Anti-pd-1 antibodies and uses thereof

Abstract

The present disclosure relates to isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to PD-1 and block the interaction between PD-1 and PD-L1 / PD-L2. The present disclosure also relates to antibodies that are chimeric antibodies, humanized antibodies, bispecific antibodies, derivatized antibodies, single-chain antibodies, portions of fusion proteins, or bispecific antibodies. Nucleic acid molecules encoding the antibodies, hybridomas, and methods for expressing the antibodies are also provided. Pharmaceutical compositions comprising the antibodies are also provided. The present disclosure also provides uses of these antibodies for enhancing T cell function and upregulating cell-mediated immune responses for the treatment and prevention of various diseases.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 836,177, filed April 19, 2019. The entire contents of the aforementioned U.S. Provisional Application are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to anti-PD-1 (programmed cell death 1) antibodies, antigen-binding fragments, and uses thereof. Background Art

[0004] Protein programmed death 1 (PD-1 or PDCD1) is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al., supra; Okazaki et al. (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 1 70: 711-8). The PD-1 gene is a 55kDa type I transmembrane protein, which is part of the Ig gene superfamily (Agata et al. (1996) Int Immunol 8: 765-72). PD-1 contains a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM) (Thomas, ML (1995) J Exp Med 181:1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18:286-91). Although structurally similar to CTLA-4, PD-1 lacks the MYPPY motif that is crucial for B7-1 and B7-2 binding.

[0005] T cells receive both positive secondary co-stimulatory signals and negative secondary co-stimulatory signals. The regulation of such positive and negative signals is crucial for maximizing the protective immune response of the host while maintaining immune tolerance and preventing autoimmunity. Negative secondary signals seem to be necessary for inducing T cell tolerance, while positive signals promote T cell activation. Although the simple two-signal model provides an effective explanation for primary lymphocytes, the host's immune response is a dynamic process, and co-stimulatory signals can also be provided to antigen-exposed T cells. PD-1 is an inhibitory member of the CD28 family expressed on activated B cells, T cells, and bone marrow cells (Agata et al., see above; Okazaki et al. (2002) Current Immunology Viewpoint 14:391779-82; Bennett et al. (2003) Journal of Immunology 1 70:711-8) and provides a negative signal.

[0006] Two ligands of PD-1, PD-L1 and PD-L2, have been identified. The ligands have been shown to downregulate T cell activation when bound to PD-1 (Freeman et al. (2000) Journal of Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members. PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat Med 8:787-9). The interaction between PD-1 and PD-L1 leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T-cell receptor-mediated proliferation, and immune evasion of cancer cells (Dong et al. (2003) J. Mal. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and this effect is additive when the interaction of PD-1 with PD-L2 is also blocked (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

[0007] There is a need in the art for antibodies or antigen-binding portions thereof, including chimeric and humanized antibodies, that neutralize / block PD-1 negative signaling and stimulate an immune response and that can be used to treat cancer and many other diseases. Summary of the Invention

[0008] The present disclosure provides monoclonal antibodies, or antigen-binding portions thereof, that specifically bind to and neutralize human PD-1. Monoclonal antibodies generated in mice can be immunogenic in humans. The present disclosure also provides humanized antibodies redesigned from the mouse antibodies to reduce immunogenicity in humans. These antibodies have improved efficacy and safety in humans.

[0009] In one aspect, provided herein is an antibody or antigen-binding fragment thereof that binds to PD-1 (programmed cell death protein 1), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH), the VH comprising complementarity determining regions (CDRs) 1, 2, and 3. In some embodiments, the VH CDR1 region comprises an amino acid sequence at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL), the VL comprising CDR1, 2, and 3. In some embodiments, the VL CDR1 region comprises an amino acid sequence at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL CDR3 amino acid sequence.

[0010] In some embodiments, the selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of the following:

[0011] (1) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 8, 9, and 10, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 5, 6, and 7, respectively;

[0012] (2) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, and 24, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, and 21, respectively;

[0013] (3) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 74, 75, and 76, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 71, 72, and 73, respectively;

[0014] (4) the selected VH CDR1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 34, 35, and 36, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 31, 32, and 33, respectively;

[0015] (5) the selected VH CDR1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 54, 55, and 56, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 51, 52, and 53, respectively;

[0016] (6) the selected VH CDR1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 64, 65, and 66, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 61, 62, and 63, respectively;

[0017] (7) The selected VH CDR1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 44, 45, and 46, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are listed in SEQ ID NOs: 41, 42, and 43, respectively.

[0018] In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences listed in SEQ ID NOs: 8, 9, and 10, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences listed in SEQ ID NOs: 5, 6, and 7, respectively.

[0019] In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences listed in SEQ ID NOs: 22, 23, and 24, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences listed in SEQ ID NOs: 19, 20, and 21, respectively.

[0020] In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences listed in SEQ ID NOs: 74, 75, and 76, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences listed in SEQ ID NOs: 71, 72, and 73, respectively.

[0021] In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences listed in SEQ ID NOs: 34, 35, and 36, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences listed in SEQ ID NOs: 31, 32, and 33, respectively.

[0022] In some embodiments, the antibody or antigen-binding fragment specifically binds to human PD-1.

[0023] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0024] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv).

[0025] In one aspect, a nucleic acid is provided, comprising a polynucleotide encoding a polypeptide comprising:

[0026] (1) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the complementarity determining regions comprise the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively, and in some embodiments, the VH binds to PD-1 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NOs: 2, 16, 28, 38, 48, 58, or 68;

[0027] (2) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively, and in some embodiments, the VL binds to PD-1 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NOs: 4, 18, 30, 40, 50, 60, or 70;

[0028] (3) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, and in some embodiments, the VH binds to PD-1 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NOs: 2, 16, 28, 38, 48, 58, or 68;

[0029] (4) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and in some embodiments, the VL binds to PD-1 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NOs: 4, 18, 30, 40, 50, 60, or 70;

[0030] (5) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 74, 75, and 76, respectively, and in some embodiments, the VH binds to PD-1 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NOs: 2, 16, 28, 38, 48, 58, or 68;

[0031] (6) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 71, 72, and 73, respectively, and in some embodiments, the VL binds to PD-1 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NOs: 4, 18, 30, 40, 50, 60, or 70;

[0032] (7) an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively, and in some embodiments, the VH binds to PD-1 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NOs: 2, 16, 28, 38, 48, 58, or 68;

[0033] (8) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, wherein CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and in some embodiments, the VL binds to PD-1 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NOs: 4, 18, 30, 40, 50, 60, or 70;

[0034] (9) an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 54, 55, and 56, respectively, and in some embodiments, the VH binds to PD-1 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NOs: 2, 16, 28, 38, 48, 58, or 68;

[0035] (10) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 51, 52, and 53, respectively, and in some embodiments, the VL binds to PD-1 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NOs: 4, 18, 30, 40, 50, 60, or 70;

[0036] (11) an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively, and in some embodiments, the VH binds to PD-1 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NOs: 2, 16, 28, 38, 48, 58, or 68;

[0037] (12) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, wherein CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and in some embodiments, the VL binds to PD-1 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NOs: 4, 18, 30, 40, 50, 60, or 70;

[0038] (13) an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 44, 45, and 46, respectively, and in some embodiments, the VH binds to PD-1 when paired with a VL comprising the amino acid sequence set forth in SEQ ID NOs: 2, 16, 28, 38, 48, 58, or 68;

[0039] (14) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 41, 42, and 43, respectively, and in some embodiments, the VL binds to PD-1 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 4, 18, 30, 40, 50, 60, or 70.

[0040] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively.

[0041] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively.

[0042] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.

[0043] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.

[0044] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 74, 75, and 76, respectively.

[0045] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 71, 72, and 73, respectively.

[0046] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, wherein the immunoglobulin heavy chain or fragment thereof comprises a VH, wherein the VH comprises CDR1, 2 and 3, and wherein the CDR1, 2 and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 34, 35 and 36, respectively.

[0047] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, wherein the immunoglobulin light chain or fragment thereof comprises a VL, wherein the VL comprises CDR1, 2 and 3, and wherein the CDR1, 2 and 3 comprise the amino acid sequences set forth in SEQ ID NOs: 31, 32 and 33, respectively.

[0048] In some embodiments, the VH specifically binds to human PD-1 when paired with the VL, or the VL specifically binds to human PD-1 when paired with the VH.

[0049] In some embodiments, the immunoglobulin heavy chain or the fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a humanized immunoglobulin light chain or a fragment thereof.

[0050] In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv).

[0051] In some embodiments, the nucleic acid is cDNA.

[0052] In one aspect, provided herein is a vector comprising one or more of the nucleic acids as described herein.

[0053] In one aspect, a vector is provided herein, comprising two of the nucleic acids described herein. In some embodiments, the vector encodes the VL region and the VH region that together bind to PD-1.

[0054] In one aspect, a vector pair is provided herein. In some embodiments, each vector comprises one of the nucleic acids described herein. In some embodiments, the vector pair together encodes the VL region and the VH region that together bind to PD-1.

[0055] In one aspect, provided herein is a cell comprising the vector as described herein or the vector pair as described herein.

[0056] In some embodiments, the cells are CHO cells.

[0057] In one aspect, provided herein is a cell comprising one or more of the nucleic acids as described herein.

[0058] In one aspect, provided herein is a cell comprising two of the nucleic acids as described herein.

[0059] In some embodiments, the two nucleic acids together encode the VL region and the VH region that together bind to PD-1.

[0060] In one aspect, provided herein is a method of producing an antibody or antigen binding fragment thereof, the method comprising (a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen binding fragment; and (b) collecting the antibody or the antigen binding fragment produced by the cell.

[0061] In one aspect, provided herein is an antibody or antigen binding fragment thereof that binds to PD-1, the antibody or antigen binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, the VL comprising an amino acid sequence that is at least 90% identical to a selected VL sequence. In some embodiments, the selected VH sequence is selected from SEQ ID NO: 4, 18, 30, 40, 50, 60, or 70, and the selected VL sequence is selected from SEQ ID NO: 2, 16, 28, 38, 48, 58, or 68.

[0062] In some embodiments, the VH comprises the sequence of SEQ ID NO: 4, and the VL comprises the sequence of SEQ ID NO: 2.

[0063] In some embodiments, the VH comprises the sequence of SEQ ID NO: 18, and the VL comprises the sequence of SEQ ID NO: 16.

[0064] In some embodiments, the VH comprises the sequence of SEQ ID NO: 70, and the VL comprises the sequence of SEQ ID NO: 68.

[0065] In some embodiments, the VH comprises the sequence of SEQ ID NO: 30, and the VL comprises the sequence of SEQ ID NO: 28.

[0066] In some embodiments, the VH comprises the sequence of SEQ ID NO: 50, and the VL comprises the sequence of SEQ ID NO: 48.

[0067] In some embodiments, the VH comprises the sequence of SEQ ID NO:60, and the VL comprises the sequence of SEQ ID NO:58.

[0068] In some embodiments, the VH comprises the sequence of SEQ ID NO:40, and the VL comprises the sequence of SEQ ID NO:38.

[0069] In some embodiments, the antibody or antigen-binding fragment specifically binds to human PD-1.

[0070] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0071] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv).

[0072] In one aspect, provided herein is an antibody or antigen-binding fragment thereof comprising the VH CDR1, 2, 3 and the VL CDR1, 2, 3 of the antibody or antigen-binding fragment thereof as described herein.

[0073] In one aspect, provided herein is an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof as described herein covalently bound to a therapeutic agent.

[0074] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0075] In one aspect, provided herein is a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein or the antibody-drug conjugate as described herein.

[0076] In some embodiments, the subject has a solid tumor or a hematological cancer (eg, lymphoma).

[0077] In some embodiments, the cancer is melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, relapsed or refractory classical Hodgkin lymphoma, squamous cell lung cancer, renal cell carcinoma, or squamous cell carcinoma of the skin.

[0078] In some embodiments, the cancer is urothelial carcinoma, Merkel cell carcinoma, or mesothelioma.

[0079] In one aspect, provided herein is a method of reducing tumor growth rate, the method comprising contacting an immune cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein or the antibody-drug conjugate as described herein, administered to a subject.

[0080] In one aspect, provided herein is a method of killing tumor cells, comprising contacting an immune cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein or the antibody-drug conjugate as described herein, administered to a subject.

[0081] In one aspect, provided herein is a method of treating or reducing the risk of an infectious disease, the method comprising administering to a subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein.

[0082] In some embodiments, provided herein are methods further comprising administering a vaccine to the subject.

[0083] In one aspect, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier.

[0084] In another aspect, provided herein is a pharmaceutical composition comprising the antibody drug conjugate as described herein and a pharmaceutically acceptable carrier.

[0085] In some embodiments, the antibody is an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody. In some embodiments, the antibody is a human IgG1 antibody.

[0086] In one aspect, an isolated monoclonal antibody, or antigen-binding portion thereof, comprises: (a) a light chain variable domain CDR1 comprising SEQ ID NO: 5; (b) a light chain variable domain CDR2 comprising SEQ ID NO: 6; and (c) a light chain variable domain CDR3 comprising SEQ ID NO: 7; (d) a heavy chain variable domain CDR1 comprising SEQ ID NO: 8; (e) a heavy chain variable domain CDR2 comprising SEQ ID NO: 9; and (f) a heavy chain variable domain CDR3 comprising SEQ ID NO: 10.

[0087] In one aspect, the monoclonal antibody or antigen-binding portion thereof blocks the interaction of PD-1 with both PD-L1 and PD-L2. Thus, the antibody or antigen-binding portion thereof can stimulate an anti-tumor immune response.

[0088] In yet another aspect, the present disclosure further provides a monoclonal antibody, or an antigen-binding portion thereof, comprising: a light chain variable domain comprising SEQ ID NO: 2 and a heavy chain variable domain comprising SEQ ID NO: 4.

[0089] The antibodies of the present disclosure can be further engineered into forms suitable for human therapeutics by modifications that minimize immunogenicity. Suitable antibodies include, but are not limited to, chimeric antibodies and humanized antibodies. The affinity, stability, and specificity of the disclosed antibodies can also be further optimized by techniques known to those skilled in the art. Other forms can include oligomerization, drug conjugation, and fusion of the disclosed antibodies with other functional proteins.

[0090] In yet another aspect, the humanized monoclonal antibody or antigen-binding portion thereof comprises a light chain variable domain amino acid sequence that is at least 95% identical to SEQ ID NO: 2 and a heavy chain variable domain amino acid sequence that is at least 95% identical to SEQ ID NO: 4.

[0091] In yet another aspect, the present disclosure further provides an isolated monoclonal antibody or antigen-binding portion thereof comprising (a) a light chain variable domain CDR1 comprising SEQ ID NO: 5; (b) a light chain variable domain CDR2 comprising SEQ ID NO: 6; (c) a light chain variable domain CDR3 comprising SEQ ID NO: 7; and a heavy chain variable domain amino acid sequence that is at least 95% identical to SEQ ID NO: 4.

[0092] In yet another aspect, the present disclosure further provides an isolated monoclonal antibody or antigen-binding portion thereof comprising (a) a heavy chain variable domain CDR1 comprising SEQ ID NO: 8; (b) a heavy chain variable domain CDR2 comprising SEQ ID NO: 9; (c) a heavy chain variable domain CDR3 comprising SEQ ID NO: 10; and a light chain variable domain amino acid sequence that is at least 95% identical to SEQ ID NO: 2.

[0093] The antibodies of the present disclosure can be, for example, full-length antibodies, such as IgGl isotype, IgG2 isotype, IgG3 isotype, or IgG4 isotype. Alternatively, the disclosed antibodies can be antibody fragments, such as Fab fragments, Fab' fragments, and F(ab')2 fragments, diabodies, triabodies, tetrabodies, single-chain variable region fragments (scFv), disulfide-stabilized variable region fragments (dsFv), and half-antibodies. Alternatively, the disclosed antibodies can be bispecific antibodies.

[0094] In another aspect of the disclosure, an isolated monoclonal antibody or antigen binding portion thereof comprises a light chain comprising SEQ ID NO: 11 and a heavy chain comprising SEQ ID NO: 12. In preferred embodiments, the isolated monoclonal antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 13 and a heavy chain having the amino acid sequence of SEQ ID NO: 14.

[0095] In another aspect of the disclosure, a pharmaceutical composition comprising an isolated monoclonal antibody or antigen binding portion thereof and a pharmaceutically acceptable carrier is also provided. A composition comprising an immunoconjugate of the disclosure and a pharmaceutically acceptable carrier is also provided.

[0096] In another aspect of the disclosure, an isolated nucleic acid molecule encoding an antibody or antigen binding portion thereof and a host cell comprising a sequence of a nucleic acid molecule encoding an antibody or antigen binding portion thereof are also provided. In preferred embodiments, the isolated nucleic acid molecule further comprises a sequence encoding a signal peptide. In preferred embodiments, the isolated nucleic acid molecule encoding an antibody or antigen binding portion thereof comprises the nucleic acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 3. In preferred embodiments, the host cell is a hybridoma cell.

[0097] The disclosure further provides a method of stimulating an immune response using an anti-PD-1 antibody of the disclosure. For example, in one embodiment, the disclosure provides a method for treating a subject in need thereof, the method comprising the step of administering to the subject an effective amount of an antibody or antigen binding portion as described herein.

[0098] In another aspect, the disclosure provides a method for treating a cancer in a human, the method comprising the step of administering to the human an antibody or antigen binding portion as described herein in an amount effective to treat the cancer.

[0099] In another aspect, the disclosure provides a method for treating an infectious disease in a human, the method comprising the step of administering to the human the antibody or antigen binding portion as described herein in an amount effective to treat the infectious disease.

[0100] In one aspect, the antibody or portion that specifically binds to human PD-1 blocks the interaction between PD-1 and PD-L1.

[0101] In one aspect, provided herein is a pharmaceutical composition comprising the antibody or antigen binding portion thereof as described herein and a pharmaceutically acceptable carrier.

[0102] In one aspect, provided herein is a method of stimulating an immune response in a subject, comprising the step of administering to the subject the pharmaceutical composition as described herein in an amount effective to stimulate an immune response in the subject.

[0103] In one aspect, provided herein is a method of treating an infectious disease in a subject, comprising the step of administering to the subject the pharmaceutical composition as described herein in an amount effective for treating the infectious disease.

[0104] In one aspect, provided herein is a method of treating cancer in a subject, comprising the step of administering to said subject said pharmaceutical composition as described herein in an amount effective to treat said cancer.

[0105] In some embodiments, the monoclonal antibody, or antigen-binding portion thereof, as described herein is a Fab fragment, a F(ab')2 fragment, a Fv fragment, a single chain antibody, or a bispecific antibody.

[0106] In some embodiments, the monoclonal antibodies as described herein are chimeric or humanized antibodies.

[0107] In some embodiments, the monoclonal antibodies as described herein are immunoglobulin G (IgG) molecules, IgM molecules, IgE molecules, IgA molecules or IgD molecules or derivatives thereof. In some embodiments, the monoclonal antibodies described herein are IgG1, IgG2, IgG3 or IgG4 or derivatives thereof.

[0108] In some embodiments, the monoclonal antibodies, or antigen-binding portions thereof, as described herein block the interaction between PD-1 and PD-L2.

[0109] In one aspect, provided herein is a monoclonal antibody comprising: a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4.

[0110] In certain embodiments, the monoclonal antibodies as described herein are immunoglobulin G (IgG) molecules, IgM molecules, IgE molecules, IgA molecules or IgD molecules or derivatives thereof. In certain embodiments, the monoclonal antibodies as described herein are IgG1, IgG2, IgG3 or IgG4 or derivatives thereof.

[0111] In some embodiments, the monoclonal antibodies as described herein are chimeric antibodies.

[0112] In some embodiments, the monoclonal antibody, or antigen-binding portion thereof, as described herein comprises a light chain variable domain amino acid sequence that is at least 95% identical to SEQ ID NO:2 and a heavy chain variable domain amino acid sequence that is at least 95% identical to SEQ ID NO:4.

[0113] In one aspect, provided herein is an isolated nucleic acid molecule encoding the antibody, or antigen-binding portion thereof, as described herein.

[0114] In one aspect, provided herein is a host cell comprising the sequence of the nucleic acid molecule as described herein.

[0115] In one aspect, provided herein is an immunoconjugate comprising the antibody, or antigen-binding portion thereof, as described herein linked to a therapeutic agent.

[0116] In one aspect, provided herein is a monoclonal antibody, or an antigen-binding portion thereof, comprising: a light chain comprising SEQ ID NO: 11; and a heavy chain comprising SEQ ID NO: 12.

[0117] In one aspect, the methods described herein can also be used to treat T cell dysfunction, including infections (eg, acute and chronic) and tumor immunity.

[0118] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention pertains. Methods and materials for use with the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0119] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Exemplary embodiments are illustrated in the accompanying drawings to which reference is made. It is intended that the embodiments and drawings disclosed herein be regarded as illustrative rather than restrictive.

[0121] Figure 1Western blot analysis with anti-PD-1 antibody (clone #11) is shown. HEK293T cells were transfected with pCMV6-ENTRY control (left lane) or pCMV6-ENTRY PDCD1 (RC210364, right lane) cDNA for 48 hours and then lysed. Equivalent cell lysates (5 μg per lane) were separated by SDS-PAGE and immunoblotted with anti-PD-1 antibody (1:2000).

[0122] Figure 2A Immunofluorescence staining of HEK293T cells stably transfected with PDCD1 (RC210364) is shown. PD-1 was labeled with a mouse monoclonal anti-PD-1 antibody, and cell nuclei were labeled with Hoechst 33342.

[0123] Figure 2B Shown are immunofluorescence staining of HEK293T cells as a negative control (1:100).

[0124] Figure 3A Shown are immunocytochemical staining of cells stably expressing PD-1 using an anti-PD-1 mouse monoclonal antibody (left).

[0125] Figure 3B Shown are immunocytochemical staining of 293T cells as a negative control (1:900).

[0126] Figure 4 Shown are flow cytometric analyses of cells stably expressing PD-1 using an anti-PD-1 antibody (F02) compared to a nonspecific negative control antibody (1:50).

[0127] Figure 5 Figure 2 shows flow cytometric analysis of cells stably expressing PD-L1 (RC213071) using anti-PD-1 antibody from hybridoma clone #11 (F02) or 0.3 ug / ml PD1-Fc fusion protein (TP700199), or both, and detected by anti-Fc (human) IgG-FITC (1:50). The binding of PD-L1 to PD-1 was completely blocked by the anti-PD-1 antibody from hybridoma clone #11.

[0128] Figure 6Figure 2 shows flow cytometric analysis of HEK293T cells transiently transfected with PD-L2 (RC224141) using anti-PD-1 antibody from hybridoma clone #11 (F02) or 1 μg / ml PD1-Fc fusion protein (TP700199), or both, and detected by anti-Fc (human) IgG-FITC (1:50). The binding of PD-L2 to PD-1 was completely blocked by the anti-PD-1 antibody from hybridoma clone #11.

[0129] Figure 7A Figure 2 shows the binding curve between the F02 anti-PD-1 antibody and recombinant human PD-1 protein (rhPD-1). The binding affinity of F02 for rhPD-1 was determined by ELISA. The Kd value was determined to be 0.14 nM.

[0130] Figure 7B Figure 2 shows the binding curve between h11 anti-PD-1 antibody and rhPD-1. The binding affinity of h11 for rhPD-1 was determined by ELISA. The Kd value was determined to be 0.29 nM.

[0131] Figure 7C The binding curve between Ab2 anti-PD-1 antibody and rhPD-1 is shown.

[0132] Figure 7D The binding curve between Ab4 anti-PD-1 antibody and rhPD-1 is shown.

[0133] Figure 7E The binding curve between Ab5 anti-PD-1 antibody and rhPD-1 is shown.

[0134] Figure 7F The binding curve between Ab7 anti-PD-1 antibody and rhPD-1 is shown. The Kd value was determined to be 0.68 nM.

[0135] Figure 7G The binding curve between Ab8 anti-PD-1 antibody and rhPD-1 is shown. The Kd value was determined to be 0.32 nM.

[0136] Figure 8A Figure 2 shows the binding curve between the F02 anti-PD-1 antibody and the human PD-1 protein (hPD-1) on the cell surface. The binding affinity of F02 for hPD-1 on the cell surface was determined by flow cytometry. The Kd value was determined to be 0.07 nM.

[0137] Figure 8BFigure 2 shows the binding curve between h11 anti-PD-1 antibody and human PD-1 protein (hPD-1) on the cell surface. The binding affinity of h11 to hPD-1 on the cell surface was determined by flow cytometry. The Kd value was determined to be 0.55 nM.

[0138] Figure 8C The binding curve between Ab2 anti-PD-1 antibody and human PD-1 protein (hPD-1) on the cell surface is shown. The Kd value was determined to be 3.24 nM.

[0139] Figure 8D The binding curve between Ab4 anti-PD-1 antibody and human PD-1 protein (hPD-1) on the cell surface is shown. The Kd value was determined to be 41 nM.

[0140] Figure 8E The binding curve between Ab5 anti-PD-1 antibody and human PD-1 protein (hPD-1) on the cell surface is shown. The Kd value was determined to be 3.69 nM.

[0141] Figure 8F The binding curve between Ab7 anti-PD-1 antibody and human PD-1 protein (hPD-1) on the cell surface is shown. The Kd value was determined to be 2.3 nM.

[0142] Figure 8G The binding curve between Ab8 anti-PD-1 antibody and human PD-1 protein (hPD-1) on the cell surface is shown. The Kd value was determined to be 0.65 nM.

[0143] Figure 9 Sequences described in this disclosure are listed. DETAILED DESCRIPTION

[0144] The present disclosure relates to isolated monoclonal antibodies that bind to PD-1 and block the interaction between PD-1 and PD-L1. In certain embodiments, the monoclonal antibodies or antigen-binding portions thereof also block the interaction between PD-1 and PD-L2. In certain embodiments, the antibodies of the present disclosure are derived from the identified heavy chain and light chain germline sequences and / or include identified structural features such as CDR regions that include identified amino acid sequences. The present disclosure provides isolated antibodies, methods for preparing such antibodies, and antigen-binding portions thereof of the present disclosure. The present disclosure also relates to methods of stimulating an immune response using the antibodies, such as the anti-PD-1 antibodies of the present disclosure, alone or in combination with other immunostimulatory antibodies. Therefore, methods of using the anti-PD-1 antibodies of the present disclosure, for example, including but not limited to, methods of treating cancer in the human body, are also provided.

[0145] Therapeutic mouse antibodies can sometimes elicit a human anti-mouse antibody (HAMA) response. In this case, the therapeutic antibody may lose its efficacy. The present disclosure also provides humanized antibodies that reduce the HAMA response and prolong the therapeutic efficacy.

[0146] In certain embodiments, the humanized antibodies can be used for anticancer therapy. In certain embodiments, the humanized antibodies can be used as independent therapeutic agents. In certain embodiments, the humanized antibodies can be used in combination with cell therapy. In certain embodiments, the humanized antibodies described herein can be further refined into single-chain variable fragments (e.g., scFv).

[0147] As used herein, the term "comprising" or "comprises" is used with reference to compositions, methods, and one or more corresponding components thereof that are useful for the embodiments but include unspecified elements (whether useful or not). Those skilled in the art will understand that, in general, the terms used herein are generally intended to be "open-ended" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "include" should be interpreted as "including but not limited to," etc.).

[0148] Unless otherwise indicated, the terms "a" and "an" and "said" and similar indicators used in the context of describing a particular embodiment of the present application (particularly in the context of the claims) may be interpreted as covering both the singular and the plural. Reference to a range of values ​​herein is intended only to serve as a simplified method of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if each individual value were individually quoted herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided with respect to certain embodiments herein is intended only to better illustrate the present application and does not limit the scope of the present application claimed in other ways. The abbreviation "e.g., for example" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Therefore, the abbreviation "e.g., for example" is synonymous with the term "for example". Any language in the specification should not be interpreted as indicating that any unclaimed element is necessary for practicing the present application.

[0149] As used herein, the term "about" refers to a measurable value such as an amount, a duration, and the like, and encompasses variations that can exist in the values that are designated. For example, "about 10%" can include 10% ± 20%, 10% ± 10%, 10% ± 5%, 10% ± 1%, 10% ± 0.5%, or 10% ± 0.1%.

[0150] As used herein, the term "epitope" can include any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants are usually composed of chemically active surface groupings of molecules such as amino acids or sugar side chains and are usually located on the surface of a molecule. Antibodies are said to specifically bind an antigen when the equilibrium dissociation constant is < 1 μM, preferably < 100 nM, and most preferably < 10 nM.

[0151] As used herein, the term "immune response" can refer to the actions by lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble molecules produced by the above cells or liver, including antibodies, cytokines, and complement, that result in the selective damage, destruction or elimination of invading pathogens, cells or tissues infected with a pathogen, cancerous cells, or normal organism cells or tissues in the context of autoimmunity or pathological inflammation from a biological organism.

[0152] As used herein, the term "antigen-specific T cell response" can refer to a response by a T cell that results from stimulation of the T cell with an antigen specific to the T cell. Non-limiting examples of responses by a T cell to antigen-specific stimulation include, but are not limited to, proliferation and cytokine production (e.g., production of IL-2).

[0153] As used herein, the term "antibody" refers to an intact immunoglobulin, or to a monoclonal or polyclonal antigen-binding fragment having an Fc (fragment crystallizable) region or Fc region FcRn binding fragment (referred to herein as an "Fc fragment" or "Fc domain"). Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single-domain antibodies, chimeric antibodies, diabodies, and polypeptides that contain enough of the immunoglobulin to confer specific antigen-binding properties on the polypeptide. Fc domains comprise portions of both heavy chains that contribute to class two or class three antibodies. Fc domains can be produced by recombinant DNA techniques, or by enzymatic (e.g., papain cleavage) or by chemical cleavage of intact antibodies.

[0154] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a protein fragment that comprises only a portion of an intact antibody, typically comprising the antigen-binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) a Fab fragment, which has a VL, CL, VH, and CH1 domain; (ii) a Fab' fragment, which is a Fab fragment with one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) an Fd fragment, which has a VH and CH1 domain; (iv) an Fd' fragment, which has a VH and CH1 domain and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) an Fv fragment, which has the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists of a VH binding site. domain composition; (vii) isolated CDR regions; (viii) a F(ab')2 fragment, a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region; (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies" with two antigen-binding sites, comprising a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 547858). 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); (xi) “linear antibodies” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

[0155] As used herein, a "single-chain variable fragment," "single-chain antibody variable fragment," or "scFv" antibody refers to a form of antibody comprising only the variable regions of a heavy chain (VH) and a light chain (VL) connected by a linker peptide. scFv can be expressed as a single-chain polypeptide. scFv retains the specificity of the intact antibody from which it is derived. The light and heavy chains can be in any order, for example, VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for the target antigen is retained.

[0156] As used herein, an "isolated antibody" can refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a PD-1 protein can be substantially free of antibodies that specifically bind antigens other than a PD-1 protein). An isolated antibody specific for a human PD-1 protein, however, can have cross-reactivity to other antigens, such as PD-1 proteins from other species. Moreover, an isolated antibody can be substantially free of other cellular material and / or chemicals.

[0157] Anti-PD-1 antibody-producing cells, such as hybridomas, can be selected, cloned, and further screened for desirable characteristics, including robust growth, high antibody production, and desirable antibody characteristics. Hybridomas can be expanded in syngeneic and immunodeficient animals (e.g., nude mice) or in vitro in cell culture. Methods of selecting, cloning, and expanding hybridomas are well known to those of ordinary skill in the art.

[0158] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" can refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0159] As used herein, the term "recombinant human antibody" can refer to all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described herein below), (b) antibodies isolated from a host cell transformed to express human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, can not naturally occur within the human antibody germline repertoire in vivo.

[0160] As used herein, the term "isotype" can refer to the class of antibody (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.

[0161] A "humanized antibody" has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that when the humanized antibody is administered to a human subject, the humanized antibody is less likely to induce an anti-antibody immune response and / or induces a less severe anti-antibody immune response than the non-human species antibody. In some embodiments, certain amino acids in the framework domains and constant domains of the heavy and / or light chains of a non-human species antibody are mutated to produce a humanized antibody. In some embodiments, one or more constant domains from a human antibody are fused to one or more variable domains of a non-human species. In some embodiments, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the potential immunogenicity of the non-human antibody when the non-human antibody is administered to a human subject, wherein the altered amino acid residues are not critical for the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes such that the binding of the humanized antibody to the antigen is not significantly worse than that of the non-human antibody to the antigen. Examples of how to make humanized antibodies can be found in US Pat. Nos. 6,054,297, 5,886,152, and 5,877,293, which are incorporated herein by reference in their entirety.

[0162] As used herein, the term "chimeric antibody" may refer to an antibody in which the variable region sequence may be derived from one species and the constant region sequence may be derived from another species, such as an antibody in which the variable region sequence may be derived from a mouse antibody and the constant region sequence may be derived from a human antibody.

[0163] As used herein, an antibody that "specifically binds to human PD-1" may refer to an antibody that binds to human PD-1 protein (and possibly PD-1 protein from one or more non-human species) but does not substantially bind to non-PD-1 proteins. In some embodiments, the antibody binds to human PD-1 protein with "high affinity," for example, with an EC50 of 1×10 -7 M or less, more preferably 5×10 -8 M or less, more preferably 3×10 -8 M or less, more preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less or even more preferably 1×10 -9 M or less. The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0164] As used herein, the term "substantially does not bind to a protein or cell" can mean that it does not bind to a protein or cell with high affinity or does not bind to a protein or cell with high affinity, i.e., binds to a protein or cell with an EC50 of 2 x 10 -6 M or more, more preferably 1 x 10 -5 M or more, more preferably 1 x 10 -4 M or more, more preferably 1 x 10 -3 M or more, even more preferably 1 x 10 -2 M or more.

[0165] As used herein, the term "high affinity" for an IgG antibody can refer to an antibody with a Kd of 1 x 10 -6 M or less, more preferably 1 x 10 -7 M or less, more preferably 1 x 10 -8 M or less, more preferably 1 x 10 -9 M or less, more preferably 1 x 10 -10 M or less. However, "high affinity" binding can differ for other antibody isotypes.

[0166] As used herein, the term "pharmaceutical formulation" refers to a preparation which is in a form suitable for administration to a subject to be treated and which is in a form which is effective for the biological activity of the active ingredient contained therein and which does not contain additional components which contribute unacceptable toxicity to the subject to which the formulation is administered.

[0167] As used herein, the term "inhibit" refers to any reduction in, for example, a particular action, function or interaction. For example, a biological function, such as the function of a protein and / or the binding of one protein to another, is inhibited if it is reduced compared to an equivalent reference state, such as a control like a wild-type state or a state where no agent is applied. For example, the binding of a PD-1 protein to one or more of its ligands, such as PD-L1 and / or PD-L2, and / or the resulting PD-1 signaling and immune effects are inhibited or deficient if the binding, signaling and other immune effects are reduced as a result of contact with an agent, such as an anti-PD-1 antibody, compared to when the PD-1 protein is not in contact with the agent. Such inhibition or deficiency can be induced, for example, by application of the agent at a particular time and / or location, or can be constitutive, such as by continuous administration. Such inhibition or deficiency can also be partial or complete, for example, substantially no measurable activity compared to a reference state, such as a control like a wild-type state. Substantially complete inhibition or deficiency is referred to as blocking.

[0168] As used herein, the term "subject" can refer to any human or non-human animal. A subject can be male or female, and can be at any suitable age, including infant subjects, juvenile subjects, adolescent subjects, adult subjects, and geriatric subjects. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, rabbits, mice, rats, amphibians, and reptiles, but is preferably a mammal, such as a non-human primate, sheep, dog, cat, cow, and horse.

[0169] PD-1 and the immune system

[0170] The immune system can distinguish between normal cells in the body and those that are considered "foreign," which allows the immune system to attack foreign cells while leaving normal cells alone. This mechanism sometimes involves proteins known as immune checkpoints. Immune checkpoints are molecules in the immune system that either enhance a signal (co-stimulatory molecules) or dampen a signal.

[0171] Checkpoint inhibitors can prevent the immune system from attacking normal tissue, and thus prevent autoimmune diseases. Many tumor cells also express checkpoint inhibitors. These tumor cells escape immune surveillance by co-opting certain immune checkpoint pathways, particularly in T cells specific for tumor antigens (Creelan, Benjamin C. "Update on immune checkpoint inhibitors in lung cancer." Cancer Control 21.1 (2014): 80-89). Because many immune checkpoints are triggered by ligand-receptor interactions, they can be easily blocked by antibodies directed against the ligand and / or its receptor.

[0172] PD-1 (Programmed cell death 1; PDCD1; or Programmed death 1) is an immune checkpoint, and prevents autoimmunity by a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while reducing apoptosis in regulatory T cells (anti-inflammatory suppressor T cells).

[0173] PD-1 is primarily expressed on the surface of T cells and primary B cells; two ligands of PD-1 (PD-L1 and PD-L2) are widely expressed in antigen-presenting cells (APCs). The interaction of PD-1 with its ligands plays an important role in the negative regulation of immune responses. Inhibiting the binding between PD-1 and its ligands can expose tumor cells to the killing effects of the immune system, and thus can achieve the effect of killing tumor tissue and treating cancer.

[0174] PD-L1 (CD274) is expressed on neoplastic cells of many different cancers. PD-L1 expression is a major mechanism by which tumor cells can evade immune attack, by binding to PD-1 on T cells leading to their inhibition. PD-L1 overexpression can be conceptually due to 2 mechanisms, intrinsic and adaptive. Intrinsic expression of PD-L1 on cancer cells is associated with cellular / genetic aberrations in these neoplastic cells. Activation of cellular signaling including AKT and STAT pathways increases PD-L1 expression. In primary mediastinal B-cell lymphoma, gene fusion of MHC class II transactivator (CIITA) with PD-L1 or PD-L2 occurs, leading to overexpression of these proteins. Amplification of chromosome 9p23-24 where PD-L1 and PD-L2 are located leads to increased expression of both proteins in classical Hodgkin lymphoma. Adaptive mechanisms are associated with induction of PD-L1 expression in the tumor microenvironment. PD-L1 can be induced on neoplastic cells in response to interferon gamma. In microsatellite instability colon cancer, PD-L1 is expressed primarily on myeloid cells in the tumor, which then suppress cytotoxic T cell function.

[0175] The use of PD-1 blockade to enhance anti-tumor immunity stems from observations in chronic infection models, where prevention of PD-1 interaction reversed T cell exhaustion. Similarly, blockade of PD-1 prevents T cell PD-1 / tumor cell PD-L1 or T cell PD-1 / tumor cell PD-L2 interaction, resulting in restoration of T cell mediated anti-tumor immunity.

[0176] Detailed descriptions of PD-1 and the use of anti-PD-1 antibodies for the treatment of cancer are described in, for example, Topalian, Suzanne L., et al. "Safety, activity, and immune correlates of anti-PD-1 antibody in cancer." New England Journal of Medicine 366.26 (2012): 2443-2454; Hirano, Fumiya, et al. "Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity." Cancer research 65.3 (2005): 1089-1096; Raedler, Lisa A. "Keytruda (pembrolizumab): first PD-1 inhibitor approved for previously treated unresectable or metastatic melanoma." American health & drug benefits 8. Spec Feature (2015): 96; Kwok, Gerry, et al. "Pembrolizumab (Keytruda)." (2016): 2777-2789; US 20170247454; US 9,834,606 B; and US 8,728,474; each of which is incorporated by reference in its entirety.

[0177] The present disclosure provides anti-PD-1 antibodies, antigen-binding fragments thereof, and methods of using these anti-PD-1 antibodies and antigen-binding fragments to inhibit tumor growth and treat cancer.

[0178] Anti-PD-1 antibodies and antigen-binding fragments

[0179] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to PD-1. The antibodies and antigen-binding fragments described herein are capable of binding to PD-1. In some embodiments, these antibodies can block the PD-1 signaling pathway, thereby increasing the immune response. In some embodiments, these antibodies can elicit complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).

[0180] The present disclosure provides, for example, mouse anti-PD-1 antibodies (e.g., F02) and chimeric antibodies and humanized antibodies thereof (e.g., H11, Ab8, Ab2, Ab5, Ab7, Ab4).

[0181] The CDR sequences of F02 and F02-derived antibodies or antigen-binding fragments thereof comprise a VH CDR1, a VH CDR2, and a VH CDR3 comprising or consisting of SEQ ID NOs: 8, 9, 10, respectively, and a VL CDR1, a VL CDR2, and a VL CDR3 comprising or consisting of SEQ ID NOs: 5, 6, 7, respectively.

[0182] The CDR sequences of H11 and H11-derived antibodies or antigen-binding fragments thereof comprise a VH CDR1, a VH CDR2, and a VH CDR3 comprising or consisting of SEQ ID NOs: 22, 23, 24, respectively, and a VL CDR1, a VL CDR2, and a VL CDR3 comprising or consisting of SEQ ID NOs: 19, 20, 21, respectively.

[0183] The CDR sequences of Ab8 and Ab8-derived antibodies or antigen-binding fragments thereof comprise a VH CDR1, a VH CDR2, and a VH CDR3 comprising or consisting of SEQ ID NOs: 74, 75, 76, respectively, and a VL CDR1, a VL CDR2, and a VL CDR3 comprising or consisting of SEQ ID NOs: 71, 72, 73, respectively.

[0184] The CDR sequences of Ab2 and Ab2-derived antibodies or antigen-binding fragments thereof comprise a VH CDR1, a VH CDR2, and a VH CDR3 comprising or consisting of SEQ ID NOs: 34, 35, 36, respectively, and a VL CDR1, a VL CDR2, and a VL CDR3 comprising or consisting of SEQ ID NOs: 31, 32, 33, respectively.

[0185] The CDR sequences of Ab5 and Ab5-derived antibodies or antigen-binding fragments thereof comprise VH CDR1, VH CDR2, and VH CDR3 comprising or consisting of SEQ ID NOs: 54, 55, 56, respectively, and VL CDR1, VL CDR2, and VL CDR3 comprising or consisting of SEQ ID NOs: 51, 52, 53, respectively.

[0186] The CDR sequences of Ab7 and Ab7-derived antibodies or antigen-binding fragments thereof comprise VH CDR1, VH CDR2, and VH CDR3 comprising or consisting of SEQ ID NOs: 64, 65, 66, respectively, and VL CDR1, VL CDR2, and VL CDR3 comprising or consisting of SEQ ID NOs: 61, 62, 63, respectively.

[0187] The CDR sequences of Ab8 and Ab8-derived antibodies or antigen-binding fragments thereof comprise VH CDR1, VH CDR2, and VH CDR3 comprising or consisting of SEQ ID NOs: 44, 45, 46, respectively, and VL CDR1, VL CDR2, and VL CDR3 comprising or consisting of SEQ ID NOs: 41, 42, 43, respectively.

[0188] The amino acid sequence of the heavy chain variable region of the F02 antibody is set forth in SEQ ID NO: 4. The amino acid sequence of the light chain variable region of the F02 antibody is set forth in SEQ ID NO: 2.

[0189] The amino acid sequence of the heavy chain variable region of the H11 antibody is set forth in SEQ ID NO: 18. The amino acid sequence of the light chain variable region of the H11 antibody is set forth in SEQ ID NO: 16.

[0190] The amino acid sequence of the heavy chain variable region of the Ab8 antibody is set forth in SEQ ID NO: 70. The amino acid sequence of the light chain variable region of the Ab8 antibody is set forth in SEQ ID NO: 68.

[0191] The amino acid sequence of the heavy chain variable region of Ab2 antibody is set forth in SEQ ID NO: 30. The amino acid sequence of the light chain variable region of Ab2 antibody is set forth in SEQ ID NO: 28.

[0192] The amino acid sequence of the heavy chain variable region of the Ab5 antibody is set forth in SEQ ID NO: 50. The amino acid sequence of the light chain variable region of the Ab5 antibody is set forth in SEQ ID NO: 48.

[0193] The amino acid sequence of the heavy chain variable region of the Ab7 antibody is set forth in SEQ ID NO: 60. The amino acid sequence of the light chain variable region of the Ab7 antibody is set forth in SEQ ID NO: 58.

[0194] The amino acid sequence of the heavy chain variable region of Ab4 antibody is set forth in SEQ ID NO: 40. The amino acid sequence of the light chain variable region of Ab4 antibody is set forth in SEQ ID NO: 38.

[0195] Also provided are the amino acid sequences of the heavy chain variable region and light variable region of humanized antibodies. Due to the presence of different ways to humanize mouse antibodies (e.g., different amino acid substitutions can be used to modify the sequence), the heavy chain and light chain of the antibody can have more than one version in the humanized sequence. In certain embodiments, the humanized heavy chain variable region is at least 80% of SEQ ID NO: 4, 18, 70, 30, 50, 60 or 40, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. In certain embodiments, the humanized light chain variable region is at least 80% of SEQ ID NO: 2, 16, 68, 28, 48, 58 or 38, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. The heavy chain variable region sequence can be paired with the light chain variable region sequence, and the heavy chain variable region sequence and the light chain variable region sequence together bind to PD-1.

[0196] Percent humanization means the percent identity of the heavy or light chain variable region sequence to human antibody sequences in the international immunogenetics information system (IMGT) database. Top hit means that the heavy or light chain variable region sequence is closer to a particular species than to other species. For example, a top hit to human means that the sequence is closer to human than to other species. A top hit to human and Macaca fascicularis means that the sequence has the same percent identity to the human sequence and the Macaca fascicularis sequence, and that these percent identities are the highest compared to sequences of other species. In some embodiments, the percent humanization is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of how to determine percent humanization and how to determine top hit are known in the art and are described in, for example, Jones et al. “The INNs and outs of antibody nonproprietary names” MAbs Journal, Volume 8, Issue 1. Taylor and Francis, 2016, which is incorporated herein by reference in its entirety. A high percent humanization generally has various advantages, for example, safer and more effective in humans, better tolerated by human subjects, and / or less likely to have side effects.

[0197] In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 8-10, 22-24, 74-76, 34-36, 54-56, 64-66, and 44-46; and / or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 5-7, 19-21, 71-73, 31-33, 51-53, 61-63, and 41-43.

[0198] In some embodiments, the antibody can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibody can have a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are shown in Table 1. Figure 9

[0199] In some embodiments, the antibody or antigen-binding fragment described herein can have a heavy chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 8 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 10 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0200] In some embodiments, the antibody or antigen-binding fragment described herein can have a heavy chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 22 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0201] ​In some embodiments, an antibody or antigen-binding fragment described herein can comprise a heavy chain variable domain comprising one, two, or three of the CDRs of SEQ ID NO: 74 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 75 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 76 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0202] In some embodiments, an antibody or antigen-binding fragment described herein can comprise a heavy chain variable domain comprising one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0203] In some embodiments, an antibody or antigen-binding fragment described herein can comprise a heavy chain variable domain comprising one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0204] In some embodiments, an antibody or antigen-binding fragment described herein can comprise a heavy chain variable domain comprising one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0205] In some embodiments, an antibody or antigen-binding fragment described herein can comprise a heavy chain variable domain comprising one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0206] In some embodiments, an antibody or antigen-binding fragment described herein can contain a heavy chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one, or two amino acid insertions, deletions, or substitutions; and a light chain variable domain that contains one, two, or three of the CDRs of SEQ ID NO: 2 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0207] Insertions, deletions, and substitutions can be within a CDR sequence, or at one or both ends of a CDR sequence. In some embodiments, CDRs are determined based on the Kabat numbering scheme.

[0208] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to PD-1. The antibody or antigen-binding fragment thereof contains a heavy chain variable region (VH) that includes or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) that includes or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 4, and the selected VL sequence is SEQ ID NO: 2. In some embodiments, the selected VH sequence is SEQ ID NO: 18, and the selected VL sequence is SEQ ID NO: 16. In some embodiments, the selected VH sequence is SEQ ID NO: 70, and the selected VL sequence is SEQ ID NO: 68. In some embodiments, the selected VH sequence is SEQ ID NO: 30, and the selected VL sequence is SEQ ID NO: 28. In some embodiments, the selected VH sequence is SEQ ID NO: 50, and the selected VL sequence is SEQ ID NO: 48. In some embodiments, the selected VH sequence is SEQ ID NO: 60, and the selected VL sequence is SEQ ID NO: 58. In some embodiments, the selected VH sequence is SEQ ID NO: 40, and the selected VL sequence is SEQ ID NO: 38.

[0209] The present disclosure also provides nucleic acids that include a polynucleotide that encodes a polypeptide that includes an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or the immunoglobulin light chain includes a CDR, or has a sequence as shown in Figure 9 The paired polypeptide binds to PD-1 (e.g., human PD-1) when the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region).

[0210] The anti-PD-1 antibodies and antigen-binding fragments can also be antibody variants, including derivatives and conjugates, of antibodies or antibody fragments and multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific antibodies (multimeric antibodies, e.g., bispecific antibodies), human antibodies, chimeric antibodies (e.g., human murine chimeras), single-chain antibodies, intracellularly manufactured antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass. In some embodiments, the antibodies or antigen-binding fragments thereof are IgG antibodies or antigen-binding fragments thereof.

[0211] Fragments of antibodies are suitable for use in the provided methods so long as they exhibit the desired affinity and specificity of the full-length antibody. Thus, fragments of antibodies that bind to PD-1 will retain the ability to bind to PD-1. Fv fragments are the smallest unit of an antibody that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight association which, in nature, can be covalent, e.g., in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, these six CDRs, or subsets thereof, confer antigen-binding specificity to an antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although generally at a lower affinity than the intact binding site. Single-chain Fv or (scFv) antibody fragments include the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0212] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any of the antibodies or antigen-binding fragments as described herein. Cross-competition assays are known in the art and are described, for example, in Moore et al.,“Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 external envelope glycoprotein,” Journal of virology 70.3 (1996): 1863-1872, which is incorporated by reference herein in its entirety. In an aspect, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any of the antibodies or antigen-binding fragments described herein. Epitope binning assays are known in the art and are described, for example, in Estep et al.,“High throughput solution-based measurement of antibody-antigen affinity and epitope binning,” MAbs 5.2 (2013): 237- 249, which is incorporated by reference herein in its entirety.

[0213] In an aspect, the present disclosure provides an isolated human PD-1 binding monoclonal antibody or antigen-binding portion thereof, comprising: (a) a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 2; and (b) a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NO: 4; wherein the antibody or portion blocks the interaction between PD-1 and PD-L1 and the interaction between PD-1 and PD-L2.

[0214] In another aspect, the present disclosure provides an isolated monoclonal antibody or antigen binding portion thereof, comprising: (a) a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 2; and (b) a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NO: 4; wherein the antibody specifically binds to PD-1 and blocks the interaction between PD-1 and PD-L1. In some embodiments, the monoclonal antibody or antigen binding portion thereof stimulates an anti-tumor immune response. In some embodiments, the monoclonal antibody can be a chimeric antibody.

[0215] In another aspect, the present disclosure provides an antibody comprising the heavy and light chain CDRl, CDR2, and CDR3 of Clone #11. The amino acid sequence of the VL CDRl of Clone #11 is set forth in SEQ ID NO: 5. The amino acid sequence of the VL CDR2 of Clone #11 is set forth in SEQ ID NO: 6. The amino acid sequence of the VL CDR3 of Clone #11 is set forth in SEQ ID NO: 7. The amino acid sequence of the VH CDRl of Clone #11 is set forth in SEQ ID NO: 8. The amino acid sequence of the VH CDR2 of Clone #11 is set forth in SEQ ID NO: 9. The amino acid sequence of the VH CDR3 of Clone #11 is set forth in SEQ ID NO: 10. The CDR regions can be delineated using the Kabat system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0216] In another aspect, the present disclosure provides polynucleotide sequences encoding the light chain variable domain (VL) and the heavy chain variable domain (VH) of monoclonal antibody Clone #11. The VL polynucleotide sequence is set forth in SEQ ID NO: 1. The VH polynucleotide sequence is set forth in SEQ ID NO: 3.

[0217] Antibodies can be affinity matured by light chain shuffling, either in combination with or not in combination with random mutagenesis of their heavy chain variable domains, and panning against PD-1. The VL CDRl, VL CDR2, and VL CDR3 of the antibodies mentioned in the present disclosure can be optimized with light chain shuffling to generate other anti-PD-1 binding molecules of the present disclosure.

[0218] Engineered antibodies can be further prepared using an antibody having one or more of the VH and / or VL sequences disclosed herein as starting material, which can have different properties than the starting antibody. Antibodies can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and / or VL), e.g., within one or more CDR regions and / or within one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues within one or more constant regions, e.g., to alter one or more effector functions of the antibody.

[0219] In certain embodiments, CDR grafting can be used to engineer the variable regions of an antibody. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs).

[0220] Because CDR sequences can be responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody having different properties (see, e.g., Riechmann et al. (1998) Nature 332:323-327; Jones et al. (1986) Nature 321 :522-525; Queen et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

[0221] Accordingly, another embodiment of the disclosure relates to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10, respectively. Accordingly, such antibodies contain the VH and VL CDR sequences of the anti-PD-1 monoclonal antibody from hybridoma clone #11, and can contain different framework sequences than these antibodies.

[0222] Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "Vbase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase) and in the following references: Kabat et al. (1991) cited above; Tomlinson et al. (1992) "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops," J. Mol. Biol. 227:776-798; and Cox et al. (1994) "A Directory of Human Germ-line VH Segments Reveals a Strong Bias in their Usage." Usage)" Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences of human heavy and light chain variable region genes can be found in the GenBank database. For example, the following heavy chain germline sequences found in HCo7HuMAb mice are available in the accompanying GenBank accession numbers 1-69 (NG_0010109, NT_024637 & BC070333), 3-33 (NG_0010109 & NT_024637), and 3-7 (NG_0010109 & NT_024637). As another example, the following heavy chain germline sequences found in the HCo12 HuMAb mouse are available in the accompanying GenBank accession numbers 1-69 (NG_0010109, NT_024637 & BC070333), 5-51 (NG_0010109 & NT_024637), 4-34 (NG_0010109 & NT_024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).

[0223] The antibody protein sequence was compared to a compiled protein sequence database using one of the sequence similarity search methods known as Gapped BLAST (Altschul et al. (1997)), which are well known to those skilled in the art. The compositions and methods of the present disclosure are not limited to variants of the exemplary sequences disclosed herein, but rather encompass sequences having a sequence identity of at least 90%, at least 95%, and at least 99% to the exemplary sequences disclosed herein.

[0224] Antibodies and antigen-binding fragments

[0225] The present disclosure provides various antibodies and antigen-binding fragments thereof derived from the anti-PD-1 antibodies described herein. Typically, antibodies (also referred to as immunoglobulins) are made of two classes of polypeptide chains, i.e., light chains and heavy chains. A non-limiting example of an antibody of the present disclosure can be a complete four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype comprising IgM, IgG, IgE, IgA or IgD or a subisotype comprising IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. The antibody can include two identical copies of a light chain and two identical copies of a heavy chain. Each contains a variable domain (or variable region V H ) and multiple constant domains (or constant regions) are bound to each other by disulfide bonds within their constant domains to form the "stem" of the antibody. L Each light chain consists of a single heavy chain (or constant region) and a single constant domain (or constant region), each bound to a single heavy chain by disulfide bonding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0226] These hypervariable regions, known as complementarity determining regions (CDRs), form loops that comprise the antigen-binding surface of the antibody. The four framework regions largely adopt a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

[0227] Methods for identifying CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and multiple definitions of CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described in, e.g., Martin, “Protein sequence and structure analysis of antibody variable domains” in Antibody engineering, Springer Berlin Heidelberg, 2001, 422-439; Abhinandan et al. “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”, Molecular immunology 45.14 (2008): 3832-3839; Wu, T.T., and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68(1-3): 9-16 (Oct, 1997); Morea et al., J. Mol. Biol. 275(2): 269-94 (Jan, 1998); Chothia et al., Nature 342(6252): 877-83 (Dec, 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated by reference herein in its entirety. The Kabat definition is used in the present disclosure, unless otherwise noted.

[0228] CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule that is capable of being specifically bound by an antigen binding domain of an antibody. The minimum size of an epitope can be about three, four, five, six, or seven amino acids, but these amino acids need not be located in a contiguous linear sequence of the primary structure of an antigen, as the epitope can depend on the three-dimensional configuration of the antigen based on its secondary and tertiary structure.

[0229] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgGl, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly in their hinge and upper CH2 domains. Sequences and differences of IgG subclasses are known in the art and described in, e.g., Vidarsson et al., “IgG subclasses and allotypes: from structure to effector functions” Frontiers in immunology 5 (2014); Irani et al. “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases” Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated by reference herein in its entirety.

[0230] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camelids). Antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising immunoglobulin binding domains fused with another polypeptide. The term "antigen binding domain" or "antigen binding fragment" refers to a part of the specific binding activity of an antibody that retains a complete antibody, that is, any part of an antibody that can specifically bind to the epitope on the target molecule of a complete antibody. It includes, for example, Fab, Fab', F(ab')2 and variants of these fragments. Therefore, in some embodiments, an antibody or its antigen binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide comprising a binding domain as an antibody binding domain or a binding domain homologous to an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs from the heavy or light chain of an intact antibody.

[0231] Also provided are fragments of antibodies suitable for use in the methods described herein. Fab fragments contain the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain. F(ab')2 antibody fragments include pairs of Fab fragments typically covalently linked by hinge cysteines near their carboxyl termini. Other chemical couplings of antibody fragments are also known in the art.

[0232] Diabodies are small antibody fragments with two antigen-binding sites, comprising a VH linked to a VL in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0233] Linear antibodies include a tandem Fd segment pair (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0234] The antibodies and antibody fragments of the disclosure can be modified in the Fc region to provide desired effector function or serum half-life.

[0235] Antibody multimerization can be achieved by natural aggregation of antibodies or chemical or recombinant ligation techniques known in the art. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0236] Alternatively, antibody homodimers can be formed by chemical connection techniques known in the art. For example, heterologous bifunctional cross-linkers including but not limited to SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acetylthioacetate) can be used to form antibody multimers. An exemplary scheme for forming antibody homodimers is described in Ghetie et al. (Proceedings of the National Academy of Sciences of the United States of America 94:7509-7514, 1997). Antibody homodimers can be converted into Fab'2 homodimers by digestion with pepsin. Another way to form antibody homodimers is by using the autophilic T15 peptide described in Zhao et al. (Journal of Immunology 25:396-404, 2002).

[0237] In certain embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be prepared by engineering the interface between antibody molecules to maximize the percentage of the heterodimer recovered from recombinant cell culture. For example, the interface can contain at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (for example, tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (for example, alanine or threonine), compensatory " cavity " of the same or similar size as one or more large side chains is produced on the interface of the second antibody molecule. This provides a mechanism for improving the productive rate of heterodimers compared to the undesirable final products such as homodimers. This method is described in, for example, WO96 / 27011, and the document is incorporated to its entirety by reference.

[0238] Bispecific antibodies include cross-linked antibodies or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other can be coupled to biotin. Heteroconjugate antibodies can also be prepared using any convenient cross-linking method. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0239] Methods for producing bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229:81, 1985) described a procedure for proteolytic cleavage of intact antibodies to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiol groups and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'TNB derivatives is then converted back into a Fab' thiol by reduction with mercaptoethylamine and mixed with another Fab'TNB derivative in an equimolar amount to form a bispecific antibody.

[0240] Any of the antibodies or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life of the antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans) or extend its biological activity.

[0241] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent.Antibody-drug conjugates comprising an antibody or antigen-binding fragment thereof can be covalently or non-covalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids (e.g., DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D). D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and the like).

[0242] In some embodiments, antigen binding fragment can form a part of chimeric antigen receptor (CAR).In some embodiments, chimeric antigen receptor is a fusion of a single chain variable fragment (scFv) as described herein with a CD3-ζ transmembrane domain and an internal domain.In some embodiments, chimeric antigen receptor also includes intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS).In some embodiments, chimeric antigen receptor includes multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to increase effectiveness.Therefore, on the one hand, the present disclosure further provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0243] In some embodiments, the scFV has one heavy chain variable domain and one light chain variable domain. In some embodiments, the scFV has two heavy chain variable domains and two light chain variable domains.

[0244] Antibody characteristics

[0245] The antibodies or antigen-binding fragments thereof described herein can block the binding between PD-1 and a PD-1 ligand (e.g., PD-L1 or PD-L2). In some embodiments, by binding to PD-1, the antibodies can inhibit the PD-1 signaling pathway. In some embodiments, the antibodies can upregulate the immune response.

[0246] In some embodiments, an antibody or antigen-binding fragment thereof as described herein can increase the immune response, activity, or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen-presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0247] In some embodiments, the antibody (or antigen-binding fragment thereof) is activated in less than 0.1 s -1 , less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 The dissociation rate (koff) of the antibody specifically binds to PD-1 (e.g., human PD-1 (SEQ ID NO: 77), rhesus monkey PD-1 (NP_001107830.1), mouse PD-1 (NP_032824.1) and / or chimeric PD-1). In some embodiments, the dissociation rate (koff) is greater than 0.01s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0248] In some embodiments, the kinetic association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1×10 5 / Ms, less than 1×106 / Ms or less than 1 x 10 7 / Ms.

[0249] Affinity can be derived from the quotient of the kinetic rate constants (KD= koff / kon). In some embodiments, the KD (Kd) is less than 1 x 10 -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M or less than 1 x 10 -10 M. In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In some embodiments, the KD is greater than 1 x 10 -7 M, greater than 1 x 10 -8 M, greater than 1 x 10 -9 M, greater than 1 x 10 -10 M, greater than 1 x 10 -11 M or greater than 1 x 10 -12 M.

[0250] General techniques for measuring affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human PD-1 (SEQ ID NO: 77).

[0251] In some embodiments, the antibody binds to monkey PD-1 (e.g., NP_001271065.1 from cynomolgus monkey), chimeric PD-1, and / or mouse PD-1 (e.g., NP_032824.1). In some embodiments, the antibody does not bind to monkey PD-1, chimeric PD-1, and / or mouse PD-1.

[0252] In some embodiments, the antibody or antigen-binding fragment thereof as described herein is a PD-1 antagonist. In some embodiments, the antibody or antigen-binding fragment reduces PD-1 signaling in a target cell expressing PD-1.

[0253] In some embodiments, the antibody or antigen-binding fragment can enhance APC (e.g., DC cell) function, for example, induce surface expression of costimulatory molecules and MHC molecules, induce production of proinflammatory cytokines, and / or enhance T cell priming function.

[0254] In some embodiments, the Fc region is human IgGl, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgGl antibody.

[0255] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a Fv fragment. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering).

[0256] Binding of the antibodies as described herein to PD-1 can also be assessed using one or more techniques well established in the art. For example, in preferred embodiments, antibodies can be tested by ELISA assays, e.g., using recombinant PD-1 protein. Still other suitable binding assays include, but are not limited to, flow cytometry assays in which antibodies are reacted with cell lines expressing human PD-1, such as HEK293T cells that have been transfected to express PD-1 (e.g., human PD-1) on their cell surface. Additionally or alternatively, binding of antibodies, including binding kinetics (e.g., KD values) can be tested in BIAcore binding assays, and the like.

[0257] Preferably, the antibodies of the present disclosure bind to PD-1 protein with an EC50 of 5 x 10 -8 M or less, to PD-1 protein with an EC50 of 2 x 10 -8 M or less, to PD-1 protein with an EC50 of 5 x 10 -9 M or less, to PD-1 protein with an EC50 of 4 x 10 -9 M or less, to PD-1 protein with an EC50 of 3 x 10 -9 M or less, to PD-1 protein with an EC50 of 2 x 10 -9 M or less, to PD-1 protein with an EC50 of 1 x 10 -9 M or less, to PD-1 protein with an EC50 of 1 x 10

[0258] Methods of making anti-PD-1 antibodies

[0259] Isolated fragments of human PD-1 can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody production. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. The animal can be injected with the antigenic peptide or protein more than once (e.g., twice, three times, or four times).

[0260] The full-length polypeptide or protein can be used as an immunogen, or alternatively, an antigenic peptide fragment thereof can be used as an immunogen. An antigenic peptide of a protein includes at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of PD-1 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immunocomplex with the protein. As noted above, the full-length sequence of human PD-1 is known in the art (SEQ ID NO: 77).

[0261] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly expressed polypeptide or a chemically synthesized polypeptide (e.g., a fragment of human PD-1). The preparation can further comprise an adjuvant, such as Freund's complete or incomplete adjuvant or a similar immunostimulatory agent.

[0262] As described above, polyclonal antibodies can be prepared by immunizing a suitable subject with a PD-1 polypeptide or an antigenic peptide thereof (e.g., a portion of PD-1) as an immunogen. Antibody titers in immunized subjects can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized PD-1 polypeptide or peptide. If desired, the antibody molecules can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques (e.g., protein A, protein G chromatography) to obtain an IgG fraction. At an appropriate time after immunization, for example, when specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques such as the hybridoma technique originally described by Kohle et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983), the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (see generally, Current Protocols in Immunology, 1994, Coligan et al. (eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies are detected by screening hybridoma culture supernatants for antibodies that bind to the polypeptide or epitope of interest, eg, using a standard ELISA assay.

[0263] Nucleic acid molecules encoding the heavy chain or the entire light chain of an anti-PD-1 antibody or a portion thereof can be isolated from any source that produces such antibodies. In various embodiments, nucleic acid molecules are isolated from B cells isolated from animals immunized with PD-1 or from immortalized cells derived from such B cells expressing anti-PD-1 antibodies. Methods for isolating mRNA encoding antibodies are well known in the art. See, for example, Sambrook et al. The mRNA can be used to produce cDNA for polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In a preferred embodiment, nucleic acid molecules are isolated from hybridomas having human immunoglobulin-producing cells from non-human transgenic animals as one of their fusion partners. In another embodiment, nucleic acids can be isolated from non-human non-transgenic animals. Nucleic acid molecules isolated from non-human non-transgenic animals can be used, for example, for humanized antibodies.

[0264] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human antibodies, humanized antibodies or chimeric antibodies or their antigen-binding fragments described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence of the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for target proteins such as PD-1. Any combination of deletions, insertions and / or combinations can be performed to obtain antibodies or their antigen-binding fragments with increased binding affinity for the target. Amino acid changes introduced into antibodies or antigen-binding fragments can also change the antibodies or antigen-binding fragments or introduce new post-translational modifications into the antibodies or antigen-binding fragments, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are connected by enzymes present in the cell) or introducing new glycosylation sites.

[0265] The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically engineered to produce human antibodies.

[0266] Human antibodies and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences (or having an amino acid sequence identical to an amino acid sequence derived from human germline immunoglobulin sequences). Human antibodies may include, for example, amino acid residues in the CDRs that are 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).

[0267] Humanized antibodies generally have a human framework (FR) that is transplanted with non-human CDRs. Therefore, humanized antibodies have one or more amino acid sequences that are introduced into them from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from "import" variable domains.

[0268] Typically, amino acid sequence variants of human, humanized, or chimeric anti-PD-1 antibodies will contain an amino acid sequence that has a percent identity of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to the sequence present in the light or heavy chain of the original antibody.

[0269] Identity or homology with respect to a reference sequence is typically the percentage of amino acid residues in the candidate sequence that are identical with the residues in the sequence present in the human antibody, humanized antibody, or chimeric anti-PD-1 antibody or fragment after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity.

[0270] Additional modifications can be made to the anti-PD-1 antibody or antigen binding fragment. For example, one or more cysteine residues can be introduced into the Fc region, thereby allowing for interchain disulfide bond formation in this region. The resulting homodimeric antibody can have any increased half-life in vitro and / or in vivo. Homodimeric antibodies with increased half-life in vitro and / or in vivo can also be prepared using heterobifunctional cross-linking reagents as described in Wolff et al. (“Monoclonal antibody homodimers: enhanced antitumor activity in nude mice” Cancer research 53.11 (1993): 2560-2565). Alternatively, antibodies with dual Fc regions can be engineered.

[0271] In some embodiments, the anti-PD-1 antibody or antigen binding fragment thereof can be covalently modified. These covalent modifications can be made by either chemical synthesis or enzymatic synthesis, or by enzymatic cleavage or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that is capable of reacting with selected side chains or N-terminal or C-terminal residues.

[0272] In certain embodiments, antibody variants are provided as having a carbohydrate structure lacking the fucose connected to the Fc region (directly or indirectly). For example, the amount of fucose in such antibody compositions can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn 297 as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in antibodies, Asn297 can also be located upstream or downstream of position 297 approximately ± 3 amino acids, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0273] In certain embodiments, in order to promote production efficiency by avoiding Fab arm exchanges, the Fc region of antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). Detailed description of S228 mutation is described below, such as Silva et al. " S228P mutation prevents in vivo and in vitro IgG4 Fab arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation" Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated by reference in its entirety.

[0274] recombinant vector

[0275] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), host cells into which the combination vectors have been introduced (i.e., so that the host cells contain the polynucleotide and / or the vector comprising the polynucleotide), and recombinant antibody polypeptides or fragments thereof produced by recombinant techniques.

[0276] As used herein, a "vector" is any construct that is capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked to regulatory elements such as promoters, enhancers, and / or poly-A tails within the vector or in the genome of the host cell at or near or flanking the site of integration of the polynucleotide of interest, such that the polynucleotide of interest will be translated in a host cell into which the expression vector has been introduced.

[0277] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0278] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which can involve the use of a non-pathogenic (defective), replication-competent virus, or a replication-defective virus can be used. In some embodiments, lentivirus is used to introduce nucleic acids into cells.

[0279] To express, a DNA insert including a polynucleotide or polypeptide-encoding polynucleotide of an antibody disclosed herein can be operatively linked to a suitable promoter (e.g., a heterologous promoter), such as the bacteriophage lambda PL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct can further contain sites for transcription initiation, termination, and, in the case of transcription into RNA, ribosome binding sites. The coding portion of a mature transcript expressed by the construct can include translation initiation at an ATG start codon and translation termination at an appropriate stop codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0280] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase resistance or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli cells, Streptomyces cells, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate media and conditions for the cultivation of host cells described herein are known in the art.

[0281] Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated by reference herein in its entirety.

[0282] Transcription of DNA encoding an antibody of the present disclosure by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act to increase the transcription of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located in the region from base pairs 100 to 270 of the SV40 genome, the cytomegalovirus early promoter enhancer, the mulbiman enhancer, and adenovirus enhancers.

[0283] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. The signal can be endogenous to the polypeptide, or it can be a heterologous signal. In some embodiments, the signal peptide is SEQ ID NO: 78 or SEQ ID NO: 79.

[0284] Polypeptides (e.g., antibodies) can be expressed in a modified form, such as a fusion protein (e.g., GST-fusion) or with a histidine tag, and can contain not only a secretion signal, but also additional heterologous functional regions. For example, additional amino acids, particularly regions of charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or during subsequent processing and storage. Similarly, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. Adding peptide moieties to polypeptides to cause secretion or excretion, improve stability, and facilitate purification (among other things) is well known and conventional in the art.

[0285] In one aspect, the disclosure provides a nucleic acid that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1, 3, 15, 17, 27, 29, 37, 39, 47, 49, 47, 49, 67 or 69.

[0286] The present disclosure also provides nucleic acid sequences that are at least 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any of the nucleotide sequences described herein. %, 98%, 99% identical to any of the amino acid sequences described herein. In some embodiments, the present disclosure relates to nucleotide sequences encoding any of the peptides described herein, or any amino acid sequence encoded by any of the nucleotide sequences described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0287] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any of the sequences described herein. In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any of the sequences described herein.

[0288] In order to determine the percent identity of two amino acid sequences or two nucleotide sequences, sequence is compared for the purpose of best comparison (for example, can introduce room to carry out best comparison in one or both of the first amino acid or nucleotide sequence and the second amino acid or nucleotide sequence, and for comparison purposes, non-homologous sequences can be ignored).Then the amino acid residue or the nucleotide at corresponding amino acid position or nucleotide position are compared.When the position in the first sequence is occupied by the amino acid residue or nucleotide identical with the corresponding position in the second sequence, then molecule is identical at said position.The percent identity between two sequences is the function of the quantity of the same position shared by said sequences having considered the quantity of room and the length of each room, needs to introduce said function to carry out the best comparison of two sequences.For the purpose of illustration, the determination of the percent identity between the comparison of sequence and two sequences can be completed using the Blossum 62 scoring matrix with wherein gap penalty of 12, gap extension penalty of 4 and frameshift gap penalty of 5.

[0289] Treatment

[0290] The antibodies, antibody compositions and methods disclosed herein have many in vitro and in vivo utilities, the effects of which relate to, for example, detecting PD-1 or enhancing immune responses by blocking PD-1. In preferred embodiments, the antibodies disclosed herein are human antibodies. For example, these molecules can be administered to cells in culture in vitro or ex vivo, or to human subjects in vivo, for example, to enhance immunity in various situations. Therefore, in one aspect, the present disclosure provides a method for modifying an immune response in a subject, the method comprising administering an antibody or antigen-binding portion thereof to a subject, thereby modifying the immune response of the subject. Preferably, the response is enhanced, stimulated or upregulated.

[0291] As used herein, the term "subject" is intended to include humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles, but preferably mammals such as non-human primates, sheep, dogs, cats, cows and horses. Preferred subjects include human patients in need of an increased immune response. The method is particularly suitable for treating human patients with conditions that can be treated by enhancing a T cell-mediated immune response. In a specific embodiment, the method is particularly suitable for in vivo treatment of cancer cells. In order to achieve antigen-specific enhancement of immunity, anti-PD-1 antibodies can be administered together with the antigen of interest. When the PD-1 antibody is administered together with another agent, the two can be administered sequentially or simultaneously.

[0292] The present disclosure further provides a method for detecting the presence of human PD-1 antigen in a sample or measuring the amount of human PD-1 antigen, the method comprising contacting the sample and a control sample with a human monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to human PD-1 under conditions that allow formation of a complex between the antibody or portion thereof and human PD-1, and then detecting the formation of the complex, wherein differential complex formation between the sample and the control sample indicates the presence of human PD-1 antigen in the sample.

[0293] Blockade of PD-1 by antibodies can enhance the immune response to cancer cells in patients. In one aspect, the present disclosure relates to treating a subject in vivo with an anti-PD-1 antibody such that the growth of a cancerous tumor is inhibited. An anti-PD-1 antibody can be used alone to inhibit the growth of a cancerous tumor. Alternatively, an anti-PD-1 antibody can be used in combination with other immunogenic agents, standard cancer treatments, or other antibodies, as described below.

[0294] Thus, in one embodiment, the present disclosure provides a method of inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding portion thereof. Preferably, the antibody is a human anti-PD-1 antibody (such as any of the human anti-human PD-1 antibodies described herein). Additionally or alternatively, the antibody may be a chimeric anti-PD-1 antibody or a humanized anti-PD-1 antibody.

[0295] Preferred cancers whose growth can be inhibited using the antibodies of the present disclosure include cancers that typically respond to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). Additionally, the present disclosure includes refractory or recurrent malignancies whose growth can be inhibited using the antibodies of the present disclosure.

[0296] Examples of other cancers that can be treated using the methods of the present disclosure include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including The present invention relates to a group of cancers that are classified as comprising acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), solid tumors of children, lymphocytic lymphoma, bladder cancer, kidney cancer or ureter cancer, renal pelvis cancer, central nervous system tumors (CNS), primary CNS lymphoma, tumor vascular, spinal cord axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal cancer, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (including cancer induced by asbestos) and combinations thereof. The present invention can also be used to treat metastatic cancers, particularly metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144).

[0297] Optionally, antibodies to PD-1 can be combined with immunogenic agents, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MARTI, and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below). In humans, some tumors have been shown to be immunogenic, such as melanoma. It is expected that by raising the threshold for T cell activation through PD-1 blockade, it is expected that tumor responses in the host can be activated. PD-1 blockade may be most effective when combined with a vaccination regimen. Many experimental strategies for vaccination against tumors have been envisioned (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D., 2000, ASCO Educational Book Spring: 414-428; Poon, K., 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Chapter 61, pp. 3023-3043 in De Vita, V. et al. (eds.), 1997, Cancer: Principles and Practice of Oncology 5th ed.). In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cell vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be an effective activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90:3539-43).

[0298] PD-1 blockade can also be combined with standard cancer treatment. PD-1 blockade can be effectively combined with chemotherapy regimens. In these cases, the dose of the chemotherapeutic agent administered may be reduced (Mokyr, M. et al. (1998) Cancer Research 58: 5301-5304). An example of this combination is the combination of anti-PD-1 antibodies and dacarbazine for the treatment of melanoma. Another example of this combination is the combination of anti-PD-1 antibodies and interleukin 2 (IL-2) for the treatment of melanoma. The scientific principle behind the combined use of PD-1 blockade and chemotherapy drugs is that cell death, as a result of the cytotoxic effects of most chemotherapy compounds, should cause the level of tumor antigens to increase in the antigen presentation pathway. Other combination therapies that may cause synergy with PD-1 blockade through cell death are radiation, surgery, and hormone deprivation. Each of these regimens produces a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with PD-1 blockade. Inhibition of angiogenesis causes tumor cell death, which can feed tumor antigens into the host antigen presentation pathway.

[0299] PD-1 blocking antibodies can also be used in combination with bispecific antibodies that target Fcα or Fcγ receptor-expressing effector cells to tumor cells (see, for example, U.S. Patents Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to the site of the tumor. This targeting can more effectively activate tumor-specific responses. The T cell arm of these responses will be enhanced by using PD-1 blocking. Alternatively, antigens can be delivered directly to DCs using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0300] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins expressed by the tumor that are immunosuppressive. Among these mechanisms are TGF-β (Kehrl, J. et al. (1986) Journal of Experimental Medicine 163:1037-1050), IL-10 (Howard, M. and O'Garra, A. (1992) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies to each of these entities can be used in combination with the anti-PD-1 antibodies of the present disclosure to counteract the effects of immunosuppressants and facilitate the tumor immune response by the host.

[0301] Other antibodies that can be used to activate host immune responsiveness can be used in combination with anti-PD-1. These antibodies contain molecules that activate DC function and antigen presentation on the surface of dendritic cells. Anti-CD40 antibodies can effectively replace T cell helper activity (Ridge, J. et al. (1998) Nature 393:474-478) and can be used in combination with PD-1 antibodies (Ito, N. et al. (2000) Immunobiology 201(5)527-40). Activation of T cell co-stimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg, A. et al. (2000) Immunol. 164:2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Med. 3:682-685 (1997)), and ICOS (Hutloff, A. et al. (1999) Nature 397:262-266)) may also increase the level of T cell activation.

[0302] Bone marrow transplantation is currently being used to treat various tumors caused by hematopoietic origin. Although graft-versus-host disease is a result of this treatment, therapeutic benefits can be obtained from the graft-versus-tumor response. PD-1 blocking can be used to improve the effectiveness of tumor-specific T cells transplanted with donors. There are also several experimental treatment options that involve ex vivo activation and amplification of antigen-specific T cells and adoptive transfer of these cells into recipients as antigen-specific T cells for tumors (Greenberg, R. and Riddell, S. (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious sources (such as CMV). It can be expected that ex vivo activation in the presence of anti-PD-1 antibodies will increase the frequency and activity of adoptively transferred T cells.

[0303] In one aspect, the present disclosure provides methods for treating cancer in a subject, methods for reducing the rate at which the volume of a tumor in a subject increases over time, methods for reducing the risk of developing metastasis, or methods for reducing the risk of developing additional metastasis in a subject. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can reduce the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0304] In some embodiments, the antibody has a tumor growth inhibition percentage (TGI%) of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be determined, for example, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days after the start of treatment, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the start of treatment. As used herein, tumor growth inhibition percentage (TGI%) is calculated using the following formula:

[0305] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100

[0306] Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0307] In some embodiments, the tumor inhibition effect of the antibodies or antigen-binding fragments thereof as described herein is comparable to that of pembrolizumab, nivolumab or cemiplimab. In some embodiments, the tumor inhibition effect of the antibodies or antigen-binding fragments thereof as described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold or 5-fold greater than that of pembrolizumab, nivolumab or cemiplimab.

[0308] In one aspect, the disclosure features a method comprising administering to a subject in need thereof (e.g., a subject having or identified as having or diagnosed with a cancer, e.g., a breast cancer (e.g., triple negative breast cancer), a benign tumor cancer, a cervical cancer, an endometrial cancer, a glioma, a head and neck cancer, a liver cancer, a lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, a colorectal cancer, a gastric cancer, a testicular cancer, a thyroid cancer, a bladder cancer, a urethral cancer, or a hematological malignancy) a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the cancer is an unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is a squamous cell carcinoma of the head and neck (SCCHN), a renal cell carcinoma (RCC), a triple negative breast cancer (TNBC), or a colorectal cancer. In some embodiments, the subject has Hodgkin’s lymphoma. In some embodiments, the subject has a triple negative breast cancer (TNBC), a gastric cancer, a urothelial cancer, a Merkel cell carcinoma, or a head and neck cancer. In some embodiments, the cancer is a melanoma, a pancreatic cancer, a mesothelioma, a hematological malignancy, in particular a non-Hodgkin’s lymphoma, a lymphoma, a chronic lymphocytic leukemia, or an advanced solid tumor.

[0309] In some embodiments, the compositions and methods disclosed herein can be used to treat a patient at risk of developing a cancer. Patients having a cancer can be identified using various methods known in the art.

[0310] In one aspect, the disclosure provides methods for treating, preventing, or reducing the risk of having a disorder associated with an aberrant or unwanted immune response, e.g., an autoimmune disorder.

[0311] As used herein, “effective amount” means the amount or dose sufficient to effectuate a beneficial or desired result, including halting, slowing, delaying, or inhibiting the progression of a disease, e.g., a cancer. The effective amount will vary depending on, e.g., the age and weight of the subject to be administered the antibody, antigen-binding fragment, antibody-encoding polynucleotide, vector comprising a polynucleotide, and / or composition thereof, the severity of the symptoms, and the route of administration, and thus administration can be determined on an individual basis.

[0312] In some embodiments, one or more additional therapeutic agents can be administered to the subject. In some embodiments, the additional therapeutic agent can include one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, a fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0313] In some embodiments, the additional therapeutic agent can include one or more anti-cancer drugs (e.g., chemotherapeutic drugs) selected from the group consisting of Abemaciclib, Abiraterone Acetate, Abraxane, Acalabrutinib, Actemra (Tocilizumab), Adcetris (Brentuximab Vedotin), Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran, Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ameluz (Aminolevulinic Acid Hydrochloride), Amifostine, Aminolevulinic Acid Hydrochloride, Anastrozole, Apalutamide, Aprepitant, Aranesp (Darbepoetin Alfa), Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Anastrozole), Arimidex (Disodium)), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Asparlas (Calaspargase Pegol-mknl), Atezolizumab, Avastin (Bevacizumab), Avelumab, Axicabtagene Ciloleucel, Axitinib, Azacitidine, Azedra (Iobenguane I 131), Bavencio (Avelumab), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, Bendeka (Bendamustine Hydrochloride), Besponsa (Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, Bicalutamide, BiCNU (Carmustine), Binimetinib, Bleomycin Sulfate, Blenoxane (Mitoxantrone), Bortezomib, Bosentan, Bortezomib, Boswellia, Braftovi (Encorafenib), BraftoVib (Encorafenib), Brivudine, Brodalumab, Brodalumab, Buparlisib, Busulfan, Cabazitaxel, Cabozantinib, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, Cabozantinib S-malate, CabozSulfate), Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Braftovi (Encorafenib), Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cablivi (Caplacizumab-yhdp), Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S-Malate, Calaspargase Pegol-mknl, Calquence (Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, Caplacizumab-yhdp, Carac (Fluorouracil- topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmustine, Carmustine Implant, Casodex (Bicalutamide), Cemiplimab-rwlc, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cetuximab, Chimeric Monoclonal Antibody Trastuzumab, Cisplatin, Clafen (Endoxan) (Cyclophosphamide), Clolar (Clofarabine), Clofarabine, Colorectal Cancer Vaccine, Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Amercian Cancer Society), Cosmegen (Hydrochloride)), Cervarix (recombinant HPV bivalent vaccine), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE, Cisplatin, Cladribine, Clofarabine, Clolar (Clofarabine), Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, Copiktra (Duvelisib), Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, Cyclophosphamide, Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dacomitinib, Dactinomycin, Daratumumab, Darbepoetin Alfa, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Daurismo (Glasdegib Maleate), Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Diflucan (Fluconazole), Dihydrotachysterol, Dihydrotachysterol Sodium, Diligene (Dinutuximab), Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride and Cytarabine Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), Droxia (Hydroxyurea), Duligene (Dinutuximab), Duloxetine Hydrochloride, Duvelisib, Eculizumab, Efudix (Fluorouracil), Efudix (Fluorouracil), Eflomithine Hydrochloride, Eflomithine Hydrochloride and Fluorouracil, Eflomithine Hydrochloride and Fluorouracil, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), Enbrel (Etanercept), EnbrelHydrochloride), Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), Durvalumab, Duvelixib, Efudex (Fluorouracil Topical), Eligard (Leuprolide Acetate), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine), Elzonris (Tagraxofusp-erzs), Emapalumab-lzsg, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Encorafenib, Enzalutamide, Epirubicin Hydrochloride, Epoetin Alfa, Epogen (Epoetin alfa), Erbitux (Cetuximab), Eribulin Mesylate Mesylate), Erivedge (Vismodegib), Erleada (Apalutamide), Erlotinib Hydrochloride, Erwinaze (Erwinia chrysanthemi asparaginase), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposomal), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride),Hydrochloride), Exemestane, 5-FU (Fluorouracil), Fareson (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), Femara (Letrozole), Filgrastim, Firmagon (Degarelix), Fludarabine Phosphate, Fluorouracil (Fluorouracil Topical), Fluorouracil Injection, Fluorouracil Topical, Flutamide, Folotyn (Pralatrexate), Fostamatinib Disodifum, Fulvestrant, Fusilev (Leucovorin Calcium), Gamifant (Emapalumab-lzsg), ​​Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Indole Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemtuzumab Ozoglucam Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Maleate), Gilteritinib Fumarate, Glevitra Maleate, Gleevec (Imatinib Mesylate), Gliadel Wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Granisetron, Granisetron Hydrochloride, Granix (Filgrastim), Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride),Hydrochloride), Herceptin Hylecta (Trastuzumab and Hyaluronidase-oysk), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant HPV Vaccine (Valentine), Recombinant HPV Quadrivalent Vaccine, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Dexamethasone (Hyper-CVAD), Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, Iclusig (Ponatinib Hydrochloride), Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Ensidib Mesylate), Ifex (Ifosfamide), Ifosfamide, IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab, Interferon alpha-2b, Recombinant Interleukin-2 (Aldesleukin), Interferon A (Recombinant Interferon alpha-2b), Iodobenzylguanidine 131. Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ivosidenib, Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate)Phosphate)), Jevtana (Cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Larotrectinib Sulfate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Oncovin (Chlorambucil), Leuprolide Acetate, Levulan Kerastik (Aminolevulinic Acid Hydrochloride), Libtayo (Cemiplimab-rwlc), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lorbrena (Lorlatinib), Lorlatinib, Lumoxiti (Moxetumomab Pasudotox-tdfk), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lutathera (Lutetium Lu 177-Dotatate), Lutetium Lu 177-Dotatate, Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Methotrexate, Methotrexate Sodium, Methotrexate Sodium, Mitoxantrone Hydrochloride, Mylotarg (Gemtuzumab Ozogamicin), Navelbine (Vinorelbine), Neulasta (Pegfilgrastim), Neumega (Oprelvekin), Neupogen (Filgrastim), Neutrexin (Trimetrexate Glucuronate), Nexavar (Sorafenib), Nilotinib, Nplate (Romiplostim), Novantrone (Plicamycin), Ofatumumab, Olaparib, Oncaspar (Pegaspargase), Ontak (Denileukin Diftitox), Oprelvekin, Orathecin (Liposomal Vinorelbine), Oxaliplatin, Paclitaxel, Paclitaxel Protein-bound, Pamidronate Disodium, Panitumumab, Panobacumab, Panitumumab, Panobacumab, Paraplatin (Carboplatin), Pazopanib Hydrochloride, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, Pegvisomant, Pembrolizumab, Pentostatin, Pemetrexed Disodium, Pentostatin, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Pemetrexed Disodium, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Pemetrexed Disodium, Pemetrexed Disodium,Acetate, Mekinist (Trametinib), Mektovi (Binimetinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methotrexate, Methylnaltrexone Bromide, Midostaurin, Mitomycin C C), Mitoxantrone Hydrochloride, Mogamulizumab-kpkc, Pacitumomab-tdfk, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Myleran (Busulfan), Mylotarg (Gemtuzumab), Nanoparticle Paclitaxel (Albumin-Stabilized Nanoparticle Formulation of Paclitaxel), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neratinib Maleate Maleate), Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib TosylateMonohydrate), Nivolumab, Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), Ofatumumab, Olaparib, Olamab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan liposomal hydrochloride), Ontak (Denileukin Diftitox), Opdivo (nivolumab), Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Pazopanib Hydrochloride, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Interferon (PEG-interferon alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Nexituzumab), Poteligeo (Mogamulizumab-kpkc), Pralatrexate, Prednisone, Procarbazine Hydrochloride, Procrit (Epoetin alfa), Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (EltrombopagOlamine)), propranolol hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, Ravulizumab-cwvz, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Interferon alfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), Retacrit (Epoetin Alfa), Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and Hyaluronidase Human, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Rusoceptin Phosphate, Rydapt (Midostaurin), Sancuso (Granisetron), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Soliris, Sorafenib Tosylate, Sprycel (Dasatinib), Sterile TalcPowder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sustol (Granisetron), Sutent (Sunitinib Malate), Sylatron (Pegylated Interferon Alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), Tafinlar (Dabrafenib), Tagraxofusp-erzs, Tagrisso (Osimertinib), Talazoparib Tosylate Tosylate), Talimogene Laherparepvec, Talzenna (Talazopali Tosylate), Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Tavalisse (Tavalisse Disodium Salt), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolumab), Temodar (Temozolomide), Temozolomide Amine, Temsirolimus, Thalidomide, Thalidomide (thalidomide), Thioguanine, Thiotepa, Tibsovo (ivosidenib), Tisagenlecleucel, Tocilizumab, Tolak (fluorouracil - topical), Topotecan hydrochloride, Toremifene, Torisel (temsirolimus), Totect (dexrazoxane hydrochloride),Hydrochloride), Trabectedin, Trametinib, Trastuzumab, Trastuzumab and Hyaluronidase-oysk, Treanda (Bendamustine Hydrochloride), Trexall (Methotrexate), Trifluridine and Dipifrin Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Ultomiris (Ravulizumab-cwvz), Unituxin (Dalutuximab), Uridine Triacetate Triacetate, Valrubicin, Valstar (Valrubicin), Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velcade (Bortezomib), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Vidaza (Azacitidine), Vinblastine Sulfate, Vincristine Sulfate, Vincristine Sulfate Liposomal, Vinorelbine Tartrate, Vismodegib, Vistogard (Uridine Triacetate) Triacetate), Vitrakvi (larotrectinib sulfate), Vizimpro (dacomitinib), Vorazaze (glutarpidase), Vorinostat, Pazopanib (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposomal), Xalkori (crizotinib), Xeloda (capecitabine), Xgeva (denosumab), Xofigo (radium-223 dichloride), Xospata (gilteritinib fumarate),Fumarate), Xtandi (enzalutamide), Yervoy (ipilimumab), Yescarta (Axonoxomycin), Yondelis (trabectedin), Zaltrap (aflibercept), Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), Zoledronic acid ( Acid), Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Ceritinib, or Zytiga (abiraterone acetate). Many of these anticancer drugs, including their dosage information, are described on the National Cancer Institute's website, which is incorporated by reference in its entirety.

[0314] In some embodiments, the chemotherapeutic agent is 5-fluorouracil, bleomycin, capecitabine, cisplatin, cyclophosphamide, dacarbazine, doxorubicin, etoposide, folinic acid, methotrexate, oxaliplatin, prednisolone, procarbazine, vinblastine, vinorelbine, docetaxel, epirubicin, or mustine.

[0315] In some embodiments, the methods described herein can increase the efficacy of an anticancer drug (e.g., a chemotherapy drug) by at least 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, or 5-fold (e.g., as compared to the efficacy when the anticancer drug alone is administered to the subject at the same dose). Because the efficacy of the anticancer drug has been improved, in some embodiments, the therapeutically effective dose of the anticancer drug (e.g., chemotherapeutic agent) is about or at least 10%, 20%, 30%, 40%, or 50% lower than the typical dose of the anticancer drug (e.g., an FDA-approved dose), thereby minimizing side effects. In addition, because the efficacy of the anticancer drug has been improved, in some embodiments, a therapeutically effective dose of the anticancer drug can be administered to the subject less frequently. For example, during the treatment period, the intervals between administrations can be increased by about or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, or 5-fold compared to the intervals between administrations in the treatment plan for an FDA-approved anticancer drug. In some embodiments, the total number of administrations can be reduced during the treatment period. For example, the total number of administrations can be reduced by about or at least 10%, 20%, 30%, 40%, or 50%. In some embodiments, due to improved efficacy, the length of the treatment period can be shortened by, for example, about or at least 10%, 20%, 30%, 40%, or 50% compared to the length of the FDA-approved treatment plan.

[0316] In certain embodiments, other therapeutic agents can be adoptive cell therapy.Adoptive cell therapy includes giving T cells (a type of immune cell) to patients to help the body resist a type of immunotherapy for diseases such as cancer. In cancer therapy, T cells are usually taken from the patient's own blood or tumor tissue, grown in large quantities in the laboratory, and then returned to the patient to help the immune system resist cancer. In certain embodiments, adoptive cell therapy includes chimeric antigen receptor T cell (CAR T cell) therapy, tumor infiltrating lymphocytes (TIL) therapy, engineered TCR therapy or natural killer (NK) cell therapy. In certain embodiments, adoptive cell therapy can be referred to as adoptive cell transfer, cell adoptive immunotherapy or T cell transfer therapy.

[0317] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of: an adjuvant, a TLR agonist, tumor necrosis factor (TNF) α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a therapeutic target CX3CL1, a therapeutic target CXCL9, a therapeutic target CXCL10, a therapeutic target CCL5, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0318] In some embodiments, the patient is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0319] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

[0320] Treating infectious diseases

[0321] The present disclosure also provides a method for treating a patient who has been exposed to a toxin or pathogen. Therefore, another aspect of the present disclosure provides a method for treating an infectious disease in a subject, the method comprising administering an anti-PD-1 antibody or an antigen-binding portion thereof to the subject, such that the subject is treated for the infectious disease. Preferably, the antibody is a human anti-human PD-1 antibody (such as any human anti-PD-1 antibody among the human anti-PD-1 antibodies described herein). Additionally or alternatively, the antibody may be a chimeric antibody or a humanized antibody.

[0322] Similar to the above uses, antibody-mediated PD-1 blocking can be used alone or in combination with a vaccine as an adjuvant to stimulate an immune response to pathogens, toxins, and self-antigens. Examples of pathogens for which this treatment method can be used include, for example, pathogens for which there is currently no effective vaccine or pathogens for which conventional vaccines are not fully effective. These pathogens include, but are not limited to, HIV, hepatitis (type A, type B, or type C), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa. PD-1 blocking is particularly useful for resisting established infections caused by pathogens such as HIV that present mutant antigens during infection. When anti-human PD-1 antibodies are administered to a subject, these new epitopes can be recognized by the immune system, thereby stimulating a strong T cell response that is not suppressed by negative signals through PD-1.

[0323] Some examples of pathogenic viruses that cause infections that can be treated by the methods of the present disclosure include HIV, hepatitis (type A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSY-II and CMY, human herpesvirus type 4), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0324] Some examples of pathogenic bacteria that cause infections that can be treated by the methods of the present disclosure include Chlamydia, Rickettsial bacteria, Mycobacteria, Staphylococci, Streptococci, Pneumococci, Meningococci and Gonorrhea, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis, and Lyme disease bacteria.

[0325] Some examples of pathogenic fungi that cause infections that can be treated by the methods of the present disclosure include Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Mucor, Abreus, such as Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0326] Some examples of pathogenic parasites that cause infections that can be treated by the methods of the present disclosure include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia Zambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

[0327] In all of the above approaches, PD-1 blockade can be combined with other forms of immunotherapy such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provide enhanced presentation of tumor antigens (see, e.g., Bolliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

[0328] Bispecific antibodies or antigen-binding fragments can be produced by a variety of methods, including fusion of hybridomas or linking Fab' fragments. See, e.g., Songsivilai and Lachmarm, Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al., J. Immunol. 148: 1547-1553 (1992). Additionally, bispecific antibodies can be formed as "diabodies" or "Janusins." In some embodiments, the bispecific antibody binds to two different epitopes of PD-1. In some embodiments, the bispecific antibody has a first heavy chain and a first light chain from monoclonal antibody from hybridoma clone #11, and a further antibody heavy chain and light chain. In some embodiments, the further light chain and heavy chain are also from one of the above-identified monoclonal antibodies, but can be different from the first heavy chain and first light chain.

[0329] Pharmaceutical formulations

[0330] In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, containing one or a combination of the monoclonal antibodies of the present disclosure, or one or more antigen-binding portions thereof, formulated with a pharmaceutically acceptable carrier. Such compositions can include one or a combination (e.g., two or more different) of the antibodies of the present disclosure, or immunoconjugates or bispecific molecules. For example, a pharmaceutical composition of the present disclosure can include a combination of antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on the target antigen or have complementary activities.

[0331] Pharmaceutical compositions of the present disclosure can also be administered in combination therapy, i.e., combined with other agents. For example, a combination therapy can include an antibody to PD-1 of the present disclosure in combination with at least one other anti-inflammatory agent or immunosuppressive agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on uses of the antibodies of the present disclosure.

[0332] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0333] Pharmaceutical compositions of the present disclosure may include one or more pharmaceutically acceptable salts." pharmaceutically acceptable salts " refer to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects (see, for example, Berge, SM et al. (1977) "Journal of Pharmaceutical Science (J.Pharm.Sci.)" 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from nontoxic inorganic acids and salts from nontoxic organic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc., and nontoxic organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic sulfonic acids and aromatic sulfonic acids, etc. Base addition salts include salts derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as salts derived from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0334] The pharmaceutical composition of the present disclosure may further comprise a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0335] Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0336] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersants. By the above-mentioned sterilization procedures and by comprising various antibacterial agents and antifungal agents (for example, parabens, chlorobutanol, phenol sorbic acid etc.), both can ensure the existence of prevention microorganisms. It may also be desirable that isotonic agents, such as sugar, sodium chloride etc., are included in these compositions. In addition, the absorption of the extension of injectable drug form can be realized by comprising the medicament (such as aluminum monostearate and gelatin) that delays absorption.

[0337] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and medicaments for pharmaceutically active substances is known in the art. Except for the case where any conventional media or medicaments are incompatible with the active compound, its use in the pharmaceutical compositions of the present disclosure is contemplated. Supplementary active compounds can also be incorporated into the composition. The therapeutic composition must generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion and by using a surfactant. In many cases, it will be preferred to include an isotonic agent in the composition, such as a sugar, a polyol (e.g., mannitol, sorbitol), or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

[0338] Sterile injectable solutions can be prepared by incorporating the active compound of a desired amount and one or a combination thereof into a suitable solvent as required, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other desired components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of the active component plus any other desired component from a previously aseptically filtered solution thereof.

[0339] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of the composition that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the combination of the active ingredient and the pharmaceutically acceptable carrier.

[0340] The dosage regimen is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, a dosage unit refers to a physically discrete unit suitable as a single dose for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure are subject to and directly depend on: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art caused by individual sensitivity to such active compounds for compounding for treatment.

[0341] For administration of the antibody, the dosage range is about 0.0001-100 mg / kg of the host body weight, and more typically 0.01-5 mg / kg. For example, the dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight or in the range of 1-10 mg / kg. An exemplary treatment regimen requires administration as follows: once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three months to every 6 months. A preferred dosage regimen for the anti-PD-1 antibody of the present disclosure comprises 1 mg / kg body weight or 3 mg / kg body weight administered intravenously, wherein the antibody is administered using one of the following dosing regimens: (i) six doses every four weeks, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once, then 1 mg / kg body weight every three weeks.

[0342] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the range indicated. Antibodies are typically administered on a variety of occasions. The intervals between single doses can be, for example, weekly, monthly, every three months, or annually. Intervals can also be irregular, as indicated by measuring blood levels of the antibody to the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml and in some methods about 25-300 μg / ml.

[0343] Alternatively, the antibody can be used as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary according to the half-life of the antibody in the patient. Typically, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages are sometimes required at relatively short intervals until disease progression reduces or terminates, and preferably until the patient demonstrates partial improvement in symptoms of the disease or complete improvement. After this, a preventive regimen can be administered to the patient.

[0344] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present disclosure can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the specific composition of the present disclosure employed, or its ester, salt, or amide; the route of administration; the time of administration; the rate of absorption of the specific compound employed; the duration of treatment; other drugs, compounds, and / or materials used in combination with the specific composition employed; the age, sex, weight, condition, general health, and previous medical history of the patient being treated; and similar factors well known in the medical arts.

[0345] A "therapeutically effective dose" of the anti-PD-1 antibodies disclosed herein preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of injury or disability due to the disease affliction. For example, for the treatment of tumors, a "therapeutically effective dose" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%, relative to an untreated subject. The ability of a compound to inhibit tumor growth can be assessed in an animal model system that is predictive of efficacy in human tumors. Alternatively, this property of a composition can be assessed by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to skilled practitioners. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. One of ordinary skill in the art will be able to determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0346] In another aspect, as described herein, the present disclosure provides a pharmaceutical kit of parts comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody. The kit can further comprise instructions for treating a hyperproliferative disease, such as a cancer as described herein. In another embodiment, the anti-PD-1 antibody and the anti-CTLA-4 antibody can be co-packaged in a unit dosage form.

[0347] In certain embodiments, two or more monoclonal antibodies having different binding specificities (e.g., anti-PD-1 and anti-CTLA-4) are administered simultaneously, in which case the dosage of each antibody falls within the ranges indicated. The antibodies can be administered in a single dosage, or more usually can be administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, or yearly. Intervals can also be irregular, as indicated by measuring blood levels of the antibody in the patient's system. In some methods, dosages are adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, and in some methods about 25-300 μg / ml.

[0348] Compositions of the present disclosure can be administered using one or more of a variety of methods known in the art by one or more routes of administration. As will be appreciated by those of ordinary skill in the art, the route and / or mode of administration will vary depending upon the desired results. Preferred routes of administration of the antibodies of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0349] Alternatively, the antibodies of the present disclosure can be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., nasal administration, oral administration, vaginal administration, rectal administration, sublingual administration or topical administration.

[0350] Active compounds can be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0351] Therapeutic compositions can be administered with medical devices known in the art. For example, in preferred embodiments, the therapeutic compositions of the present disclosure can be administered with needle-free hypodermic injection devices, such as those disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules that can be used with the present disclosure include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapy administration apparatus for administering medicine through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chambered compartment; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Numerous other such implants, delivery systems, and modules are known to those skilled in the art.

[0352] In certain embodiments, human monoclonal antibodies of the present disclosure can be formulated to ensure proper distribution in vivo. For example, the blood brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that a therapeutic compound of the present disclosure crosses the BBB, if desired, the compound can be formulated, for example, in a liposome. See, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331 for methods of manufacturing liposomes. The liposomes can include one or more moieties that are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); a mannose

[0353] Also provided herein are pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical compositions can be formulated in any manner known in the art.

[0354] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods of manufacturing the antibodies or antigen-binding fragments thereof for the various uses as described herein.

[0355] Examples

[0356] The application is further described in the following examples, which do not limit the scope of the application described in the claims.

[0357] Example 1. Generation of mouse anti-PD-1 antibody

[0358] Experiments were performed to generate mouse antibodies against human PD-1. Mice were immunized with human PD-1. Spleen cells were collected. Hybridomas were screened for binding affinity. A hybridoma (clone #11) was selected for further experiments. The sequence of the antibody was determined by sequencing (F02).

[0359] Western blot analysis was performed with the anti-PD-1 antibody obtained from the selected hybridoma (clone #11). Figure 1 ) HEK-293T cells were purchased from the American Type Culture Collection (ATCC). HEK-293T cells were transfected with pCMV6-ENTRY control (left lane) or pCMV6-ENTRY with PDCD1 cDNA (Origene, Cat# RC210364; right lane) for 48 hours and lysed. Equivalent amounts of cell lysates (5 μg per lane) were separated by SDS-PAGE and immunoblotted with the anti-PD-1 antibody (1 :2000). The results show that the anti-PD-1 antibody can bind to human PD-1 with high affinity.

[0360] Example 2. Immunofluorescence staining

[0361] Immunofluorescence staining was performed on cells expressing human PD-1. 293T-PD-1 cells were generated by lentiviral transduction of HEK-293T cells with a vector expressing human PD-1. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

[0362] Figure 2A Immunofluorescence staining of PDCD1 on HEK293T cells stably transfected with RC210364 is shown with mouse monoclonal antibody anti-PD-1. Nuclei were labeled with Hoechst 33342. The same experiment was performed on HEK293T cells as a negative control (1 : 100). Figure 2B

[0363] Figure 3A Immunocytochemical staining of cells stably expressing PD1 using anti-PD-1 mouse monoclonal antibody is shown. Figure 3B Immunocytochemical staining of 293T cells as a negative control (1 : 900) is shown. ​

[0364] Flow cytometry analysis of cells expressing PD-1 was also performed using an anti-PD-1 antibody and a non-specific control antibody (1 :50) (Figure 2). Figure 4 ).

[0365] Figure 5 Flow cytometry analysis of cells stably expressing PD-L1 (R&D Systems, Cat# RC213071) using either the anti-PD-1 antibody from hybridoma clone #11 (F02) or 0.3 ug / ml PD1-Fc fusion protein (TP700199) or both and detected by anti-Fc (human) IgG-FITC (1 :50) is shown. Binding of PD-L1 to PD-1 was completely blocked by the anti-PD-1 antibody from hybridoma clone #11. The results show that the anti-PD-1 antibody can effectively block the binding of PD-L1 to PD-1.

[0366] Figure 6 Flow cytometry analysis of HEK293T cells transiently transfected with PD-L2 (R&D Systems, Cat# RC224141) using either the anti-PD-1 antibody from hybridoma clone #11 or 1 ug / ml PD1-Fc fusion protein (TP700199) or both and detected by anti-Fc (human) IgG-FITC (1 :50) is shown. Binding of PD-L2 to PD-1 was completely blocked by the anti-PD-1 antibody from hybridoma clone #11. The results show that the anti-PD-1 antibody can effectively block the binding of PD-L2 to PD-1.

[0367] Example 3. Humanization of antibodies

[0368] Typically, the antibody specific sequences (CDRs) from the mouse parental antibody are grafted onto human donor sequences. The donor sequences are chosen based on bioinformatics software that calculates a unique humanness score for each combination. These antibodies are cloned into expression vectors and transfected into cell lines for recombinant protein expression. The antibodies are tested in vitro for affinity to the target protein, PD-1, by ELISA and cell-based binding assays.

[0369] Humanization of antibodies is achieved by CDR grafting strategies and surface remodelling strategies. In addition, deimmunization strategies are also used. Bioinformatics tools including antibody modelling and identification of key framework residues are utilized to design humanized heavy and light chains. Those antibodies with the highest humanization score are combined to obtain a complete antibody. Many of these antibodies will have an affinity that can be included by the original antibody.

[0370] Humanized VH and VL are cloned into transient expression vectors. Final construct is confirmed by sequencing. In addition, candidate pairs of heavy chain and light chain (comprising a chimeric pair for comparison) are cotransfected into CHO or HEK293 cells to carry out transient expression. Expressed antibodies are purified, and their epitope specificity and affinity are measured by ELISA or cell-based binding assays.

[0371] Example 4. Binding affinity of anti-PD1 to recombinant human PD-1 (rhPD-1)

[0372] Microwells were coated with 5 μg / ml anti-PD-1 antibody in 100 μl of coating buffer overnight at 4°C. Following the ELISA protocol, 5-fold serial dilutions of recombinant biotinylated human PD-1 were added to the assay plate pre-coated with anti-PD-1 antibody. Bound human PD-1 protein was then detected using Sav-HRP reagent. Results were analyzed using Prism 7, and Kd values ​​were determined using the program.

[0373] like Figures 7A-7G As shown, most anti-PD1 antibodies can bind to recombinant human PD-1 with Kd values ​​in the nM range as determined by ELISA. For example, the estimated Kd for h11 was 0.29 nM, the estimated Kd for Ab7 was 0.68 nM, and the estimated Kd for Ab8 was 0.32 nM. The h11, Ab7, and Ab8 antibodies exhibited Kd values ​​comparable to the original mouse clone F02.

[0374] Example 5. Binding affinity of anti-PD1 to human PD-1 (hPD-1) on the cell surface

[0375] 293T-PD-1 cells (0.03 × 10 6 The cells were incubated with 4% paraformaldehyde (4% paraformaldehyde) per well for 60 minutes. The cells were then stained with secondary antibodies (humanized APC anti-Hu IgG Fc and F02 anti-FITC anti-mouse IgG F(ab')2) for 30 minutes (1:50) and analyzed by flow cytometry. The results were analyzed using Prism 7, and K values ​​were determined using the program.

[0376] like Figures 8A-8GAs shown, all anti-PD1 antibodies can bind to the PD-1 protein expressed on 293T-PD-1 cells. The Kd values ​​of all antibodies determined by flow cytometry were in the nM range. For example, the estimated Kd of h11 was 0.55 nM, the estimated Kd of Ab2 was 3.24 nM, the estimated Kd of Ab4 was 41 nM, the estimated Kd of Ab5 was 3.69 nM, the estimated Kd of Ab7 was 2.3 nM, and the estimated Kd of Ab8 was 0.65 nM.

[0377] Other embodiments

[0378] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to programmed cell death protein 1 (PD-1), comprising: a heavy chain variable region VH, comprising complementarity determining regions CDR1, 2, and 3, wherein the amino acid sequence of the VH CDR1 region is identical to the selected VH CDR1 amino acid sequence, the amino acid sequence of the VH CDR2 region is identical to the selected VH CDR2 amino acid sequence, and the amino acid sequence of the VH CDR3 region is identical to the selected VH CDR3 amino acid sequence; and a light chain variable region VL, said VL comprising CDR1, 2, and 3, wherein the amino acid sequence of the VL CDR1 region is identical to the selected VL CDR1 amino acid sequence, the amino acid sequence of the VL CDR2 region is identical to the selected VL CDR2 amino acid sequence, and the amino acid sequence of the VL CDR3 region is identical to the selected VL CDR3 amino acid sequence, wherein the selected VH CDR1, 2 and 3 and the selected VL CDR1, 2 and 3 are amino acid sequences based on one of the following: (1) the selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 22, 23, and 24, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 19, 20, and 21, respectively; (2) the selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 8, 9, and 10, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 5, 6, and 7, respectively; (3) the selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 74, 75, and 76, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 71, 72, and 73, respectively; and (4) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 34, 35, and 36, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 31, 32, and 33, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequences of CDR1, 2, and 3 of the VH are shown in SEQ ID NOs: 8, 9, and 10, respectively, and the amino acid sequences of CDR1, 2, and 3 of the VL are shown in SEQ ID NOs: 5, 6, and 7, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequences of CDR1, 2, and 3 of the VH are shown in SEQ ID NOs: 22, 23, and 24, respectively, and the amino acid sequences of CDR1, 2, and 3 of the VL are shown in SEQ ID NOs: 19, 20, and 21, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequences of CDR1, 2, and 3 of the VH are shown in SEQ ID NOs: 74, 75, and 76, respectively, and the amino acid sequences of CDR1, 2, and 3 of the VL are shown in SEQ ID NOs: 71, 72, and 73, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequences of CDR1, 2, and 3 of the VH are shown in SEQ ID NOs: 34, 35, and 36, respectively, and the amino acid sequences of CDR1, 2, and 3 of the VL are shown in SEQ ID NOs: 31, 32, and 33, respectively. The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody or antigen-binding fragment thereof specifically binds to human PD-1.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

9. A nucleic acid comprising a polynucleotide encoding a polypeptide comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. The nucleic acid of claim 9, wherein the nucleic acid encodes a single-chain variable fragment (scFv).

11. The nucleic acid of claim 9, wherein the nucleic acid is cDNA.

12. A vector comprising the nucleic acid according to any one of claims 9 to 11.

13. A vector comprising two of the nucleic acids according to any one of claims 9 to 11, wherein the vector encodes a VL region and a VH region that together bind to PD-1.

14. A vector pair, each comprising the nucleic acid of any one of claims 9 to 11, wherein the vector pair together encodes a VL region and a VH region that together bind to PD-1.

15. A cell comprising the vector according to claim 12.

16. The cell of claim 15, wherein the cell is a CHO cell.

17. A cell comprising one or more of the nucleic acids according to any one of claims 9 to 11.

18. A cell comprising two of the nucleic acids according to any one of claims 9 to 11.

19. The cell of claim 18, wherein two of the nucleic acids together encode a VL region and a VH region that together bind to PD-1.

20. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising The cell of claim 17 is cultured under conditions sufficient for the cell to produce the antibody or antigen-binding fragment thereof.

21. An antibody or antigen-binding fragment thereof that binds to PD-1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL; in: The amino acid sequence of the VH is SEQ ID NO: 4, and the amino acid sequence of the VL is SEQ ID NO: 2; The amino acid sequence of the VH is SEQ ID NO: 18, and the amino acid sequence of the VL is SEQ ID NO: 16; The amino acid sequence of the VH is SEQ ID NO: 70, and the amino acid sequence of the VL is SEQ ID NO: 68; The amino acid sequence of the VH is SEQ ID NO: 30, and the amino acid sequence of the VL is SEQ ID NO: 28; The amino acid sequence of the VH is SEQ ID NO: 50, and the amino acid sequence of the VL is SEQ ID NO: 48; The amino acid sequence of the VH is SEQ ID NO: 60, and the amino acid sequence of the VL is SEQ ID NO: 58; or The amino acid sequence of the VH is SEQ ID NO:40, and the amino acid sequence of the VL is SEQ ID NO:

38.

22. The antibody or antigen-binding fragment thereof according to claim 21, wherein the antibody or antigen-binding fragment thereof specifically binds to human PD-1.

23. The antibody or antigen-binding fragment thereof according to claim 21, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

24. The antibody or antigen-binding fragment thereof of claim 21, wherein the antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

25. An antibody or antigen-binding fragment thereof, comprising CDR1, 2, 3 of the VH and CDR1, 2, 3 of the VL of the antibody or antigen-binding fragment thereof according to claim 21.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 21 to 25 and a pharmaceutically acceptable carrier.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 21 to 25, wherein the antibody is an IgG1 antibody, an IgG2 antibody or an IgG4 antibody.

28. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 21 to 25, wherein the antibody is a human IgG1 antibody.

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