Antibodies to human programmed death receptor PD-1

Abstract

To provide PD-1 / PD-L1 pathway based disease treatment with antibodies that can effectively bind human PD-1 and block its binding with PD-L1 or PD-L2 to improve upon some of the challenges of existing therapies.SOLUTION: The present invention relates to an antibody or antigen-binding portion thereof that can bind to human programmed death 1 (PD-1). The antibody according to the present invention is further used in the preparation of a drug for treating disease in which expression of PD-1 is detrimental.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an antibody, or an antigen-binding portion thereof, capable of binding to human programmed cell death protein 1 (PD-1). According to the invention, the antibody is further used in the preparation of a medicament for treating a disease in which expression of PD-1 is detrimental. [Background technology]

[0002] PD-1 (CD279) is a 288 amino acid protein inhibitory receptor expressed on activated T and B cells, natural killer cells, and monocytes. PD-1 is a member of the CD28 / CTLA-4 (cytotoxic T lymphocyte antigen) / ICOS (inducible costimulator) family of T cell co-inhibitory receptors. The PD-1 receptor has two ligands, the protein death ligand 1 (PD-L1) and the protein death ligand 2 (PD-L2). PD-L1 (CD274, B7H1) is expressed broadly on both lymphoid and non-lymphoid tissues, including CD4 and CD8 T cells, macrophage lineage cells, peripheral tissues, as well as tumor cells, virus-infected cells, and autoimmune tissue cells. PD-L2 (CD273, B7-DC) is more restricted in expression than PD-L1 and is expressed on activated dendritic cells and macrophages (1). PD-L1 is expressed in the majority of human cancers, including melanoma, glioma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, leukemia, pancreatic cancer, renal cell carcinoma, and hepatocellular carcinoma, and can be induced in nearly all cancer types (2). Upon binding to its ligand, PD-1 reduces T-cell proliferation and cytokine secretion, suppressing humoral and cellular immune responses, exacerbating disease states that would otherwise be ameliorated by active immune responses. This immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when PD-1 interaction with PD-L2 is also blocked (3, 4).

[0003] Blockade of PD-1 with antagonists, including monoclonal antibodies, has been investigated in the treatment of cancer and chronic viral infections ( 5 ).

[0004] Monoclonal antibodies against PD-1 are known in the art and have been described, for example, in patent documents WO2006121168, WO2009114335, WO2008156712, WO2012145493, WO2015036394, WO2015112800, WO2016015685 and WO2018128939.

[0005] Three antibodies targeting human PD-1 are commercially available for the treatment of various cancers in combination with conventional drugs: nivolumab, pembrolizumab, and cemiplimab.

[0006] Despite the clinical success of anti-PD-1 antibodies, these therapeutic antibodies suffer from several drawbacks, including high cost, limited half-life, and immunogenicity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2006121168 [Patent Document 2] WO2009114335 [Patent Document 3] WO2008156712 [Patent Document 4] WO2012145493 [Patent Document 5] WO2015036394 [Patent Document 6] WO2015112800 [Patent Document 7] WO2016015685 [Patent Document 8] WO2018128939 [Patent Document 9] WO2007 / 017903 [Patent Document 10] WO2012 / 046255A2 Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there is a continuing need in the field of PD-1 / PD-L1 pathway-based disease treatment with antibodies that can effectively bind to human PD-1 and block its binding to PD-L1 or PD-L2, while also ameliorating some of the problems of existing therapies. The present invention discloses such novel antibodies. [Means for solving the problem]

[0009] The present invention provides novel anti-PD-1 antibodies having one or more improved characteristics compared to known anti-PD-1 antibodies used, for example, for therapy. The anti-PD-1 antibodies, or antigen-binding portions thereof, of the present invention bind to human PD-1 with high affinity. The amino acid sequence of the constant region of the anti-PD-1 antibody comprises that of an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, preferably IgG1 or IgG4. Furthermore, one or more of the anti-PD-1 antibodies of the present invention have altered, reduced, or absent ADCC and / or CDC activity. In some embodiments, the present invention provides anti-PD-1 antibodies with high ADCC and / or CDC activity, which may cause lysis of PD-1-expressing cells. The anti-PD-1 antibodies, or antigen-binding portions thereof, of the present invention have a 10 -10 M or less, more preferably 10 -11 M or less, and even more preferably, 10 -12 K against PD-1 antigen below M D K DThe value is a measure of the binding affinity of the antibody to its target antigen. The anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention can be used to treat diseases in which the interaction of PD-1 with PDL1 and / or PDL2 is involved in modifying the disease state, such as in infectious diseases and various cancers. In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention block the interaction of the PD-1 receptor with its natural ligands PD-L1 and / or PD-L2. In another embodiment of the invention, the anti-PD-1 antibodies, or antigen-binding portions thereof, kill T cells expressing PD-1. The invention also provides methods for preparing novel anti-PD-1 antibodies and pharmaceutical compositions comprising same. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows a general vector map of the pZRCIIhyg anti-PD-1 LC-HC vector construct used to prepare full-length monoclonal anti-PD-1 antibodies of the invention. [Diagram 2] FIG. 1 shows a general vector map of pSEX83 used for library generation to generate the anti-PD-1 Fab of the present invention. [Diagram 3] FIG. 1 shows a general vector map of the pZRCIIIhyg anti-PD-1 LC-HC vector construct used to prepare the anti-PD-1 Fab full-length monoclonal anti-PD-1 antibody of the invention. [Figure 4] Figure 1 shows the results of flow cytometry experiments demonstrating that the anti-PD-1 antibodies of the invention IP-H4.2L1, IP-H4L1(IgG1), and IP-H4L1(IgG4), directed against human PD-1, bind to PD-1 on the cell surface of human PBMCs. [Diagram 5] 1 is a graph showing the results of flow cytometry experiments using a HEK293T cell line demonstrating that the anti-PD-1 antibodies of the invention IP-H4.2L1, IP-H4.19L1, IP-H4.36L1, directed against human PD-1, bind to cell surface PD-1. [Figure 6]1 is a graph showing the results of flow cytometry experiments using a HEK293T cell line demonstrating that the anti-PD-1 antibodies of the invention N5NL, N6NL, N7NL, N9NL and N10NL, which are directed against human PD-1, bind to cell surface PD-1. [Figure 7] 1 is a graph showing the results of experiments demonstrating that the anti-PD-1 antibodies of the invention IP-H4.2L1, IP-H4.19L1, and IP-H4.36L1, which are directed against human PD-1, promote interleukin-2 (IL-2) secretion in a mixed lymphocyte reaction assay. [Figure 8] 1 is a graph depicting the results of experiments demonstrating that the anti-PD-1 antibodies of the invention IP-H4.2L1, IP-H4.19L1, and IP-H4.36L1, which are directed against human PD-1, promote IFN-gamma (IFN-γ) secretion in a mixed lymphocyte reaction assay. [Figure 9] 1 is a graph showing the results of experiments demonstrating that the anti-PD-1 antibodies of the invention N5NL, N6NL, N9NL and N10NL directed against human PD-1 promote IL-2 secretion in a mixed lymphocyte reaction assay. [Figure 10] 1 is a graph showing the results of experiments demonstrating that the anti-PD-1 antibodies of the invention N5NL, N6NL, N9NL and N10NL directed against human PD-1 promote IFN-γ secretion in a mixed lymphocyte reaction assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] definition The term "antibody" as referred to herein includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may be involved in binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., immune effector cells) and the first component (C1q) of the classical complement system.

[0012] The term "operably linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of controlling the transcription and translation of the antibody gene.

[0013] The terms "Ka" and Kd are well known to those of skill in the art, where "Ka" is the on-rate of a particular antibody-antigen interaction, while "Kd" is the off-rate of a particular antibody-antigen interaction. DThe term "affinity rate constant" is the ratio of Kd to Ka. It can be measured by using surface plasmon resonance techniques that are well known in the art. K D The "K" value is a measure of the binding affinity of an antibody for its target antigen. D The term " is also defined in WO2006121168, which is incorporated herein by reference.

[0014] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0015] The term "bispecific antibodies" refers to homogeneous antibody populations involved in the highly specific recognition and binding of two different antigenic determinants, or epitopes.

[0016] The term "recombinant antibody" according to the present invention includes monoclonal antibodies that are recombinantly produced using synthetic heavy and light chain genes. The recombinant antibodies of the present invention are monoclonal antibodies (mAbs) that are not produced using traditional hybridoma-based technology and do not require hybridomas or animals in the production process.

[0017] The term "immune effector function" as used herein refers to a biochemical event that occurs as a result of the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. This term also refers to physiological events such as the circulating half-life of a drug or the targeting of a drug to a specific cell or tissue type.

[0018] The term "ADCC" or "antibody-dependent cell-mediated cytotoxicity," as used herein, is a cellular reaction in which non-specific cytotoxic cells expressing FcγR recognize antibody bound to a target cell, and subsequently cause lysis of the target cell.

[0019] The term "ADCP" or "antibody-dependent cellular phagocytosis," as used herein, is a cellular reaction in which non-specific cytotoxic cells expressing FcγR recognize antibody bound to a target cell, and subsequently cause phagocytosis of the target cell.

[0020] The term "immune effector cells," as used herein, are cells that express one or more Fc receptors and mediate one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γδ T cells, and may be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys.

[0021] The term "Fc" fragment reflects its ability to crystallize readily. In human IgG molecules, the Fc region is generated by papain cleavage from the N-terminus to Cys226. The Fc region is central to the immune effector functions of the antibody.

[0022] The term "Fc protein," as used herein, refers to the portion of a single immunoglobulin heavy chain beginning at the hinge region just upstream of the papain cleavage site and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least a portion of the hinge (e.g., upper, middle, and / or lower hinge region), CH2, and CH3 domains.

[0023] The term "pharmaceutical formulation" refers to a preparation that is in such a form as to allow the biological activity of the active ingredient to be clearly effective. The terms "pharmaceutical formulation" or "pharmaceutical composition" or "composition" may be used interchangeably herein.

[0024] The term "excipient" refers to an agent that is added to a formulation to stabilize the active drug substance in the formulated form, and adjusts and maintains the osmolality and pH of the pharmaceutical preparation. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers. A "pharmaceutical acceptable" excipient is one that can be reasonably administered to a target mammal to provide an effective amount of the active ingredient utilized.

[0025] The term "treatment" or "therapy" as used herein refers to any treatment of a disease in a mammal, particularly a human, including (a) preventing the disease from occurring in a subject who may be susceptible to or at risk for the disease, but who has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) ameliorating the disease, i.e., causing the reversal of the disease.

[0026] The terms "patient" and "subject" are used interchangeably and in the conventional sense to refer to an organism suffering from or susceptible to a condition that can be prevented or treated by administering the compositions of the present invention, including both human and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees and other ape and monkey species; livestock animals, such as cows, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats and guinea pigs; domestic, wild and game birds, including chickens, turkeys and other pheasants, ducks, geese, etc. The term does not refer to a particular age. Thus, adult, juvenile and newborn individuals are subjects.

[0027] An "effective amount" of an antibody of the invention, or composition thereof, is that amount delivered to a mammalian subject in a single dose, or as part of a series, effective to induce an immune response to the target antigen in said subject. This amount will vary according to the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined by routine trials.

[0028] A "pharmaceutical effective dose" or "therapeutically effective dose" is the dose required to treat or prevent or alleviate one or more PD-1-associated disorders or symptoms, preferably, in the present invention, cancer or an infectious disease or an autoimmune disease, in a subject. A pharmaceutical effective dose depends, among other things, on the particular compound administered (which herein is an anti-PD-1 antibody or a combination or composite or bispecific thereof), the severity of the symptoms, the susceptibility of the subject to side effects, the type of disease, the composition used, the route of administration, the species of mammal being treated, the physical characteristics of the particular mammal under consideration, such as health and physical condition, co-administered drugs, the degree of protection desired, and other factors that one of ordinary skill in the medical arts would recognize.

[0029] The amino acid abbreviations used in this application are set out in the table below.

[0030] [Table 1]

[0031] Other abbreviations used in this application: ACT: Adoptive cell transfer ADCC: Antibody-dependent cytotoxicity ADCP: Antibody-dependent cell phagocytosis CDC: Complement-dependent cytotoxicity CDR: Complementarity determining region CFU: colony forming unit CH: constant region of the heavy chain cHL: classical Hodgkin lymphoma CL: constant region of the light chain dNTP: deoxyribonucleotide triphosphate ESCC: Esophageal squamous cell carcinoma FACS: Fluorescence-activated cell sorting HC: Heavy chain HCC: Hepatocellular carcinoma HCVR: Heavy chain variable region HEK: Human Embryonic Kidney hyg: hygromycin IFN-γ: IFN-gamma LC: light chain LCVR: Light chain variable region mAb: Monoclonal antibody MCC: Merkel cell carcinoma MLR: mixed lymphocyte reaction MOI: Multiplicity of infection NSCLC: Non-small cell lung cancer PBMC: Peripheral blood mononuclear cells PD-1 / PD 1: Programmed cell death receptor 1 PD-L1 / PD L1: Programmed cell death ligand 1 PD-L2 / PD L2: Programmed cell death ligand 2 Pfx: Proofreading DNA polymerase, Pfx™ from Invitrogen. PMBCL: Primary mediastinal large B-cell lymphoma sRCA: Selective rolling circle amplification RCC: Renal cell carcinoma rpm: revolutions per minute SCCHN: Squamous cell carcinoma of the head and neck SCLC: small cell lung cancer SEQ / seq: sequence SPR: Surface plasmon resonance VH: variable region of the heavy chain VL: variable region of the light chain

[0032] EMBODIMENTS OF THE PRESENTINVENTION The present disclosure relates to novel anti-PD-1 antibodies that can be used therapeutically.

[0033] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention bind to human PD-1 with high affinity.

[0034] In one embodiment, the amino acid sequence of the constant region of the anti-PD-1 antibody comprises that of an IgG1, IgG2, IgG3, IgG4, IgG2 / G4, IgA, IgE, IgM, or IgD constant region, preferably, IgG1 or IgG4.

[0035] In another embodiment, one or more of the anti-PD-1 antibodies of the present invention have altered, reduced, or absent ADCC and / or CDC activity. In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, has a reduced potential to cause ADCC and CDC safety issues. In another embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, has ADCC and / or CDC activity.

[0036] In another embodiment, one or more of the anti-PD-1 antibodies of the invention have ADCP activity.

[0037] In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention binds to the PD-1 antigen at a concentration of 10 -8 M or less, more preferably 10 -10 M or less, and even more preferably, 10 -11 M or less, and even more preferably, 10 -12 K below M D K D The value is a measure of the binding affinity of the antibody for its target antigen.

[0038] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention cross-react with PD-1 of a species other than human.

[0039] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention have higher binding specificity for human PD-1.

[0040] In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention has an extended half-life in a subject.

[0041] In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention blocks a function of PD-1.

[0042] In one embodiment, an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention interferes with the binding of PD-1 to a PD-L1-expressing target cell.

[0043] In one embodiment, an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention interferes with the binding of PD-1 to a PD-L2-expressing target cell.

[0044] In another embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention kill T cells that express PD-1.

[0045] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention have improved circulating half-life.

[0046] In another embodiment, the anti-PD-1 antibodies or antigen-binding portions of the invention are capable of binding to monkey PD-1, and can facilitate drug development by providing relevant animal pharmacology and toxicology models.

[0047] In one embodiment, the invention provides a nucleic acid sequence encoding an anti-PD-1 antibody, or an antigen-binding portion thereof, of the invention.

[0048] In one embodiment, the invention provides an expression vector comprising a nucleic acid sequence encoding an anti-PD-1 antibody, or an antigen-binding portion thereof, of the invention.

[0049] In one embodiment, the invention provides a host cell comprising an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding an anti-PD-1 antibody, or antigen-binding portion thereof, of the invention.

[0050] In one embodiment, the invention provides a composition comprising an anti-PD-1 antibody, or an antigen-binding portion thereof, that specifically binds to human PD-1, and an acceptable carrier.

[0051] In one embodiment, the invention provides an immunoconjugate comprising an anti-PD-1 antibody, or an antigen-binding portion thereof, linked to a therapeutic agent.

[0052] In one embodiment, the invention provides a bispecific molecule comprising an anti-PD-1 antibody, or an antigen-binding portion thereof, linked to a second functional moiety that has a binding specificity different from the antibody.

[0053] In one embodiment, the invention provides a combination comprising at least two or more antibodies, or antigen-binding portions thereof, wherein at least one antibody, or antigen-binding portion thereof, is an anti-PD-1 antibody, or antigen-binding portion thereof, of the invention.

[0054] In another embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention can be used to treat diseases in which PD-1 activity is detrimental, such as various cancers, various infectious diseases, etc. In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention can be used to treat autoimmune disorders.

[0055] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention can be used to treat a cancer selected from NSCLC, SCLC, RCC, cHL, SCCHN, urothelial carcinoma, colorectal cancer, ESCC, primary mediastinal large B-cell lymphoma, microsatellite instability-high cancer, gastric cancer, cervical cancer, Merkel cell carcinoma, endometrial cancer, and tumor mutational burden-high (TMB-H) cancer in which PD-1 activity is enhanced.

[0056] Detailed Description of the Invention In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention bind to human PD-1 with high affinity.

[0057] In one embodiment, the CDR1 of the heavy chain of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention (hereinafter referred to as CDRH1) has the general formula (I): 1a -A 1a -X 2a -A 2a wherein: X 1a is an amino acid selected from asparagine, glycine and threonine; A 1a is a dipeptide selected from Tyr-Tyr and Ile-Thr, X 2a is an amino acid selected from isoleucine, leucine, valine, phenylalanine, methionine and alanine, A 2a is a single amino acid or a dipeptide or a tripeptide or a tetrapeptide selected from tyrosine, asparagine, serine, glycine, Asn-Ser, Asn-Ser-Gly and Ser-Asn-Ser-Gly, with the proviso that X 1a Asparagine and X 2a Methionine is not present together with .

[0058] In a preferred embodiment, the CDRH1 of the heavy chain of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention has the structure represented by formula (Ia): 1a -YYX 2a -Y, wherein X 1a is an amino acid selected from asparagine and threonine; X 2a is an amino acid selected from isoleucine, leucine, valine, methionine and alanine, with the proviso that X 1a Asparagine and X 2a Methionine is not present together with .

[0059] In a further preferred embodiment, CDRH1 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is TYYIY.

[0060] In another preferred embodiment, CDRH1 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is GITFSNSG.

[0061] In one embodiment, the CDR2 of the heavy chain of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention (hereinafter referred to as CDRH2) has the general formula (II): A 1b -X 1b -A 2b -X 2b -X 3b -A 3b -X 4b -X 5b -X 6b -X 7b -X 8b -A 4b wherein: A 1b is optionally present and, if present, represents glycine; X 1b is an amino acid selected from methionine, isoleucine, leucine, glycine, valine, and alanine; A 2b is a tripeptide or tetrapeptide selected from Asn-Pro-Ser-Asn or Trp-Tyr-Asp, X 2b and X 3b each independently represents an amino acid selected from glycine and serine; A 3b is a single amino acid or dipeptide selected from lysine or Thr-Asn, X 4b is an amino acid selected from tyrosine, arginine and phenylalanine; X 5b is an amino acid selected from serine, tyrosine and asparagine; X 6b represents an amino acid selected from glutamic acid and glutamine, which may optionally be present and, if present, X 7b represents an amino acid selected from asparagine and lysine, which may optionally be present and, if present, X 8b represents an amino acid selected from tyrosine and phenylalanine, which may optionally be present and, if present, A 4b is optionally present and, if present, represents lysine.

[0062] In a preferred embodiment, the CDRH2 of the heavy chain of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention has the formula (IIa): 1b -NPSNX 2b -X 3b -TNX 4b -X 5b -X 6b -X 7b -X 8b -K, wherein X 1b is an amino acid selected from methionine, isoleucine, leucine, glycine, valine, and alanine; X 2b and X 3b each independently represents an amino acid selected from glycine and serine; X 4b and X 8b each independently represents an amino acid selected from tyrosine and phenylalanine; X 5b is an amino acid selected from serine and asparagine, X 6b is an amino acid selected from glutamic acid and glutamine, X 7b is an amino acid selected from asparagine and lysine.

[0063] In a further preferred embodiment, CDRH2 of the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention is selected from GVNPSNGGTNYNENYK, GVNPSNGGTNYNQNYK, or GVNPSNSGTNYNENYK.

[0064] In another preferred embodiment, CDRH2 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is IWYDGSKRY.

[0065] In one embodiment, the CDR3 of the heavy chain of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention (hereinafter referred to as CDRH3) has the general formula (III): A 1c -X 1c -A 2c -X 2c -A 3c -X 3c -A 4c -X 4c -X 5c -A 5c wherein: A 1c is optionally present and, if present, represents arginine; X 1c is an amino acid selected from aspartic acid, asparagine, and glutamic acid; A 2c is an amino acid or dipeptide selected from asparagine, serine and threonine or Tyr-Arg; X 2c is an amino acid selected from tyrosine, histidine, aspartic acid, glutamic acid, glycine and phenylalanine; A 3c is an amino acid selected from aspartic acid, tyrosine, isoleucine, phenylalanine, histidine and tryptophan; X 3c represents an amino acid selected from isoleucine, leucine, valine, alanine, glutamine and methionine, which may optionally be present and, if present, A 4c is optionally present and, when present, represents glycine; X 4c represents an amino acid selected from tyrosine, histidine and phenylalanine, which may optionally be present and, if present, X 5crepresents an amino acid which may optionally be present and, if present, is selected from aspartic acid and glutamic acid; A 5c is optionally present and, when present, represents tyrosine.

[0066] In one embodiment, the CDRH3 of the heavy chain of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention has the formula (IIIa):RX 1c -YRX 2c -DX 3c -GX 4c -X 5c -Y, wherein X 1c and X 5c each independently represents an amino acid selected from aspartic acid and glutamic acid; X 2c and X 4c each independently represents an amino acid selected from tyrosine, histidine, and phenylalanine; X 3c is an amino acid selected from isoleucine, leucine, valine, alanine, glutamine and methionine.

[0067] In a further preferred embodiment, CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is selected from RDYRYDMGFDY, RDYRYDMGYDY, or RDYRYDMGHDY.

[0068] In another preferred embodiment, CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is ESEY or NNDI or NSDF or NSDH or NSDY or NSGY or NTDW or NTDY.

[0069] In one embodiment, the CDR1 of the light chain of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention (hereinafter referred to as CDRL1) has the general formula (IV):A 1d -X 1d -A 2d -X 2d -X 3d -A 3d -X4d -A 4d -X 5d -A 5d wherein: A 1d is a dipeptide or tripeptide selected from Gln-Ser or Arg-Ala-Ser, X 1d is an amino acid selected from valine, glutamic acid and lysine; A 2d is an amino acid selected from glycine and serine; X 2d is an amino acid selected from isoleucine, leucine, valine, alanine, serine and methionine; X 3d is an amino acid selected from serine, tyrosine and glutamic acid; A 3d represents an amino acid which is optionally present and, if present, is selected from threonine, X 4d represents an amino acid that is optionally present and, if present, is selected from serine, aspartic acid and glutamic acid; A 4d is optionally present and, when present, represents the tetrapeptide Gly-Tyr-Ser-Tyr, X 5d represents an amino acid selected from isoleucine, leucine, valine, alanine and methionine, which may optionally be present and, if present, A 5d is optionally present and, if present, represents histidine.

[0070] In a preferred embodiment, CDRL1 of the light chain of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is represented by formula (IVa): 1d -GX 2d -X 3d -TX 4d -GYSYX 5d -H, wherein X 1dis an amino acid selected from glutamic acid and lysine; X 2d and X 5d each independently represents an amino acid selected from isoleucine, leucine, valine, alanine, and methionine; X 3d is an amino acid selected from serine and glutamic acid; X 4d is an amino acid selected from serine, aspartic acid and glutamic acid.

[0071] In a further preferred embodiment, CDRL1 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is RASKGVSTSGYSYLH.

[0072] In another preferred embodiment, CDRL1 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is QSVSSY.

[0073] In one embodiment, the CDRL2 of the light chain of the anti-PD-1 antibody, or antigen-binding portion thereof (hereinafter referred to as CDRL2), has the general formula (V): A 1e -X 1e -A 2e -X 2e -A 3e wherein: A 1e is optionally present and, when present, represents the dipeptide Leu-Ala, X 1e is an amino acid selected from serine, aspartic acid and glutamic acid; A 2e is an amino acid selected from tyrosine or alanine, X 2e is an amino acid selected from isoleucine, leucine, valine, alanine, serine and methionine; A 3e is optionally present and, when present, represents the dipeptide Glu-Ser.

[0074] In a preferred embodiment, CDRL2 of the light chain of the anti-PD-1 antibody, or antigen-binding portion thereof, has the formula (Va): 1e -YX 2e -ES, wherein X 1e is an amino acid selected from serine, aspartic acid and glutamic acid; X 2e is an amino acid selected from isoleucine, leucine, valine, alanine and methionine.

[0075] In a further preferred embodiment, CDRL2 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is LASYLES.

[0076] In another preferred embodiment, CDRL2 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is DAS.

[0077] In one embodiment, the CDR3 of the light chain of the anti-PD-1 antibody, or antigen-binding portion thereof (hereinafter referred to as CDRL3) has the general formula (VI): A 1f -X 1f -A 2f -X 2f -X 3f -X 4f -X 5f -X 6f wherein: A 1f is a dipeptide selected from Gln-His and Gln-Gln, X 1f is an amino acid sequence selected from serine, arginine, aspartic acid and glutamic acid, A 2f is an amino acid selected from arginine and serine; X 2f is an amino acid selected from aspartic acid, asparagine, and glutamic acid; X 3f and X 5feach independently represents an amino acid sequence selected from isoleucine, leucine, valine, alanine, tryptophan, arginine, and methionine; X 4f is proline, X 6f is threonine.

[0078] In a preferred embodiment, the CDRL3 of the light chain of the anti-PD-1 antibody, or antigen-binding portion thereof, has the formula (VIa): QHX 1f -RX 2f -X 3f -PX 4f -T, wherein X 1f is an amino acid sequence selected from serine, arginine, aspartic acid and glutamic acid, X 2f is an amino acid selected from aspartic acid and glutamic acid; X 3f and X 4f each independently represents an amino acid sequence selected from isoleucine, leucine, valine, alanine, and methionine.

[0079] In a further preferred embodiment, CDRL3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is QHSRDLPLT.

[0080] In another preferred embodiment, CDRL3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention is QQSSNWPRT.

[0081] In a preferred embodiment, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are selected from the amino acid sequences set out in Table 1 below.

[0082] [Table 2A]

[0083] [Table 2B]

[0084] [Table 2C]

[0085] [Table 2D]

[0086] [Table 2E]

[0087] In one embodiment, the present invention provides (a) General formula (I):X 1a -A 1a -X 2a -A 2a CDRH1; (b) General formula (II):A 1b -X 1b -A 2b -X 2b -X 3b -A 3b -X 4b -X 5b -X 6b -X 7b -X 8b -A 4b CDRH2; (c) General formula (III):A 1c -X 1c -A 2c -X 2c -A 3c -X 3c -A 4c -X 4c -X 5c -A 5c CDRH3; (d) General formula (IV): A 1d -X 1d -A 2d -X 2d -X 3d -A3d -X 4d -A 4d -X 5d -A 5d CDRL1; (e) General formula (V):A 1e -X 1e -A 2e -X 2e -A 3e CDRL2 and (f) General formula (VI): A 1f -X 1f -A 2f -X 2f -X 3f -X 4f -X 5f -X 6f CDRL3 wherein X 1a is an amino acid selected from asparagine, glycine and threonine; A 1a is a dipeptide selected from Tyr-Tyr and Ile-Thr, X 2a is an amino acid selected from isoleucine, leucine, valine, phenylalanine, methionine and alanine, A 2a is a single amino acid or a dipeptide or tripeptide or tetrapeptide selected from tyrosine, asparagine, serine, glycine, Asn-Ser, Asn-Ser-Gly and Ser-Asn-Ser-Gly, A 1b is optionally present and, if present, represents glycine; X 1b is an amino acid selected from methionine, isoleucine, leucine, glycine, valine, and alanine; A 2b is a tripeptide or tetrapeptide selected from Asn-Pro-Ser-Asn or Trp-Tyr-Asp, X 2b and X 3b each independently represents an amino acid selected from glycine and serine; A 3bis a single amino acid or dipeptide selected from lysine or Thr-Asn, X 4b is an amino acid selected from tyrosine, arginine and phenylalanine; X 5b is an amino acid selected from serine, tyrosine and asparagine; X 6b represents an amino acid selected from glutamic acid and glutamine, which may optionally be present and, if present, X 7b represents an amino acid selected from asparagine and lysine, which may optionally be present and, if present, X 8b represents an amino acid selected from tyrosine and phenylalanine, which may optionally be present and, if present, A 4b is optionally present and, if present, represents lysine; A 1c is optionally present and, if present, represents arginine; X 1c is an amino acid selected from aspartic acid, asparagine, and glutamic acid; A 2c is an amino acid or dipeptide selected from asparagine, serine and threonine or Tyr-Arg; X 2c is an amino acid selected from tyrosine, histidine, aspartic acid, glutamic acid, glycine and phenylalanine; A 3c is an amino acid selected from aspartic acid, tyrosine, isoleucine, phenylalanine, histidine and tryptophan; X 3c represents an amino acid selected from isoleucine, leucine, valine, alanine, glutamine and methionine, which may optionally be present and, if present, A 4c is optionally present and, when present, represents glycine; X 4crepresents an amino acid selected from tyrosine, histidine and phenylalanine, which may optionally be present and, if present, X 5c represents an amino acid which may optionally be present and, if present, is selected from aspartic acid and glutamic acid; A 5c is optionally present and, when present, represents tyrosine; A 1d is a dipeptide or tripeptide selected from Gln-Ser or Arg-Ala-Ser, X 1d is an amino acid selected from valine, glutamic acid and lysine; A 2d is an amino acid selected from glycine and serine; X 2d is an amino acid selected from isoleucine, leucine, valine, alanine, serine and methionine; X 3d is an amino acid selected from serine, tyrosine and glutamic acid; A 3d represents an amino acid which is optionally present and, if present, is selected from threonine, X 4d represents an amino acid that is optionally present and, if present, is selected from serine, aspartic acid and glutamic acid; A 4d is optionally present and, when present, represents the tetrapeptide Gly-Tyr-Ser-Tyr, X 5d represents an amino acid selected from isoleucine, leucine, valine, alanine and methionine, which may optionally be present and, if present, A 5d is optionally present and, if present, represents histidine; A 1e is optionally present and, when present, represents the dipeptide Leu-Ala, X 1e is an amino acid selected from serine, aspartic acid and glutamic acid; A 2e is an amino acid selected from tyrosine or alanine, X 2e is an amino acid selected from isoleucine, leucine, valine, alanine, serine and methionine; A 3e is optionally present and, when present, represents the dipeptide Glu-Ser, A 1f is a dipeptide selected from Gln-His and Gln-Gln, X 1f is an amino acid sequence selected from serine, arginine, aspartic acid and glutamic acid, A 2f is an amino acid selected from arginine and serine; X 2f is an amino acid selected from aspartic acid, asparagine, and glutamic acid; X 3f and X 5f each independently represents an amino acid sequence selected from isoleucine, leucine, valine, alanine, tryptophan, arginine, and methionine; X 4f is proline, X 6f is threonine, However, X 1a Asparagine and X 2a The present invention provides an anti-PD-1 antibody, or antigen-binding portion thereof, which is free of methionine as the amino acid sequence.

[0088] In a preferred embodiment, the present invention comprises: (a) Formula (Ia): X 1a -YYX 2a -CDRH1 containing the amino acid sequence of Y; (b) Formula (IIa): GX 1b -NPSNX 2b -X 3b -TNX 4b -X 5b -X 6b -X 7b -X8b CDRH2 containing the amino acid sequence of -K, (c) Formula (IIIa):RX 1c -YRX 2c -DX 3c -GX 4c -X 5c -CDRH3 containing the amino acid sequence of Y; (d) Formula (IVa): RASX 1d -GX 2d -X 3d -TX 4d -GYSYX 5d -CDRL1 comprising the amino acid sequence of H; (e) Formula (Va): LAX 1e -YX 2e -CDRL2 containing the amino acid sequence of ES; (f) Formula (VIa): QHX 1f -RX 2f -X 3f -PX 4f CDRL3 containing the amino acid sequence -T wherein X 1a is an amino acid selected from asparagine and threonine; X 2a is an amino acid selected from isoleucine, leucine, valine, methionine and alanine, X 1b is an amino acid selected from methionine, isoleucine, leucine, glycine, valine, and alanine; X 2b and X 3b each independently represents an amino acid selected from glycine and serine; X 4b and X 8b each independently represents an amino acid selected from tyrosine and phenylalanine; X 5b is an amino acid selected from serine and asparagine, X 6b is an amino acid selected from glutamic acid and glutamine, X 7bis an amino acid selected from asparagine and lysine; X 1c and X 5c each independently represents an amino acid selected from aspartic acid and glutamic acid; X 2c and X 4c each independently represents an amino acid selected from tyrosine, histidine, and phenylalanine; X 3c is an amino acid selected from isoleucine, leucine, valine, alanine, glutamine and methionine; X 1d is an amino acid selected from glutamic acid and lysine; X 2d and X 5d each independently represents an amino acid selected from isoleucine, leucine, valine, alanine, and methionine; X 3d is an amino acid selected from serine and glutamic acid; X 4d is an amino acid selected from serine, aspartic acid and glutamic acid; X 1e is an amino acid selected from serine, aspartic acid and glutamic acid; X 2e is an amino acid selected from isoleucine, leucine, valine, alanine and methionine; X 1f is an amino acid sequence selected from serine, arginine, aspartic acid and glutamic acid, X 2f is an amino acid selected from aspartic acid and glutamic acid; X 3f and X 4f each independently represents an amino acid sequence selected from isoleucine, leucine, valine, alanine, and methionine; However, X 1a Asparagine and X 2aThe present invention provides an anti-PD-1 antibody, or antigen-binding portion thereof, which is free of methionine as the amino acid sequence.

[0089] In a preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of an anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are TYYIY, GVNPSNGGTNYNENYK, and RDYRYDMGFDY, respectively.

[0090] In another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are TYYIY, GVNPSNGGTNYNENYK, and RDYRYDMGYDY, respectively.

[0091] In yet another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are TYYIY, GVNPSNGGTNYNENYK, and RDYRYDMGHDY, respectively.

[0092] In another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are TYYIY, GVNPSNSGTNYNENYK, and RDYRYDMGFDY, respectively.

[0093] In yet another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are TYYIY, GVNPSNSGTNYNENYK, and RDYRYDMGYDY, respectively.

[0094] In yet another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are TYYIY, GVNPSNSGTNYNENYK, and RDYRYDMGHDY, respectively.

[0095] In another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are TYYIY, GVNPSNGGTNYNQNYK, and RDYRYDMGFDY, respectively.

[0096] In another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are TYYIY, GVNPSNGGTNYNQNYK, and RDYRYDMGYDY, respectively.

[0097] In yet another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are TYYIY, GVNPSNGGTNYNQNYK, and RDYRYDMGHDY, respectively.

[0098] In another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are GITFSNSG, IWYDGSKRY, and NSDF, respectively.

[0099] In another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are GITFSNSG, IWYDGSKRY, and NSDH, respectively.

[0100] In a preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of an anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are GITFSNSG, IWYDGSKRY, and NSDY, respectively.

[0101] In another preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are GITFSNSG, IWYDGSKRY, and NTDW, respectively.

[0102] In a preferred embodiment, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of an anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are GITFSNSG, IWYDGSKRY, and NTDY, respectively.

[0103] In a preferred embodiment, the amino acid sequences of CDRL1, CDRL2, and CDRL3 of an anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are RASKGVSTSGYSYLH, LASYLES, and QHSRDLPLT, respectively.

[0104] In another preferred embodiment, the amino acid sequences of CDRL1, CDRL2, and CDRL3 of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are QSVSSY, DAS, and QQSSNWPRT, respectively.

[0105] In another embodiment, the present invention provides (a) a CDRH1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7; (b) a CDRH2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30; (c) a CDRH3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55; (d) a CDRL1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, and 81; (e) a CDRL2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, and 112; and (f) a CDRL3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, and 136; wherein the antibody specifically binds to PD-1, preferably human PD-1.

[0106] In one embodiment, the HCVR and LCVR of the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention are selected from the amino acid sequences set out in Table 2 below.

[0107] [Table 3A]

[0108] [Table 3B]

[0109] [Table 3C]

[0110] In one embodiment, the variable region of the heavy chain of an anti-PD-1 antibody, or antigen-binding portion thereof, of the invention comprises CDRH1, CDRH2, and CDRH3 comprising an amino acid sequence selected from Table 3 shown below.

[0111] [Table 4]

[0112] In one embodiment, the variable region of the light chain of an anti-PD-1 antibody, or antigen-binding portion thereof, of the invention comprises CDRL1, CDRL2, and CDRL3 comprising an amino acid sequence selected from Table 4 shown below.

[0113] [Table 5]

[0114] Accordingly, the present invention provides an anti-PD-1 antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, (a) the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, and 150; (b) the light chain variable region comprises the amino acid sequence of SEQ ID NOs: 151 and 152; Anti-PD-1 antibodies, or antigen-binding portions thereof, are provided.

[0115] Preferred combinations of CDRs: Preferred combinations of CDRs for the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include the following: (a) a heavy chain variable region CDRH1 comprising SEQ ID NO:4; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:8; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO: 32; (d) a light chain variable region CDRL1 comprising SEQ ID NO:77; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 108; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 132.

[0116] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: (a) a heavy chain variable region CDRH1 comprising SEQ ID NO:4; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:9; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO:32; (d) a light chain variable region CDRL1 comprising SEQ ID NO:77; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 108; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 132.

[0117] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: (a) a heavy chain variable region CDRH1 comprising SEQ ID NO:4; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:26; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO: 32; (d) a light chain variable region CDRL1 comprising SEQ ID NO:77; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 108; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 132.

[0118] Further preferred combinations of CDRs of the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include the following: (a) a heavy chain variable region CDRH1 comprising SEQ ID NO:4; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:8; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO: 33; (d) a light chain variable region CDRL1 comprising SEQ ID NO:77; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 108; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 132.

[0119] Further preferred combinations of CDRs of the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include the following: (a) a heavy chain variable region CDRH1 comprising SEQ ID NO:4; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:8; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO:47; (d) a light chain variable region CDRL1 comprising SEQ ID NO:77; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 108; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 132.

[0120] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: (a) a heavy chain variable region CDRH1 comprising SEQ ID NO:4; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:9; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO: 33; (d) a light chain variable region CDRL1 comprising SEQ ID NO:77; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 108; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 132.

[0121] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: (a) a heavy chain variable region CDRH1 comprising SEQ ID NO:4; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:9; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO:47; (d) a light chain variable region CDRL1 comprising SEQ ID NO:77; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 108; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 132.

[0122] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: (a) a heavy chain variable region CDRH1 comprising SEQ ID NO:4; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:26; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO: 33; (d) a light chain variable region CDRL1 comprising SEQ ID NO:77; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 108; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 132.

[0123] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: (a) a heavy chain variable region CDRH1 comprising SEQ ID NO:4; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:26; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO:47; (d) a light chain variable region CDRL1 comprising SEQ ID NO:77; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 108; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 132

[0124] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: a) a heavy chain variable region CDRH1 comprising SEQ ID NO:7; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:30; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO:50; (d) a light chain variable region CDRL1 comprising SEQ ID NO: 81; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 112; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 136.

[0125] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: a) a heavy chain variable region CDRH1 comprising SEQ ID NO:7; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:30; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO:51; (d) a light chain variable region CDRL1 comprising SEQ ID NO: 81; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 112; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 136.

[0126] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: a) a heavy chain variable region CDRH1 comprising SEQ ID NO:7; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:30; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO:52; (d) a light chain variable region CDRL1 comprising SEQ ID NO: 81; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 112; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 136.

[0127] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: a) a heavy chain variable region CDRH1 comprising SEQ ID NO:7; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:30; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO:54; (d) a light chain variable region CDRL1 comprising SEQ ID NO: 81; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 112; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 136.

[0128] Other suitable combinations of CDRs of an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention include: a) a heavy chain variable region CDRH1 comprising SEQ ID NO:7; (b) a heavy chain variable region CDRH2 comprising SEQ ID NO:30; (c) a heavy chain variable region CDRH3 comprising SEQ ID NO:55; (d) a light chain variable region CDRL1 comprising SEQ ID NO: 81; (e) a light chain variable region CDRL2 comprising SEQ ID NO: 112; and (f) a light chain variable region CDRL3 comprising SEQ ID NO: 136.

[0129] Preferred combinations of variable regions: Preferred combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include the following: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 137; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151.

[0130] Preferred combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include the following: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 138; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151.

[0131] Preferred combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include the following: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 139; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151.

[0132] Preferred combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include the following: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151.

[0133] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151.

[0134] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151.

[0135] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 143; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151.

[0136] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 144; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151.

[0137] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 145; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151.

[0138] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 146; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152.

[0139] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 147; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152.

[0140] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 148; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152.

[0141] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 149; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152.

[0142] Other suitable combinations of HCVRs and LCVRs of anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 150; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152.

[0143] In one embodiment, an antibody, or antigen-binding portion thereof, of the invention comprises at least one of the following features: (a) cross-reacts with PD-1 from species other than human; (b) higher binding specificity for human PD-1; (c) promoting IL-2 cytokine secretion; (d) promotes higher IFN-gamma secretion; (e) blocking the interaction of the PD-1 receptor with its natural ligands PD-L1 and / or PD-L2; (f) promoting T cell proliferation; and (g) has an improved circulating half-life.

[0144] In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, binds to the PD-1 antigen at a concentration of 10 -10 M or less, more preferably 10 -11 K below M D In a preferred embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, of the invention has a -10 M or less, preferably 10 -11 K below M D It binds to human PD-1 at K DThe value is a measure of the binding affinity of an antibody to its target antigen. Such binding affinity and binding kinetics of an anti-PD-1 antibody can be examined by SPR analysis. The above analysis is very well known in the art for measuring the affinity of an antibody or an antigen-binding portion thereof to its target antigen, and the skilled artisan is well aware of the techniques for performing such analysis.

[0145] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention have high specificity for PD-1. The anti-PD-1 antibodies according to the invention have high affinity for the human PD-1 antigen / epitope. In another embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention have high binding specificity for human PD-1. The binding specificity is investigated by SPR. "High specificity for PD-1 or for human PD-1" means that the antibodies, or antigen-binding portions thereof, do not bind with the same affinity to other T cell co-inhibitory receptors.

[0146] In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, cross-reacts with sufficient affinity with PD-1 of other species, such as cynomolgus monkeys.

[0147] In another embodiment, an anti-PD-1 antibody, or antigen-binding portion thereof, of the invention blocks the interaction of PD-1 with its ligand in a surface plasmon resonance assay.

[0148] In another embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention kill T cells that express PD-1.

[0149] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention promote IL-2 cytokine secretion, hi such an embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention, when available during an MLR, increase the production of IL-2 as measured by ELISA.

[0150] In one embodiment, the anti-PD-1 antibody or antigen-binding portion thereof according to the present invention promotes higher IFN-gamma secretion. The term "higher" is referred to herein with reference to known anti-PD-1 antibodies. In another embodiment, the anti-PD-1 antibody or antigen-binding portion thereof according to the present invention increases the production of IFN-gamma as measured by ELISA when available during MLR. Bone marrow transplantation experiments using IFN-γR- / - mice implicated IFN-γ as a critical center for T cell suppression of PD-1-associated tumor infiltration. It has also been evaluated and confirmed that other IFN-γ-inducible chemokines may also be involved in the synergistic effect of anti-PD-1 on ACT. (7) Furthermore, IFN-γ is reported as an important marker for predicting response to immune checkpoint blockade (8).

[0151] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention promote T cell proliferation. In such an embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention, when available during an MLR, increase T cell proliferation as measured by a proliferation measurement assay. Such assays are known to those of skill in the art.

[0152] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, according to the invention have improved pharmacokinetic properties.

[0153] In one embodiment, the anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention has an extended half-life in humans.

[0154] The pharmacokinetic profile of the anti-PD1 of the present invention is confirmed in cynomolgus monkeys. Since the intended route of administration in patients is intravenous, the study involves intravenous administration of anti-PD1 antibody in buffer to animals. Briefly, the study includes 3 animals / sex / group, which are administered at a dose level of up to 50mg / Kg. The PD effects of anti-PD1 antibody, such as peripheral immune cell population regulation and absolute lymphocyte count (ALC) changes, are examined.

[0155] In one embodiment, an anti-PD-1 antibody, or antigen-binding portion thereof, according to the invention blocks the interaction of the PD-1 receptor with its natural ligands PD-L1 and / or PD-L2. Anti-PD-1 antibodies of the invention are tested for the ability to block the binding of ligands PD-L1 and PD-L2 to PD-1 expressed on cells by using a mixed lymphocyte reaction (MLR). Anti-PD-1 antibodies of the invention are tested for the ability to block the binding of ligands PD-L1 and PD-L2 to PD-1 by SPR.

[0156] In one embodiment, the effect of the binding of the anti-PD-1 antibody, or antigen-binding portion thereof, of the present invention to PD-1 on T cell activation is analyzed by a suitable assay known in the art. The anti-PD-1 antibody, or antigen-binding portion thereof, of the present invention binds to PD-1 expressed on a PD-1-expressing transfected cell line. Furthermore, the anti-PD-1 antibody, or antigen-binding portion thereof, of the present invention binds to peripheral blood mononuclear cells (PBMCs).

[0157] Antibodies according to the invention may be full-length (e.g. IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g. Fab, F(ab')2 or scFv fragments) and may optionally be modified to impart functionality, e.g. to remove residual effector functions such as ADCC and CDC activity.

[0158] In one embodiment, the amino acid sequence of the constant region of the anti-PD-1 antibody is an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, IgD constant region or a hybrid of the mentioned constant regions, e.g., IgG2 / IgG4, preferably IgG1 or IgG4, more preferably IgG4.

[0159] In another embodiment, the Fc region of an antibody of the invention is modified to alter the effector functions of the antibody by replacing at least one amino acid residue with a different amino acid residue.

[0160] In a preferred embodiment, the amino acid sequence of the heavy chain constant region of the anti-PD-1 antibody of the invention has a mutation in the hinge region, preferably a serine to proline mutation in the hinge region of the IgG4 constant region of the antibody. Human IgG4 antibodies may exist in two forms associated with hinge heterogeneity. In one form, the antibody comprises a stable four-chain construct of approximately 150-160 kDa, the dimer of which is held together by inter-heavy chain disulfide bonds. In the second form, the dimer is not linked by inter-chain disulfide bonds, forming a molecule of approximately 75-80 kDa composed of covalently linked light and heavy chains (half antibodies). The latter form has been extremely difficult to separate from the complete antibody, even after affinity purification. The frequency of occurrence of the second form in the various complete IgG4 isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form (6) to the level commonly observed using a human IgG1 hinge. A full-length antibody comprising the CDR or variable region of the invention further comprises the single amino acid substitution (i.e., S228P) when it is developed in the IgG4 format.

[0161] In one embodiment, the anti-PD-1 antibody of the invention has ADCC and / or CDC activity. In a preferred embodiment, the anti-PD-1 antibody of the invention with altered, reduced or absent ADCC and / or CDC activity is an anti-PD-1 antibody having an IgG1 or IgG4 or IgG2 / IgG4 constant region. In another embodiment, the Fc region of the antibody of the invention is modified to enhance ADCC and / or CDC activity by replacing at least one amino acid residue with a different amino acid residue. In a preferred embodiment, the anti-PD-1 antibody of the invention with enhanced ADCC and / or CDC activity is an IgG1 or an anti-PD-1 antibody having an IgG1 constant region with at least one altered amino acid residue. The IgG1 constant region with at least one altered amino acid residue referred to herein is an altered IgG1, which may result in higher ADCC and / or CDC activity compared to an IgG1 without such alteration.

[0162] In another preferred embodiment, the anti-PD-1 antibody of the invention with reduced or no ADCC and / or CDC activity is an IgG1 or anti-PD-1 antibody with an IgG1 constant region with at least one modified amino acid residue. The IgG1 constant region with at least one modified amino acid residue referred to herein is an modified IgG1, which may or may not result in reduced ADCC and / or CDC activity compared to an IgG1 without such modification.

[0163] In another preferred embodiment, the anti-PD-1 antibody of the invention with reduced or no ADCC and / or CDC activity is an IgG4 or anti-PD-1 antibody with an IgG4 constant region with at least one modified amino acid residue. The IgG4 constant region with at least one modified amino acid residue referred to herein is an modified IgG4, which may or may not result in reduced ADCC and / or CDC activity compared to an IgG4 without such modification.

[0164] In another aspect, the anti-PD-1 antibodies according to the invention have increased FcRn binding and extended half-life, and have altered or reduced or no ADCC and / or CDC activity. In one embodiment, the anti-PD-1 antibodies according to the invention have the amino acid sequence of an IgG1 or IgG4 constant region with the P329G and / or M428L & N434S mutations. The constant regions of anti-PD-1 antibodies with all three mentioned mutations in the IgG1 and IgG4 constant regions are referred to herein as IgG1(GLS) and IgG4(GLS), respectively.

[0165] In a preferred embodiment, the anti-PD-1 antibodies according to the invention comprise a single amino acid substitution selected from S228P, P329G, M428L, N434S, and suitable combinations thereof.

[0166] In one embodiment, the anti-PD-1 antibody according to the invention is a monoclonal antibody or a bispecific antibody or a polyclonal antibody, preferably a monoclonal antibody.

[0167] In one embodiment, an antibody or antigen-binding portion thereof that targets the PD-1 antigen according to the present invention is murine, chimeric, recombinant or humanized in nature, preferably recombinant in nature.

[0168] Immunoconjugates and Bispecific Antibodies In one embodiment, the anti-PD-1 antibody of the present invention is linked to a drug, preferably a cytotoxic agent, optionally via a linker. The present invention also provides immunoconjugates comprising the antibody of the present invention, or an antigen-binding portion thereof, linked to another therapeutic agent, such as a cytotoxin or a radioisotope. Such immunoconjugates prepared according to the present invention specifically bind to and cause the lysis of PD1-expressing immune cells. Such immune cells may include T cells, NK cells, B cells, monocytes, etc., that express PD-1. Thus, the immunoconjugates prepared according to the present invention help treat diseases in which the activity of PD-1-expressing immune cells is detrimental.

[0169] In one embodiment, an anti-PD-1 antibody of the invention is used to generate a bispecific molecule. A bispecific molecule comprising an antibody of the invention, or an antigen-binding portion thereof, has two unique antigen-binding arms or functional moieties, one that binds to PD-1 and the other that has a different binding specificity. In one embodiment, the second functional moiety according to the invention is capable of binding to an antigen selected from CTLA-4, PD-L1, LAG-3, TIGIT, and HER2.

[0170] Nucleic acid molecules encoding anti-PD-1 antibodies, vectors and host cells The present invention provides nucleic acid molecules or sequences encoding the antibodies, or antigen-binding portions thereof, of the present invention, as well as expression vectors comprising such nucleic acid sequences and host cells comprising such expression vectors. In the present application, pZRCII and pZRCIII vectors are used for cloning and expressing the anti-PD-1 antibodies of the present invention. The pZRCII vector (pZRCIIhyg) and pZRCIII vectors are described in patent documents WO2007 / 017903 and WO2012 / 046255A2, respectively. The host cell according to the present invention is a prokaryotic or eukaryotic cell, preferably the host cell is an E. coli cell or a mammalian cell, such as a CHO cell or an NS0 cell or a CHO-GS cell or a CHO-S cell or a CHO-K1 cell.

[0171] Antibody production In the present invention, the anti-PD-1 antibody or antigen-binding portion thereof is produced using recombinant techniques. In the recombinant techniques according to the present invention, a suitable expression vector is used. The vector for producing the antibody of the present invention can be any vector readily known to one skilled in the art. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more commonly, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods known to those skilled in the art (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present). For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into the expression vector such that the gene is operably linked to transcriptional and translational control sequences. In addition to the antibody chain genes and control sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The heavy chain variable regions of the antibodies described herein are expressed as V H Segment is C HThe segments can be used to generate full-length antibody genes of any antibody isotype by inserting them operably linked into an expression vector already encoding the heavy chain constant region and full-length light chain of the desired isotype. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. For expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques, such as lipid-mediated transfection, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including DHFR-CHO cells), NSO myeloma cells, COS cells, and SP2 cells. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system. Antibodies produced according to the invention may be further produced by known cell culture techniques for large-scale antibody production. Antibodies can be recovered from the culture medium using standard protein purification methods. When using recombinant techniques, the antibodies may be produced intracellularly in the periplasmic space or directly secreted into the medium. If the antibodies are produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. The antibodies of the invention may be tested for binding to PD-1, for example, by standard ELISA.

[0172] The anti-PD-1 antibodies of the present invention can also be made using a phage display library approach after generating a Fab library using a site-directed mutagenesis approach, as exemplified herein, in which an anti-PD-1 Fab region is generated using a phage library approach, which is further converted to a full-length form using expression vector-based recombinant techniques, as described in this application.

[0173] Combination of Anti-PD-1 Agents of the Invention with Other Drugs The invention provides a combination comprising at least two or more antibodies or antigen-binding portions thereof, where at least one antibody or antigen-binding portion thereof is an anti-PD-1 antibody of the invention. A combination according to the invention may comprise a second antibody or antigen-binding portion thereof selected from an anti-LAG3 antibody, an anti-CTLA4 antibody, an anti-HER2 antibody, an anti-VEGF antibody, an anti-PDL1 antibody, an anti-PD-1 antibody, in combination with the anti-PD-1 antibody or antigen-binding portion thereof of the invention. In another embodiment, the invention provides a combination comprising an anti-PD-1 antibody or antigen-binding portion thereof and a peptide, or a combination comprising an anti-PD-1 antibody or antigen-binding portion thereof and a cytokine, preferably an interleukin.

[0174] Pharmaceutical Compositions Pharmaceutical compositions may be developed that comprise one or a combination of monoclonal antibodies or antigen-binding portions thereof, or a combination of anti-PD-1 antibodies or antigen-binding portions thereof and a peptide, or a combination of anti-PD-1 antibodies or antigen-binding portions thereof and a cytokine (preferably an interleukin) of the invention, formulated with a pharmaceutically acceptable carrier. The invention also provides compositions, e.g., pharmaceutical compositions, that comprise one or a combination of monoclonal antibodies or antigen-binding portions thereof, or a combination of anti-PD-1 antibodies or antigen-binding portions thereof and a peptide, or a combination of anti-PD-1 antibodies or antigen-binding portions thereof and a cytokine (preferably an interleukin) of the invention, formulated with a pharmaceutically acceptable carrier. Such compositions may comprise a combination of one or more (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules of the invention. For example, a pharmaceutical composition of the invention may comprise a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on their target antigen or different epitopes on different target antigens, or that have complementary activities.

[0175] In one embodiment, the anti-PD-1 antibodies, or antigen-binding portions thereof, may be combined with other drugs suitable for the treatment of various cancers, infectious diseases, or autoimmune disorders.

[0176] The anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention may be combined or used in combination with other suitable chemically synthesized therapeutic agents, which may be anti-cancer or anti-infective agents or combinations of such agents.

[0177] The anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention may be combined with chemotherapeutic agents in a clinical setting.

[0178] The anti-PD-1 antibodies, or antigen-binding portions thereof, of the invention may also be used in combination with vaccines, in both prophylactic and therapeutic settings, to elicit T and B cell immune responses, optionally by prolonging vaccine-induced T or B cell proliferation.

[0179] therapeutic use In one embodiment, the anti-PD-1 antibodies or antigen-binding portions thereof, or combinations, conjugates, or bispecific antibodies of the invention can be used to treat a disease in which PD-1 activity or expression is detrimental. Such diseases will be readily apparent to one of skill in the art.

[0180] The anti-PD-1 antibodies or antigen-binding portions thereof or combinations or conjugates or bispecific antibodies of the invention may be used to treat NSCLC, SCLC, RCC, cHL, SCCHN, breast cancer, urothelial carcinoma, colorectal cancer, ESCC, primary mediastinal large B-cell lymphoma, microsatellite instability high carcinoma, gastric cancer, ovarian cancer, prostate cancer, glioma, glioblastoma, astrocytoma, glioblastoma multiforme, Banayan-Zonana syndrome, Cowden disease, Lhermitte-Dube disease, and other conditions. Crohn's disease, inflammatory breast cancer, Wilms' tumor, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, kidney cancer, liver cancer, melanoma, pancreatic cancer, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid cancer, lymphocytic T-cell leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia, acute myeloid leukemia, AML, chronic neutrophilic leukemia, acute lymphocytic T-cell leukemia, plasmacytoma, immunoblastic large cell leukemia It can be used to treat various cancers such as myeloid leukemia, myeloma, myeloma, acute large cell leukemia, mantle cell leukemia, multiple myeloma, megakaryoblastic leukemia, multiple myeloma, acute megakaryoblastic leukemia, promyelocytic leukemia, erythroleukemia, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic T cell lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, cervical cancer, Merkel cell carcinoma, endometrial cancer, tumor mutation burden high (TMB-H) cancer, vulvar cancer, salivary gland cancer, nasopharyngeal cancer, buccal cancer, oral cancer, GIST (gastrointestinal stromal tumor), and testicular cancer.

[0181] The anti-PD-1 antibodies or antigen-binding portions thereof or combinations or conjugates or bispecific antibodies of the invention can be used to treat various infectious diseases. Examples of such infectious diseases may be infectious diseases caused by pathogens selected from HIV, Hepatitis (A, B, and C), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa, SARS-CoV, MERS-CoV, SARS-CoV-2, Ebola, etc.

[0182] The anti-PD-1 antibodies or antigen-binding portions thereof or combinations or conjugates or bispecific antibodies of the invention can be used to treat a variety of autoimmune disorders. Examples of such autoimmune disorders include rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, Crohn's disease, ulcerative colitis, sepsis, atopic diseases such as asthma, eczema, allergies, multiple sclerosis, systemic lupus erythematosus, vasculitis, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, sepsis, autoimmune hemolytic anemia, pernicious anemia, idiopathic thrombocytopenic purpura, Goodpasture's syndrome, bullous pemphigoid, pemphigus vulgaris, Hashimoto's thyroiditis, and rheumatoid arthritis. These include: diabetes, insulin-dependent diabetes mellitus (IDDM), CIDP diabetes, anti-anemia agents (beta thalassemia), hematopoietic agents, eye drops, bone diseases, hereditary neuropathy, age-related macular degeneration, diabetic retinopathy, macular diseases, hereditary disorders of the eye, hemophilia B, (clotting factor IX(9) deficiency) drugs for cardiovascular disease, type 2 diabetes, immunosuppressants, treatment of transplant rejection, myelodysplastic syndrome treatment, retinopathy, aplastic anemia, analgesics, hereditary blood disorders, multisystem genetic disorders, osteoarthritis, scleroderma, treatment of autoimmune diseases, treatment of gout, urticaria, heart failure treatment, lymphoma treatment, nephritis, respiratory disorders and treatment of congenital metabolic disorders.

[0183] In a preferred embodiment, the invention provides an effective amount of the anti-PD-1 antibodies or antigen-binding portions thereof disclosed herein that are capable of reducing PD-1-associated immunosuppression. These antibodies can be administered by conventional routes and dosages, e.g., a "pharmaceutical effective dose" or a "therapeutically effective dose." EXAMPLES

[0184] The following examples are illustrative and should not be construed as limiting the scope of the disclosure in any way, since many variations and equivalents will become apparent to those skilled in the art upon reading this disclosure. All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety.

[0185] Example 1 Construction of pZRCII hyg anti-PD-1 light and heavy chain vectors Construction of pZRCII hygIgG4 vector Chemically synthesized human IgG4 constant region (SEQ ID NO: 156) was amplified by 30 cycles of PCR using 0.2 pM of specific oligonucleotide primers containing the above restriction sites in a volume of 50 μl containing 1×Pfx buffer, 1.5 mM MgSO4, 2.5 μM of each of the four dNTPs and 1 unit of Pfx polymerase to incorporate the restriction enzymes SalI, ApaI overhang at the 5′ end and NotI overhang at the 3′ end. Each PCR amplification cycle consisted of incubation at 95° C. for 30 s (denaturation), 55° C. for 45 s (annealing) and 72° C. for 1 min (extension). The amplification products of the PCR reaction were separated on a 1% agarose gel. The desired fragment with a size of approximately 980 base pairs was excised from the gel and purified using a Qiagen Gel extraction kit. The purified PCR fragment and the pZRCII vector carrying the hygromycin resistance gene were digested with the restriction enzymes SalI and NotI. The pZRCII vector was made as described in patent publication WO2007 / 017903. The approximately 7692 base pair vector fragment was purified from the gel by using a Qiagen Gel extraction kit. The digested PCR fragment was also purified using a Qiagen Gel extraction kit. The digested and purified PCR product and the pZRCII hyg vector were ligated and the ligation product was transformed into E. coli Top 10F' strain. The transformed cells were scored based on kanamycin resistance. Plasmid DNA isolated from such colonies was analyzed for the presence of the IgG4 constant region fragment by restriction digestion using SalI and NotI restriction enzymes. One of the plasmids showing the expected restriction profile with the IgG4 constant region fragment incorporated into the pZRCII vector was named pZRC II IgG4 constant. In the same manner, a chemically synthesized IgG1 Fc (SEQ ID NO: 157) was cloned into the pZRCII vector and named pZRCIIhyg.IgG1 vector.

[0186] Construction of pZRCIIhyg anti-PD-1 LC-HC vector construct Anti-PD1 light and heavy chain genes were chemically synthesized and cloned into the cloning vector pMA (Geneart, Germany). To clone the light chain gene into the pZRCIIhyg.IgG4 constant region vector, the pMA and pZRCIIhyg.IgG4 vectors containing the light chain were digested with XhoI and kpnI restriction enzymes. The light chain (SEQ ID NO: 163) of approximately 737 bp and the pZRCIIhyg.IgG4 vector of approximately 8672 base pairs in size were excised from the gel and purified using a Qiagen Gel extraction kit. The digested and purified light chain and pZRCIIhyg.IgG4 vector were ligated and transformed into E. coli Top 10F' strain. Transformants were selected on the basis of kanamycin resistance. Plasmid DNA isolated from approximately 20 such colonies was analyzed for the presence of the light chain gene by restriction digestion with various restriction enzymes. One such plasmid with a light chain gene integrated in the first transcriptional assembly of the pZRCIIhyg.IgG4 vector was selected to generate the pZRCIIhyg.LC-HC vector. To clone the heavy chain gene, the pZRCII hyg.LC-IgG4 vector with chemically synthesized heavy chain gene variable fragments (SEQ ID NOs: 158, 159, 160, 161 and 162), all approximately 446 base pairs in size, and the light chain of SEQ ID NO: 163, was digested with SalI and ApaI. The digested and purified heavy chain gene variable region fragments (SEQ ID NOs: 158, 159, 160, 161 and 162) were individually ligated into the pZRCIIhyg anti-PD-1 LC IgG4 vector and transformed into E. coli Top 10F'. Transformants were selected on the basis of kanamycin resistance. Plasmid DNA isolated from the transformed cells was analyzed for the presence of the heavy chain gene by restriction digestion with SalI and NotI. The final vectors containing a light chain of SEQ ID NO: 163 and a heavy chain of SEQ ID NO: 158, a light chain of SEQ ID NO: 163 and a heavy chain of SEQ ID NO: 159, a light chain of SEQ ID NO: 163 and a heavy chain of SEQ ID NO: 160, a light chain of SEQ ID NO: 163 and a heavy chain of SEQ ID NO: 161, a light chain of SEQ ID NO: 163 and a heavy chain of SEQ ID NO: 162 were confirmed by nucleotide sequencing of the heavy and light chains.The final constructs containing various combinations of heavy and light chains were named IP-H4L1, IP-H4.2L1, IP-H4.2(YH)L1, IP-H4.19L1, and IP-H4.36L1, respectively. The various constructs above encode the amino acid sequences of the HCVR and LCVR of the anti-PD-1 antibodies of the invention as set out in Table 5.

[0187] [Table 6]

[0188] A general vector map of the above construct is shown in Figure 1. IP-H4L1 is also formed of the IgG1 isotype as shown herein using a chemically synthesized human IgG1 constant region (SEQ ID NO: 157).

[0189] The amino acid sequences of the heavy and light chain variable regions are shown in Table 2, and the IgG1, IgG4 and light chain constant regions are shown at the end of the specification. In the same manner, the nucleotide sequences of the heavy chain variable regions, IgG1 and IgG4 constant regions and light chain are also shown at the end of the specification.

[0190] Example 2 Formation of anti-PD-1 mAb synthesized using pZRCII vector To form materials for proof of concept studies, the various anti-PD-1 antibody constructs (IP-H4L1, IP-H4.2L1, IP-H4.2(YH)L1, IP-H4.19L1, and IP-H4.36L1) generated in Example 1 were expressed using the ExpiCHO™ Expression System (Gibco). The day before transfection, ExpiCHO-S™ cells were cultured at 3×10 6 The cells were seeded at a final density of viable cells / mL into shake flasks / culture tubes and grown overnight. The following day, cells were counted and resuspended at 6×10 using fresh ExpiCHO™ Expression Medium pre-warmed to 37° C. 6The final density was adjusted to 10000 viable cells / mL. A complex of ExpiFectamine™ CHO transfection reagent and plasmid DNA was prepared. The solution was slowly added to the cell suspension with gentle mixing. The cells were incubated on an orbital shaker in a humidified atmosphere and 5% CO2 incubator at 37°C. The temperature was brought to 32°C on day 1. ExpiFectamine™ CHO enhancer and feed were added on day 1 post-transfection, followed by a second feed addition on day 5. The cultures were harvested (days 8-12) and one-step purified using an agarose-Protein A matrix (MAbselect SuRe antibody purification resin, GE). Bound antibodies were eluted using glycine buffer, pH 2.5, and neutralized to pH 7 with Tris buffer pH 9. The protein concentration of the purified antibodies was determined by Protein A HPLC method.

[0191] Example 3 Phage display Fab library construction (a) Fab plasmid construction For library construction, E. coli -optimized Fab regions with EcoRI and BamHI overhangs were chemically synthesized and cloned into pMA / pMK vectors obtained from Geneart, Germany.

[0192] The Fab genes (SEQ ID NO: 164) were isolated from the pMA vector construct after digestion with EcoRI and BamHI. The amino sequences of the cloned Fab genes are shown herein as SEQ ID NO: 165 for VH and SEQ ID NO: 166 for VL.

[0193] The pSEX81 (Cat. No. PR3005, Progen) phagemid vector was modified by cutting out the pIII gene of the phagemid vector, digesting with EcoRI and BamHI, and ligating the linearized vector with the digested Fab gene. The resulting modified vector is referred to herein as pSEX83. The vector map of pSEX83 is shown herein as FIG. 2. The digested Fab and pSEX83 vector fragments were analyzed on an agarose gel and purified from the gel using a QIAquick Gel Extraction Kit (Qiagen Cat. No. 28706). The ligation products of both the Fab (insert) and pSEX83 (vector) were transformed into E. coli TG1 electrocompetent cells (Cat. No. 60502-1, Lucigen). The transformed cells were plated on 2xYT agar plates containing ampicillin (100 μg / mL). Clones were analyzed by EcoRI and BamHI restriction digestion and DNA sequencing using the Sanger method.

[0194] (b) Library construction Two steps were taken to generate the Fab phage display library.

[0195] Step 1: PCR primer-based site-directed mutagenesis in CDRH1 Here, the CDRH1 region of the Fab was targeted to generate variants. Mutations were incorporated into the selected region by using PCR with the respective primer pairs (NI-CDRH1F and NI-CDRH1R) carrying a SapI restriction site (Table 6). Each primer pair was designed in such a way that they amplified the complete linear plasmid, which after restriction digestion with SapI followed by ligation with T4 DNA ligase resulted in a circular plasmid carrying the mutation incorporated by the primer pair.

[0196] 100 ng of template DNA of Fab genes in pSEX83 vector was used for amplification using the primer pairs mentioned in Table 6. Once the template was amplified using random primers, the products were digested with SapI to obtain sticky ends. After PCR purification, the digestion products were ligated overnight at 16°C. The ligated DNA was then transformed into freshly generated electrocompetent cells (TG1). The transformed cells were cultured for phage production. Phage production was performed separately for each library as previously described (9).

[0197] [Table 7A]

[0198] [Table 7B]

[0199] Step 2: Kunkel mutagenesis and sRCA Kunkel mutagenesis was performed using sRCA according to Huovinen et al., 2012. Small cultures of CJ236 cells were infected with phages carrying plasmids of CDRH1 mutant Fab genes. Infected cells were grown in 2xYT containing uridine (6 μg / ml) and carbenicillin antibiotic (100 μg / ml). After superinfection with VCS M13 helper phage (Agilent technologies, USA), phages were produced and purified by precipitation with PEG6000 (4%) and NaCl (500 mM). Single-stranded uridylated DNA (ss(U)DNA) was extracted from purified phages by using the M13 purification kit of the EZNA® M13 DNA Mini Kit (OMEGA bio-tek, USA) according to the manufacturer's instructions.

[0200] This ss(U)DNA was used as a template in Kunkel mutagenesis, and various primers with random mutations were used to generate libraries. Primers targeting different CDRs of Nivolumab Fab, namely CDRH2, CDRH3, and CDRL3 (Table 6), were separately hybridized to the ss(U)DNA template and extended by Kunkel mutagenesis. The products were UDG treated and selectively amplified by RCA (10). The RCA products were digested with HindIII, circularized using T4 DNA ligase, and transformed into SS320 cells. Phages (secondary libraries) were generated separately for each mutagenic primer by superinfection with helper phage as shown below.

[0201] Example 4 Mutant Fab phage production and purification Cells from the glycerol stocks of the above libraries were cultured in flasks containing 2xYT medium containing carbenicillin or ampicillin (100 μL / mL) to an OD of 0.06. 600 The cultures were grown at 37 °C with 250 rpm shaking until they reached a maximum OD of approximately 0.4–0.6. 600 The cultures were then grown to a multiplicity of infection (MOI) of 20. Helper phage, either VCSM13 (Agilent, Cat. No. 200251) or M13KO7 (GE healthcare, Cat. No. 27152401), was then added to the cultures, first incubated at 37°C for 40 min without shaking, followed by another 40 min at 37°C with shaking. Kanamycin was then added to the medium, and the cultures were grown overnight at 26°C and 150 rpm. The phage overnight cultures were centrifuged at 4000g, and the cell pellet was discarded. A 1:5 ratio of PEG (20%) / NaCl (2.5M) solution was added to the supernatant to precipitate the phage. The resuspended solution was incubated on ice for 20 min, followed by centrifugation at 14,000g for 15 min at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 mL of sterile PBS containing 0.01% sodium azide. The phages were stored at 4° C. until further use.

[0202] Example 5 Biopanning of phages expressing high affinity Fab variants The library prepared in Example 4 (1 × 10 12 pfu) were screened for high affinity Fab binders to the PD1 receptor. The first round of panning against recombinant PD1 receptor antigen (Sino biological, Cat. No. 10377-H08H) at pH 7.4 was performed using antigen-immobilized immunotubes (Quidel, USA) prepared by incubation with 5 μg / mL PD1 protein solution in carbonate buffer (0.1 M, pH 9.6) overnight at 4 °C. The immunotubes were washed three times with PBS and then incubated with phages in PBS at 25 °C for 2 h with constant rotation. The tubes were washed 10 times with 4 mL PBS containing Tween 20 (0.1%) and then 10 times with PBS. Bound phages were eluted with glycine·HCL, pH 2.1 (0.1 M). Eluted phages were rescued by infection of plated TG1 E. coli cells and phages were generated as described in Example 4.

[0203] The second and third rounds of panning were performed using the same plasmid p53 as the first round (1 × 10 11 CFU) and second (1 × 10 10 Output phages from panning rounds 100-150 CFU were run on PD1 antigen-coated immunotubes in such a manner that they were used as input phages for the second and third panning rounds, respectively. Phages after the third panning round were infected into TG1 cells as described above, and phagemid DNA was isolated for anti-PD-1 cloning in a suitable expression vector.

[0204] Example 6 Screening of individual anti-PD-1 clones as soluble Fab proteins Cloning into expression vectors and protein production The anti-PD-1 genes from the enriched libraries generated after three rounds of panning were cloned into the expression vector pOPE101 (carbenicillin resistant) (Progen, Germany) so that individual anti-PD-1 clones could be generated as His-tagged fusion products. Both the vector DNA (pOPE101) and the phagemid DNA were digested with restriction enzymes (EcoRI and BamHI) to isolate the vector and anti-PD-1 gene, respectively. Both the restriction digested vector and anti-PD-1 gene were ligated and transformed into TG1 electrocompetent cells. The transformed cells were plated on 2xYT agar plates containing carbenicillin antibiotic. Individual clones were picked from the 2xYT agar plates and cultured overnight at 32°C, 200 rpm in 15 mL tubes containing 5 mL of 2xYT medium containing carbenicillin or ampicillin (100 μL / mL). The following day, the cultures were re-inoculated into fresh 2xYT medium containing carbenicillin (100 μg / mL) and glucose (0.1%) in a volume ratio of 1:200. The cultures were cultured at OD 600 The cells were grown until the β-D-1-thiogalactopyranoside (IPTG) was approximately 0.6–0.8. 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was then added to the culture and grown overnight at 30°C, 200 rpm. The overnight culture was spun at 10,800 g for 15 min and the supernatant was removed. The cell pellet was resuspended in 1 / 20 volume of the original culture in 1× PBS buffer, pH 7.4, containing 2 mg / ml lysozyme, 0.1% Triton-X and 1 U / 100 mL benzonase and incubated at 37°C for 1 h. The resuspended pellet was centrifuged at 10,800 g for 15 min and the supernatant was collected as the cell lysate containing the soluble His-tagged Fab variants. This cell lysate fraction was used in an immunoassay to investigate anti-PD-1 binding after quantification of soluble Fab in the periplasmic fraction.

[0205] Measurement of the concentration of soluble anti-PD-1 Fab in the periplasmic fraction Clones were cultured individually for the generation of soluble Fab variants as described above. Total Fab in cell lysates or periplasmic fractions was quantified by immobilizing soluble Fab on maxisorp plates precoated with (Fab-specific) goat anti-human IgG antibody (Sigma-Aldrich, Cat. No. I5260). Coating was performed overnight with (Fab-specific) goat anti-human IgG antibody (Sigma-Aldrich, Cat. No. I5260) at a concentration of 1 μg / mL (100 μl / well) in coating buffer (0.1 M NaHCO3, pH 9.6). After washing the plates twice, 100 μL of cell lysate (1 / 20 v / v in 1×PBS) was added and incubated for 1 h at 25°C. Serially diluted human Fabs of known concentrations were used as standards for the calculation of the amount of Fab protein in the cell lysates. Plates were washed three times with PBST (0.1% Tween 20 in 1×PBS). 100 μl (1:10000) of (Fab-specific) HRP-conjugated goat anti-human IgG antibody (Sigma-Aldrich, Cat. No. A0293) was added to each well of the plate and incubated for 1 hour. The plate was then washed 5 times with PBST, followed by addition of the substrate o-phenylenediamine dihydrochloride (OPD) (100 μL / well) for 15 minutes at 37°C. The reaction was stopped using 1N H2SO4 (100 uL / well) and the optical density (OD) was measured at 450 nm using a TECAN INFINITE® M1000pro. The concentration of Fab in the cell lysate was then calculated using the reference standard.

[0206] Qualitative analysis of anti-PD-1 Fab binders Recombinant PD1 antigen in coating buffer (0.1M NaHCO3, pH 9.6) was coated onto polystyrene plates (100ng / 100μL / well) overnight at 4°C. After washing the plates twice, 100ng of Fab binder diluted in 100μl of PBS, pH 6.0 was added to each well and incubated at 25°C for 1 hour. The plates were washed 4 times with PBST (0.1% Tween 20 in 1x PBS, pH 6.0) followed by addition of 100μl (1:10000) of (Fab-specific) HRP-conjugated goat anti-human IgG antibody (Sigma-Aldrich, Cat. No. A0293) and incubation at 25°C for 1 hour. The plates were then washed 5 times with PBST followed by addition of the substrate o-phenylenediamine dihydrochloride (OPD) (100μL / well) for 15 minutes at 37°C. The reaction was stopped using 1N H2SO4 (100 uL / well) and absorbance was measured at 450 nm using a TECAN INFINITE® M1000pro. Individual clones with relatively high OD signals compared to the parental Fab were shortlisted for further characterization.

[0207] Example 7 Construction of pZRCIII hyg anti-PD-1 light and heavy chain vectors Based on the results from the qualitative analysis of the anti-PD-1 Fab binders, five clones were shortlisted, and these five clones are referred to herein as N5, N6, N7, N9, and N10 based on the HCVR regions of the Fab binders.

[0208] Genes encoding the heavy chain variable regions of N5, N6, N7, N9 and N10 (amino acid sequences SEQ ID NOs: 146-150 and nucleotide sequences SEQ ID NOs: 169-173, respectively) and the light chain genes (amino acid and nucleotide sequences SEQ ID NOs: 167 and 174, respectively) were chemically synthesized and cloned into pMA vector (GeneArt, Germany). To clone the light chains into mammalian expression vectors, the pZRCIII hyg.IgG4 vector was digested with XhoI and KpnI restriction enzymes. The light chain inserts digested with the same restriction enzymes were ligated with XhoI and KpnI digested pZRCIII hyg.IgG4 vector and transformed into E. coli Top 10F' strain. Transformants were selected on the basis of kanamycin resistance. Plasmid DNA isolated from such colonies was analyzed for the presence of the light chain genes by restriction digestion with XhoI and KpnI restriction enzymes. One of the plasmids showing the expected restriction profile was further digested with SalI and ApaI restriction enzymes to insert the genes encoding the heavy chain variable regions of N5, N6, N7, N9 and N10 (amino acid sequences SEQ ID NOs: 146-150 and nucleotide sequences SEQ ID NOs: 169-173, respectively) in frame with the IgG4 constant region of the pZRCIII hyg.IgG4 vector (amino acid and nucleotide sequences SEQ ID NOs: 168 and 175, respectively). The SalI and ApaI digested vector and insert were ligated and transformed into E. coli Top 10F' strain. Transformants were selected on the basis of kanamycin resistance. Plasmid DNA from the transformed cells was digested with SalI and ApaI restriction enzymes to confirm the presence of each variable region. The final vectors containing a light chain of SEQ ID NO: 174 and a heavy chain of SEQ ID NO: 169; a light chain of SEQ ID NO: 174 and a heavy chain of SEQ ID NO: 170; a light chain of SEQ ID NO: 174 and a heavy chain of SEQ ID NO: 171; a light chain of SEQ ID NO: 174 and a heavy chain of SEQ ID NO: 172; and a light chain of SEQ ID NO: 174 and a heavy chain of SEQ ID NO: 173 were confirmed by nucleotide sequencing of the heavy and light chains. These final vectors were named N5NL, N6NL, N7NL, N8NL, N9NL and N10NL, respectively.

[0209] The various constructs above encode the amino acid sequences of the HCVR and LCVR of the anti-PD-1 antibodies of the invention as set out in Table 7.

[0210] [Table 8]

[0211] A general vector map of the above construct is shown in FIG.

[0212] The amino acid sequences of the heavy and light chain variable regions are shown in Table 2, and the IgG1, IgG4 and light chain constant regions are shown at the end of the specification. In the same manner, the nucleotide sequences of the heavy chain variable regions, IgG1 and IgG4 constant regions and light chain are also shown at the end of the specification.

[0213] Example 8 Formation of anti-PD-1 mAb synthesized using pZRCIII vector To form material for proof of concept studies, the various anti-PD-1 antibody constructs (N5NL, N6NL, N7NL, N9NL, and N10NL) generated in Example 7 were expressed in CHO-S cells (Invitrogen). Cells were uniformly cultured in Lonza's CDM medium. Cells were seeded approximately 24 hours prior to transfection. Transfection was performed by electroporation using the Neon Transfection system (Invitrogen) with pre-optimized conditions. After transfection, cells were seeded in 24-well plates containing 1 ml of pre-warmed serum-free medium. Cells were incubated in a humidified incubator at 37°C in the presence of 5% CO2. Selection of transfected pools was performed in serum-free medium in the presence of 600 μg / ml hygromycin. During the selection process, the cell numbers of the pools were monitored regularly over a period of 2-3 weeks, and regular medium changes were performed. Anti-PD-1 expressing stable transfectant pools were expanded and antibody was produced using fed-batch cultures. 0.3 × 10 6 Cells were seeded in chemically defined medium at a density of 1000000000 cells / ml. Flasks were incubated in a humidified Kuhner incubator shaker at a temperature of 37°C, 5% CO2 level, and a shaking speed of 110 RPM. A fixed daily feeding regime was followed during the entire culture period. Chemically defined feed from Hyclone, GE was used. Feeding was started 72 hours after culture and continued until culture harvest. Cultures were harvested (days 8–12) and subjected to one-step purification using an agarose-Protein A matrix (MAbselect SuRe antibody purification resin, GE). Bound antibodies were eluted using glycine buffer, pH 2.5, and neutralized to pH 7 with Tris buffer pH 9. Protein concentrations of purified antibodies were determined by Protein A HPLC method.

[0214] Example 9 Affinity analysis of anti-human PD-1 antibodies using surface plasmon resonance (SPR) The affinity rate constants for the binding of anti-PD-1 antibody candidates (IP-H4L1 (IgG4), IP-H4L1 (IgG1), IP-H4.2L1, IP-H4.19L1, IP-H4.36L1, N5NL, N6NL, N7NL, N9NL, and N10NL) to human PD-1 were measured using Proteon XPR (Biorad).

[0215] Human PD-1 (AcroBiosystems) was directly immobilized on a GLC chip (ProteOn) using the amine coupling method to obtain a ligand immobilization level of approximately 500 RU. Kinetic measurements were performed using 10 mM phosphate-buffered saline (PBS) (10 mM phosphate buffer, pH 7.4, 300 mM NaCl, 0.005% surfactant P20) as the running buffer. All of the one-step purified antibody samples were diluted in the above running buffer at suitable concentrations in a series of concentrations from 10 nM to 0.09 nM. These samples were injected onto the chip at a flow rate of 50 μL / min, with an association time of 300 s and a dissociation time of 1800 s. After each sample run, the chip surface was regenerated with 10 mM glycine buffer, pH 1.5 for 18 s at a flow rate of 100 μL / min.

[0216] K D The value (rate constant) was calculated using ProteOn Manager software v3.1.0.6 as the ratio of the dissociation rate (kd) to the association rate (ka), i.e., K D = kd / ka.

[0217] The results are shown in Table 8. The data was analyzed using the Langmuir binding model.

[0218] [Table 9]

[0219] Example 10 Analysis of anti-PD-1 antibodies using a flow cytometry assay in PBMC cells Monocyte-depleted human peripheral blood mononuclear cells (PBMCs) were activated with plate-coated anti-CD3 antibody (clone UCHT-1, BD Biosciences) for 3 days. After washing, cells were incubated with different concentrations of anti-PD-1 antibodies IP-H4.2L1, IP-H4L1 (IgG1) and IP-H4L1 (IgG4). Fcγ fragment-specific R-phycoerythrin-conjugated F(ab) was used as the secondary reagent. 2 Samples were evaluated using a flow cytometer (BD FACS Canto™ II) to assess binding of these anti-PD-1 antibodies to PD1 expressed on activated cells using fragment goat anti-human IgG. Binding data are shown in Figure 4, which shows the binding specificity of IP-H4.2L, IP-H4L1(IgG1) and IP-H4L1(IgG4) to human PD1 expressed on cells.

[0220] Example 11 Analysis of anti-PD-1 antibodies using flow cytometry assay in HEK-293T cells HEK293T cells stably transfected to express human PD-1 were incubated with different concentrations of anti-PD-1 antibodies IP-H4.19L1, IP-H4.2L1, and IP-H4.36L1. Binding of these antibodies to PD1 on HEK-293T cells was assessed using a flow cytometer (BD FACS Canto™ II) with Fcγ fragment-specific R-phycoerythrin-conjugated F(ab) as the secondary reagent. 2 Fragment goat anti-human IgG was used to assess the binding specificity of IP-H4.19L1, IP-H4.2L1 and IP-H4.36L1 to human PD1 expressed on cells. The data are shown in Figure 5.

[0221] In a similar manner, the other anti-PD-1 antibodies N5NL, N6NL, N7NL, N9NL and N10NL were analyzed using a flow cytometry assay in HEK-293T cells, and the data are shown in Figure 6.

[0222] Example 12 Analysis of anti-PD-1 antibodies in mixed lymphocyte reactions Dendritic cells (DCs) were formed by culturing monocytes isolated from PBMCs in vitro for 7 days with 50 ng / mL interleukin-4 (IL-4) and 100 ng / mL GM-CSF (11). Monocyte-depleted PBMCs (5 × 10 4 ) and the same type of DC (1 × 10 4 ) were co-cultured. After 5 days, culture supernatants were collected and analyzed for IL2 and IFN-gamma secretion by ELISA (BD Biosciences). The increase in either IL-2 or IFN-gamma secretion by a given anti-PD-1 antibody was calculated after subtracting the value obtained with the IgG4 isotype control for the respective group. The results for IL2 and IFN-gamma are shown in Figures 7 and 8, respectively. The results obtained show that the anti-PD-1 antibodies of the invention are able to promote the secretion of IL-2 and IFN-gamma by T cells in an MLR.

[0223] In a similar manner, the other anti-PD-1 antibodies N5NL, N6NL, N9NL and N10NL were analyzed in a mixed lymphocyte reaction for analysis of increased IL2 and IFN-gamma secretion by ELISA (BD Biosciences). The IL2 and IFN-gamma results are shown in Figures 9 and 10, respectively.

[0224] List of amino acid sequences used in the present invention: SEQ ID NO:153 - IgG4 constant region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:154 - IgG1 constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 155 - Light chain constant region of IP-L1 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:165 - Heavy chain variable region of Nivolumab (synthetic) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC SEQ ID NO: 166 - Light chain of Nivolumab (synthetic) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 167 - Light chain of NL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:168 - IgG4 constant region PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0225] List of nucleotide sequences used in the present invention: SEQ ID NO: 156 - IgG4 constant region GCCTCCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCCTCCAGCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCCCCCTGCCCTGCCCCTGAATTTCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCTCGGCTGACCGTGGACAAGTCCCGGTGGCAGGAAGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCTGGGTAAGTGATGA SEQ ID NO:157 - IgG1 constant region GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Heavy chain variable region of SEQ ID NO: 158 - IP-H4 CAGGTGCAGCTGGTGCAGTCCGGCGTGGAAGTGAAGAAACCTGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCACCTACTACATCTACTGGGTCCGACAGGCCCCAGGCCAGGGCCTGGAATGGATGGGCGGCGTGAACCCTTCCAACGGCGGCACCAACTACAACGAGAACTACAAGAACAGAGTGACCCTGACCACCGACTCCTCCACCACCACCGCCTACATGGAACTGAAGTCCCTGCAGTTCGACGACACCGCCGTGTACTACTGCGCCAGACGGGACTACAGATACGACATGGGCTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCT Heavy chain variable region of SEQ ID NO: 159 - IP-H4.2 CAGGTTCAATTGGTGCAGTCCGGCGTGGAAGTGAAGAAACCTGGCGCTTCTGTGAAGGTGTCCTGCAAGGCCTCTGGCTACACCTTTACCACCTACTACATCTACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCGTGAACCCTTCTAACGGCGGCACCAACTACAACGAGAACTACAAGAACAGAGTGACCCTGACCACCGACTCCTCTACCACCACCGCCTACATGGAACTGAAGTCCCTGCAGTTCGACGACACCGCCGTGTACTACTGCGCCAGAAGAGACTACAGATACGACATGGGCTACGACTACTGGGGCCAGGGCACAACAGTGACCGTGTCCTCT Heavy chain variable region of SEQ ID NO: 160 - IP-H4.2(Y.H) CAGGTGCAGCTGGTGCAGTCCGGCGTGGAAGTGAAGAAACCTGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCACCTACTACATCTACTGGGTCCGACAGGCCCCAGGCCAGGGCCTGGAATGGATGGGCGGCGTGAACCCTTCCAACGGCGGCACCAACTACAACGAGAACTACAAGAACAGAGTGACCCTGACCACCGACTCCTCCACCACCACCGCCTACATGGAACTGAAGTCCCTGCAGTTCGACGACACCGCCGTGTACTACTGCGCCAGACGGGACTACAGATACGACATGGGCCATGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCTGCCTCCACCAAGGGCCC Sequence number 161 - Heavy chain variable region of IP-H4.19 CAGGTTCAATTGGTGCAGTCCGGCGTGGAAGTGAAGAAACCTGGCGCTTCTGTGAAGGTGTCCTGCAAGGCCTCTGGCTACACCTTTACCACCTACTACATCTACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCGTGAACCCTTCCAACTCCGGCACCAACTACAACGAGAACTACAAGAACAGAGTGACCCTGACCACCGACTCCTCTACCACCACCGCCTACATGGAACTGAAGTCCCTGCAGTTCGACGACACCGCCGTGTACTACTGCGCCAGAAGAGACTACAGATACGACATGGGCTTCGACTACTGGGGCCAGGGCACAACAGTGACCGTGTCCTCT Sequence number 162 - Heavy chain variable region of IP-H4.36 CAGGTTCAATTGGTGCAGTCCGGCGTGGAAGTGAAGAAACCTGGCGCTTCTGTGAAGGTGTCCTGCAAGGCCTCTGGCTACACCTTTACCACCTACTACATCTACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCGTGAACCCTTCTAACGGCGGCACCAACTACAACCAGAACTACAAGAACAGAGTGACCCTGACCACCGACTCCTCTACCACCACCGCCTACATGGAACTGAAGTCCCTGCAGTTCGACGACACCGCCGTGTACTACTGCGCCAGAAGAGACTACAGATACGACATGGGCTTCGACTACTGGGGCCAGGGCACAACAGTGACCGTGTCCTCT Sequence number 163 - Light chain of IP-L1 GAGATCGTGCTGACCCAGTCCCCCGCCACCCTGTCTCTGTCTCCCGGCGAGAGAGCCACCCTGAGCTGCAGAGCCTCCAAGGGCGTGTCCACCTCCGGCTACTCCTACCTGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCTACCTGGCCTCCTACCTGGAATCCGGCGTGCCCGCCAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCACTCCCGGGACCTGCCCCTGACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGCTAATGAGGTACC Sequence number 164 - Fab gene of nivolumab SEQ ID NO:169 - Heavy chain variable region of N5 CAGGTTCAATTGGTTGAATCTGGCGGCGGAGTGGTGCAGCCTGGAAGAAGTCTGAGACTGGATTGCAAGGCCTCCGGCATCACCTTTCTCCAACTCTGGCATGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGGTCGCCGTGATTTGGTACGACGGCTCTAAGCGGTACT ACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTTTCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCACCAACTCTGATTTTTGGGGCCAGGGCACACTGGTCACCGTGTCCTCTGCTTCTACAAAGGGCCC SEQ ID NO:170 - Heavy chain variable region of N6 CAGGTTCAATTGGTTGAATCTGGCGGCGGAGTGGTGCAGCCTGGAAGAAGTCTGAGACTGGATTGCAAGGCCTCCGGCATCACCTTTCTCCAACTCTGGCATGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGGTCGCCGTGATTTGGTACGACGGCTCTAAGCGGTACT ACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTTTCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCACCAATTCTGATCATTGGGGCCAGGGCACACTGGTCACCGTGTCCTCTGCTTCTACAAAGGGCCC SEQ ID NO:171 - Heavy chain variable region of N7 CAGGTTCAATTGGTTGAATCTGGCGGCGGAGTGGTGCAGCCTGGAAGAAGTCTGAGACTGGATTGCAAGGCCTCCGGCATCACCTTCTCCAACTCTGGCATGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGGTCGCCGTGATTTGGTACGACGGCTCTAAGCGGTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTTTCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCACCAACTCTGATTATTGGGGCCAGGGCACACTGGTCACCGTGTCCTCTGCTTCTACAAAGGGCCC Sequence number 172 - Heavy chain variable region of N9 CAGGTTCAATTGGTTGAATCTGGCGGCGGAGTGGTGCAGCCTGGAAGAAGTCTGAGACTGGATTGCAAGGCCTCCGGCATCACCTTCTCCAACTCTGGCATGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGGTCGCCGTGATTTGGTACGACGGCTCTAAGCGGTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTTTCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCACAAATACCGATTGGTGGGGCCAGGGCACACTGGTCACAGTGTCCTCTGCTTCCACAAAGGGCCC Sequence number 173 - Heavy chain variable region of N10 CAGGTTCAATTGGTTGAATCTGGCGGCGGAGTGGTGCAGCCTGGAAGAAGTCTGAGACTGGATTGCAAGGCCTCCGGCATCACCTTCTCCAACTCTGGCATGCACTGGGTCCGACAGGCCCCTGGAAAAGGACTGGAATGGGTCGCCGTGATTTGGTACGACGGCTCTAAGCGGTACTACGCCGACTCCGTGAAGGGCAGATTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTTTCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCACCAATACCGATTATTGGGGCCAGGGCACACTGGTCACCGTGTCCTCTGCTTCTACAAAGGGCCC Sequence number 174 - Light chain of NL GAGATCGTGCTGACCCAGTCCCCCGCCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCTCCCAGTCCGTGTCCTCCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCTACGACGCCTCCAACCGGGCCACCGGCATCCCTGCCAGATTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTCCTCCAACTGGCCCCGGACCTTCGGCCAGGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTCTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGCTGATGA Sequence number 175 - IgG4 constant region GGGCCCCTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCCTCCAGCCTGGGCACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCCCCCTGCCCTGCCCCTGAATTTCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCTCCAGCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCTCGGCTGACCGTGGACAAGTCCCGGTGGCAGGAAGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCTGGGCAAGTGATGA

[0226] References incorporated into this patent application: (References) [Table 10]

[0227] Incorporation by Reference The entire disclosure of each patent document and scientific article referred to herein is incorporated by reference for all purposes.

[0228] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative and not limiting of the invention described herein. Thus, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. An anti-PD-1 antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 149 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

152.

2. 2. The anti-PD-1 antibody, or antigen-binding portion thereof, of claim 1 , wherein the anti-PD-1 antibody, or antigen-binding portion thereof, comprises an IgG4 constant region having the amino acid sequence of SEQ ID NO: 153, which contains a single amino acid substitution selected from S228P, P329G, M428L, and N434S.

3. The anti-PD-1 antibody, or antigen-binding portion thereof, has at least one of the following characteristics: (a) cross-reacts with PD-1 from species other than human; (b) higher binding specificity for human PD-1; (c) promoting IL-2 cytokine secretion; (d) promotes higher IFN-gamma secretion; (e) blocking the interaction of the PD-1 receptor with its natural ligands PD-L1 and / or PD-L2; (f) promoting T cell proliferation; (g) an extended half-life in the subject; 2. The anti-PD-1 antibody, or antigen-binding portion thereof, of claim 1 , comprising:

4. 4. A nucleic acid sequence encoding the anti-PD-1 antibody, or antigen-binding portion thereof, of any one of claims 1 to 3, or an expression vector comprising said nucleic acid sequence.

5. A medicament for the treatment of a disease modulated by the interaction of PD-1 with PDL1 and / or PDL2, comprising an anti-PD-1 antibody or an antigen-binding portion thereof according to any one of claims 1 to 3.

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