, an antibody-like protein or a fragment thereof. Preferably, the antibody is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, single-chain variable fragment antibodies, diabodies, Fab fragments, F(ab)2 fragments, antibody mimetics, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies and IgG4 antibodies.

[0341] In another preferred embodiment of the third aspect, the antagonist 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, the antagonist antigen binding protein that specifically binds to LAG-3 binds to LAG-3 with a Kd of 5 pM to 100 nM, 5 pM to 50 nM, 5 pM to 10 nM, 5 pM to 1 nM, 5 pM to 100 pM, e.g., 5 pM to 10 pM.

[0342] In another preferred embodiment of the third aspect, the antagonist antigen binding protein that specifically binds to LAG-3 can block the binding of the ligand to LAG-3 with an IC50 of less than 10 nM, less than 1 nM or less than 200 pM. For example, the antagonist antigen binding protein that specifically binds to LAG-3 can block the binding of the ligand to LAG-3 with an IC50 of 200 pM to 10 nM, such as 200 pM to 1 nM.

[0343] In another preferred embodiment of the third aspect, the antagonist antigen binding protein that specifically binds to LAG-3 stimulates T cell proliferation.

[0344] In another preferred embodiment of the third aspect, the antagonist antigen binding protein that specifically binds to LAG-3 induces lymphocyte proliferation.

[0345] In another preferred embodiment of the third aspect, the antagonist antigen binding protein that specifically binds to LAG-3 induces the secretion of IL-2 and / or IFNγ.

[0346] In another preferred embodiment of the third aspect, the antagonist antigen binding protein that specifically binds LAG-3 reduces tumor volume by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a baseline value.

[0347] In a fourth aspect, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises any one of the following

[0348] (i) a combination of a light chain variable domain and a heavy chain variable domain, the combination being selected from:

[0349] A light chain variable domain comprising a sequence having at least 90% identity to SEQ ID NO: 3 and

[0350] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 2;

[0351] A light chain variable domain comprising a sequence having at least 90% identity to SEQ ID NO: 12 and

[0352] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 11;

[0353] (ii) a combination of a heavy chain complementarity determining region 3 (CDRH3) and a light chain complementarity determining region 3 (CDRL3), the combination being selected from the group consisting of:

[0354] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and

[0355] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9;

[0356] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17 and

[0357] A CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:18.

[0358] In a preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising a sequence having at least 90% identity with SEQ ID NO: 3, and the heavy chain variable domain comprising a sequence having at least 90% identity with SEQ ID NO: 2.

[0359] In another preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises a combination of CDRH3 and CDRL3, wherein the CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 8, and the CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0360] In a preferred embodiment of the fourth aspect of the present invention, the antagonist antigen binding protein specifically binds to PD-1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain selected from the following:

[0361] A light chain variable domain comprising a sequence having at least 95% identity to SEQ ID NO: 3 and

[0362] a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 2;

[0363] A light chain variable domain comprising a sequence having at least 95% identity to SEQ ID NO: 12 and

[0364] A heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 11.

[0365] In a more preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising a sequence having at least 95% identity with SEQ ID NO: 3, and the heavy chain variable domain comprising a sequence having at least 95% identity with SEQ ID NO: 2.

[0366] In a more preferred embodiment of the fourth aspect of the present invention, the antagonist antigen binding protein specifically binds to PD-1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain selected from the following:

[0367] A light chain variable domain comprising a sequence having at least 99% identity to SEQ ID NO: 3 and

[0368] a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 2;

[0369] A light chain variable domain comprising a sequence having at least 99% identity to SEQ ID NO: 12 and

[0370] A heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 11.

[0371] In a more preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 3, and the heavy chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 2.

[0372] In a more preferred embodiment of the fourth aspect of the present invention, the antagonist antigen binding protein specifically binds to PD-1, wherein the antigen binding protein comprises a combination of a heavy chain complementarity determining region 3 (CDRH3) and a light chain complementarity determining region 3 (CDRL3) selected from the following:

[0373] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and

[0374] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9;

[0375] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17 and

[0376] A CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:18.

[0377] In another preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises a combination of CDRH3 and CDRL3, the CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and the CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9. Preferably, the antagonist antigen binding protein specifically binds to PD-1, wherein the antigen binding protein further comprises one or more selected from the following: CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4 or 13, CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 6 or 15, CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or 14, and CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 16.

[0378] For example, an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises

[0379] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and

[0380] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9;

[0381] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4 and

[0382] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 5;

[0383] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 6 and

[0384] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:7.

[0385] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0386] For example, an antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises

[0387] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17 and

[0388] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 18;

[0389] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 13 and

[0390] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 14;

[0391] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and

[0392] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:16.

[0393] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0394] In a more preferred embodiment of the fourth aspect, the antagonist antigen binding protein that specifically binds to PD-1 is an antibody, an antibody-like protein or a fragment thereof. Preferably, the antibody is selected from human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, single-chain variable fragment antibodies, double antibodies, Fab fragments, F(ab)2 fragments, antibody mimetics, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies and IgG4 antibodies.

[0395] In another preferred embodiment of the fourth aspect, the antagonist antigen binding protein that specifically binds to PD-1 binds to PD-1 with a Kd of less than 100nM, less than 50nM, less than 10nM, less than 1nM, less than 100pM, less than 10pM or less than 5pM. For example, the antagonist antigen binding protein that specifically binds to PD-1 binds to PD-1 with a Kd of 5pM to 100nM, 5pM to 50nM, 5pM to 10nM, 5pM to 1nM, 5pM to 100pM, for example, 5pM to 10pM.

[0396] In another preferred embodiment of the fourth aspect, the antagonist antigen binding protein that specifically binds to PD-1 can block the binding of the ligand to PD-1 with an IC50 of less than 10 nM, less than 1 nM, or less than 200 pM. For example, the antagonist antigen binding protein that specifically binds to PD-1 can block the binding of the ligand to PD-1 with an IC50 of 200 pM to 10 nM, such as 200 pM to 1 nM.

[0397] In another preferred embodiment of the fourth aspect, the antagonist antigen binding protein that specifically binds to PD-1 stimulates T cell proliferation.

[0398] In another preferred embodiment of the fourth aspect, the antagonist antigen binding protein that specifically binds to PD-1 induces lymphocyte proliferation.

[0399] In another preferred embodiment of the fourth aspect, the antagonist antigen binding protein that specifically binds to PD-1 induces the secretion of IL-2 and / or IFNγ.

[0400] In another preferred embodiment of the fourth aspect, the antagonist 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 a baseline value.

[0401] In a fifth aspect, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises any one of the following

[0402] (i) a combination of a light chain variable domain and a heavy chain variable domain, the combination being selected from:

[0403] A light chain variable domain comprising a sequence having at least 90% identity to SEQ ID NO: 21 and

[0404] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 20;

[0405] A light chain variable domain comprising a sequence having at least 90% identity to SEQ ID NO: 30 and

[0406] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 29;

[0407] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 84 and

[0408] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 83;

[0409] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 93 and

[0410] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 92;

[0411] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 102 and

[0412] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 101;

[0413] (ii) a combination of a heavy chain complementarity determining region 3 (CDRH3) and a light chain complementarity determining region 3 (CDRL3), the combination being selected from the group consisting of:

[0414] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and

[0415] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27;

[0416] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and

[0417] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36;

[0418] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 89 and

[0419] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 90;

[0420] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and

[0421] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 99;

[0422] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 107 and

[0423] A CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:108.

[0424] In a preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising a sequence having at least 90% identity with SEQ ID NO: 30, and the heavy chain variable domain comprising a sequence having at least 90% identity with SEQ ID NO: 29.

[0425] In another preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises a combination of CDRH3 and CDRL3, the CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 35, and the CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 36.

[0426] In a preferred embodiment of the fifth aspect of the present invention, the antagonist antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain selected from the following:

[0427] A light chain variable domain comprising a sequence having at least 95% identity to SEQ ID NO: 21 and

[0428] a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 20;

[0429] A light chain variable domain comprising a sequence having at least 90% identity to SEQ ID NO: 30 and

[0430] a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 29;

[0431] A light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 84 and

[0432] a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 83;

[0433] A light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 93 and

[0434] a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 92;

[0435] A light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 102 and

[0436] A heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 101.

[0437] In a more preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises a combination of a light chain variable domain comprising a sequence that is at least 95% identical to SEQ ID NO: 30 and a heavy chain variable domain comprising a sequence that is at least 95% identical to SEQ ID NO: 29.

[0438] In a more preferred embodiment of the fifth aspect of the present invention, the antagonist antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain selected from the group consisting of:

[0439] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 21 and

[0440] a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 20;

[0441] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 30 and

[0442] a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 29;

[0443] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 84 and

[0444] a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 83;

[0445] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 93 and

[0446] a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 92;

[0447] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 102 and

[0448] A heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 101.

[0449] In a preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 30, and the heavy chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 29.

[0450] In a more preferred embodiment of the fifth aspect of the invention, the antagonist antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein comprises a combination of a heavy chain complementarity determining region 3 (CDRH3) and a light chain complementarity determining region 3 (CDRL3) selected from the following:

[0451] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and

[0452] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27;

[0453] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and

[0454] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36;

[0455] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 89 and

[0456] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 90;

[0457] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and

[0458] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 99;

[0459] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 107 and

[0460] A CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:108.

[0461] In another preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises a combination of CDRH3 and CDRL3, the CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 35, and the CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 36.

[0462] Preferably, the antagonist antigen binding protein specifically binds to PD-L1, wherein the antigen binding protein further comprises one or more selected from the following: a CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22 or 31, a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or 33, a CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23 or 32, and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 25 or 34.

[0463] For example, an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises

[0464] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and

[0465] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 27;

[0466] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 22 and

[0467] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 23;

[0468] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 24 and

[0469] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:25.

[0470] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0471] For example, an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises

[0472] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and

[0473] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 36;

[0474] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 31 and

[0475] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 32;

[0476] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 33 and

[0477] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:34.

[0478] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0479] For example, an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises

[0480] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 89 and

[0481] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 90;

[0482] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 85 and

[0483] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 86;

[0484] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 87 and

[0485] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:88.

[0486] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0487] For example, an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises

[0488] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 98 and

[0489] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 99;

[0490] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 94 and

[0491] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 95;

[0492] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 96 and

[0493] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:97.

[0494] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0495] For example, an antagonist antigen binding protein that specifically binds to PD-L1, wherein the antigen binding protein comprises

[0496] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 107 and

[0497] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 108;

[0498] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 103 and

[0499] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 104;

[0500] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 105 and

[0501] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:106.

[0502] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0503] In a more preferred embodiment of the fifth aspect, the antagonist antigen binding protein that specifically binds to PD-L1 is an antibody, an antibody-like protein or a fragment thereof. Preferably, the antibody is selected from human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, single-chain variable fragment antibodies, double antibodies, Fab fragments, F(ab)2 fragments, antibody mimetics, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies and IgG4 antibodies.

[0504] In another preferred embodiment of the fifth aspect, the antagonist 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 antagonist antigen binding protein that specifically binds to PD-L1 binds to PD-L1 with a Kd of 5 pM to 100 nM, 5 pM to 50 nM, 5 pM to 10 nM, 5 pM to 1 nM, 5 pM to 100 pM, for example, 5 pM to 10 pM.

[0505] In another preferred embodiment of the fifth aspect, the antagonist antigen binding protein that specifically binds to PD-L1 can block the binding of the ligand to PD-L1 with an IC50 of less than 10 nM, less than 1 nM, or less than 200 pM. For example, the antagonist antigen binding protein that specifically binds to PD-L1 can block the binding of the ligand to PD-L1 with an IC50 of 200 pM to 10 nM, such as 200 pM to 1 nM.

[0506] In another preferred embodiment of the fifth aspect, the antagonist antigen binding protein that specifically binds to PD-L1 stimulates T cell proliferation.

[0507] In another preferred embodiment of the fifth aspect, the antagonist antigen binding protein that specifically binds to PD-L1 induces lymphocyte proliferation.

[0508] In another preferred embodiment of the fifth aspect, the antagonist antigen binding protein that specifically binds to PD-L1 induces the secretion of IL-2 and / or IFNγ.

[0509] In another preferred embodiment of the fifth aspect, the antagonist 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 a baseline value.

[0510] In a sixth aspect, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises any one of the following

[0511] (i) a combination of a light chain variable domain and a heavy chain variable domain, the combination being selected from:

[0512] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 39 and

[0513] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 38;

[0514] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 48 and

[0515] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 47;

[0516] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 57 and

[0517] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO:56;

[0518] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 66 and

[0519] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 65;

[0520] A light chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 75 and

[0521] a heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO: 74;

[0522] (ii) a combination of a heavy chain complementarity determining region 3 (CDRH3) and a light chain complementarity determining region 3 (CDRL3) selected from the group consisting of:

[0523] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and

[0524] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45;

[0525] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and

[0526] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54;

[0527] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and

[0528] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 63;

[0529] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71 and

[0530] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 72;

[0531] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 80 and

[0532] A CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:81.

[0533] In a preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of a light chain variable domain comprising a sequence that is at least 90% identical to SEQ ID NO: 39 and a heavy chain variable domain comprising a sequence that is at least 90% identical to SEQ ID NO: 38.

[0534] In another preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of CDRH3 and CDRL3, wherein the CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 45.

[0535] In a more preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of a light chain variable domain comprising a sequence that is at least 90% identical to SEQ ID NO: 48 and a heavy chain variable domain comprising a sequence that is at least 90% identical to SEQ ID NO: 47.

[0536] In another preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of CDRH3 and CDRL3, wherein the CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 53, and the CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 54.

[0537] In a preferred embodiment of the sixth aspect of the invention, the antagonist antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain selected from the group consisting of:

[0538] A light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 39 and

[0539] a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 38;

[0540] A light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 48 and

[0541] a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 47;

[0542] A light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 57 and

[0543] a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO:56;

[0544] A light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 66 and

[0545] a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 65;

[0546] A light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 75 and

[0547] A heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO:74.

[0548] In a more preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of a light chain variable domain comprising a sequence that is at least 95% identical to SEQ ID NO: 39 and a heavy chain variable domain comprising a sequence that is at least 95% identical to SEQ ID NO: 38.

[0549] In a more preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of a light chain variable domain comprising a sequence that is at least 95% identical to SEQ ID NO: 48 and a heavy chain variable domain comprising a sequence that is at least 95% identical to SEQ ID NO: 47.

[0550] In a more preferred embodiment of the sixth aspect of the invention, the antagonist antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain selected from the group consisting of:

[0551] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 39 and

[0552] a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 38;

[0553] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 48 and

[0554] a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 47;

[0555] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 57 and

[0556] a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO:56;

[0557] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 66 and

[0558] a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 65;

[0559] A light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 75 and

[0560] A heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO:74.

[0561] In a more preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of a light chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 39 and a heavy chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 38.

[0562] In a more preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of a light chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 48 and a heavy chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 47.

[0563] In a more preferred embodiment of the sixth aspect of the invention, the antagonist antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of a heavy chain complementarity determining region 3 (CDRH3) and a light chain complementarity determining region 3 (CDRL3) selected from the group consisting of:

[0564] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and

[0565] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45;

[0566] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and

[0567] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54;

[0568] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and

[0569] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 63;

[0570] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71 and

[0571] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 72;

[0572] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 80 and

[0573] A CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO:81.

[0574] In another preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of CDRH3 and CDRL3, wherein the CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 45.

[0575] In another preferred embodiment, the present invention relates to an antagonist antigen binding protein that specifically binds to LAG-3, wherein the antigen binding protein comprises a combination of CDRH3 and CDRL3, wherein the CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 53, and the CDRL3 comprises or consists of the amino acid sequence of SEQ ID NO: 54.

[0576] Preferably, the antagonist antigen binding protein specifically binds to LAG-3, wherein the 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: 40, 49, 58, 67 or 76, a CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 42, 51, 60, 69 or 78, a CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 41, 50, 59, 68 or 77, and a CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 43, 52, 61, 70 or 79.

[0577] For example, an antagonist antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein comprises

[0578] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and

[0579] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 45;

[0580] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 40 and

[0581] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 41;

[0582] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 42 and

[0583] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:43.

[0584] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0585] For example, an antagonist antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein comprises

[0586] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and

[0587] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 54;

[0588] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 49 and

[0589] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 50;

[0590] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 51 and

[0591] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:52.

[0592] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0593] For example, an antagonist antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein comprises

[0594] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 62 and

[0595] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 63;

[0596] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 58 and

[0597] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 59;

[0598] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 60 and

[0599] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:61.

[0600] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0601] For example, an antagonist antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein comprises

[0602] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71 and

[0603] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 72;

[0604] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 67 and

[0605] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 68;

[0606] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 69 and

[0607] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:70.

[0608] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0609] For example, an antagonist antigen binding protein specifically binds to LAG-3, wherein the antigen binding protein comprises

[0610] A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 80 and

[0611] CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 81;

[0612] CDRH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 76 and

[0613] CDRL1 comprising or consisting of the amino acid sequence of SEQ ID NO: 77;

[0614] CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 78 and

[0615] A CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO:79.

[0616] Preferably, the antagonist antigen binding proteins as described above comprising one amino acid exchange in CDRH1, CDRL1, CDRH2 or CDRL2 also fall within the scope of the present invention.

[0617] In a more preferred embodiment of the sixth aspect, the antagonist antigen-binding protein that specifically binds to LAG-3 is an antibody, an antibody-like protein or a fragment thereof. Preferably, the antibody is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, single-chain variable fragment antibodies, diabodies, Fab fragments, F(ab)2 fragments, antibody mimetics, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies and IgG4 antibodies.

[0618] In another preferred embodiment of the sixth aspect, the antagonist 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, the antagonist antigen binding protein that specifically binds to LAG-3 binds to LAG-3 with a Kd of 5 pM to 100 nM, 5 pM to 50 nM, 5 pM to 10 nM, 5 pM to 1 nM, 5 pM to 100 pM, e.g., 5 pM to 10 pM.

[0619] In another preferred embodiment of the sixth aspect, the antagonist antigen binding protein that specifically binds to LAG-3 can block the binding of the ligand to LAG-3 with an IC50 of less than 10 nM, less than 1 nM or less than 200 pM. For example, the antagonist antigen binding protein that specifically binds to LAG-3 can block the binding of the ligand to LAG-3 with an IC50 of 200 pM to 10 nM, such as 200 pM to 1 nM.

[0620] In another preferred embodiment of the sixth aspect, the antagonist antigen binding protein that specifically binds to LAG-3 stimulates T cell proliferation.

[0621] In another preferred embodiment of the sixth aspect, the antagonist antigen binding protein that specifically binds to LAG-3 induces lymphocyte proliferation.

[0622] In another preferred embodiment of the sixth aspect, the antagonist antigen binding protein that specifically binds to LAG-3 induces the secretion of IL-2 and / or IFNγ.

[0623] In another preferred embodiment of the sixth aspect, the antagonist antigen binding protein that specifically binds LAG-3 reduces tumor volume by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a baseline value.

[0624] In a seventh aspect, the present invention relates to a nucleic acid encoding the antagonist antigen binding protein of any one of aspects 1 to 6 of the present invention.

[0625] In an eighth aspect, the present invention relates to a recombinant expression vector comprising the nucleic acid molecule of the seventh aspect.

[0626] In a ninth aspect, the present invention relates to a host cell comprising the vector of the eighth aspect of the present invention.

[0627] In a tenth aspect, the present invention relates to a method for preparing the antagonist antigen binding protein of any one of aspects 1 to 6 of the present invention, comprising the step of preparing the antigen binding protein from a host cell expressing the antigen binding protein.

[0628] In an eleventh aspect, the present invention relates to an antagonist antigen binding protein prepared by expressing a recombinant DNA in a host cell of the ninth aspect of the present invention.

[0629] In the twelfth aspect, the present invention relates to a pharmaceutical composition comprising at least one antagonist antigen binding protein according to any one of aspects 1 to 6 of the present invention, a nucleic acid according to the seventh aspect of the present invention, or a vector according to the eighth aspect of the present invention and a pharmaceutically acceptable carrier.

[0630] In a preferred embodiment of the twelfth aspect, the pharmaceutical composition is suitable for parenteral administration. Pharmaceutical compositions suitable for parenteral administration include aqueous sterile injectable solutions or suspensions and non-aqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, antibacterial agents and solutes, so that the composition is substantially isotonic with the blood of the intended recipient. Other components that may be present in such compositions include, for example, water, alcohol, polyols, glycerol and vegetable oils. Compositions suitable for parenteral administration may be present in unit dose containers or multidose containers, such as sealed ampoules and vials, and may be stored in freeze drying (lyophilization) conditions of sterile saline solutions for injection, such as only needing to be added before immediate use. Temporary injection solutions and suspensions may be prepared by sterile powders, granules and tablets.

[0631] In a preferred embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to a conventional method. Usually, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Usually, the composition is provided separately or mixed together in a unit dose form, such as in a sealed container such as an ampoule or a medicine bag indicating the amount of the active agent as a dry lyophilized powder or anhydrous concentrate. When the composition is administered by infusion, it can be distributed with 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 composition can be mixed before administration.

[0632] In a preferred embodiment of the twelfth aspect of the present invention, the pharmaceutical composition comprises at least one other active agent. Preferably, at least one other active agent is selected from another antagonistic antigen binding protein according to any one of aspects 1 to 6 of the present invention, a checkpoint inhibitor, a chemotherapeutic drug, a radiotherapeutic drug, an anti-tumor drug, an anti-angiogenic agent, a cancer vaccine, and an oncolytic virus. For example, at least one other active agent is selected from carboplatin-paclitaxel, trastuzumab, pertuzumab, erlotinib, lapatinib, imatinib, vemurafenib, dabrafenib, trametinib, bevacizumab, sunitinib, and pazopanib.

[0633] In the thirteenth aspect, the present invention relates to a kit comprising a pharmaceutical composition according to the twelfth aspect of the present invention and optionally at least one other active agent. Preferably, at least one other active agent is selected from another antagonistic antigen binding protein according to any one of aspects 1 to 6 of the present invention, a checkpoint inhibitor, a chemotherapeutic drug, a radiotherapeutic drug, an anti-tumor drug, an anti-angiogenic agent, a cancer vaccine, and an oncolytic virus. For example, at least one other active agent is selected from carboplatin-paclitaxel, trastuzumab, pertuzumab, erlotinib, lapatinib, imatinib, vemurafenib, dabrafenib, trametinib, bevacizumab, sunitinib, and pazopanib.

[0634] In a fourteenth aspect, the present invention relates to an antagonist antigen-binding protein according to any one of aspects 1 to 6 of the present invention, the nucleic acid according to the seventh aspect of the present invention, the vector according to the eighth aspect of the present invention, or the pharmaceutical composition according to the twelfth aspect of the present invention, for use in treating cancer and / or chronic infectious diseases.

[0635] In a preferred embodiment of the fourteenth aspect of the present invention, the cancer is selected from

[0636] (a) malignant neoplasm of the lips, oral cavity and pharynx; and / or

[0637] (b) malignant tumor of digestive organs; and / or

[0638] (c) malignant tumours of the respiratory and intrathoracic organs; and / or

[0639] (d) malignant tumors of bone and articular cartilage; and / or

[0640] (e) melanoma and other malignant tumors of the skin; and / or

[0641] (f) malignant tumors of mesothelial and soft tissues; and / or

[0642] (g) malignant neoplasm of the breast; and / or

[0643] (h) malignant tumor of the female reproductive organs; and / or

[0644] (i) malignant tumor of male reproductive organs; and / or

[0645] (j) urinary tract malignancy; and / or

[0646] (k) malignant tumors of the eye, brain or other parts of the central nervous system; and / or

[0647] (l) malignant tumors of the thyroid gland and other endocrine glands; and / or

[0648] (m) Malignant tumors of lymphatic, hematopoietic and related tissues.

[0649] Preferably, the cancer is selected from 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.

[0650] In another preferred embodiment of the fourteenth aspect of the present invention, the chronic infectious disease is selected from HBV, HCV, HIV, HSV, HPV, EBV, CMV and Chlamydia. Preferably, the chronic infectious disease is caused by HBV, HCV, HIV and HPV.

[0651] In another preferred embodiment of the fourteenth aspect of the present invention, at least one other active agent is administered to a subject in need thereof. Preferably, at least one other active agent of any one of aspects 1 to 6 of the present invention and the antagonist binding protein, the nucleic acid of the seventh aspect of the present invention, the vector of the eighth aspect of the present invention, or the pharmaceutical composition of the twelfth aspect of the present invention are administered simultaneously or sequentially or in combination.

[0652] The following examples are intended to illustrate the present invention only and should not be construed in any way to limit the scope of the present invention as indicated by the appended claims.

[0653] Example

[0654] Materials and methods

[0655] Antibodies and human recombinant proteins

[0656] 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). 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 Purple 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; human IgG1-Fc (all from R&D Systems).

[0657] Cell culture

[0658] In Dulbecco's modified Eagle's medium (Gibco TMMDA-MB-231 cells were cultured in DMEM (Thermo Fisher Scientific). TM MCF7 cells were cultured in MEM (Thermo Fisher Scientific). The culture medium was supplemented with 10% (vol / vol) heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, MO, USA), 50 IU ml -1 Penicillin, 50 µg ml -1 Streptomycin, 2 nM L-glutamine (both from Gibco TM , ThermoFisher The cell line was purchased from the American Type Culture Collection (ATCC) and was maintained at 37°C with 5% CO 2 Cultivated in a humid environment.

[0659] Isolation of human peripheral blood mononuclear cells (hPBMC)

[0660] Human peripheral blood mononuclear cells (hPBMCs) were isolated from the blood of healthy donors using the 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. TM Cellular Technology Limited, Cleveland, OH, USA) and 100 U / ml Benzonase (Merck Millipore, Billerica, MA, USA) in RPMI 1640 medium (Gibco TM , Thermo Fisher Scientific). The collected hPBMCs were then washed by centrifugation, plated, and incubated overnight at 37°C in R10 medium 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 (both from Gibco TM , RPMI1640 (Gibco TMAfter overnight standing, hPBMCs were collected into PBS and counted using a Muse® cell analyzer (Merck Millipore) at a rate of 1×10 6 Resuspend at a density of 10 cells / ml.

[0661] FACS analysis of expression levels of immune checkpoints on hPBMCs

[0662] The rescued and counted human peripheral blood mononuclear cells (hPBMC) were treated with 1·10 3 Dynabeads® Human T-Activator CD3 / CD28 (Gibco TM , Thermo Fisher Scientific). After 24 to 96 hours of activation, cells were seeded into round-bottom 96-well plates (1x10 6 Cells / well) were added and then centrifuged at 1200 rpm for 5 minutes to remove the supernatant. Unlabeled anti-LAG-3, anti-PD-1 (nivolumab), or anti-PD-L1 primary antibodies were added to each well at a concentration of 10 μg / ml and incubated at room temperature for 90 minutes with gentle shaking. After extensive washing, the cells were stained with 100 μl APC / anti-human IgG Fc antibody and 10 μg / ml PE / anti-human CD2 antibody (BioLegend) in FACS buffer (PBS, 1% FBS) at room temperature for 45 minutes with gentle shaking. The 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 Purple 510 TM Anti-mouse / rat / human CD27, APC / anti-human / mouse / rat CD278 (ICOS), FITC / anti-human TIGIT antibodies were added to each well at a concentration of 10 μg / ml and incubated with gentle shaking at room temperature for 90 minutes. After washing twice with FACS buffer, the cells were resuspended in PBS, transferred to a 5 ml polystyrene round-bottom tube (BD Biosciences), and analyzed on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA).

[0663] Flow cytometric binding assay of mAbs to hPBMCs

[0664] Human peripheral blood mononuclear cells (hPBMCs) isolated from healthy donors were thawed in RPMI 1640 medium (Gibco) supplemented with 2 mM L-glutamine, 10% (vol / vol) CTL wash supplement (CTL, Shaker Heights, Ohio, USA), 100 U / ml Benzonase (Merck Millipore, Burlington, Massachusetts, United States). After centrifugation at 1200 rpm for 10 minutes, they were resuspended in complete RPMI medium supplemented with 10% (vol / vol) heat-inactivated fetal bovine serum (FBS, Sigma), 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin (Gibco), HEPES 10 mM (Gibco). After 16 hours of resting at 37°C in a humidified atmosphere with 5% CO2, hPBMCs were counted, resuspended in complete RPMI medium (as described above) at a concentration of 10^6 viable cells / ml, and activated with Dynabeads Human T-Activator CD3 / CD28 (Gibco) at a concentration of 25 μl beads / 10^6 viable cells. 24 hours after activation, cells were centrifuged and plated at 4×10 5 10 cells / well were seeded in PBS in a 96-well plate with a round bottom. After washing once with PBS, they were mixed with 50 μl of LIVE / DEAD TM Fixable dead cell dye (Invitrogen) was incubated at +4°C for 30 minutes and then washed again. mAbs diluted in 100 μl PBS at 48 pM and 9.6 pM concentrations were added to the cells and incubated for 1 hour 30 minutes at room temperature with gentle shaking in the dark. As before, the cells were washed twice and then incubated with 5 μl PE-conjugated anti-human CD2 (BD Biosciences) and 1:2000 APC-conjugated anti-human IgG, Fcγ specific antibody (Jackson ImmunoResearch), diluted in 100 μl FACS buffer (PBS1X 1% (vol / vol) FBS). After incubation for 45 minutes under the same conditions as before, two washes were performed with FACS buffer and the cells were resuspended in 150 μl PBS1X for acquisition on a CytoFLEX flow cytometer (Beckman Coulter).

[0665] Selection of scFv phage clones

[0666] Phagemid particles were recovered from the library by using M13-K07 helper phage (Invitrogen, Thermo Fisher Scientific) as described previously (De Lorenzo C, Clinical Cancer Research, 2002). For each round of selection, phages were blocked with 5% (weight / volume) skim milk powder (Fluka Analytical, Sigma-Aldrich) in PBS (10 13 For the first round of selection, blocked phage were mixed with activated lymphocytes (1x10 6 ) were incubated with rotation at 4°C overnight for one round of selection. After extensive washing with PBS, bound phages were eluted from 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 phages were amplified by infecting E. coli TG1 cells to prepare phages for the next round of selection for purification of chimeric proteins. To this end, 0.05 M NaHCO 3 The selected recombinant chimeric protein was coated on Nunc TM Polypropylene tubes (Fisher Scientific, Thermo Fisher Scientific) were placed at 4°C for 72 hours. The blocked phages were incubated in tubes coated with rhIgG1-Fc protein at 4°C for 2 hours with rotation, followed by two rounds of negative selection. The unbound phages recovered in the supernatant were then incubated in coated tubes prepared as described above for positive selection, rotated at 4°C overnight, and eluted as described above. Alternatively, trypsin was used for elution. Briefly, after extensive washing with PBS, the bound phages were incubated with 1 mM CaCl 2 The chimeric protein was incubated with 50 mM Tris⋅HCl (pH 8.0) buffer at 4°C for 1 hour with rotation, and then eluted with trypsin (1 μg / ml). The chimeric protein was incubated at 25°C for another 15 minutes with gentle shaking. The protein was then eluted with protease inhibitors (cOmplete TM The reaction was blocked with EDTA-free protease inhibitor cocktail, Sigma-Aldrich). Phages were then collected and stored at 4°C until use.

[0667] DNA fragment preparation and high-throughput sequencing

[0668] For each sublibrary, the phagemid double-stranded DNA containing scFv was purified from the culture of repeatedly infected Escherichia coli TG1 cells using Endo free Plasmid Maxi Kit (Qiagen). Full-length scFv was cut with restriction enzymes BamHI and HindIII (New England Biolabs) and purified from 1.2% agarose gel with Wizard® SV Gel and PCR Clean-Up System (Promega). A second restriction digest was performed with NcoI and XhoI (New England Biolabs), and VH was separated from the previously purified material and then extracted from 1.4% agarose gel. Library preparation, sequencing and preliminary bioinformatics analysis of NGS data were performed at the Center for Translational Genomics and Bioinformatics of San Raffaele Hospital in Milan, Italy. VH extracted from the sublibrary was barcoded by TruSeq ChIP Sample Preparation Kit (Illumina). A barcode supplementation scheme was implemented to obtain deep and appropriate sequencing of VH mixtures of multiple sub-cycles. Subcycles 2 and 3 for each target were mixed together in a dedicated run. The first universal cycle 1 was sequenced separately to cover greater complexity. Barcoded samples were diluted to a final concentration of 10 pM and sequenced on an Illumina MiSeq instrument with the 2 × 300 nt SBS kit v3.

[0669] scFv recovery

[0670] Target clones were isolated from the sublibraries at cycle 3 of the corresponding target. For advanced clones, clones were replicated using the QuickChange II XL site-directed mutagenesis kit (Agilent Technologies) according to step 26 described previously, using overlapping primers designed in the corresponding HCDR3 region. Briefly, the extension reaction was performed as follows: 50 ng to 250 ng template; 2 / 5 μL QuickSolution reagent; 1 μL Pfu ultra-high fidelity DNA polymerase (2.5 U / μL); 5 μL 10x reaction buffer; 1 μL dNTPmix; 125 ng forward primer; 125 ng reverse primer; add H2O to a final volume of 50 μL. Template DNA was removed by restriction with 1 μL DpnI enzyme as recommended by the kit provider. An appropriate amount of the reaction was used to transform XL10-GOLD supercompetent cells (Agilent Technologies), which were then plated on LB / agar containing 100 μg / ml ampicillin. Some 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 in the corresponding HCDR3 region and in the upstream and downstream plasmid constant regions of VH and VL, respectively, using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). In the second step, the PCR fragments corresponding to each clone were mixed and extended to obtain the complete scFv. The reaction was performed as follows: 150 ng of template for VH and VL fragment amplification and 10 ng of template for complete scFv amplification (VH and VL fragments); 0.5 μL Phusion DNA polymerase (0.02 U / μl); 10 μL 5x Phusion HF buffer; 1 μL dNTP mixture; 0.5 μM forward primer; 0.5 μM reverse primer; 1.5 μL DMSO; add H 2 O to a final volume of 50 μl.

[0671] Antibody preparation and purification

[0672] The target scFv was converted into a complete IgG4 antibody by cloning the corresponding VH and VL cDNA in the pEU vectors 8.2VH and 4.2VL, and the constant antibody heavy and light chains were expressed respectively (Paciello R, J Gen Virol., 2016). In brief, VH and VL were amplified using specific primers under standard conditions by CloneAmp HiFi PCR premix and purified from 1.3% agarose gel using Wizard SV® Gel and PCR Clean-Up System (Promega). In-FusionHD cloning kit (Clontech Laboratories, Mountain View, CA, USA) was used to clone VH in pEU8.2VH vector linearized by BamHI and BssHII (New England Biolabs) and VL in pEU4.2VL vector linearized by ApaLI and AvrII (New England Biolabs). The obtained vector was used to transform the Stellar Competent Cell (Stellar Competent Cell, Clontech Laboratories, Inc, Mountain View, CA, USA), and the colonies were screened by digestion and sequence analysis. The correct preparation was co-transfected in HEK293-EBNA using Lipofectamine Transfection Reagent (Life Technologies, Inc.) and grown for about 10 days at 37°C in chemically defined CD CHO medium (Gibco, Life Technologies, Inc.), which was supplemented with 5ml 200mM L-glutamine (Gibco, Life Technologies), 5ml 10000 U / mL to 10 mg / mL penicillin-streptomycin (Sigma-Aldirch) in 6-well plates or 150mm Corning® tissue culture treated culture dishes. The conditioned medium was collected and the antibodies were purified by using Protein A HP SpinTrap (GE Healthcare Life Sciences, New York, USA). By SDS-PAGE NuPAGE TM The purity of the final product was assessed by 4% to 12% Bis-Tris protein gel, 1.0 mm, 10 well (Thermo Fisher Scientific), which was then stained with Coomassie blue solution (Biorad) for 20 min and then washed with 7% CH 3 Decolorization was performed with COOH and 20% ethanol.

[0673] Enzyme-linked immunosorbent assay (ELISA)

[0674] To confirm the binding specificity of the purified mAb, ELISA analysis was performed on 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 untreated or activated hPBMCs. 3 5 μg / ml rhPD-1, rhPD-1 and rhLAG-3 recombinant proteins in solution coated Nunc TM Flat-bottomed 96-well plates (Fisher Scientific, Thermo Fisher Scientific) were used for ELISA analysis of the coated chimeric proteins for 72 hours. After blocking the coated 96-well plates with 5% skim milk powder in PBS at 37°C for 1 hour, the purified mAbs were added to the plates in 2.5% skim milk powder in PBS at increasing concentrations (10 nM to 200 nM). Incubation was performed for 2 hours with gentle shaking at room temperature. After washing thoroughly with PBS, the plates were incubated with (HRP)-conjugated anti-human IgG (Fab') 2 goat monoclonal antibodies (Abcam, Cambridge, UK) for 1 hour, washed again and incubated with TMB reagent for 10 minutes, and then quenched with an equal volume of 1N HCl.

[0675] The cells were seeded in round-bottom 96-well plates (2 × 10 5 cells or 2×10 5 Lymphocytes) and incubated with increasing concentrations (0.5 nm to 200 nm) of mAb in 2.5% skim milk powder for 2 hours at room temperature under gentle stirring for cell ELISA assay. The plate was then centrifuged, the cell pellet washed with PBS, and incubated with anti-human IgG goat polyclonal antibody conjugated with HRP for 1 hour at room temperature. After additional washing in PBS 1X, TMB reagent was added for 10 minutes and then quenched with an equal volume of 1N HCl. The absorbance at 450 nm was measured with an Envision plate reader (Perkin Elmer, Waltham, MA, USA).

[0676] Competitive ELISA assay

[0677] To determine whether the novel anti-PD-1 mAbs recognize different epitopes from nivolumab, a competitive ELISA assay was performed with coated PD-1 / Fc chimeric proteins (5 µg / ml). After blocking with 5% skim milk powder in PBS, the coated 96-well plates were incubated with saturating concentrations of each unlabeled mAb (400 nM) in 2.5% skim milk powder in PBS at room temperature for 2 hours with stirring. After washing with extensive PBS, increasing concentrations of biotinylated nivolumab mAb were added. To detect binding, the plates were incubated with HRP-conjugated streptavidin (Biorad) at room temperature for 30 minutes with stirring, washed again, and analyzed as described above.

[0678] Analysis of antibody binding to hPBMCs by FACS

[0679] Human peripheral blood mononuclear cells (hPBMCs) were rescued and counted the day after thawing and then cultured with Dynabeads® Human T Activator CD3 / CD28 at 1x10 3 Beads / ml concentration (Gibco TM Thermo Fisher Scientific) for activation. After 24 to 96 hours of activation, cells were seeded into round-bottom 96-well plates (4x10 5 Cells / well) were added and then centrifuged at 1200 rpm / min for 5 minutes to remove the supernatant. Anti-PD-L1 monoclonal antibodies PD-L1_A and PD-L1_C were added at a concentration of 10 μg / ml and incubated with gentle shaking at room temperature for 90 minutes. After thorough washing, the cells were stained with 100 μl APC / anti-human IgG Fc antibody and 10 μg / ml PE / anti-human CD2 antibody (BioLegend) in FACS buffer (PBS, 1% FBS) at room temperature for 45 minutes. After washing twice with FACS buffer, the cells were resuspended in PBS, transferred to a polystyrene round-bottom tube (BD Biosciences), and analyzed on a CytoFLEX flow cytometer (Beckman Coulter).

[0680] Lymphocyte proliferation assay

[0681] Lymphocyte proliferation assay was performed by using hPBMCs, which were counted as described above and expressed as 2×10 6 The cells were resuspended in pre-warmed 0.1% BSA / PBS solution at a density of 10 cells / ml. For staining, a pre-warmed 0.1% BSA / PBS solution containing 10 μM CFDA-SE (Vybrant® Cell Tracer Kit, Invitrogen TM, Thermo Fisher Scientific) was used to measure 1x10 6 hPBMCs were resuspended at a density of 10 cells / ml and incubated at 37°C for 10 minutes. The cells were then permeabilized using ice-cold R10 medium by incubating on ice for another 5 minutes. The cells were then washed 3 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, the cells were centrifuged at 1 × 10 6 The cells were resuspended in R10 at a density of 1x10 cells / ml and seeded into 48-well plates (1x10 6 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 the selected anti-LAG-3 mAb (10 µg / ml). Then, the plates were incubated at 37°C for 5 days. At the end of the treatment, each sample was recovered, resuspended in 100 µl PBS, and transferred to a round-bottom 96-well plate. First, the cells were incubated with purple LIVE / DEAD solution (Thermo Fisher Scientific) at 4°C for 30 minutes. After several washes, the samples were incubated with anti-human CD3 (APC), anti-CD4 (PE), and anti-CD8 (PerCP) antibodies (10 μl / sample) for 1 hour at 4°C. Finally, cells were washed extensively, resuspended in 200 µl PBS, and transferred to 5 ml polystyrene round-bottom tubes (BD Biosciences) for cell counting on a CytoFLEX flow cytometer (BeckmanCoulter).

[0682] Example 1: Selection of scFv on activated human T lymphocytes

[0683] Our goal was to generate a human antibody library against immune checkpoints (ICs). Since many ICs are expressed on the surface of T lymphocytes and their expression increases when T cells are exposed to antigen-dependent or antigen-independent stimulation, we sought to screen a human single-chain antibody fragment library (scFvs) using unfractionated human peripheral blood mononuclear cells (hPBMCs). To optimize the selection process, we first evaluated the kinetics and expression levels of ten different ICs (shown in Table 1) after in vitro activation with anti-CD3 / CD28 beads. As shown in Table 1, the peak expression measured by flow cytometric analysis varied between different immunomodulators, however, most stimulators reached maximum display levels 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 that BTLA maintained the same surface display level between untreated hPBMCs and activated hPBMCs, and TIGIT only slightly increased its expression level. Based on this analysis, we selected 96h as the time point for activated lymphocytes for the selection of human scFv libraries.

[0684] Table 1 - Percent expression of each target on untreated or activated human lymphocytes at different time intervals.

[0685]

[0686] As described in the Experimental Procedure section (Selection Cycle 1), approximately one million phage particles were selected by panning the library on activated hPBMCs. This phage library may represent a large collection of many different ICs and is hereafter referred to as the "immune library". To facilitate the determination of binders to a specific IC, we used Fc-fused recombinant proteins in sequential panning to perform ten different parallel selections of the immune panel library to generate IC-specific libraries.

[0687] Example 2: Determination of scFv binders by next generation sequencing

[0688] To identify individual phage clones selected by the combined in vitro / in vitro approach, we sequenced the VH regions of the IC-specific libraries by massively parallel sequencing on the MiSeq Illumina platform (see Materials and Methods for details). To ensure efficient enrichment of target-specific phages, we performed two subsequent selection cycles (cycles 2 and 3) for Fc-fusion recombinant proteins, starting from the immunization group library (cycle 1). Sequence analysis of the entire selection sequence showed that enrichment had indeed occurred after cycle 2, but that 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, which could be due to the enrichment of Fc binders common to the 10 biochemical baits. Similarly, nonspecific bioenriched clones with stop codons within the scFv coding sequence were removed from the list of potential binders. To obtain the most specific clones related to each target, we ultimately set the threshold of the lists of different selection cycles 3 to 85 per million. In this way, we captured the details of the best potential binders for 9 out of 10 targets, as shown in Figure 2. Figure 1 The TIGIT selection was indeed removed from the analysis as it did not show significant sequence enrichment, likely due to its low expression on activated hPBMCs (see Table 1).

[0689] Binders with specificity for three targets, LAG-3, PD-1, and PD-L1, were selected for further study because antibodies specific for them, such as anti-PD-1 nivolumab, have been previously developed and widely used in the clinic by demonstrating therapeutic benefit, so they can be used for comparison in bioassays. We rescued the best scFvs from the LAG-3, PD-1, and PD-L1 IC-specific libraries for conversion to fully human IgG4s with the goal of further characterizing at least five potent antibodies against each of the three targets.

[0690] Example 3: Human IgG produced by selected binders has high binding affinity and receptor / ligand competitive activity

[0691] Human IgG4 antibodies generated from optimal LAG-3, PD-1, and PD-L1 binders have been shown to recognize activated PBMC proteins and recombinant proteins with low nanomolar or subnanomolar affinities ( Figure 2 and the tables therein). In particular, some anti-PD-1 mAbs (i.e., PD-1_A and PD-1_B) exhibited comparable or better apparent affinity than the clinically validated nivolumab. Most anti-PD-1 and anti-PD-L1 antibodies, including nivolumab, showed stronger binding to the recombinant proteins compared to activated hPBMCs, while the opposite was true for anti-LAG3 antibodies. Since anti-PD-L1 antibodies have been shown to provide clinical benefits by blocking the interaction between PD-L1 expressed on cancer cells and PD-1 displayed by T cells, we tested whether anti-PD-L1 antibodies can also recognize their targets on the surface of cancer cells. All anti-PD-L1 antibodies also showed high affinity for tumor cells expressing PD-L1, such as breast MDA-MB-231 tumor cells, and the grading of binding activity to tumor cells was different from that observed with activated hPBMCs ( Figure 2In addition, we showed that mAbs PD-L1_A and PD-L1_B were able to interfere with the recognition of PD-L1 by its two receptors PD-1 and B7.1 in a competition ELISA assay.

[0692] In addition, the selected antibodies were tested for binding to the murine ortholog. In contrast to nivolumab, one anti-PD-1 antibody was found to cross-react with murine PD-L1. Therefore, PD-1_A might recognize a different epitope than nivolumab, which was also confirmed from competition ELISA analysis performed by measuring the binding of biotinylated nivolumab to PD-1 in the absence or presence of saturating concentrations of unlabeled PD-1_A mAb. The simultaneous binding of PD-1_A and nivolumab suggests that the two mAbs do not interfere with their interaction with the receptor.

[0693] Example 4: High-affinity IgG against immune checkpoint molecules has T cell immunostimulatory activity and effector function

[0694] Previous reports have shown that the use of Staphylococcal enterotoxin B (SEB) or phytohemagglutinin (PHA) can induce proliferation of primary resting cells of CD3-positive in unfractionated hPBMCs in vitro and that this activity is regulated by CI (Maçon-Lemaître L, Immunology, 2005; Wang C, Cancer Immunol Res., 2014; Selby MJ, PlosOne, 2016). Therefore, we used the above lymphocyte proliferation assay to test whether the selected antibodies against LAG-3, PD-1, or PD-L1 were able to 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 resulted in a 50% increase in proliferation activity. The anti-PD-1 antibodies PD-1_A and PD-1_B were also able to effectively stimulate T cell proliferation, with the former showing higher activity than the clinically activated nivolumab ( Figure 3 ). Likewise, all three antibodies against PD-L1 and one of the three LAG-3 antibodies also induced proliferation to varying degrees. The ability to stimulate proliferation did not always correlate with binding ability, suggesting that different antibodies may have different ways of interacting with their targets.

[0695] Interestingly, the antibodies PD-1_A and PD-L1_A, which were the best activating antibodies on human lymphocytes, also demonstrated this ability on mouse lymphocytes, thus indicating that they cross-react 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 inhibit the inhibitory effect of PD-1 / PD-L1 interaction on lymphocyte proliferation when both cell types were co-cultured. Figure 4 As shown, mAbs PD-L1_A and PD-1_A induced lymphocyte proliferation in a dose-dependent manner, while only weak effects were detected on lymphocytes treated with mAbs PD-L1_B and PD-1_B. Also in this assay, mAbs PD-L1_A and PD-1_A showed higher activity than nivolumab, while 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, the ability to stimulate proliferation did not correlate with the highest apparent affinity ( Figure 2 and Figure 2 of the table).

[0696] Three additional anti-PD-L1 antibodies were identified and characterized relative to PD-L1_A. The three antibodies, PD-L1_C, PD-L1_D and PD-L1_E, were evaluated for binding to human PBMCs that had been previously activated to induce PD-L1 surface expression. A higher percentage of lymphocytes were stained by the three antibodies relative to PD-L1_A (see Figure 7 ).

[0697] Example 5: Effects of novel antibodies on cytokine production by stimulated hPBMCs

[0698] Herein, hPBMCs (1x10 6 Cells were stimulated with 2.5 μg / mL PHA-L or 50 ng / ml Staphylococcal enterotoxin B (SEB, Sigma-Aldrich) for 18, 42, and 66 hours. Nivolumab was tested as a positive control in parallel under the same conditions. The levels of IL-2 or IFNγ in the cell culture supernatant were measured by ELISA assay (DuoSet ELISA, R&D Systems) according to the manufacturer's recommendations.

[0699] Immunomodulatory antibodies such as nivolumab and ipilimumab have been shown to improve T cell effector function, and this property may increase their clinical benefit (Maçon-Lemaître L, Immunology, 2005; Wang C, Cancer Immunol Res., 2014; Selby MJ, Plos One, 2016). Therefore, we tested the five antibodies with the highest ability to induce T cell proliferation (LAG-3_A, PD-1_A, PD-1_B, PD-L1_A, and PD-L1_B) in the trial previously reported on the characterization of nivolumab (Wang C, Cancer Immunol Res., 2014). Figure 5 As shown, all tested antibodies were able to increase IL-2 and IFNγ secretion from hPBMCs stimulated with PHA or SEB. After adding different antibodies to the cell culture mixture, the secretion of cytokines increased over time. The ability of PD-1_1 mAb to stimulate the secretion of both cytokines appeared to be consistently more effective than all other tested antibodies. Also in this assay, the newly identified antibodies had an advantage over nivolumab, further supporting the conclusion that the immune repertoire we generated is rich in binders and has good potential for development for clinical use.

[0700] Two additional anti-PD-L1 antibodies (PD-L1_C and PD-L1_D) identified and characterized against PD-L1_A were also evaluated in the described cytokine secretion assay relative to nivolumab. Figure 8 As shown, all antibodies tested were able to increase IL-2 and IFNγ secretion from hPBMCs stimulated with PHA or SEB.

[0701] Example 6: In vivo antitumor activity

[0702] Six-week-old female BalBC mice (Envigo) were used for in vivo studies. Mice were implanted subcutaneously on the right flank with 2x10 5 Cells were added (day 0) and then 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 by calipers every 3 to 4 days using the formula LxW2 / 2 (L is the maximum diameter of the tumor and W is the minimum diameter of the tumor). Once signs of discomfort appeared or the tumor volume exceeded 2000mm 3 When, the animals were killed.

[0703] Given their cross-reactivity with mouse PD-1 and PD-L1, two antibodies, PD-1_A and PD-L1_A, were also tested in vivo on the CT26 colon cancer model. Mice were implanted with CT-26 cells (day 0) and then treated with PD-1_A and PD-L1 antibodies (day 3, day 6, day 10). Two commercially available antibodies (α-mPD-1 and α-mPD-L1) that react with mouse PD-1 and PD-L1 and have been previously validated in vivo (reference) were used as positive controls. The growth rate of tumors in untreated mice was very rapid and uncontrolled, with most tumors reaching >650mm on day 21. 3 The size of the tumors was reduced in mice treated with PD-1_A, but a reduction in tumor volume was found in mice treated with PD-1_A (p=0.03). The activity of the cross-reactive anti-PD-1 antibody was comparable to that of the commercially available anti-murine PD-1 antibody. A trend towards comparable activity was also observed for the two anti-PD-L1 antibodies ( Figure 6 ). In response to PD-1 and PD-L1 blockade, the treated mice consistently showed two different treatment outcomes dichotomously, responding mice and non-responding mice, as well described for this and other cancer models. Therefore, the biological and functional activities of these two novel mAbs were also confirmed in relevant in vivo models.

[0704] Experimental Summary

[0705] The inventors customized phage display technology to allow rapid determination of a large number of antibodies that recognize several different ICs. To this end, live activated hPBMCs were selected as the selected objects to generate an unbiased IC binder library, an immune group library, from which scFvs that specifically recognize a given receptor can be extracted by subsequent affinity selection cycles using recombinant proteins, peptides or other target-specific baits. The rationale for using hPBMCs as the starting point for the selection process is also to ensure that antibodies that can recognize natural receptors with correct post-translational modifications as shown in living human cells are selected. Consistent with this working hypothesis, human IgG4 generated using selected scFvs can recognize cell-displayed receptors with an apparent affinity as low as 0.1nM in the low nanomolar range. This binding affinity is equal to or better than the binding affinity shown for clinically active checkpoint-specific antibodies, and is better than the binding affinity of anti-PD-1 mAb nivolumab, which has been approved for the treatment of various types of cancer (Wang C, Cancer Immunol Res., 2014). To improve the selection efficiency of hPBMCs, we increased the expression level of target ICs by anti-CD3 cross-linking of T cells.

[0706] To facilitate the identification of binders recognizing specific receptors, we used ten recombinant proteins to further affinity select the immunogenomic library. To develop a general protocol for the selection of antibodies against cell surface displayed proteins, we performed two subsequent selection cycles on recombinant Fc fusion proteins. This strategy represents a compromise between selection efficiency and the generation of an IC-specific library with significant diversity, allowing the identification of antibodies that bind to different regions of the target IC and have different biological activities.

[0707] All selections achieved enrichment levels ranging from 100-fold to 1000-fold, with four (PD-1, 4-1BB, CD27, and OX40) reaching higher enrichment levels. Comparative sequence analysis of different selection cycles confirmed that in most selections (i.e., seven of nine), enrichment of clones after the second cycle was significantly improved (i.e., at least tenfold), while selections for LAG3 and OX40 did not appear to improve after the second cycle.

[0708] Our results support the conclusion that the ex vivo / in vitro combined selection method used in the context of the present invention is faster and more efficient than the combined selection on living cells and the method of determining specific binders by direct screening of the selected targets we previously adopted (Monaci P, Plos One, 2008). A total of sixteen IgGs (five anti-LAG3, five anti-PD-L1 and six anti-PD-1) with confirmed binding, eleven of which showed lower nanomolar apparent affinity to their cognate receptors expressed on hPBMCs, of which three (PD-L1_2, PD-1_1 and PD-1_2) had sub-nanomolar apparent affinity and had good affinity compared to the apparent affinity of nivolumab. Among them, at least 2 to 3 of the 5 converted antibodies for each target were highly specific to recombinant purified targets and activated lymphocytes, and had no obvious binding to untreated lymphocytes or Fc, so they were selected for further biological and functional analysis.

[0709] When 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) showed higher biological activity than nivolumab. In co-cultures with the tumor cell line MDA-MB-231, which highly expresses PD-L1, PD-1_A also showed significant activity in promoting T cell proliferation. In this assay, the PD-L1_A mAb almost tripled T cell proliferation and had the ability to interfere with the PD-L1 / PD-1 interaction. The most active antibodies do not always correspond to antibodies with stronger binding, for example, mAb LAG-3_A has a lower apparent affinity than mAb LAG-3_C in binding to hPBMCs, but it is able to stimulate T cell proliferation, while the latter cannot. These results also indirectly indicate that the selected antibodies recognize different epitopes on the target protein. We could show that some of the antibodies identified in this work can stimulate T cells to secrete IL2 and IFNγ. PD-1_1 mAbs consistently showed the highest activity in stimulating cytokine secretion and extended the observation that some of the antibodies identified in this work exhibited similar binding affinity and biological properties to the clinically validated nivolumab. In fact, some of the new antibodies showed even higher activity when lymphocytes were co-cultured with APCs or tumor cells in cell-based assays compared to nivolumab. These assays can better represent the in vivo situation because not only the negative effects of the interaction between the receptor and its ligand (i.e.: PD-1 / PD-L1) can be reproduced, but there are other potential biological effects that the new antibodies may have antagonistic effects. This could explain why the new anti-PD-1 and anti-PD-L1 antibodies have a stronger stimulatory effect on T cell proliferation and cytokine secretion than nivolumab, whose activity may mainly rely on the inhibition of the interaction between PD-1 and PD-L1. The interesting finding also supports the hypothesis that the new anti-PD-1 antibodies recognize different epitopes than nivolumab and can therefore act with different mechanisms of action. In fact, PD-1_A was able to cross-react with mouse lymphocytes in a similar manner to PD-L1_A, distinguishing PD-1_A from nivolumab. Due to this property, the two novel antibodies were also tested in mice bearing CT26 colon cancer and found to be effective in controlling tumor growth.

[0710] Overall, the data presented in this work support the conclusion that the selection procedure employed generates antibodies that specifically recognize the target receptor in its native conformation with high affinity without the need for further affinity maturation, resulting in antibodies that specifically bind checkpoint inhibitors with properties superior to those of prior art antibodies.

Claims

1. An antagonist antigen binding protein that specifically binds to PD-1, wherein the antigen binding protein comprises: a. 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, CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 6 and CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, A CDRH3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and CDRL3 comprising or consisting of the amino acid sequence of SEQ ID NO: 9; or b. 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, CDRH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and CDRL2 comprising or consisting of the amino acid sequence of SEQ ID NO: 16, 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.

2. The antagonist antigen binding protein according to claim 1, which specifically binds to PD-1, wherein the antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain, and the combination is selected from: (i) a light chain variable domain comprising a sequence that is at least 90% identical to SEQ ID NO: 3 and A heavy chain variable domain comprising a sequence that is at least 90% identical to SEQ ID NO: 2; or (ii) a light chain variable domain comprising a sequence that is at least 90% identical to SEQ ID NO: 12 and A heavy chain variable domain comprising a sequence at least 90% identical to SEQ ID NO:

11.

3. The antagonistic antigen binding protein according to claim 1 or 2, wherein (a) the antigen binding protein is an antibody or an antigen binding fragment thereof; and / or (b) The antigen binding protein has at least one of the following characteristics: (i) binds to PD-1 with a Kd of less than 100 nM; (ii) Able to produce an IC less than 10 nM 50 Blocking the binding of ligand to PD-1; (iii) stimulate T cell proliferation; (iv) inducing lymphocyte proliferation; (v) inducing the secretion of IL-2 and / or IFNγ; and / or (vi) Reduction of tumor volume by at least 50% compared to baseline values.

4. A nucleic acid encoding the antagonist antigen binding protein according to any one of claims 1 to 3. A recombinant expression vector comprising the nucleic acid according to claim 4 . A host cell comprising the vector according to claim 5 .

7. A method for preparing the antagonist antigen-binding protein according to any one of claims 1 to 3, comprising the step of preparing the antigen-binding protein from a host cell expressing the antigen-binding protein.

8. An antagonistic antigen binding protein prepared by expressing a recombinant DNA in the host cell of claim 6.

9. A pharmaceutical composition comprising at least one antagonist antigen binding protein 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.

10. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition comprises at least one other active agent. A kit comprising the pharmaceutical composition according to claim 9 or 10.

12. Use of the antagonist antigen binding protein according to any one of claims 1 to 3 or 8, the nucleic acid according to claim 4, the vector according to claim 5 or the pharmaceutical composition according to claim 9 or 10 in the preparation of a medicament for treating cancer, wherein: (i) the cancer is selected from: (a) malignant neoplasm of the lips, oral cavity and pharynx; and / or (b) malignant tumor of digestive organs; and / or (c) malignant tumours of the respiratory and intrathoracic organs; and / or (d) malignant tumors of bone and articular cartilage; and / or (e) melanoma and other malignant tumors of the skin; and / or (f) malignant tumors of mesothelial and soft tissues; and / or (g) malignant neoplasm of the breast; and / or (h) malignant tumor of the female reproductive organs; and / or (i) malignant tumor of male reproductive organs; and / or (j) urinary tract malignancy; and / or (k) malignant tumors of the eye, brain or other parts of the central nervous system; and / or (l) malignant tumors of the thyroid and other endocrine glands; and / or (ii) administering at least one other active agent to a subject in need thereof, wherein the at least one other active agent and the antagonist antigen binding protein according to any one of claims 1 to 3 or 8, the nucleic acid according to claim 4, the vector according to claim 5 or the pharmaceutical composition according to claim 9 or 10 are administered simultaneously or sequentially.

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