Human antibodies to PD-1

Abstract

To provide antibodies that bind to T-cell co-inhibitor programmed death-1 (PD-1) protein, and methods of use.SOLUTION: In various embodiments of the invention, the antibodies are fully human antibodies that bind to PD-1. In certain embodiments, the present invention provides multi-specific antigen-binding molecules comprising a first binding specificity that binds to PD-1 and a second binding specificity that binds to an autoimmune tissue antigen, another T-cell co-inhibitor, an Fc receptor, or a T-cell receptor. In some embodiments, the antibodies of the invention are useful for inhibiting or neutralizing PD-1 activity, thus providing a means of treating a disease or disorder such as cancer or a chronic viral infection. In other embodiments, the antibodies are useful for enhancing or stimulating PD-1 activity, thus providing a means of treating, for example, an autoimmune disease or disorder.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to human antibodies and antigen-binding fragments of human antibodies that specifically bind to the immunoregulatory receptor Programmed Cell Death 1 (PD-1) and to therapeutic and diagnostic methods using the antibodies. [Background technology]

[0002] Programmed cell death 1 (PD-1) (also called CD279) is a 288 amino acid protein 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 costimulatory molecule) family of T cell co-inhibitory receptors (Non-Patent Document 1). The main function of PD-1 is to attenuate immune responses (Non-Patent Document 2). PD-1 has two ligands, PD-ligand 1 (PD-L1) and PD-L2. PD-L1 (CD274, B7H1) is widely expressed on both lymphoid and non-lymphoid tissues, such as CD4 and CD8 T cells, macrophage lineage cells, peripheral tissues, and tumor cells, virally 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 (Non-Patent Document 3). PD-L1 is expressed in most 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 is inducible in almost all cancer types (Non-Patent Document 4). When PD-1 binds to its ligand, T cell proliferation and cytokine secretion are reduced, and humoral and cellular immune responses are impaired in diseases such as cancer, viral infection, and autoimmune diseases. Blockade of PD-1 binding to reverse immune suppression has been investigated in autoimmune, viral, and tumor immunotherapy (Non-Patent Document 5, Non-Patent Document 6, Non-Patent Document 7).

[0003] T cell costimulatory and co-inhibitory molecules (collectively named co-signaling molecules) play a crucial role in controlling T cell activation, subset differentiation, effector function and survival (Non-Patent Document 1). After recognition of cognate peptide-MHC complexes on antigen-presenting cells by T cell receptors, co-signaling receptors coexist with T cell receptors in the immune synapse, where they cooperate with TCR signaling to promote or inhibit T cell activation and function (Non-Patent Document 8). The final immune response is controlled by the balance between costimulatory and co-inhibitory signals ("immune checkpoints") (Non-Patent Document 9). PD-1 functions as one such "immune checkpoint" in mediating peripheral T cell tolerance and avoiding autoimmunity. PD-1 binds to PD-L1 or PD-L2 and inhibits T cell activation. The ability of PD1 to inhibit T cell activation is exploited by chronic viral infections and tumors to evade immune responses. In chronic viral infections, PD-1 is highly expressed on virus-specific T cells, and these T cells become "exhausted" and lose effector function and proliferation ability (Non-Patent Document 10). PD-L1 is expressed on a wide range of tumors, and studies in animal models have shown that PD-L1 on tumors inhibits T cell activation and tumor cell lysis, leading to increased death of tumor-specific T cells. The PD-1:PD-L1 system also plays an important role in induced T regulatory (Treg) cell development and in maintaining Treg function (Non-Patent Document 11).

[0004] PD-1 plays an important role in autoimmunity, tumor immunity, and infection immunity, making it an ideal target for immunotherapy. Blockade of PD-1 with antagonists, including monoclonal antibodies, has been investigated in the treatment of cancer and chronic viral infections (Non-Patent Document 12).

[0005] Monoclonal antibodies against PD-1 are known in the art and are described, for example, in U.S. Patent No. 5,393,633, U.S. Patent No. 5,393,663, U.S. Patent No. 5,393,675, U.S. Patent No. 5,393,685, U.S. Patent No. 5,393,696, and in U.S. Patent No. 5,403,363, U.S. Patent No. 5,403,675, U.S. Patent No. 5,403,363 ... and U.S. Patent No. 5,403,363. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 8008449 [Patent Document 2] U.S. Patent No. 8,168,757 [Patent Document 3] U.S. Patent Application Publication No. 20110008369 [Patent Document 4] U.S. Patent Application Publication No. 20130017199 [Patent Document 5] U.S. Patent Application Publication No. 20130022595 [Patent Document 6] WO2006121168 [Patent Document 7] WO20091154335 [Patent Document 8] WO2012145493 [Patent Document 9] WO2013014668 [Patent Document 10] WO2009101611 [Patent Document 11] EP2262837 [Patent Document 12] EP2504028 [Non-patent literature]

[0007] [Non-Patent Document 1] Chen et al. 2013, Nat. Rev. Immunol. 13:227-242 [Non-Patent Document 2] Riley2009, Immunol.Rev.229:114~125 [Non-Patent Document 3] Dong et al. 1999, Nature Med. [Non-Patent Document 4] Zou and Chen 2008, Nat. Rev. Immunol. 8:467-77 [Non-Patent Document 5] Ribas2012, NEJM366:2517~2519 [Non-Patent Document 6] Watanabe et al. 2012, Clin.Dev.Immunol. Volume 2012, Article ID: 269756 [Non-Patent Document 7] Wang et al. 2013, J. Viral Hep. 20:27-39 [Non-Patent Document 8] Flies et al. 2011, Yale J. Biol. Med. 84:409-421 [Non-Patent Document 9] Pardoll 2012, Nature 12: pp. 252-264. [Non-Patent Document 10] Freeman2008, PNAS105:10275~10276 [Non-Patent Document 11] Francisco et al. 2010, Immunol. Rev. 236:219-242 [Non-Patent Document 12] Sheridan 2012, Nature Biotechnology 30: 729-730. Summary of the Invention [Means for solving the problem]

[0008] The present invention provides antibodies, and antigen-binding fragments thereof, that bind to PD-1. The antibodies of the invention are useful, inter alia, for targeting T cells that express PD-1 and for modulating PD-1 activity. In certain embodiments, the antibodies of the invention are useful for inhibiting or neutralizing PD-1 activity and / or inhibiting, for example, T cell-mediated killing. The antibodies are useful for stimulating T cell activation under circumstances where it is beneficial or desirable. In an alternative embodiment, the antibodies are used to enhance PD-1 binding and / or activity and inhibit T cell activation. The anti-PD-1 antibodies or antigen-binding portions thereof of the present invention are included as part of a multispecific antigen-binding molecule, for example, to regulate immune responses and / or to target the antibodies to specific cell types, such as tumor cells, autoimmune tissue cells, or virally infected cells. The antibodies are useful in treating diseases or disorders, such as cancer, viral infections, and autoimmune diseases.

[0009] The antibodies of the invention are full length (e.g., IgG1 or IgG4 antibodies) or contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and are modified to affect functionality, e.g., to remove residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, the antibodies are bispecific.

[0010] In a first aspect, the present invention provides an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to PD-1. In certain embodiments, the antibody is fully human. Exemplary anti-PD-1 antibodies of the present invention are listed in Tables 1-3 herein. Table 1 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) of exemplary anti-PD-1 antibodies. Table 2 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-PD-1 antibodies. Table 3 lists the amino acid sequence identifiers of the heavy and light chain sequences of exemplary anti-PD-1 antibodies.

[0011] The present invention provides antibodies or antigen-binding fragments thereof comprising an HCVR that comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0012] The present invention also provides antibodies or antigen-binding fragments thereof comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0013] The present invention also provides antibodies, or antigen-binding fragments thereof, that comprise an HCVR and LCVR amino acid sequence pair (HCVR / LCVR), comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, that comprise an HCVR / LCVR amino acid sequence pair contained in any of the exemplary anti-PD-1 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 202, 218 / 202, 226 / 202, 234 / 202, 242 / 202, 250 / 202, 258 / 202, 266 / 202, 274 / 202, 282 / 202, 290 / 202, 298 / 186, 306 / 186, and 314 / 186. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 130 / 138 (e.g., H2M7795N), 162 / 170 (e.g., H2M7798N), 234 / 202 (e.g., H4xH9048P), or 314 / 186 (e.g., H4xH9008P).

[0014] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0015] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0017] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0018] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0019] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0020] The present invention also provides antibodies or antigen-binding fragments thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-PD-1 antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 136 / 144 (e.g., H2M7795N), 168 / 176 (e.g., H2M7798N), 240 / 208 (e.g., H4xH9048P), and 320 / 192 (e.g., H4xH9008P).

[0021] The present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain comprising an amino acid sequence selected from any of the HC amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0022] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain comprising an amino acid sequence selected from any of the LC amino acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0023] The present invention also provides antibodies or antigen-binding fragments thereof comprising a HC and LC amino acid sequence pair (HC / LC), comprising any of the HC amino acid sequences listed in Table 3 paired with any of the LC amino acid sequences listed in Table 3. According to certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising an HC / LC amino acid sequence pair contained in any of the exemplary anti-PD-1 antibodies listed in Table 3. In certain embodiments, the HC / LC amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 330 / 331, 332 / 333, 334 / 335, and 336 / 337.

[0024] The present invention also provides antibodies or antigen-binding fragments thereof that comprise a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-PD-1 antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 132-134-136-140-142-144 (e.g., H2M7795N); 164-166-168-172-174-176 (e.g., H2M7798N); 236-238-240-204-206-208 (e.g., H4xH9048P); and 316-318-320-188-190-192 (e.g., H4xH9008P).

[0025] In a related embodiment, the invention provides an antibody or antigen-binding fragment thereof that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-PD-1 antibodies listed in Table 1. For example, the invention includes an antibody or antigen-binding fragment thereof that comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained in an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 130 / 138 (e.g., H2M7795N); 162 / 170 (e.g., H2M7798N); 234 / 202 (e.g., H4xH9048P); and 314 / 186 (e.g., H4xH9008P). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Typical conventions used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological See, "Academic Press, 1999," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0026] The present invention includes anti-PD-1 antibodies with modified glycosylation patterns. In certain embodiments, modifications are useful to remove undesirable glycosylation sites or antibodies lacking fucose moieties present on the oligosaccharide chains, e.g., to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications are made to modify complement-dependent cytotoxicity (CDC).

[0027] The present invention also provides the CDRs of the HCVR and the CDRs of the LCVR for specific binding to PD-1. Also provided are antibodies and antigen-binding fragments thereof that compete with an antibody or antigen-binding fragment thereof comprising the CDRs, wherein the HCVR and LCVR, respectively, have amino acid sequences selected from the HCVR and LCVR sequences listed in Table 1.

[0028] The invention also provides isolated antibodies and antigen-binding fragments thereof that block PD-1 binding to PD-L1 or PD-L2. In one embodiment, the antibodies or antigen-binding fragments thereof that block PD-1 binding to PD-L1 bind to the same epitope on PD-1 as PD-L1 or bind to a different epitope on PD-1 as PD-L1.

[0029] In alternative embodiments, the invention provides antibodies or antigen-binding fragments thereof that agonize PD-1 binding to PD-L1. In certain embodiments, the invention provides isolated antibodies or antigen-binding fragments thereof that bind to PD-1, wherein the antibodies or antigen-binding fragments thereof enhance PD-1 binding to PD-L1. In certain embodiments, the isolated antibodies or antigen-binding fragments thereof comprise a CDR of an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 98, and 250, and a CDR of an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 106, and 202. In certain embodiments, the isolated antibodies or antigen-binding fragments thereof comprise a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 (e.g., H1M7789N), 98 / 106 (e.g., H2M7791N), and 250 / 202 (e.g., H4H9068P2).

[0030] The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to PD-1 from humans or other species, hi certain embodiments, the antibodies bind to human PD-1 and / or to cynomolgus PD-1.

[0031] The invention also provides antibodies, and antigen-binding fragments thereof, that cross-compete with a reference antibody, or antigen-binding fragment thereof, comprising the CDRs of an HCVR and the CDRs of an LCVR, for binding to PD-1, where the HCVR and LCVR, respectively, have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0032] In one embodiment, the invention provides an isolated antibody or antigen-binding fragment having one or more of the following characteristics: (a) blocks binding of PD-1 to PD-L1 or PD-L2; (b) specifically binds to human PD-1 and / or cynomolgus PD-1; (c) blocks PD-1-induced T cell downregulation and rescues T cell signaling; (d) suppresses tumor growth and increases survival in subjects with colorectal cancer; (e) inhibits T cell proliferation in a mixed lymphocyte reaction (MLR) assay; and (f) increases IL-2 and / or interferon-gamma secretion in an MLR assay.

[0033] In certain embodiments, the antibodies or antigen-binding fragments thereof specifically bind to PD-1 in an agonistic manner, i.e., enhance or stimulate PD-1 binding and / or activity; in other embodiments, the antibodies specifically bind to PD-1 in an antagonistic manner, i.e., block PD-1 from binding to its ligands.

[0034] In certain embodiments, the antibodies or antigen-binding fragments of the invention are bispecific, comprising a first binding specificity to PD-1 and a second binding specificity for a second target epitope. The second target epitope is another epitope on PD-1 or on a different protein. In certain embodiments, the target epitope is a different epitope on a different cell, including a different T cell, B cell, tumor cell, autoimmune tissue cell, or virally infected cell. It's on top.

[0035] In a second aspect, the invention provides nucleic acid molecules encoding anti-PD-1 antibodies or portions thereof. For example, the invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0036] The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0037] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0038] The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0039] The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0040] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0041] The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0042] The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0043] The present invention also provides nucleic acid molecules encoding HCVRs, where the HCVRs comprise a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), where the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-PD-1 antibodies listed in Table 1.

[0044] The present invention also provides nucleic acid molecules encoding LCVRs, where the LCVRs comprise a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), where the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti-PD-1 antibodies listed in Table 1.

[0045] The invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, where the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and where the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and an LCVR, where both the HCVR and the LCVR are from the same anti-PD-1 antibody listed in Table 1.

[0046] The present invention provides nucleic acid molecules that encode any of the heavy chain amino acid sequences listed in Table 3. The present invention also provides nucleic acid molecules that encode any of the light chain amino acid sequences listed in Table 3.

[0047] The present invention also provides nucleic acid molecules encoding both a heavy chain (HC) and a light chain (LC), where the HC comprises any of the amino acid sequences of the HC amino acid sequences listed in Table 3, and where the LC comprises any of the amino acid sequences of the LC amino acid sequences listed in Table 3.

[0048] In a related embodiment, the invention provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-PD-1 antibody. For example, the invention includes recombinant expression vectors comprising any of the above-mentioned nucleic acid molecules, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences set forth in Table 1. The invention also provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain of an anti-PD-1 antibody. For example, the invention includes recombinant expression vectors comprising any of the above-mentioned nucleic acid molecules, i.e., nucleic acid molecules encoding any of the heavy or light chain sequences set forth in Table 3. Also within the scope of the invention are host cells into which such vectors are introduced, as well as methods for producing an antibody or portion thereof by culturing the host cell under conditions that allow for the production of the antibody or antibody fragment, and recovering the antibodies and antibody fragments produced.

[0049] In a third aspect, the present invention provides multispecific antigen-binding molecules, and antigen-binding fragments thereof, comprising a first antigen-binding specificity that specifically binds to PD-1, and a second antigen-binding specificity that specifically binds to an antigen selected from the group consisting of a tumor cell-specific antigen, an autoimmune tissue-specific antigen, an infected cell-specific antigen, a T cell co-inhibitory molecule, a T cell receptor, an Fc receptor, PD-L1, and PD-1. In certain embodiments, the first antigen-binding specificity binds to an HCVR having an amino acid sequence selected from the HCVR sequences in Table 1. The multispecific antigen-binding molecule comprises three CDRs derived from PD-L1 and three CDRs derived from an LCVR having an amino acid sequence selected from the LCVR sequences in Table 1. In one embodiment, the first antigen-binding specificity comprises the extracellular domain of PD-L1. The second antigen-binding specificity targets an antigen on the same cell as PD-1, or on a different cell of the same tissue type or a different tissue type. For example, the multispecific antigen-binding molecule binds to T cells, where the first antigen-binding specificity specifically binds to PD-1 and the second antigen-binding specificity binds to a T cell receptor on the T cell. Alternatively, in another embodiment, the first antigen-binding specificity specifically binds to PD-1 on the T cell, and the second antigen-binding specificity is targeted to an antigen / receptor on a B cell, or a macrophage, or an antigen-presenting cell. In certain embodiments, the second antigen-binding specificity is for an antigen associated with an autoimmune tissue. In one embodiment, the first antigen-binding specificity comprises the extracellular domain of PD-L1 and the second antigen-binding specificity binds to a different epitope on PD-1. In certain embodiments, the first antigen-binding specificity binds with a lower affinity, e.g., 10 -7 Greater than M, 10 -6 Greater than M, 10 -5 Greater than M or 10 -4 K is bigger than M D It binds to PD-1.

[0050] In a fourth aspect, the invention provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to PD-1, and a pharma- ceutically acceptable carrier. In a related aspect, the invention features a composition that is a combination of an anti-PD-1 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-PD-1 antibody. Exemplary agents that are advantageously combined with an anti-PD-1 antibody include, but are not limited to, other agents that bind to PD-1 and / or modulate PD-1 signaling (including other antibodies or antigen-binding fragments thereof, etc.), and / or agents that do not directly bind PD-1, but nevertheless modulate immune cell activation. Additional combination therapies and co-formulations that include the anti-PD-1 antibodies of the invention are disclosed elsewhere herein.

[0051] In a fifth aspect, the invention provides a method of modulating an immune response in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof of the invention. In certain embodiments, the invention provides a method of enhancing an immune response in a subject, comprising administering to the subject an effective amount of an antibody or fragment thereof of the invention that binds to PD-1 and blocks PD-1 binding to PD-L1. In one embodiment, the invention provides a method of stimulating or enhancing T cell stimulation in a subject. In one embodiment, the invention provides a method of inhibiting T regulatory (Treg) cells in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a blocking antibody or antigen-binding fragment thereof of the invention. In certain embodiments, the subject in need thereof suffers from a disease or disorder, such as cancer or a viral infection. In an alternative embodiment, the invention provides a method of inhibiting or suppressing T cell activation in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an activating antibody or fragment thereof of the invention. In one embodiment, the subject suffers from an autoimmune disease or disorder.

[0052] In a sixth aspect, the invention provides a therapeutic method of treating a disease or disorder, such as cancer, an autoimmune disease, or a viral infection in a subject using an anti-PD-1 antibody or antigen-binding portion of the antibody of the invention, wherein the therapeutic method comprises administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antibody fragment of the invention. The disorder to be treated is any disease or condition that is ameliorated, ameliorated, inhibited, or prevented by stimulation or inhibition of PD-1 activity or signaling. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent may be another The second therapeutic agent is selected from the group consisting of an antibody against a T cell co-inhibitory molecule, an antibody against a tumor cell antigen, an antibody against a T cell receptor, an antibody against an Fc receptor, an antibody against an epitope on a virally infected cell, an antibody against an autoimmune tissue antigen, an antibody against PD-L1, a cytotoxic agent, an anti-cancer drug, an anti-viral drug, an anti-inflammatory drug (e.g., a corticosteroid), a chemotherapeutic agent, radiation therapy, an immunosuppressant, and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent is an agent that helps counter or reduce any potential side effects associated with the antibody or antigen-binding fragment thereof of the invention, if such side effects occur.

[0053] In certain embodiments, the present invention provides a method of suppressing tumor growth. In certain embodiments, the present invention provides a method of enhancing survival of cancer patients. Examples of cancer include, but are not limited to, primary and / or recurrent cancers, including brain cancer (e.g., glioblastoma multiforme), lung cancer (e.g., non-small cell lung cancer), squamous cell carcinoma of the head and neck, renal cell carcinoma, melanoma, multiple myeloma, prostate cancer, and colorectal cancer. The method includes administering a pharmaceutical composition comprising a therapeutically effective amount of an anti-PD-1 antibody of the present invention in combination with a second therapeutic agent selected from the group consisting of a vascular endothelial growth factor (VEGF) antagonist (e.g., aflibercept, bevacizumab), an angiopoietin 2 (Ang2) inhibitor (e.g., an anti-Ang2 antibody such as nesbacumab), a lymphocyte activation gene 3 (LAG-3) inhibitor, a cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor (e.g., ipilimumab), a chemotherapeutic agent, and radiation therapy. Further examples of additional therapies / therapeutics for use in combination with the anti-PD-1 antibodies of the invention for use in the treatment of cancer are described elsewhere herein.

[0054] The antibody or fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. The antibody or fragment thereof is administered at a dose of about 0.1 mg / kg to about 100 mg / kg of body weight of the subject.

[0055] The invention also includes the use of an anti-PD-1 antibody, or antigen-binding fragment thereof, of the invention in the manufacture of a medicament for treating a disease or disorder that benefits from blocking or enhancing PD-1 binding and / or signaling.

[0056] Other embodiments will become apparent from the following detailed description. [Brief description of the drawings]

[0057] [Figure 1-1]Schematic diagram of the luciferase-based PD-1 bioassay described in Example 8 herein. Panel A: inactive Jurkat cells; Panel B: Jurkat cells are activated by T cell receptor (TCR) clustering via a CD3xCD20 bispecific antibody; Panel C: PD-1 activation attenuates responses in activated Jurkat cells; Panel D: blocking PD-1 rescues responses in activated Jurkat cells. [Figure 1-2] Figure 1-1 continued [Diagram 2] FIG. 1 illustrates tumor growth and survival results for mice implanted with Colon-26 tumor cells on day 0 and treated with the indicated molecular combinations by injection on days 3, 6, 10, 13, and 19 ("Early Treatment Tumor Model"). The graph shows tumor volume (mm3) for the different experimental groups at various time points after implantation. The upward arrows along the X-axis indicate the timing of treatment injections. As described elsewhere herein, "mIgG2a" is an IgG2 isotype control; "Fc" is a human Fc control; "VEGF Trap" is aflibercept; "anti-PD-1" is an anti-mouse PD-1 clone RPMI-14; and "anti-PD-L1" is an anti-PD-L1 monoclonal antibody. [Diagram 3] FIG. 1 illustrates tumor growth and survival results for mice implanted with Colon-26 tumor cells on day 0 and treated with the indicated molecular combinations by injection on days 3, 6, 10, 13, and 19 ("Early Treatment Tumor Model"). Graphs show individual mouse tumor volumes (mm3) in each experimental group 28 days after implantation. As described elsewhere herein, "mIgG2a" is an IgG2 isotype control; "Fc" is a human Fc control; "VEGF Trap" is aflibercept; "anti-PD-1" is an anti-mouse PD-1 clone RPMI-14; and "anti-PD-L1" is an anti-PD-L1 monoclonal antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Before the method is described, it should be understood that the invention is not limited to the particular methods and experimental conditions described, as the methods and conditions themselves may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, the preferred methods and materials are described here. All publications mentioned herein are incorporated herein by reference in their entirety.

[0060] The term "PD-1" refers to programmed cell death 1 protein, a T cell co-inhibitory molecule, also known as CD279. The amino acid sequence of full-length PD-1 is provided in GenBank under accession number NP_005009.2, and is also referred to herein as SEQ ID NO: 327. The term "PD-1" also includes protein variants of PD-1 having the amino acid sequence of SEQ ID NO: 321, 322, 323, or 324. The term "PD-1" includes recombinant PD-1 or fragments thereof. The term also encompasses PD-1 or fragments thereof linked to, for example, a histidine tag, mouse or human Fc, or a signal sequence such as ROR1. For example, the term includes sequences exemplified by SEQ ID NO: 323 or 324, which include mouse Fc (mIgG2a) or human Fc (hIgG1) at the C-terminus linked to amino acid residues 25-170 of full-length PD-1 with a C93S modification. The protein variant exemplified by SEQ ID NO:321 contains a C-terminal histidine tag linked to full-length PD-1 amino acid residues 25 to 170. Unless specified to be from a non-human species, the term "PD-1" refers to human PD-1.

[0061] PD-1 is a member of the CD28 / CTLA-4 / ICOS family of T cell co-inhibitory molecules. PD-1 is a 288 amino acid protein with an IgV-like extracellular N-terminal domain, a transmembrane domain, and an intracellular domain containing immunoreceptor tyrosine-based inhibitory (ITIM) motifs and immunoreceptor tyrosine-based switch (ITSM) motifs (Chattopadhyay et al. 2009, Immunol. Rev.). The PD-1 receptor has two ligands, PD-ligand-1 (PD-L1) and PD-L2.

[0062] The term "PD-L1" refers to the ligand of the PD-1 receptor, also known as CD274 and B7H1. The amino acid sequence of full-length PD-L1 is provided in GenBank under Accession No. NP_054862.1, and is also referred to herein as SEQ ID NO: 328. The term also encompasses PD-L1 or fragments thereof linked to, for example, a histidine tag, mouse or human Fc, or a signal sequence, such as ROR1. For example, the term includes PD-L1 or fragments thereof linked to amino acid residues 19-239 at the C-terminus of full-length PD-L1. PD-L1 includes sequences exemplified by SEQ ID NO: 325 or 326, including mouse Fc (mIgG2a) or human Fc (hIgG1). PD-L1 is a 290 amino acid protein with an extracellular IgV-like domain, a transmembrane domain, and a highly conserved intracellular domain of approximately 30 amino acids. PD-L1 is constitutively expressed on many cells, such as antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells), as well as on hematopoietic and non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islets, and sites of immune privilege). PD-L1 is also expressed on a wide range of tumors, virally infected cells, and autoimmune tissues, and is a component of the immunosuppressive environment (Ribas 2012, NEJM 366:2517-2519).

[0063] The term "T cell co-inhibitory molecule" as used herein refers to a ligand and / or receptor that regulates immune response through T cell activation or suppression. The term "T cell co-inhibitory molecule", also known as T cell co-signaling molecule, includes, but is not limited to, lymphocyte activation gene 3 protein (LAG-3, also known as CD223), cytotoxic T lymphocyte antigen 4 (CTLA-4), B and T lymphocyte attenuator (BTLA), CD-28, 2B4, LY108, T cell immunoglobulin and mucin 3 (TIM3), T cell immunoreceptor with immunoglobulin and ITIM (TIGIT; also known as VSIG9), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1; also known as CD305), inducible T cell co-stimulatory molecule (ICOS; also known as CD278), V domain Ig inhibitory molecule of T cell activation (VISTA), and CD160.

[0064] The term "Fc receptor" as used herein refers to a surface receptor protein found on immune cells, including B lymphocytes, natural killer cells, macrophages, basophils, neutrophils, and mast cells, and has binding specificity for the Fc region of antibody.The term "Fc receptor" includes, but is not limited to, Fcγ receptors [e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b)], Fcα receptors (e.g., FcαRI or CD89), and Fcε receptors [e.g., FcεRI, and FcεRII (CD23)].

[0065] The term "antibody" as used herein is intended to refer to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (i.e., an "intact antibody molecule"), as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain comprises a heavy chain variable region ("HCVR" or "V H "), and the heavy chain constant region (domain C H 1. C H 2, and CH Each light chain comprises a light chain variable region ("LCVR" or "V L "), and the light chain constant region (C L ) is included. V H and V L The regions are further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H And V L comprises three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen-binding fragment thereof) are identical to human germline sequences or are naturally or artificially modified. The amino acid contiguous sequence is defined based on a side-by-side analysis of two or more CDRs.

[0066] Substitution of one or more CDR residues or elimination of one or more CDRs is also possible. Antibodies that bind despite the omission of one or two CDRs have been described in the scientific literature. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact areas between an antibody and its antigen based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also noted that have found many antibodies in which one or two CDRs have no amino acids in contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0067] CDR residues that do not contact the antigen are identified by molecular modeling and / or experimentally from regions of the Kabat CDRs outside the Chothia CDRs in previous studies (e.g., residues H60-H65 in CDRH2 are often not required). If a CDR or its residues are excluded, it is usually replaced with an amino acid that occupies the corresponding position in another human antibody sequence or a common such sequence. The positions for substitution within the CDR and the amino acids to be substituted are also selected experimentally. The experimental substitutions are conservative or non-conservative substitutions.

[0068] The fully human anti-PD-1 monoclonal antibodies disclosed herein contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations are readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence alterations are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of skill in the art will readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll of the framework and / or CDR residues in the domain are mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues, for example, mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues found in CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residues in a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody is originally derived). Furthermore, the antibodies of the present invention contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations are readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

[0069] The invention also includes fully human anti-PD-1 monoclonal antibodies that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the invention includes anti-PD-1 antibodies that have HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0070] The term "human antibody" as used herein is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the present invention contain, for example, in the CDRs, particularly CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis, or in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) are grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in cells of non-human mammals. The term is not intended to include antibodies isolated from or raised in human subjects.

[0071] The term "recombinant" as used herein refers to an antibody or antigen-binding fragment thereof of the present invention that is made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term refers to antibodies expressed in non-human mammalian (including transgenic non-human mammalian, e.g., transgenic mice) or cellular (e.g., CHO cell) expression systems, or isolated from a recombinant combinatorial human antibody library.

[0072] The term "multispecific antigen-binding molecule" as used herein refers to bispecific, trispecific, or multispecific antigen-binding molecules, and antigen-binding fragments thereof. Multispecific antigen-binding molecules contain antigen-binding domains that are specific for different epitopes of one target polypeptide or specific for epitopes of more than one target polypeptide. Multispecific antigen-binding molecules are single multifunctional polypeptides or multimeric complexes of two or more polypeptides that are covalently or non-covalently linked to each other. The term "multispecific antigen-binding molecule" includes the antibodies of the present invention that are bound to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof is functionally linked (e.g., by chemical cross-linking, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as a protein or a fragment thereof to create a bispecific or multispecific antigen-binding molecule with a second binding specificity. According to the present invention, the term "multispecific antigen-binding molecule" also includes bispecific, trispecific, or multispecific antibodies or antigen-binding fragments thereof. In certain embodiments, an antibody of the invention is operatively linked to another antibody or antigen-binding fragment thereof to create a bispecific antibody having a second binding specificity. Bispecific and multispecific antibodies of the invention are described elsewhere herein.

[0073] The terms "specifically bind," or "specifically bind to," and the like, mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1×10 -8 Characterized by an equilibrium dissociation constant M or less (e.g., K D(A smaller β indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, an antibody is identified by surface plasmon resonance, e.g., BIACORE™, that specifically binds to PD-1. Furthermore, a multispecific antibody that binds to one domain in PD-1 and one or more additional antigens, or a bispecific antibody that binds to two different regions of PD-1, are nevertheless considered to be an antibody that "specifically binds" as used herein.

[0074] The term "high affinity" antibody refers to antibodies that are capable of being detected by surface plasmon resonance, e.g., BIACORE™ or or solution affinity ELISA. -7 M; preferably 10 -8 M; more preferably, 10 -9 M, and even more preferably 10 -10 M, and even more preferably 10 -11 K of M D The term refers to those mAbs that have binding affinity for PD-1, expressed as

[0075] The term "slow off rate", "Koff", or "kd" refers to a slow off rate of 1×10 as determined by surface plasmon resonance, e.g., BIACORE™. -3 s -1 or less, preferably 1×10 -4 s -1 It means an antibody that dissociates from PD-1 with a rate constant equal to or lower than that.

[0076] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms "antigen-binding fragment" of an antibody, or "antibody fragment" refer to one or more fragments of an antibody that retain the ability to bind to PD-1.

[0077] In certain embodiments, the antibodies or antibody fragments of the invention are conjugated to a moiety such as a ligand or therapeutic moiety (an "immunoconjugate"), such as an antibody against another antigen, such as a tumor-specific antigen, an autoimmune tissue antigen, a virally infected cell antigen, an Fc receptor, a T cell receptor, or a T cell co-inhibitory molecule, or an immunotoxin, or any other therapeutic moiety useful for treating a disease or condition, including cancer, an autoimmune disease, or a chronic viral infection.

[0078] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds to PD-1 is substantially free of Abs that specifically bind to antigens other than PD-1).

[0079] As used herein, a "blocking antibody" or "neutralizing antibody" (or an "antibody that neutralizes PD-1 activity" or an "antagonist antibody") is intended to refer to an antibody whose binding to PD-1 results in the inhibition of at least one biological activity of PD-1. For example, the antibodies of the invention prevent or block PD-1 binding to PD-L1.

[0080] As used herein, an "activating antibody" or "enhancing antibody" (or "agonist antibody") is intended to refer to an antibody whose binding to PD-1 results in an increase or stimulation of at least one biological activity of PD-1. For example, an antibody of the invention increases PD-1 binding to PD-L1.

[0081] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biomolecular interactions by detection of changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0082] As used herein, the term "K D " is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0083] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, also known as a paratope. A single antigen may contain more than one epitope. Each epitope has a specific structure. Thus, different antibodies bind to different areas on an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes are defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have their residues that are directly involved in the affinity of the interaction. Epitopes are also conformational, i.e., they contain non-linear amino acids. In certain embodiments, epitopes contain determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and in certain embodiments have specific three-dimensional structural features, and / or specific alteration features.

[0084] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is a nucleotide sequence identity of at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule, in certain instances, encodes a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0085] The term "substantial similarity" or "substantially similar" as applied to polypeptides means that two peptide sequences, when optimally aligned, for example by the programs GAP or BESTFIT with default gap weights, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Preferably, residue positions are not identical but differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity is adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45 (incorporated herein by reference). A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0086] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software can be used to match homologous polypeptides from different species of organisms or between wild-type polypeptides. These include programs such as GAP and BESTFIT, used with default parameters, to determine sequence homology or sequence identity between closely related polypeptides, such as between a native protein and its mutant protein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG Version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) produces alignment and percent sequence identity of the regions of optimal overlap between the query and search sequences (Pearson, 2000, supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410, and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0087] By the phrase "therapeutically effective amount" is meant an amount that produces the desired effect in those to whom it is administered. The exact amount will depend on the purpose of the treatment and will be ascertainable by those skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0088] The term "subject" as used herein refers to an animal, preferably a mammal, in need of amelioration, prevention, and / or treatment of a disease or disorder, such as a chronic viral infection, cancer, or an autoimmune disease.

[0089] As used herein, "anticancer drug" refers to any agent useful for treating cancer, including, but not limited to, cytotoxins, antimetabolites, alkylating agents, anthracyclines, antibiotics, mitotic inhibitors, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), biologics (e.g., antibodies, and interferons), and radioactive agents. As used herein, "cytotoxins or cytotoxic agents" also refers to chemotherapeutic agents, and any agent that is harmful to cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbilastine, coitisin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.

[0090] The term "antiviral drug" as used herein refers to any drug or therapy that is used to treat, prevent, or improve viral infection in a host subject.The term "antiviral drug" includes, but is not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics, and corticosteroids.In the context of the present invention, viral infection includes long-term or chronic infection caused by viruses, including, but not limited to, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV).

[0091] The antibodies and antigen-binding fragments of the invention specifically bind to PD-1 and modulate the interaction of PD-1 with PD-L1. Anti-PD-1 antibodies may be used with high or low affinity to PD-1. In certain embodiments, the antibodies of the present invention are blocking antibodies, where the antibodies bind to PD-1 and block the interaction of PD-1 with PD-L1. In certain embodiments, the blocking antibodies of the present invention block the binding of PD-1 to PD-L1 and / or stimulate or enhance T cell activation. In certain embodiments, the blocking antibodies are useful for stimulating or enhancing immune responses and / or treating subjects suffering from cancer or chronic viral infections. When administered to a subject in need thereof, the antibodies reduce chronic infections in the subject with viruses such as HIV, LCMV, or HBV. They are used to inhibit the growth of tumor cells in the subject. They are used alone or as adjunctive therapy with other therapeutic moieties or modalities known in the art for treating cancer or viral infections.

[0092] In other embodiments, the antibodies of the invention are activating antibodies, where the antibodies bind to PD-1 and enhance the interaction of PD-1 with PD-L1. In certain embodiments, the activating antibodies enhance the binding of PD-1 to PD-L1 and / or inhibit or suppress T cell activation. The activating antibodies of the invention are useful for inhibiting an immune response in a subject and / or for treating an autoimmune disease.

[0093] In certain embodiments, the anti-PD-1 antibodies are multispecific antigen-binding molecules, wherein they comprise a first binding specificity for PD-1 and a second binding specificity for an antigen selected from the group consisting of another T cell co-inhibitory molecule, an autoimmune tissue antigen, a T cell receptor, an Fc receptor, a T cell receptor, PD-L1, and a different epitope of PD-1.

[0094] In certain embodiments, antibodies of the invention are obtained from mice immunized with a first immunogen such as full-length PD-1 [see GenBank Accession No. NP_005009.2 (SEQ ID NO: 327)], or a recombinant form of PD-1 or a modified human PD-1 fragment (SEQ ID NO: 321, 323, or 324), or a modified cynomolgus PD-1 fragment (SEQ ID NO: 322), and subsequently immunized with a second immunogen, or an immunogenically active fragment of PD-1.

[0095] The immunogen is a biologically active and / or immunogenic fragment of PD-1, or DNA encoding the active fragment. The fragment is derived from the N-terminal or C-terminal domain of PD-1. In certain embodiments of the invention, the immunogen is a fragment of PD-1 ranging from amino acid residues 25 to 170 of SEQ ID NO: 327 having a C93S alteration.

[0096] Peptides are modified to include the addition or substitution of certain residues for tagging or for purposes of conjugation to a carrier molecule such as KLH, e.g., cysteines are added to either the N- or C-terminus of the peptide, or linker sequences are added, to prepare the peptide for conjugation to KLH for immunization.

[0097] The full-length amino acid sequence of full-length human PD-1 is set forth as SEQ ID NO:327.

[0098] In certain embodiments, antibodies that specifically bind to PD-1 are prepared using fragments of the regions indicated above, or peptides extending beyond the designated regions by about 5 to about 20 amino acid residues from either the N- or C-terminus of the regions described herein, or both. In certain embodiments, any combination of the above indicated regions or fragments thereof is used in the preparation of PD-1 specific antibodies. In certain embodiments, any one or more of the above indicated regions of PD-1, or fragments thereof, are used to prepare monospecific, bispecific, or multispecific antibodies.

[0099] Certain anti-PD-1 antibodies of the invention bind to PD-1 and neutralize the activity of PD-1 as determined by in vitro or in vivo assays. The ability of the antibodies of the invention to bind to PD-1 and neutralize the activity of PD-1 may be measured using any standard method known to those of skill in the art, including the binding assays or activity assays described herein.

[0100] Non-limiting exemplary in vitro assays for measuring binding activity are described in the Examples herein. In Example 3, the binding affinity and kinetic constants of human anti-PD-1 antibodies for human PD-1 and cynomolgus PD-1 were determined by surface plasmon resonance, and measurements were performed on a Biacore4000 or T200 instrument. In Examples 4 and 5, a blocking assay was used to determine the ability of anti-PD-1 antibodies to block the PD-L1-binding ability of PD-1 in vitro. In Example 6, a blocking assay was used to determine cross-competition between anti-PD-1 antibodies. Example 7 describes the binding of antibodies to cells overexpressing PD-1. In Example 8, a luciferase assay was used to determine the ability of anti-PD-1 antibodies to antagonize PD-1 / PD-L1 signaling in T cells.

[0101] In certain embodiments, the antibodies of the invention can enhance or stimulate T cell activation in vitro and in subjects with cancer or infected with a virus such as LCMV, hi certain embodiments, the antibodies of the invention are used in combination with a second therapeutic agent, such as an antibody against a second T cell co-inhibitory molecule, to enhance the immune response and inhibit tumor growth in a subject.

[0102] Antibodies specific for PD-1 do not contain additional labels or moieties, or they contain N-terminal or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if any) determines the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin is oriented such that the C-terminal portion of the peptide is distal to the surface. In one embodiment, the label is a radionuclide, a fluorescent dye, or an MRI-detectable label. In certain embodiments, such labeled antibodies are used in diagnostic assays, including imaging assays.

[0103] Antigen-binding fragment of an antibody Unless specifically indicated otherwise, the term "antibody" as used herein will be understood to encompass an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule"), as well as antigen-binding fragments thereof. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to PD-1. Antibody fragments include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. In such embodiments, the term "antigen-binding fragment" includes, for example, the extracellular domain of PD-L1 that specifically binds to PD-1. Antigen-binding fragments of antibodies can be obtained from whole antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or available, for example, from commercially available sources. The DNA may be readily available from a source, a DNA library (including, for example, a phage-antibody library), or may be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, e.g., to place one or more variable and / or constant domains in the appropriate position, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids.

[0104] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides), or limited FR3-CDR3-FR4 peptides. Domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragment" as used herein.

[0105] Antigen-binding fragments of antibodies typically contain at least one variable domain. A variable domain may be of any size or amino acid composition and generally contains at least one CDR and is flanked or in-frame by one or more framework sequences. L V connected to the domain H In an antigen-binding fragment having a domain, H and V L The domains may be positioned relative to one another in any suitable arrangement. For example, the variable region may be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of the antibody contains a monomeric V H or V L Contains a domain.

[0106] In certain embodiments, an antigen-binding fragment of an antibody contains at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains found within an antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any arrangement of the variable and constant domains, including any of the exemplary arrangements listed above, the variable and constant domains are either directly linked to each other or linked by a complete or partial hinge or linker region. A hinge region consists of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids and provides a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention can be linked (e.g., by disulfide bonds) to each other and / or to one or more monomeric V domains. H Or V L The invention includes homo- or hetero-dimers (or other multimers) of any of the above listed variable and constant domain arrangements non-covalently associated with the domain.

[0107] Like whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats discussed above, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention, using routine techniques available in the art.

[0108] Preparation of human antibodies Methods for producing human antibodies in transgenic mice are known in the art. Any such known method is used in the context of the present invention to make human antibodies that specifically bind to PD-1.

[0109] Antibodies to PD-1 are generated using an immunogen comprising any one of the following: In certain embodiments, antibodies of the invention are obtained from mice immunized with full-length native PD-1 (see GenBank Accession No. NP_005009.2) (SEQ ID NO: 327), or recombinant PD-1 peptides. Alternatively, PD-1 or a fragment thereof is engineered and modified using standard biochemical techniques (SEQ ID NOs: 321-324) and used as an immunogen.

[0110] In certain embodiments, the immunogen is a peptide derived from the N-terminus or C-terminus of PD-1. In one embodiment, the immunogen is the extracellular domain or IgV-like domain of PD-1. In certain embodiments of the invention, the immunogen is a fragment of PD-1 ranging from about amino acid residues 25 to 170 of SEQ ID NO: 327 having a C93S alteration.

[0111] In one embodiment, the immunogen is a recombinant PD-1 peptide expressed in Escherichia coli (E. coli) or in any other eukaryotic or mammalian cell, such as Chinese Hamster Ovary (CHO) cells.

[0112] In certain embodiments, antibodies that specifically bind to PD-1 are prepared using fragments of the regions indicated above, or peptides extending beyond the designated regions by about 5 to about 20 amino acid residues from either the N- or C-terminus of the regions described herein, or both. In certain embodiments, any combination of the regions indicated above, or fragments thereof, are used in the preparation of PD-1-specific antibodies.

[0113] Using VELOCIMMUNE® technology (e.g., US6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®), or any other known method of making monoclonal antibodies, a high affinity chimeric antibody against PD-1 with a human variable region and a mouse constant region is first isolated. VELOCIMMUNE® technology involves the creation of a transgenic mouse with a genome that includes human heavy and light chain variable regions operably linked to endogenous mouse constant region sites, such that the mouse produces antibodies that include human variable regions and mouse constant regions in response to antigenic stimulation. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0114] biological equivalent The anti-PD-1 antibodies and antibody fragments of the invention include proteins having amino acid sequences which differ from those of the described antibodies but which retain the ability to bind to PD-1. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity which is substantially equivalent to that of the described antibodies. Similarly, antibodies encoding the DNA sequences of the invention can be prepared using the disclosed The term "antibody" includes sequences that encode antibodies or antibody fragments that contain one or more additions, deletions, or substitutions of nucleotides when compared to the sequences described herein, but that are substantially biologically equivalent to the antibodies or antibody fragments of the present invention.

[0115] Two antigen-binding proteins, or antibodies, are considered bioequivalents if, for example, their rate and extent of absorption are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences when administered under similar experimental conditions at the same molar, single or multiple doses. An antibody is considered to be equivalent in its extent of absorption but not in its rate of absorption, and would still be considered to be bioequivalent because such differences in rate of absorption are intentional, reflected in the label, and are not essential, for example, to achieving effective body drug concentrations during chronic use, and are not considered to be pharmaceutical significant for the particular drug product being studied.

[0116] In one embodiment, two antigen binding proteins are biologically equivalent if there are no clinically meaningful differences in the safety, purity, or efficacy of the two antigen binding proteins.

[0117] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without expecting a clinically significant change in immunogenicity or an increased risk of side effects, including impaired efficacy, compared to continuing therapy without switching.

[0118] In one embodiment, two antigen binding proteins are biologically equivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, so long as such mechanisms are known.

[0119] Bioequivalence is demonstrated by in vivo and / or in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo studies in humans or other mammals where the concentration of the antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with and sufficiently predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals where the relevant acute pharmacological effects of the antibody (or its target) are measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0120] Biological equivalents of the antibodies of the invention are constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity are deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, biological equivalent antibodies include antibody variants that contain amino acid changes to modify the glycosylation characteristics of the antibody, for example mutations that remove or eliminate glycosylation.

[0121] Anti-PD-1 Antibodies Containing Fc Variants According to certain embodiments of the invention, there are provided anti-PD-1 antibodies comprising an Fc domain that contains one or more mutations that enhance or impair antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the invention provides an Fc domain that contains one or more mutations that enhance or impair antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C H The present invention also includes anti-PD-1 antibodies that contain mutations in the three regions, where the mutations increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). When such Fc modifications are made, they result in an increase in the serum half-life of the antibody. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W, or T), 254 (e.g., S, or T), and 256 (e.g., S / R / Q / E / D, or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q, or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification; a 428L, a 259I (e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g., H433K), and a 434 (e.g., 434Y) modification; a 252, 254, and a 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and a 428L modification (e.g., T250Q, and M428L); and a 307 and / or a 308 modification (e.g., 308F, or 308P). In yet another embodiment, the modifications include a 265A (e.g., D265A), and / or a 297A (e.g., N297A) modification.

[0122] For example, the present invention provides 250Q and 248L (e.g., T250Q, and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L, and N434S); 257I and 311I (e.g., P257I, and Q311I); 257I and 434H (e.g., P257I, and N434H); 376V, and 434H (e.g., D376V, and N434H); 307A, 380A, and 434A (e.g., T307A, E380A, and N434A); and 433K, and 434F (e.g., H433K, and N434F). In one embodiment, the invention includes an anti-PD-1 antibody comprising an Fc domain that comprises an S108P mutation in the hinge region of IgG4 to promote dimer stabilization. All possible combinations of the foregoing Fc domain mutations and other mutations in the antibody variable domains disclosed herein are considered within the scope of the invention.

[0123] The present invention also relates to chimeric heavy chain constant (C H ) region, wherein the chimeric C H A region may contain more than one C H For example, the antibodies of the present invention may comprise segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H C derived from a human IgG1, human IgG2, or human IgG4 molecule, combined with some or all of the three domains H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibodies of the invention comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge comprises an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1, or human IgG4 upper hinge, and amino acid residues derived from a human IgG2 lower hinge. The chimeric hinges described herein may be H Antibodies comprising the regions, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., USSN. 14 / 170,166, filed Jan. 31, 2014, which is incorporated by reference in its entirety.)

[0124] Antibody Biology Generally, the antibodies of the invention function by binding to PD-1. The invention includes anti-PD-1 antibodies and antigen-binding fragments thereof that bind soluble monomeric or dimeric PD-1 molecules with high affinity. For example, the invention includes antibodies that have a K of less than about 50 nM (e.g., at 25° C. or at 37° C.) as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein. D In certain embodiments, the antibodies or antigen-binding fragments thereof have a K of less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, or less than about 1 nM, as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. D binds to monomeric PD-1.

[0125] The present invention also relates to a method for treating pulmonary circulation disorders, comprising administering to a patient a therapeutically effective amount of a pulmonary artery disease (PAH) having a K of less than about 400 pM (e.g., at 25° C. or at 37° C.) as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein. D In certain embodiments, the antibodies or antigen-binding fragments thereof have a K of less than about 300 pM, less than about 250 pM, less than about 200 pM, less than about 100 pM, or less than about 50 pM, as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. D and binds to dimeric PD-1.

[0126] The present invention also relates to a method for treating pulmonary circulation disorders, comprising administering to a patient a therapeutically effective amount of a pulmonary circulation inhibitor having a K of less than about 35 nM (e.g., at 25° C. or at 37° C.) as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein. D The present invention relates to a method for the treatment of cynomolgus monkey (Macaca fascicularis) PD-1-related diseases, comprising administering to the patient a therapeutically effective amount of the antibody or antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment thereof has a K of less than about 30 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, or less than about 5 nM, as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. D It binds to cynomolgus monkey PD-1.

[0127] The present invention also relates to a method for producing a medicament having a dissociation half-life (t ) of greater than about 1.1 minutes, as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. 1 / 2In certain embodiments, the antibodies or antigen-binding fragments of the invention have a t of greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes as measured by surface plasmon resonance at 25° C. or 37° C. using, for example, an assay format defined in Example 3 herein (e.g., mAb capture or antigen capture format), or a substantially similar assay. 1 / 2 It binds to PD-1.

[0128] The present invention also provides an IC of less than about 3 nM, as determined, for example, using the ELISA-based immunoassay assay shown in Example 4, or a substantially similar assay. 50 The present invention also includes antibodies or antigen-binding fragments thereof that bind to PD-1 and enhance binding of PD-1 to PD-L1. Also included are antigen-binding fragments of the

[0129] In certain embodiments, antibodies of the invention bind to the extracellular domain of PD-1, or a fragment of that domain. In certain embodiments, antibodies of the invention bind to more than one domain (cross-reactive antibodies). In certain embodiments, antibodies of the invention bind to an epitope located in the extracellular domain comprising amino acid residues 21-171 (SEQ ID NO:327) of PD-1. In one embodiment, the antibody binds to an epitope comprising one or more amino acids selected from the group consisting of amino acid residues 1-146 of SEQ ID NOs:321-324.

[0130] In certain embodiments, the antibodies of the invention function by blocking or inhibiting PD-L1-binding activity associated with PD-1 by binding to any other region or fragment of the full-length protein whose amino acid sequence is set forth in SEQ ID NO: 327. In certain embodiments, the antibodies attenuate or modulate the interaction between PD-1 and PD-L1.

[0131] In certain embodiments, the antibodies of the present invention are bispecific antibodies. Bispecific antibodies of the present invention bind to one epitope in one domain and also bind to a second epitope in a different domain of PD-1. In certain embodiments, bispecific antibodies of the present invention bind to two different epitopes in the same domain. In one embodiment, a multispecific antigen-binding molecule comprises a first binding specificity that comprises the extracellular domain of PD-L1 or a fragment thereof, and a second binding specificity for another epitope of PD-1.

[0132] In one embodiment, the invention provides an isolated fully human monoclonal antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 218, 226, 234, 242, 250, 258, 266, 274, 282, 290, 298, 300, 302, 304, 306, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372 (ii) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, and 202, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 312, and 320, or a sequence substantially similar thereto having at least 90%, at least 95%, at least an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, and 208, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;(iv) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 220, 228, 236, 244, 252, 260, 268, 276, 284, 292, 300, 308, and 316, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; SEQ ID NO: 6; HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, and 204, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, and 206, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) a multispecific antigen-binding molecule comprising a first binding specificity for PD-1 and a second binding specificity for an antigen selected from the group consisting of PD-1, a tumor-specific antigen, an autoimmune tissue-specific antigen, a virally infected cell antigen, a different T cell co-inhibitory molecule, a T cell receptor, and an Fc receptor; (vi) a K D (vii) binds to cynomolgus monkey PD-1 with a K of about 3 nM to about 7.5 μM; D (viii) binds PD-1 to PD-L1 with an IC of about 3.3 nM or less; 50(ix) block PD-1 induced T cell downregulation and / or rescue T cell signaling in a T cell / APC luciferase reporter assay; (x) stimulate T cell proliferation and activity in a mixed lymphocyte reaction (MLR) assay; (xi) induce IL-2 and / or IFNγ production in an MLR assay; and (xii) suppress tumor growth and increase survival in subjects with cancer.

[0133] In one embodiment, the invention provides an isolated fully human monoclonal antibody or antigen-binding fragment thereof that blocks PD-1 binding to PD-L1, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 130, 162, 234, and 314, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 138, 17 (iii) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 136, 168, 240, and 320, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iv) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 176, 192, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iv) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 132, 164, 236, and 316, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134, 166, 238, and 3 HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 140, 172, 188, and 204, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 142, 174, 190, and 206, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) a multispecific antigen-binding molecule comprising a first binding specificity for PD-1 and a second binding specificity for an antigen selected from the group consisting of a different epitope of PD-1, a tumor-specific antigen, an autoimmune tissue-specific antigen, a virally infected cell antigen, a different T cell co-inhibitory molecule, a T cell receptor, and an Fc receptor; (vi) a multispecific antigen-binding molecule comprising an LCDR2 domain having an amino acid sequence selected from the ... -9 K below M D (vii) binds to cynomolgus monkey PD-1 at 10 -8 K below M D (viii) binding of PD-1 to PD-L1 at 10 -10 IC below M 50 (ix) block PD-1 induced T cell downregulation and / or rescue T cell signaling in a T cell / APC luciferase reporter assay; (x) stimulate T cell proliferation and activity in a mixed lymphocyte reaction (MLR) assay; (xi) induce IL-2 and / or IFNγ production in an MLR assay; and (xii) suppress tumor growth and increase survival in subjects with cancer.

[0134] The antibodies of the invention have one or more of the above biological characteristics, or any combination thereof. Other biological characteristics of the antibodies of the invention will be apparent to those skilled in the art from the present disclosure, including the Examples herein.

[0135] Species selectivity and species cross-reactivity According to certain embodiments of the invention, the anti-PD-1 antibodies bind to human PD-1 but do not bind to PD-1 from other species. Alternatively, the anti-PD-1 antibodies of the invention, in certain embodiments, bind to human PD-1 and PD-1 from one or more non-human species. For example, the anti-PD-1 antibodies of the invention bind to human PD-1 and either do or do not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee PD-1. In certain embodiments, the anti-PD-1 antibodies of the invention bind to human and cynomolgus monkey PD-1 with the same affinity or with different affinities, but do not bind to rat and mouse PD-1.

[0136] Epitope mapping and related techniques The invention includes anti-PD-1 antibodies that interact with one or more amino acids found within one or more domains of the PD-1 molecule, including, for example, the extracellular (IgV-like) domain, the transmembrane domain, and the intracellular domain containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs). The epitope to which the antibody binds can consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located in any of the aforementioned domains of the PD-1 molecule (e.g., a linear epitope in a domain). Alternatively, the epitope can consist of a number of non-contiguous amino acids (or sequences of amino acids) located in either or both of the aforementioned domains of the PD-1 molecule (e.g., a conformational epitope).

[0137] A variety of techniques known to those of skill in the art can be used to determine whether an antibody interacts with one or more amino acids within a polypeptide or protein. Exemplary techniques include routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis, and crystallography. , and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens are utilized (Tomer (2000) Prot. Sci. 9:487-496). Another method used to identify amino acids in polypeptides with which antibodies interact is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo deuterium-to-hydrogen back exchange at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface retain deuterium and therefore present a relatively large mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to proteolytic enzyme cleavage and mass spectrometry, thereby revealing deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0138] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. B cell epitopes are formed from both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation.

[0139] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) directed against the same antigen by the similarity of the binding profile of each antibody to a chemically or enzymatically modified antigen surface (US2004 / 0101920, incorporated herein by reference in its entirety). Each category reflects a unique epitope that is either distinct or partially overlapping with the epitope represented by another category. This technique allows for rapid sorting of genetically matched antibodies so that characterization can be focused on genetically defined antibodies. When applied to hybridoma screening, MAP facilitates the identification of rare hybridoma clones that produce mAbs with desired characteristics. Using MAP, the antibodies of the present invention are classified into groups of antibodies that bind to different epitopes.

[0140] In certain embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof binds to an epitope within any one or more of the regions exemplified in PD-1, either in its native form as exemplified in SEQ ID NO:327, or recombinantly produced as exemplified in SEQ ID NOs:321-324, or a fragment thereof. In certain embodiments, an antibody of the invention binds to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 21-171 of PD-1. In certain embodiments, an antibody of the invention binds to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 1-146 of cynomolgus PD-1, as exemplified by SEQ ID NO:322.

[0141] In certain embodiments, the antibodies of the invention, as set forth in Table 1, comprise at least one amino acid residue selected from the group consisting of: an amino acid residue ranging from about position 21 to about position 136 of SEQ ID NO:327; or an amino acid residue ranging from about position 136 to about position 171 of SEQ ID NO:327. These regions are partially exemplified in SEQ ID NOs: 321-324.

[0142] The present invention includes anti-PD-1 antibodies that bind to the same epitope, or portion of an epitope, as any of the specific exemplary antibodies described in Table 1 herein, or antibodies having the CDR sequences of any of the exemplary antibodies described in Table 1. Similarly, the present invention also includes anti-PD-1 antibodies that compete for binding to PD-1 or a PD-1 fragment with any of the specific exemplary antibodies described in Table 1 herein, or antibodies having the CDR sequences of any of the exemplary antibodies described in Table 1. For example, the present invention includes anti-PD-1 antibodies that cross-compete for binding to PD-1 with one or more antibodies defined in Example 6 herein (e.g., H2aM7788N, H4xH8992P, H4xH8999P, H1M7799N, H2aM7780N, H1M7800N, H2aM7794N, H2aM 7798N, H4xH9145P2, H4H9057P2, H4xH9120P2, H4xH9128P2, H4H9019P, H4xH9119P2, H4xH9135P2, H4xH9034P, H2aM7790N, H4xH9035P, H4xH9037P, H4xH9045P, and H2aM7795N).

[0143] By using routine methods known in the art, one can easily determine whether an antibody binds to the same epitope as a reference anti-PD-1 antibody or competes with the reference anti-PD-1 antibody for binding. For example, to determine whether a test antibody binds to the same epitope as a reference anti-PD-1 antibody of the present invention, the reference antibody is allowed to bind to PD-1 protein or peptide under saturating conditions. The ability of the test antibody to bind to the PD-1 molecule is then evaluated. If the test antibody is able to bind to PD-1 after saturation binding with the reference anti-PD-1 antibody, it is concluded that the test antibody binds to a different epitope than the reference anti-PD-1 antibody. On the other hand, if the test antibody is unable to bind to the PD-1 protein after saturation binding with the reference anti-PD-1 antibody, then the test antibody binds to the same epitope as the epitope bound by the reference anti-PD-1 antibody of the present invention.

[0144] To determine whether an antibody competes with a reference anti-PD-1 antibody for binding, the binding methodology described above is performed in two ways. In the first approach, the reference antibody is allowed to bind to the PD-1 protein under saturating conditions, and then the binding of the test antibody to the PD-1 molecule is evaluated. In the second approach, the test antibody is allowed to bind to the PD-1 molecule under saturating conditions, and then the binding of the reference antibody to the PD-1 molecule is evaluated. In both approaches, if only the first (saturating) antibody is capable of binding to the PD-1 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to PD-1. As will be understood by one of skill in the art, an antibody that competes with a reference antibody for binding will not necessarily bind to the same epitope as the reference antibody, but will sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0145] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990, 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0146] Further routine experiments (e.g., peptide mutations and binding analysis) are then performed to determine whether the observed lack of binding of the test antibody is due in fact to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This screening experiment is performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0147] immune complex The present invention encompasses human anti-PD-1 monoclonal antibodies ("immunoconjugates") conjugated to a therapeutic moiety, such as a cytotoxin or a chemotherapeutic agent for treating cancer. As used herein, the term "immunoconjugate" refers to an antibody that is chemically or biologically conjugated to a cytotoxin, a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a toxin, a peptide or protein, or a therapeutic agent. The antibody is conjugated to a cytotoxin, a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a toxin, a peptide, or a therapeutic agent at any position along the molecule so long as it is capable of binding to its target. Examples of immunoconjugates include antibody drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent is a second, different antibody against PD-1. In certain embodiments, the antibody is conjugated to an agent specific for tumor cells or virally infected cells. The type of therapeutic moiety conjugated to the anti-PD-1 antibody takes into consideration the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art, see, for example, WO05 / 103081.

[0148] multispecific antibody The antibody of the present invention can be monospecific, bispecific, or multispecific.Multispecific antibodies contain antigen-binding domains that are specific to different epitopes of one target polypeptide or specific to more than one target polypeptide.See, for example, Tutt et al., 1991, J.Immunol.147:60-69; Kufer et al., 2004, Trends Biotechnol.22:238-244.

[0149] In one aspect, the invention includes a multispecific antigen-binding molecule, or antigen-binding fragment thereof, where one specificity of the immunoglobulin is specific for the extracellular domain of PD-1, or a fragment thereof, and the other specificity of the immunoglobulin is specific for binding to the extracellular domain of PD-1, or a second therapeutic target, or is conjugated to a therapeutic moiety. In certain embodiments, the first antigen-binding specificity comprises PD-L1 or PD-L2, or a fragment thereof. In certain embodiments of the invention, one specificity of the immunoglobulin is specific for an epitope comprising amino acid residues 21-171 of PD-1 (SEQ ID NO: 327), or a fragment thereof, and the other specificity of the immunoglobulin is specific for a second target antigen. The second target antigen is on the same cell as PD-1 or on a different cell. In one embodiment, the second target cell is on an immune cell other than a T cell, such as a B cell, an antigen-presenting cell, a monocyte, a macrophage, or a dendritic cell. In certain embodiments, the second target antigen is present on a tumor cell, or on an autoimmune tissue cell, or on a virally infected cell.

[0150] In another embodiment, the invention provides a multispecific antigen-binding molecule or antigen-binding fragment thereof comprising a first antigen-binding specificity that binds to PD-1 and a second antigen-binding specificity that binds to a T cell receptor, a B cell receptor, or an Fc receptor. In a related embodiment, the invention provides a multispecific antigen-binding molecule or antigen-binding fragment thereof comprising a first antigen-binding specificity that binds to PD-1 and a second antigen-binding specificity that binds to a different T cell co-inhibitory molecule, such as LAG-3, CTLA-4, BTLA, CD-28, 2B4, LY108, TIGIT, TIM3, LAIR1, ICOS, and CD160. The present invention provides multispecific antigen-binding molecules or antigen-binding fragments thereof that contain specificities.

[0151] In another aspect, the present invention provides a multispecific antigen-binding molecule or antigen-binding fragment thereof comprising a first antigen-binding specificity that binds to PD-1 and a second antigen-binding specificity that binds to an autoimmune tissue-specific antigen. In certain embodiments, the antibody is an activating or agonist antibody.

[0152] Any of the multispecific antigen-binding molecules of the present invention, or variants thereof, are constructed using standard molecular biology techniques (eg, recombinant DNA, and protein expression techniques) that will be known to those skilled in the art.

[0153] In certain embodiments, PD-1-specific antibodies are made in a bispecific format ("bispecific") in which variable regions that bind to separate domains of PD-1 are linked together to confer dual-domain specificity within a single binding molecule. Suitably, the engineered bispecific enhances overall PD-1 inhibitory potency through both increased specificity and avidity. Variable regions with specificity for individual domains (e.g., segments of the N-terminal domain) or that bind to different regions within one domain are paired on a structural scaffold that allows each region to simultaneously bind to a distinct epitope, or different regions within one domain. In one example of a bispecific, a heavy chain variable region (V) from a binder with specificity for one domain is combined with a heavy chain variable region (V H) with a light chain variable region (V) from a series of binders with specificity for the second domain. L ) and combine it again to get the V H Without breaking the original singularity of H Paired with non-cognate V L In this way, a single V L Segment (e.g., V L 1) is two different V H Domain (e.g., V H 1, and V H 2) to form two binding "arms" (V H 1-V L 1, and V H 2-V L 1) produces a bispecific antibody containing a single V L The use of segments reduces the complexity of the system, thereby simplifying and increasing the efficiency of the cloning, expression, and purification methods used to generate bispecifics (see, e.g., USSN13 / 022759, and US2010 / 0331527).

[0154] Alternatively, more than one domain and an antibody that binds to a second target, such as, but not limited to, a second different anti-PD-1 antibody, can be prepared in a bispecific format using the techniques described herein or other techniques known to those skilled in the art. Antibody variable regions that bind to separate regions can be combined together, for example, with variable regions that bind to related sites on the extracellular domain of PD-1, to ensure dual-antigen specificity within a single binding molecule. Suitably, an engineered bispecific of this nature performs dual functions. A variable region with specificity for the extracellular domain is combined with a variable region with specificity for the outside of the extracellular domain, and paired on a structural scaffold that allows each variable region to bind to a different antigen.

[0155] An exemplary bispecific antibody format for use in the context of the present invention is H 3 domain, and the second Ig CH 3 domains, where the first and second Ig C H The three domains differ from each other by at least one amino acid, where the at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H The 3 domain binds to protein A and the second Ig C H The third domain contains a mutation that reduces or abolishes protein A binding, such as the H95R modification (according to the IMGT exon numbering; H435R by EU numbering). H 3 further contains a Y96F modification (by IMGT; Y436F by EU). H Further modifications found in 3 are: D In the case of IgG2 antibodies, N44S, K52N, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (by IMGT; N384S, K392N, and V422I by EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU). Variations in the bispecific antibody formats described above are considered to be within the scope of the present invention.

[0156] Other exemplary bispecific formats used in the context of the present invention include, but are not limited to, for example, scFv-based or bispecific antibody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (such as common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific formats include (see, e.g., Klein et al., 2012, mAbs 4:6, pp. 1-11, and references cited therein, for a description of such formats). Bispecific antibodies have also been constructed using peptide / nucleic acid linkages, such as where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide linkages that then self-assemble into multimeric complexes with defined composition, valency, and geometry (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0157] Therapeutic Administration and Formulation The present invention provides therapeutic compositions comprising the anti-PD-1 antibodies or antigen-binding fragments thereof of the present invention. The therapeutic compositions are administered in accordance with the present invention with suitable carriers, excipients, and other agents that are incorporated into the formulation to effect transport, delivery, improved tolerance, and the like. A large number of suitable formulations can be found in a formulary known to any medicinal chemist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Powell et al., "Compendium of excipients for See also "parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0158] The dose of the antibody varies depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. When the antibody of the present invention is used to treat a disease or disorder in an adult patient or to prevent such a disease, it is advantageous to administer the antibody of the present invention in a single dose of usually about 0.1 to about 60 mg per kg of body weight, more preferably about 5 to about 60, about 10 to about 50, or about 20 to about 50 mg per kg of body weight. Depending on the severity of the condition, the frequency and duration of the treatment are adjusted. In certain embodiments, the antibody or antigen-binding fragment of the present invention is administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, about 100 mg, or about 50 mg. In certain embodiments, the initial dose is followed by an amount that is approximately the same as or less than that of the initial dose. A second or multiple subsequent doses of the antibody or antigen-binding fragment thereof are administered, where subsequent doses are separated by at least 1 day to 3 days; at least 1 week; at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0159] A variety of delivery systems are known and may be used to administer the pharmaceutical compositions of the invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration may be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see, eg, Langer (1990) Science 249:1527-1533).

[0160] The use of nanoparticles to deliver the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles are used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and preparation and use methods are described in detail in Arruebo, M. et al., 2009 ("Antibody-conjugated nanoparticles for biomedical applications" in J. Nanomat., Vol. 2009, Article No. 439389, p. 24, doi:10.1155 / 2009 / 439389), which is incorporated herein by reference. Nanoparticles have been developed and conjugated to antibodies contained in pharmaceutical compositions for targeting tumor cells, or autoimmune tissue cells, or virally infected cells. Nanoparticles for drug delivery are also described in, for example, US8257740, or US8246995, each of which is incorporated herein by reference in its entirety.

[0161] In some situations, pharmaceutical compositions are delivered in controlled release systems. In one embodiment, pumps are used. In another embodiment, polymeric materials are used. In yet another embodiment, the controlled release system is placed close to the target of the composition, thus requiring only a small systemic dose.

[0162] The injectable preparations include intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injection forms, drip infusion forms, etc. These injectable preparations are prepared by publicly known methods. For example, the injectable preparations are prepared by dissolving, suspending or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium that is usually used for injection. As the aqueous medium for injection, for example, physiological saline, isotonic solution containing glucose, and other auxiliary agents are present, and are used in combination with suitable dissolving agents such as alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), non-ionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, for example, sesame oil, soybean oil, etc. are used in combination with dissolving agents such as benzyl benzoate, benzyl alcohol, etc. The injections prepared are preferably filled into suitable ampoules.

[0163] The pharmaceutical compositions of the present invention may be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device may be used to administer the pharmaceutical compositions of the present invention. Pen delivery devices are easily adapted for delivery of pharmaceutical compositions. Such pen delivery devices are either reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered, the cartridge is emptied and the empty cartridge is easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device is then reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition that is held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0164] A number of reusable pens and autoinjector delivery devices are applicable in the subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), the HUMALOG MIX75 / 25™ pen, the HUMALOG™ pen, the HUMALIN70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN™, the HUMALOG™, the HUMALOG™, the HUMALOG™, the HUMALIN ... Examples of disposable pen delivery devices that are applicable in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are certainly not limited to, the SOLOSTAR pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), SURECLICK Autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0165] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in unit doses suitable for the dosage of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is generally about 5 to about 500 mg per dosage form in unit doses; in particular, about 5 to about 100 mg for injections, and about 10 to about 250 mg for other dosage forms.

[0166] Therapeutic Uses of Antibodies The antibodies of the invention are useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by PD-1 expression, signaling, or activity, or treatable by blocking the interaction between PD-1 and a PD-1 ligand (e.g., PD-L1, or PD-L2), or otherwise inhibiting PD-1 activity and / or signaling. For example, the invention provides methods of treating cancer (tumor growth inhibition), chronic viral infection, and / or autoimmune disease by administering to a patient in need of treatment an anti-PD-1 antibody (or a pharmaceutical composition comprising an anti-PD-1 antibody) described herein. The antibodies of the invention can be used to treat a disease, or disorder, or condition, such as cancer, an autoimmune disease, or a viral infection. and / or are useful for treating, preventing, and / or ameliorating, and / or at least one symptom associated with such disease, disorder, or condition. In the context of the methods of treatment described herein, the anti-PD-1 antibodies are administered as monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents, examples of which are described elsewhere herein.

[0167] In certain embodiments of the invention, the antibodies described herein are useful for treating subjects suffering from primary or recurrent cancers, including, but not limited to, renal cell carcinoma, colorectal cancer, non-small cell lung cancer, brain cancer (e.g., glioblastoma multiforme), squamous cell carcinoma of the head and neck, gastric cancer, prostate cancer, ovarian cancer, kidney cancer, breast cancer, multiple myeloma, and melanoma.

[0168] The antibodies are used to treat symptoms of early or late stage cancer. In one embodiment, the antibodies or fragments thereof of the present invention are used to treat metastatic cancer. The antibodies are useful in reducing, inhibiting, or shrinking tumor growth, both solid tumors and blood cancers. In certain embodiments, treatment with the antibodies or antigen-binding fragments thereof of the present invention leads to greater than 50% regression, greater than 60% regression, greater than 70% regression, greater than 80% regression, or greater than 90% regression of tumors in subjects. In certain embodiments, the antibodies are used to prevent tumor recurrence. In certain embodiments, the antibodies are useful in extending overall survival in subjects with cancer. In certain embodiments, the antibodies are useful in reducing toxicity resulting from chemotherapy or radiation therapy while maintaining long-term survival in patients suffering from cancer.

[0169] In certain embodiments, the antibodies of the invention are useful for treating subjects suffering from chronic viral infections. In certain embodiments, the antibodies of the invention are useful in reducing viral titers in a host and / or rescuing exhausted T cells. In certain embodiments, the antibodies of the invention or fragments thereof are used to treat chronic viral infections caused by lymphocytic choriomeningitis virus (LCMV). In certain embodiments, the antibodies of the invention or antigen-binding fragments thereof are administered in therapeutic doses to patients with infections caused by human immunodeficiency virus (HIV), or human papillomavirus (HPV), or hepatitis B / C virus (HBV / HCV). In a related embodiment, the antibodies of the invention or antigen-binding fragments thereof are used to treat infections caused by simian immunodeficiency virus (SIV) in monkey subjects, such as cynomolgus monkeys.

[0170] In certain embodiments, the blocking antibodies of the invention are administered in therapeutically effective amounts to a subject suffering from cancer or a viral infection.

[0171] In certain embodiments, the antibodies of the present invention are useful for treating autoimmune diseases, including, but not limited to, alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes, eosinophilic fasciitis, eosinophilic gastroenteritis, Goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and Wegener's granulomatosis. In certain embodiments, the activating antibodies of the present invention are used to treat subjects suffering from autoimmune diseases.

[0172] One or more antibodies of the invention are administered to alleviate or prevent or reduce the severity of one or more of the symptoms or conditions of a disease or disorder.

[0173] Also contemplated herein is the prophylactic use of one or more antibodies of the invention in patients at risk of developing a disease or disorder, such as cancer, autoimmune diseases, and chronic viral infections.

[0174] In a further embodiment of the invention, the antibody is used for the preparation of a pharmaceutical composition for treating a patient suffering from cancer, an autoimmune disease, or a viral infection. In another embodiment of the invention, the antibody is used as an adjunct therapy with any other agent or any other therapy known to the skilled artisan useful for treating cancer, an autoimmune disease, or a viral infection.

[0175] Combination Therapies and Formulations The combination therapy comprises an anti-PD-1 antibody of the invention and any additional therapeutic agent that is advantageously combined with the antibody of the invention, or a biologically active fragment of the antibody of the invention.

[0176] The antibodies of the present invention are synergistically combined with one or more anti-cancer drugs or therapies used to treat cancers including, for example, renal cell carcinoma, colorectal cancer, glioblastoma multiforme, squamous cell carcinoma of the head and neck, non-small cell lung cancer, colon cancer, ovarian cancer, adenocarcinoma, prostate cancer, glioma, and melanoma. It is also contemplated herein to use the anti-PD-1 antibodies of the present invention in combination with immunostimulatory and / or immunosupportive therapies to inhibit tumor growth and / or enhance survival of cancer patients. Immunostimulatory therapies include direct immunostimulatory therapies to enhance immune cell activity, either by "releasing the brakes" on suppressed immune cells or by "stepping on the gas" to activate immune responses. Examples include targeting other checkpoint receptors, vaccinations, and adjuvants. Immunosuppressive modalities have other direct effects of increasing tumor antigenicity or promoting anti-tumor immune responses by promoting immunogenic cell death, inflammation. Examples include radiation, chemotherapy, anti-antagonism, and surgery.

[0177] In various embodiments, one or more antibodies of the invention are selected from the group consisting of an antibody against PD-L1, a secondary antibody against PD-1 (e.g., nivolumab), a LAG-3 inhibitor, a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an antagonist of another T cell co-inhibitory molecule or ligand (e.g., an antibody against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), an indoleamine -2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists (e.g., "VEGF-Trap" such as aflibercept, or other VEGF inhibitory fusion proteins described in US 7,087,411, or anti-VEGF antibodies, or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab), or small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesvacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), costimulatory receptor agonists (e.g., glucocorticoid-induced TNFR-released protein agonists), tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin Antibodies against leukocytes (e.g., erythrocyte colony-stimulating factor), vaccines (e.g., Bacillus Calmette-Guerin, cancer vaccines), adjuvants to enhance antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3×CD20 bispecific antibody, PSMA×CD3 bispecific antibody), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, cyclophosphamide ... In certain embodiments, the anti-PD-1 antibodies of the present invention are used in combination with cancer vaccines, including dendritic cell vaccines, oncolytic viruses, tumor necrosis factor receptor agonists, platins, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine, cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADCs, and anti-DS6-DM4 ADCs), anti-inflammatory drugs (e.g., corticosteroids, and nonsteroidal anti-inflammatory drugs), nutritional supplements such as antioxidants, or symptomatic care to treat any cancer. In certain embodiments, the anti-PD-1 antibodies of the present invention are used in combination with cancer vaccines, including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, and the like, to enhance anti-tumor responses. Examples of cancer vaccines that may be used in combination with the anti-PD-1 antibodies of the invention include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., Rindopepimut) for brain cancers (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancers).

[0178] In certain embodiments, the anti-PD-1 antibodies of the present invention are administered in combination with radiation therapy in a manner that produces durable anti-tumor responses and / or enhances survival of patients with cancer. In some embodiments, the anti-PD-1 antibodies of the present invention are administered prior to, simultaneously with, or after administration of radiation therapy to a cancer patient. For example, radiation therapy is administered in one or more doses to a tumor lesion, followed by administration of one or more doses of an anti-PD-1 antibody of the present invention. In some embodiments, radiation therapy is administered locally to a tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and / or to kill tumor cells (ablative irradiation), followed by systemic administration of an anti-PD-1 antibody of the present invention. For example, intracranial radiation is administered to a patient with brain cancer (e.g., glioblastoma multiforme) in combination with systemic administration of an anti-PD-1 antibody of the present invention. In certain embodiments, the anti-PD-1 antibodies of the invention are administered in combination with radiation therapy and a chemotherapeutic agent (eg, temozolomide) or a VEGF antagonist (eg, aflibercept).

[0179] In certain embodiments, the anti-PD-1 antibody of the present invention is administered in combination with one or more antiviral drugs to treat chronic viral infections caused by LCMV, HIV, HPV, HBV, or HCV. Examples of antiviral drugs include, but are not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, and corticosteroids. In some embodiments, the anti-PD-1 antibody of the present invention is administered in combination with any antagonist of LAG3 inhibitor, CTLA-4 inhibitor, or another T cell co-inhibitory molecule to treat chronic viral infections.

[0180] In certain embodiments, the anti-PD-1 antibodies of the present invention are combined with antibodies against Fc receptors on immune cells for the treatment of autoimmune diseases. In one embodiment, the antibodies of the present invention or fragments thereof are administered in combination with antibodies or antigen-binding proteins targeted to antigens specific to autoimmune tissues. In certain embodiments, the antibodies of the present invention or antigen-binding fragments thereof are administered in combination with antibodies or antigen-binding proteins targeted to T cell receptors or B cell receptors, including, but not limited to, Fcα (e.g., CD89), Fcγ (e.g., CD64, CD32, CD16a, and CD16b), CD19, and the like. The antibody or fragment thereof of the present invention can be used to treat alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes, and osteoporosis. It may be used in combination with any drug or therapy known in the art to treat an autoimmune disease or disorder, including, but not limited to, eosinophilic fasciitis, eosinophilic gastroenteritis, Goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and Wegener's granulomatosis (e.g., corticosteroids, and other immunosuppressants).

[0181] The additional therapeutically active agent / component is administered prior to, concomitantly with, or following administration of an anti-PD-1 antibody of the invention. For purposes of this disclosure, such administration regimens will be considered administration of an anti-PD-1 antibody "in combination with" a second therapeutically active ingredient.

[0182] The additional therapeutically active ingredient is administered to the subject prior to administration of the anti-PD-1 antibody of the invention. For example, a first ingredient is considered to be administered "prior to" the second ingredient if the first ingredient is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of the second ingredient. In other embodiments, the additional therapeutically active ingredient is administered to the subject after administration of the anti-PD-1 antibody of the invention. For example, a first component is considered to be administered "after" the second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours after administration of the second component. In still other embodiments, an additional therapeutically active component is administered to a subject simultaneously with administration of an anti-PD-1 antibody of the invention. For purposes of this invention, "concurrent" administration includes administration of an anti-PD-1 antibody and an additional therapeutically active component to a subject, for example, in a single dosage form (e.g., co-formulated), or in separate dosage forms that are administered to the subject within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form is administered via the same route (e.g., both the anti-PD-1 antibody and the additional therapeutically active ingredient are administered intravenously, subcutaneously, etc.); or, each dosage form is administered via a different route (e.g., the anti-PD-1 antibody is administered intravenously and the additional therapeutically active ingredient is administered subcutaneously). In any event, the administration of the ingredients in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes will all be considered "co-administration" for purposes of this disclosure. For purposes of this disclosure, administration of an anti-PD-1 antibody "prior to," "concurrent with," or "following" the administration of an additional therapeutically active ingredient (as those terms are defined herein above) will be considered administration of the anti-PD-1 antibody "in combination with" the additional therapeutically active ingredient.

[0183] The present invention includes pharmaceutical compositions in which the anti-PD-1 antibodies of the invention are co-formulated with one or more additional therapeutically active ingredients, as described elsewhere herein, using a variety of dosage combinations.

[0184] In typical embodiments where an anti-PD-1 antibody of the invention is administered in combination with a VEGF antagonist (e.g., a VEGF trap such as aflibercept), including administration of a co-formulation comprising an anti-PD-1 antibody and a VEGF antagonist, the individual components are administered to the subject and / or co-formulated using various dosage combinations. For example, the anti-PD-1 antibody is administered to a subject and / or contained in a co-formulation in an amount selected from the group consisting of 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, and 10.0 mg; and the VEGF antagonist (e.g., a VEGF trap such as aflibercept) is administered to a subject and / or contained in a co-formulation in an amount selected from the group consisting of 0.1 mg, 0.2 mg, 0.

[0033] The compound may be administered to a subject and / or contained in a co-formulation in an amount selected from the group consisting of: 3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, and 3.0 mg. The combination / co-formulation is administered to a subject according to any of the dosing regimens disclosed elsewhere herein, including, for example, twice a week, once every week, once every two weeks, once every three weeks, once every month, once every two months, once every three months, once every four months, once every five months, once every six months, etc.

[0185] Treatment regimen According to certain embodiments of the invention, multiple doses of an anti-PD-1 antibody (or a pharmaceutical composition comprising an anti-PD-1 antibody and any combination of additional therapeutically active agents as mentioned herein) are administered to a subject over a defined time course. The method according to this aspect of the invention comprises sequentially administering to a subject multiple doses of an anti-PD-1 antibody of the invention. As used herein, "sequentially administering" means that each dose of an anti-PD-1 antibody is administered to a subject at different points in time, e.g., on different days separated by a predetermined interval (e.g., hour, day, week, or month). The invention includes methods comprising sequentially administering to a patient a single initial dose of an anti-PD-1 antibody, followed by one or more secondary doses of an anti-PD-1 antibody, optionally followed by one or more tertiary doses of an anti-PD-1 antibody. The anti-PD-1 antibody is administered at a dose between 0.1 mg / kg and 100 mg / kg.

[0186] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal sequence of administration of the anti-PD-1 antibody of the present invention. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"); a "secondary dose" is a dose administered after the initial dose; and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses all contain the same amount of anti-PD-1 antibody, but generally differ from one another in terms of frequency of administration. However, in certain embodiments, the amount of anti-PD-1 antibody contained in the initial, secondary, and / or tertiary doses differ from one another during the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0187] In certain exemplary embodiments of the invention, each secondary and / or tertiary dose is administered 1 to 26 (e.g., 1, 11 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 As used herein, the phrase "the immediately preceding dose" refers to the dose of an anti-PD-1 antibody that is administered to a patient prior to administration of the exact next dose in an uninterrupted dose sequence in a multiple dose sequence.

[0188] The methods according to this aspect of the invention include administering to the patient any number of secondary and / or tertiary doses of an anti-PD-1 antibody. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, In certain embodiments, only a single tertiary dose is administered to the patient, hi other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0189] In embodiments comprising multiple secondary doses, each secondary dose is administered at the same frequency as the other secondary doses. For example, each secondary dose is administered 1-2 weeks or 1-2 months after the immediately preceding dose. Similarly, in embodiments comprising multiple tertiary doses, each tertiary dose is administered at the same frequency as the other tertiary doses. For example, each tertiary dose is administered 2-12 weeks after the immediately preceding dose. In certain embodiments of the invention, the frequency at which the secondary and / or tertiary doses are administered to a patient varies over the course of a treatment regimen. The frequency of administration is also adjusted during the course of treatment by a physician depending on the needs of an individual patient according to clinical testing.

[0190] The invention includes dosing regimens in which 2-6 additive doses are administered to a patient at a first frequency (e.g., once per week, once every 2 weeks, once per 3 weeks, once per month, once every 2 months, etc.), followed by 2 or more maintenance doses administered to the patient on a less frequent basis. For example, according to this aspect of the invention, if additive doses are administered, for example, once per month (e.g., 2, 3, 4, or more additive doses administered once per month), then maintenance doses are administered to the patient once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 10 weeks, once every 12 weeks, etc.

[0191] Diagnostic Uses of Antibodies The anti-PD-1 antibodies of the invention are used to detect and / or measure PD-1 in a sample, e.g., for diagnostic purposes. Certain embodiments contemplate the use of one or more of the antibodies of the invention in assays to detect a disease or disorder, such as cancer, an autoimmune disease, or a chronic viral infection. A typical diagnostic assay for PD-1 involves, for example, contacting a sample obtained from a patient with an anti-PD-1 antibody of the invention, where the anti-PD-1 antibody is labeled with a detectable label or reporter molecule, or used as a capture ligand to selectively isolate PD-1 from the patient sample. Alternatively, an unlabeled anti-PD-1 antibody is used in diagnostic applications in combination with a second antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 The specific assays used to detect or measure PD-1 in a sample include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence-activated cell sorting (FACS).

[0192] The samples used in the PD-1 diagnostic assays according to the invention include any tissue or body fluid sample that can be obtained from a patient and that contains a detectable amount of PD-1 protein, or either a fragment thereof, under normal or pathological conditions. In general, the level of PD-1 in a particular sample obtained from a healthy patient (e.g., a patient not suffering from cancer or an autoimmune disease) is measured to first establish a baseline, or standard, PD-1 level. This baseline level of PD-1 is then compared to the level of PD-1 measured in a sample obtained from an individual suspected of having a cancer-related condition, or a symptom associated with such a condition.

[0193] The antibodies specific for PD-1 do not contain an additional label or moiety, or they contain an N-terminal or C-terminal label or moiety. The moiety is biotin. In binding assays, the location of the label (if any) determines the orientation of the peptide with respect to the surface to which it is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface.

[0194] Aspects of the invention relate to the use of the disclosed antibodies as markers for predictive diagnosis of cancer or autoimmune disorders in patients. The antibodies of the invention are used in diagnostic assays to assess cancer prognosis and to predict survival in patients. EXAMPLES

[0195] The following examples are provided to provide those skilled in the art with a complete disclosure or description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Attempts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric. EXAMPLES

[0196] Generation of human antibodies against PD-1 A fragment of PD-1 ranging from approximately amino acids 25-170 of GenBank accession NP_005009.2 (SEQ ID NO: 327) with a C93S alteration was used to generate human antibodies against PD-1. The immunogen was administered directly to VELOCIMMUNE® mice, which contain DNA encoding human immunoglobulin heavy and kappa light chain variable regions, along with an adjuvant to stimulate an immune response. Antibody immune responses were monitored by PD-1-specific immunoassays. Once the desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to retain viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines producing PD-1-specific antibodies. Using this technique, and the immunogens described above, several anti-PD-1 chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were generated; exemplary antibodies generated in this manner were designated H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7794N, H2M7795N, H2M7796N, and H2M7798N.

[0197] Anti-PD-1 antibodies were also isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in US2007 / 0280945A1 (specifically incorporated herein by reference in its entirety). Using this method, several fully human anti-PD-1 antibodies (i.e., antibodies having human variable domains and human constant domains) have been generated; exemplary antibodies generated by this method are designated as follows: H4H9019P, H4xH9034P2, H4xH9035P2, H4xH9037P2, H4xH9045P2, H4xH9048P2, H4H9057P2, H4H9068P2, H4xH9119P2, H4xH9120P2, H4xH9128P2, H4xH9135P2, H4xH9145P2, H4xH8992P, H4xH8999P, and H4xH9008P.

[0198] The biological properties of exemplary antibodies generated according to the methods of this Example are described in detail in the Examples set forth below. EXAMPLES

[0199] Heavy and light chain variable region amino acid and nucleotide sequences Table 1 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-PD-1 antibodies of the invention. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0200] [Table 1]

[0201] [Table 2]

[0202] Antibodies are typically referred to herein by the following nomenclature: an Fc prefix (e.g., "H4xH", "H1M", "H2M", etc.), followed by a numeric identifier (e.g., "7789", "7799", etc., as shown in Table 1), followed by a "P", "P2", "N", or "B" suffix. Thus, according to this nomenclature, antibodies are referred to herein as, for example, "H1H7789N", "H1M7799N", "H2M7780N", etc. The H4xH, H1M, H2M, and H2aM prefixes on antibody names used herein indicate the particular Fc region isotype of the antibody. For example, an "H4xH" antibody has a human IgG4 Fc with two or more amino acid changes as disclosed in US20100331527, an "H1M" antibody has a murine IgG1 Fc, and an "H2M" antibody has a murine IgG2 Fc (a or b isotype) (all variable (The regions are fully human, as indicated by the initial "H" in the antibody name.) As one of skill in the art will appreciate, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG1 Fc can be converted to an antibody having a human IgG4 Fc, etc.), but in each case the variable domains (including the CDRs) indicated by the numerical identifiers shown in Table 1 remain the same, and the antigen binding characteristics are expected to be consistent or substantially similar regardless of the nature of the Fc domain.

[0203] In certain embodiments, selected antibodies with mouse IgG1 Fc were converted to antibodies with human IgG4 Fc. In one embodiment, the IgG4 Fc domain contains a serine to proline mutation (S108P) in the hinge region to promote dimer stabilization. Table 3 lists the amino acid sequence identifiers of the heavy and light chain sequences of selected anti-PD-1 antibodies with human IgG4 Fc.

[0204] [Table 3]

[0205] Each heavy chain sequence in Table 3 is H or HCVR; including HCDR1, HCDR2, and HCDR3), and a constant region (C H 1. C H 2, and C H Each light chain sequence in Table 3 contained a variable region (V L or LCVR; including LCDR1, LCDR2, and LCDR3), and a constant region (C L). SEQ ID NO:330 contained an HCVR comprising amino acids 1-117, and a constant region comprising amino acids 118-444. SEQ ID NO:331 contained an LCVR comprising amino acids 1-107, and a constant region comprising amino acids 108-214. SEQ ID NO:332 contained an HCVR comprising amino acids 1-122, and a constant region comprising amino acids 123-449. SEQ ID NO:333 contained an LCVR comprising amino acids 1-107, and a constant region comprising amino acids 108-214. SEQ ID NO:334 contained an HCVR comprising amino acids 1-119, and a constant region comprising amino acids 120-446. SEQ ID NO:335 contained an LCVR comprising amino acids 1-108, and a constant region comprising amino acids 109-215. SEQ ID NO:336 contained an HCVR comprising amino acids 1-121, and a constant region comprising amino acids 122-448. SEQ ID NO:337 contained an LCVR comprising amino acids 1-108, and a constant region comprising amino acids 109-215. EXAMPLES

[0206] Antibody binding to PD-1 determined by surface plasmon resonance The binding and dissociation rate constants (k, respectively) for antigen binding to purified anti-PD1 antibodies were determined using real-time surface plasmon resonance biosensor assays on a Biacore 4000 or Biacore T200 instrument. a and k d ), the equilibrium dissociation constant, and the dissociation half-life (K D and t 1 / 2 The Biacore sensor surface was stained with a polyclonal rabbit anti-mouse antibody (GE, no. BR-1008-38), or a monoclonal mouse anti-human Fc antibody (GE, no. BR-1008-39) to capture approximately 100-900 RU of anti-PD-1 monoclonal antibodies expressed with either mouse Fc or human Fc, respectively. The PD-1 reagents tested for binding to anti-PD-1 antibodies included recombinant human PD-1 expressed with a C-terminal myc-myc-hexahistidine tag (hPD-1-MMH; SEQ ID NO: 321), recombinant cynomolgus monkey PD-1 expressed with a C-terminal myc-myc-hexahistidine tag (MfPD-1-MMH; SEQ ID NO: 322), recombinant human PD-1 dimer expressed with a C-terminal mouse IgG2a Fc tag (hPD-1-mFc; SEQ ID NO: 323), or recombinant human PD-1 dimer expressed with a C-terminal human IgG1 Fc tag (hPD1-hFc; SEQ ID NO: 324), and monkey PD-1 with mFc (SEQ ID NO: 329). Different concentrations of PD-1 reagent, ranging from 200 nM to 3.7 nM, were injected across the anti-PD-1 monoclonal antibody capture surface at a flow rate of 30 μL / min on the Biacore 4000 or 50 μL / min on the Biacore T200. Binding of the PD-1 reagent to the captured monoclonal antibody was monitored for 3–5 min, while their dissociation from the antibody was monitored for 7–10 min in HBST running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20). Experiments were performed at 25°C and 37°C. Kinetic binding (k a ) and dissociation (k d ) rate constant was determined. Next, from the kinetic rate constant, K D (M)=k d / k a , and t 1 / 2 (min) = [ln2 / (60 * k d )] and the binding / dissociation equilibrium constant (K D ), and dissociation half-life (t 1 / 2) was calculated. The binding kinetic parameters for different anti-PD-1 monoclonal antibodies binding to different PD-1 reagents at 25° C. and 37° C. are tabulated in Tables 4-11.

[0207] [Table 4]

[0208] [Table 5]

[0209] [Table 6]

[0210] [Table 7]

[0211] [Table 8]

[0212] [Table 9]

[0213] [Table 10]

[0214] [Table 11]

[0215] As shown in Table 4, at 25°C, 28 of the 29 anti-PD-1 antibodies of the invention have K values ​​in the range of 2.1 nM to 291 nM. DAll of the 29 anti-PD-1 antibodies of the invention bound to hPD-1-MMH with K values ​​ranging from 3.79 nM to 1.51 μM at 37° C. One antibody, H4H9068P2, showed no measurable binding to hPD-1-MMH at 25° C. As shown in Table 5, at 37° C., 26 of the 29 anti-PD-1 antibodies of the invention bound to hPD-1-MMH with K values ​​ranging from 3.79 nM to 1.51 μM. D All 29 anti-PD-1 antibodies of the invention bound to hPD-1-MMH with K values ​​ranging from 65.5 pM to 59.4 nM at 25°C. The three antibodies of the invention did not show significant binding to hPD-1-MMH at 37°C. As shown in Table 6, at 25°C, all 29 anti-PD-1 antibodies of the invention bound to hPD-1-MMH with K values ​​ranging from 65.5 pM to 59.4 nM. D As shown in Table 7, at 37°C, all 27 anti-PD-1 antibodies of the invention bound to hPD-1 dimeric protein with K values ​​ranging from 3.09 pM to 551 nM. D As shown in Table 8, at 25°C, 27 of the 29 anti-PD-1 antibodies of the invention bound to hPD-1 dimeric protein with K values ​​ranging from 3.09 nM to 551 nM. D The two antibodies of the invention bound to MfPD-1-MMH with K values ​​ranging from 7.00 nM to 7.54 μM at 37° C. Two of the antibodies of the invention showed no significant binding to MfPD-1-MMH at 25° C. As shown in Table 9, at 37° C., 25 of the 29 anti-PD-1 antibodies of the invention bound to MfPD-1-MMH with K values ​​ranging from 7.00 nM to 7.54 μM. D The four antibodies of the present invention bound to MfPD-1-MMH at 37° C. As shown in Table 10, at 25°C, all 18 anti-PD-1 antibodies of the invention tested had K values ​​ranging from 137 pM to 54.2 nM. D As shown in Table 11, at 37°C, all 18 tested anti-PD-1 antibodies of the invention bound to the MfPD-1 dimer with K values ​​ranging from less than 49 pM to 86.3 nM. D It bound to MfPD-1 dimers at 100 ng / mL. EXAMPLES

[0216] Blockade of PD-1 binding to PD-L1 as determined by ELISA The ability of anti-PD-1 antibodies to block human PD-1 binding to its ligand, the PD-L1 receptor, was measured using a three-way competitive sandwich ELISA format. Dimeric human PD-L1 protein, comprising a portion of the human PD-L1 extracellular domain expressed with either a C-terminal human Fc tag (hPD-L1-hFc; SEQ ID NO: 325) or a C-terminal mouse Fc tag (hPD-L1-mFc; SEQ ID NO: 326), or dimeric human PD-L2, comprising the human PD-L2 extracellular domain produced with a C-terminal human Fc tag (hPD-L2-hFc; R&D Systems, no. 1224-PL), were separately coated onto 96-well microtiter plates at a concentration of 2 μg / mL in PBS overnight at 4° C. Nonspecific binding sites were subsequently blocked using a 0.5% (w / v) BSA solution in PBS. In the first competitive format, a constant concentration of 1.5 nM of dimeric human PD-1 protein containing a human PD-1 extracellular domain expressed with a C-terminal mouse Fc tag (hPD-1-mFc; SEQ ID NO: 323) was added to a dilution series of anti-PD-1 antibodies or isotype control antibodies such that the final antibody concentrations ranged from 0 to 200 nM. In the second competitive format, a constant concentration of 200 pM of dimeric biotinylated human PD-1 protein containing a human PD-1 extracellular domain expressed with a C-terminal human Fc tag (biot-hPD-1-hFc; SEQ ID NO: 323) was similarly added to a dilution series of anti-PD-1 antibodies or isotype controls such that the final antibody concentrations ranged from 0 to 50 nM. In the third competitive format, a constant concentration of 100 pM of dimeric hPD-1-mFc protein was similarly added to serial dilutions of anti-PD-1 antibodies or isotype controls, with final antibody concentrations ranging from 0 to 100 nM. These antibody and protein complexes were then incubated for 1 hour at room temperature (RT). Antibody and protein complexes with a given hPD-1-mFc were transferred to a microtiter plate coated with hPD-L1-hFc, antibody and protein complexes with a given 200 pM biot-hPD-1-hFc were transferred to a hPD-L1-mFc coated plate, and antibody and protein complexes with a given 100 pM hPD-1-mFc were transferred to a microtiter plate coated with hPD-L2-hFc. After incubation at RT for 1 hour, the wells were washed and plate-bound hPD-1-mFc was detected with an anti-mFc polyclonal antibody conjugated with horseradish peroxidase (HRP) (Jackson ImmunoResearch Inc., no. 115-035-164), and plate-bound biot-hPD-1-hFc was detected with streptavidin conjugated with HRP (Thermo Scientific, no. N200). Samples were developed with TMB solution (BD Biosciences, no. 51-2606KC, and no. 51-2607KC) to produce a colorimetric reaction, and color development was then stabilized by the addition of 1 M sulfuric acid before measuring absorbance at 450 nm in a VictorX5 plate reader. Data analysis was performed using a sigmoidal dose-response model in Prism™ software (GraphPad). The calculated IC, defined as the concentration of antibody required to reduce 50% of human PD-1 binding to human PD-L1 or PD-L2, was calculated as the concentration of antibody required to reduce 50% of human PD-1 binding to human PD-L1 or PD-L2. 50 The value was used as an index of blocking efficacy. Percent maximal blockade was calculated as a measure of the ability of the antibody to completely block human PD-1 binding to human PD-L1 or PD-L2 on the plate, as determined from the dose curve. This percent maximal blockade was compared to the difference between the signal observed for a sample of human PD-1 containing no anti-PD-1 antibody (0% blockade) and the background signal from the HRP-conjugated secondary antibody alone (100% blockade). The percent reduction in signal observed in the presence of the highest test concentration for each antibody was calculated by subtracting it from 100%.

[0217] Percent maximal block and calculated IC for antibodies blocking hPD-1 binding signal by more than 35% 50 Values ​​are shown in Tables 12-14. Antibodies that showed a 35% or less reduction in hPD-1 binding signal were defined as non-blockers. Antibodies that showed a 35% or greater increase in human PD-1 binding signal were characterized as non-blockers / enhancers. The theoretical assay bottom, defined as the minimum antibody concentration theoretically required to occupy 50% of the binding sites of human PD-1 in the assay, was 0.75 nM for formats with a constant 1.5 nM hPD-1-mFc, 100 pM for formats with a constant 200 pM biot-hPD-1-hFc, and 50 pM for formats with a constant 100 pM hPD-1-mFc, which corresponds to the lower calculated IC 50 values ​​did not represent quantitative protein-antibody site binding. For this reason, the calculated IC values ​​were less than 0.75 nM in assays using hPD-1-mFc constant and hPD-L1 coated, less than 100 pM in assays using biot-hPD-1-hFc constant and hPD-L1 coated, and less than 50 pM in assays using hPD-1-mFc constant and hPD-L2 coated. 50 Antibodies with values ​​reported in Tables 12-14 as less than 7.5E-10 M, less than 1.0E-10 M, and less than 5.0E-11 M, respectively.

[0218] [Table 12]

[0219] [Table 13]

[0220] [Table 14]

[0221] As shown in Table 12, in the first assay format, 23 of the 27 anti-PD-1 antibodies had IC values ​​ranging from 190 pM to 3.3 nM, with maximum percent blockage ranging from 67% to 100%. 50 Blocked hPD-1-mFc binding to hPD-L1-hFc at a value of 1.5 nM. One antibody, H2aM7796N, was identified as a non-blocker. Three anti-PD-1 antibodies (H4H9068P2, H1M7789N, and H2aM7791N) were identified as non-blockers / enhancers.

[0222] As shown in Table 13, in the second assay format, 23 of the 27 anti-PD-1 antibodies had IC values ​​ranging from 59 pM to 1.3 nM, with maximum percent blockage ranging from 60% to 101%. 50 Blocked biot-hPD-1-hFc binding to hPD-L1-mFc at a value of 200 pM. One antibody, H1M7789N, was identified as a non-blocker. Three anti-PD-1 antibodies (H4H9057P2, H4H9068P2, and H2aM7791N) were identified as non-blockers / enhancers.

[0223] Four anti-PD-1 antibodies of the invention, as well as an isotype control, were tested in the third assay format shown in Table 14. All four anti-PD-1 antibodies of the invention had IC values ​​ranging from 0.13 nM to 1.3 nM. 50 values, and blocked 100 pM (fixed concentration) of hPD-1-mFc from binding to hPD-L2-hFc coated on the plate, with maximum percent blockage ranging from 94% to 100%. EXAMPLES

[0224] Blockade of PD-1 binding to PD-L1 as determined by biosensor assay and by surface plasmon resonance The inhibition of human PD-1 binding to human PD-L1 by different anti-PD-1 monoclonal antibodies was studied using a real-time biolayer interferometry assay on either an Octet Red96 biosensor instrument (Fortebio Inc.) or a real-time surface plasmon resonance biosensor assay on a Biacore3000 instrument.

[0225] Inhibition studies for anti-PD-1 monoclonal antibodies expressed with mouse Fc were performed on an Octet Red96 instrument. First, 100 nM of recombinant human PD-1 expressed with a C-terminal mouse IgG2a Fc tag (hPD-1-mFc; SEQ ID NO: 323) was incubated with 500 nM of each anti-PD-1 monoclonal antibody for at least 1 hour, and then the inhibition assay was performed. The concentrations ranged from approximately 0.8 nM to 1.2 nM. Recombinant human PD-L1 expressed with a human IgG1 Fc tag at the C-terminus of M (hPD-L1-hFc; SEQ ID NO: 325) was captured using an anti-human IgG Fc capture Octet biosensor. The hPD-L1-hFc coated Octet biosensor was then immersed into wells containing a mixture of hPD-1-mFc and different anti-PD-1 monoclonal antibodies. The entire experiment was performed at 25°C in Octet HBST buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v surfactant P20, 0.1mg / mL BSA) with the plate shaking at a speed of 1000 rpm. Between each step of the experiment, the biosensor was washed in Octet HBST buffer. The real-time binding response was monitored throughout the entire course of the experiment and the binding response at the end of each step was recorded. The binding of hPD-1-mFc to captured hPD-L1-hFc was compared in the presence and absence of different anti-PD-1 monoclonal antibodies and used to determine the inhibitory activity of the antibodies tested, as shown in Table 15.

[0226] [Table 15]

[0227] As shown in Table 15, 9 of the 11 anti-PD-1 antibodies tested on the Octet Red 96 instruments demonstrated strong blocking of hPD-1-mFc binding to hPD-L1-hFc, ranging from 86% blockage to complete blockage of binding. One anti-PD-1 antibody tested (H1M7789N) demonstrated weak blocking of hPD-1-mFc binding to hPD-L1-hFc, with 29% blockage. One antibody tested (H2aM7791N) demonstrated the ability to enhance binding of hPD-1-mFc to hPD-L1-hFc.

[0228] Next, inhibition studies of anti-PD-1 monoclonal antibodies expressed with human Fc were performed on a Biacore3000 instrument. First, 100 nM of recombinant human PD-1 expressed with a C-terminal human IgG1 Fc tag (hPD-1-hFc; SEQ ID NO:3) was used. 24) was incubated with 500 nM of each anti-PD-1 monoclonal antibody for at least 2 hours, after which the inhibition assay was performed. The CM5 Biacore sensor surface was first derivatized with a polyclonal rabbit anti-mouse antibody (GE catalog no. BR-1008-38) using standard EDC-NHS chemistry. Approximately 730 RU were then detected. Recombinant human PD-L1 expressed with a C-terminal mouse IgG2a Fc tag (hPD-L1.mFc; SEQ ID NO: 326) was captured, followed by injection of 100 nM hPD-1.hFc for 3 minutes at a flow rate of 25 μL / min in the presence and absence of different anti-PD-1 monoclonal antibodies. The entire experiment was performed at 25°C in a running buffer containing 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant Tween-20 (HBS-ET running buffer). Real-time binding responses were monitored throughout the course of the experiment and binding responses were recorded at the end of every step. Binding of hPD-1-hFc to captured hPD-L1-mFc was compared in the presence and absence of different anti-PD-1 monoclonal antibodies and used to determine the inhibitory activity of the tested antibodies, as shown in Table 16.

[0229] [Table 16]

[0230] As shown in Table 16, the anti-PD-1 antibodies of the invention tested on 18 of the 20 Biacore3000 instruments demonstrated strong blocking of hPD-1-hFc binding to hPD-L1-mFc, with blocking ranging from 96% to 100%. One antibody demonstrated the ability to enhance binding of hPD-1-hFc to hPD-L1-mFc. In this study, one of the tested antibodies of the invention (H4H9057P2) demonstrated non-specific background binding to the anti-mouse Fc capture surface. EXAMPLES

[0231] Octet cross-competition between anti-PD-1 antibodies Binding competition between anti-PD-1 monoclonal antibodies was determined using a real-time label-free biolayer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp.). The entire experiment was performed at 25°C in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDT A, 0.05% v / v detergent Tween-20, 0.1 mg / mL BSA (Octet HBS-ET buffer) with plate shaking at a speed of 1000 rpm. To assess whether two antibodies could compete with each other for binding to their respective epitopes on recombinantly expressed human PD-1 with a C-terminal myc-myc-hexahistidine tag (hPD-1-MMH; SEQ ID NO: 321), approximately 0.1 nM hPD-1-MMH was first captured onto an Octet biosensor chip (Pall ForteBio Corp., #18-5079) coated with anti-penta-His antibody by submerging the chip in a well containing a 50 μg / mL hPD-1-MMH solution for 5 minutes. The antigen-captured biosensor chip was then saturated with the primary anti-PD-1 monoclonal antibody (hereafter referred to as mAb-1) by submerging it in a well containing a solution of 50 μg / mL mAb-1 for 5 minutes. The biosensor chip was then subsequently immersed in a well containing a solution of 50 μg / mL secondary anti-PD-1 monoclonal antibody (hereafter referred to as mAb-2). Between each step of the experiment, the biosensor chip was washed in Octet HBS-ET buffer. The real-time binding response was monitored over the course of the experiment and the binding response at the end of each step was recorded. The response of mAb-2 binding to hPD-1-MMH pre-complexed with mAb-1 was compared to determine the competitive / non-competitive behavior of the different anti-PD-1 monoclonal antibodies. The results are summarized in Table 17 ( * Self-competing mAb2 was not listed).

[0232] [Table 17]

[0233] [Table 18]

[0234] Secondary binding competition was determined between a panel of selected anti-PD-1 monoclonal antibodies using a real-time label-free biolayer interferometry assay on an Octet HTX biosensor (Pall ForteBio Corp.). The entire experiment was performed at 25°C in 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v surfactant Tween-20, 0.1mg / mL BSA (Octet HBS-ET buffer) with plate shaking at a speed of 1000 rpm. The two antibodies bound to their respective epitopes on hPD-1-MMH. To assess whether the antibodies could compete with each other for binding to the same peptide, anti-penta-His antibody-coated Octet biosensor chips (Fortebio) were incubated with 10 μg / mL hPD-1-MMH for 150 s by submerging the chip in a well containing a solution of 10 μg / mL hPD-1-MMH. Approximately 0.25 nM of hPD-1-MMH was first captured on a mAb-1 antibody (Absorbance Assay Kit, Inc., no. 18-5079). The antigen-captured biosensor chip was then saturated with a primary anti-PD-1 monoclonal antibody (hereafter referred to as mAb-1) by immersion in a well containing a solution of 100 μg / mL mAb-1 for 5 minutes. The biosensor chip was then subsequently immersed in a well containing a solution of 100 μg / mL secondary anti-PD-1 monoclonal antibody (hereafter referred to as mAb-2) for 4 minutes. All biosensors were washed in Octet HBS-ET buffer between each step of the experiment. Real-time binding responses were monitored during the course of the experiment and the binding responses at the end of each step were recorded as shown in FIG. 2. The responses of mAb-2 binding to hPD-1-MMH pre-complexed with mAb-1 were compared to determine the competitive / non-competitive behavior of the different anti-PD-1 monoclonal antibodies. The results are summarized in Table 18 ( * Self-competing mAb2 was not listed).

[0235] [Table 19]

[0236] Under the experimental conditions disclosed in this example, H4H7795N2 cross-competed with H4H7798N; H4H7798N cross-competed with H4H7795N2 and H4H9008P; H4H9008P cross-competed with H4H7798N and H4H9068P2; H4H9068P2 cross-competed with H4H9008P and H4H9048P2. EXAMPLES

[0237] Antibody binding to cells overexpressing PD-1 Binding of anti-PD-1 antibodies to a human embryonic kidney cell line (HEK293; ATCC, no. CRL-1573) stably transfected with full-length human PD-1 (amino acids 1–289 of accession no. NP_005009.2) (HEK293 / hPD-1) was determined by FACS.

[0238] For the assay, adherent cells were detached using trypsin or enzyme-free dissociation buffer and blocked in complete medium. Cells were centrifuged and diluted to 2.5–6 × 10 in cold PBS containing 2% FBS. 6 The cells were resuspended at a concentration of 100 nM cells / mL. The HEK293 parental and HEK293 / hPD-1 cells were then incubated with 100 nM of each anti-PD-1 antibody on ice for 15–30 min. Unbound antibodies were removed by washing with D-PBS containing 2% FBS, and the cells were subsequently incubated for 15–30 min on ice with allophycocyanin-conjugated secondary F(ab')2 recognizing either human Fc (Jackson ImmunoResearch, no. 109-136-170) or mouse Fc (Jackson ImmunoResearch, no. 115-136-146). The cells were washed with D-PBS containing 2% FBS, and unbound secondary F(ab')2 was subsequently incubated for 15–30 min on ice. The plate was then stripped and fluorescence measurements were taken using either a HyperCyte (IntelliCyt, Inc.) flow cytometer or an Accuri flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).

[0239] [Table 20]

[0240] As shown in Table 19, 25 of the 27 anti-PD-1 antibodies of the invention showed stronger binding to HEK293 / hPD-1 cells compared to binding in the parental HEK293 line. Two of the antibodies of the invention (H2aM7795N, and H4H9068P2) bound weaker to cells expressing human PD-1 compared to the other antibodies tested.

[0241] To further characterize the anti-PD1 antibodies of the present invention, full-length human PD-1 (accession no. NP_ Dose-dependent binding to a human embryonic kidney cell line (HEK293; ATCC, no. CRL-1573) stably transfected with 005009.2 (amino acids 1-289) (HEK293 / hPD-1) was determined by FACS.

[0242] For the assay, adherent cells were detached using trypsin and blocked with complete medium. Cells were centrifuged and plated at 6 × 10 in staining buffer (1% FBS in PBS). 6 The cells were resuspended at a concentration of 1000 cells / mL. EC of anti-PD1 antibody 50 and E maxTo determine , 90 μL of cell suspension was incubated for 30 min on ice with serial dilutions of anti-PD-1 antibodies and controls diluted to final concentrations ranging from 5 pM to 100 nM in staining buffer (a sample without mAb was included as a negative control). Cells were then centrifuged and the pellet washed once with staining buffer to remove unbound antibody. Cells were subsequently incubated for 30 min on ice with either allophycocyanin-conjugated secondary F(ab')2 recognizing human Fc (Jackson ImmunoResearch, no. 109-136-170), or mouse Fc (Jackson ImmunoResearch, no. 115-136-071). Cells were centrifuged and the pellet washed once with wash buffer to remove unbound secondary F(ab')2, then incubated overnight with Cytofix (BD Cells were fixed with a 1:1 dilution of 1:1 IgG4-associated IgG4 (Biosciences, no. 554655) and staining buffer. The following day, cells were centrifuged and pellets were washed once with staining buffer, resuspended, and filtered. Fluorescence measurements were acquired on a Hypercyt® cytometer and analyzed in ForeCyt™ (IntelliCyt; Albuquerque, NM) to determine mean fluorescence intensity (MFI). EC 50 The E values ​​for each antibody were calculated. max was defined as the binding at the highest antibody dose tested (100 nM).

[0243] [Table 21]

[0244] [Table 22]

[0245] As shown in Table 20, 25 of the 28 anti-PD1 antibodies of the invention have an EC value in the range of 33.18 pM to 2.59 nM.50 values, and E ranging from 37,789 to 11,368 MFI. max The three anti-PD1 antibodies of the present invention did not show strong binding to HEK293 / hPD-1 cells and therefore had no significant EC 50 It was not possible to determine a value. None of the isotype controls showed measurable binding in this assay.

[0246] As shown in Table 21, three of the six anti-PD1 antibodies of the invention have EC values ​​in the range of 509 pM to 4.81 nM. 50 values, and E ranging from 39,774 to 14,111 MFI. max The three tested antibodies of the invention bound to HEK293 / hPD-1 cells but did not reach a plateau. Therefore, their exact EC 50 The EC values ​​could not be determined and 50 Values ​​were referred to as inconclusive. None of the isotype controls showed measurable binding in this assay. EXAMPLES

[0247] Blockade of PD-1-induced T cell downregulation in a T cell / APC luciferase reporter assay T cell activation occurs by stimulating the T cell receptor (TcR), which recognizes specific peptides presented by major histocompatibility complex class I or II proteins on antigen-presenting cells (APCs). Activated TcR then initiates a cascade of signaling events that can be monitored by reporter genes driven by transcription factors such as activator-protein 1 (AP-1), nuclear factor of activated T cells (NFAT), or nuclear factor kappa-light-chain-enhancer of activated B cells (NFκb). T cell responses are regulated through the engagement of co-receptors that are either constitutively or inducibly expressed on T cells. One such receptor is PD-1, a negative regulator of T cell activity. PD-1 acts to deliver an inhibitory signal by interacting with its ligand, PD-L1, expressed on APCs or target cells, including cancer cells, and recruiting phosphatases to the TcR signalosome, resulting in the suppression of positive signaling.

[0248] The ability of anti-PD-1 antibodies to antagonize PD-1 / PD-L1 mediated signaling through the PD-1 receptor in human T cell lines was evaluated using an in vitro cell-based assay shown in Figure 1. A bioassay was developed to investigate the ability of anti-PD-1 antibodies to antagonize PD-1 / PD-L1 mediated signaling through the PD-1 receptor in human T cell lines. Mixed cultures derived from Jurkat cells (an immortalized T cell line) and Raji cells (a B cell line) were utilized to measure T cell signaling induced by interactions between APCs and T cells. For the first component of the bioassay, Jurkat clone E6-1 cells (ATCC, no. TIB-152) were transduced with Cignal Lenti AP-1 Luc Reporter (Qiagen-Sabiosciences, no. CLS-011L) according to the manufacturer's instructions. The lentivirus encoded the firefly luciferase gene under the control of a minimal CMV promoter, tandem repeats of a TPA-inducible transcription response element (TRE), and a puromycin resistance gene. The engineered Jurkat cell line was subsequently transduced with a PD-1 chimera containing the extracellular domain of human PD-1 (amino acids 1-170 of human PD1; accession number NP_005009.2) and the transmembrane and cytoplasmic domains of human CD300a (amino acids 181-299 of human CD300a; accession number NP_009192.2). The resulting stable cell line (Jurkat / AP1-Luc / hPD1-hCD300a) was selected and maintained in RPMI / 10% FBS / penicillin / streptomycin / glutamine supplemented with 500 μg / mL G418 + 1 μg / mL puromycin.

[0249] For the second component of the bioassay, Raji cells (ATCC, no. CCL-86) were transfected with the lentiviral (pLEX) vector system (Thermo Scientific Cells were transduced with the cloned human PD-L1 gene (amino acids 1-290 of accession no. NP_054862.1) into IgG4-dependent PD-L1 (Cell Signaling Technology, Inc., Biosystems, no. OHS4735). PD-L1 positive Raji cells (Raji / hPD-L1) were isolated by FACS using a PD-L1 antibody and maintained in Iscove / 10% FBS / penicillin / streptomycin / glutamine supplemented with 1 μg / mL puromycin.

[0250] To stimulate APC / T cell interaction, a bispecific antibody (CD3xCD20 bispecific antibody; e.g., as disclosed in US20140088295) was utilized, which contains one Fab arm that binds to CD3 on T cells and another that binds to CD20 on Raji cells. The presence of the bispecific molecule in the assay resulted in activation of T cells and APCs by binding the CD3 subunit on T cells to the endogenously expressed CD20 on Raji cells. Ligation of CD3 with anti-CD3 antibodies was shown to lead to activation of T cells. In this bioassay, antibodies blocking PD1 / PD-L1 interaction rescued T cell activity by abolishing inhibitory signaling and subsequently leading to increased AP1-Luc activation.

[0251] In a luciferase-based bioassay, RPMI1640 supplemented with 10% FBS and penicillin / streptomycin / glutamine was used as the assay medium to prepare cell suspensions and antibody dilutions to screen anti-PD1 monoclonal antibodies (mAbs). On the day of screening, the EC of anti-PD1 mAbs in the presence of a fixed concentration of CD3×CD20 bispecific antibody (30 pM) was measured. 50 values, as well as the EC of the bispecific antibody alone 50 The following order of cells and reagents were added to a 96-well white flat-bottom plate: anti-PD1 mAb EC 50 For the determination of the EC, a fixed concentration of CD3xCD20 bispecific antibody (30 pM final) was first prepared and added to the wells of a microtiter plate. Then, 12 serial dilutions of anti-PD1 mAb and controls were added (final concentrations ranging from 1.7 pM to 100 nM; extra wells contained assay medium only). The EC of the bispecific antibody (alone) was 50 For determination, final concentrations of bispecific antibodies ranging from 0.17 pM to 10 nM (extra wells containing assay medium only) were added to wells of a microtiter plate. 6A 10 mL Raji / hPD-L1 cell suspension was prepared and 20 μL was added per well (final cell number / well 5 × 10 4 Leave the plate at room temperature (15-20 min) while incubating 2.5 × 10 6 Prepare a suspension of 100 µg / mL Jurkat / AP1-Luc / hPD1(ecto)-hCD300a (TM-Cyto). Jurkat suspension (final cell number / well 5 × 10 4 20 μL of 100 mM NaCl (cells) were added per well. Plates containing co-cultures were incubated for 5-6 hours at 37° C. / 5% CO2. Samples were tested in duplicate and luciferase activity was detected after addition of ONE-Glo™ (Promega, no. E6051) reagent and relative light units (RLU) were measured in a Victor luminometer.

[0252] The RLU values ​​for each screened antibody were normalized by setting the assay condition containing a fixed (30 pM) concentration of CD3 / CD20 bispecific antibody but no anti-PD-1 antibody as 100%. This condition corresponded to the maximum AP1-Luc response elicited by the bispecific molecule in the presence of PD-1 / PD-L1 inhibitory signals. Upon addition of anti-PD-1 antibody, the inhibitory signal was suppressed, and the increased stimulation was now defined as E max The results are presented as the percentage increase in signal in the presence of the highest antibody dose tested (100 nM). To compare the efficacy of the anti-PD1 antibodies tested, GraphPad Prism was used to determine the antibody concentration at which the normalized RLU value reached 150% activation from a four parameter logistic equation across the 12 response curves. The results are summarized in Table 22 and Table 23, respectively.

[0253] [Table 23]

[0254] [Table 24]

[0255] As shown in Table 22, 25 of the 31 tested anti-PD-1 antibodies of the invention had E values ​​ranging from 239 to 163. max Six of the 31 anti-PD-1 antibodies of the invention did not demonstrate substantial blockade of the PD1 / PD-L1 interaction when tested in this assay.

[0256] As shown in Table 23, two of the four tested anti-PD-1 antibodies of the invention had E of 150 and 343%, respectively. max Two of the four anti-PD-1 antibodies of the invention did not demonstrate substantial blockade of the PD1 / PD-L1 interaction when tested in this assay. EXAMPLES

[0257] In vivo efficacy of anti-PD-1 antibodies To determine the efficacy of a select number of anti-PD-1 antibodies of the invention in a relevant in vivo model, three MC38.ova tumor growth studies involving subcutaneous injection of tumor cells and initiated on different days were performed in mice homozygous for expression of the extracellular domain of human PD-1 in place of the extracellular domain of mouse PD-1 (PD-1 HuMiN mice) in a 75% C57 / Bl6 / 25% 129 strain background.

[0258] For the studies, mice were evenly divided by body weight into five treatment or control groups (5 mice per group) for Study 1, eight treatment or control groups (5 mice per group) for Study 2, and five treatment or control groups (7 mice per group) for Study 3. On day 0, mice were anesthetized by isoflurane inhalation and then injected with 5 × 10 MC38.ova cells in 100 μL of a suspension of DMEM for Study 1. 5 , or for Study 2 and Study 3, 1 x 10 MC38.ova cells in a suspension of 100 µL of DMEM 6were subcutaneously injected into the right flank. For Study 1, treatment groups were intraperitoneally injected with 200 μg of either one of three anti-PD-1 antibodies of the invention, or an isotype control antibody of irrelevant specificity, on days 3, 7, 10, 14, and 17 of the experiment, while one group of mice was left untreated. For Study 2, treatment groups were intraperitoneally injected with either one of three anti-PD-1 antibodies of the invention at 10 mg / kg or 5 mg / kg per dose, one antibody of the invention (H4H7795N2) at 10 mg / kg per dose, or an isotype control antibody of irrelevant specificity at 10 mg / kg, on days 3, 7, 10, 14, and 17 of the experiment. For Study 3, treatment groups were injected intraperitoneally with either one of two anti-PD-1 antibodies of the invention at 5 mg / kg or 2.5 mg / kg per dose, or an isotype control antibody of irrelevant specificity at 5 mg / kg on days 3, 7, 10, 14, and 17 of the experiment. The experimental dosing and treatment protocol for the groups of mice is shown in Table 2. 4.

[0259] [Table 25]

[0260] For the studies, the mean tumor volume, as determined by caliper measurement and percent survival, was recorded for each treatment group on days 14 or 17, and days 23 or 24 of each experiment. In addition, the number of tumor-free mice at the end of the study (day 42 for Study 1, and day 31 for Study 2 and Study 3) was also assessed. Mean tumor volume (mm 3 The results, expressed as mean mean sigma-dopa (±SD), percent survival, and number of tumor-free mice, are shown in Table 23 for study 1, Table 3 for study 2, and Table 4 for study 3.

[0261] [Table 26]

[0262] As shown in Table 25 for Study 1, one antibody of the invention, H4H7798N Treated mice did not develop detectable tumors over the course of the study. Mice treated with H4H9008P showed sustained tumor volume reduction compared to controls on days 17 and 24 of the study, with 3 out of 5 mice or 4 out of 5 mice tumor-free at the end of the experiment, respectively. In contrast, treatment with one of the anti-PD1 antibodies, H4H7795N2, showed no significant efficacy in reducing tumor volume in this study compared to controls. By day 23 of the study, 1 out of 5 mice in the H4H7795N2 group had died, and 2 out of 5 mice in the isotype control-treated group had died. In the untreated group as well as in the isotype control group, some mice showed spontaneous regression of tumors (1 out of 5 mice and 2 out of 5 mice, respectively).

[0263] [Table 27]

[0264] As shown in Table 26 for Study 2, mice treated with 10 mg / kg of one antibody of the invention, H4H7798N, did not develop detectable tumors over the course of the study. Groups of mice treated with either 10 mg / kg H4H9008P, or H4H9048P2 showed a substantial reduction in tumor volume compared to controls on days 17 and 24 of the study. Four out of five mice in each group treated with either 10 mg / kg H4H9008P, or H4H9048P2 were tumor-free at day 31, while only one out of five mice in the isotype control-treated group was tumor-free as a result of spontaneous tumor regression. One antibody tested at 10 mg / kg, H4H7795N2, showed a substantial reduction in tumor volume compared to controls on days 17 and 24 of the study, but this antibody was the least effective anti-PD1 antibody, with only 2 of 5 mice surviving at the end of the experiment.

[0265] A dose-dependent response of tumor suppression was observed in the groups treated with H4H7798N, H4H9008P, and H4H9048P2 at the doses tested (5 mg / kg and 10 mg / kg). The 5 mg / kg H4H7798N or H4H9008P therapy was less effective, with 4 out of 5 tumor-free mice at the end of the experiment on day 21, while 5 out of 5 mice remained tumor-free in both the 10 mg / kg H4H7798N and H4H9008P dose groups.

[0266] Dunnett's test in a two-way ANOVA multiple comparisons revealed that the difference in tumor growth between the group treated with 10 mg / kg isotype control antibody as reference and the group treated with either H4H7798N, H4H9008P, or H4H9048P2 at 10 mg / kg was statistically significant with a p value of less than 0.005. The difference in tumor growth between groups treated with either H4H9008P, or H4H9048P2 was also statistically significant with a p-value of less than 0.05.

[0267] [Table 28]

[0268] As shown in Table 27 for Study 3, 6 of 7 mice treated with 5 mg / kg of one antibody of the invention, H4H7798N, or another antibody of the invention, H4H9008P, were tumor-free at the end of the experiment, while 10 of the mice in the isotype control group were tumor-free. There were no tumors. One tumor-bearing mouse in the IgG4 control group died 17 days after implantation. Only 4 of 7 mice treated with the 2.5 mg / kg dose of H4H9008P remained tumor-free at the end of the experiment. At 21 days, differences in tumor volume between the tested anti-PD-1 antibodies and the isotype control group were determined by one-way ANOVA with Dunnett's multiple comparison post-test and were statistically significant at p < 0.01. All four anti-PD-1 antibodies were equally more effective at the 5 mg / kg dose than at the 2.5 mg / kg dose. EXAMPLES

[0269] Antitumor effect of a combination of anti-PD-1 antibody and VEGF antagonist in a mouse early stage tumor model An early treatment tumor model was developed to test the efficacy of anti-PD-1 antibody and VEGF antagonist combination. In this model, the combination therapy was administered immediately after tumor implantation. Experiments were also performed with anti-PD-L1 antibody alone and in combination with VEGF antagonist. The anti-PD-1 antibody used in this experiment was the anti-mouse PD-1 clone "RPMI-14" (Bio X Cell, West Lebanon, NH) with rat IgG2b. The VEGF antagonist used in this experiment was aflibercept (VEGF receptor-based chimeric molecule also known as "VEGF-trap", or "VEGFR1R2-FcΔC1(a)", a full description of which is provided elsewhere herein). The anti-PD-L1 antibody used in this experiment was the V-type of antibody "YW243.55S70" from US20100203056A1 (Genentech, Inc.) with mouse IgG2a. H / V L It is an anti-PD-L1 monoclonal antibody with the same sequence, and was cross-reactive with mouse PD-L1.

[0270] For this experimental model, 1.0 × 10 Colon-26 tumor cells were 6were implanted subcutaneously into BALB / c mice on day 0. Beginning on day 3, prior to the establishment of measurable tumors, mice were treated with one of the single or combination therapies, or a control combination, as described in Table 28.

[0271] [Table 29]

[0272] The various regimens were administered at five different time points over a two week period (ie, injections on days 3, 6, 10, 13, and 19).

[0273] Animals in each treatment group were evaluated in terms of tumor incidence, tumor volume, median survival time, and number of tumor-free animals at 50 days. The extent of tumor growth is shown in Figure 2 (Tumor growth curves). The results are summarized in Figure 2 (line), and Figure 3 (tumor volume at 28 days). The results are also summarized in Table 29.

[0274] [Table 30]

[0275] Compared to treatment regimens with either therapeutic agent alone, tumor growth was substantially reduced in animals treated with the combination of VEGF Trap + anti-PD-1 antibody (see Figures 2 and 3). Furthermore, survival was substantially increased in the VEGF Trap + anti-PD-1 antibody group, with 70% of animals surviving at least 50 days after tumor implantation. In contrast, survival to 50 days was only 40% and 30% for the anti-PD-1 and VEGF Trap monotherapy groups, respectively (see Figure 3 and Table 29). EXAMPLES

[0276] A clinical trial study of repeated dosing with anti-PD-1 antibodies as monotherapy and in combination with other anticancer therapies in patients with advanced malignancies. This is a dose escalation study of anti-PD-1 antibodies alone or in combination with radiation therapy, cyclophosphamide, or both in patients with advanced malignancies. The exemplary anti-PD-1 antibody ("mAb") used in this example contained a HCVR of SEQ ID NO:162, and a LCVR of SEQ ID NO:170.

[0277] Research purpose The primary objective of the study is to characterize the safety, tolerability, and DLTs of the mAb administered IV as monotherapy or in combination with targeted radiation (with the aim of having this serve as an immune stimulant rather than a primary tumor-removal therapy), low-dose cyclophosphamide (a therapy shown to inhibit regulatory T cell responses), or both in patients with advanced malignancies.

[0278] The secondary objectives of the study are: (1) to determine the recommended phase 2 dose (RP2D) of the mAb as monotherapy and in combination with other anticancer therapies (targeted radiation, low-dose cyclophosphamide, or both); (2) to describe the preliminary antitumor activity of the mAb alone and with each combination partner; (3) to characterize the PK of the mAb as monotherapy and in combination with other anticancer therapies (targeted radiation, low-dose cyclophosphamide, or both); and (4) to evaluate the immunogenicity of the mAb.

[0279] research design Safety will be evaluated in separate standard 3+3 dose escalation cohorts (monotherapy, combination with radiation therapy, combination with cyclophosphamide, and combination with radiation therapy and cyclophosphamide). Selection is based on the investigator's assessment of the best choice of therapy for the individual patient in consultation with the sponsor. To be enrolled in the radiation therapy cohort, patients must have lesions that can be safely irradiated and for which radiation at the anticipated limited symptomatic dose is deemed medically appropriate, and at least one other lesion suitable for response evaluation. Patients will be allowed to enroll only if there is space in the cohort for the chosen treatment.

[0280] Patients undergo a screening method to determine eligibility within 28 days prior to their first dose of mAb. After enrollment of patients into the mAb monotherapy cohort, enrollment into subsequent cohorts is determined by the occurrence of DLTs in the previous cohort (i.e., no DLTs in the 3 patient cohort or less than 1 DLT in the 6 patient expansion cohort) and patient availability. Planned monotherapy dosing levels are 1, 3, or 10 mg / kg administered IV every 14 days (2 weeks).

[0281] Upon completion of one or both of the 1 mg / kg or 3 mg / kg mAb monotherapy cohort DLT observation periods with no DLTs in the cohorts of 3 patients or with less than one DLT in the expansion cohorts of 6 patients, patients may be enrolled in cohorts combining cyclophosphamide or radiation therapy with the mAb at that monotherapy dose level. Upon completion of the DLT observation periods for both the cohort for that mAb dose level + cyclophosphamide and the cohort for that mAb dose level + the same radiation therapy treatment regimen with no DLTs in the cohorts of 3 patients or with less than one DLT in the expansion cohorts of 6 patients, patients may be enrolled in the combination mAb + cyclophosphamide / radiotherapy cohorts.

[0282] Once the 3 mg / kg mAb monotherapy cohort DLT observation period has been completed with no DLTs in the cohort of 3 patients or less than 1 DLT in the expansion cohort of 6 patients, the 10 mg / kg mAb monotherapy cohort may be enrolled.

[0283] The 3 mg / kg and 10 mg / kg mAb monotherapy cohorts will be enrolled only after the required number of patients in the previous monotherapy dose cohorts (i.e., 1 mg / kg and 3 mg / kg, respectively) have passed the 28-day DLT observation period without showing the maximum tolerated dose (MTD) for that dose level. The 1 mg / kg mAb combination treatment cohort will be enrolled only after the completion of the DLT observation period for the 1 mg / kg monotherapy cohort. The combination cohorts receiving 3 mg / kg mAb will be enrolled only if the required number of patients in each 1 mg / kg mAb combination cohort have passed the DLT observation period without showing the MTD. The three combination cohorts combining the mAb with cyclophosphamide and radiation treatment regimens will be enrolled only if the required number of patients in both of the corresponding two combination cohorts of that dosage level have passed the DLT observation period without showing the MTD.

[0284] Table 30 summarizes the dose escalation cohorts into which patients will be enrolled.

[0285] [Table 31]

[0286] DLTs are defined as being either: non-hematological toxicity (e.g., uveitis, or any other irAE), or hematological toxicity (e.g., neutropenia, thrombocytopenia, febrile neutropenia).

[0287] The maximum tolerated dose (MTD) is defined as the highest dose at which less than one-third of the expanded cohort of six patients experience DLT during the first cycle of treatment. Therefore, the MTD is defined as the dose level just below the level at which dosing is stopped due to the occurrence of two or more DLTs in the expanded cohort of six patients. If dose escalation is not stopped due to the occurrence of DLTs, the MTD is considered not to have been determined. In this study, the MTD may not be defined for either the monotherapy group or the individual combination therapy groups. In addition, the mAb MTD may differ between monotherapy and each combination treatment plan.

[0288] Research period Patients will receive treatment for up to 48 weeks, followed by a 24-week follow-up period. Patients will receive treatment until the 48-week treatment period is completed or until disease progression, unacceptable toxicity, withdrawal of consent, or another study withdrawal criteria meeting. After a minimum of 24 weeks of treatment, patients with a confirmed complete response (CR) may choose to discontinue treatment and continue with all relevant study evaluations (e.g., efficacy evaluations). After a minimum of 24 weeks of treatment, patients with a tumor burden assessment of stable disease (SD) or partial response (PR) that is unchanged for three sequential tumor assessments may also choose to discontinue treatment and continue with all relevant study evaluations (e.g., efficacy evaluations).

[0289] Study population The target population for this study includes patients with advanced malignancies who are not candidates for standard therapy, who are unwilling to undergo standard therapy, or who do not expect available therapies to provide clinical benefit; as well as patients with malignancies that are incurable and who have not responded to standard therapy or have shown tumor progression despite standard therapy.

[0290] Inclusion Criteria: Patients must meet the following criteria to qualify for inclusion in this study: (1) have demonstrated solid tumor progression without alternative, available standard-of-care treatment options; (2) have at least one lesion for response evaluation. Patients assigned to radiation therapy must have at least one additional lesion for which radiation, at a presumed limited symptomatic dose, is deemed medically appropriate, and can be safely irradiated without irradiation of the index lesion. (3) Eastern Cooperative Oncology Group (ECOG) performance status 1 or less; (4) Age >18 years; (5) Liver function: a. Total bilirubin 1.5 × upper limit of normal (ULN; if liver metastases 3 × ULN or less), b. Transaminases 3 × ULN or less (or if liver metastases 5.0 × ULN or less), c. Alkaline phosphatase (ALP) 2.5 × ULN or less (or if liver metastases 5.0 × ULN); (6) Renal function: Serum creatinine 1.5 × ULN or less; (7) Neutrophil count (ANC) 1.5 × 10 9 / L or more, c. Platelet count 75×10 9 / L or greater; (8) ability to provide signed informed consent; and (9) ability and willingness to comply with visit schedules, treatment plans, laboratory tests, and other study-related procedures.

[0291] Exclusion Criteria: Patients who meet any of the following criteria will be excluded from the study: (1) significant autoimmune disease progression or recent (within 5 years) evidence of significant autoimmune disease requiring treatment with systemic immunosuppressive treatment indicating risk of irAEs; (2) prior treatment with an agent that blocks the PD-1 / PD-L1 pathway; (3) prior treatment with another immunomodulatory agent for less than 4 weeks or 4 half-lives (whichever is longer) prior to the first dose of mAb; (4) examples of immunomodulatory agents include inhibitors of CTLA-4, 4-1BB (CD137), OX-40, therapeutic vaccines, or cytokine treatment; (5) untreated brain metastases that are considered active. Patients with previously treated brain metastases may be enrolled, provided that they are stable (i.e., no evidence of progression by imaging for at least 4 weeks prior to the first dose of study treatment and any neurological symptoms have returned to baseline) and there is no evidence of new or enlarging brain metastases; (6) immunosuppressant corticosteroid medication (>10 mg daily prednisone, or equivalent) within 4 weeks prior to the first dose of mAb; (7) deep vein thrombosis, pulmonary embolism (including asymptomatic pulmonary embolism identified on imaging), or other thromboembolic event within 6 months prior to the first dose of mAb; (8) active infection requiring therapy, including known infection with human immunodeficiency virus or active infection with hepatitis B or C virus; (9) history of interstitial pneumonia within the last 5 years; (10) any of the following within 30 days prior to the first dose of mAb: (11) history of documented allergic or acute hypersensitivity reactions due to antibody therapy in general or treatment with agents specifically used in the study; (12) known allergy to doxycycline or tetracycline (caution due to the presence of trace components in the mAb); (13) breast-feeding; (14) positive serum pregnancy test; (15) history within the last 5 years of invasive malignancies other than those treated in this study, except for limited / excised basal or squamous cell carcinoma of the skin or cervical intraepithelial neoplasia or other localized tumors that were deemed cured by local treatment; (16) acute or chronic psychiatric problems that, as assessed by the investigator, would disqualify the patient from participation; and (17) ongoing sexual activity in men or women of childbearing potential who are not willing to practice reasonable contraception during the study.

[0292] Study Procedures The mAb is supplied as a liquid in a sterile, single-use vial. Each vial contains sufficient volume to withdraw 10 mL of mAb at a concentration of 25 mg / mL. Dose adjustment instructions are provided in the Study Reference Manual. The mAb is administered in an outpatient setting as a 30-minute IV infusion. Each patient's dose depends on their individual body weight. The dose of mAb must be adjusted each cycle for changes in body weight of 10% or more. The mAb is administered alone and in combination with radiation and or cyclophosphamide.

[0293] Monotherapy The mAb will be administered in an outpatient setting by IV infusion over 30 minutes every 14 days for 48 weeks (i.e., days 1, 15±3, 29±3, and 43±3 of a 56-day cycle). The planned monotherapy treatment regimens to be assigned are: (i) 1 mg / kg IV infusion over 30 minutes every 14 days for 48 weeks; (ii) 3 mg / kg infusion over 30 minutes every 14 days for 48 weeks; (iii) a 10 mg / kg infusion over 30 minutes every 14 days for 48 weeks; and (iv) a 0.3 mg / kg infusion over 30 minutes every 14 days for 48 weeks (if the MTD is determined to be less than 1 mg / kg).

[0294] Combination therapy Concomitant radiation therapy and cyclophosphamide will be provided throughout the regimen and their use, dose, dose modification, reduction, or delay, and any potential AEs resulting in their use will be tracked along with those of the mAb.

[0295] Concurrent administration of mAb and radiation: mAb will be administered by IV infusion over 30 minutes every 14 days for 48 weeks in combination with radiation treatment for 8-12 days. The planned combined mAb and radiation therapy treatment plan is: 1 mg / kg mAb infusion over 30 minutes every 14 days for 48 weeks, plus 30 Gy of radiation therapy (6 Gy x 5 doses / week; preferably daily for 1 week after the first dose of mAb) 1 mg / kg mAb infusion over 30 minutes every 14 days for 48 weeks, plus 27 Gy of radiation therapy (9 Gy x 3 doses / week; given for 1 week after the first dose of mAb, preferably not on consecutive days) 3 mg / kg mAb infusion over 30 minutes every 14 days for 48 weeks, plus 30 Gy of radiation therapy (6 Gy x 5 doses / week; preferably daily for 1 week after the first dose of mAb) 3 mg / kg mAb infusion over 30 minutes every 14 days for 48 weeks, plus 27 Gy of radiation therapy (9 Gy x 3 / week; given 1 week after the first dose of mAb, preferably not on consecutive days).

[0296] Patients will receive either 30 Gy given as 6 Gy in five doses administered daily starting one week after the first dose of mAb, or 27 Gy given as 9 Gy in three doses administered every other day starting one week after the first dose of mAb. Lesions selected for radiation should be those that can safely receive focal radiation while tolerating the index lesion, and those that are deemed medically appropriate with symptomatic medication taken into account to limit radiation. Target doses for patients should be based on cohort assignment and follow normal tissue requirements according to standard radiation oncology practice. Treatment with protocol-specified dosing regimens will be permitted only if normal tissue criteria are met. If normal tissue criteria are not met for any of the protocol-specified radiotherapy regimens, the patient will not be eligible for enrollment in the concomitant radiation treatment cohort in this study.

[0297] Coadministration of mAb and cyclophosphamide: 200 mg / m every 14 days for four doses. 2 The mAb will be administered by IV infusion over 30 minutes every 14 days (2 weeks) for 48 weeks in combination with cyclophosphamide (days -1, 14, 28, and 42 of the first 56-day cycle) with each of the four cyclophosphamide doses being administered 1 day prior to each of the first four mAb doses.

[0298] Cyclophosphamide has been used successfully in combination with other drugs, but the rate of metabolism and leukopenic activity of cyclophosphamide are reportedly increased by chronic administration of large doses of phenobarbital. Cyclophosphamide treatment causes a marked and sustained inhibition of cholinesterase activity, thus enhancing the effect of succinylcholine chloride. The combined mAb and cyclophosphamide treatment schedule planned to be administered is as follows: 200 mg / m every 14 days (days -1, 14, 28, and 42 of the first 56-day cycle) for a total of 4 doses 2 of cyclophosphamide, plus 3mg / kg mAb infusion over 30 minutes every 14 days for 48 weeks (but below the MTD) (monotherapy dose of 3 mg / kg; if 3 mg / kg is higher than the MTD, the dose is 1 mg / kg).

[0299] Concomitant administration of mAb, radiation, and cyclophosphamide: The planned combination mAb, radiation, and cyclophosphamide treatment regimens include: 200 mg / m every 14 days (days -1, 14, 28, and 42 of the first 56-day cycle) for a total of 4 doses 2 of cyclophosphamide, plus 27 Gy of radiation therapy (9 Gy x 3 fractions / week; for a period of 1 week after the first dose of mAb, preferably not on consecutive days); or 30 Gy of radiation therapy (6 Gy x 5 fractions / week; preferably daily for one week after the first dose of mAb), plus 3mg / kg mAb infusion over 30 minutes every 14 days for 48 weeks (but with a monotherapy dose of 3mg / kg below the MTD; if 3mg / kg is higher than the MTD, the dose is 1mg / kg).

[0300] Study variables Primary Variables: Primary safety variables include the incidence of DLTs, incidence and severity of treatment-emergent adverse events (TEAEs), and abnormal laboratory findings through 48 weeks of treatment.

[0301] Secondary Variables: Key secondary variables include: Serum concentration and pharmacokinetics (PK) of mAbs Antitumor activity assessed using appropriate criteria for the following endpoints: Response Evaluation Criteria in Solid Tumors (RECIST) measured by computed tomography (CT) or magnetic resonance imaging (MRI) For specific tumors where RECIST measurements are not standard, other evaluation criteria should be used. - Immune-related response criteria (irRC) applied to RECIST measurements. In all cases, the irRC is a management tool to determine progressive disease (PD), SD, CR, or PR. For informational purposes, standard RECIST data will also be collected. ·Anti-mAb antibody.

[0302] Research method The following methods will be performed in screening for the purposes of determining study eligibility or characterizing the baseline population: (i) serum β-HCG (results must be within 72 hours prior to first dose); (ii) collection of accomplished tumor material: after the patient has given informed consent, the patient will be asked to provide any available previously collected tumor sample; (iii) brain MRI: a brain MRI will be required at screening if not done in the previous 60 days; and (iv) chest x-ray: a chest x-ray will be required at screening if not done in the previous 60 days.

[0303] Efficacy Methods: CT or MRI for tumor assessment will be performed at the screening visit (within 28 days prior to infusion) and at 56±3 days during all cycles (approximately every 8 weeks), and if disease progression is suspected. In addition, for patients who do not progress on study, tumor assessment will be performed at follow-up visits 3, 5, and 7. If CT scan or MRI is chosen to be used, it will be subsequently evaluated using the same modality.

[0304] Tumor response assessment will be performed according to immune-related response criteria (irRC; Nishino 2013). Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (Eisenha Assessment by IRRC (Uer 2009) will also be performed as a secondary exploration; however, the primary determination of disease progression for individual patients will be made according to irRC. Measurable lesions selected as target lesions for RECIST assessment will also be included as index lesions for irRC evaluation.

[0305] Safety Methods: Vital signs will also be collected, including temperature, resting blood pressure, pulse, and respirations. Vital signs should also be measured prior to clinical laboratory assessments, PK, or exploratory sample collections, if scheduled at visits concurrent with other methods. During Cycle 1, vital signs will be recorded on treatment days prior to treatment, at the end of infusion, every 30 minutes for the first 4 hours post-infusion, and at 6 and 8 hours after study drug administration. In subsequent cycles, vital signs will be assessed and recorded on treatment days prior to infusion, every 30 minutes, for the first 2 hours, then hourly until 4 hours after study drug administration.

[0306] At the visit, a thorough complete or limited physical examination will be performed. A complete physical examination will include examination of the skin, head, eyes, nose, throat, neck, joints, lungs, heart, pulse, abdomen (including liver and spleen), lymph nodes, and extremities, as well as a brief neurological examination. A limited physical examination will include the lungs, heart, abdomen, and skin.

[0307] A standard 12-lead ECG will be performed. Any ECG finding that is deemed a clinically significant change (worsening) by the investigator compared to baseline values ​​will be considered an AE and will be recorded and monitored.

[0308] Immunosafety assays consist of rheumatoid factor (RF), thyroid stimulating hormone (TSH), C-reactive protein (CRP), and antinuclear antibody (ANA) titers and patterns. If a 4-fold or greater increase from baseline in RF or ANA, or abnormal levels of TSH or CRP is observed over the course of the study, the following tests will also be performed: anti-DNA antibodies, anti-Sjogren's syndrome A antigen (SSA) antibodies (Ro), anti-Sjogren's syndrome B antigen (SSB) antibodies (La), anti-thyroglobulin antibodies, anti-LKM antibodies, antiphospholipid antibodies, anti-islet cell antibodies, antineutrophil cytoplasmic antibodies, C3, C4, CH50. Activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be analyzed by the site local laboratory.

[0309] safety An adverse event (AE) is any untoward medical occurrence in a patient administered a study drug that may or may not be causally related to the study drug. Therefore, an AE is any untoward and unintended sign (including abnormal laboratory findings), symptom, or disease that is temporally related to the use of the study drug, whether or not it is considered related to the study drug. AE also includes any worsening of a pre-existing condition (i.e., any clinically significant change in frequency and / or intensity) that is temporally related to the use of the study drug. Progression of the underlying malignancy is not considered an AE if it clearly matches the typical progression pattern of the underlying cancer (including time course, organs affected, etc.). Clinical symptoms of progression are reported as AEs if they are not determined to be exclusively attributable to progression of the underlying malignancy or do not fit the expected progression pattern for the disease under study.

[0310] A serious adverse event (SAE) is any untoward medical occurrence at any dose that results in death, is life-threatening, requires hospitalization of a patient or an extension of an existing hospital stay, results in persistent or significant incapacity / incapacity (substantial disruption of one's ability to perform normal life functions), or is a congenital abnormality / birth defect.

[0311] Patient information for all AEs and SAEs will be recorded.

[0312] Statistical Planning The study dose escalation is based on a traditional 3+3 design with 3 to 6 patients assigned per dose level. The exact number of patients enrolled in the study will depend on the number of protocol-defined DLTs observed, the need to expand the currently defined dose level, or initiate open additional cohorts at lower dose levels. After the required initial enrollment for the next cohort in the dose escalation, enrollment in each of the previous cohorts below the MTD for that treatment will be expanded to a total of 6 patients (unless already expanded during escalation).

[0313] Data will be summarized using descriptive statistics only. Data will generally be summarized by dose level and composition. Safety summaries and analyses will be performed in the Safety Analysis Set (SAF). The primary analysis of safety will be based on treatment-emergent AEs (TEAEs).

[0314] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that competes with a reference antibody or antigen-binding fragment thereof for binding to human programmed cell death 1 (PD-1) protein, the antibody or antigen-binding fragment thereof comprising: a complementarity determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1; and a CDR of a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1: (a) IC of less than 3 nM as measured in a competitive sandwich ELISA assay at 25°C ５０ the property of blocking human PD-1 protein binding to PD-L1; (b) a binding-dissociation equilibrium constant (K) of less than about 50 nM as measured in a surface plasmon resonance assay at 37° C. Ｄ ) to bind monomeric human PD-1; (c) a K of less than about 12 nM in a surface plasmon resonance assay at 25° C. Ｄ the property of binding to monomeric human PD-1 at (d) a K of less than about 8.5 nM in a surface plasmon resonance assay at 25° C. Ｄ Binding to monomeric cynomolgus PD-1 at (e) a dissociation half-life (t) of greater than about 6.3 minutes as measured in a surface plasmon resonance assay at 25° C. １／２ ) to monomeric human PD-1; and (f) a dissociation half-life (t) of greater than about 0.9 minutes as measured in a surface plasmon resonance assay at 37° C. １／２ ) and its ability to bind to monomeric human PD-1 The isolated antibody or antigen-binding fragment thereof,

2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the reference antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair set forth in Table 1.

3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the reference antibody comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 130 / 138, 162 / 170, 234 / 202, and 314 / 186.

4. An isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human PD-1, the isolated human monoclonal antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained in any one of the HCVR sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in any one of the LCVR sequences listed in Table 1.

5. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, comprising an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1.

6. 6. The isolated antibody or antigen-binding fragment thereof of claim 4 or 5, comprising an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

7. The isolated antibody or antigen-binding fragment thereof of any one of claims 4 to 6, comprising: (a) an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1; and (b) an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

8. (a) SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 14 8, 164, 180, 196, 212, 220, 228, 236, 244, 252, 260, 268, 276, 284, 292, 300, 308, and 316; (b) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 222, 230, 238, 246, 254, 262, 270, 278, 286, 294, 302, 310, and 318; (c) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 312, and 320; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, and 204; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, and 206; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, and 208. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 7, comprising:

9. 1. An isolated antibody or antigen-binding fragment thereof that blocks PD-1 binding to PD-L1, comprising: a CDR of HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 34, 50, 66, 82, 114, 130, 162, 178, 194, 210, 218, 226, 234, 242, 258, 266, 274, 282, 290, 298, 306, and 314; and a CDR of LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 42, 58, 74, 90, 122, 138, 170, 186, and 202.

10. 10. The isolated antibody or antigen-binding fragment thereof of claim 9, comprising a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 130 / 138, 162 / 170, 234 / 202, and 314 / 186.

11. An isolated antibody or antigen-binding fragment thereof that binds to human PD-1, wherein the antibody or antigen-binding fragment thereof enhances PD-1 binding to PD-L1 as measured by a competitive sandwich ELISA assay at 25°C.

12. The isolated antibody or antigen-binding fragment thereof of claim 11, wherein the antibody comprises a CDR of HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 98, and 250; and a CDR of LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 106, and 202.

13. 13. The isolated antibody or antigen-binding fragment thereof of claim 12, wherein the antibody comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 98 / 106, and 250 / 202.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody is a multispecific antigen-binding molecule.

15. 15. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to PD-1 according to any one of claims 1 to 14, and a pharma- ceutically acceptable carrier or diluent.

16. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of the antibody of any one of claims 1 to 13.

17. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of the antibody of any one of claims 1 to 13.

18. A vector comprising the polynucleotide sequence of claim 16 or 17.

19. A cell expressing the vector of claim 18.

20. A multispecific antigen-binding molecule, or antigen-binding fragment thereof, comprising a first antigen-binding specificity that specifically binds to PD-1, and a second antigen-binding specificity that comprises the extracellular domain of PD-L1 or PD-L2, or a fragment thereof.

21. A multispecific antigen-binding molecule, or antigen-binding fragment thereof, comprising a first antigen-binding specificity that specifically binds to PD-1 and a second antigen-binding specificity that specifically binds to a T cell co-inhibitory molecule.

22. The multispecific antigen-binding molecule or fragment thereof of claim 20 or 21, wherein the first antigen-binding specificity comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained in any one of the HCVR sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in any one of the LCVR sequences listed in Table 1.

23. The multispecific antigen-binding molecule or fragment thereof according to any one of claims 20 to 22, wherein the first antigen-binding specificity comprises an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1; and an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

24. 24. The multispecific antigen-binding molecule or fragment thereof of claim 23, wherein the first antigen-binding specificity comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 130 / 138, 162 / 170, 234 / 202, and 314 / 186.

25. The multispecific antigen-binding molecule or fragment thereof of claim 21, wherein the second antigen-binding specificity specifically binds to a T cell co-inhibitory molecule selected from the group consisting of LAG3, TIM3, B7-1, CTLA-4, BTLA, CD28, 2B4, LY108, TIGIT, ICOS, and CD160.

26. The multispecific antigen-binding molecule or fragment thereof according to any one of claims 20 to 25, for use in the treatment of a cancer selected from the group consisting of renal cell carcinoma, colorectal cancer, ovarian cancer, prostate cancer, breast cancer, colon cancer, non-small cell lung cancer, and melanoma.

27. by a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV). A multispecific antigen-binding molecule or fragment thereof according to any one of claims 20 to 25 for use in the treatment of a chronic viral infection caused by a virulent agent.

28. A multispecific antigen-binding molecule, or fragment thereof, comprising a first antigen-binding specificity that specifically binds to PD-1, and a second antigen-binding specificity that specifically binds to an antigen selected from the group consisting of an autoimmune tissue-specific antigen, a T cell receptor, and an Fc receptor.

29. The multispecific antigen-binding molecule or fragment thereof of claim 28, wherein the first antigen-binding specificity comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained in any one of the HCVR sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in any one of the LCVR sequences listed in Table 1.

30. The multispecific antigen-binding molecule or fragment thereof of claim 28 or 29, wherein the first antigen-binding specificity comprises an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1; and an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

31. 31. The multispecific antigen-binding molecule or fragment thereof of claim 30, wherein the first antigen-binding specificity comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 130 / 138, 162 / 170, 234 / 202, and 314 / 186.

32. 29. The multispecific antigen-binding molecule or fragment thereof of claim 28, wherein the first antigen-binding specificity comprises the extracellular domain of PD-L1 and / or PD-L2, or a fragment thereof.

33. The multispecific antigen-binding molecule or fragment thereof according to any one of claims 28 to 32, wherein the second antigen-binding specificity specifically binds to an autoimmune tissue-specific antigen.

34. 34. The multispecific antigen-binding molecule or fragment thereof of claim 33, wherein the autoimmune tissue specific antigen is associated with alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes, eosinophilic fasciitis, eosinophilic gastroenteritis, Goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, or Wegener's granulomatosis.

35. The multispecific antigen-binding molecule or fragment thereof according to any one of claims 28 to 32, wherein the second antigen-binding specificity specifically binds to one of a T cell receptor, an Fcα receptor, an Fcγ receptor, or CD19.

36. 36. The multispecific antigen-binding molecule or fragment thereof according to any one of claims 28 to 35, for use in the treatment of an autoimmune disease or disorder selected from the group consisting of alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes mellitus, eosinophilic fasciitis, eosinophilic gastroenteritis, Goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

37. 26. A method of enhancing an immune response in a subject, comprising administering a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 13; or a multispecific antigen-binding molecule or fragment thereof according to any one of claims 20 to 25.

38. 26. A method of inhibiting T regulatory (Treg) cells in a subject, comprising administering a pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof according to any one of claims 1 to 13; or a multispecific antigen-binding molecule or fragment thereof according to any one of claims 20 to 25.

39. A method for enhancing T cell activation in a subject, the method comprising administering a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13; or a multispecific antigen-binding molecule or fragment thereof according to any one of claims 20 to 25.

40. 40. The method of any one of claims 37 to 39, wherein the subject has a disease or disorder selected from the group consisting of brain cancer, renal cell carcinoma, ovarian cancer, prostate cancer, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, and melanoma.

41. The method of any one of claims 37 to 39, wherein the subject has a chronic viral infection caused by a virus selected from the group consisting of HIV, HCV, HBV, HPV, LCMV, and SIV.

42. A method for inhibiting the growth of a tumor or tumor cells, comprising contacting the tumor or tumor cells with a therapeutically effective amount of an antibody according to any one of claims 1 to 13 or 20 to 25.

43. 36. A method for inhibiting T cell activation in a subject, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13; or a multispecific antigen-binding molecule or fragment thereof according to any one of claims 28 to 35.

44. 44. The method of claim 43, wherein the subject has an autoimmune disease or disorder selected from the group consisting of alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes, eosinophilic fasciitis, eosinophilic gastroenteritis, Goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

45. The method of any one of claims 37 to 44, wherein the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, is administered to the subject in combination with a second therapeutic agent.

46. The second therapeutic agent consists of NSAIDs, corticosteroids, antibodies against different T cell co-inhibitory molecules, antibodies against tumor specific antigens, antibodies against autoimmune tissue antigens, antibodies against virally infected cell antigens, antibodies against PD-L1, nutritional supplements such as antioxidants, VEGF antagonists, chemotherapeutic agents, cytotoxic agents, antiviral agents, radiation, and any other therapy useful in ameliorating at least one symptom associated with the disease or disorder.

46. ​​The method of claim 45, selected from the group:

47. The method of any one of claims 37 to 46, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

48. 48. The method of any one of claims 37 to 47, wherein the antibody or antigen-binding fragment is administered at a dose of about 0.1 mg / kg to about 60 mg / kg of body weight of the subject.

49. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, which competes with a reference antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain for binding to human PD-1, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 330, 332, 334, and 336.

50. 2. The isolated antibody or antigen-binding fragment of claim 1, which competes with a reference antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain for binding to human PD-1, wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 331, 333, 335, and 337.

51. 2. The isolated antibody or antigen-binding fragment of claim 1, which competes for binding to human PD-1 with a reference antibody or antigen-binding fragment thereof comprising a heavy chain / light chain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 330 / 331, 332 / 333, 334 / 335, and 336 / 337.

52. 52. A pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof that binds to PD-1 according to any one of claims 49 to 51, and a pharma- ceutically acceptable carrier or diluent.

53. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the heavy chain of the antibody according to any one of claims 49 to 51.

54. 52. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the light chain of the antibody according to any one of claims 49 to 51.

55. 55. A vector comprising the polynucleotide sequence of claim 53 or 54.

56. A cell expressing the vector of claim 55.

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