Engineered PD-1 antibodies and uses thereof

Abstract

To provide engineered PD-1 antibodies and uses thereof.SOLUTION: In some aspects, provided herein are antibodies that bind to PD-1. Antibodies provided herein, in some cases, agonize PD-1 signaling. Antibodies provided herein, in some cases, have modified Fc region. In other aspects, provided herein are compositions, methods of use, methods of making, and kits relating to antibodies that bind to PD-1. In some aspects, provided herein are methods of suppressing immune cells expressing programmed cell death 1 (PD-1), the methods comprising contacting the immune cells with an antibody that specifically binds to PD-1 and enhances an interaction between PD-1 and PD-L1 on a surface of the immune cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 281,404, filed November 19, 2021, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on November 7, 2022, is named 56270_718601_SL.xml and is 19,479 bytes in size. Summary of the Invention

[0003] Disclosed herein in some aspects are methods of inhibiting immune cells that express programmed cell death 1 (PD-1), comprising contacting the immune cells with an antibody that specifically binds to PD-1 and agonizes PD-1 signaling in the immune cells, wherein the antibody comprises an Fc region that comprises an amino acid substitution, wherein the amino acid substitution results in reduced antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells in a subject compared to the parent molecule lacking the amino acid substitution, and wherein the antibody has the same or greater agonistic effect on PD-1 signaling in the immune cells compared to the parent molecule.

[0004]

[0003] In some aspects, disclosed herein are methods of inhibiting immune cells that express programmed cell death 1 (PD-1), comprising contacting the immune cells with an antibody that specifically binds to PD-1 and enhances the interaction between PD-1 and PD-L1 on the surface of the immune cells. In some cases, the antibody comprises an Fc region, and the Fc region comprises an amino acid substitution. In some cases, the amino acid substitution results in reduced antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells in a subject compared to the parent molecule lacking the amino acid substitution, and the antibody has the same or greater agonistic effect on PD-1 signaling in immune cells compared to the parent molecule.

[0005] In some embodiments of the method, ADCC against PD-1-expressing regulatory T cells is reduced as determined by the natural killer cell activation assay described in Example 7.

[0006] In some embodiments of the method, the antibody does not result in significant ADCC against PD-1-expressing regulatory T cells as determined by the natural killer cell activation assay described in Example 7.

[0007] In some embodiments of the method, the antibody does not activate natural killer (NK) cells.

[0008] In some embodiments of the method, the antibody comprises a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region, and in some embodiments of the method, the heavy chain variable region comprises a complementarity determining region (CDR) comprising a sequence set forth in one or more of SEQ ID NOs: 1-3 with 0-3 amino acid modifications.

[0009] In some embodiments of the method, the Fc region is derived from IgG1 and comprises an aspartic acid (D) at position 238, numbered according to the EU index.

[0010]

[0010] In some aspects, disclosed herein are methods of inhibiting immune cells that express programmed cell death 1 (PD-1), comprising contacting the immune cells with an antibody comprising a heavy chain, a light chain, and an Fc region, wherein (i) the heavy chain comprises a heavy chain variable region comprising a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 1-3, with 0-3 amino acid modifications; (ii) the light chain comprises a light chain variable region comprising a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4-6, with 0-3 amino acid modifications; and (iii) the Fc region is derived from IgG1 and comprises an aspartic acid (D) at position 238, numbered according to the EU index.

[0011] In some embodiments of the method, the light chain variable region comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4-6, with 0-3 amino acid modifications.

[0012] In some embodiments of the method, the heavy chain variable region comprises heavy chain complementarity determining region 1 (CDRH1), CDRH2, and CDRH3, wherein CDRH1, CDRH2, and CDRH3 each comprise the sequence set forth in SEQ ID NOs: 1-3 with 0-3 amino acid modifications.

[0013] In some embodiments of the method, the light chain variable region comprises light chain complementarity determining region 1 (CDRL1), CDRL2, and CDRL3, wherein CDRL1, CDRL2, and CDRL3 each comprise the sequence set forth in SEQ ID NOs: 4-6 with 0-3 amino acid modifications.

[0014] In some embodiments of the method, the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3, wherein CDRH1, CDRH2, and CDRH3 comprise the sequences set forth in SEQ ID NOs: 1-3, respectively.

[0015] In some embodiments of the method, the light chain variable region comprises CDRL1, CDRL2, and CDRL3, wherein CDRL1, CDRL2, and CDRL3 comprise the sequences set forth in SEQ ID NOs: 4-6, respectively.

[0016] In some embodiments of the method, the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in any one of SEQ ID NOs: 7-11.

[0017] In some embodiments of the method, the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in any one of SEQ ID NOs: 12-16.

[0018] In some embodiments of the method, the heavy chain comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in SEQ ID NO:18.

[0019] In some embodiments of the method, the light chain comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in SEQ ID NO:19.

[0020] In some embodiments of the method, the Fc region is derived from human IgG1. In some embodiments of the method, the Fc region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in SEQ ID NO: 17.

[0021] In some embodiments of the method, the heavy chain variable region and the light chain variable region form a structure selected from the group consisting of an scFv, a sc(Fv)2, a dsFv, a Fab, a Fab', a (Fab')2, and a diabody.

[0022] In some embodiments of the method, the heavy chain variable region and the light chain variable region form a single chain variable fragment (ScFv) operably linked to an Fc region.

[0023] In some embodiments of the method, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, and a multispecific antibody. In some embodiments of the method, the antibody is monoclonal.

[0024] In some embodiments of the method, the antibody reduces immune cell activation by at least about 10%, 15%, 20%, 25%, 30%, 40%, or 50%.

[0025] In some embodiments of the method, the antibody reduces immune cell activation by about 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 10% to 15%, 20% to 50%, 20% to 40%, or 20% to 30%.

[0026] In some embodiments of the method, the immune cells comprise T cells, B cells, or macrophages. In some embodiments of the method, the immune cells comprise antigen-specific T cells.

[0027] In some embodiments of the method, the Fc region selectively binds to FcγR2B. In some embodiments of the method, the antibody binds to human FcγR2B with a KD of less than 5 μM, 4 μM, 3 μM, or 2 μM, as determined by surface plasmon resonance at 37° C. In some embodiments of the method, the antibody binds to human FcγR2A (131R allotype) with a KD of greater than 5 μM or 10 μM, as determined by surface plasmon resonance at 37° C. In some embodiments of the method, the antibody binds to human FcγR2A (131R allotype) with a KD of at least 15 μM, as determined by surface plasmon resonance at 37° C. In some embodiments of the method, the antibody binds to human FcγR2A (131H allotype) with a KD of at least 50 μM, as determined by surface plasmon resonance at 37° C. In some embodiments of the method, the antibody binds to human FcγR2A (131H allotype) with a KD of at least 80 μM as determined by surface plasmon resonance at 37° C. In some embodiments of the method, the ratio of the binding affinity of the antibody for human FcγR2B to the binding affinity of the antibody for human FcγR2A (131R allotype) is at least 2:1, 3:1, 4:1, 5:1, or 6:1. In some embodiments of the method, the ratio of the binding affinity of the antibody for human FcγR2B to the binding affinity of the antibody for human FcγR2A (131R allotype) is at least 6:1. In some embodiments of the method, the ratio of the binding affinity of the antibody for human FcγR2B to the binding affinity of the antibody for human FcγR2A (131R allotype) is about 6:1. In some embodiments of the method, the ratio of the antibody's binding affinity for human FcγR2B to the antibody's binding affinity for human FcγR2A (131H allotype) is at least 10:1, 15:1, 20:1, 40:1, or 50:1. In some embodiments of the method, the ratio of the antibody's binding affinity for human FcγR2B to the antibody's binding affinity for human FcγR2A (131H allotype) is at least 40:1. In some embodiments of the method, the ratio of the antibody's binding affinity for human FcγR2B to the antibody's binding affinity for human FcγR2A (131H allotype) is about 40:1.In some embodiments of the method, the ratio is determined by surface plasmon resonance at 37°C.

[0028] In some aspects, disclosed herein is an isolated antibody that specifically binds programmed cell death 1 (PD-1) and agonizes PD-1 signaling, wherein the antibody comprises a heavy chain, a light chain, and an Fc region, wherein the heavy chain comprises a heavy chain variable region, the light chain comprises a light chain variable region, and the Fc region comprises an amino acid substitution that reduces antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells compared to a parent molecule lacking that substitution, and wherein the antibody has the same or greater agnostic effect on PD-1 signaling in immune cells compared to its parent molecule.

[0029] In some aspects, disclosed herein are isolated antibodies that specifically bind to programmed cell death 1 (PD-1), wherein the antibodies comprise a heavy chain, a light chain, and an Fc region, wherein the heavy chain comprises a heavy chain variable region and the light chain comprises a light chain variable region, and wherein the antibodies enhance the interaction of PD-1 expressed on the surface of immune cells with PD-L1. In some cases of the antibodies, the Fc region comprises an amino acid substitution that results in decreased antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells in a subject compared to the parent molecule lacking the amino acid substitution, and the antibodies have the same or greater agnostic effect on PD-1 signaling in immune cells compared to the parent molecule.

[0030] In some antibody embodiments, the heavy chain variable region comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 1-3 with 0-3 amino acid modifications. In some antibody embodiments, the light chain variable region comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4-6 with 0-3 amino acid modifications. In some antibody embodiments, the Fc region is derived from IgG1 and comprises an aspartic acid (D) at position 238, numbered according to the EU index.

[0031]

[0010] In some aspects, disclosed herein is an isolated antibody that specifically binds to programmed cell death 1 (PD-1), comprising a heavy chain, a light chain, and an Fc region, wherein the heavy chain comprises a heavy chain variable region and the light chain comprises a light chain variable region, and wherein (i) the heavy chain variable region comprises a CDR comprising a sequence as set forth in one or more of SEQ ID NOs: 1-3, with 0-3 amino acid modifications; (ii) the light chain variable region comprises a CDR comprising a sequence as set forth in one or more of SEQ ID NOs: 4-6, with 0-3 amino acid modifications; and (iii) the Fc region is derived from IgG1 and comprises an aspartic acid (D) at position 238, numbered according to the EU index.

[0032] In some antibody embodiments, the antibody enhances the interaction between PD-1 and PD-L1 expressed on the surface of immune cells.

[0033] In some antibody embodiments, the interaction between PD-1 and PD-L1 is enhanced as determined by an assay such as that described in Example 10.

[0034] In some antibody embodiments, the antibody induces decreased antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells compared to an otherwise identical molecule comprising an IgG1 Fc region, and the antibody has the same or a greater agnostic effect on PD-1 signaling in immune cells compared to an otherwise identical molecule. In some antibody embodiments, ADCC against PD-1-expressing regulatory T cells is reduced as determined by the natural killer cell activation assay described in Example 7. In some antibody embodiments, the antibody does not result in significant ADCC against PD-1-expressing regulatory T cells as determined by the natural killer cell activation assay described in Example 7.

[0035] In some embodiments of the antibody, the antibody does not activate natural killer (NK) cells.

[0036] In some antibody embodiments, the heavy chain variable region comprises heavy chain complementarity determining region 1 (CDRH1), CDRH2, and CDRH3, wherein CDRH1, CDRH2, and CDRH3 each comprise the sequence set forth in SEQ ID NOs: 1-3 with 0-3 amino acid modifications.

[0037] In some antibody embodiments, the light chain variable region comprises light chain complementarity determining region 1 (CDRL1), CDRL2, and CDRL3, wherein CDRL1, CDRL2, and CDRL3 each comprise the sequence set forth in SEQ ID NOs: 4-6 with 0-3 amino acid modifications.

[0038] In some antibody embodiments, the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3, wherein CDRH1, CDRH2, and CDRH3 comprise the sequences set forth in SEQ ID NOs: 1-3, respectively.

[0039] In some embodiments of the antibody, the light chain variable region comprises CDRL1, CDRL2, and CDRL3, wherein CDRL1, CDRL2, and CDRL3 comprise the sequences set forth in SEQ ID NOs: 4-6, respectively.

[0040] In some antibody embodiments, the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in any one of SEQ ID NOs: 7-11.

[0041] In some antibody embodiments, the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in any one of SEQ ID NOs: 12-16.

[0042] In some embodiments of the antibody, the heavy chain comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in SEQ ID NO:18.

[0043] In some embodiments of the antibody, the light chain comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in SEQ ID NO:19.

[0044] In some antibody embodiments, the Fc region is derived from human IgG1. In some antibody embodiments, the Fc region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in SEQ ID NO: 17.

[0045] In some embodiments of the antibody, the heavy chain variable region and the light chain variable region form a structure selected from the group consisting of an scFv, a sc(Fv)2, a dsFv, a Fab, a Fab', a (Fab')2, and a diabody.

[0046] In some embodiments of the antibody, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region operably linked to an Fc region, and the light chain comprises a light chain variable region.

[0047] In some antibody embodiments, the heavy chain variable region and the light chain variable region form a single chain variable fragment (ScFv) operably linked to an Fc region.

[0048] In some embodiments of the antibody, the antibody is a humanized antibody.

[0049] In some embodiments of the antibody, the antibody is a human antibody.

[0050] In some embodiments of the antibody, the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, and a multispecific antibody.

[0051] In some embodiments of the antibody, the antibody is monoclonal.

[0052] In some antibody embodiments, the antibody binds to human PD-1 with a KD of less than 200 nM, 100 nM, 80 nM, 60 nM, or 40 nM as determined by surface plasmon resonance (SPR) at 37°C.

[0053] In some antibody embodiments, the antibody binds to human PD-1 with a KD of less than 60 nM as measured by surface plasmon resonance (SPR) at 37°C.

[0054] In some antibody embodiments, the antibody binds to human PD-1 with a KD of less than 40 nM as measured by surface plasmon resonance (SPR) at 37°C.

[0055] In some antibody embodiments, the antibody binds to cynomolgus PD-1 with a KD of less than 5000 nM, 4000 nM, 2000 nM, 1000 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, or 200 nM as determined by surface plasmon resonance (SPR) at 37°C.

[0056] In some antibody embodiments, the antibody binds to cynomolgus PD-1 with a KD of less than 600 nM as measured by surface plasmon resonance (SPR) at 37°C.

[0057] In some antibody embodiments, the antibody binds to cynomolgus PD-1 with a KD of less than 300 nM as measured by surface plasmon resonance (SPR) at 37°C.

[0058] In some antibody embodiments, the antibody agonizes human PD-1 expressed on the surface of immune cells.

[0059] In some antibody embodiments, the immune cell is a T cell.

[0060] In some antibody embodiments, binding of the antibody to human PD-1 expressed on the surface of an immune cell reduces proliferation of the cell compared to a comparable immune cell not bound by the antibody. In some antibody embodiments, the cell is a T cell. In some antibody embodiments, the reduction in cell activation is measured by the NFAT reporter assay described in Example 4. In some antibody embodiments, the reduction in cell activation is measured by the tetanus toxoid activation assay or the viral peptide activation assay described in Example 5. In some antibody embodiments, the reduction in cell proliferation is measured by the anti-CD3 / 28 activation assay described in Example 6. In some antibody embodiments, the reduction in cell proliferation is measured when the immune cell is in proximity to a PD-L1-expressing cell. In some antibody embodiments, the reduction in cell proliferation is measured by the assay described in Example 8. In some antibody embodiments, the reduction in cell proliferation is measured in vitro or in vivo. In some antibody embodiments, the reduction in cell proliferation is at least about 10%, 15%, 20%, 25%, 30%, 40%, or 50%. In some embodiments of the antibody, the reduction in cell proliferation is about 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 10% to 15%, 20% to 50%, 20% to 40%, or 20% to 30%.

[0061] In some antibody embodiments, the Fc region selectively binds to FcγR2B. In some antibody embodiments, the antibody binds to human FcγR2B with a KD of less than 5 μM, 4 μM, 3 μM, or 2 μM as determined by surface plasmon resonance at 37° C. In some antibody embodiments, the antibody binds to human FcγR2B with a KD of at least 2 μM, 1 μM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 80 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, or 5 nM. In some antibody embodiments, the antibody binds to human FcγR2B with a KD of 200 nM to 5 μM, 400 nM to 4 μM, 500 nM to 3.5 μM, 800 nM to 3 μM, 1 μM to 5 μM, 1 μM to 4.5 μM, 1 μM to 4 μM, 1 μM to 3.5 μM, 1 μM to 3 μM, 1 μM to 2.5 μM, or 1 μM to 2 μM. In some antibody embodiments, the antibody binds to human FcγR2A (131R allotype) with a KD of greater than 5 μM or 10 μM as determined by surface plasmon resonance at 37° C. In some antibody embodiments, the antibody binds to human FcγR2A (131R allotype) with a KD of at least 15 μM as determined by surface plasmon resonance at 37° C. In some antibody embodiments, the antibody binds to human FcγR2A (131H allotype) with a KD of at least 50 μM as determined by surface plasmon resonance at 37° C. In some antibody embodiments, the antibody binds to human FcγR2A (131H allotype) with a KD of at least 80 μM as determined by surface plasmon resonance at 37° C. In some antibody embodiments, the ratio of the antibody's binding affinity for human FcγR2B to the antibody's binding affinity for human FcγR2A (131R allotype) is at least 2:1, 3:1, 4:1, 5:1, or 6:1. In some antibody embodiments, the ratio of the antibody's binding affinity for human FcγR2B to the antibody's binding affinity for human FcγR2A (131R allotype) is at least 6:1. In some embodiments of the antibody, the ratio of the antibody's binding affinity for human FcγR2B to its binding affinity for human FcγR2A (131R allotype) is about 6:1.In some antibody embodiments, the ratio of the antibody's binding affinity for human FcγR2B to the antibody's binding affinity for human FcγR2A (131H allotype) is at least 10:1, 15:1, 20:1, 40:1, or 50:1. In some antibody embodiments, the ratio of the antibody's binding affinity for human FcγR2B to the antibody's binding affinity for human FcγR2A (131H allotype) is at least 40:1. In some antibody embodiments, the ratio of the antibody's binding affinity for human FcγR2B to the antibody's binding affinity for human FcγR2A (131H allotype) is about 40:1. In some antibody embodiments, the ratio is determined by surface plasmon resonance at 37°C.

[0062] In some aspects, disclosed herein is an isolated nucleic acid comprising one or more nucleotide sequences encoding a polypeptide capable of forming an antibody disclosed herein.

[0063] In some aspects, disclosed herein are vectors that include one or more nucleotide sequences that encode polypeptides capable of forming the antibodies disclosed herein.

[0064] Disclosed herein, in some aspects, are host cells comprising one or more nucleic acid molecules encoding heavy and light chain amino acid sequences that, when expressed, can form the antibodies disclosed herein.

[0065] In some aspects, disclosed herein are methods comprising culturing a host cell disclosed herein under conditions for the production of an antibody.

[0066] In some aspects, disclosed herein are methods comprising: (a) providing a host cell comprising one or more nucleic acid molecules encoding heavy and light chain amino acid sequences that, when expressed, can form an antibody disclosed herein; (b) culturing the host cell that expresses the encoded amino acid sequences; and (c) isolating the antibody.

[0067] Disclosed herein, in some aspects, is an immunoconjugate comprising an antibody disclosed herein conjugated to an agent.

[0068] In some aspects, disclosed herein is a pharmaceutical composition comprising a therapeutically effective amount of an antibody disclosed herein or an immunoconjugate disclosed herein and at least one pharmaceutically acceptable excipient.

[0069] In some aspects, disclosed herein is a pharmaceutical composition for use in treating a disease or condition, comprising a therapeutically effective amount of an antibody disclosed herein or an immunoconjugate disclosed herein, and at least one pharmaceutically acceptable excipient.

[0070] In some aspects, disclosed herein is a kit comprising an antibody disclosed herein or an immunoconjugate disclosed herein in a container.

[0071] In some cases of the kit, the kit further comprises informational material containing instructions for use of the antibodies disclosed herein or the immunoconjugates disclosed herein.

[0072] In some aspects, disclosed herein are methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody disclosed herein or an immunoconjugate disclosed herein, or a pharmaceutical composition disclosed herein. In some cases of the methods, the disease or condition comprises a PD-1-associated disease or condition. In some cases, the disease or condition is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), Addison's disease, allergies, alopecia areata, amyotrophic lateral sclerosis, ANCA vasculitis, ankylosing spondylitis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, Behçet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, celiac disease, Crohn's disease, Cushing's syndrome, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, gallbladder disease, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, IgG4-related disease, inflammatory bowel disease (IGBD), and others. IBD), inflammatory fibrosis, irritable bowel syndrome, juvenile arthritis, Kawasaki disease, leukemia, lupus nephritis, Lyme arthritis, lymphoma, lymphoproliferative disorders, meningoencephalitis, multiple sclerosis, myasthenia gravis, myeloma, non-radiographic axial spondyloarthritis (nr-AxSpA), neuromyelitis optica, osteoarthritis, pelvic inflammatory disease, pemphigus, peritonitis, pilonidal cyst disease, polymyositis, primary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjogren's syndrome, systemic lupus erythematosus, systemic sclerosis, Takayasu's arteritis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease. In some cases, the subject is a human subject.

[0073] In some aspects, disclosed herein are methods of downregulating an immune response in a subject comprising administering to the subject an antibody disclosed herein, administering to the subject an immunoconjugate disclosed herein, or administering to the subject a pharmaceutical composition disclosed herein.

[0074]

[0003] In some aspects, disclosed herein are methods of inhibiting immune cells that express PD-1, comprising contacting the immune cells with an antibody disclosed herein or an immunoconjugate disclosed herein. In some cases, the immune cells comprise T cells, B cells, or macrophages. In some cases, the immune cells comprise antigen-specific T cells. In some cases, the subject is a human subject.

[0075] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0076] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0077] [Figure 1A] Included is a schematic diagram (left) showing FcR-expressing aAPCs (artificial antigen-presenting cells) and opposing cell membranes displaying PD-1 and TCR (T cell receptor), and a graph (right) demonstrating that in the presence of FcR-expressing aAPCs, treatment with the exemplary antibody clone 19 mIgG1 resulted in T cell inhibition, as indicated by a decrease in luciferase signal, whereas treatment with the mIgG1 isotype control did not significantly affect T cell activation. In this experiment, NFAT-luciferase reporter Jurkat cells were co-cultured with stimulator cells expressing mouse FcγR2B together with increasing concentrations of anti-PD-1 clone 19 mIgG1 or isotype control, and luminescence was measured as a readout of T cell activation.

[0078] [Figure 1B]Included are a schematic diagram of aAPCs (artificial antigen presenting cells) that do not express FcRs and opposing cell membranes displaying PD-1 and TCRs (T cell receptors; left), and a graph (right) showing that treatment with exemplary antibody clone 19 mIgG1 in the presence of aAPCs that do not express FcRs had no effect on T cell activation, as indicated by a steady state in the luciferase signal, similar to the effect following treatment with the mIgG1 isotype control. In this experiment, the same assay as shown in Figure 1A was performed using stimulator cells that do not express any Fc receptors.

[0079] [Figure 2A] Graphs demonstrating the effect of exemplary antibodies on T cell activation, as determined by NFAT signaling in a Jurkat reporter assay, are shown. The figure shows that treatment of cells with all PD-1 antibodies and the P238D mutant version of humCL19v1 (see Table 2), but not the isotype control, significantly suppressed T cell activation; no significant difference was detected between the wild-type IgG1 version of the humCL19v1 antibody and the P238D mutant version of the humCL19v1 antibody. In this experiment, PD-1-expressing NFAT-luciferase Jurkat reporter cells were co-cultured with human FcγR2B-expressing stimulator cells in the presence of various anti-PD-1 antibodies at a single concentration of 10 μg / ml for 6 hours, and then NFAT activity was measured by luminescence quantification.

[0080] [Figure 2B] 1 shows a graph demonstrating the effect of exemplary antibodies on T cell activation, as determined by NFAT signaling, in another T cell reporter assay in which human HEK293T stimulator cells expressing anti-CD3 "T cell stimulator" were used to stimulate Jurkat T cell activation. The graph shows that treatment of cells with the P238D mutant version of humCL19v1 significantly suppressed T cell activation, whereas the P238D isotype control did not.

[0081] [Figure 3A]Figure 1 shows a graph demonstrating the effect of exemplary antibodies on T cell activation, as determined by IFNγ release in a tetanus toxoid (TT) activation assay. IFNγ release by peripheral blood mononuclear cells following a tetanus toxoid activation assay in the presence of a PD-L1 / 2-blocking antibody was significantly more suppressed following treatment with PD-1 antibodies and the P238D mutant version compared to IgG1 isotype control treatment. In this experiment, human PBMCs from six healthy donors were stimulated with tetanus toxoid in the presence of various PD-1 antibodies at a single dose of 1 μg / ml. IFNγ production was assessed 96 hours later by ELISA of supernatants and, for each donor, normalized to IFNγ levels in cells stimulated in the absence of the test antibody. *p<0.05 versus isotype control using one-way ANOVA.

[0082] [Figure 3B] 1 shows a graph demonstrating the effect of exemplary antibodies on T cell activation, as determined by IFNγ production in a viral peptide activation assay. IFNγ production by peripheral blood mononuclear cells after stimulation with CEF HLA class I peptides in the presence of Brefeldin A was significantly suppressed after treatment with P238D mutant humCL19v1 compared to IgG1 isotype control treatment.

[0083] [Figure 4] This figure shows a graph demonstrating the effect of exemplary antibodies on CD25 expression after CD25 induction by anti-CD3 and anti-CD28 stimulation of peripheral blood mononuclear cells. Unlike the isotype control, the P238D mutant humCL19v1 antibody effectively suppressed primary T cell activation (CD25) expression, similar to the IgG1 antibody. In this experiment, human PBMCs from three healthy donors were stimulated with soluble anti-CD3 and anti-CD28 antibodies in the presence of various PD-1 antibodies at a single dose of 1 μg / ml. CD25 expression on CD4 T cells was assessed 72 hours later by flow cytometry, and for each donor, data were normalized to the CD25 expression level in cells stimulated in the absence of the test antibody. *p<0.05 vs. isotype control using one-way ANOVA.

[0084] [Figure 5] Figure 1 shows in vitro degranulation by natural killer cells (antibody-dependent cellular cytotoxicity, or ADCC) induced by co-culture of purified NK cells with regulatory T cells at a 1:5 ratio in the presence of PD-1 antibodies. Unlike the IgG1 isotype control, all IgG1 isotype anti-PD-1 antibodies, but the P238D mutant PD-1 antibody (humCL19v1 P238D), resulted in significant ADCC killing of regulatory T cells and thus cell degranulation. In this experiment, 20,000 healthy donor NK cells were incubated with the indicated antibodies at 1 μg / ml and regulatory T cells. Data are shown from two separate studies using one Treg donor and three different NK cell donors per study. Each different icon represents a different NK cell donor, and NK cell degranulation is normalized to that donor's no-antibody setting. *p<0.05 vs. isotype control using one-way ANOVA.

[0085] [Figure 6] These results demonstrate that only the humCLV19v1 P238D PD-1 antibody, but not all other anti-PD-1 agonist antibodies, was able to inhibit T cell activation in the presence of high PD-L1 levels, as measured by NFAT signaling in a Jurkat reporter assay. When PD-L1-expressing cells containing a T cell stimulator construct were incubated with PD-1-expressing Jurkat reporter cells to test the effect of the P238D mutant humCL19v1 compared with other PD-1 antibodies, only the mutant P238D PD-1 antibody significantly suppressed T cell activation. In this experiment, PD-1-expressing Jurkat reporter cells were co-cultured with PD-L1-expressing stimulator cells in the presence of various PD-1 antibodies, and T cell activation was assessed by luciferase production.

[0086] [Figure 7A]Figures 7A and 7B show the effects of exemplary PD-1 agonist antibodies on T cell activation in RA PBMC and fibroblast cocultures, as measured by CD25 expression (Figure 7A), ICOS expression (Figure 7B), IFNγ (Figure 7C, labeled "IFNγ"), IL-17F (Figure 7D), and TNFα (Figure 7E, labeled "TNFα"). In this experiment, PBMCs from RA patients were cocultured with fibroblast-like synoviocytes and stimulated with anti-CD3 and anti-CD28 in the presence of different PD-1 antibodies or isotype controls. Cells and supernatants were assessed at 72 hours. CD25 and ICOS expression on CD4 T cells was assessed by flow cytometry. IFNγ, IL-17F, and TNFα levels in culture supernatants were assessed by cytometric bead array. Each symbol represents a different PBMC donor normalized to that donor's antibody-free setting. *p<0.05 versus isotype control using one-way ANOVA. [Figure 7B] Figures 7A and 7B show the effects of exemplary PD-1 agonist antibodies on T cell activation in RA PBMC and fibroblast cocultures, as measured by CD25 expression (Figure 7A), ICOS expression (Figure 7B), IFNγ (Figure 7C, labeled "IFNγ"), IL-17F (Figure 7D), and TNFα (Figure 7E, labeled "TNFα"). In this experiment, PBMCs from RA patients were cocultured with fibroblast-like synoviocytes and stimulated with anti-CD3 and anti-CD28 in the presence of different PD-1 antibodies or isotype controls. Cells and supernatants were assessed at 72 hours. CD25 and ICOS expression on CD4 T cells was assessed by flow cytometry. IFNγ, IL-17F, and TNFα levels in culture supernatants were assessed by cytometric bead array. Each symbol represents a different PBMC donor normalized to that donor's antibody-free setting. *p<0.05 versus isotype control using one-way ANOVA. [Figure 7C]Figures 7A and 7B show the effects of exemplary PD-1 agonist antibodies on T cell activation in RA PBMC and fibroblast cocultures, as measured by CD25 expression (Figure 7A), ICOS expression (Figure 7B), IFNγ (Figure 7C, labeled "IFNγ"), IL-17F (Figure 7D), and TNFα (Figure 7E, labeled "TNFα"). In this experiment, PBMCs from RA patients were cocultured with fibroblast-like synoviocytes and stimulated with anti-CD3 and anti-CD28 in the presence of different PD-1 antibodies or isotype controls. Cells and supernatants were assessed at 72 hours. CD25 and ICOS expression on CD4 T cells was assessed by flow cytometry. IFNγ, IL-17F, and TNFα levels in culture supernatants were assessed by cytometric bead array. Each symbol represents a different PBMC donor normalized to that donor's antibody-free setting. *p<0.05 versus isotype control using one-way ANOVA. [Figure 7D] Figures 7A and 7B show the effects of exemplary PD-1 agonist antibodies on T cell activation in RA PBMC and fibroblast cocultures, as measured by CD25 expression (Figure 7A), ICOS expression (Figure 7B), IFNγ (Figure 7C, labeled "IFNγ"), IL-17F (Figure 7D), and TNFα (Figure 7E, labeled "TNFα"). In this experiment, PBMCs from RA patients were cocultured with fibroblast-like synoviocytes and stimulated with anti-CD3 and anti-CD28 in the presence of different PD-1 antibodies or isotype controls. Cells and supernatants were assessed at 72 hours. CD25 and ICOS expression on CD4 T cells was assessed by flow cytometry. IFNγ, IL-17F, and TNFα levels in culture supernatants were assessed by cytometric bead array. Each symbol represents a different PBMC donor normalized to that donor's antibody-free setting. *p<0.05 versus isotype control using one-way ANOVA. [Figure 7E]Figures 7A and 7B show the effects of exemplary PD-1 agonist antibodies on T cell activation in RA PBMC and fibroblast cocultures, as measured by CD25 expression (Figure 7A), ICOS expression (Figure 7B), IFNγ (Figure 7C, labeled "IFNγ"), IL-17F (Figure 7D), and TNFα (Figure 7E, labeled "TNFα"). In this experiment, PBMCs from RA patients were cocultured with fibroblast-like synoviocytes and stimulated with anti-CD3 and anti-CD28 in the presence of different PD-1 antibodies or isotype controls. Cells and supernatants were assessed at 72 hours. CD25 and ICOS expression on CD4 T cells was assessed by flow cytometry. IFNγ, IL-17F, and TNFα levels in culture supernatants were assessed by cytometric bead array. Each symbol represents a different PBMC donor normalized to that donor's antibody-free setting. *p<0.05 versus isotype control using one-way ANOVA.

[0087] [Figure 8] 1 is a graph showing the effect of exemplary PD-1 agonist antibodies on PDL1-Fc binding to PD-1-expressing Jurkat cells preincubated with various PD-1 antibodies. Only humCL19v1 P238D was shown to increase PDL1-Fc binding to PD-1-expressing Jurkat cells. In this experiment, PD-1-expressing Jurkat cells were preincubated at 10 μg / ml on ice for 1 hour and then stained with increasing concentrations of AF647-conjugated PDL1-Fc.

[0088] [Figure 9A]These results demonstrate that genes downregulated by PD-1 agonists are associated with autoimmunity. Figure 9A is a graph showing activation of Jurkat T cells in the absence of PD-L1 but in the presence of an exemplary PD-1 agonist antibody (clone 19 mIgG1) or an isotype control, as measured by luciferase activity. PD-1-expressing Jurkat reporter cells were co-cultured with FcR-expressing stimulator cells in the presence of 5 μg / ml clone 19 mIgG1 or an isotype control, and a portion of cells from each well was harvested to assess luciferase production as a readout of T cell activation. Figure 9B shows representative flow cytometry plots of activated Jurkat cells before and after magnetic sorting. Jurkat cells were separated from stimulator cells by negative selection using magnetic beads, and the purity of the purified Jurkats was assessed by flow cytometry. Figure 9C is a volcano plot showing differentially expressed genes for Jurkats activated in the presence of PD-1 agonist versus isotype control, as determined by bulk RNA sequencing of purified Jurkat cells using GeneWiz. Figure 9D shows the signature of genes significantly downregulated by PD-1 agonism. To identify enrichment of genes associated with different traits, genes were mapped to the EBI GWAS catalog. [Figure 9B]These results demonstrate that genes downregulated by PD-1 agonists are associated with autoimmunity. Figure 9A is a graph showing activation of Jurkat T cells in the absence of PD-L1 but in the presence of an exemplary PD-1 agonist antibody (clone 19 mIgG1) or an isotype control, as measured by luciferase activity. PD-1-expressing Jurkat reporter cells were co-cultured with FcR-expressing stimulator cells in the presence of 5 μg / ml clone 19 mIgG1 or an isotype control, and a portion of cells from each well was harvested to assess luciferase production as a readout of T cell activation. Figure 9B shows representative flow cytometry plots of activated Jurkat cells before and after magnetic sorting. Jurkat cells were separated from stimulator cells by negative selection using magnetic beads, and the purity of the purified Jurkats was assessed by flow cytometry. Figure 9C is a volcano plot showing differentially expressed genes for Jurkats activated in the presence of PD-1 agonist versus isotype control, as determined by bulk RNA sequencing of purified Jurkat cells using GeneWiz. Figure 9D shows the signature of genes significantly downregulated by PD-1 agonism. To identify enrichment of genes associated with different traits, genes were mapped to the EBI GWAS catalog. [Figure 9C]These results demonstrate that genes downregulated by PD-1 agonists are associated with autoimmunity. Figure 9A is a graph showing activation of Jurkat T cells in the absence of PD-L1 but in the presence of an exemplary PD-1 agonist antibody (clone 19 mIgG1) or an isotype control, as measured by luciferase activity. PD-1-expressing Jurkat reporter cells were co-cultured with FcR-expressing stimulator cells in the presence of 5 μg / ml clone 19 mIgG1 or an isotype control, and a portion of cells from each well was harvested to assess luciferase production as a readout of T cell activation. Figure 9B shows representative flow cytometry plots of activated Jurkat cells before and after magnetic sorting. Jurkat cells were separated from stimulator cells by negative selection using magnetic beads, and the purity of the purified Jurkats was assessed by flow cytometry. Figure 9C is a volcano plot showing differentially expressed genes for Jurkats activated in the presence of PD-1 agonist versus isotype control, as determined by bulk RNA sequencing of purified Jurkat cells using GeneWiz. Figure 9D shows the signature of genes significantly downregulated by PD-1 agonism. To identify enrichment of genes associated with different traits, genes were mapped to the EBI GWAS catalog. [Figure 9D]These results demonstrate that genes downregulated by PD-1 agonists are associated with autoimmunity. Figure 9A is a graph showing activation of Jurkat T cells in the absence of PD-L1 but in the presence of an exemplary PD-1 agonist antibody (clone 19 mIgG1) or an isotype control, as measured by luciferase activity. PD-1-expressing Jurkat reporter cells were co-cultured with FcR-expressing stimulator cells in the presence of 5 μg / ml clone 19 mIgG1 or an isotype control, and a portion of cells from each well was harvested to assess luciferase production as a readout of T cell activation. Figure 9B shows representative flow cytometry plots of activated Jurkat cells before and after magnetic sorting. Jurkat cells were separated from stimulator cells by negative selection using magnetic beads, and the purity of the purified Jurkats was assessed by flow cytometry. Figure 9C is a volcano plot showing differentially expressed genes for Jurkats activated in the presence of PD-1 agonist versus isotype control, as determined by bulk RNA sequencing of purified Jurkat cells using GeneWiz. Figure 9D shows the signature of genes significantly downregulated by PD-1 agonism. To identify enrichment of genes associated with different traits, genes were mapped to the EBI GWAS catalog.

[0089] [Figure 10A] 10A-10D are graphs showing the effect of an exemplary PD-1 agonist antibody, Clone 19, in a mouse model of SLE, as measured by total anti-histone IgG levels (FIG. 10A), levels of anti-dsDNA IgG in serum at day 35 after cell transfer (FIG. 10B), Tfh cell frequency (CXCR5+ICOS+ as a percent of total CD4) in the spleen at day 35 (FIG. 10C), and spleen weight at the end of the study at day 35 (FIG. 10D). Levels of anti-dsDNA IgG were assessed by ELISA and quantified as arbitrary units using a standard curve of pooled serum. [Figure 10B]10A-10D are graphs showing the effect of an exemplary PD-1 agonist antibody, Clone 19, in a mouse model of SLE, as measured by total anti-histone IgG levels (FIG. 10A), levels of anti-dsDNA IgG in serum at day 35 after cell transfer (FIG. 10B), Tfh cell frequency (CXCR5+ICOS+ as a percent of total CD4) in the spleen at day 35 (FIG. 10C), and spleen weight at the end of the study at day 35 (FIG. 10D). Levels of anti-dsDNA IgG were assessed by ELISA and quantified as arbitrary units using a standard curve of pooled serum. [Figure 10C] 10A-10D are graphs showing the effect of an exemplary PD-1 agonist antibody, Clone 19, in a mouse model of SLE, as measured by total anti-histone IgG levels (FIG. 10A), levels of anti-dsDNA IgG in serum at day 35 after cell transfer (FIG. 10B), Tfh cell frequency (CXCR5+ICOS+ as a percent of total CD4) in the spleen at day 35 (FIG. 10C), and spleen weight at the end of the study at day 35 (FIG. 10D). Levels of anti-dsDNA IgG were assessed by ELISA and quantified as arbitrary units using a standard curve of pooled serum. [Figure 10D] 10A-10D are graphs showing the effect of an exemplary PD-1 agonist antibody, Clone 19, in a mouse model of SLE, as measured by total anti-histone IgG levels (FIG. 10A), levels of anti-dsDNA IgG in serum at day 35 after cell transfer (FIG. 10B), Tfh cell frequency (CXCR5+ICOS+ as a percent of total CD4) in the spleen at day 35 (FIG. 10C), and spleen weight at the end of the study at day 35 (FIG. 10D). Levels of anti-dsDNA IgG were assessed by ELISA and quantified as arbitrary units using a standard curve of pooled serum.

[0090] [Figure 11A]Figure 11 shows that the exemplary PD-1 agonist antibody, Clone 19, prevents the expansion of Tfh cells rather than depleting them in a mouse model of SLE. Figure 11A shows the Tfh cell frequency in the spleen at day 30. Figure 11B shows the spleen weight at day 30 after administration of Clone 19 on days 0, 14, or 28 after immune cell transfer. [Figure 11B] Figure 11 shows that the exemplary PD-1 agonist antibody, Clone 19, prevents the expansion of Tfh cells rather than depleting them in a mouse model of SLE. Figure 11A shows the Tfh cell frequency in the spleen at day 30. Figure 11B shows the spleen weight at day 30 after administration of Clone 19 on days 0, 14, or 28 after immune cell transfer.

[0091] [Figure 12A] Figure 12A shows that the exemplary PD-1 agonist antibody, Clone 19, inhibits delayed-type hypersensitivity in mice. Figure 12A shows the effect of Clone 19 on keyhole limpet hemocyanin (KLH)-induced delayed-type hypersensitivity (DTH). Mice were immunized with KLH antigen on day 0, 1 hour after treatment with 10 mg / kg Clone 19 or an isotype control antibody, and then challenged intradermally in one ear on day 5. The difference in biopsy weights of challenged and unchallenged ears measured on day 6 in the different treatment groups is shown in the figure. Figure 12B shows the results of another experiment in which mice were treated similarly but with various doses of Clone 19. Each dot represents an individual mouse. *The above groups represent p<0.05 versus isotype control using the Kruskal-Wallis, Dunn's multiple comparison test. [Figure 12B]Figure 12A shows that the exemplary PD-1 agonist antibody, Clone 19, inhibits delayed-type hypersensitivity in mice. Figure 12A shows the effect of Clone 19 on keyhole limpet hemocyanin (KLH)-induced delayed-type hypersensitivity (DTH). Mice were immunized with KLH antigen on day 0, 1 hour after treatment with 10 mg / kg Clone 19 or an isotype control antibody, and then challenged intradermally in one ear on day 5. The difference in biopsy weights of challenged and unchallenged ears measured on day 6 in the different treatment groups is shown in the figure. Figure 12B shows the results of another experiment in which mice were treated similarly but with various doses of Clone 19. Each dot represents an individual mouse. *The above groups represent p<0.05 versus isotype control using the Kruskal-Wallis, Dunn's multiple comparison test.

[0092] [Figure 13A] Figure 13 shows that the exemplary PD-1 agonist antibody, humCL19v1 P238D, ameliorates symptoms of graft-versus-host disease in a mouse model. Irradiated mice were injected with human peripheral blood mononuclear cells (PBMCs) and then treated with 10 mg / kg of humCL19v1 P238D or the P238D mutant hIgG1 isotype on days 0, 7, 14, and 21 after PBMC injection. humCL19v1 P238D was shown to significantly reduce spleen weight (Figure 13A), human immune cell proliferation in the spleen (Figure 13B), and human immune cell proliferation in the liver (Figure 13C), as well as serum inflammatory cytokine levels (Figure 13D), compared with the isotype control. Figure 13E shows that humCL19v1 P238D also reduced CD4 and CD8 cytokine production on a per-cell basis, as assessed by intracellular flow cytometry of human immune cells in the liver and spleen. [Figure 13B]Figure 13 shows that the exemplary PD-1 agonist antibody, humCL19v1 P238D, ameliorates symptoms of graft-versus-host disease in a mouse model. Irradiated mice were injected with human peripheral blood mononuclear cells (PBMCs) and then treated with 10 mg / kg of humCL19v1 P238D or the P238D mutant hIgG1 isotype on days 0, 7, 14, and 21 after PBMC injection. humCL19v1 P238D was shown to significantly reduce spleen weight (Figure 13A), human immune cell proliferation in the spleen (Figure 13B), and human immune cell proliferation in the liver (Figure 13C), as well as serum inflammatory cytokine levels (Figure 13D), compared with the isotype control. Figure 13E shows that humCL19v1 P238D also reduced CD4 and CD8 cytokine production on a per-cell basis, as assessed by intracellular flow cytometry of human immune cells in the liver and spleen. [Figure 13C] Figure 13 shows that the exemplary PD-1 agonist antibody, humCL19v1 P238D, ameliorates symptoms of graft-versus-host disease in a mouse model. Irradiated mice were injected with human peripheral blood mononuclear cells (PBMCs) and then treated with 10 mg / kg of humCL19v1 P238D or the P238D mutant hIgG1 isotype on days 0, 7, 14, and 21 after PBMC injection. humCL19v1 P238D was shown to significantly reduce spleen weight (Figure 13A), human immune cell proliferation in the spleen (Figure 13B), and human immune cell proliferation in the liver (Figure 13C), as well as serum inflammatory cytokine levels (Figure 13D), compared with the isotype control. Figure 13E shows that humCL19v1 P238D also reduced CD4 and CD8 cytokine production on a per-cell basis, as assessed by intracellular flow cytometry of human immune cells in the liver and spleen. [Figure 13D]Figure 13 shows that the exemplary PD-1 agonist antibody, humCL19v1 P238D, ameliorates symptoms of graft-versus-host disease in a mouse model. Irradiated mice were injected with human peripheral blood mononuclear cells (PBMCs) and then treated with 10 mg / kg of humCL19v1 P238D or the P238D mutant hIgG1 isotype on days 0, 7, 14, and 21 after PBMC injection. humCL19v1 P238D was shown to significantly reduce spleen weight (Figure 13A), human immune cell proliferation in the spleen (Figure 13B), and human immune cell proliferation in the liver (Figure 13C), as well as serum inflammatory cytokine levels (Figure 13D), compared with the isotype control. Figure 13E shows that humCL19v1 P238D also reduced CD4 and CD8 cytokine production on a per-cell basis, as assessed by intracellular flow cytometry of human immune cells in the liver and spleen. [Figure 13E] Figure 13 shows that the exemplary PD-1 agonist antibody, humCL19v1 P238D, ameliorates symptoms of graft-versus-host disease in a mouse model. Irradiated mice were injected with human peripheral blood mononuclear cells (PBMCs) and then treated with 10 mg / kg of humCL19v1 P238D or the P238D mutant hIgG1 isotype on days 0, 7, 14, and 21 after PBMC injection. humCL19v1 P238D was shown to significantly reduce spleen weight (Figure 13A), human immune cell proliferation in the spleen (Figure 13B), and human immune cell proliferation in the liver (Figure 13C), as well as serum inflammatory cytokine levels (Figure 13D), compared with the isotype control. Figure 13E shows that humCL19v1 P238D also reduced CD4 and CD8 cytokine production on a per-cell basis, as assessed by intracellular flow cytometry of human immune cells in the liver and spleen. DETAILED DESCRIPTION OF THE INVENTION

[0093] In some embodiments, disclosed herein are antibodies that specifically bind to programmed cell death 1 (PD-1) and agonize PD-1 signaling. In some cases, the PD-1 antibodies disclosed herein can act as agonists of PD-1, thereby modulating immune responses regulated by PD-1.

[0094] In some cases, the agonist anti-PD-1 antibodies disclosed herein comprise an Fc region with an Fc region amino acid substitution that results in decreased antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells in a subject compared to a parent molecule lacking the Fc region amino acid substitution, while maintaining or enhancing the agonistic effect of the antibody on PD-1 signaling compared to the parent molecule. In some cases, the Fc region amino acid substitution of the anti-PD-1 antibodies disclosed herein results in improved binding selectivity for FcγR2B, e.g., higher binding affinity for FcγR2B compared to other types of Fc receptors, with the difference in binding affinity being greater than that of the parent molecule lacking the amino acid substitution. The terms "FcγR2B," "FcgR2B," "FcγR2B," and "FcγRIIB" are used interchangeably herein to refer to the same subtype of Fc receptor.

[0095] Without wishing to be bound by any particular theory, it is believed that the amino acid substitutions in the Fc region of the anti-PD-1 antibodies disclosed herein can increase the binding selectivity of the antibodies to FcγR2B. In humans, one inhibitory Fc gamma receptor (FcγR2B) exists, but all other Fc gamma receptors can deliver immune activation signals (e.g., FcγR1, FcγR2A, FcγR3A, and FcγR3B). These activating FcRs can contribute to antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), which can lead to the depletion of target-expressing cells. It is believed that increasing the selectivity of Fc binding to FcγR2B can enhance the efficacy of the PD-1 agonist antibodies disclosed herein in suppressing immune responses without inducing inflammatory FcR signaling and without depleting PD-1-expressing regulatory T cells. Furthermore, in some cases, selective binding to FcγR2B can promote bidirectional inhibitory signaling via PD-1 on PD-1-expressing cells and FcγR2B on FcγR2B-expressing cells, which can enhance the immunosuppressive effects of the antibody. Conversely, in some cases, very high affinity for FcγR2B can be achieved with a K of 7.74 nM. DThis can adversely affect antibody half-life due to receptor turnover in liver sinusoidal epithelial cells, as demonstrated by the FcγR2B-enhanced IgG1 antibody XmAb7195, which binds to FcγR2B in liver (Ganesan et al. J Immunol. 2012 Nov 15;189(10):4981-8. doi:10.4049 / jimmunol.1202017) (Chu et al. J Allergy Clin Immunol. 2012 Apr;129(4):1102-15. doi:10.1016 / j.jaci.2011.11.029). Xencor demonstrated a mean half-life of approximately 21 days compared to wild-type IgG1 in a Phase 1a study (American Thoracic Society (ATS) 2016 International Conference in San Francisco, CA-A6476: Poster Board Number 407) reported a mean in vivo half-life of 3.9 days (Morell et al. J Clin Invest. 1970;49(4):673-680. doi:10.1172 / JCI106279). Thus, while sufficient binding to support selectivity and agonism for FcγR2B may be desirable for a PD-1 agonist antibody, excessively high affinity for FcγR2B may be undesirable in a therapeutic setting, as the potentially resulting shortened half-life may require more frequent administration. Without wishing to be bound by theory, the amino acid substitutions in the Fc region of the anti-PD-1 antibodies disclosed herein may avoid excessively high affinity for FcγR2B and increase the binding selectivity of the antibody for FcγR2B while maintaining the desired half-life of the antibody.

[0096] In some cases, the disclosed agonist anti-PD-1 antibodies are more effective than current antibodies at promoting inhibitory signaling to immune cells and / or the immune system and down-regulating immune cell responses. In some cases, the PD-1 antibodies disclosed herein enhance the binding of PD-1 to PD-L1. In some cases, the PD-1 antibodies disclosed herein promote PD-1 signaling in immune cells, even in the vicinity of PD-L1. The PD-1 antibodies disclosed herein may be particularly useful in treating immune system-mediated and / or PD-1-associated disorders, or diseases caused by aberrant immune pathologies or diseases with cancerous origins. PD-1-associated disorders can include disorders that exhibit as one of their symptoms dysregulated PD-1 expression and / or activity in one or more types of immune cells, or disorders caused by dysregulated PD-1 expression and / or activity in one or more types of immune cells.

[0097] In several aspects, methods, systems, pharmaceutical compositions, compositions, methods of treatment, kits, and methods of manufacture related to PD-1 antibodies are disclosed herein.

[0098] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be applied to any aspect unless the content clearly dictates otherwise. Moreover, various embodiments may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows.

[0099] definition As used herein, the terms "agonist," "agonist," "agonize," and other grammatical equivalents refer to or relate to an agent that can bind to a receptor or any other protein target and activate or enhance the activity of, or help initiate the activation of, the receptor or protein target. In some cases, an agonist can promote the receptor or other protein target to which it binds, inducing a biological response, such as signal transduction or other change in cellular activity. As used herein, a PD-1 agonist antibody (or antibody fragment) refers to an antibody (or antibody fragment) that binds to PD-1 expressed on the surface of immune cells and enhances its inhibitory signal on immune cells, including, but not limited to, T cells, macrophages, and / or B cells.

[0100] In this disclosure, whenever an embodiment is described herein with the word "comprising," it may also be expressed as "consisting of" and / or "consisting essentially of." Other similar embodiments described in the term "(essentially of)" are also provided. All definitions set forth herein should be construed to refer to the definitions used throughout this specification and the appended claims, whether or not specifically referred to.

[0101] Throughout this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0102] In this disclosure, one, some, or all of the characteristics of the various embodiments described herein may be applied to any aspect, unless the content clearly dictates otherwise. Furthermore, various embodiments may be combined to form other embodiments of the invention. These and other aspects of the invention will be apparent to those skilled in the art. These and other embodiments of the invention are further explained by the detailed description herein.

[0103] Unless otherwise defined throughout this specification and the appended claims, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0104] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes.

[0105] The numbering of amino acids in antibody variable domains, CDRs, and framework regions (FRs) follows the definition of Kabat as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise indicated.

[0106] The term "about" or "approximately" means within an acceptable error range of a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold or within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0107] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified, either naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Because the polypeptides described herein are based on antibodies, it is understood that the polypeptides can occur as single chains or associated chains.

[0108] The term "amino acid" refers to natural, unnatural, and synthetic amino acids, including, but not limited to, the D or L optical isomers, and both amino acid analogs and peptidomimetics. Amino acids are designated using standard one-letter or three-letter codes.

[0109] A "variant," as applied to a protein, is a protein having sequence homology with a naturally occurring biologically active protein that retains at least a portion of the therapeutic and / or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 99%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity compared to a reference biologically active protein, or any range between at least 70% and 75%. As used herein, the term "biologically active protein portion" includes proteins that have been accidentally modified, for example, by site-directed mutagenesis, synthesis, insertion, or mutation of the encoding gene.

[0110] In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in the polypeptide from amino to carboxyl terminal, in which adjacent residues in the sequence are contiguous in the primary structure of the polypeptide. A "subsequence" is the linear sequence of a portion of a polypeptide that is known to contain additional residues in one or both directions.

[0111] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to a polymer of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as modifications with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and modifications with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), modifications containing pendant moieties, such as modifications containing proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine lysine, etc.), those containing intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylators, those with modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Additionally, any hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH may be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxo sugars, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0112] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda, MD)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). As used herein, CDRs may refer to CDRs defined by either approach or a combination of both approaches.

[0113] A "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0114] A "host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector containing an exogenous polynucleotide. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of this disclosure.

[0115] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from the amino acid residue at position Pro230, to the carboxyl-terminus thereof. The numbering of residues in the Fc region is that of the EU index as in Kabat. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3.

[0116] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, and retains at least one effector function of the native sequence Fc region. In some cases, a variant Fc region comprises at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, e.g., about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of a parent polypeptide. In some cases, a variant Fc region herein has at least about 80% sequence identity to a native sequence Fc region and / or the Fc region of a parent polypeptide. In some cases, a variant Fc region herein has at least about 90% sequence identity to a native sequence Fc region and / or the Fc region of a parent polypeptide. In some cases, the variant Fc region herein has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity, and within that range, with a native sequence Fc region and / or the Fc region of the parent polypeptide.

[0117] An "individual" or "subject" is a mammal, such as a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0118] As used herein, "vector" refers to a construct capable of delivering and expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0119] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to produce a beneficial or desired result. A therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, etc., and these can be readily determined by one of ordinary skill in the art. The term "effective amount" also applies to a dose that provides an image for detection by an appropriate imaging method. Specific doses may vary depending on one or more of the particular agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried. An effective amount of an active agent may be administered in a single dose or multiple doses. A therapeutically effective amount of an antibody ranges from about 0.001 to about 25 mg / kg body weight, e.g., from about 0.01 to about 25 mg / kg body weight, from about 0.1 to about 20 mg / kg body weight, or from about 1 to about 10 mg / kg body weight. Dosage amounts may be adjusted, if necessary, to suit the observed effects of treatment and / or to be most effective in providing a cure, prevention, symptom control, etc., as determined by one of ordinary skill in the art. Appropriate doses will be selected based on the clinical indication by the treating physician or skilled artisan. Components may be described herein as having at least an effective amount, or at least an effective amount, to produce a desired result, such as any described herein, associated with a particular goal or objective. Desired therapeutic results herein may include, but are not limited to, treating, alleviating, or curing any symptoms from a disorder, cancer, immune-related disease, PD-1-associated disorder, and / or immune-related pathology, etc., as described herein and / or in the appended claims.

[0120] As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows that ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline. In some cases, compositions containing such carriers are formulated by well-known conventional methods (e.g., Remington's Pharmaceuticals, Inc., New York, NY, USA). Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy. (See 20th Ed. Mack Publishing, 2000).

[0121] Throughout this specification and the appended claims, the methods and systems of the present disclosure described herein may employ, unless otherwise indicated, conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, microarray and sequencing techniques that are within the skill of those in the art. Such conventional techniques include polymer array synthesis, oligonucleotide hybridization and ligation, oligonucleotide sequencing, and detection of hybridization using labels. Specific illustrations of suitable techniques can be obtained by reference to the examples herein. However, equivalent conventional procedures can, of course, also be used. Such prior art techniques and explanations are found in Green, et al., Eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler, Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis. (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006); and Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012) (all from Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4th Ed.) WH Freeman, NY (1995); Gait, "Oligonucleotide Synthesis: A Practical Approach" IRL Press, London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 6 th Ed.,WHFreeman Pub.,New York(2012);RIFreshney,Culture of Animal Cells:A Manual of Basic Technique and Specialized Applications,6 th Ed., Wiley-Blackwell (2010); and Berg et al., Biochemistry, 5 th Ed., W.H. Freeman Pub., New York (2002), all of which are incorporated herein by reference in their entirety for all purposes. Before the compositions, research tools, and systems and methods of the present invention are described, it is to be understood that this disclosure is not limited to the particular systems and methods, compositions, targets, and uses described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims.

[0122] As used herein, unless otherwise specified, the term "anti-PD-1 antibody" or molecule refers to either an antibody or a binding fragment thereof capable of specific binding to PD-1.

[0123] In this disclosure, "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes immunoglobulin molecules that specifically bind to an antigen and contain an FcR binding site, which may or may not be functional. As used in this disclosure, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, diabodies), Fv fragments, and single-chain (ScFv) variants that contain an antigen recognition or binding site and are capable of binding to an antigen. Antigen-binding antibody or immunoglobulin fragments are well known in the art, and such fragments may have a functional or non-functional Fc receptor binding site. Furthermore, as used herein, the term is not limited to intact polyclonal or monoclonal antibodies, multispecific antibodies such as bispecific or multispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, single chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted antibodies, human antibodies, and any other modified immunoglobulin molecule that contains an antigen-binding site, so long as the antibody exhibits the desired biological activity.

[0124] There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Unless context dictates otherwise, antibodies of the present invention may be derived from one of these classes or subclasses of antibodies. The heavy-chain constant domains corresponding to the different classes of antibodies are typically designated by the corresponding lowercase Greek letters α, δ, ε, γ, and μ, respectively. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0125] Throughout this specification and the appended claims, "Fc receptor" and "FcR" describe receptors that bind to the Fc region of an antibody. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).

[0126] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. Generally, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or may be made by recombinant DNA methods as described in U.S. Pat. No. 4,816,567. Monoclonal antibodies may also be produced by methods described, for example, in McCafferty et al. Alternatively, the vector may be isolated from a phage library generated using the techniques described in Wang et al., 1990, Nature 348:552-554.

[0127] As used herein, "antibody-dependent cellular cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on target cells, resulting in subsequent lysis of the target cells. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337, or in Example 7 of the present disclosure. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively, or additionally, ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.

[0128] "Complement-dependent cytotoxicity" and "CDC" refer to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, for example, a CDC assay as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) can be performed.

[0129] The term "specifically binds" to an epitope is well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular cell, protein, or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than with alternative cells, proteins, or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to PD-1 is an antibody that binds this epitope with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other epitopes. As a further example, an antibody (or other moiety) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[0130] A "fragment," as applied to a protein, is a truncated form of a native biologically active protein that may or may not retain at least some of its therapeutic and / or biological activity. As used herein, the terms "antibody fragment," "antigen-binding fragment thereof," and "fragment" are used interchangeably when referring to antibodies.

[0131] Sequence identity Sequence identity with respect to an anti-PD-1 antibody sequence or any other amino acid sequence identified herein is defined as the percentage of amino acid residues in a query sequence that are identical to those of a second reference polypeptide sequence, or portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. Percent identity can be measured over the length of the entire defined polypeptide sequence, or over a shorter length, for example, over the length of a fragment derived from the larger defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. It is understood that such lengths are exemplary only, and that any fragment length supported by the sequences set forth herein in the tables, figures, or sequence listing may be used to describe the length over which percent identity may be measured. In some embodiments, percent identity is determined with respect to the full length of the reference sequence referred to, such as the sequences provided herein. For example, sequence comparisons between two amino acid sequences (or shorter lengths thereof) of the present disclosure may be performed using the National Center for Biotechnology Information. This can be done by the computer program Blastp (protein-protein BLAST) provided online by the National Center for Biological Information (NCBI). The percentage amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having a certain % amino acid sequence identity to a given amino acid sequence B) is calculated by the following formula:

number

[0132] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence. Comparisons between two sequences are typically performed over a comparison window to identify and compare local regions of sequence similarity.

[0133] Programmed cell death 1 (PD-1) In some aspects, provided herein are compositions and methods related to antibodies or antigen-binding fragments thereof that bind to and agonize PD-1, a receptor that can be present on the surface of activated lymphocytes, including T cells, natural killer cells, B cells, and monocytes, and on the surface of myeloid cells. The PD-1 pathway can be an important immune checkpoint for regulating the responses of PD-1-expressing immune cells.

[0134] Without wishing to be bound by any particular theory, activation of the PD-1 pathway may result in the inhibition of immune cell activation. Antibodies that block PD-1 signaling are used in cancer patients to promote anti-tumor immune responses.

[0135] Programmed cell death protein 1 (PD-1 or CD279) is an immunoglobulin superfamily (IgSF) protein and a member of the B7-CD28 family. It can consist of a single extracellular IgV-like domain, a single-pass transmembrane region, and a cytoplasmic tail containing ITIMs and ITSMs. In some cases, it is monomeric on the cell surface due to the lack of extracellular cysteines that allow these molecules to form covalent homodimers, as found in CD28, CTLA4, and ICOS. In some cases, PD-1 is also expressed on cells throughout the immune system, including CD4 T cells, CD8 T cells, B cells, NKT cells, monocytes, macrophages, and dendritic cells. It can be upregulated briefly in acutely activated T cells and persistently in exhausted T cells. PD-1 can bind to two ligands, termed PD-L1 (PD-L1, CD279, or B7-H1) and PDL-2 (CD273, or B7-DC), each of which contains two IgSF domains in their extracellular regions. PD-L1 can be constitutively expressed on professional antigen-presenting cells (APCs) such as DCs, macrophages, and B cells, and can be induced on non-hematopoietic cells during inflammation to limit tissue damage, but is often upregulated on cancer cells, allowing them to attenuate antitumor immune responses.

[0136] Upon binding to its ligand, PD-1's intracellular tyrosine motif is phosphorylated, and the phosphorylated ITSM can recruit the protein phosphatase SHP2 (and possibly SHP1). Once recruited to the cell surface, SHP2 negatively regulates cell signaling by dephosphorylating ITAMs of activating receptors (especially CD28) and other downstream mediators of activation signaling. PD-1 signaling not only suppresses T cell activation but can also play a role in the generation of regulatory T cells (Tregs). As used herein, the term "PD-1 signaling" may refer to one or more of the phosphorylation of PD-1's intracellular tyrosine motif, the recruitment of protein phosphatases SHP2 and / or SHP1, or the dephosphorylation of ITAMs of activating receptors or other downstream mediators of activation signaling. The antibodies disclosed herein, in some cases, promote PD-1 signaling, e.g., promoting one or more of the phosphorylation of intracellular tyrosine motifs on a PD-1 molecule to which the antibody binds or on a PD-1 molecule that is not bound by the antibody but that binds to another PD-1 molecule expressed on the same cell surface, recruitment of SHP2 and / or SHP1, or dephosphorylation of ITAMs of activating receptors or other downstream mediators of activation signaling.

[0137] In some aspects, provided herein are antibodies, compositions, uses thereof, and methods of making thereof that can avoid some of the aforementioned and other problems known in the art associated with existing anti-PD-1 antibodies. In some embodiments, provided herein are PD-1 agonist antibodies that can trigger PD-1 signaling to bind to PD-L1 on effector T cells without depleting PD-1-expressing regulatory T cells or with minimal depleting effects on PD-1-expressing regulatory T cells.

[0138] Without wishing to be bound by any particular theory, in autoimmune diseases, PD-L1 expression may be upregulated by the inflammatory environment (Keir et al., 2008) (Garcia-Diaz et al., 2017), raising the possibility that PD-1 is already fully engaged under these pathological conditions, thus providing a limited scope for the further benefit of agonistic antibodies. However, in some aspects, provided herein are PD-1 agonistic antibodies that can provide an additional inhibitory signal even in the presence of receptor binding by its natural ligand, PD-L1. In some embodiments, the PD-1 agonistic antibodies disclosed herein have an inhibitory effect on PD-1-expressing immune cells that have PD-1 receptors in the vicinity of, in contact with, or engaged with PD-L1, or on PD-L1-expressing cells or PD-L1 itself.

[0139] Antibody sequence The present disclosure provides compositions, therapeutic agents, kits, vectors, nucleic acid sequences, manufacture, culture, and / or methods for producing PD-1 agonist antibodies, or antigen-binding or functional fragments thereof, with mutations in the Fc region (FcR), which enhance selectivity for the inhibitory Fc receptor FcγR2B (CD23B), thereby enhancing the biological effect of PD-1 activation, e.g., inhibiting the activity or proliferation of immune cells expressing a PD-1 molecule to which the antibody binds, or promoting downregulation of PD-1-expressing immune cell responses. In some cases, the PD-1 agonist antibody enhances the interaction between PD-1 and PD-L1. In some cases, the PD-1 agonist antibody promotes downstream signaling of PD-1 triggered by PD-L1 binding. In some cases, the PD-1 agonist antibody promotes downstream signaling of PD-1 without increasing or enhancing the interaction between PD-1 and PD-L1. In some cases, the PD-1 agonist antibody activates or enhances PD-1 signaling in the absence of PD-L1 binding to PD-1.

[0140] In some embodiments, the antibodies or antigen-binding fragments (e.g., isolated antibodies) provided herein specifically bind to PD-1 and enhance the interaction between PD-1 and PD-L1 expressed on the surface of immune cells.

[0141] In some embodiments, an antibody or antigen-binding fragment (e.g., an isolated antibody) provided herein is a PD-1 antibody comprising a heavy chain variable region, a light chain variable region, and an Fc region, wherein the Fc region of the PD-1 antibody comprises amino acid substitutions that enhance selectivity for the inhibitory Fc receptor FcγR2B, thereby enhancing PD-L1-mediated triggering of PD-1, increasing PD-1 / PD-L1 interaction, and decreasing antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells compared to the parent molecule lacking the substitutions.

[0142] In some embodiments, an antibody or antibody fragment (e.g., an isolated antibody) provided herein is a PD-1 antibody comprising a heavy chain variable region, a light chain variable region, and an Fc region. In some embodiments, the heavy chain variable region of the PD-1 antibody comprises a CDR comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in one or more of SEQ ID NOs: 1-3, with 0-3 amino acid modifications. In some embodiments, the heavy chain variable region of the PD-1 antibody comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 1-3, with 0-3 amino acid modifications. In some embodiments, the light chain variable region of the PD-1 antibody comprises a CDR comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in one or more of SEQ ID NOs: 4-6, with 0-3 amino acid modifications. In some embodiments, the light chain variable region of the PD-1 antibody comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4-6, with 0-3 amino acid modifications. In some embodiments, the Fc region of the PD-1 antibody is derived from an IgG1 molecule (e.g., a human IgG1 molecule) and comprises an amino acid substitution of proline (P) to aspartic acid (D) at position 238, numbered according to the EU index.

[0143] In some cases, the CDR of the heavy chain variable region of the antibody (or antigen-binding fragment (hereinafter referred to as "antibody") refers to a full-length antibody or an antigen-binding fragment of the antibody) comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to a sequence set forth in one or more of SEQ ID NOs: 1-3. In some cases, the CDR of the heavy chain variable region of the antibody comprises a sequence set forth in one or more of SEQ ID NOs: 1-3. In some cases, the CDR of the antibody comprises a sequence set forth in one or more of SEQ ID NOs: 1-3 with one amino acid modification. In some cases, the CDR of the heavy chain variable region of the antibody comprises a sequence set forth in one or more of SEQ ID NOs: 1-3 with two amino acid modifications. In some cases, the CDR of the heavy chain variable region of the antibody comprises a sequence set forth in one or more of SEQ ID NOs: 1-3 with three amino acid modifications.

[0144] In some cases, an antibody provided herein comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (CDRHs), wherein CDRH1 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1; CDRH2 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2; and CDRH3 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3, with 0 to 3 amino acid modifications. In some cases, an antibody provided herein comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (CDRHs), wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 2, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 3, with 0 to 3 amino acid modifications. In some cases, in the heavy chain variable region of the antibody, CDRH1 has the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 2, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 3, with one amino acid modification. In some cases, in the heavy chain variable region of the antibody, CDRH1 has the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 2, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 3, with two amino acid modifications. In some cases, in the heavy chain variable region of the antibody, CDRH1 has the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 2, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 3, with three amino acid modifications.

[0145] In some cases, the antibodies provided herein comprise a light chain variable region comprising a CDR having an amino acid sequence as set forth in one or more of SEQ ID NOs: 4-6, with zero to three amino acid modifications. In some cases, the CDR of the light chain variable region of the antibody comprises a sequence set forth in one or more of SEQ ID NOs: 4-6. In some cases, the CDR of the light chain variable region of the antibody comprises a sequence set forth in one or more of SEQ ID NOs: 4-6, with one amino acid modification. In some cases, the CDR of the light chain variable region of the antibody comprises a sequence set forth in one or more of SEQ ID NOs: 4-6, with two amino acid modifications. In some cases, the CDR of the light chain variable region of the antibody comprises a sequence set forth in one or more of SEQ ID NOs: 4-6, with three amino acid modifications.

[0146] In some cases, an antibody provided herein comprises a light chain variable region comprising three light chain complementarity determining regions, wherein CDRL1 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:4; CDRL2 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:5; and CDRL3 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:6, with 0 to 3 amino acid modifications. In some cases, the antibodies provided herein comprise a light chain variable region comprising three light chain complementarity determining regions, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 has the amino acid sequence set forth in SEQ ID NO:6, with 0 to 3 amino acid modifications. In some cases, the light chain variable region of the antibody, CDRL1 has the amino acid sequence set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 has the amino acid sequence set forth in SEQ ID NO:6. In some cases, the light chain variable region of the antibody, CDRL1 has the amino acid sequence set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 has the amino acid sequence set forth in SEQ ID NO:6, with one amino acid modification. In some cases, the light chain variable region of the antibody, CDRL1 has the amino acid sequence set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 has the amino acid sequence set forth in SEQ ID NO:6, with two amino acid modifications. In some cases, in the light chain variable region of the antibody, CDRL1 has the amino acid sequence set forth in SEQ ID NO:4, CDRL2 has the amino acid sequence set forth in SEQ ID NO:5, and CDRL3 has the amino acid sequence set forth in SEQ ID NO:6, with three amino acid modifications.

[0147] In some cases, an antibody provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions (CDRHs), wherein CDRH1 has the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 2, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 3 with 0-3 amino acid modifications; and the light chain variable region comprises three light chain complementarity determining regions, wherein CDRL1 has the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 has the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 has the amino acid sequence set forth in SEQ ID NO: 6 with 0-3 amino acid modifications. In some cases, CDRH1 has the amino acid sequence set forth in SEQ ID NO: 1, CDRH2 has the amino acid sequence set forth in SEQ ID NO: 2, and CDRH3 has the amino acid sequence set forth in SEQ ID NO: 3, the light chain variable region comprises three light chain complementarity determining regions, and CDRL1 has the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 has the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 has the amino acid sequence set forth in SEQ ID NO: 6.

[0148] In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, 85%, 90%, 95%, or 99% or 100% identity to the sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0149] In some cases, an antibody provided herein (e.g., hereinafter isolated antibody or "antibody") comprises a light chain variable region (VL) comprising a sequence having at least 80%, 85%, 90%, 95%, or 99% or 100% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, an antibody provided herein comprises a light chain variable region (VL) comprising a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, an antibody provided herein comprises a light chain variable region (VL) comprising a sequence having at least 85% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, the antibodies provided herein comprise a light chain variable region (VL) comprising a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, the antibodies provided herein comprise a light chain variable region (VL) comprising a sequence having at least 95% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, the antibodies provided herein comprise a light chain variable region (VL) comprising a sequence having at least 99% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, the antibodies provided herein comprise a light chain variable region (VL) comprising a sequence having at least 100% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, the antibodies provided herein comprise a light chain variable region (VL) comprising a sequence having any range between at least 80% and up to 100% identity to any one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

[0150] In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% or 100% identity to the sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and the VL comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% or 100% identity to the sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 85% identity to the sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 95% identity to the sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 99% identity to the sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 100% identity to the sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% or 100% identity to the sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises a sequence having at least 85% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises a sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises a sequence having at least 95% identity to the sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises a sequence having at least 99% identity to a sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises a sequence having at least 100% identity to a sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0151] In some cases, an antibody (e.g., an isolated antibody) provided herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and the VL comprises the sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

[0152] In certain embodiments, the heavy or light chains disclosed herein and in the appended claims may also comprise a constant region. If the molecule is a full-length IgG-type antibody molecule, the heavy chain may comprise three constant domains.

[0153] In one aspect, an antibody provided herein comprises an Fc region, wherein the Fc region is derived from human IgG1. In some cases, the Fc region of an antibody provided herein comprises an amino acid substitution in which a proline is replaced with an aspartic acid (D) at position 238, as numbered according to the EU index. In one aspect, the Fc region of an antibody provided herein comprises an amino acid substitution in which a proline is replaced with an aspartic acid (D) at position 238, as numbered according to the EU index, and further comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in SEQ ID NO: 17. In other aspects, the Fc region of an antibody provided herein comprises an amino acid substitution in which a proline is replaced with an aspartic acid (D) at position 238, as numbered according to the EU index, and further comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 17. In one aspect, the Fc region of an antibody provided herein comprises an amino acid substitution where a proline is replaced with an aspartic acid (D) at position 238, numbered according to the EU index, and further comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 17. In one aspect, the Fc region of an antibody provided herein comprises an amino acid substitution where a proline is replaced with an aspartic acid (D) at position 238, numbered according to the EU index, and further comprises a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 17. In one aspect, the Fc region of an antibody provided herein comprises an amino acid substitution where a proline is replaced with an aspartic acid (D) at position 238, numbered according to the EU index, and further comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 17. In one aspect, the Fc region of an antibody provided herein comprises an amino acid substitution at position 238, numbered according to the EU index, where proline is replaced with aspartic acid (D), and further comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 17.In one aspect, the Fc region of an antibody provided herein comprises an amino acid substitution at position 238, numbered according to the EU index, where a proline is replaced with an aspartic acid (D), and further comprises a sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 17. In one aspect, the Fc region comprises a sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 17.

[0154] In some cases, an antibody disclosed herein comprises a heavy chain variable region and an Fc region comprising an amino acid substitution, wherein the heavy chain variable region comprises a CDR comprising a sequence as set forth in one or more of SEQ ID NOs: 1-3 with 0-3 amino acid modifications. In some cases, an antibody comprises a heavy chain variable region (VH) and an Fc region comprising an amino acid substitution, wherein the VH comprises a CDR comprising a sequence as set forth in one or more of SEQ ID NOs: 1-3. In some cases, an agonist antibody comprises a VH and an Fc region comprising an amino acid substitution, wherein the VH comprises a CDR comprising a sequence as set forth in one or more of SEQ ID NOs: 1-3 with one amino acid modification. In some cases, an antibody comprises a VH and an Fc region comprising an amino acid substitution, wherein the VH comprises a CDR comprising a sequence as set forth in one or more of SEQ ID NOs: 1-3 with two amino acid modifications. In some cases, an antibody comprises a VH and an Fc region comprising an amino acid substitution, wherein the VH comprises a CDR comprising a sequence as set forth in one or more of SEQ ID NOs: 1-3 with three amino acid modifications.

[0155] In some cases, the agonist antibody comprises a light chain variable region (VL) and an Fc region comprising an amino acid substitution, wherein the light chain variable region comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4-6 with 0-3 amino acid modifications. In one aspect of this embodiment, the agonist antibody comprises a VL and an Fc region comprising an amino acid substitution, wherein the VL region comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4-6. In one aspect of the embodiment, the agonist antibody comprises a VL and an Fc region comprising an amino acid substitution, wherein the VL region comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4-6 with one amino acid modification. In one aspect of the embodiment, the agonist antibody comprises a VL and an Fc region comprising an amino acid substitution, wherein the VL region comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4-6 with two amino acid modifications. In one aspect of the embodiment, the agonist antibody comprises a VL and an Fc region comprising an amino acid substitution, wherein the VL region comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4 to 6 with three amino acid modifications.

[0156] In some cases, the antibodies or antigen-binding fragments disclosed herein comprise a heavy chain variable region and a light chain variable region that form a structure selected from the group consisting of an scFv, sc(Fv)2, dsFv, Fab, Fab', (Fab')2, and a diabody.

[0157] Effector Function In some cases, the antibodies disclosed herein agonize PD-1, e.g., human PD-1 expressed on the surface of an immune cell, such as a T cell, a B cell, or a macrophage. In some cases, the antibodies disclosed herein bind to PD-1 expressed on the surface of an effector immune cell and reduce the activation and / or proliferation of the effector immune cell compared to a comparable immune cell that is not bound to the antibody.

[0158] In some cases, the antibodies disclosed herein bind to PD-1 expressed on immune cells and inhibit cellular activation and / or proliferation. In some embodiments, the antibodies disclosed herein bind to PD-1 expressed on immune cells and reduce immune cell activation and / or proliferation when the immune cells are in proximity to PD-L1-expressing cells. In some cases, the antibodies disclosed herein have an agonistic effect on PD-1 signaling in immune cells in the presence of abundant PD-L1 in the surrounding environment. Without wishing to be bound by any particular theory, in some cases, in autoimmune diseases, PD-L1 expression is upregulated by an inflammatory environment (Keir et al., Annu, 2002). Rev Immunol. 2008;26:677-704. doi:10.1146 / annurev.immunol.26.021607.090331; Garcia-Diaz et al. Cell Rep. 2017 May 9;19(6):1189-1201. doi:10.1016 / j.celrep.2017.04.031), and thus, in such inflammatory environments, PD-1 is already fully engaged with surrounding PD-L1, potentially limiting the scope for further benefit of known agonistic antibodies targeting PD-1. Antibodies according to some embodiments of the present disclosure can further promote PD-1 signaling in immune cells even in the vicinity of PD-L1-expressing cells, suggesting that the antibodies disclosed herein may be particularly useful for downregulating immune responses, e.g., in the context of autoimmune disease, and thus may be useful for treating disorders associated with an excessive immune response, e.g., autoimmune disease.

[0159] In some embodiments, the antibodies disclosed herein bind to PD-1 expressed on immune cells and reduce immune cell activation and / or proliferation in the absence of PD-L1 binding to the PD-1 molecule to which the antibody binds.

[0160] In some cases, the inhibitory effect of an antibody disclosed herein on immune cell activation and / or proliferation can be measured by an NFAT reporter assay, such as that described in Example 4. For example, an NFAT reporter assay can be performed using Jurkat T cells engineered to express luciferase under the control of an NFAT response element. In some cases, Jurkat T cells expressing luciferase under the control of an NFAT response element are cultured with stimulator cells configured to stimulate the Jurkat T cells, for example, murine BW5147 cells expressing an anti-CD3 "T cell stimulator" (TCS) construct as previously described (Leitner et al., 2010) and expressing human FcγR2B, or HEK293T cells expressing an anti-CD3 "T cell stimulator" (TCS) construct as previously described (Leitner et al., 2010) and expressing human FcγR2B. Jurkat T cells are co-cultured with stimulator cells and either a test antibody (e.g., an exemplary PD-1 antibody according to some embodiments of the present disclosure), or an isotype control, or some other control PD-1 antibody for a period of time. After incubation with the test antibody or control for a period of time (e.g., 3 hours, 6 hours, 9 hours, or 12 hours), the cells can be harvested for a luciferase assay. The luminescent signal can be quantified as an indicator of Jurkat T cell activation.

[0161] In some cases, the inhibitory effect of an antibody disclosed herein on immune cell (e.g., T cell) activation and / or proliferation can be measured by an immune cell activation assay (e.g., a tetanus toxoid activation assay or a viral peptide activation assay), such as that described in Example 5. For example, immune cells such as human peripheral blood mononuclear cells (PBMCs) can be collected and stimulated with tetanus toxoid (e.g., 0.5 μg / mL) or a viral peptide (e.g., a commercially available pooled mixture of CEF HLA class I peptides—peptides derived from cytomegalovirus, Epstein-Barr virus, and influenza) in the presence of a PD-L1 / 2 blocking antibody (e.g., 5 μg / mL each) and a fixed concentration of a test PD-1 antibody (e.g., an exemplary antibody according to some embodiments herein), or an isotype control, or some other control antibody. Interferon (e.g., IFNγ) release from cells (e.g., in the supernatant or cell culture medium) can be assessed by ELISA or any other suitable method after incubation with the test antibody for a period of time (e.g., 24 hours, 48 ​​hours, 72 hours, 96 hours, or 1 week). Interferon release can be measured as an indicator of immune cell activation. In some cases, interferon production without stimulatory treatment (e.g., tetanus toxoid treatment or viral peptide treatment) can also be subtracted as unstimulated background from interferon production with treatment.

[0162] In some cases, the inhibitory effect of an antibody disclosed herein on immune cell (e.g., T cell) activation and / or proliferation can be measured by an anti-CD3 / 28 activation assay, such as that described in Example 6. For example, immune cells, such as human peripheral blood mononuclear cells (PBMCs), can be collected and stimulated with soluble anti-CD3 and anti-CD28 antibodies (e.g., at a final concentration of 0.5 ng / mL each) in the presence of a test PD-1 antibody (e.g., an exemplary antibody according to some embodiments herein), or an isotype control, or some other control antibody at a specific concentration. CD25 expression on CD4 T cells can be assessed by flow cytometry or any other method as a marker of T cell activation after incubation with the antibody for a period of time (e.g., 24 hours, 48 ​​hours, 72 hours, or 96 hours).

[0163] In some cases, the inhibitory effect of the antibodies disclosed herein on immune cell (e.g., T cell) activation and / or proliferation can be measured by cell proliferation, cytokine production, chemokine production, or any other activation marker of immune cells. The percentage inhibition described herein is measured by normalizing the readout of the immune cell activation marker in otherwise identical cells treated with the antibody of interest but with an isotype control or not treated with the antibody of interest. In some embodiments, the antibodies disclosed herein bind to PD-1 expressed on immune cells and reduce immune cell activation and / or proliferation by at least about 10%, 15%, 20%, 25%, 30%, 40%, or 50%.

[0164] In some cases, with respect to inhibiting immune cell activation (e.g., T cell activation), the antibodies disclosed herein have an IC50 of at most about 0.5 nM, at most about 0.2 nM, at most about 0.15 nM, at most about 0.1 nM, at most about 0.09 nM, at most about 0.08 nM, at most about 0.07 nM, at most about 0.06 nM, at most about 0.05 nM, at most about 0.04 nM, at most about 0.03 nM, at most about 0.02 M, or at most about 0.01 nM. In some cases, with respect to inhibiting immune cell activation (e.g., T cell activation), the antibodies disclosed herein have an IC50 of about 0.5 nM, about 0.2 nM, about 0.15 nM, about 0.1 nM, about 0.09 nM, about 0.08 nM, about 0.07 nM, about 0.06 nM, about 0.05 nM, about 0.04 nM, about 0.03 nM, about 0.02 M, or about 0.01 nM. The IC50 of the antibodies disclosed herein with respect to inhibiting immune cell activation (e.g., T cell activation) can be measured in immune cell assays such as those described above and in the Examples.

[0165] In some cases, provided herein are methods of suppressing immune cells that express PD-1 using the antibodies disclosed herein. The methods can include contacting immune cells that express PD-1 with the antibody and reducing immune cell activation and / or proliferation by about 10% to 50%. In some cases, the methods result in about a 10% to 40% decrease in immune cell activation and / or proliferation. In some cases, the methods result in about a 10% to 30% decrease in immune cell activation and / or proliferation. In some cases, the methods result in about a 10% to 20% decrease in immune cell activation and / or proliferation. In some cases, the methods result in about a 10% to 15% decrease in immune cell activation and / or proliferation. In some cases, the methods result in about a 20% to 50% decrease in immune cell activation and / or proliferation. In some cases, the methods result in about a 20% to 40% decrease in immune cell activation and / or proliferation. In some cases, the methods result in about a 20% to 30% decrease in immune cell activation and / or proliferation.

[0166] In some cases, the antibodies disclosed herein enhance the interaction between PD-1 and PD-L1 expressed on the surface of an immune cell. For example, in some cases, the antibodies disclosed herein enhance the binding of PD-L1 to a PD-1 molecule to which the antibody binds. In some cases, the antibodies disclosed herein enhance the binding of PD-L1 to one or more PD-1 molecules on the surface of an immune cell that are not bound by the antibody, while the antibody binds to other PD-1 molecules on the surface of the immune cell. In one embodiment, the antibodies disclosed herein enhance the interaction between PD-1 and PD-L1 expressed on the surface of an immune cell, as determined by an assay such as the assay described in Example 10. In some cases, the antibodies disclosed herein enhance the interaction between PD-1 and PD-L1 expressed on the surface of an immune cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 300%, 400%, 500%, or more.

[0167] In some cases, the antibodies disclosed herein induce reduced antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells compared to an otherwise identical molecule comprising an unmodified Fc region of IgG1, while having the same or enhanced agonistic effect on PD-1 signaling compared to an otherwise identical molecule comprising an unmodified Fc region of IgG1. In some cases, the antibodies disclosed herein have reduced ADCC against PD-1-expressing regulatory T cells as measured by a natural killer cell activation assay (such as the assay described in Example 7). In some cases, the antibodies disclosed herein do not result in significant ADCC against PD-1-expressing regulatory T cells as measured by either an in vitro assay such as that described in Example 7 or in vivo when the antibody is administered to a subject. In some cases, the antibodies disclosed herein do not cause natural killer cell degranulation, or cause reduced levels of degranulation compared to an otherwise identical molecule comprising an unmodified Fc region of IgG1. In some examples, the antibodies disclosed herein do not cause regulatory T cell death or cause a reduced level of regulatory T cell death compared to an otherwise identical molecule comprising an unmodified IgG1 Fc region. In some embodiments, the antibodies disclosed herein do not cause natural killer cell degranulation or regulatory T cell death or cause reduced natural killer cell degranulation or regulatory T cell death compared to an otherwise identical molecule comprising an unmodified IgG1 Fc region. The effect on natural killer cell degranulation and / or regulatory T cell death can be measured in vivo or in vitro, for example, by the assays described in Example 7.

[0168] binding affinity The binding affinity of the humanized anti-PD-1 antibody variant to the human or cynomolgus monkey PD-1 receptor or to PD-1 of another animal is k a , k d or K D As used herein, "k aThe term "k" is intended to refer to the rate constant for the association of an antibody with an antigen. d The term "K" is intended to refer to the rate constant for dissociation of an antibody from the antibody / antigen complex. D The term "KD" or "KD" is intended to refer to the equilibrium dissociation constant of an antibody-antigen interaction. For purposes of this disclosure, K D are the two rate constants k a / k d The smaller the equilibrium dissociation constant, the tighter the binding between the antibody of interest and PD-1.

[0169] In some cases, the antibodies disclosed herein have a K of less than 200 nM, less than 100 nM, less than 80 nM, less than 60 nM, or less than 40 nM as measured by surface plasmon resonance (SPR) at 37°C. D In one aspect, the antibodies disclosed herein bind to human PD-1 with a K of less than 5000 nM, 4000 nM, 2000 nM, 1000 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, or 200 nM as determined by surface plasmon resonance (SPR) at 37°C. D It binds to cynomolgus monkey PD-1.

[0170] In some cases, the antibodies or antigen-binding fragments thereof disclosed herein have increased binding to FcγR2B and decreased binding to one or more activating Fcγ receptors, such as FcγR2A (e.g., the 131R or 131H allotype) or FcγR1A, compared to the parent molecule lacking the Fc region substitution.

[0171] In some cases, the antibodies or antigen-binding fragments thereof disclosed herein have an increased binding ratio to FcγR2B / FcγR2A (e.g., the 131R allotype or the 131H allotype) compared to the parent molecule lacking the Fc region amino acid substitution. In some cases, the increased ratio of binding FcγR2B / FcγR2A (e.g., the 131R allotype or the 131H allotype) is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.8, 2, 2.2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 fold compared to the parent molecule lacking the Fc region substitution.

[0172] In some cases, the Fc region of an antibody or antigen-binding fragment thereof disclosed herein binds to FcγR2B with higher affinity compared to a comparable control antibody comprising an Fc region lacking the above-described amino acid substitutions. In some cases, the antibody has a dissociation constant (K) of at most about 5 μM, e.g., between about 5 μM and 0.1 μM, as determined by surface plasmon resonance (SPR). D ) binds to FcγR2B.

[0173] In some cases, the Fc region (FcR) of an antibody or antigen-binding fragment disclosed herein selectively binds to FcγR2B. In some cases, the antibody has a K of up to 5 μM as determined by surface plasmon resonance (SPR). D It binds to FcγR2B.

[0174] In some cases, the antibodies or antigen-binding fragments disclosed herein have a K of less than 5 μM, 4 μM, 3 μM, or 2 μM as determined by surface plasmon resonance at 37° C. D In some cases, the antibodies or antigen-binding fragments disclosed herein bind to human FcγR2B with a K of at least 2 μM, 1 μM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 80 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, or 5 nM. DIn some cases, the antibodies or antigen-binding fragments disclosed herein bind to human FcγR2B with a K of 200 nM to 5 μM, 400 nM to 4 μM, 500 nM to 3.5 μM, 800 nM to 3 μM, 1 μM to 5 μM, 1 μM to 4.5 μM, 1 μM to 4 μM, 1 μM to 3.5 μM, 1 μM to 3 μM, 1 μM to 2.5 μM, or 1 μM to 2 μM. D It binds to human FcγR2B.

[0175] In some cases, the antibody binds to FcγR2A (e.g., the 131R or 131H allotype) with lower or equal affinity compared to the parent molecule, which is an equivalent antibody lacking Fc substitutions that confer increased binding to FcγR2B and therefore enhanced FcγR2B signaling to the antibody molecule.

[0176] In some cases, when an antibody comprises a P238D substitution, the antibody binds to FcγR2A (131R allotype) with less or equal affinity compared to a comparable control antibody comprising an Fc region that comprises a proline at position 238 (EU index).

[0177] In some cases, the antibody has a K of greater than 5 μM as determined by surface plasmon resonance (SPR) at 37° C. D In some cases, the antibody binds to FcγR2A (131R allotype) with a K of greater than 10 μM as determined by surface plasmon resonance (SPR) at 37° C. D In some cases, the antibody binds to FcγR2A (131R allotype) with a K of at least 15 μM as determined by surface plasmon resonance (SPR) at 37° C. D In some cases, the antibody binds to FcγR2A (131R allotype) with a K of at least 20 μM as determined by surface plasmon resonance (SPR) at 37° C. D In some cases, the antibody binds to FcγR2A (131R allotype) with a K of about 15 μM to 25 μM as determined by surface plasmon resonance (SPR) at 37° C. DIt binds to FcγR2A (131R allotype) at

[0178] In some cases, the antibody has a K of at least 50 μM as determined by surface plasmon resonance (SPR) at 37° C. D In some cases, the antibody binds to FcγR2A (131H allotype) with a K of at least 75 μM as determined by surface plasmon resonance (SPR) at 37° C. D In some cases, the antibody binds to FcγR2A (131H allotype) with a K of at least 80 μM as determined by surface plasmon resonance (SPR) at 37° C. D In some cases, the antibody binds to FcγR2A (131H allotype) with a K of at least 90 μM as determined by surface plasmon resonance (SPR) at 37° C. D In some cases, the antibody binds to FcγR2A (131H allotype) with a K of about 50 μM to about 100 μM as determined by surface plasmon resonance (SPR) at 37° C. D In some cases, the antibody binds to FcγR2A (131H allotype) with a K of about 75 μM to about 125 μM as determined by surface plasmon resonance (SPR) at 37° C. D It binds to FcγR2A (131H allotype) at

[0179] In some cases, the antibody has a K of 3 or more, e.g., at least 4, 5, 6, 7, 8, 9, or 10 [K of antibody to FcγR2A(131R)]. D value / K of antibody against FcγR2B D Preferably, the value is determined by surface plasmon resonance (SPR).

[0180] In some cases, the antibody has a K of 10 or more, e.g., at least 15, 20, 25, 30, 35, 40, 45, or 50 [K of antibody to FcγR2A(131H)]. D value] / [K of antibody against FcγR2B D Preferably, the value is determined by surface plasmon resonance (SPR).

[0181] In some cases, the antibody has a K of 3 or more, e.g., at least 4, 5, 6, 7, 8, 9, or 10 [K of antibody to FcγR2A(131R)]. D value / K of antibody against FcγR2B D value], and / or 10 or more, e.g., at least 15, 20, 25, 30, 35, 40, 45, or 50 [K value of antibodies against FcγR2A(131H)] D value] / [K of antibody against FcγR2B D Preferably, the value is determined by surface plasmon resonance (SPR).

[0182] In some cases, the antibodies or antigen-binding fragments thereof disclosed herein have an increased binding ratio for FcγR2B / FcγR1A relative to the wild-type sequence as compared to the parent molecule lacking the Fc region substitution. In some cases, the increased ratio of binding FcγR2B / FcγR1A is at least 1.1, 1.2, 1.5, 2, 5, 10, 50, 100, 150, 200, 250-fold as compared to the parent molecule lacking the Fc region substitution.

[0183] "Compared to a parent molecule lacking the Fc region substitution(s)" means compared to an antibody molecule having the same amino acid sequence except for the amino acids recited in the claims that represent the Fc substitution(s) relative to the wild-type Fc. Thus, binding of antibody molecules with or without the recited Fc substitution(s) to FcγR2B can be measured, and optionally binding of antibody molecules with or without the recited Fc substitution(s) to an activating Fcγ receptor such as FcγR2A (e.g., the 131R or 131H allotype) or FcγR1A can be measured, e.g., by SPR.

[0184] In some cases, the antibodies or antigen-binding fragments thereof disclosed herein have a lower K for binding of FcγR1A compared to the parent molecule lacking the Fc region substitution than the wild-type sequence. D value] / [K for FcγR2B binding DIn some cases, the ratio of [K for FcγR1A binding] for the variant molecules is increased. D value] / [K for FcγR2B binding D The ratio of [K for FcγR1A binding] to [K for FcγR1A binding] for the parent molecule lacking the Fc region substitution D value] / [K for FcγR2B binding D value].

[0185] In some cases, the antibodies or antigen-binding fragments thereof disclosed herein exhibit a lower K for binding of FcγR2A(131R) compared to the parent molecule lacking the Fc region substitution than the wild-type sequence. D value] / [K for FcγR2B binding D In some cases, the ratio of [K for binding of FcγR2A(131R)] for the variant molecules is increased. D value] / [K for FcγR2B binding D The ratio of [K for FcγR1A binding] to [K for FcγR1A binding] for the parent molecule lacking the Fc region substitution D value] / [K for FcγR2B binding D The ratio is at least 1.1, 1.2, 1.5, 2, 5, 10, 50, or 100 times the ratio of [value of the nucleotides in the nucleotide sequence] to [value of the nucleotide sequence].

[0186] In some embodiments provided herein, the binding affinity of a humanized variant of an anti-PD-1 antibody to human PD-1, cynomolgus monkey PD-1, or PD-1 from another animal is measured by surface plasmon resonance. A Biacore® surface plasmon resonance (SPR) system (GE Healthcare, Chicago, IL) can be used to measure the binding affinity of the antibody of interest. Exemplary SPR analysis systems include the Biacore X100, Biacore T200, and Biacore X100. Examples of suitable Biacore systems include, but are not limited to, Biacore 3000 or Biacore 4000 instruments, and commercially available sensor chip series. In a typical application of a Biacore system, interaction kinetics are analyzed by monitoring the interaction as a function of time over a range of analyte concentrations and then fitting the entire data set to a mathematical model describing the interaction. The association phase (during sample injection) contains information about both the association and dissociation processes, while only dissociation occurs during the dissociation phase (after sample injection, when buffer flow removes dissociated analyte molecules). Those skilled in the art can select or determine appropriate parameters and / or conditions for performing binding affinity assays according to the manufacturer's instructions. In some embodiments, the binding affinity of a subject antibody is determined by surface plasmon resonance at 37°C. In some cases, the binding affinity and kinetics of a humanized antibody variant to human or cynomolgus PD-1 are determined by surface plasmon resonance (SPR) using a Biacore 8K (Cytiva), as disclosed in Example 2.

[0187] antibody manipulation In some cases, the antibodies disclosed herein comprise human antibodies. In some embodiments, the antibodies disclosed herein comprise monoclonal humanized antibodies, chimeric antibodies, or multispecific antibodies. In some cases, the antibodies disclosed herein comprise monoclonal antibodies.

[0188] The antibodies embodied herein may be monoclonal, chimeric, human, or humanized. As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences. The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody. In some embodiments, the antibodies provided herein are monoclonal antibodies.

[0189] The subject antibodies can be prepared by a hybridoma process or a recombinant DNA process. The antibody-producing cells used in the cell fusion process to prepare hybridomas are spleen cells, lymph node cells, peripheral blood leukocytes, etc., from animals (e.g., mice, rats, hamsters, rabbits, monkeys, and goats) immunized with an antigen (PD-1, its partial peptides, or cells expressing them), as described by Kohler and Milstein (Nature, 256:495 (1975)). Alternatively, antibody-producing cells obtained by exposing the above-mentioned cells or lymphocytes previously isolated from a non-immunized animal to an antigen in a culture medium can also be used. Various known cell lines can be used as myeloma cells. The antibody-producing cells and myeloma cells can be derived from different animal species, as long as they are fusible with each other. In some cases, they are derived from the same animal species. Hybridomas can be produced, for example, by cell fusion between spleen cells obtained from an antigen-immunized mouse and mouse myeloma cells, followed by screening to obtain hybridomas that produce monoclonal antibodies against PD-1. Monoclonal antibodies against PD-1 can be produced by culturing hybridomas or from the ascites fluid of a mammal administered with the hybridoma.

[0190] In some embodiments, the antibodies disclosed herein include humanized antibodies. In generating humanized antibodies, the selection of framework residues can be important for maintaining high binding affinity. In principle, the framework sequence from any HuAb can serve as a template for CDR grafting; however, it has been demonstrated that direct CDR substitution into such a framework can result in a significant loss of binding affinity for the antigen. Glaser et al. (1992) J. Immunol. 149:2606; Tempest et al. (1992) Biotechnology 9:266; and Shalaby et al. (1992) J. Exp. Med. 17:217. The more homologous the HuAb is to the original muAb, the less likely it is that the human framework will introduce distortions into the murine CDRs that could reduce affinity. Based on sequence homology searches against antibody sequence databases, HuAb IC4 provides good framework homology to muM4TS.22, but other highly homologous HuAbs are suitable as well, particularly kappa light chains from human subgroup I or heavy chains from human subgroup III. Kabat et al. (1987). Various computer programs, such as ENCAD (Levitt et al. (1983) J. Mol. Biol. 168:595), are available for predicting ideal V-region sequences. Thus, the present invention encompasses HuAbs with different V-regions. Determining appropriate V-region sequences and optimizing these sequences is within the skill of one of ordinary skill in the art. Methods for obtaining antibodies with reduced immunogenicity are also described in U.S. Pat. No. 5,270,202 and European Patent No. 699,755.

[0191] In some embodiments, the antibodies disclosed herein comprise a heavy chain variable region, and the light chain variable region forms a structure selected from the group consisting of an scFv, an sc(Fv)2, a dsFv, an Fab, an Fab', an (Fab')2, and a diabody.

[0192] In one aspect, an antibody disclosed herein comprises a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain variable region operably linked to the Fc region, and the light chain comprises the light chain variable region. In one feature, an antibody disclosed herein comprises a humanized antibody. In one aspect, an antibody disclosed herein comprises a human antibody. In another embodiment, an antibody disclosed herein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, and a multispecific antibody. In some cases, an antibody disclosed herein is a monoclonal antibody.

[0193] Humanization In some embodiments, provided herein are antibody variants comprising any potential combination of humanized VH and VL domains. In some embodiments, the antibodies provided herein comprise a humanized variant of the VH of a PD-1 agonist antibody comprising human framework sequences. In some embodiments, the antibody or antigen-binding fragment comprises a humanized variant of the VL of a PD-1 agonist antibody comprising human framework sequences.

[0194] Humanized antibodies can retain high affinity for antigens and other favorable biological properties. To achieve this goal, in one example, PD-1 humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the possible role of the residues in the function of the candidate immunoglobulin sequence and residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from consensus and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0195] In some cases, the variable heavy (VH) chain comprises the amino acid sequence set forth in SEQ ID NO: 7. In some cases, the humanized VH chain comprises the human framework IGHV1-24 * In some embodiments, the humanized VH chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the humanized VH chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 9. In yet other embodiments, the humanized VH chain is comprised of the human framework IGHV7-4-1. * 02. In some embodiments, the humanized VH chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the humanized VH chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 11.

[0196] In some cases, the variable light (VL) chain comprises the amino acid sequence set forth in SEQ ID NO: 12. In some cases, the humanized VL chain comprises the human framework IGKV1-39 * 01. In some embodiments, the humanized VL chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 13. In some cases, the humanized VL chain comprises a human framework IGKV3-11 *01. In some embodiments, the humanized VL chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 14. In some embodiments, the humanized VL chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the humanized VL chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 16.

[0197] mutation In some embodiments provided herein, the PD-1 antibodies described herein may have one or more mutations or modifications relative to a reference sequence. The mutations or modifications may be deletions, insertions, or additions, or substitutions or replacements of amino acid residues. A "deletion" refers to a change in the amino acid sequence resulting from the absence of one or more amino acid residues. An "insertion" or "addition" refers to a change in the amino acid sequence that results in the addition of one or more amino acid residues compared to the reference sequence. A "substitution" or "substitution" refers to the replacement of one or more amino acids with different amino acids. In the context of the present disclosure, the mutations of a subject antibody or a fraction thereof relative to a reference sequence may be determined by comparing the subject antibody or a fraction thereof to the reference sequence. Optimal alignment of sequences for comparison may be performed according to any method known in the art.

[0198] Mutations can be identified by their mutation site. The mutation site is the position on the reference sequence where a modification, such as a deletion, addition, or substitution, occurs. Amino acid residues on the reference sequence are numbered from the N-terminus to the C-terminus, and the mutation site is the number of the amino acid residue where the deletion, addition, or substitution occurs. For example, position 26 on the reference sequence is the position of the 26th amino acid residue counting from the N-terminus.

[0199] antibody conjugates In some embodiments, the antibodies or fragments thereof disclosed herein are fused to serum albumin. Fusion to serum albumin can improve the pharmacokinetics of the subject antibodies as described herein. For example, the subject antibodies or fragments thereof can be fused to serum albumin. Serum albumin is a globular protein that is the most abundant blood protein in mammals. Serum albumin is produced in the liver and constitutes approximately half of serum proteins. It is monomeric and soluble in blood. In some embodiments, the subject antibodies or fragments thereof can be fused to serum albumin. In further embodiments, the serum albumin is human serum albumin (HSA).

[0200] In some embodiments, the antibodies or fragments thereof disclosed herein are fused to an albumin-binding peptide that exhibits serum albumin binding activity and extends the half-life of the subject antibody or fragment thereof. Albumin-binding peptides that can be used herein include, but are not limited to, those described in Dennis et al., J. Biol. Chem. 277:35035-35043, 2002 and Miyakawa et al., J. Pharm. Sci. 102:3110-3118, 2013. In some embodiments, the albumin-binding peptide is genetically fused to a subject antibody or fragment thereof described herein. In further embodiments, the albumin-binding peptide is attached to a subject antibody or fragment thereof described herein by chemical means, such as chemical conjugation. In some embodiments, the albumin-binding peptide may be fused to the N-terminus or C-terminus of a subject antibody or fragment thereof described herein. The C-terminus of the albumin-binding peptide may be fused directly to the N-terminus of the subject antibody via a peptide bond. Alternatively, the N-terminus of the albumin-binding peptide may be fused directly to the C-terminus of the subject antibody or fragment thereof via a peptide bond. In a further embodiment, the C-terminal carboxylic acid of the albumin-binding peptide may be fused to an internal amino acid residue of the subject antibody or fragment thereof using conventional chemical conjugation techniques.

[0201] In some embodiments, the PD-1 antibodies or fragments thereof disclosed herein are fused to a polymer, e.g., polyethylene glycol (PEG). The antibodies or fragments thereof can be PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody or fragment thereof. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. In some cases, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. Methods for pegylating proteins can be used, such as those disclosed in European Patent No. 0 154 316 by Nishimura et al. and European Patent No. 0 401 384 by Ishikawa et al. In some embodiments, a polymer, such as PEG, can be covalently attached to a subject antibody or fragment thereof described herein at either the N-terminus or C-terminus or an internal position using conventional chemical methods, such as chemical conjugation. Without being bound by theory, PEG moieties, when attached to an antibody described herein, may contribute to water solubility, high mobility in solution, lack of toxicity and low immunogenicity, extended circulatory life, increased stability, provision for clearance from the body, and altered biodistribution.

[0202] Other half-life extension technologies that can be used to extend the serum half-life of the subject antibodies or fragments thereof include, but are not limited to, XTEN (Schellenberger et al., Nat. Biotechnol. 27:1186-1192, 2009) and Albu tag (Trussel et al., Bioconjug Chem. 20:2286-2292, 2009).

[0203] In some embodiments, the aPD-1 antibodies or fragments thereof disclosed herein are conjugated to a chemically functional moiety. Typically, the moiety is a label capable of generating a detectable signal. These conjugated antibodies or fragments thereof are useful in detection systems such as, for example, tumor burden quantification, metastatic imaging, and tumor imaging. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, substrates, cofactors, and inhibitors. For examples of patents describing the use of such labels, see U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. The moiety may be covalently bound to an antibody or fragment thereof described herein, recombinantly bound, or conjugated to the antibody or fragment thereof via a secondary reagent, such as a second antibody, protein A, or biotin-avidin complex.

[0204] Other functional moieties include signal peptides, agents that enhance or reduce immunological reactivity, agents that facilitate coupling to solid supports, vaccine carriers, biological response modifiers, paramagnetic labels, and drugs. A signal peptide is a short amino acid sequence that guides newly synthesized proteins through a cell membrane, usually the endoplasmic reticulum of eukaryotic cells, and either the inner membrane or both the inner and outer membranes of bacteria. Signal peptides are typically located at the N-terminal portion of a polypeptide and are typically enzymatically removed between biosynthesis and secretion of the polypeptide from the cell. Such peptides can be incorporated into a subject antibody or fragment thereof to enable secretion of the synthesized molecule.

[0205] Agents that enhance immunological reactivity include, but are not limited to, bacterial superantigens. Agents that facilitate coupling to solid supports include, but are not limited to, biotin or avidin. Immunogen carriers include, but are not limited to, any physiologically acceptable buffer. Biological response modifiers include cytokines, particularly tumor necrosis factor (TNF), interleukin-2, interleukin-4, granulocyte-macrophage colony-stimulating factor, and gamma-interferon.

[0206] Agents that reduce immunological reactivity include, but are not limited to, anti-inflammatory agents and immunosuppressants. Anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. NSAIDs include salicylates such as acetylsalicylic acid; diflunisal, salicylic acid, and salsalate; propionic acid derivatives such as ibuprofen; naproxen; dexibuprofen, dexketoprofen, flurbiprofen, oxaprozin, fenoprofen, loxoprofen, and ketoprofen; acetic acid derivatives such as indomethacin, diclofenac, tolmetin, aceclofenac, sulindac, nabumetone, etodolac, and ketorolac; piroxicam, lorhexidine glucan, and lorhexidine glucan. Examples of suitable anti-inflammatory drugs include, but are not limited to, enolic acid derivatives such as cicam, meloxicam, isoxicam, tenoxicam, phenylbutazone, and droxicam; anthranilic acid derivatives such as mefenamic acid, flufenamic acid, meclofenamic acid, and tolfenamic acid; selective COX-2 inhibitors such as celecoxib, lumiracoxib, rofecoxib, etoricoxib, valdecoxib, firocoxib, and parecoxib; sulfonanilides such as nimesulide; and others such as clonixin and licofelone. Corticosteroids include, but are not limited to, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone. Immunosuppressants include, but are not limited to, hydroxychloroquine, sulfasalazine, leflunomide, etanercept, infliximab, adalimumab, D-penicillamine, oral gold compounds, injectable gold compounds (intramuscular injection), minocycline, gold sodium thiomalate, auranofin, D-penicillamine, lobenzarit, bucillamine, actarit, cyclophosphamide, azathioprine, methotrexate, mizoribine, cyclosporine, and tacrolimus.

[0207] Suitable drug moieties include anti-neoplastic agents, non-limiting examples of which are radioactive isotopes, vinca alkaloids such as vinblastine, vincristine, and vindesine sulfate, adriamycin, bleomycin sulfate, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, lomustine, mechloroethamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbazepe hydrochloride, streptozotocin, taxol, thioguanine, and uracil mustard.

[0208] Immunotoxins, including single-chain molecules, can be produced by recombinant means. A variety of immunotoxins are available, and methods can be found, for example, in "Monoclonal Antibody-toxin Conjugates: Aiming the Magic Bullet," Thorpe et al. (1982) Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190; Vitatta (1987) Science 238:1098-1104; and Winter and Milstein (1991) Nature 349:293-299. Suitable toxins include, but are not limited to, ricin, radionuclides, Chenopodium antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal toxins such as restrictocin, and phospholipase enzymes. See generally, "Chimeric Toxins," Olsnes and Pihl, Pharmac. Ther. 15:355-381 (1981); and "Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985).

[0209] Chemically functional moieties can be produced recombinantly, for example, by creating a fusion gene encoding the antibody and the functional moiety. Alternatively, the antibody or fragment thereof can be chemically linked to the moiety by any of a variety of well-established chemical procedures. For example, when the moiety is a protein, various coupling agents can be used, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al. Cancer Research, 52:127-131 (1992)) can be used. The moiety can be covalently linked or conjugated via a secondary reagent such as a second antibody, protein A, or biotin-avidin complex. For examples of paramagnetic moieties and their conjugation to antibodies, see, e.g., Miltenyi et al. (1990) Cytometry 11:231-238.

[0210] In some embodiments, the PD-1 antibodies or fragments thereof disclosed herein are bispecific antibodies. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. The bispecific antibodies described herein may be bispecific antibodies that recognize different epitopes on PD-1, or bispecific antibodies in which one antigen-binding site recognizes PD-1 and the other antigen-binding site recognizes an antigen other than PD-1.

[0211] nucleic acid molecule In some embodiments, the antibodies described herein are encoded by one or more nucleic acid molecules. In one case, the antibody is encoded by a single nucleic acid molecule. In other cases, the antibody is encoded by two or more nucleic acid molecules. For example, because the antigen-binding site is formed by the heavy chain variable polypeptide region together with the light chain variable polypeptide region, the two variable (heavy and light) polypeptide regions are encoded by separate nucleic acid molecules. Alternatively, for example, in the case of an ScFv, they are encoded by the same nucleic acid molecule.

[0212] According to some aspects of the present disclosure, there are provided one or more nucleic acid molecules encoding an antibody or antigen-binding fragment thereof according to some embodiments of the present disclosure.

[0213] From the primary amino acid sequence of a polypeptide encoding an antibody provided herein, one skilled in the art can determine an appropriate nucleotide sequence encoding the polypeptide, and one that has been codon-optimized, if desired (see, e.g., Mauro and Chappell. Trends Mol Med. 20(11):604-613, 2014).

[0214] According to some aspects of the present disclosure, an isolated nucleic acid is provided comprising a nucleotide sequence encoding a heavy chain variable region polypeptide or a light chain variable region polypeptide of the present disclosure. A heavy chain variable polypeptide or a light chain variable polypeptide of the present disclosure refers to an individual polypeptide chain comprising amino acids that form part of an antigen-binding site. In some cases, the polypeptide also comprises other domains, such as a constant domain, a hinge region, and an Fc region, e.g., one that comprises one or more Fc receptor binding sites.

[0215] According to some aspects of the present disclosure, there is provided an isolated nucleic acid comprising one or more nucleotide sequences encoding a polypeptide capable of forming an antibody or antigen-binding fragment of the present disclosure. In certain embodiments, the polypeptide may comprise a constant domain, a hinge region, and other domains of an Fc region (such as one comprising one or more Fc receptor binding sites).

[0216] In one case, the nucleic acid molecule encodes only the polypeptide sequence comprising the VL domain of the antibody or fragment thereof. In some cases, the nucleic acid molecule encodes only the polypeptide sequence comprising the VH domain of the antibody or fragment thereof. In other cases, the nucleic acid molecule encodes both the VH domain-containing polypeptide sequence and the VL domain-containing polypeptide sequence that can form an antibody or antibody fragment thereof of the disclosure.

[0217] Nucleic acid molecules encoding an antibody or antigen-binding fragment thereof of the present disclosure can be, or can be part of, a vector (e.g., a plasmid, cosmid, or viral vector, or an artificial chromosome), which can include other functional regions (elements), such as one or more promoters, one or more origins or replication, one or more selectable markers, and one or more other elements typically found in expression vectors. The cloning and expression of nucleic acids encoding proteins, including antibodies, is well established and well within the skill of one of ordinary skill in the art.

[0218] vector According to some aspects of the present disclosure, there is provided a vector comprising a nucleic acid according to some embodiments of the present disclosure. In certain embodiments, the vector is a plasmid vector, a cosmid vector, a viral vector, or an artificial chromosome.

[0219] Nucleic acids of the present disclosure, including vector nucleic acids comprising a nucleotide sequence encoding a polypeptide capable of forming an antibody or antigen-binding fragment thereof of the present disclosure, can be in purified / isolated form.

[0220] Isolated / purified nucleic acids encoding antibodies or antigen-binding fragments thereof of the present disclosure, when such preparation is by recombinant DNA techniques practiced in vitro or in vivo, are free or substantially free of materials with which they are naturally associated, e.g., other proteins or nucleic acids with which they are found in their natural environment or the environment in which they are prepared (e.g., cell culture).

[0221] In some embodiments, the nucleic acids of the disclosure are more than 80% pure, such as more than 90%, more than 95%, more than 97%, and more than 99% pure.

[0222] Thus, according to some aspects of the present disclosure, there is provided a vector comprising a nucleic acid or nucleotide sequence encoding a heavy chain variable polypeptide or a light chain variable polypeptide of the present disclosure. In certain embodiments, the vector comprises nucleic acid encoding both a heavy chain variable region and a light chain variable region. In some embodiments, the polypeptide comprises a constant domain, a hinge region, and other domains, such as an Fc region, e.g., one or more Fc receptor binding sites.

[0223] In some embodiments, the nucleic acids and / or vectors of the present disclosure are introduced into host cells. For eukaryotic cells, suitable techniques include, for example, calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia, or, in the case of insect cells, baculovirus. In one aspect, introducing nucleic acids into host cells, particularly eukaryotic cells, uses a virus- or plasmid-based system. In some cases, the plasmid system is maintained episomally. In other cases, the plasmid system is integrated into the host cell or into an artificial chromosome. In certain embodiments, integration is by random integration of one or more copies at a single or multiple loci. In some embodiments, integration is by targeted integration of one or more copies at a single or multiple loci. For bacterial cells, suitable techniques include, for example, calcium chloride transformation, electroporation, and transfection using bacteriophage.

[0224] In one embodiment, the nucleic acid of the present disclosure is integrated into the genome (e.g., chromosome) of the host cell. In certain embodiments, integration is facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

[0225] host cell A further aspect of the present disclosure provides a host cell containing a nucleic acid disclosed herein. In some embodiments, such a host cell is in vitro. In some embodiments, such a host cell is in culture.

[0226] In some cases, the host cell is from any species, such as bacteria or yeast, hi other cases, the host cell is a mammalian cell, such as a human cell or a rodent cell, e.g., a HEK293T cell or a CHO-K1 cell.

[0227] Thus, according to some aspects of the present disclosure, there is provided a host cell comprising a nucleic acid sequence or vector according to some embodiments of the present disclosure.

[0228] In some cases, the host cells are treated to cause or allow expression of a protein of the disclosure from the nucleic acid, for example, by culturing the host cells under conditions for expression of the encoding nucleic acid. In some embodiments, purification of the expression product is achieved by methods known to those of skill in the art.

[0229] In some embodiments, a nucleic acid of the present disclosure, including a vector nucleic acid comprising a nucleotide sequence encoding a polypeptide for an antibody or antigen-binding fragment thereof of the present disclosure, is present in an isolated host cell. In some cases, the host cell is part of a clonal population of host cells. As used herein, reference to a host cell also encompasses a clonal population of cells. A clonal population is one that has been propagated from a single parent host cell. In some cases, the host cell is derived from any suitable organism. In some cases, the host cell is, for example, a bacterial, fungal, or mammalian cell.

[0230] In some embodiments, the host cell facilitates amplification of the vector nucleic acid (e.g., using a plasmid). In certain embodiments, the host cell serves as a biological factory for expressing the polypeptides of the disclosure that form the PD-1 antibodies or fragments thereof described herein. In one example, a suitable host for amplifying the vector nucleic acid is a bacterial or fungal cell, such as an Escherichia coli cell or a Saccharomyces cerevisiae cell. In other cases, a suitable host for expressing the proteins of the disclosure (i.e., the polypeptides that form the PD-1 antibodies or fragments thereof of the disclosure) is a mammalian cell, such as an HEK293T or CHO-K1 cell. In certain embodiments, the host cell is a mammalian cell, such as an HEK293T or CHO-K1 cell.

[0231] A variety of host-expression vector systems are suitable for expressing the PD-1 antibodies or fragments thereof described herein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system is selected to ensure the correct modification and processing of the proteins of the disclosure. In some embodiments, eukaryotic host cells are used that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, HEK, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0, CRL7O3O, and HsS78Bst cells.

[0232] Antibody production The PD-1 antibodies or fragments thereof disclosed herein can be produced as recombinant antibodies by cloning DNA encoding the antibody or peptide of interest from hybridomas or B-cells, or any type of antibody and / or antibody fragment library, inserting the clone into an appropriate vector, and transfecting the vector into host cells (e.g., P. J. Delves, Antibody Production: Essential Techniques, 1997; WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; Vandamme, A. M. et al., Eur. J. Biochem. 192:767-775 (1990)). Accordingly, in one aspect, provided herein is an isolated polynucleotide encoding an antibody or fragment thereof of the present disclosure.

[0233] Nucleotide sequences corresponding to various regions of the light or heavy chains of existing antibodies can be readily obtained and sequenced using conventional techniques, including, but not limited to, hybridization, PCR, and DNA sequencing. Hybridoma cells producing monoclonal antibodies serve as one source of antibody nucleotide sequences. A vast number of hybridoma cells producing a range of monoclonal antibodies are available from public or private repositories. The largest depository is the American Type Culture Collection, which provides a diverse collection of well-characterized hybridoma cell lines. Alternatively, antibody nucleotides can be obtained from immunized or non-immunized rodents or humans, as well as from organs such as spleen and peripheral blood lymphocytes. Specific techniques applicable for extracting and synthesizing antibody nucleotides are described in Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837, Larrick et al. (1989) Biochem. Biophys. Res. Commun. 160:1250-1255; Sastry et al. (1989) Proc. Natl. Acad. Sci., USA 86:5728-5732; and U.S. Patent No. 5,969,108.

[0234] The PD-1 antibody nucleotide sequence can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant regions for the homologous non-human sequences, thus preparing chimeric antibodies that retain the binding specificity of the original antibody.

[0235] Furthermore, polynucleotides encoding the heavy and / or light chains of PD-1 antibodies or functional fragments thereof can be subjected to codon optimization to achieve optimized expression of the subject antibodies or functional fragments thereof in desired host cells. For example, one method of codon optimization involves replacing naturally occurring codons with the most frequently occurring codons from a reference set of genes, designed to increase the rate of codon translation for each amino acid. Further exemplary methods for generating codon-optimized polynucleotides for expressing desired proteins, which can be applied to the heavy and / or light chains of PD-1 antibodies or functional fragments thereof, are described in Kanaya et al., Gene, 238:143-155 (1999), Wang et al., Mol. Biol. Evol., 18(5):792-800 (2001), U.S. Patent No. 5,795,737, U.S. Patent Publication No. 2008 / 0076161, and WO 2008 / 000632.

[0236] The PD-1 antibody polynucleotides of the present disclosure include those that encode functional equivalents of the exemplified polypeptides and fragments thereof. Functional equivalents may be polypeptides with conservative amino acid substitutions, analogs, including fusions, and variants.

[0237] Due to the degeneracy of the genetic code, there may be considerable variation in the nucleotides of the L and H sequences and heterodimerization sequences suitable for constructing the polynucleotides and vectors of this disclosure. These variations are encompassed by this disclosure.

[0238] If desired, the recombinant polynucleotide can contain heterologous sequences that facilitate detection of expression and purification of the gene product. Examples of such sequences include those encoding reporter proteins such as β-galactosidase, β-lactamase, chloramphenicol acetyltransferase (CAT), luciferase, green fluorescent protein (GFP), and their derivatives. Other heterologous sequences that facilitate purification can encode epitopes such as Myc, HA (derived from influenza virus hemagglutinin), His-6, FLAG, or the Fc portion of immunoglobulin, glutathione S-transferase (GST), and maltose-binding protein (MBP).

[0239] Polynucleotides can be conjugated to a variety of chemically functional moieties, as described above. Commonly used moieties include labels capable of producing a detectable signal, signal peptides, agents that enhance or reduce immunological reactivity, agents that facilitate coupling to solid supports, vaccine carriers, biological response modifiers, paramagnetic labels, and drugs. Moieties can be covalently attached to polynucleotides recombinantly or by other means known in the art.

[0240] The polynucleotide can include additional sequences, such as additional coding sequences within the same transcription unit, control elements such as promoters, ribosome binding sites and polyadenylation sites, additional transcription units under the control of the same promoter or different promoters, sequences allowing for cloning, expression and transformation of host cells, and any such constructs as may be desired according to any of the various embodiments described herein.

[0241] Polynucleotides can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Using the sequence data provided herein, one skilled in the art can obtain the desired polynucleotide by using a DNA synthesizer or by ordering from a commercial service.

[0242] A polynucleotide containing a desired sequence can be inserted into an appropriate vector, which can then be introduced into an appropriate host cell for replication, amplification, and expression. Thus, in one aspect, various vectors containing one or more of the polynucleotides of the present disclosure are provided herein. Also provided is a selectable library of expression vectors, each containing at least one vector encoding a target antibody.

[0243] In some aspects, provided herein are polynucleotide sequences encoding at least a portion of a heavy or light chain of an antibody or fragment thereof disclosed herein, hi some aspects, provided herein are vectors comprising a polynucleotide sequence disclosed herein.

[0244] The vectors of the present disclosure are generally classified as cloning vectors and expression vectors. Cloning vectors are useful for obtaining replicate copies of the polynucleotides they contain or as a means of storing polynucleotides in a depository for future retrieval. Expression vectors (and host cells containing these expression vectors) can be used to obtain polypeptides produced from the polynucleotides they contain. Suitable cloning and expression vectors include any known in the art, such as those for use in bacterial, mammalian, yeast, insect, and phage display expression systems.

[0245] Suitable cloning vectors can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. While the cloning vector selected can vary depending on the host cell intended for use, useful cloning vectors generally possess the ability to autonomously replicate, have a single target for a particular restriction endonuclease, or may carry a marker gene. Suitable examples include plasmids and bacterial viruses, e.g., pBR322, pMB9, ColE1, pCR1, RP4, pUC18, mp18, mp19, phage DNA (including filamentous and non-filamentous phage DNA), and shuttle vectors, e.g., pSA3 and pAT28. These and other cloning vectors are available from commercial suppliers such as Clontech, BioRad, Stratagene, and Invitrogen.

[0246] Expression vectors containing these nucleic acids are useful for creating host-vector systems for producing proteins and polypeptides. Typically, these expression vectors are replicable in the host organism either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include plasmids, viral vectors including phagemids, adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the like. Numerous expression vectors suitable for expression in eukaryotic cells, including yeast, avian, and mammalian cells, are available. One example of an expression vector is pcDNA3 (Invitrogen, San Diego, CA), in which transcription is driven by the cytomegalovirus (CMV) early promoter / enhancer. Two types of expression vectors that are particularly useful for expressing the subject antibodies described herein are phage display vectors and bacterial display vectors.

[0247] The vectors of the present disclosure can include transcriptional or translational control sequences necessary for expression of the encoded antibody. Suitable transcriptional or translational control sequences include, but are not limited to, an origin of replication, a promoter, an enhancer, a repressor binding region, a transcription initiation site, a ribosome binding site, a translation initiation site, and a termination site for transcription and translation.

[0248] The expression vector can be introduced into host cells, and the transfected cells can then be cultured to produce the antibody of interest or a functional fragment thereof. Thus, in one aspect, a host cell is provided herein containing a polynucleotide encoding the antibody of interest or a functional fragment thereof operably linked to a heterologous promoter. The host cell can be co-transfected with two expression vectors: a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. The two vectors can contain identical selectable markers that enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used that encodes and expresses both heavy and light chain polypeptides. In such situations, the light chain can be placed before the heavy chain to avoid excess non-toxic heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199).

[0249] A variety of host-expression vector systems can be utilized to express the subject antibodies or functional fragments thereof (e.g., 5,807,715). Such host-expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the subject antibody molecule in situ. These include bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequence; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequence; These include, but are not limited to, insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; those transformed with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) with recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter); and microorganisms such as (vaccinia virus 7.5K promoter). For example, mammalian cells such as Chinese hamster ovary cells (CHO) are effective expression systems for antibodies in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio / Technology 8:2). In some embodiments, the antibody or fragment thereof is produced in CHO cells.

[0250] In bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use of the expressed antibody molecule. For example, when producing large quantities of such antibodies or fragments thereof for the generation of pharmaceutical compositions of the antibody molecule, vectors directing the expression of high levels of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Uther et al., 1983, EMBO 12:1791), in which the antibody coding sequence can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0251] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. This virus is grown in Spodoptera frugiperda cells. The antibody or functional fragment coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0252] Numerous viral-based expression systems are available for mammalian host cells. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation signals can also be used to ensure efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0253] For plant cells, various vector delivery techniques are available in the art. Host cells can be in the form of whole plants, isolated cells, or protoplasts. Exemplary procedures for introducing vectors into plant cells include Agrobacterium-mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs, and injection into immature embryos. As will be apparent to those skilled in the art, each of these methods has distinct advantages and disadvantages. Therefore, one particular method for introducing a vector into a particular plant species may not necessarily be the most effective for another plant species.

[0254] Additionally, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the antibody or functional fragment. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NSO (a murine myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, and HsS78Bst cells.

[0255] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express antibodies or functional fragments thereof can be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibody molecules.

[0256] These include, but are not limited to, systems that use herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (owy et al., 1980, Cell 22:8-17) genes in tk-, hgprt-, or aprt- cells, respectively. Additionally, antimetabolite resistance can be used as the basis of selection for the following genes: dhf, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA. 77(6):3567-70; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); glutamine synthetase (GS), which is involved in the biosynthesis of glutamine using glutamate and ammonia (Bebbington et al., 1992, Biotechnology 10:169); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); and neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy). 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215; and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Recombinant DNA technology methods can be applied to select the desired recombinant clones, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated herein by reference in their entireties. Expression levels of antibody molecules can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). If the marker in the vector system expressing an antibody or functional fragment thereof is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene, and since the amplified region is associated with the antibody gene, antibody production will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[0257] Once an antibody molecule is produced by recombinant expression, it can be purified by any suitable method for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen following protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Furthermore, the subject antibodies or functional fragments thereof can be fused to heterologous polypeptide sequences provided herein or otherwise known in the art to facilitate purification. For example, the subject antibodies or functional fragments thereof can be purified by recombinantly adding a poly-histidine tag (His tag), a FLAG tag, a hemagglutinin tag (HA tag), or a myc tag, which are commercially available, and utilizing appropriate purification methods.

[0258] Treatment method In another aspect, provided herein are methods of using an antibody or functional fragment thereof disclosed herein to suppress immune cells in vitro, ex vivo, or in vivo. In some cases, the immune cell is a T cell, a B cell, a macrophage, or any other immune cell. In some cases, the immune cell is an effector T cell. In some cases, the immune cell is an antigen-specific T cell. In some cases, the methods disclosed herein are applicable to treating a subject in need thereof. In some cases, the method includes administering an antibody or fragment thereof disclosed herein to a subject in need thereof. In some cases, the method includes suppressing immune cells in vitro and transferring the immune cells into a subject in need thereof.

[0259] In another embodiment, the antibodies of the invention can be used as targeting agents for delivery of another therapeutic or cytotoxic agent (e.g., a toxin) to cells expressing PD-1. The method includes administering an anti-PD-1 antibody coupled to a therapeutic or cytotoxic agent or under conditions that allow binding of the antibody to PD-1 expressed on the cell surface.

[0260] In another aspect, provided herein are methods of treating a disease or condition in a subject in need thereof using the antibodies or functional fragments thereof disclosed herein. In some cases, the disease or condition to which the subject methods are applicable is associated with PD-1 or PD-L1 signaling. In some cases, the disease or condition is an inflammatory disorder, an autoimmune disorder, and / or is associated with an excessive or unwanted immune response. In some embodiments, the disclosure provides a method of treating an inflammatory disorder in a mammal, e.g., a human in need thereof, comprising administering to the mammal a therapeutically effective amount of an antibody of the disclosure. In some cases, the inflammatory disorder is multiple sclerosis. In other cases, the inflammatory disorder is an autoimmune disease. In some cases, the disease or condition is adult-onset Still's disease; alcoholic hepatitis, alcoholic steatohepatitis, alcoholic liver disease, asthma including allergen-induced asthma, bullous pemphigoid (BP) asthma, non-allergen-induced asthma, allergies and allergic conditions, such as allergic bronchopulmonary aspergillosis, allergic conjunctivitis, allergic encephalomyelitis and allergic neuritis, food allergies, allograft rejection, alcoholic steatohepatitis (ASH), ANCA vasculitis, anti-glomerular basement membrane disease (anti-GBM), antiphospholipid syndrome, aphthous stomatitis, appendicitis, arthritis, autoimmune diseases. Diseases, atrophic thyroiditis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune polyendocrine deficiency, autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), autoimmune hepatitis, pernicious anemia (Addison's disease) and autoimmune thyroid disorders, autoinflammatory diseases, autosomal dominant polycystic kidney disease (ADPKD), ankylosing spondylitis (AS), acute respiratory distress syndrome (ARDS), Behcet's disease or syndrome, bee sting-induced inflammation, Blau syndrome, bursitis, Barrett's esophagus, bleomycin-induced pulmonary fibrosis, obliterative bronchiolitis;Cardiac hypertrophy, gluten-sensitive enteropathy (celiac disease), chemical irritant-induced inflammation, chorioretinitis, chronic atypical neutrophilic dermatosis with lipodystrophy and hypertension (CANDLE) syndrome, chronic obstructive pulmonary disease (COPD), chronic pancreatitis, chronic prostatitis, chronic recurrent multifocal osteomyelitis, cicatricial alopecia, colitis, complex regional pain syndrome, chronic intrahepatic or extrahepatic cholestatic disease, conjunctivitis, connective tissue disease, connective tissue disease-associated interstitial lung disease (CTD-ILD), corneal ulcer, cryopyrin-associated periodic syndrome, cutaneous lupus erythematosus Death (CLE), cystic fibrosis, interleukin-1 receptor antagonist (DIRA) deficiency, IL36R antagonist (DITRA) deficiency, dermatitis, diabetic kidney disease (DKD) (diabetic nephropathy), diverticulitis, discoid lupus erythematosus, drug-induced delayed allergic dermatitis, encephalitis, eosinophilic gastrointestinal disorders (EGID), such as eosinophilic esophagitis (EoE), eosinophilic gastroenteritis, eosinophilic colitis; familial cold urticaria, familial Mediterranean fever, fistulous Crohn's disease, giant cell arteritis, glomerulonephritis, pain Gout, gouty arthritis, graft-versus-host disease (GVHD), granulomatous hepatitis, Guillain-Barré syndrome (GBS), Graves' disease, Hashimoto's thyroiditis; Henoch-Schönlein purpura, hidradenitis suppurativa (HS), hyaline membrane disease, hyperinflammatory response, hypereosinophilic syndrome (HES), hyperimmunoglobulinemia D with recurrent fever (HIDS), hypersensitivity pneumonitis (HP), immunoglobulin A (IgA) nephropathy, IgG4-related disease, immune complex nephritis, immune thrombocytopenic purpura (ITP), inflammation, CNS inflammation, inflammatory bowel disease (IBD), inflammatory diseases of the airways (upper or lower), such as inflammatory lung disease, bronchitis, sinusitis, inflammatory-ischemic events such as stroke or cardiac arrest, inflammatory liver disease, inflammatory myopathy, inflammatory neuropathy, inflammatory pain, insect sting-induced inflammation, interstitial cystitis, iritis, irritant inflammation, juvenile arthritis, juvenile rheumatoid arthritis, keratitis, kidney transplant rejection, kidney disease, renal fibrosis, renal failure, leukocyte adhesion deficiency, Löffler's syndrome, lupus, lupus nephritis (LN), liver fibrosis, fatty liver; liver ischemia; lipid and lipoprotein disorders;Mast cell activation syndrome, mastocytosis, meningitis, microscopic colitis, mixed connective tissue disease, morphea or morphea variant, Muckle-Wells syndrome (urticaria deafness amyloidosis), mucositis, myelitis, myocarditis, myositis, necrotizing enterocolitis, neonatal-onset multisystem inflammatory disease (NOMID), nasal polyps, neovascular glaucoma, neuritis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), axial spondyloarthritis not meeting radiographic criteria (nr-AxSpA) , non-cystic fibrosis bronchiectasis (non-CFB), obstructive or chronic inflammatory disorders of the liver; ocular allergies, optic neuritis, organ transplant rejection, osteoarthritis (OA), otitis, pancreatitis, pancolitis, pelvic inflammatory disease, pemphigus vulgaris (PV), bullous pemphigoid (BP), pericarditis, periodontitis, PFAPA (periodic fever, aphthous stomatitis, pharyngitis, adenitis), plant irritant-induced inflammation, Pneumocystis infection, pneumonia, pneumonitis, poison ivy / urushiol oil-induced inflammation, polyarteritis nodosa, polychondritis, polyarteritis nodosa Proctitis, rectal scleritis, polymyalgia rheumatica (PCKD), polymyalgia rheumatica, polymyositis, pouchitis, proctitis, rectal scleritis, psoriatic arthritis (PsA), pulmonary arterial hypertension (PAH), pulmonary fibrosis, suppurative aseptic arthritis, pruritus, reperfusion injury and transplant rejection, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), Raynaud's disease, Reiter's disease, reactive arthritis, renal transplant rejection, reperfusion injury, rheumatic carditis, rheumatic diseases, rheumatic fever, rheumatoid arthritis (RA), rhinitis, psoriatic rhinitis, sarcoidosis idiopathies, Schnitzler's syndrome, scleritis, sclerosis, e.g. systemic sclerosis (SSc), seborrhea, sepsis, septic shock, Sjogren's syndrome, inflammatory skin diseases or conditions, e.g. acne, alopecia areata, atopic dermatitis, rosacea, eczema, dermatitis, dermatitis endotoxemia, dermatomyositis, stasis dermatitis, Stevens-Johnson syndrome (SJS), skin irritation, skin rash, skin sensitization (contact dermatitis or allergic contact dermatitis), scleroderma, psoriasis, plaque psoriasis, psoriatic arthritis;spinal stenosis, spondyloarthropathy, synovial inflammation, systemic inflammatory response syndrome (SIRS), systemic lupus erythematosus (SLE), systemic mast cell disease (SMCD), systemic vasculitis, systemic-onset juvenile idiopathic arthritis, temporal arteritis, tendinitis, tenosynovitis, thyroiditis, transplant rejection, tubulointerstitial nephritis, renal tubular dysfunction, Takayasu's arteritis, toxic epidermal necrolysis, urticaria, uterine fibroids, uveitis, retinal uveitis, vasculitis, vasculitis (NHLBI), vitiligo, or Wegener's granulomatosis. In some cases, the disease or condition is acne, acid-induced lung injury, Addison's disease, adrenal hyperplasia, adrenal insufficiency, age-related macular degeneration, aging, alcoholic liver disease, Alzheimer's disease, angina, angiofibroma, hidradenitis ectodermal dysplasia, ascites, aspergillosis, atherosclerosis, atherosclerotic plaque, amyloidosis, amyotrophic lateral sclerosis (ALS), angioedema, acute myocardial infarction; antigen-antibody complex-mediated disease, alpha 1-antitrypsin deficiency; back pain, anthrax infection, Bell's palsy, beryllium disease, bone pain, burns, bullous pemphigoid, cancer, carpal tunnel syndrome, Castleman's disease, catabolic disorders, cataracts, cerebral aneurysms, complications of organ transplants, corneal graft neovascularization, cryptococcal polyposis, glaucoma ... ulcerative colitis, non-malignant hyperproliferative disorders; malignant hyperproliferative disorders; hepatocellular carcinoma; colon adenoma; polyposis; colon adenocarcinoma; breast cancer; pancreatic adenocarcinoma; chronic heart failure, chronic lung disease of prematurity, cardiometabolic syndrome, cardiovascular disease, cutaneous T-cell lymphoma, diabetic macular edema, dyslipidemia; endometriosis, endotoxemia, eosinophilic GI disease (EGID), eosinophilic esophagitis (EoE), eosinophilic pneumonia, epicondylitis, epidermolysis bullosa, erythema multiforme, erythroblastopenia, familial amyloidotic polyneuropathy, fetal growth retardation, fibromyalgia, glaucoma, glioblastoma, glomerular disease, enteropathy, growth plate injury, hair loss, herpes zoster and simplex, hypoplastic and other anemias, head injury, hepatitis A, B, C, D and E, herpesHeadache, hearing loss, heart disease, hemangiomas, hemophilic joints, hereditary periodic fever syndromes, hereditary disorders of connective tissue, Hodgkin's disease, Huntington's disease, hyperammonemia, hypercalcemia, hypercholesterolemia, hemolytic anemia, hepatitis, hip replacement, hypertonic bone formation, hypersensitivity pneumonitis, hereditary fructose intolerance, hypertension, hyperuricemia, idiopathic demyelinating polyneuropathy, infectious diseases including viral diseases such as AIDS (HIV infection), ichthyosis, incontinentia pigmenti (IP, Bloch-Siemens syndrome), idiopathic thrombocytopenic purpura, infectious diseases Mononucleosis, ischemia / reperfusion, insulin resistance, joint replacement, kidney damage caused by parasitic infections, leptospirosis, lichen sclerosus (LS), lichen planus, Lambert-Eaton myasthenic syndrome, Lyme disease, liver failure including acute liver failure, muscle atrophy, muscular dystrophy, Marfan syndrome (MFS), meningioma, mesothelioma, multiple organ injury syndrome, myasthenia gravis (MG), myelodysplastic syndrome, metabolic syndrome, multiple sclerosis, renal syndrome, neuropathological disorders, nuclear factor kappa B essential modulator (NEMO) deficiency syndrome, obesity, Osler-Weber syndrome, osteogenesis imperfecta, osteonecrosis, osteoporosis, congenital pachydermopathy, Paget's disease, Paget's disease of bone, Parkinson's disease, periodic fever, whooping cough, primary pulmonary hypertension, pyoderma gangrenosum, pyogenic granulomas, retrolental fibroplasia, peritoneal endometriosis, nodular pruritus, psychotic stress disorders, lung disease, pulmonary hypertension, respiratory distress syndrome, kidney disease, retinal disease, retrolental fibroplasia, renal transplant rejection, renal protection against drugs inducing Fanconi syndrome, respiratory tract disease caused by respiratory syncytial virus, rhinosinusitis; radiation Radiation-induced fibrosis, sarcoidosis, severe pain, sleep apnea, scoliosis, sickle cell anemia, sports injuries, sprains and strains, sunburn, spinal cord injury, Sézary syndrome, silica-induced disease (silicosis), subarachnoid hemorrhage, tuberculosis, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), thrombosis; traumatic brain injury, tissue transplant, complications from type 1 or type 2 diabetes, toxoplasmosis, thrombocytopenia, trachoma, vascular restenosis, ventilator-induced lung injury; Whipple's disease or 2,8-dihydroxyadenine nephropathy;

[0261] Examples of diseases or conditions that the subject antibodies can treat include acute disseminated encephalomyelitis (ADEM), Addison's disease, allergies, alopecia areata, amyotrophic lateral sclerosis, ANCA vasculitis, ankylosing spondylitis, antiphospholipid syndrome, asthma (including allergic asthma), atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, Behcet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, celiac disease, Crohn's disease, Cushing's syndrome, dermatomyositis, type 1 diabetes, eosinophilic granulomatosis with polyangiitis, graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, IgG Related diseases include, but are not limited to, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and liver cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lupus nephritis, lymphoma, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, non-radiographic axial spondyloarthritis (nr-AxSpA), neuromyelitis optica, osteoarthritis, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, systemic sclerosis, Takayasu's arteritis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease. In some cases, the disease or condition includes rheumatoid arthritis. In some cases, the disease or condition includes multiple sclerosis.

[0262] In some cases, a method of treating a disease or condition in a subject in need thereof comprises administering to the subject a therapeutically effective amount of an agonist antibody disclosed herein; or administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody or immunoconjugate disclosed herein and at least one pharmaceutically acceptable excipient, wherein the disease or condition is selected from the group consisting of infection, infection-associated endotoxic shock, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic-onset juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), systemic sclerosis, asthma, atopic dermatitis, pelvic inflammatory disease, Alzheimer's disease, Crohn's disease, ulcerative colitis, and irritable bowel disease. syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal cyst disease, psoriasis, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme arthritis, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, autoimmune disorders, pancreatitis, surgical trauma, graft-versus-host disease, transplant rejection, heart disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic / pulmonary fibrosis, periodontitis, hypochlorhydria, solid tumors (renal cell carcinoma), liver cancer, multiple myeloma, prostate cancer, bladder cancer, pancreatic cancer, neurological cancer, and B-cell malignancies (e.g., Castleman's disease, certain lymphomas, chronic lymphocytic leukemia, and multiple myeloma), lupus nephritis, and osteoarthritis. In some cases, the disease or condition includes Sjogren's syndrome. In some cases, the disease or condition comprises inflammatory bowel disease (IBD). In some cases, the disease or condition comprises systemic lupus erythematosus (SLE). In some cases, the disease or condition comprises lupus nephritis (LN). In some cases, the disease or condition comprises vasculitis, such as antineutrophil cytoplasmic antibody (ANCA)-associated (ANCA) vasculitis. In some cases, the disease or condition comprises graft-versus-host disease (GvHD). In some cases, the disease or condition comprises type 1 diabetes. In some cases, the disease or condition comprises Behcet's syndrome. In some cases, the disease or condition comprises sepsis. In some cases, the disease or condition comprises osteoarthritis (OA). In some cases, the disease or condition comprises systemic sclerosis (SSc).In some cases, the disease or condition comprises dermatomyositis. In some cases, the disease or condition comprises psoriatic arthritis (PsA). In some cases, the disease or condition comprises IgG4-related disease. In some cases, the disease or condition comprises non-radiographic axial spondyloarthritis (nr-AxSpA). In some cases, the disease or condition comprises polymyositis. In some cases, the disease or condition comprises Takayasu's arteritis.

[0263] In some embodiments, the present disclosure provides a method of treating cancer in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of an antibody of the present disclosure. In some cases, the cancer is hepatocellular carcinoma. In other cases, the cancer is acute myeloid leukemia, thymus, brain, lung, squamous cell, skin, eye, retinoblastoma, intraocular melanoma, oral and oropharynx, bladder, stomach, pancreas, bladder, breast, cervix, head and neck, kidney, liver, ovarian, prostate, colorectal, esophagus, testis, gynecological, thyroid, CNS, PNS, AIDS-related (e.g., lymphoma and Kaposi's sarcoma), or virus-induced cancer.

[0264] In some embodiments, the subject to be treated is a mammal, such as a human. In some embodiments, the subject to be treated is a human. In other cases, the mammal is a mouse, rat, cat, dog, rabbit, pig, sheep, horse, cow, goat, gerbil, hamster, guinea pig, monkey, or any other mammal. Many such mammals may be subjects known in the art as preclinical models of particular diseases or disorders, including inflammatory diseases, solid tumors, and / or other cancers (e.g., Talmadge et al., 2007 Am. J. Pathol. 170:793; Kerbel, 2003 Canc. Biol. Therap. 2(4 Suppl 1):S134; Man et al., 2007 Canc. Met. Rev. 26:737; Cespedes et al., 2006 Clin. TransL Oncol. 8:318).

[0265] In another aspect, the present disclosure provides methods of using a PD-1 antibody of the present disclosure to treat a disease or condition in a mammal in conjunction with a second agent. The second agent can be administered together with, before, or after the antibody. In some embodiments, the second agent is an agent that acts to alleviate the symptoms of an inflammatory condition described herein. Anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. NSAIDs include salicylates, such as acetylsalicylic acid; diflunisal, salicylic acid, and salsalate; propionic acid derivatives, such as ibuprofen; naproxen; dexibuprofen, dexketoprofen, flurbiprofen, oxaprozin, fenoprofen, loxoprofen, and ketoprofen; acetic acid derivatives, such as indomethacin, diclofenac, tolmetin, aceclofenac, sulindac, nabumetone, etodolac, and ketorolac; piroxicam, lorhexidine glucan, and lorhexidine glucan. Examples of suitable anti-inflammatory drugs include, but are not limited to, enolic acid derivatives such as cicam, meloxicam, isoxicam, tenoxicam, phenylbutazone, and droxicam; anthranilic acid derivatives such as mefenamic acid, flufenamic acid, meclofenamic acid, and tolfenamic acid; selective COX-2 inhibitors such as celecoxib, lumiracoxib, rofecoxib, etoricoxib, valdecoxib, firocoxib, and parecoxib; sulfonanilides such as nimesulide; and others such as clonixin and licofelone. Corticosteroids include, but are not limited to, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone.

[0266] In some embodiments, the second agent is an immunosuppressant. Immunosuppressants that can be used in combination with a subject antibody include, but are not limited to, hydroxychloroquine, sulfasalazine, leflunomide, etanercept, infliximab, adalimumab, D-penicillamine, oral gold compounds, injectable gold compounds (intramuscular injection), minocycline, gold sodium thiomalate, auranofin, D-penicillamine, lobenzarit, bucillamine, actarit, cyclophosphamide, azathioprine, methotrexate, mizoribine, cyclosporine, and tacrolimus.

[0267] In some embodiments, the second agent is useful for the treatment and / or prevention of rheumatoid arthritis. Non-limiting examples of such agents include disease-modifying antirheumatic drugs (DMARDS), such as hydroxychloroquine, sulfasalazine, methotrexate, and leflunomide; TNF inhibitors (e.g., etanercept, adalimumab, infliximab, golimumab, certolizumab pegol), T cell costimulation inhibitors (e.g., abatacept), IL-6 receptor inhibitors (e.g., tocilizumab, sarilumab), anti-CD20 antibodies (e.g., rituximab); and JAK inhibitors (e.g., tofacitinib, baricitinib, upadacitinib); NSAIDs, such as ibuprofen, naproxen, and diclofenac; These include OX-2 inhibitors, such as celecoxib and etoricoxib; steroids and corticosteroids, such as prednisolone and cortisone; and biologic agents known for the treatment and / or prevention of such conditions, such as etanercept (e.g., ENBREL), infliximab (e.g., REMICADE), adalimumab (e.g., HUMIRA), anakinra (e.g., KINARET), abatacept (ORENCIA), rituximab (e.g., RITUXAN), certolizumab (e.g., CIMZIA), golimumab (e.g., SIMPONI), and tocilizumab (e.g., Actemra). In some embodiments, a compound of the present disclosure is administered with two additional therapeutic agents useful for the treatment and / or prevention of a rheumatic condition. In some embodiments, agents useful for the treatment and / or prevention of rheumatic conditions include a compound of the present disclosure and two additional therapeutic agents, for example, methotrexate plus leflunomide, methotrexate plus sulfasalazine, methotrexate plus cyclosporine, methotrexate plus hydroxychloroquine, and the triple therapy treatments hydroxychloroquine plus sulfasalazine plus methotrexate, hydroxychloroquine plus sulfasalazine plus leflunomide.

[0268] In some embodiments, the second agent is useful for the treatment and / or prevention of systemic lupus erythematosus (SLE) or lupus nephritis (LN). Non-limiting examples of such agents include immunosuppressants that inhibit the activity of the immune system and agents approved for the treatment of SLE, such as hydroxychloroquine, steroids and corticosteroids (e.g., prednisone, methylprednisolone), belimumab, azathioprine, methotrexate, cyclophosphamide, mycophenolate and mycophenolate mofetil, cyclosporine, leflunomide, voclosporin, abatacept, anifrolumab, rituximab, NSAIDs, such as naproxen sodium and ibuprofen, antimalarials, such as hydroxychloroquine, calcineurin inhibitors, and tacrolimus.

[0269] In some embodiments, the second agent is useful in the treatment of LN, such as prednisone plus a mycophenolic acid analog, prednisone plus mycophenolate sodium, prednisone plus cyclophosphamide, prednisone plus tacrolimus, prednisone plus voclosporin, prednisone plus belimumab plus a mycophenolic acid analog, prednisone plus belimumab plus cyclophosphamide, or prednisone plus rituximab.

[0270] In some embodiments, the second agent is useful in the treatment of LN, such as prednisone plus a mycophenolic acid analog, prednisone plus mycophenolate sodium, prednisone plus azathioprine, prednisone plus tacrolimus, prednisone plus cyclosporine, or prednisone plus mizoribine.

[0271] In some embodiments, the second agent is useful for the treatment and / or prevention of osteoarthritis (OA). Non-limiting examples of such agents include nonsteroidal anti-inflammatory drugs (NSAIDs), topical capsaicin, intra-articular glucocorticoid injections, acetaminophen, duloxetine, tramadol, and injectable corticosteroids such as methylprednisolone acetate, triamcinolone acetate, betamethasone acetate and betamethasone phosphate sodium acetate, triamcinolone hexacetonide, and dexamethasone.

[0272] In some embodiments, the second agent is useful for the treatment and / or prevention of a gastrointestinal condition, such as ulcerative colitis (UC) or Crohn's disease (CD). Non-limiting examples of such agents include corticosteroids such as infliximab, adalimumab, golimumab, vedolizumab, tofacitinib, ustekinumab, natalizumab, mesalamine, diazo-conjugated 5-ASA, sulfasalazine, balsalazide, olsalazine, budesonide, hydrocortisone, methylprednisolone, and prednisone; immunosuppressants or immunomodulators such as azathioprine and 6-mercaptopurine, cyclosporine, and methotrexate.

[0273] In some embodiments, the second agent is useful for the treatment and / or prevention of a pulmonary condition, such as idiopathic pulmonary fibrosis (IPF) or interstitial lung disease (ILD). Non-limiting examples of such agents include corticosteroids, such as nitendanib, pirfenidone, and prednisone; other rheumatic drugs, including mycophenolate (e.g., CellCept®), azathioprine (e.g., Imuran®), leflunomide (e.g., ARAVA®), rituximab (e.g., RITUXAN®), cyclophosphamide (e.g., CYTOXAN®), and tacrolimus (e.g., PROGRAF®); and medications that reduce stomach acid, such as H-2 receptor antagonists or proton pump inhibitors, such as lansoprazole (e.g., PREVACID® 24HR), omeprazole (e.g., Prilosec OTC), and pantoprazole (e.g., PROTONIX®).

[0274] In some embodiments, the second agent is useful for the treatment and / or prevention of a hepatic or renal condition, such as NAFLD, NASH, DKD, or CKD. Non-limiting examples of such agents include metformin, sodium-glucose cotransporter-2 inhibitors (SGLT2i), medications for glycemic control, DPP-4 inhibitors, insulin, sulfonylureas, TZDs (thiazolidinedione), α-glucosidase inhibitors, SGLT2 inhibitors (e.g., empagliflozin, canagliflozin, dapagliflozin), glucagon-like peptide-1 receptor agonist (GLP- 1 Medications used to treat hypertension, such as antihypertensive drugs (RAs) (e.g., lixisenatide, liraglutide, semaglutide, exenatide, albiglutide, dulaglutide), DPP-4 inhibitors (e.g., saxagliptin, alogliptin, sitagliptin, linagliptin), angiotensin-converting enzyme (ACE) inhibitors, and angiotensin 2 receptor blockers (ARBs) In addition to the above drugs, drugs to support weight loss or drugs for controlling blood sugar, cholesterol-lowering drugs (e.g., statins), fine lenone, and drugs for the treatment of diabetes mellitus, such as α-glucosidase inhibitors (e.g., acarbose, miglitol, voglibose).

[0275] In some embodiments, the second agent is useful for the treatment and / or prevention of skin conditions such as atopic dermatitis (AD). Non-limiting examples of such agents include topical corticosteroids (TCS) (e.g., desonide, hydrocortisone, fluocinolone, triamcinolone, betamethasone dipropionate), topical calcineurin inhibitors (TCIs) (e.g., tacrolimus, pimecrolimus), topical antibacterial agents and antiseptics, cyclosporine, methotrexate, mycophenolate mofetil, interferon gamma, phosphodiesterase 4 (PDE4) inhibitors such as crisaborole, JAK inhibitors (e.g., ruxolitinib, upadatinib, abrocitinib), systemic glucocorticoids (e.g., prednisone), dupilumab, and anti-IL-13 antibodies (e.g., tralokinumab).

[0276] Yet another aspect of the invention provides the use of the disclosed antibodies to detect the presence of PD-1 in a biological sample. The amount of PD-1 detected can be correlated with the expression level of PD-1, which in turn correlates with the activation state of immune cells (e.g., activated T cells, B cells, and monocytes) in the subject.

[0277] The specific dose of an antibody disclosed herein administered to a subject for treatment can vary depending on the particular antibody selected, the dosing regimen to be followed, whether it is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system it is carried in. In some embodiments, the antibody is administered at a dose of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 In some embodiments, the dose ranges from 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg to be administered to the subject.

[0278] In some embodiments, the antibody is administered to the subject in an amount that averages more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg per day over the course of a treatment cycle. For example, the antibody is administered to the subject in an amount that averages about 6-10 mg, about 6.5-9.5 mg, about 6.5-8.5 mg, about 6.5-8 mg, or about 7-9 mg per day over the course of a treatment cycle.

[0279] In some embodiments, a single dose of antibody administered to a subject is in the range of about 0.01 mg / kg to 50 mg / kg, e.g., about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 5 mg / kg kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg, or about less thereof, is administered to a subject. In some embodiments, a single dose of the antibody is in the range of about 0.01 mg / kg to 10 mg / kg, e.g., 0.01 mg / kg to 0.1 mg / kg, 0.01 mg / kg to 1 mg / kg, 0.01 mg / kg to 0.5 mg / kg, 0.05 mg / kg to 0.1 mg / kg, 0.05 mg / kg to 0.5 mg / kg, 0.05 mg / kg to 1 mg / kg, 0.05 mg / kg to 5 mg / kg, 0.1mg / kg to 0.5mg / kg, 0.1mg / kg to 1mg / kg, 0.1mg / kg to 5mg / kg, 0.1mg / kg to 10mg / kg, 0.5mg / kg to 1mg / kg, 0.5mg / kg to 5mg / kg, 0.5mg / kg to 10mg / kg, 1mg / kg to 5mg / kg, 1mg / kg to 10mg, or 5mg / kg to 10mg / kg.The dose of antibody may be about, at least about, or at most about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 , 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000 mg or mg / kg, or any range derivable therein. A mg / kg dosage is considered to refer to mg of antibody per kg of total subject body weight. It is contemplated that when multiple doses are administered to a patient, the doses may be different amounts or they may be the same.

[0280] In some embodiments, the antibody is administered in the range of about 0.01 mg / kg to 50 mg / kg per day, e.g., about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 5 mg / kg per day. / kg, 6mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 25mg / kg, 30mg / kg, 35mg / kg, 40mg / kg, 45mg / kg, or 50mg / kg, or less, is administered to the subject. In some embodiments, the antibody is administered to a subject within the range of about 0.1 mg / kg to 400 mg / kg per week, e.g., about, less than, or greater than about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 100 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, or 400 mg / kg per week.In some embodiments, the antibody is administered at a dose in the range of about 0.4 mg / kg to 1500 mg / kg per month, e.g., about 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 100 mg / kg, 150 mg / kg, 2 ...35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 100 mg / kg, 150 mg / kg, 20 mg / kg, 35 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 100 mg / kg, 150 mg / kg, 20 mg / kg, 35 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, In some embodiments, the antibody is administered to a subject at a dose of about 0 mg / m per week, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 950 mg / kg, or 1000 mg / kg, or less or more. In some embodiments, the antibody is administered to a subject at a dose of about 0.1 mg / m per week. 2 ~200mg / m 2 , e.g., about 1 mg / m per week 2 , 5 mg / m 2 , 10 mg / m 2 ,15mg / m 2 , 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , 50 mg / m 2 , 55 mg / m 2 , 60 mg / m 2 , 65 mg / m 2 , 70 mg / m 2 , 75 mg / m 2 , 100 mg / m 2 , 125 mg / m 2 , 150 mg / m 2 , 175 mg / m 2 , or 200 mg / m 2, or less, or more than that, is administered to the subject. The target dose may be administered in a single dose. Alternatively, the target dose may be administered in about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more doses. For example, a dose of about 1 mg / kg / week may be delivered weekly over the course of a week at a dose of about 1 mg / kg every week, about 2 mg / kg administered every two weeks, or about 4 mg / kg administered every four weeks. The administration schedule may be repeated according to any regimen described herein, including any of the administration schedules described herein. In some embodiments, the antibody is administered in a dose of about 0.1 mg / m 2 ~500mg / m 2 , for example, about 1 mg / m 2 , 5 mg / m 2 , 10 mg / m 2 , 15 mg / m 2 , 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , 50 mg / m 2 , 55 mg / m 2 , 60 mg / m 2 , 65 mg / m 2 , 70 mg / m 2 , 75 mg / m 2 , 100 mg / m 2 , 130 mg / m 2 , 135 mg / m 2 , 155 mg / m 2 , 175 mg / m 2 , 200 mg / m 2 , 225 mg / m 2 , 250 mg / m 2 , 300 mg / m 2 , 350 mg / m 2 , 400 mg / m 2 , 420 mg / m 2 , 450 mg / m 2 , or 500 mg / m 2 , or less than, or more than, is administered to the subject.

[0281] Pharmaceutical Composition In another aspect, provided herein are pharmaceutical compositions comprising an anti-PD-1 antibody or functional fragment thereof disclosed herein and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers or excipients may include, but are not limited to, inert solid diluents and fillers, diluents, sterile aqueous solutions and various organic solvents, permeation enhancers, solubilizers, and adjuvants. These compositions can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in several sources that are well known and readily available to those skilled in the art. For example, see Remington's Pharmaceutical Sciences (Martin E. W., Easton Pennsylvania, Mack Publishing Company, 1999). th ed., 1995) describe formulations that may be used in connection with the present invention.

[0282] The pharmaceutical compositions disclosed herein may be in a form suitable for oral administration, for example, as tablets, capsules, pills, powders, sustained-release formulations, solutions, or suspensions; parenteral injection, for example, as sterile solutions, suspensions, or emulsions; topical administration, for example, as ointments or creams; or rectal administration, for example, as suppositories. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing an antibody, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations allow for release of molecules over a period of weeks to months or even years. In some embodiments, a subject pharmaceutical composition releases a subject antibody described herein for at least several weeks, e.g., at least 1, 2, 3, or 4 weeks. In further embodiments, a subject pharmaceutical composition releases a subject antibody described herein for several months, e.g., at least 1, 2, 3, 4, 5, or 6 months.

[0283] The pharmaceutical compositions disclosed herein may be in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical composition may further comprise an antibody or functional fragment thereof as an active ingredient and may include conventional pharmaceutical carriers or excipients. Additionally, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc.

[0284] Exemplary parenteral dosage forms include solutions or suspensions of the active polypeptide and / or PEG-modified polypeptide in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose, which may be suitably buffered, if desired, with salts such as histidine and / or phosphate salts.

[0285] Formulations suitable for administration include sterile aqueous solutions for injection which may contain, for example, antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0286] The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried (lyophilized) condition requiring only the form of a sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, and the like.

[0287] In some embodiments, the present disclosure provides injectable pharmaceutical compositions containing a subject antibody or functional fragment thereof and a pharmaceutical excipient suitable for injection. Exemplary components and amounts of agents in such compositions are as described herein.

[0288] Forms into which the compositions of the present disclosure can be incorporated for administration by injection include aqueous or oily suspensions or emulsions (with sesame oil, corn oil, cottonseed oil, or peanut oil), as well as elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles.

[0289] Aqueous solutions of physiological saline can be used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, etc. (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be used. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

[0290] Sterile injectable solutions can be prepared by incorporating the antibody or its functional fragment of the present disclosure in the desired amount in a suitable solvent containing various other ingredients listed above, followed by filtration sterilization.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other ingredients.For the preparation of sterile powder for sterile injectable solutions, a particular preferred preparation method is vacuum drying and freeze-drying technology, which obtains a powder of active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0291] In some embodiments, the present disclosure provides a pharmaceutical composition for oral administration comprising an antibody or functional fragment thereof of the present disclosure and a pharmaceutical excipient suitable for oral administration.

[0292] In some embodiments, provided herein is a solid pharmaceutical composition for oral administration containing (i) an effective amount of an antibody or functional fragment thereof of the present disclosure; optionally (ii) an effective amount of a second agent; and (iii) a pharmaceutical excipient suitable for oral administration. In some embodiments, the composition further comprises (iv) an effective amount of a third agent.

[0293] In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition suitable for oral ingestion. Pharmaceutical compositions suitable for oral administration can be provided in discrete dosage forms, such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient as a powder or granules, in a solution or suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion, or as a liquid or aerosol spray. Such dosage forms can be prepared by any of the methods of pharmacy and typically include the step of bringing the active ingredient into association with the carrier, which constitutes one or more necessary ingredients. Generally, the compositions are prepared by uniformly and intimately admixing the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired form.

[0294] Because water can accelerate the degradation of some polypeptides, the present disclosure also encompasses anhydrous pharmaceutical compositions and dosage forms comprising active ingredients. For example, water can be added (e.g., 5%) in pharmaceutical manufacturing as a means of simulating long-term storage to determine properties such as shelf life or the stability of a formulation over time. Anhydrous pharmaceutical compositions and dosage forms can be prepared using anhydrous or low-moisture containing ingredients and low-moisture or low-humidity conditions. Pharmaceutical compositions and dosage forms containing lactose can be made anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is expected. Anhydrous pharmaceutical compositions can be prepared and stored such that their anhydrous nature is maintained. Thus, anhydrous compositions can be packaged using materials known to prevent exposure to water, such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit-dose containers, blister packs, and strip packs.

[0295] The antibodies of the present disclosure can be intimately mixed and combined with pharmaceutical carriers according to conventional pharmaceutical compounding techniques. Carriers can take a wide variety of forms depending on the form of preparation desired for administration. When preparing compositions for oral dosage forms, any of the usual pharmaceutical media can be used as a carrier, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. for oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols. Alternatively, carriers such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrants can be used for oral solid preparations, in some embodiments without the use of lactose. For example, suitable carriers include powders, capsules, and tablets, including solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

[0296] Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethylcellulose, sodium carboxymethylcellulose), polyvinylpyrrolidone, methylcellulose, pregelatinized starch, hydroxypropyl methylcellulose, microcrystalline cellulose, and mixtures thereof.

[0297] Examples of fillers suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof.

[0298] Disintegrants can be used in the composition to provide tablets that disintegrate when exposed to an aqueous environment. Too much disintegrant can result in tablets that may disintegrate in the bottle. Too little can be insufficient for disintegration to occur, thereby altering the release rate and extent of the active ingredient from the dosage form. Therefore, dosage forms can be formed using a sufficient amount of disintegrant that is neither too little nor too much to adversely alter the release of the active ingredient. The amount of disintegrant used can vary based on the type of formulation and mode of administration, and is readily discernible to those skilled in the art. About 0.5 to about 15% by weight of disintegrant, or about 1 to about 5% by weight of disintegrant, may be used in the pharmaceutical composition. Disintegrants that can be used to form pharmaceutical compositions and dosage forms include, but are not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pregelatinized starch, other starches, clays, other algins, other celluloses, gums, or mixtures thereof.

[0299] Lubricants that can be used to form pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, or mixtures thereof. Additional lubricants include, for example, syloid silica gel, coagulated aerosol of synthetic silica, or mixtures thereof. Lubricants can optionally be added in an amount of less than about 1% by weight of the pharmaceutical composition.

[0300] When aqueous suspensions and / or elixirs are desired for oral administration, the active ingredient therein may be combined with diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof, along with various sweetening or flavoring agents, dyes or dyes, and, if desired, emulsifying and / or suspending agents.

[0301] Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used. Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0302] Surfactants that can be used to form pharmaceutical compositions and dosage forms include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof, i.e., a mixture of hydrophilic surfactants can be used, a mixture of lipophilic surfactants can be used, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant can be used.

[0303] Surfactants with a low HLB value are more lipophilic or hydrophobic and have a higher solubility in oil, while surfactants with a high HLB value are more hydrophilic and have a higher solubility in aqueous solution.Hydrophilic surfactants are generally considered to be compounds with an HLB value greater than about 10, and anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable.Similarly, lipophilic (i.e., hydrophobic) surfactants are compounds with an HLB value of about 10 or less.However, the HLB value of surfactants is only a rough guideline that is generally used to enable the formulation of industrial, medical, and cosmetic emulsions.

[0304] The hydrophilic surfactant can be either ionic or nonionic. Suitable ionic surfactants include alkylammonium salts, fusidate salts, fatty acid derivatives of amino acids, oligopeptides, and polypeptides, glyceride derivatives of amino acids, oligopeptides, and polypeptides, lecithin and hydrogenated lecithin, lysolecithin and hydrogenated lysolecithin, phospholipids and derivatives thereof, lysophospholipids and derivatives thereof, carnitine fatty acid ester salts, alkyl sulfate salts, fatty acid salts, docusate sodium, acyl lactylates, mono- and diacetylated tartaric acid esters of mono- and diglycerides, succinylated mono- and diglycerides, citrate esters of mono- and diglycerides, and mixtures thereof.

[0305] Within the aforementioned group, ionic surfactants include, for example, lecithin, lysolecithin, phospholipids, lysophospholipids, and derivatives thereof, carnitine fatty acid ester salts, salts of alkyl sulfates, fatty acid salts, sodium docusate, acyl lactylates; mono- and diacetylated tartaric acid esters of mono- and diglycerides, succinylated mono- and diglycerides, citrate esters of mono- and diglycerides, and mixtures thereof.

[0306] Ionic surfactants include lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylethanolamine, PVP-phosphatidylethanolamine, lactic acid esters of fatty acids, stearoyl-2-lactylate, stearoyl lactylate , succinylated monoglycerides, mono / diacetylated tartaric acid esters of mono / diglycerides, citrate esters of mono / diglycerides, cholyl sarcosine, caproate, caprylate, caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate, linolenate, stearate, lauryl sulfate, tetracecyl sulfate, docusate, ionized forms of lauroylcarnitine, palmitoylcarnitine, myristoylcarnitine, and salts and mixtures thereof.

[0307] Hydrophilic nonionic surfactants may include, but are not limited to, alkyl glucosides, alkyl maltosides, alkyl thioglucosides, lauryl macrogolglycerides, polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers, polyoxyalkylene alkylphenols such as polyethylene glycol alkylphenols, polyoxyalkylene alkylphenol fatty acid esters such as polyethylene glycol fatty acid monoesters and polyethylene glycol fatty acid diesters, polyethylene glycol glycerol fatty acid esters, polyglycerol fatty acid esters, polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters, hydrophilic interesterification products of polyols with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols, polyoxyethylene sterols, derivatives, and analogs thereof, polyoxyethylated vitamins and derivatives thereof, polyoxyethylene-polyoxypropylene block copolymers and mixtures thereof, polyethylene glycol sorbitan fatty acid esters, and hydrophilic interesterification products of polyols with at least one member of the group consisting of triglycerides, vegetable oils, and hydrogenated vegetable oils. The polyol can be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or a saccharide.

[0308] Other hydrophilic nonionic surfactants include PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32 distearate, PEG-40 stearate, PEG-1 00 Stearate, PEG-20 Dilaurate, PEG-25 Glyceryl Trioleate, PEG-32 Dioleate, PEG-20 Glyceryl Laurate, PEG-30 Glyceryl Laurate, PEG-20 Glyceryl Stearate, PEG-20 Glyceryl Oleate, PEG-30 Glyceryl Oleate, PEG-30 Glyceryl Laurate, PEG-40 Glyceryl Laurate, PEG-40 Palm Kernel Oil, PEG-50 Hydrogenated Castor Oil, PEG-40 Castor Oil, PEG -35 Castor Oil, PEG-60 Castor Oil, PEG-40 Hydrogenated Castor Oil, PEG-60 Hydrogenated Castor Oil, PEG-60 Corn Oil, PEG-6 Caprate / Caprylate Glyceride, PEG-8 Caprate / Caprylate Glyceride, Polyglyceryl-10 Laurate, PEG-30 Cholesterol, PEG-25 Phytosterol, PEG-30 Soy Sterol, PEG-20 Trioleate, PEG-40 Sorbitan Oleate, PEG-80 Sorbitan Laurate, Polysol Examples of suitable glycerin-containing polymers include, but are not limited to, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopheryl PEG-100 succinate, PEG-24 cholesterol, polyglyceryl-10 oleate, Tween® 40, Tween® 60, sucrose monostearate, sucrose monolaurate, sucrose monopalmitate, PEG 10-100 nonylphenol series, PEG 15-100 octylphenol series, and poloxamer.

[0309] Suitable lipophilic surfactants include, by way of example only, fatty alcohols, glycerol fatty acid esters, acetylated glycerol fatty acid esters, lower alcohol fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, polyethylene glycol sorbitan fatty acid esters, sterols and sterol derivatives, polyoxyethylated sterols and sterol derivatives, polyethylene glycol alkyl ethers, sugar esters, sugar ethers, lactic acid derivatives of mono- and diglycerides, the hydrophobic transesterification product of polyols with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols, oil-soluble vitamins / vitamin derivatives, and mixtures thereof.In this group, exemplary lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or the hydrophobic transesterification product of polyols with at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils, and triglycerides.

[0310] In some cases, the composition contains a solubilizer to ensure good solubilization and / or dissolution of the compound and minimize precipitation of the compound.This may be particularly advantageous for compositions for parenteral use, such as injection compositions.Solubilizers can also be added to increase the solubility of other components, such as hydrophilic drugs and / or surfactants, or to maintain the composition as a stable or uniform solution or dispersion.

[0311] Examples of suitable solubilizers include alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediol and its isomers, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycol having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG; amides and other nitrogen-containing compounds, such as 2-pyrrolidone, 2-piperidone, ε-caprolactone, methylcellulose ... Examples of solubilizing agents include, but are not limited to, methylpropionate, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide, and polyvinylpyrrolidone; esters such as ethyl propionate, tributyl citrate, acetyltriethyl citrate, acetyltributyl citrate, triethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, ε-caprolactone and its isomers, δ-valerolactone and its isomers, β-butyrolactone and its isomers; and other solubilizing agents known in the art, such as dimethylacetamide, dimethylisosorbide, N-methylpyrrolidone, monooctanoin, diethylene glycol monoethyl ether, and water.

[0312] Mixtures of solubilizers may also be used. Examples include, but are not limited to, triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcyclodextrin, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Exemplary solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol, and propylene glycol.

[0313] The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer can be limited to a biologically acceptable amount, which can be easily determined by one of ordinary skill in the art. In some situations, it may be advantageous to include an amount of solubilizer that far exceeds the biologically acceptable amount, for example, to maximize the drug concentration, with the excess solubilizer being removed using conventional techniques such as distillation or evaporation before providing the composition to a subject. Thus, if present, the solubilizer can be present in an amount of 10%, 25%, 50%, 100%, or up to about 200% by weight, based on the total weight of the drug and other excipients. If necessary, very small amounts of solubilizer, such as 5%, 2%, 1%, or less, can also be used. Typically, the solubilizer may be present in an amount of about 1% to about 100% by weight, more typically about 5% to about 25% by weight.

[0314] The composition may further comprise one or more pharmaceutically acceptable additives and excipients, including, but not limited to, anti-adherents, anti-foaming agents, buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers, tonicity agents, flavors, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

[0315] Additionally, acids or bases can be incorporated into the compositions to facilitate processing, enhance stability, or for other reasons. Examples of pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium bicarbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS), and the like. Also suitable are bases that are salts of pharmaceutically acceptable acids, such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, and uric acid. Salts of polybasic acids, such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate, can also be used. When the base is a salt, the cation can be any convenient, pharmaceutically acceptable cation, such as ammonium, an alkali metal, or an alkaline earth metal. Examples include, but are not limited to, sodium, potassium, lithium, magnesium, calcium, and ammonium.

[0316] Suitable acids are pharmaceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, etc. Examples of suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, parabromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, toluenesulfonic acid, etc.

[0317] In another aspect of the present disclosure, a kit is provided that includes a unit dose containing an antibody composition of the present disclosure and instructions for use. The kit can further include one or more unit doses containing one or more additional reagents, such as the immunosuppressant reagents described above, or one or more additional antibodies described herein (e.g., human antibodies with complementary activity that bind to an epitope in an antigen different from that of the first human antibody). The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material that is attached to, supplied with, or otherwise accompanies the kit.

[0318] Kits of the present disclosure may also include diagnostic and / or other therapeutic agents. In some cases, the kit includes an antibody of the present disclosure and a diagnostic agent that can be used in a diagnostic method for diagnosing the status or presence of a disease, condition, or disorder in a subject as described herein. Medical Use

[0319] In another aspect, provided herein is an antibody or antigen-binding fragment or immunoconjugate of the present disclosure for use in therapy, or a pharmaceutical composition comprising the antibody or antigen-binding fragment or immunoconjugate of the present disclosure. Suitably, provided herein is an antibody or antigen-binding fragment or immunoconjugate of the present disclosure, or a pharmaceutical composition comprising the antibody or antigen-binding fragment or immunoconjugate of the present disclosure, for use in the methods of treatment disclosed herein.

[0320] In another aspect, provided herein is the use of an antibody or antigen-binding fragment or immunoconjugate of the present disclosure, or a pharmaceutical composition comprising an antibody or antigen-binding fragment or immunoconjugate of the present disclosure, in the manufacture of a medicament for use in therapy, e.g., a medicament for use in the methods of treatment disclosed herein. Sequence Listing SEQ ID NO: 1, CDRH1, Molecule type: protein, Organism: synthetic construct TYPIE SEQ ID NO: 2, CDRH2, Molecule type: protein, Organism: synthetic construct NFHPYNDDTKYNEKFQG SEQ ID NO: 3, CDRH3, Molecule type: protein, Organism: synthetic construct ENYGSHGGFVY SEQ ID NO: 4, CDRL1, Molecule type: protein, Biology: synthetic construct RASSSVISSYLH SEQ ID NO:5, CDRL2, Molecule type: protein, Biology: synthetic construct STSNLAS SEQ ID NO: 6, CDRL3, Molecule type: protein, Biology: synthetic construct QQYNSYPLT SEQ ID NO: 17, Fc of human IgG1 with P238D mutation, Molecule type: protein, Biology: synthetic construct THTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 18, heavy chain, molecule type: protein, organism: synthetic construct QVQLVQSGAEVKKPGASVKVSCKAFGYTFTTYPIEWMRQAPGKGLEWIGNFHPYNDDTKYNEKFQGRVTLTVDKSSTTVYMELSSLRSEDTAVYYCARENYGSHGGFVYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 19, light chain, molecule type: protein, biological: synthetic construct ENQLTQSPSSLSASVGDRVTITCRASSSVISSYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [Example]

[0321] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure. By their illustrative nature, it will be understood that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0322] Example 1: Effect of Fc receptor interactions on the agonist function of exemplary anti-PD-1 antibodies The signaling effects of an exemplary anti-human PD-1 agonist antibody (clone 19 murine IgG1 described in U.S. Patent No. 9,181,342, issued November 10, 2015) were measured in PD-1-expressing Jurkat cells that produce luciferase under the control of NFAT response elements. T cells were investigated in reporter assays in which they were cultured with BW5147 cells expressing an anti-CD3 "T cell stimulator" (TCS) construct as previously described (Leitner et al. 2010). To examine the role of Fc receptor interactions in antibody agonism, assays were performed with either BW5147 cells expressing the TCS construct alone or BW5147 cells also transfected to express mouse FcγR2B.

[0323] 5 x 10 per well in a 96-well U-bottom plate 4 Jurkat reporter cells (Promega catalog number J1250b) were added and cultured at 5 × 10 in a total volume of 80 μL of assay buffer (RPMI 1640 + 1% FCS).4 BW5147 cells were co-cultured with either PD-1 antibody or isotype control. A nine-point dilution series of antibody was performed, starting at 200 nM and followed by a 1:3 dilution. After 6 hours of incubation in a humidified CO2 incubator at 37°C, the plate was removed from the incubator and equilibrated to room temperature for 10 minutes. The amount of luciferase produced (as a measure of T cell activation) was quantified using the Bio-Glo™ Luciferase Assay System (Promega). 80 μl of Bio-Glo™ Luciferase Assay Reagent was added to each well, and the plate was incubated at room temperature for 10 minutes. Luminescence was quantified using a CLARIOstar Plus (BMG Labtech).

[0324] When stimulator cells that expressed mouse FcγR2B were used, the PD-1 antibody clone 19 caused a significant decrease in T cell activation with an IC50 of 0.13 nM (Figure 1A). When stimulator cells that did not express Fc receptors were used, clone 19 had no effect on T cell activation (Figure 1B).

[0325] Example 2: Humanization of exemplary anti-PD-1 antibodies The VH and VL sequences of clone 19 were aligned to a database of human germline sequences, and homologous sequences were selected as frameworks for humanization. * 01 or IGHV7-4-1 * 02 as the framework for the VH domain, and IGKV1-39 * 01 or IGKV3-11 * 01 was used as the framework for the VL domain.

[0326] The VH and VL sequences were passed through a CDR-grafting algorithm to transfer the CDRs to human germline sequences selected from murine antibody clone 19. To enable structure-guided humanization, models of the clone 19 murine VH and VL were constructed, and a structure-guided approach was used to determine which framework amino acids to retain in the humanized antibody frame to preserve binding integrity. Table 1 summarizes the VH and VL sequences that were generated.

[0327] Antibody variants consisting of all possible combinations of humanized VH and VL domains were generated. The variants were produced with the human IgG1 kappa isotype. The binding affinity and kinetics of the humanized antibody variants to human or cynomolgus PD-1 were determined by surface plasmon resonance (SPR) using a Biacore 8K (Cytiva). A series S CM5 sensor chip (Cytiva) was coated with polyclonal anti-human IgG using a human antibody capture kit (Cytiva). The anti-PD-1 antibody was then captured on the biosensor surface, and an isotype control antibody was captured in the reference channel. Various concentrations of monomeric soluble human PD-1 extracellular domain or soluble cynomolgus PD-1 extracellular domain were then injected over the immobilized antibody in a single-cycle kinetic analysis in a buffer containing 10 mM Hepes, 150 mM NaCl, and 0.005% v / v surfactant P20, pH 7.4 (HBS-P) at 37°C. After reference and blank subtraction, the association and dissociation rates were fitted using BiaEvaluation Software (Cytiva) to calculate the dissociation constants. Table 2 shows the binding K of each humanized variant to human and cynomolgus monkey PD-1. D Shows. [Table 1] [Table 2]

[0328] Example 3: Effect of Fc mutations on selectivity of binding to FcγR2B The humanized variant humCL19v1 was recombinantly produced on hIgG1, hIgG4 or a range of different Fc mutant hIgG1 constant regions and their binding to human FcγR2B or two highly homologous FcγR2A allotypes was assessed by surface plasmon resonance (in buffer HBS-EP+, pH 7.4 at 37°C).

[0329] Interactions were assessed by surface plasmon resonance using a Biacore 8K with recombinantly expressed FcR (extracellular domain only) as the analyte. Briefly, recombinant human PD-1 extracellular domain was covalently immobilized to both flow cells of all channels of a CM5 Series S sensor chip using a GE Healthcare amine coupling kit. The humCL19v1 Fc variant to be evaluated was then captured on flow cell 2 of each channel (approximately 500-1000 response units). Steady-state affinity analysis was then performed by injecting various concentrations of FcR and measuring equilibrium binding. Double referencing was used (subtracting the signal from the reference Fc1 and also subtracting the signal from a blank injection of 0 concentration). K D Values ​​were calculated from Langmuir curves (equilibrium binding was plotted against analyte concentration to determine the concentration required for half-maximal binding). D The values ​​are shown in Table 3. Of the Fc variants tested, only the P238D mutation enhanced selectivity for binding to FcγR2B over both FcγR2A allotypes. This mutation resulted in a modest increase in binding to FcγR2B and a significant decrease in binding to both FcγR2A allotypes. [Table 3]

[0330] Example 4: P238D mutant PD-1 antibodies are as effective as IgG1 antibodies in suppressing T cell activation in an NFA reporter assay To assess whether the P238D mutation affects the agonist function of humCL19v1, we used a Jurkat reporter assay. We compared the unmutated IgG1 version of humCL19v1 with previously described IgG1 isotype agonist antibodies, including PD1AB6 (WO 2017 / 058859), PD1B1094 (WO 2018 / 226580), Antibody 1 (WO 2019 / 168745), and ANB030 (WO 2020 / 247628). These antibodies were recombinantly produced from the sequences provided in their respective patent applications. Jurkat T cells producing luciferase under the control of an NFAT response element expressed an anti-CD3 “T cell stimulator” (TCS) construct as previously described ( Leitner et al. 2010 ) and were cultured with BW5147 cells expressing human FcγR2B.

[0331] In a 96-well U-bottom plate, 5x10^4 Jurkat reporter cells (Promega catalog number J1250b) were added per well and co-cultured with 5x10^4 BW5147 cells and either PD-1 antibody or isotype control in a total volume of 80μL of assay buffer (RPMI 1640 + 1% FCS). A single high dose of each PD-1 antibody (10μg / ml) was tested.

[0332] After 6 hours of incubation in a humidified CO2 incubator at 37°C, the plates were removed from the incubator and allowed to equilibrate to room temperature for 10 minutes. The amount of luciferase produced (as a measure of T cell activation) was quantified using the Bio-Glo™ Luciferase Assay System (Promega). 80 μl of Bio-Glo™ Luciferase Assay Reagent was added to each well, and the plates were incubated at room temperature for 10 minutes. Luminescence was quantified using a CLARIOstar Plus (BMG Labtech).

[0333] All PD-1 antibodies tested significantly reduced T cell activation compared to isotype controls, with no significant difference between wild-type IgG1 or the P238D mutant version of humCL19v1 (Figure 2A).

[0334] In another series of experiments, we evaluated the efficacy of the P238D mutant version of humaCL19v1 using an optimized T cell reporter assay. The T cell reporter assay was similar to that described above in this example, except that mouse BW5147 stimulator cells were replaced with human HEK293T stimulator cells. Jurkat T cells producing luciferase under the control of an NFAT response element were co-cultured with HEK293T cells expressing an anti-CD3 "T cell stimulator" (TCS) construct as previously described (Leitner et al. 2010) and expressing human FcγR2B.

[0335] 4x10^4 HEK293T stimulator cells were seeded per well of a flat-bottom 96-well plate. After 16 hours, the medium was removed and 5x10^4 Jurkat reporter cells (Promega cat#J1250b) per well were added plus either PD-1 antibody or isotype control in a total volume of 80μL of assay buffer (RPMI 1640 + 1% FCS). Five-fold serial dilutions of antibody were tested, starting at 5μg / ml.

[0336] After 6 hours of incubation in a humidified CO2 incubator at 37°C, the plates were removed from the incubator and allowed to equilibrate to room temperature for 10 minutes. The amount of luciferase produced (as a measure of T cell activation) was quantified using the Bio-Glo™ Luciferase Assay System (Promega). 80 μl of Bio-Glo™ Luciferase Assay Reagent was added to each well, and the plates were incubated at room temperature for 10 minutes. Luminescence was quantified using a CLARIOstar Plus (BMG Labtech). As shown in Figure 2B, humCL19v1 P238D inhibited T cell activation by up to 84%, as assessed by NFAT-induced luminescent signal, with an IC50 of 0.0278 nM.

[0337] Example 5: P238D mutant PD-1 antibodies are as effective as IgG1 antibodies in inhibiting primary T cell activation in a T cell activation assay In one set of experiments, a tetanus toxoid activation assay was used to evaluate the inhibitory effects of exemplary antibodies on T cell activation. Whole human peripheral blood mononuclear cells (PBMCs) from healthy donors (400,000 cells per well of a 96-well U-bottom plate) were stimulated with tetanus toxoid (0.5 μg / mL) in the presence of PD-L1 / 2 blocking antibodies (5 μg / mL each) and 1 μg / mL of a PD-1 agonist antibody or isotype control. IFNγ release was assessed by ELISA of supernatants after 96 hours of incubation at 37°C and 5% CO2. Six donors were evaluated, and data were collated by normalizing each donor's IFNγ levels to cells activated with tetanus toxoid in the absence of the test antibody.

[0338] Antibodies tested included the P238D mutated humCL19v1, the unmutated IgG1 version of humCL19v1, and previously described IgG1 isotype agonist antibodies, including PD1AB6 (WO 2017 / 058859) and Antibody 1 (WO 2019 / 168745). These antibodies were recombinantly produced from the sequences provided in the respective patents.

[0339] Averaged across all donors tested, tetanus toxoid (TT) induced a roughly two-fold increase in IFNg production compared to PBMC cultures without TT. The IgG1 isotype control slightly reduced IFNg production. humCL19v1 P238D, humCL19v1 IgG1, and PD1AB6 all significantly reduced IFNg production compared to the isotype control. There was no significant difference between wild-type IgG1 or the P238D mutant version of humCL19v1 (Figure 3A).

[0340] In another series of experiments, a viral peptide activation assay was used to evaluate the inhibitory effect of exemplary antibodies on immune cell activation. Whole human peripheral blood mononuclear cells (PBMCs) from healthy donors (500,000 cells per well of a 96 U-bottom plate) were stimulated with 2 μg / mL of CEF HLA class I peptides (a pooled mixture of peptides derived from cytomegalovirus, Epstein-Barr virus, and influenza, Mabtech cat#3618-1) in the presence of 5 μg / mL brefeldin A (Biolegend catalog #420601) and 1 μg / mL of PD-1 antibody or P238D mutant hIgG1 isotype control, or no antibody. The percentage of IFNγ-producing cells within the CD8 T cell population was assessed using intracellular flow cytometry after 16 hours of incubation at 37°C and 5% CO2. Eighteen donors were evaluated. Data were collated by normalizing for each donor using the following formula: (cytokine production in the presence of antibody - unstimulated background) / (cytokine production in stimulated cells without antibody - unstimulated background).

[0341] Averaged across all donors tested, CEF peptide induced a four-fold increase in CD8 IFNg-producing T cells compared to PBMC cultures without CEF peptide. humCL19v1 P238D significantly reduced IFNg by an average of 63% compared to the no-antibody control (Figure 3B).

[0342] Example 6: P238D mutant PD-1 antibodies are as effective as IgG1 antibodies in inhibiting primary T cell activation in an anti-CD3 / 28 activation assay Whole human peripheral blood mononuclear cells (PBMCs) from healthy donors (100,000 cells per well in a 96-well U-bottom plate) were stimulated with soluble anti-CD3 and anti-CD28 antibodies (final concentration of 0.5 ng / mL each) in the presence of 1 μg / mL PD-1 agonist antibody or isotype control. CD25 expression on CD4 T cells was assessed by flow cytometry as a marker of T cell activation after 72 hours of incubation at 37°C and 5% CO2. Data from multiple donors were collated by normalizing each donor's CD25 dimer to the CD25 dimer for cells activated with anti-CD3 and anti-CD28 in the absence of test antibodies.

[0343] Antibodies tested included previously described P238D mutant humCL19v1 and IgG1 agonist antibodies, including PD1AB6 (WO 2017 / 058859) and Antibody 1 (WO 2019 / 168745). These antibodies were recombinantly produced from the sequences provided in the respective patents.

[0344] Anti-CD3 and anti-CD28 resulted in a significant increase in CD25 expression that was significantly inhibited by all PD-1 antibodies tested (Figure 4).

[0345] Example 7: IgG1 isotype anti-PD-1 antibodies, but not P238D mutant PD-1 antibodies, result in ADCC killing of regulatory T cells in vitro An in vitro NK cell degranulation assay was used to study the potential of anti-PD-1 antibodies to deplete regulatory T cells by ADCC. Tregs were purified by magnetic isolation from PBMCs of healthy donors using the Miltenyi Human CD4+CD25+CD127dim / - Regulatory T Cell Isolation Kit II (Cat. No. 130-094-775). NK cells were similarly purified using the Miltenyi Human NK Isolation Kit (Cat. No. 130-092-657).

[0346] 20,000 isolated NK cells were plated per well in a 96-well U-bottom plate containing 1 μg / ml of different anti-PD-1 antibodies or an IgG1 isotype control. Antibodies tested included the P238D mutated humCL19v1, the unmutated IgG1 version of humCL19v1, and previously described IgG1 isotype agonist antibodies, including PD1B1094 (WO 2018 / 226580) and Antibody 1 (WO 2019 / 168745). These antibodies were recombinantly produced from the sequences provided in their respective patent applications.

[0347] 100,000 Treg cells were added per well to obtain a 1:5 effector:target ratio. Finally, anti-CD107a antibody (Biolegend #328638) was added to each well for a final dilution of 1:100, along with monensin (Biolegend #420701) and brefeldin (Biolegend #420601), to obtain a final 1x concentration of each. The final well volume was 200 μl. The assay was incubated for 6 hours at 37°C in 5% CO2. The assay was then stained with an antibody panel including CD3, dead cell markers, and CD56, fixed with 1% formaldehyde, and evaluated by flow cytometry. The generated data was analyzed using FlowJo V10. Degranulated NK cells were identified as CD107+CD56+ cells. Dead Treg cells were identified as CD3+ cells positive for dead cell markers.

[0348] The IgG1 isotype anti-PD-1 antibody significantly activated NK cell degranulation (Figure 5). The P238D mutant humCL19v1 did not induce NK cell degranulation or Treg death.

[0349] Example 8: humCL19v1 P238D, but not other PD-1 agonist antibodies, can further inhibit T cell activation in the presence of high levels of PD-L1 Using a PD-1 reporter cell line, we assessed the impact of P238D mutant humCL19v1 compared with previously described PD-1 agonist antibodies in the setting of highly expressed PD-L1.

[0350] PD-L1-expressing CHOK1 cells expressing a T cell stimulator construct (Promega catalog number J1250a) were seeded at 40,000 cells / well into 96-well flat-bottom plates and incubated overnight to allow adhesion. The following day, the supernatant was removed, and 50,000 PD-1-expressing Jurkat reporter cells expressing luciferase under the control of an NFAT response element were added along with a PD-1 antibody or isotype control. A dose titration of the PD-1 antibody was assessed using 4-fold dilutions from 10 μg / ml to a total volume of 80 μl. After 6 hours of incubation at 37°C, 80 μl of Bio-Glo was added per well, incubated for 15 minutes, and then read on a Clariostar plate reader using the firefly luciferase setting to quantify luciferase production.

[0351] The antibodies tested included previously described P238D mutant humCL19v1 and IgG1 isotype agonist antibodies, including PD1AB6 (WO 2017 / 058859), PD1B1094 (WO 2018 / 226580), Antibody 1 (WO 2019 / 168745), and ANB030 (WO 2020 / 247628). These antibodies were recombinantly produced from the sequences provided in the respective patents. As a control, a biosimilar of the PD-1 blocking antibody nivolumab was also evaluated.

[0352] As expected, nivolumab, by blocking the interaction between PD-L1 and PD-1, resulted in a significant dose-responsive increase in T cell activation. Unexpectedly, only the P238D mutant humCL19v1 antibody demonstrated the ability to further suppress T cell activation (beyond the inhibition already provided by PD-L1 interaction with PD-1). None of the other PD-1 antibodies tested affected T cell activation (Figure 6).

[0353] Example 9: humCL19v1 P238D can inhibit T cell activation in RA PBMC, fibroblast co-cultures To test the effects of PD-1 agonists on T cells from rheumatoid arthritis (RA) patients, PBMCs from four donors with RA were activated in a co-culture with RA fibroblast-like synoviocytes (FLS from Tebu-bio #408RAK-05a). Because stromal cells such as fibroblasts express PD-L1 and PDL-2, this assay represents a physiological situation where PD-1 ligands are present (Dezutter-Dambuyant et al. 2016).

[0354] In a flat 96-well plate, 10,000 FLS cells were plated in 50 μl of medium and allowed to adhere for 2 hours. Next, 100,000 PBMCs were added in 50 μl of medium. Anti-PD-1 antibody (humCL19v1 P238D or antibody 1 from WO 2019 / 168745 A1) or an isotype control was added to 50 μl of medium at a final concentration of 1 μg / ml. Finally, anti-CD3 (clone OKT3) and anti-CD28 (clone CD28.2) were added to 50 μl of medium at a final concentration of 0.5 ng / ml each. After 3 days of incubation at 37°C and 5% CO2, supernatants were collected and assessed by cytometric bead array (Biolegend Th17 panel number 741032). Cells were assessed by flow cytometry using the markers CD3, CD4, CD25, and ICOS. The agonist humCL19v1 P238D resulted in a significant reduction in T cell activation markers on CD4 T cells, including CD25 (Figure 7A) and ICOS (Figure 7B), as well as a significant reduction in inflammatory cytokine production, including IFNg (Figure 7C), IL17F (Figure 7D), and TNFα (Figure 7E). The reference agonist Antibody 1 had no significant effect on any of these readouts. These data suggest that humCL19v1 P238D may be active in situations where the PD-1 ligand is expressed, whereas other described PD-1 agonists may be ineffective in these situations.

[0355] Example 10. humCL19v1 P238D enhances the interaction of PD-L1 with PD-1 PD-L1 binding to PD-1-expressing cells was assessed in the presence of various PD-1 antibodies. PD-1-expressing Jurkat T cells were incubated with PD-1 antibodies or isotype controls at a concentration of 10 μg / ml on ice for 1 hour. Cells were then washed and incubated for 30 minutes with increasing concentrations of PDL1-Fc (Biolegend No. 762506) conjugated to AF647 using a conjugation kit (Thermofisher No. A20186). Cells were then washed again and assessed by flow cytometry. Cells preincubated with humCL19v1 P238D showed brighter staining with PDL1-Fc than cells preincubated with no antibody, isotype controls, or other listed PD-1 antibodies (Figure 8). Cells preincubated with nivolumab did not show binding to PDL1-Fc, as expected due to its ligand-blocking epitope. These data suggest that binding of humCL19v1 to PD-1 enhances its interaction with PD-L1.

[0356] Example 11 Genes Downregulated by PD-1 Agonists Are Associated with Autoimmunity A transcriptional signature of PD-1 agonism by clone 19 was defined using a method similar to that described in Example 1. 1.5 million PD-1-expressing reporter Jurkats were added per well of a 6-well flat-bottom plate in 1 ml of assay buffer (RPMI 1% FCS) containing 10 μg / ml of test antibody, followed by 1.5 million FcR-expressing stimulator cells in 1 ml of assay buffer (to generate a final antibody concentration of 5 μg / ml). For static samples, 1.5 million Jurkats were added to empty wells in 2 ml of assay buffer. All conditions were performed in six technical replicates. All samples were incubated at 37°C for 18 hours. Next, 80 μl of cells from each sample were transferred to a white 96-well plate for luciferase assay (described in Example 1) (FIG. 9A), 80 μl were transferred to a 96-well plate for flow cytometry assay, and then the remaining sample stimulator cells were depleted by negative selection using Mojosort mouse CD45 nanobeads (Biolegend #480028). From the remaining cells, 80 μl were transferred to a 96-well plate for flow cytometry assay to confirm Jurkat cell purity (FIG. 9B). The remainder were pelleted by centrifugation, the supernatant was aspirated, and the cells were then frozen at -80°C. Samples were subjected to RNA extraction and sequencing in GeneWiz using a concatenated library preparation protocol with a sequencing depth target of at least 20M reads per sample.

[0357] RNA sequencing files from GeneWiz were processed using the rnaseq (v3.1) Nextflow pipeline within the nf core. FastQC was used to check sequencing quality, and Salmon was used to enumerate transcripts relative to the human genome (GRCh38 v96). An in-house differential expression pipeline was used to perform all subsequent analyses: tximport was used to generate gene counts from estimated transcript abundances, DESeq2 was used to perform differential expression between groups (without adjusting for additional covariates), and the EnrichR package was used to perform gene set enrichment analysis for gene sets with significantly higher or lower expression between groups. EnrichR was used to run various different gene set databases. Significantly higher or lower gene sets were defined based on their log-fold change in DESeq2 analysis where the gene FDR correct p-value was less than 0.05. 1,227 genes were significantly downregulated in cells activated in the presence of PD-1 agonist compared to activation in the presence of isotype control (Figure 9C). Mapping this set of downregulated genes to the EBI GWAS catalog revealed enrichment for genes associated with autoimmune diseases, particularly seropositive rheumatoid arthritis (Figure 9D). These data suggest that antibody agonism of the PD-1 pathway can downregulate inflammatory gene pathways associated with autoimmunity.

[0358] Example 12. Treatment of a mouse model of systemic lupus erythematosus The efficacy of the exemplary antibody clone 19 in treating SLE disease models was tested in a transfer model in which disease was induced by the transfer of partially MHC-II mismatched splenocytes from humanized PD-1 mice in H2-Ab1bm12 recipient mice. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the breakdown of self-tolerance and the production of autoantibodies against nuclear antigens such as chromatin and DNA. Deposition of immune complexes in various organs, including the kidneys and skin, leads to diverse clinical symptoms. SLE can affect approximately 0.1% of the population, with increased frequency in women of childbearing age and certain ethnicities, including African Americans, Asians, Hispanics, and Native Americans (Izmirly et al., 2021). Existing treatments include immunosuppressants such as anti-inflammatory agents and corticosteroids, which can help manage symptoms, but there is no cure for the disease, and more effective treatments remain needed.

[0359] The efficacy of Clone 19 in ameliorating disease was tested in a mouse model of SLE. A transfer model of SLE in which disease is induced by the transfer of unfractionated splenocytes from donor bm12 mice to C57BL / 6 recipient mice, or vice versa, has been previously described (Klarquist & Janssen, 2015). Bm12 mice differ from C57BL / 6 mice by three amino acids in the MHC class II antigen H-2A. This MHC mismatch results in an allogeneic response in which donor CD4 T cells are activated, differentiate into follicular helper cells (Tfh), and promote germinal center formation accompanied by the production of autoantibodies. To enable evaluation of the anti-human PD-1 agonist antibody Clone 19, an adapted protocol was used to transfer donor splenocytes into C57BL / 6 mice humanized at the PD-1 locus. These mice were previously described and characterized in Billur Akkaya's doctoral dissertation (Akkaya, 2012). This provides a model whereby some disease-propagating immune cells (such as recipient B cells) do not express human PD-1, but disease-initiating T cells can be targeted with anti-human antibodies.

[0360] Donor C57BL / 6 mice humanized at the PD-1 locus were sacrificed, and spleens were collected in RPMI medium + 2% FCS + P / S / N. bm12 mice were also sacrificed to provide disease-free control donor splenocytes. Spleens were processed into a single-cell suspension by pressing through a 70 μM nylon cell strainer with the plunger of a 5 ml syringe. Cells were then pelleted and resuspended in PBS at a concentration of 200 million cells / ml. 200 μl of this cell suspension was then injected intraperitoneally per recipient mouse (40 million cells per mouse). Adult bm12 mice (Jax stock number: 00116) were used as recipients. Female recipients received cells from female donors, and male recipients received cells from male donors. Test and control antibodies were diluted to 1 mg / ml in sterile PBS and injected IP. Groups were distributed among cages to avoid potential cage effects. Dexamethasone was administered to the control group in drinking water (thus, these mice had to be grouped in the same cage). An average daily intake of 0.2 ml / g was assumed to provide approximately 0.5 mg / kg / day. Dexamethasone was first reconstituted at 10 mg / ml in 100% ethanol and then diluted to 2.5 μg / ml (1 in 4000) in drinking water.

[0361] In the first study, mice were intraperitoneally administered 200 μg of test antibody or mIgG1 isotype control on days 1 and 22 after cell transfer. The control group received dexamethasone in the drinking water from day 1 until termination on day 35. Blood was collected from mice via tail vein pretreatment on day 22 for serum autoantibody ELISA and on day 35, when the study was terminated. Spleens were also collected and evaluated by flow cytometry to quantify immune cell proliferation, including the proliferation of donor Tfh cells (gated as CD4+CXCR5+ICOS+ cells). Clone 19 provided near-complete prevention of disease, as assessed by autoantibody levels (Figures 10A-B), Tfh frequency (Figure 10C), or splenomegaly (Figure 10D). The magnitude of the effect was comparable to dexamethasone administered throughout the study. There was a high degree of variability among untreated mice, with some failing to engraft donor cells. The magnitude of the effect was similar to that of dexamethasone administered throughout the study.

[0362] In another experiment, Clone 19 was administered at different time points; administration on day 0 again completely prevented the expansion of donor TfH cells and significantly reduced other markers of disease development (Figures 11A-11B). Administration on day 14 resulted in a partial reduction in disease markers. Administration on day 28 (before the end of the study on day 30) did not result in a significant reduction in any markers, including TfH cell frequency. Because Tfh cells express high levels of human PD-1, significant depletion would be expected within this 48-hour time frame if Clone 19 were acting as a depleting antibody via ADCC or CDC.

[0363] Example 13. PD-1 agonists inhibit delayed-type hypersensitivity responses in humanized mice We used C57BL / 6 mice humanized at the PD-1 locus to evaluate the effect of the PD-1 agonist Clone 19 on delayed-type hypersensitivity responses in a skin challenge model associated with autoimmune skin disease. On day 0, mice were immunized with keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (CFA). The emulsion was a mixture of KLH (Sigma) in PBS and added to CFA (BD Biosciences) at a 1:1 ratio. The final concentration of KLH was 4 mg / mL. Animals were immunized with 100 μL of the immunization emulsion injected subcutaneously at one or two sites. The unprimed control group received PBS only. Also on day 0, mice were treated intraperitoneally with either the mIgG1 isotype control (clone Mopc21) or the anti-PD-1 Clone 19 mIgG1 at a single dose of 10 mg / kg 1 hour prior to immunization. The unprimed control group received PBS only. Animals in the positive treatment control group were treated by oral gavage with CsA at a dose of 3 mg / kg once daily from days 0–5. To prepare CsA, Sandimmune Neoral Solution (Novartis) was diluted to 0.3 mg / ml with 0.5% methylcellulose 400 cp (Sigma). Five days after immunization, mice were challenged (under anesthesia) with 20 μL of 4 mg / mL antigen solution in the left ear pinna. The non-challenged control group received 20 μL of PBS in the left ear pinna. One day after ear challenge, ear thickness was measured using a digital caliper. After measuring ear thickness, animals were humanely sacrificed, and postmortem, 8 mm diameter circles were cut from the left and right ears of each animal in all groups using a biopsy punch. Ears were weighed on a precision analytical balance. Ear edema was assessed as the difference between the left (antigen-challenged) ear weight and the right (control) ear weight. The PD-1 agonist clone 19 significantly inhibited ear swelling (Figure 12A).

[0364] In another experiment, mice were treated intraperitoneally with either 10, 1, 0.1, or 0.01 mg / kg of clone 19 0 or 1 hour prior to immunization. In this study, CTLA-Ig fusion protein (Biolegend, catalog no. 591908) was used as a positive control and was administered at a dose of 10 mg / kg IP 1 hour prior to immunization on day 0. Clone 19 at doses of 10 mg / kg or 1 mg / kg resulted in significant inhibition of ear swelling, comparable to the effect of CTLA4-Ig (FIG. 12B).

[0365] Example 14. PD-1 agonists improve symptoms of graft-versus-host disease in a mouse model In one set of experiments, the effect of humCL19v1 P238D on human PBMC-driven graft-versus-host disease (GvHD) was determined in vivo. Briefly, approximately 8-10 week old female NSG mice (JAX Labs, stock no. 05557) were subjected to 2.4 Gy total body irradiation (day -1). Human peripheral blood mononuclear cells (PBMCs) were isolated from leukopaks (HemaCare products ordered from Tissue Solutions) at 25 x 10 per ml of PBS. 6 Mice were inoculated with 200 μl of cell suspension (5 × 10 6 PBMCs) were injected intravenously (IV) via tail injection one day after irradiation (day 0). Mice were treated with 10 mg / kg of humCL19v1 P238D or P238D mutant hIgG1 isotype control via intraperitoneal injection on days 0 and 7, 14, and 21. Mice were weighed periodically and euthanized when they had lost 15% weight or after 28 days. At the end of the study, peripheral blood was collected for assessment of inflammatory cytokines by cytometric bead array. Spleens were weighed, and infiltration of human PBMCs into the liver and spleen was quantified by flow cytometry using markers for hCD45, hCD4, hCD8, hCD20, hCD25, and FOXP3. IFNg production by CD8 and CD4 T cells in the spleen and liver was also assessed by intracellular flow cytometry.

[0366] Following the above procedure, humCL19v1 P238D significantly reduced spleen weight (Figure 13A), human immune cell proliferation in the spleen (Figure 13B), and liver (Figure 13C), as well as serum inflammatory cytokine levels (Figure 13D), compared with isotype controls. Furthermore, in addition to reducing overall cytokine levels, humCL19v1 P238D also reduced CD4 and CD8 cytokine production on a per-cell basis, as assessed by intracellular flow cytometry of human immune cells in the liver and spleen (Figure 13E).

[0367] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure can be used in practicing the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby. The present invention provides, for example, the following items. (Item 1) 1. A method of suppressing immune cells that express programmed cell death 1 (PD-1), comprising contacting the immune cells with an antibody that specifically binds to PD-1 and agonizes PD-1 signaling in the immune cells, wherein the antibody comprises an Fc region that comprises an amino acid substitution, wherein the amino acid substitution results in reduced antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells in the subject compared to a parent molecule lacking the amino acid substitution, and wherein the antibody has the same or greater agonistic effect on PD-1 signaling in the immune cells compared to the parent molecule. (Item 2) 1. A method of inhibiting immune cells that express programmed cell death 1 (PD-1), comprising contacting the immune cells with an antibody that specifically binds to PD-1 and enhances the interaction between the PD-1 and PD-L1 on the surface of the immune cells. (Item 3) 3. The method of claim 2, wherein the antibody comprises an Fc region and the Fc region comprises an amino acid substitution. (Item 4) 4. The method of claim 3, wherein the amino acid substitution results in reduced antibody-dependent cellular cytotoxicity (ADCC) against PD-1-expressing regulatory T cells in the subject compared to a parent molecule lacking the amino acid substitution, and the antibody has the same or greater agonistic effect on PD-1 signaling in the immune cells compared to the parent molecule. (Item 5) 5. The method of item 1 or 4, wherein ADCC against the PD-1-expressing regulatory T cells is reduced as determined by a natural killer cell activation assay described in Example 7. (Item 6) the antibody does not result in significant ADCC against the PD-1-expressing regulatory T cells as determined by the natural killer cell activation assay described in Example 7. 6. The method according to any one of 1 to 5. (Item 7) 7. The method of any one of items 1 to 6, wherein the antibody does not activate natural killer (NK) cells. (Item 8) 8. The method according to any one of items 1 to 7, wherein the antibody comprises a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region. (Item 9) 9. The method according to Item 8, wherein the heavy chain variable region comprises a complementarity-determining region (CDR) comprising a sequence set forth in one or more of SEQ ID NOs: 1 to 3, having 0 to 3 amino acid modifications. (Item 10) 10. The method according to any one of items 3 to 9, wherein the Fc region is derived from IgG1 and comprises an aspartic acid (D) at position 238, numbered according to the EU index. (Item 11) 1. A method of inhibiting an immune cell that expresses programmed cell death 1 (PD-1), comprising contacting the immune cell with an antibody comprising a heavy chain, a light chain, and an Fc region; (i) the heavy chain comprises a heavy chain variable region comprising a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 1 to 3, with 0 to 3 amino acid modifications; (ii) the light chain comprises a light chain variable region comprising a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4 to 6, with 0 to 3 amino acid modifications; (iii) A method for suppressing immune cells expressing programmed cell death 1 (PD-1), wherein the Fc region is derived from IgG1 and contains aspartic acid (D) at position 238 as numbered according to the EU index. (Item 12) 12. The method according to any one of Items 8 to 11, wherein the light chain variable region comprises a CDR comprising a sequence set forth in one or more of SEQ ID NOs: 4 to 6 having 0 to 3 amino acid modifications. (Item 13) 13. The method of any one of Aspects 8 to 12, wherein the heavy chain variable region comprises heavy chain complementarity determining region 1 (CDRH1), CDRH2, and CDRH3, and CDRH1, CDRH2, and CDRH3 each comprise the sequence set forth in SEQ ID NOs: 1 to 3 with 0 to 3 amino acid modifications. (Item 14) 14. The method of any one of Aspects 8 to 13, wherein the light chain variable region comprises light chain complementarity determining region 1 (CDRL1), CDRL2, and CDRL3, and CDRL1, CDRL2, and CDRL3 each comprise the sequence set forth in SEQ ID NOs: 4 to 6 with 0 to 3 amino acid modifications. (Item 15) 15. The method of any one of Items 8 to 14, wherein the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3, and CDRH1, CDRH2, and CDRH3 comprise the sequences set forth in SEQ ID NOs: 1 to 3, respectively. (Item 16) 16. The method according to any one of items 8 to 15, wherein the light chain variable region comprises CDRL1, CDRL2, and CDRL3, and CDRL1, CDRL2, and CDRL3 comprise the sequences set forth in SEQ ID NOs: 4 to 6, respectively. (Item 17) 17. The method of any one of Items 8 to 16, wherein the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to a sequence set forth in any one of SEQ ID NOs: 7 to 11. (Item 18) The light chain variable region has at least one amino acid sequence different from that of any one of SEQ ID NOs: 12 to 16. 18. The method of any one of items 8 to 17, comprising a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence. (Item 19) 19. The method of any one of items 8 to 18, wherein the heavy chain comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in SEQ ID NO: 18. (Item 20) 20. The method of any one of items 8 to 19, wherein the light chain comprises a sequence having at least 80%, 85%, 90%, 95%, or 99%, or 100% identity to the sequence set forth in SEQ ID NO: 19. (Item 21) 21. The method according to any one of items 3 to 20, wherein the ...

Claims

[Claim 1] The invention described in the specification.

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