Novel Anti-PD-1 antibodies

Abstract

To provide monoclonal antibodies against programmed cell death 1 (PD-1).SOLUTION: The present disclosure provides monoclonal antibodies against programmed cell death 1 (PD-1), which can block the binding of PD-1 ligands to PD-1 and therefore can block the inhibitory function of PD-1 ligands on PD-1 expressing T cells. The antibodies of disclosure provide very potent agents for treatment of multiple cancers by modulating the human immune function.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates generally to novel anti-PD-1 antibodies. [Background technology]

[0002] Increasing evidence from preclinical and clinical trial results has shown that targeting immune checkpoints is becoming the most promising approach to treat cancer patients. Programmed cell death 1, one of the immune checkpoint proteins, plays a major role in limiting the activity of T cells, which provides the main immune resistance mechanism for tumor cells to escape immune surveillance. The interaction of PD-1 expressed on activated T cells with PD-L1 expressed on tumor cells negatively regulates immune responses and weakens antitumor immunity. Expression of PD-L1 on tumors correlates with poor survival in esophageal, pancreatic and other types of cancer, highlighting this pathway as a novel promising target in cancer immunotherapy. Multiple agents targeting the PD-1 pathway are being developed by pharmaceutical companies such as Bristol-Myers Squibb (BMS), Merck, Roche and GlaxoSmithKline (GSK). Clinical trial data showed early evidence of sustained clinical activity and an encouraging safety profile in patients with various tumor types. Nivolumab, a PD-1 drug developed by BMS, is taking center stage for the next generation. Currently in six late-stage studies, treatment with nivolumab has promoted tumor shrinkage in three of the five cancer groups studied, including 18% of 72 lung cancer patients, nearly one-third of 98 melanoma patients, and 27% of 33 kidney cancer patients. Lambrolizumab, developed by Merck, is a fully human IgG4 monoclonal antibody that acts against PD-1 and has garnered FDA Breakthrough Designation after exciting data from a Phase Ib clinical trial in skin cancer. Phase Ib results showed objective antitumor responses in 51% of 85 cancer patients, with 9% of patients achieving complete responses. Rosci The company's experimental MPDL3280A showed the ability to shrink tumors in 29 of 140 (21%) patients with advanced cancer who had tumors of various sizes.

[0003] However, existing treatments may not be entirely satisfactory, and therefore new anti-PD-1 antibodies remain needed. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides novel anti-PD-1 monoclonal antibodies, particularly fully human antibodies, polynucleotides encoding the antibodies, and methods of using the antibodies.

[0005] In one aspect, the present disclosure provides a method for the production of a medicament comprising administering to a subject a therapeutically effective amount ... -8 M or less (e.g. 9x10 -9 M or less, 8x10 -9 M or less, 7x10 -9 M or less, 6x10 -9 M or less, 5x10 -9 M or less, 4x10 -9 M or less, 3x10 -9 M or less, 2x10 -9 M or less, or 10 -9 The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that can specifically bind to human PD-1 with a Kd value of M or less.

[0006] In one embodiment, the antibodies or antigen-binding fragments thereof bind to monkey PD-1 with an EC50 of 100 nM or less or 10 nM or less (e.g., 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less). In one embodiment, the antibodies or antigen-binding fragments thereof bind to monkey PD-1 but not to mouse PD-1 with a binding affinity similar to the binding affinity to human PD-1. In one embodiment, the antibody or antigen-binding fragment thereof potently inhibits the binding of human or monkey PD-1 to its ligand (e.g., PD-L1 or PD-L2) with an IC50 of 100 nM or less (e.g., 50 nM, 40 nM, 30 nM, 20 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 or less). In one embodiment, the EC50 or IC50 is measured by fluorescence activated cell sorting (FACS) analysis.

[0007] In one embodiment, the antibody or antigen-binding fragment thereof has a substantially reduced effector function, hi one embodiment, said antibody or antigen-binding fragment thereof does not mediate ADCC or CDC, or both.

[0008] In one embodiment, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain CDR sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37. include.

[0009] In one embodiment, the antibody or antigen-binding fragment thereof provided herein comprises a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, 43 and 65.

[0010] In one embodiment, the antibody or antigen-binding fragment thereof provided herein comprises one or more, two or more, three or more, four or more, five or more, or six or more CDRs selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11; or selected from the group consisting of SEQ ID NOs: 13, 15, 5, 7, 17, and 11; or selected from the group consisting of SEQ ID NOs: 1, 15, 5, 7, 17, and 19; or selected from the group consisting of SEQ ID NOs: 1, 15, 5, 7, 17, and 65; or selected from the group consisting of SEQ ID NOs: 21, 23, 25, 27, 29, and 31; or selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, 41, and 43.

[0011] In one embodiment, the antibody or antigen-binding fragment thereof provided herein is a) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:3, and / or SEQ ID NO:5; b) a heavy chain variable region comprising SEQ ID NO:13, SEQ ID NO:15, and / or SEQ ID NO:5; c) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and / or SEQ ID NO:5; d) a heavy chain variable region comprising SEQ ID NO:21, SEQ ID NO:23, and / or SEQ ID NO:25; and e) a heavy chain variable region comprising SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37. The heavy chain variable region is selected from the group consisting of:

[0012] In one embodiment, the antibody or antigen-binding fragment thereof provided herein is a) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:11; b) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:11; c) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:19; d) a light chain variable region comprising SEQ ID NO:27, SEQ ID NO:29, and / or SEQ ID NO:31; e) a light chain variable region comprising SEQ ID NO:39, SEQ ID NO:41, and / or SEQ ID NO:43; and f) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:65. The light chain variable region comprises a light chain variable region selected from the group consisting of:

[0013] In one embodiment, the antibody or antigen-binding fragment thereof provided herein is a) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:3, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:11; b) a heavy chain variable region comprising SEQ ID NO:13, SEQ ID NO:15, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:11; c) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:19; d) a heavy chain variable region comprising SEQ ID NO:21, SEQ ID NO:23, and / or SEQ ID NO:25; and a light chain variable region comprising SEQ ID NO:27, SEQ ID NO:29, and / or SEQ ID NO:31; e) a heavy chain variable region comprising SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37; and a light chain variable region comprising SEQ ID NO:39, SEQ ID NO:41, and / or SEQ ID NO:43; or f) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15 and / or SEQ ID NO:5; and A light chain variable region comprising sequence number 7, SEQ ID NO: 17, and / or SEQ ID NO: 65. Includes.

[0014] In one embodiment, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region selected from the group consisting of SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57 and SEQ ID NO:61.

[0015] In one embodiment, the antibody or antigen-binding fragment thereof provided herein comprises a light chain variable region selected from the group consisting of SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63 and SEQ ID NO:67.

[0016] In one embodiment, the antibody or antigen-binding fragment thereof provided herein is a) a heavy chain variable region comprising SEQ ID NO:45; and a light chain variable region comprising SEQ ID NO:47; b) a heavy chain variable region comprising SEQ ID NO:49; and a light chain variable region comprising SEQ ID NO:51; c) a heavy chain variable region comprising SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO:55; d) a heavy chain variable region comprising SEQ ID NO:57; and a light chain variable region comprising SEQ ID NO:59; e) a heavy chain variable region comprising SEQ ID NO: 61; and a light chain variable region comprising SEQ ID NO: 63; or f) a heavy chain variable region comprising SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO:67 Includes.

[0017] In one embodiment, the antibodies provided herein include, for example, 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb, and 1.153.7hAb.

[0018] In one embodiment, an antibody or antigen-binding fragment thereof provided herein competes for the same epitope as antibody 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb, or 1.153.7 hAb. In one embodiment, an antibody or antigen-binding fragment thereof provided herein binds to an epitope that includes at least one of the following amino acid residues of PD-1: V64, P83, D85, L128, A129, P130, K131, A132, and Q133.

[0019] In one embodiment, the antibody or antigen-binding fragment thereof is capable of inhibiting the binding of human PD-1 to its ligand, thereby inhibiting the following activities: a) the activity of inducing IL-2 production in CD4+ T cells; b) the activity of inducing IFNγ production in CD4+ T cells; c) the ability to induce proliferation of CD4+ T cells; and d) Ability to neutralize the suppressive function of regulatory T cells Provide at least one of the following:

[0020] In one embodiment, the antibody provided herein is a monoclonal antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, a bivalent antibody, or an anti-idiotypic antibody. In one embodiment, the antibody or antigen-binding fragment thereof is a fully human monoclonal antibody, optionally produced by a transgenic rat, e.g., a transgenic rat having a recombinant human immunoglobulin locus in which endogenous expression of rat immunoglobulin genes has been inactivated, the J locus has been deleted, and Cκ has been mutated.

[0021] In one embodiment, the antigen-binding fragment provided herein is a camelized single domain antibody, diabody, scFv, dimeric scFv, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, nanobody, domain antibody, or bivalent domain antibody.

[0022] In one embodiment, the antibody or antigen-binding fragment thereof provided herein further comprises an immunoglobulin constant region.

[0023] In one embodiment, the antibody or antigen-binding fragment thereof provided herein further comprises a conjugate.

[0024] In one embodiment, the conjugate may be a detectable label, a pharmacokinetic-modifying moiety, or a purification moiety.

[0025] In another aspect, the disclosure provides isolated polynucleotides encoding the antibodies or antigen-binding fragments thereof provided herein. In one embodiment, polynucleotides are provided that encode the amino acid sequence of the antibodies or antigen-binding fragments thereof disclosed herein. In another embodiment, vectors are provided that include these polynucleotides, and in another embodiment, host cells are provided that include these vectors. In one embodiment, one of the antibodies or antigen-binding fragments disclosed herein is produced by culturing a host cell under conditions in which the antibody or antigen-binding fragment encoded by the polynucleotide is expressed from the vector. Methods for expressing one or more species are provided. In one embodiment, the polynucleotides provided herein are operably linked to a promoter, such as SV40, in a vector. In one embodiment, the host cell comprising the vector provided herein is a Chinese hamster ovary cell, or a 293F cell.

[0026] In another aspect, the disclosure provides a kit comprising the antibody or antigen-binding fragment thereof.

[0027] In another aspect, the PD-1 antibodies provided herein, such as 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2 hAb, 1.139.15 hAb, and 1.153.7 hAb, are well tolerated in animals and have high in vivo anti-tumor activity. In one embodiment, animals bearing tumor cells administered a PD-1 antibody provided herein experience a 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, or 95% or greater reduction in tumor volume compared to control animals with similar baseline tumor volumes but administered vehicle only.

[0028] In another aspect, a method is provided for treating a PD-1 associated condition in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the individual has been identified as having a disorder or condition that may be responsive to a PD-1 antagonist. In one embodiment, the individual has been identified as positive for the presence of PD-L1 or elevated levels of PD-L1 in a test biological sample from the individual.

[0029] In another aspect, the disclosure provides pharmaceutical compositions comprising an antibody or antigen-binding fragment provided herein and one or more pharma- ceutically acceptable carriers, which in some of these embodiments may be, for example, diluents, antioxidants, adjuvants, excipients, or non-toxic auxiliary substances.

[0030] In another aspect, the disclosure provides a method of treating a condition in a subject that would benefit from upregulation of an immune response, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment provided herein. In certain of these embodiments, the subject has upregulated expression of PD-L1 or has been identified as being positive for expression of PD-L1.

[0031] Use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a condition that would benefit from upregulation of an immune response, in one embodiment, the condition is cancer or a chronic viral infection. The present invention provides, for example, the following items. (Item 1) A heavy chain selected from the group consisting of SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35 and 37. An isolated antibody or antigen-binding fragment thereof comprising the CDR sequences of said antibody or antigen-binding fragment. (Item 2) The antibody or antigen-binding fragment thereof according to item 1, comprising a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, 43 and 65. (Item 3) The antibody or antigen-binding fragment thereof of any one of the preceding items, a) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:3, and / or SEQ ID NO:5; b) a heavy chain variable region comprising SEQ ID NO:13, SEQ ID NO:15, and / or SEQ ID NO:5; c) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and / or SEQ ID NO:5; d) a heavy chain variable region comprising SEQ ID NO:21, SEQ ID NO:23, and / or SEQ ID NO:25; and e) a heavy chain variable region comprising SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region selected from the group consisting of: (Item 4) The antibody or antigen-binding fragment thereof of any one of the preceding items, a) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:11; b) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:11; c) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:19; d) a light chain variable region comprising SEQ ID NO:27, SEQ ID NO:29, and / or SEQ ID NO:31; e) a light chain variable region comprising SEQ ID NO:39, SEQ ID NO:41, and / or SEQ ID NO:43; and f) a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:65 An antibody or antigen-binding fragment thereof comprising a light chain variable region selected from the group consisting of: (Item 5) The antibody or antigen-binding fragment thereof of any one of the preceding items, a) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:3, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:11; b) a heavy chain variable region comprising SEQ ID NO:13, SEQ ID NO:15, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:11; c) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:19; d) a heavy chain variable region comprising SEQ ID NO:21, SEQ ID NO:23, and / or SEQ ID NO:25; and a light chain variable region comprising SEQ ID NO:27, SEQ ID NO:29, and / or SEQ ID NO:31; or e) a heavy chain variable region comprising SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37; and a light chain variable region comprising SEQ ID NO:39, SEQ ID NO:41, and / or SEQ ID NO:43; or f) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and / or SEQ ID NO:5; and A light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:65. An antibody or an antigen-binding fragment thereof comprising: (Item 6) The antibody or antigen thereof of any one of the preceding items, comprising a heavy chain variable region selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57 and SEQ ID NO: 61. Associative fragment. (Item 7) 2. The antibody or antibody of any one of the preceding items, comprising a light chain variable region selected from the group consisting of SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63 and SEQ ID NO:67. or an antigen-binding fragment thereof. (Item 8) The antibody or antigen-binding fragment thereof of any one of the preceding items, a) a heavy chain variable region comprising SEQ ID NO:45; and a light chain variable region comprising SEQ ID NO:47; b) a heavy chain variable region comprising SEQ ID NO:49; and a light chain variable region comprising SEQ ID NO:51; c) a heavy chain variable region comprising SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO:55; d) a heavy chain variable region comprising SEQ ID NO:57; and a light chain variable region comprising SEQ ID NO:59; e) a heavy chain variable region comprising SEQ ID NO:61; and a light chain variable region comprising SEQ ID NO:63; or f) a heavy chain variable region comprising SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO:67 An antibody or an antigen-binding fragment thereof comprising: (Item 9) 10 as measured by plasmon resonance binding assay -8 Specific for human PD-1 with Kd value of ≤ M Item 1. The antibody or antigen-binding fragment thereof of any one of the preceding items, capable of binding to (Item 10) The antibody or antigen-binding fragment thereof of any one of the preceding items, which binds to monkey PD-1 with an EC50 of 100 nM or less or 10 nM or less, and / or does not bind to mouse PD-1. (Item 11) The antibody or antigen-binding fragment thereof of any one of the preceding items, which is capable of inhibiting binding of human PD-1 or monkey PD-1 to a PD-1 ligand with an IC50 of 100 nM or less. (Item 12) The antibody or antigen-binding fragment thereof of any one of the preceding items, which does not substantially bind to CD28 or CTLA4. (Item 13) The antibody or antigen-binding fragment thereof of any one of the preceding items, which does not mediate ADCC or CDC or both. (Item 14) 10. The antibody or antigen-binding fragment thereof of any one of the preceding items, which is a fully human monoclonal antibody. (Item 15) 15. The antibody or antigen-binding fragment thereof according to item 14, wherein the fully human monoclonal antibody is produced by a transgenic rat. (Item 16) An antibody or antigen-binding fragment thereof that competes for the same epitope as the antibody or antigen-binding fragment thereof of any one of the preceding items. (Item 17) It is capable of inhibiting the binding of human PD-1 to the PD-1 ligand, thereby inhibiting the following activities: a) CD4 + Induction of IL-2 production in T cells; b) CD4 + Induction of IFNγ production in T cells; c) CD4 + Induction of T cell proliferation; and d) Ability to neutralize the suppressive function of regulatory T cells The antibody or antigen-binding fragment thereof of any one of the preceding items, which provides at least one of the following activities: (Item 18) The antibody or antigen-binding fragment thereof of any one of the preceding items, which is a camelized single domain antibody, diabody, scFv, dimeric scFv, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, nanobody, domain antibody, or bivalent domain antibody. (Item 19) 2. The antibody or antigen-binding fragment thereof of any one of the preceding items, further comprising an immunoglobulin constant region. (Item 20) The antibody or antigen-binding fragment thereof of any one of the preceding items, further comprising a conjugate. (Item 21) 20. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of items 1 to 19. (Item 22) 22. A vector comprising the isolated polynucleotide of item 21. (Item 23) A host cell comprising the vector according to item 22. (Item 24) A method for expressing the antibody or antigen-binding fragment thereof according to any one of Items 1 to 19, comprising culturing the host cell according to Item 23 under conditions in which the polynucleotide according to Item 21 is expressed. (Item 25) 21. A kit comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 20. (Item 26) 21. A method for treating a PD-1 associated condition in an individual, comprising administering to said individual a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 20. (Item 27) 27. The method of claim 26, wherein the individual is an individual identified as having a disorder or condition that may be responsive to a PD-1 antagonist. (Item 28) 28. The method of claim 27, wherein the individual has been identified as positive for the presence of PD-L1 or elevated levels of PD-L1 in a test biological sample from the individual. (Item 29) The antibody or antigen-binding fragment thereof according to any one of items 1 to 20 and one or more A pharmaceutical composition comprising a pharma- ceutically acceptable carrier. (Item 30) 21. A method for treating a condition that can be ameliorated by upregulation of an immune response in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 20. (Item 31) 31. The method of claim 30, wherein the subject exhibits elevated expression of PD-L1. (Item 32) 21. Use of the antibody or antigen-binding fragment according to any one of items 1 to 20 in the manufacture of a medicament for the treatment of a condition which would benefit from upregulation of an immune response. (Item 33) 33. The use according to item 32, wherein the condition is cancer or a chronic viral infection. (Item 34) 17. The antibody or antigen-binding fragment of item 16, wherein the epitope comprises at least one of the following amino acid residues of PD-1: V64, P83, D85, L128, A129, P130, K131, A132, and Q133. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 shows binding of fully human anti-PD-1 antibodies to PD-1-expressing CHO cells as measured by FACS analysis. [Diagram 2] FIG. 2 shows binding of fully human anti-PD-1 antibodies to PD-1-expressing CHO cells with an EC50 of approximately 2 nM as measured by FACS analysis. [Diagram 3] FIG. 3. Binding of fully human anti-PD-1 antibodies to PD-1 expressed on activated CD4+ cells as measured by FACS analysis. [Figure 4] Figure 4 shows that fully human anti-PD-1 antibodies inhibited PD-L1 binding to PD-1 transfected CHO cells with IC50 values ​​of approximately 3-8 nM as measured by FACS analysis. [Diagram 5] FIG. 5 shows that fully human anti-PD-1 antibodies specifically bind to PD-1, but not to family members CD28 and CTLA4, as determined by FACS analysis. [Figure 6] FIG. 6 shows that fully human anti-PD-1 antibodies against PD-1 bind to cynomolgus monkey PD-1 but not to mouse PD-1. [Figure 7] FIG. 7 shows the overall kinetics of binding affinity of PD-1 antibodies to human PD-1 ranging from 3.76E-9 mol / L to 1.76E-10 mol / L as determined by surface plasmon resonance. [Figure 8] FIG. 8 illustrates the effect of fully human anti-PD-1 antibodies on IL-2 production in a mixed lymphocyte reaction (MLR). [Figure 9] FIG. 9 illustrates the effect of fully human anti-PD-1 antibodies on IFNγ production in MLRs. [Figure 10]FIG. 10 shows that fully human anti-PD-1 antibodies promoted T cell proliferation in an MLR. [Figure 11] Figure 11 shows that fully human anti-PD-1 antibodies promoted T cell proliferation in specific T cell responses. [Figure 12] FIG. 12 shows that anti-PD-1 antibody abolished the suppressive function of regulatory T cells. [Figure 13] FIG. 13 shows that anti-PD-1 antibodies did not demonstrate ADCC against activated T cells. [Figure 14] FIG. 14 shows that anti-PD-1 antibodies did not exhibit CDC against activated T cells. [Figure 15] Figure 15 shows that 1.103.11-v2 hAb binds to the extracellular domain of human PD-1 with similar affinity as measured by ELISA in different buffers. "1.103.11-v2hAb in buffer" refers to the antibody in the formulation buffer and "1.103.11-v2hAb in PBS" refers to the antibody in 1x PBS at pH 7.4. [Figure 16] Figure 16 shows that 1.103.11-v2 hAb binds to PD-1 expressing CHO cells in different buffers with similar affinity as measured by FACS. "1.103.11-v2hAb in buffer" refers to the antibody in the formulation buffer and "1.103.11-v2hAb in PBS" refers to the antibody in 1x PBS at pH 7.4. [Figure 17] Figure 17 shows the hotspot residues (shaded areas) on the crystal structure of human PD-L1 to which the antibodies bind. A indicates the common hotspot residues; BD indicates the hotspot residues of 1.103.11 hAb, Keytruda, and 11.148.10 hAb, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description of the Invention The following description of the present disclosure is intended to merely illustrate various embodiments of the present disclosure. Therefore, the specific modifications described should not be understood as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and such equivalent embodiments are understood to be included in the present invention. All references cited in this specification, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0034] definition The term "antibody" as used herein includes any immunoglobulin, monoclonal, polyclonal, polyspecific, or bispecific (bivalent) antibody that binds to a specific antigen. A natural intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, and each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, while mammalian light chains are classified as λ or κ. Antibodies are in the shape of a "Y" with the base of the Y consisting of the second and third constant regions of two heavy chains linked together by disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain linked to the variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains generally contain three hypervariable loops called complementarity determining regions (CDRs) (the light (L) chain CDRs contain LCDR1, LCDR2, and LCDR3, and the heavy (H) chain CDRs contain HCDR1, HCDR2, and HCDR3). The boundaries of the CDRs of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5; 186(3):651-63 (1985); Chothia, C. and Lesk, A. M., J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83 (1989); Kabat E. A. et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are flanked by adjacent stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified into classes based on the amino acid sequence of the constant region of the heavy chain.The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, characterized by the presence of α, δ, ε, γ and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0035] The term "antibody-binding fragment" as used herein refers to an antibody fragment formed from a portion of an antibody that contains one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain the entire native antibody structure. Examples of antibody-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab') 2 , Fv fragment, Fv fragment stabilized by disulfide bond (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain antibody molecule (scFv), dimeric scFv (bivalent diabody), multispecific antibody, camelized single domain antibody, nanobody Antigen-binding fragments include domain antibodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment can bind to the same antigen that its parent antibody binds. In one embodiment, an antigen-binding fragment may contain one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0036] "Fab" with respect to an antibody refers to that portion of that antibody that consists of a single light chain (both the variable and constant regions) bonded by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0037] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0038] "F(ab') 2 " refers to a Fab' dimer.

[0039] "Fc" with respect to an antibody refers to the portion of that antibody that consists of the second and third constant regions of one heavy chain linked via disulfide bonds to the second and third constant regions of a second heavy chain. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not function in antigen binding.

[0040] "Fv" with respect to an antibody refers to the minimum fragment of that antibody that contains a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0041] A "single chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked together directly or via a peptide linker sequence (Huston J Setal. Proc Natl Acad Sci USA, 85:5879 (1988)).

[0042] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0043] "Camelized single domain antibodies", "heavy chain antibodies" or "HCAbs" are antibodies that consist of two V HIt refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38 (1999); Muyldermans S., JBiotechnol.Jun; 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; US ​​Patent No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae family (camels, dromedaries and llamas). Camelized antibodies lack light chains but have a full antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. Apr;54(1):39-47(2002); Nguyen VK. et al. Immunology. May;109(1):93-101 (2003)). The variable domain of heavy chain antibodies (VHH domain) is the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0044] "Nanobody" refers to an antibody fragment consisting of a VHH domain derived from a heavy chain antibody and two constant domains, CH2 and CH3.

[0045] "Diabodies" are V L Domain-bound V H Domain (V H -V L or V L -V H), a small antibody fragment that has two antigen-binding sites (see, for example, Holliger P. et al., Proc Natl Acad Sci US A. Jul 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The binding sites are for different antigens (or epitopes). may be targeted.

[0046] A "domain antibody" refers to an antibody fragment that contains only the heavy chain variable region or only the light chain variable region. H The domains are covalently linked with a peptide linker to create a bivalent or multivalent domain antibody. H The domains may target the same antigen or they may target different antigens.

[0047] In one embodiment, "(dsFv) 2 " is a peptide linker linked to two V H Two V's L It contains three peptide chains linked together by disulfide bridges in the cytoplasmic domain.

[0048] In one embodiment, the "bispecific ds diabody" is H1 and V L1 Disulfide bridge between V L1 -V H2 V bound to (peptide linker) H1 -V L2 (also linked by a peptide linker).

[0049] In one embodiment, a "bispecific dsFv" or "dsFv-dsFv'" is a VFv in which the heavy chains are linked by a peptide linker (e.g., a long, flexible linker). H1 -V H2 The part is V L1 Part and V L2It contains three peptide chains linked by disulfide bridges to each of the three portions, with each pair of disulfide-bridged heavy and light chains having a different antigen specificity.

[0050] In one embodiment, a "dimeric scFv" is a dimeric scFv having a V L -V H V dimerized with the moiety H -V L (linked by a peptide linker), thereby forming a V H The other part is V L In another embodiment, a "dimeric scFv" is a bivalent diabody or a bivalent ScFv (BsFv) that combines with V to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). L1 -V H2 (linked with a peptide linker) together with V H1 -V L2 (also linked by a peptide linker), thereby H1 and V L1 are united and V H2 and V L2 These are bispecific diabodies in which each pair has a different antigen specificity.

[0051] The term "fully human," as used herein, in relation to an antibody or antigen-binding fragment, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human individual or human immune cell, or an antibody from a non-human source, such as a non-human transgenic animal that utilizes a human antibody repertoire or other sequences that encode human antibodies. In one embodiment, a fully human antibody does not contain any amino acid residues (particularly antigen-binding residues) derived from a non-human antibody.

[0052] The term "humanized" as used herein, with respect to an antibody or antigen-binding fragment, means that the antibody or antigen-binding fragment comprises CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human. Humanized antibodies or humanized antigen-binding fragments have reduced immunogenicity in humans and, in one embodiment, are useful as human therapeutics. In one embodiment, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In one embodiment, a humanized antibody or humanized antigen-binding fragment is substantially identical to the human antibody except for the non-human CDR sequences. In one embodiment, the human-derived FR region may comprise the same amino acid sequence as the human antibody from which it is derived, or may contain amino acid changes, such as 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less amino acid changes. In some embodiments, such amino acid changes may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains. In a preferred embodiment, the humanized antibody comprises human FR1 and human FR2. ∼3 as well as human JH and human Jκ.

[0053] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment in which a portion of the heavy and / or light chain is derived from one species and the remaining portion of the heavy and / or light chain is derived from another species. As an illustrative example, a chimeric antibody may contain a constant region of human origin and a variable region of a non-human species, such as mouse.

[0054] As used herein, "PD-1" refers to a programmed cell death protein that belongs to the immunoglobulin superfamily and functions as a co-inhibitory receptor to negatively regulate the immune system. PD-1 is a member of the CD28 / CTLA-4 family and has two known ligands, including PD-L1 and PD-L2. A representative amino acid sequence of human PD-1 is disclosed under NCBI Accession No. NP_005009.2, and a representative nucleic acid sequence encoding human PD-1 is provided under NCBI Accession No. NM_005018.2.

[0055] As used herein, "PD-L1" refers to programmed cell death ligand 1 (PD-L1, see e.g., Freeman et al. (2000) J. Exp. Med. 192:1027). A representative amino acid sequence of human PD-L1 is disclosed in NCBI Accession No. NP_054862.1, and a representative nucleic acid sequence encoding human PD-L1 is set forth in NCBI Accession No. NM_014143.3. PD-L1 is expressed in the placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells. PD-L1 is involved in the regulation of activated T cells, B cells, and bone marrow. It binds to PD-1 or B7-1, the receptor for PD-L1 expressed on T cells. Binding of PD-L1 to its receptor induces signaling that suppresses TCR-mediated activation of cytokine production and T cell proliferation. Therefore, it is believed that PD-L1 plays a major role in suppressing the immune system during certain events such as pregnancy, autoimmune diseases, and tissue allografts, allowing tumor or cancer cells to evade immune checkpoints and escape the immune response.

[0056] As used herein, an "anti-PD-1 antibody" refers to an antibody that can specifically bind to PD-1 (e.g., human or monkey PD-1) with sufficient affinity to provide for diagnostic and / or therapeutic use.

[0057] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen. In one embodiment, the antibodies or antibody-binding fragments provided herein bind to human and / or monkey PD-1 at least 10 -6 M or less (e.g., 5x10 -7 M or less, 2x10 -7 M or less, 10 -7 M or less, 5x10 -8 M or less, 2x10 -8 M or less, 10 -8 M or less, 5x10 -9 M or less, 2x10 -9 M or less, 10 -9 M or less, 10 -10 Binding affinity (K D As used herein, K D is the ratio of the dissociation rate to the association rate (K off / K on ), which can be determined using surface plasmon resonance with a device such as Biacore. This can be done.

[0058] As used herein, the ability to "inhibit binding" or "compete for the same epitope" refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human PD-1 and an anti-PD-1 antibody) to any detectable extent. In one embodiment, an antibody or antigen-binding fragment that inhibits binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In one embodiment, the inhibition may be 60% or more, 70% or more, 80% or more, or 90% or more.

[0059] The term "epitope" as used herein refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, the two antibodies may bind to the same epitope in the antigen. For example, if an antibody or antigen-binding fragment disclosed herein inhibits the binding of exemplary antibodies such as 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2 hAb, 1.139.15 hAb, and 1.153.7 hAb to human PD-1, the antibody or antigen-binding fragment may be considered to bind to the same epitope as the exemplary antibodies.

[0060] Specific amino acid residues within the epitope can be mutated, for example, by alanine scanning mutagenesis, and mutations that reduce or prevent binding of the protein are identified. "Alanine scanning mutagenesis" is a method that can be performed to identify specific residues or regions of a protein that affect the interaction of the epitope with another compound or another protein that binds to the protein. A residue or group of targeted residues in the protein are substituted with a neutral or negatively charged amino acid (most preferably alanine or polyalanine or conservative amino acid substitutions). Any mutation of an amino acid residue or codon that reduces binding of the protein below a threshold value or reduces binding of the protein to the greatest extent compared to other mutations may be within the epitope bound by the protein. In one embodiment of the present disclosure, the epitope of interest for the PD-1 antibody includes at least one of V64, P83, D85, L128, A129, P130, K131, A132, and Q133.

[0061] As used herein, "1.7.3 hAb" refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO:45, a light chain variable region of SEQ ID NO:47, and a human constant region of the IgG4 isotype.

[0062] As used herein, "1.49.9 hAb" refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO:49, a light chain variable region of SEQ ID NO:51, and a human constant region of the IgG4 isotype.

[0063] As used herein, "1.103.11 hAb" refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO:53, a light chain variable region of SEQ ID NO:55, and a human constant region of the IgG4 isotype.

[0064] As used herein, "1.103.11-v2 hAb" refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO:53, a light chain variable region of SEQ ID NO:67, and a human constant region of the IgG4 isotype. This refers to monoclonal antibodies.

[0065] As used herein, "1.139.15 hAb" refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO:57, a light chain variable region of SEQ ID NO:59, and a human constant region of the IgG4 isotype.

[0066] As used herein, "1.153.7 hAb" refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO:61, a light chain variable region of SEQ ID NO:63, and a human constant region of the IgG4 isotype.

[0067] A "conservative substitution" in the context of an amino acid sequence refers to the replacement of an amino acid residue with a different amino acid residue having a side chain of similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not cause significant changes in the conformation of a protein and thus may retain the biological activity of the protein.

[0068] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences, introducing gaps, if necessary, to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available at the US National Center for Biotechnology Information (NCBI) website, see also Altschul S.F.etal, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res.,25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website, see also Higgins D.G.etal, Methods in Enzymology, 266:383-402 (1996); Larkin MA etal, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN. One skilled in the art may use the default parameters provided by the tool or may customize the parameters as appropriate for the alignment, for example by selection of a suitable algorithm.

[0069] As used herein, "T cells" refers to CD4 + T cells, CD8 + These include T cells, helper T1 type T cells, helper T2 type T cells, helper T17 type T cells and suppressor T cells.

[0070] As used herein, "effector function" refers to a biological activity resulting from binding of an antibody Fc region to an antibody effector, such as the C1 complex or an Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is induced by the interaction of an antibody with C1q on the C1 complex; antibody-mediated cytotoxicity (ADCC), which is induced by binding of an antibody Fc region to an Fc receptor on an effector cell; and phagocytosis.

[0071] As used herein, "cancer" or "cancerous condition" refers to any medical condition mediated by neoplastic or malignant cell growth, proliferation or metastasis, and includes both solid cancers and non-solid cancers such as leukemia. As used herein, "tumor" refers to a solid mass of neoplastic and / or malignant cells.

[0072] As used herein, "treating" a condition or "treatment" of a condition includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of development of a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, causing complete or partial regression of a condition, curing a condition, or some combination thereof. In the context of cancer, "treating" or "treatment" may refer to inhibiting or slowing neoplastic or malignant cell growth, cell proliferation or cell metastasis, preventing or slowing the development of neoplastic or malignant cell growth, cell proliferation or cell metastasis, or some combination thereof. In the context of tumors, "treating" or "treatment" includes eradicating all or part of the tumor, inhibiting or slowing the rate of tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0073] An "isolated" material has been altered by the hand of man from its natural state. If an "isolated" composition or material occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated", but the same polynucleotide or polypeptide is "isolated" if it is sufficiently separated from the coexisting materials in its natural state and exists in a substantially pure state. In one embodiment, the antibody or antigen-binding fragment has a purity of 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more as determined using electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis, etc.) or chromatographic methods (e.g., ion exchange chromatography or reverse phase HPLC, etc.).

[0074] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be inserted so that it functions to express the protein. A vector may be used for transformation, transduction or transduction of a host cell so that the genetic elements carried by the vector are expressed in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. Vectors Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain various elements for expression control, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, vectors may contain origins of replication. Vectors may also contain materials that aid in entry into cells, including, but not limited to, viral particles, liposomes, or protein coatings.

[0075] As used herein, the term "host" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0076] As used herein, a "PD-1-associated or related disease" refers to any condition caused by, exacerbated by, or otherwise linked to increased or decreased expression or activity of PD-1 (e.g., human PD-1).

[0077] The term "therapeutically effective amount" or "effective dose" as used herein refers to a dose or concentration of an agent effective to treat a disease or condition associated with human PD-1. For example, with respect to the use of the antibodies or antigen-binding fragments disclosed herein to treat cancer, a therapeutically effective amount is a dose or concentration of the antibody or antigen-binding fragment that allows for eradicating all or part of a tumor, inhibiting or slowing the growth of a tumor, inhibiting the growth or proliferation of cells that mediate a cancerous condition, inhibiting metastasis of tumor cells, ameliorating symptoms or markers associated with a neoplastic or cancerous condition, preventing or slowing the development of a neoplastic or cancerous condition, or some combination thereof.

[0078] The term "pharmaceutical acceptable" means that the specified carrier, vehicle, diluent, excipient, and / or salt is generally, chemically and / or materially compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0079] Anti-PD-1 antibody In one embodiment, the present disclosure provides anti-PD-1 antibodies and antigen-binding fragments thereof. PD-1, also known as CD279, is a key immune checkpoint receptor expressed by activated T cells and is known to mediate immune suppression. PD-1 ligand 1 (PD-L1) is a 40 kDa transmembrane protein expressed on various tumor cells, stromal cells, or both, and binds to PD-1. Inhibition of the interaction between PD-1 and PD-L1 can enhance T cell responses, thereby mediating anti-cancer activity.

[0080] In one embodiment, the present disclosure provides exemplary fully human monoclonal antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb, and 1.153.7 hAb, whose CDR sequences are shown in Table 1 below, and whose heavy chain variable region or light chain variable region sequences are also shown in Table 1. As shown below. [Table 1-1] [Table 1-2] [Table 1-3]

[0081] 1.7.3hAb-VH (23466-VH): (amino acid sequence is SEQ ID NO: 45, nucleic acid sequence is SEQ ID NO: 46) For CDRs 1 to 3 of the heavy chain, SEQ ID NOs: 1, 3, and 5 are the amino acid sequences, respectively, and SEQ ID NOs: 2, 4, and 6 are the nucleic acid sequences, respectively. [ka]

[0082] 1.7.3hAb-VL (23195-VL): (amino acid sequence is SEQ ID NO: 47, nucleic acid sequence is SEQ ID NO: 48) For CDRs 1 to 3 of the light chain, SEQ ID NOs: 7, 9, and 11 are the amino acid sequences, respectively, and SEQ ID NOs: 8, 10, and 12 are the nucleic acid sequences, respectively. [ka]

[0083] 1.49.9hAb-VH (20951-VH): (amino acid sequence is SEQ ID NO: 49, nucleic acid sequence is SEQ ID NO: 50) For CDRs 1 to 3 of the heavy chain, SEQ ID NOs: 13, 15, and 5 are the amino acid sequences, respectively, and SEQ ID NOs: 14, 16, and 6 are the nucleic acid sequences, respectively. [ka]

[0084] 1.49.9hAb-VL (21526-VL): (amino acid sequence is SEQ ID NO: 51, nucleic acid sequence is SEQ ID NO: 52) For CDRs 1 to 3 of the light chain, SEQ ID NOs: 7, 17, and 11 are the amino acid sequences, respectively, and SEQ ID NOs: 8, 18, and 12 are the nucleic acid sequences, respectively. [ka]

[0085] 1.103.11hAb-VH (20975-VH): (amino acid sequence is SEQ ID NO: 53, nucleic acid sequence is SEQ ID NO: 54) For CDRs 1 to 3 of the heavy chain, SEQ ID NOs: 1, 15, and 5 are the amino acid sequences, respectively, and SEQ ID NOs: 2, 16, and 6 are the nucleic acid sequences, respectively. [ka]

[0086] 1.103.11hAb-VL (21038-VL): (amino acid sequence is SEQ ID NO: 55, nucleic acid sequence is SEQ ID NO: 56) For CDRs 1 to 3 of the light chain, SEQ ID NOs: 7, 17, and 19 are the amino acid sequences, respectively, and SEQ ID NOs: 8, 18, and 20 are the nucleic acid sequences, respectively. [ka]

[0087] 1.139.15hAb-VH (23521-VH): (amino acid sequence is SEQ ID NO: 57, nucleic acid sequence is SEQ ID NO: 58) For CDR1 to 3 of the heavy chain, SEQ ID NOs: 21, 23, and 25 are the amino acid sequences, respectively, and SEQ ID NOs: 22, 24, and 26 are the nucleic acid sequences, respectively. [ka]

[0088] 1.139.15hAb-VL (22895-VL): (amino acid sequence is SEQ ID NO: 59, nucleic acid sequence is SEQ ID NO: 60) For CDR1 to 3 of the light chain, SEQ ID NOs: 27, 29, and 31 are the amino acid sequences, respectively, and SEQ ID NOs: 28, 30, and 32 are the nucleic acid sequences, respectively. [ka]

[0089] 1.153.7hAb-VH (20942-VH): (amino acid sequence is SEQ ID NO: 61, nucleic acid sequence is SEQ ID NO: 62) For CDR1 to 3 of the heavy chain, SEQ ID NOs: 33, 35, and 37 are the amino acid sequences, respectively, and SEQ ID NOs: 34, 36, and 38 are the nucleic acid sequences, respectively. [ka]

[0090] 1.153.7hAb-VL (21110-VL): (amino acid sequence is SEQ ID NO: 63, nucleic acid sequence is SEQ ID NO: 64) For CDR1 to 3 of the light chain, SEQ ID NOs: 39, 41, and 43 are the amino acid sequences, respectively, and SEQ ID NOs: 40, 42, and 44 are the nucleic acid sequences, respectively. [ka]

[0091] 1.103.11-v2hAb-VH (20975-VH): (amino acid sequence is SEQ ID NO: 53, nucleic acid sequence is SEQ ID NO: 54) For CDRs 1 to 3 of the heavy chain, SEQ ID NOs: 1, 15, and 5 are the amino acid sequences, respectively, and SEQ ID NOs: 2, 16, and 6 are the nucleic acid sequences, respectively. [ka]

[0092] 1.103.11-v2hAb-VL (21038-2-VL): (amino acid sequence is SEQ ID NO: 67, nucleic acid sequence is SEQ ID NO: 68) For CDRs 1 to 3 of the light chain, SEQ ID NOs: 7, 17, and 65 are the amino acid sequences, respectively, and SEQ ID NOs: 8, 18, and 66 are the nucleic acid sequences, respectively. [ka]

[0093] In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise heavy chain CDR sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 13, 15, 21, 23, 25, 33, 35, and 37. In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise light chain CDR sequences selected from the group consisting of SEQ ID NOs: 7, 9, 11, 17, 19, 27, 29, 31, 39, 41, 43, and 65. In one embodiment, one or more of the CDR sequences provided herein can be modified or altered such that the resulting antibody has one or more improved properties compared to the parent antibody (e.g., improved antigen binding ability, improved glycosylation pattern, reduced risk of glycosylation of the CDR residues, reduced deamination of the CDR residues, increased pharmacokinetic half-life, pH sensitivity, suitability for conjugation, etc.) and is otherwise equivalent to the parent antibody (i.e., an antibody having the same set of CDR sequences except for the modifications or changes described above) or at least substantially retains the antibody binding characteristics of the parent antibody.

[0094] In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of: a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:3, and / or SEQ ID NO:5; a heavy chain variable region comprising SEQ ID NO:13, SEQ ID NO:15, and / or SEQ ID NO:5; a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and / or SEQ ID NO:5; a heavy chain variable region comprising SEQ ID NO:21, SEQ ID NO:23, and / or SEQ ID NO:25; and a heavy chain variable region comprising SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37.

[0095] In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof are selected from the group consisting of a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:11; a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:11; a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:19; a light chain variable region comprising SEQ ID NO:27, SEQ ID NO:29, and / or SEQ ID NO:31; a light chain variable region comprising SEQ ID NO:39, SEQ ID NO:41, and / or SEQ ID NO:43; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:65. The antibody comprises a light chain variable region selected from the group consisting of:

[0096] In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise: a) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:3, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:11; b) a heavy chain variable region comprising SEQ ID NO:13, SEQ ID NO:15, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:11; c) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:19. d) a heavy chain variable region comprising SEQ ID NO:21, SEQ ID NO:23, and / or SEQ ID NO:25; and a light chain variable region comprising SEQ ID NO:27, SEQ ID NO:29, and / or SEQ ID NO:31; e) a heavy chain variable region comprising SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37; and a light chain variable region comprising SEQ ID NO:39, SEQ ID NO:41, and / or SEQ ID NO:43; or f) a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:15, and / or SEQ ID NO:5; and a light chain variable region comprising SEQ ID NO:7, SEQ ID NO:17, and / or SEQ ID NO:65.

[0097] One skilled in the art will appreciate that the CDR sequences provided in Table 1 can be modified to include one or more amino acid substitutions to provide improved biological activity, such as improved binding affinity to human PD-1. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed using phage display technology, and then screened for binding affinity to human PD-1. In another example, computer software can be used to virtually simulate the binding of an antibody to human PD-1 to identify amino acid residues on the antibody that form the binding interface. Such residues can be avoided in substitutions to prevent a decrease in binding affinity, or can be targeted for substitution to provide stronger binding. In one embodiment, one or more or all of the substitutions in the CDR sequences are conservative substitutions.

[0098] In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise one or more CDR sequences that have 80% or greater (e.g., 85% or greater, 88% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater) sequence identity to the CDR sequences listed in Table 1, while retaining a similar or greater level of binding affinity to human PD-1 as a parent antibody having substantially the same sequence except that the corresponding CDR sequence exhibits 100% sequence identity to the CDR sequences listed in Table 1.

[0099] In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof are fully human. Fully human antibodies do not suffer from the problems of immunogenicity and / or reduced binding affinity in humans that are often observed with humanized antibodies.

[0100] In some embodiments, the fully human anti-PD-1 antibodies and antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, and homologous sequences thereof having 80% or more (e.g., 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) sequence identity thereto; and / or a light chain variable region selected from the group consisting of SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:67, and homologous sequences thereof having 80% or more (e.g., 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) sequence identity thereto. These fully human antibodies preferably retain binding affinity to human PD-1 at a similar level as one of the exemplary antibodies: 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.103.11-v2 hAb, 1.139.15 hAb, and 1.153.7 hAb.

[0101] In some embodiments, fully human anti-PD-1 antibodies and antigen-binding fragments thereof comprise: a) a heavy chain variable region comprising SEQ ID NO:45; and a light chain variable region comprising SEQ ID NO:47; b) a heavy chain variable region comprising SEQ ID NO:49; and a light chain variable region comprising SEQ ID NO:51; c) a heavy chain variable region comprising SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO:55; d) a heavy chain variable region comprising SEQ ID NO:57; and a light chain variable region comprising SEQ ID NO:59; e) a heavy chain variable region comprising SEQ ID NO:61; and a light chain variable region comprising SEQ ID NO:63; or f) a heavy chain variable region comprising SEQ ID NO:53; and a light chain variable region comprising SEQ ID NO:67.

[0102] Antibodies and antigen-binding fragments thereof that compete for the same epitope as the anti-PD-1 antibodies and antigen-binding fragments thereof provided herein are also contemplated herein. In one embodiment, the antibodies are -6 Less than M, 10 -7 Less than M, 10 -7.5 Less than M, 10 -8 Less than M, 10 -8.5 Less than M, 10 -9 Less than M or 10 -10 IC less than M 50 Inhibit binding of 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb, or 1.153.7 hAb to human or monkey PD-1 with IC values ​​(i.e., 50% inhibitory concentration). 50 Values ​​are determined based on competitive assays such as ELISA assays, radioligand competitive binding assays, and FACS analysis.

[0103] In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments provided herein have a binding activity of at least 10% by mass as measured by a plasmon resonance binding assay. -6 M or less (e.g. 5x10 -7 M or less, 2x10 -7 M or less, 10 -7 M or less, 5x10 -8 M or less, 2x10 -8 M or less, 10 -8 M or less, 5x10 -9M or less, 2x10 -9 M or less, 10 -9 M or less, 10 -10 It can specifically bind to human PD-1 with a binding affinity (Kd) of 100 M or less. The binding affinity is expressed as the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (K off / K on ) is calculated as D The antigen binding affinity (e.g., K D ) can be suitably determined using any suitable method known in the art, including, for example, plasmon resonance binding assays using instruments such as Biacore (see, for example, Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006).

[0104] In one embodiment, the antibodies and antigen-binding fragments provided herein have an EC of 0.1 nM to 100 nM (e.g., 0.1 nM to 50 nM, 0.1 nM to 30 nM, 0.1 nM to 20 nM, 0.1 nM to 10 nM, or 0.1 nM to 1 nM). 50 (i.e., 50% binding concentration). Binding of the antibodies to human PD-1 can be measured by methods known in the art, for example, sandwich assays such as ELISA, Western blot, FACS or other binding assays. Illustratively, a test antibody (i.e., a first antibody) is bound to immobilized human PD-1 or human PD-1-expressing cells, and after washing away unbound antibody, a labeled second antibody is introduced that can bind to the first antibody, thereby allowing the bound first antibody to be detected. Detection is performed using a microplate reader when immobilized PD-1 is used, or using FACS analysis when human PD-1-expressing cells are used. In one embodiment, the antibodies and antigen-binding fragments thereof provided herein have an EC of 1 nM-10 nM or 1 nM-5 nM as measured by FACS analysis. 50 (i.e., 50% effective concentration) to human PD-1.

[0105] In one embodiment, the antibodies and antigen-binding fragments provided herein have an IC of 0.2 nM to 100 nM (e.g., 0.2 nM to 50 nM, 0.2 nM to 30 nM, 0.2 nM to 20 nM, 0.2 nM-10 nM, or 1 nM to 10 nM) as measured in a competitive assay. 50 inhibits the binding of human PD-1 to its ligand.

[0106] In one embodiment, the antibodies and antigen-binding fragments provided herein inhibit the binding of human PD-1 to its ligand, thereby inhibiting, for example, activated T cells (e.g., CD4 + T cells and CD8 + T cells), and activated T cells (e.g., CD4 + T cells and CD8 + The term "IL-2" refers to interleukin 2, a type of cytokine signaling molecule in the immune system that controls the activity of white blood cells (e.g., leukocytes). The term "interferon gamma (IFNγ)" refers to the signaling molecules that regulate the activity of natural killer (NK) cells, NKT cells, CD4+ cells, and CD4+ T cells. + T cells and CD8 + A cytokine produced by T cells that is an important activator of macrophages and an inducer of expression of major histocompatibility complex (MHC) molecules. Cytokine production can be measured using methods known in the art, such as ELISA. 3 Methods for detecting T cell proliferation can also be used, including [H]thymidine incorporation assays.

[0107] The anti-PD-1 antibodies and antigen-binding fragments thereof are specific for PD-1. In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof do not bind to CD28 and / or CTLA-4. For example, the binding affinity to CD28 and / or CTLA-4 is less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the binding affinity to PD-1.

[0108] In one embodiment, the antibodies and antigen-binding fragments thereof bind to monkey PD-1 with an EC50 of less than or equal to 100 nM, e.g., less than or equal to 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, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM, as measured by ELISA. In one embodiment, the antibodies and antigen-binding fragments thereof bind to monkey PD-1 with an EC50 of about 1 nM to 10 nM. Bind to.

[0109] In one embodiment, the antibodies and antigen-binding fragments thereof do not bind mouse PD-1 but bind monkey PD-1 with a binding affinity similar to that of human PD-1. For example, the binding of exemplary antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb, and 1.153.7 hAb to mouse PD-1 is not detectable by conventional binding assays such as ELISA or FACS analysis, whereas the binding of these antibodies to monkey PD-1 is similar to that of human PD-1 as measured by ELISA or FACS. with similar affinity or EC50 values.

[0110] In one embodiment, the anti-PD-1 antibody and its antigen-binding fragment have reduced or depleted effector function. In one embodiment, the anti-PD-1 antibody and its antigen-binding fragment have a constant region of IgG4 isotype with reduced or depleted effector function. Effector functions such as ADCC and CDC can lead to cytotoxicity to PD-1 expressing cells. Many cells, such as T cells, normally express PD-1. To avoid the possibility of undesired toxicity to these normal cells, one embodiment of the antibody and its antigen-binding fragment provided herein may have reduced or even depleted effector function. For example, various assays for evaluating ADCC or CDC activity, such as Fc receptor binding assay, C1q binding assay, and cytolysis assay, are known and can be easily selected by those skilled in the art. Without wishing to be bound by theory, antibodies with reduced or depleted effector functions such as ADCC or CDC may cause no or minimal cytotoxicity against PD-1 expressing cells, such as T cells, and thus be free of undesirable side effects, while at the same time blocking PD-1 enhances the immune system for the treatment of conditions such as cancer or chronic infections.

[0111] In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof provided herein exhibit reduced side effects. For example, the antibodies and antigen-binding fragments thereof provided herein can have a fully human IgG sequence and therefore have reduced immunogenicity compared to the corresponding humanized antibodies. In another example, the antibodies and antigen-binding fragments thereof provided herein can be of IgG4 type to eliminate ADCC and CDC.

[0112] In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof provided herein are advantageous in that they may be used in combination with immunogenic agents, such as tumor cells, purified tumor antigens, cells transfected with genes encoding immune stimulatory cytokines, and tumor vaccines. In addition, the anti-PD-1 antibodies and antigen-binding fragments thereof may be included in combination therapies, including standard chemotherapy and radiation therapy, targeted small molecule therapy, and emerging other immune checkpoint modulator therapies. In one embodiment, the antibodies and antigen-binding fragments thereof may be used as the basis for antibody-drug conjugates, bispecific antibodies, or multivalent antibodies. This can be done.

[0113] The anti-PD-1 antibodies or antigen-binding fragments provided herein may be monoclonal, polyclonal, fully human, humanized, chimeric, recombinant, bispecific, labeled, bivalent, or anti-idiotypic antibodies. Recombinant antibodies are antibodies prepared in vitro using recombinant techniques rather than in an animal. Bispecific or bivalent antibodies are artificial antibodies that have fragments of two different monoclonal antibodies and can bind to two different antigens. A "bivalent" antibody or "bivalent" antigen-binding fragment thereof contains two antigen-binding sites. The two antigen-binding sites may bind to the same antigen or to different antigens, in which case the antibody or antigen-binding fragment is characterized as "bispecific."

[0114] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof provided herein is a fully human antibody. In one embodiment, the fully human antibody is prepared using recombinant methods. For example, transgenic animals such as mice can be engineered to have transgenes or transchromosomes of human immunoglobulin genes, and thus be able to produce fully human antibodies after immunization with an appropriate antigen such as human PD-1. The fully human antibody can be isolated from such transgenic animals or alternatively, by fusing spleen cells of the transgenic animal with an immortalized cell line by hybridoma technology to generate hybridoma cells that secrete the fully human antibody. Exemplary transgenic animals include, but are not limited to, OmniRat, which has been engineered such that the endogenous expression of the rat immunoglobulin genes is inactivated and at the same time contains a functional recombinant human immunoglobulin locus; OmniMouse, which has been engineered such that the endogenous expression of the mouse immunoglobulin genes is inactivated and at the same time contains a recombinant human immunoglobulin locus with a deletion of the J locus and a Cκ mutation; and OmniFlic, a transgenic rat that has been engineered such that the endogenous expression of the rat immunoglobulin genes is inactivated and at the same time contains a recombinant human immunoglobulin locus with a single and common rearranged VkJk light chain and a functional heavy chain. Detailed information can be found further in Osborn M. et al, Journal of Immunology, 2013, 190: 1481-90; Ma B. et al, Journal of Immunological Methods 400-401 (2013) 78-86; Geurts A. et al, Science, 2009, 325: 433; U.S. Patent 8,907,157; European Patent 2152880B1; European Patent 2336329B1, all of which are hereby incorporated by reference in their entirety.Other suitable transgenic animals can also be used, such as HuMab mice (for details see Lonberg, N. et al. Nature 368(6474): 856 859 (1994)), Xeno mice (Mendez et al. NatGenet., 1997, 15:146-156), TransChromo mice (Ishida et al. Cloning Stem Cells, 2002, 4:91-102) and VelocImmune mice (Murphy et al. Proc Natl Acad Sci USA, 2014, 111:5153-5158), Kymouse (Leee et al. NatBiotechnol, 2014, 32:356-363), and transgenic rabbits (Flisikowska et al. PLoSOne, 2011, 6:e21045).

[0115] In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof are camelized single domain antibodies, diabodies, scFv, dimeric scFv, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragments, Fab, Fab', F(ab')2, ds diabodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0116] In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region and / or a light chain constant region. The heavy chain constant region comprises a CH1, a CH1-CH2, or a CH1-CH3 region. In some embodiments, the constant region may further comprise one or more modifications to confer desirable properties. For example, the constant region may be modified to reduce or deplete one or more effector functions to improve binding to the FcRn receptor, or may be modified to introduce one or more cysteine ​​residues.

[0117] In one embodiment, the anti-PD-1 antibodies and antigen-binding fragments thereof further comprise conjugates. It is contemplated that a variety of conjugates may be attached to the antibodies or antigen-binding fragments provided herein (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates may be attached to the antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complex formation, association, mixing, or addition, among other methods. In one embodiment, the antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside of the epitope binding site that are available for binding to one or more conjugates. For example, such sites may contain one or more reactive amino acid residues, such as, for example, cysteine ​​or histidine residues, to facilitate covalent binding to the conjugate. In one embodiment, the antibody may be indirectly attached to the conjugate or through another conjugate. For example, an antibody or antigen-binding fragment may be conjugated to biotin and then indirectly conjugated to a second conjugate that is conjugated to avidin. The detectable label may be an excitable label, a pharmacokinetic-modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, glycoxidase, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67Cu、 86 Y、 88 Y、 90 Y、 177 On, 211 Available、 186 King、 188 King、 153 Sm、 212 Wind、および 32P, other lanthanides, luminescent labels), color-producing moieties, digoxigenin, biotin / avidin, DNA molecules, or gold. In one embodiment, the conjugate may be a pharmacokinetic-modifying moiety such as PEG, which helps increase the half-life of the antibody. Other suitable polymers include carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, copolymers of ethylene glycol / propylene glycol, and the like. In one embodiment, the conjugate may be a purification moiety such as magnetic beads. A "cytotoxic moiety" may be any agent that is harmful to cells or capable of damaging or killing cells. Examples of cytotoxic moieties include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacaine ... levadine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and mitotic inhibitors (e.g., vincristine and vinblastine).

[0118] Polynucleotides and Recombinant Methods The present disclosure provides isolated polynucleotides encoding anti-PD-1 antibodies and antigen-binding fragments thereof. In one embodiment, the isolated polynucleotides comprise one or more of the nucleic acid sequences set forth in Table 1, which encode the CDR sequences provided in Table 1.

[0119] In some embodiments, the isolated polynucleotide encodes a heavy chain variable region and comprises a sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, and homologous sequences thereof having 80% or more (e.g., 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) sequence identity. In some embodiments, the isolated polynucleotide encodes a light chain variable region and comprises a sequence selected from the group consisting of SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, and homologous sequences thereof having 80% or more (e.g., 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) sequence identity. In one embodiment, the percent identity is due to the degeneracy of the genetic code, such that the encoded protein sequence remains identical.

[0120] Isolated polynucleotides encoding anti-PD-1 antibodies and antigen-binding fragments thereof (e.g., including sequences set forth in Table 1) can be inserted into vectors for further cloning (amplification of DNA) or expression using recombinant techniques known in the art. In another embodiment, antibodies may be produced by homologous recombination, as known in the art. DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0121] In one embodiment, vector systems include mammalian, bacterial, yeast systems, and the like, including plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, and the like, as well as other laboratory and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, adeno-associated viruses).

[0122] A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in a vector in the present invention are prokaryotes, yeast, or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Escherichia coli, such as E. coli, Salmonella, such as Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, such as Serratia marcescans, Shigella, Bacillus, such as Bacillus subtilis and B. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Enterobacteriaceae, such as Streptomyces.

[0123] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for vectors encoding anti-PD-1 antibodies. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, other species such as Schizosaccharomyces pombe; K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 24,178), K. spp. (ATCC 24,220 ... hosts of Kluyveromyces spp., such as K. thermotolerans, K. marxianus, Yarrowia spp. (EP 402,226); Pichia pastoris (EP 183,070); Candida spp.; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces spp., such as Schwanniomyces occidentalis; and species of Neurospora spp., Penicillium spp., Tolypocladium spp., A. nidulans and A. niger. Numerous other genera, species, and strains of filamentous fungi, such as Aspergillus hosts, are commonly available and useful in the present invention.

[0124] Suitable host cells for expressing the glycosylated antibodies or antigen fragments provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous strains and variants of baculoviruses and corresponding permissive insect host cells have been identified, such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyxmori. Various virus strains for gene transfer are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as viruses herein in accordance with the present invention, particularly for gene transfer into Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be utilized as hosts.

[0125] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney cell line CV1 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Grahametal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK cells, ATCC CCL 10); Chinese hamster ovary cells / DHFR-deficient (CHO, Urlaubetal., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and and a human hepatoma cell line (Hep G2). In a preferred embodiment, the host cell is a 293F cell.

[0126] Host cells can be transformed with the above-described expression or cloning vectors to produce the anti-PD-1 antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0127] The host cells used to produce the antibodies or antigen-binding fragments provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing the host cells. In addition, any medium described in Hamel, Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90 / 03430; WO87 / 00195; or U.S. Pat. Re. 30,985 may be used as a culture medium for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin™ agents), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those of skill in the art may also be included at appropriate concentrations. Culture conditions such as temperature, pH, etc. will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0128] When using recombinant techniques, antibodies may be produced intracellularly or in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a method for isolating antibodies secreted into the periplasmic space of Escherichia coli (E. coli). Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes or more. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the expression system supernatant is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore-Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0129] Antibodies prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, of which affinity chromatography is the preferred purification technique. The suitability of Protein A as an affinity ligand depends on the type and isotype of immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, gamma 3, or gamma 4 heavy chains (Lindmarketal., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Gussetal., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or polystyrene divinylbenzene allow for faster flow rates and shorter processing times than with agarose. If the matrix contains the CH3 domain, BakerbondABX.TM. resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other protein purification techniques such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on Heparin Sepharose.TM., chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0130] Following any primary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, preferably using an elution buffer with a low salt concentration (e.g., about 0-0.25 M salt) and a pH of between about 2.5-4.5.

[0131] kit The present disclosure provides kits comprising an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the kits are useful for detecting the presence or level of PD-1 in a biological sample. The biological sample may include cells or tissues.

[0132] In some embodiments, the kit comprises an anti-PD-1 antibody or antigen-binding fragment thereof conjugated to a detectable label. In another embodiment, the kit comprises an unlabeled anti-PD-1 antibody or antigen-binding fragment thereof, and further comprises a labeled secondary antibody capable of binding to the unlabeled anti-PD-1 antibody. The kit may further comprise instructions for use, and containers separating each component in the kit.

[0133] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is associated with a substrate or device useful in a sandwich assay, such as an ELISA, or an immunographic assay. Useful substrates or devices can be, for example, microtiter plates and test strips.

[0134] Pharmaceutical compositions and methods of treatment The disclosure further provides pharmaceutical compositions comprising an anti-PD-1 antibody, or antigen-binding fragment thereof, and one or more pharma- ceutically acceptable carriers.

[0135] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0136] Suitable components may include, for example, antioxidants, bulking agents, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickening agents, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins.Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate.As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in the compositions comprising the antibodies or antigen-binding fragments and conjugates provided herein reduces the oxidation of the antibodies or antigen-binding fragments.This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving the stability and maximizing the shelf life of the antibodies. Thus, in one embodiment, a composition is provided that includes one or more of the antibodies or antigen-binding fragments disclosed herein and one or more antioxidants, such as methionine. Further provided are methods for preventing oxidation, extending shelf life, and / or improving efficacy of the antibodies or antigen-binding fragments by combining the antibodies or antigen-binding fragments provided herein with one or more antioxidants, such as, for example, methionine.

[0137] To further illustrate, pharma- ceutically acceptable carriers may include aqueous vehicles such as, for example, sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water for injection, lactated Ringer's dextrose, non-aqueous vehicles such as, for example, fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial agents in bacteriostatic or fungistatic concentrations, isotonic agents such as, for example, sodium chloride or dextrose, buffers such as, for example, phosphate buffer or citrate buffer, antioxidants such as, for example, sodium bisulfate, local anesthetics such as, for example, procaine hydrochloride, suspending and dispersing agents such as, for example, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as, for example, polysorbate 80 (TWEEN-80), sequestering or chelating agents such as, for example, EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bistetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. The antibacterial agent used as carrier includes phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, thimerosal, benzalkonium chloride and benzethonium chloride, and may be added to the pharmaceutical composition in a multi-dose container.Suitable excipients may include, for example, water, saline, glucose, glycerol or ethanol.Suitable non-toxic auxiliary substances may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0138] The pharmaceutical compositions may be in the form of liquid solutions, suspensions, emulsions, pills, capsules, tablets, controlled release formulations, or powders. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0139] In one embodiment, the pharmaceutical composition is in the form of an injectable composition.The injectable pharmaceutical composition may be prepared in any conventional form, such as liquid solution, suspension, emulsion, or solid form suitable for making liquid solution, suspension or emulsion.The injectable preparation may include sterile and / or non-pyrogenic solution, sterile dry soluble product, such as lyophilized powder (including hypodermic tablets) that is prepared to be combined with solvent immediately before use, sterile suspension that is prepared for injection, sterile dry insoluble product that is prepared to be combined with vehicle immediately before use, and sterile and / or non-pyrogenic emulsion.

[0140] In one embodiment, unit dose parenteral preparations are packaged in ampoules, vials or syringes with needles. As is known and practiced in the art, all preparations for parenteral administration should be sterile and non-pyrogenic.

[0141] In one embodiment, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may include excipients that improve the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may include a buffer, such as citric acid, sodium or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at about neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those of skill in the art, provides the desired formulation. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-PD-1 antibody or antigen-binding fragment thereof, or a composition thereof. Overfilling the vial with a small amount (e.g., about 10%) more than is needed for a single dose or set of doses is permissible to facilitate accurate sample removal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0142] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile and / or non-pyrogenic water or other liquid suitable carrier is added to the lyophilized powder for preparation. The exact amount depends on the selected therapy being administered and can be empirically determined.

[0143] Also provided are methods of treatment comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment provided herein, thereby treating or preventing a condition or disorder associated with or related to PD-1. In another aspect, provided are methods of treating a condition in a subject that could benefit from upregulation of an immune response comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment provided herein.

[0144] The therapeutically effective amount of an antibody or antigen-binding fragment provided herein will depend on a variety of factors known in the art, such as, for example, the weight, age, past medical history, current medications, the health of the subject, as well as the potential for cross-reactivity, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of tumor development. As indicated by these and other circumstances or requirements, dosages may be proportionately reduced or increased by one skilled in the art (e.g., a physician or veterinarian).

[0145] In one embodiment, the antibodies or antigen-binding fragments provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In some of these embodiments, the antibody or antigen-binding fragment is administered at a dose of about 50 mg / kg or less, and in some of these embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In one embodiment, the dosage may vary over the course of treatment. For example, in one embodiment, an initial dosage may be higher than a later dosage. In one embodiment, the dosage may vary over the course of treatment depending on the subject's response.

[0146] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose can be given or several divided doses can be administered over time.

[0147] The antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as, for example, parenteral (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous infusion, intramuscular injection, or intradermal injection) or non-parenteral (oral, nasal, intraocular, sublingual, rectal or topical) routes.

[0148] The PD-1 associated conditions and disorders may be immune-related diseases or disorders. In one embodiment, the PD-1 associated conditions or disorders include tumors and cancers, such as non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma. The tumors and cancers include primary mediastinal large B-cell lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK-cell / T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, tumors of the central nervous system (CNS), such as primary CNS lymphoma, spinal axis tumor, and brain stem glioma. In one embodiment, the tumors and cancers are metastatic, particularly metastatic tumors that express PD-L1. In one embodiment, the PD-1-associated conditions and disorders include autoimmune diseases, such as systemic lupus erythematosus (SLE), psoriasis, systemic dermatosis, and autoimmune diabetes. In one embodiment, PD-1 associated conditions and disorders include infectious diseases such as chronic viral infections, e.g., viral infections such as hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papillomavirus, adenovirus, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus (Torquetenovirus), JC virus, or BK virus.

[0149] How to use

[0150] The disclosure further provides methods of using the anti-PD-1 antibodies or antigen-binding fragments thereof.

[0151] In one embodiment, the disclosure provides a method of treating a PD-1 associated or related condition or disorder in an individual comprising administering a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof, hi one embodiment, the individual has been identified as having a disorder or condition that is likely to respond to a PD-1 antagonist.

[0152] The presence or level of PD-L1 on a biological sample of interest may be indicative of whether the individual from whom the biological sample is derived is likely to respond to a PD-1 antagonist. Various methods may be used to determine the presence or level of PD-L1 in a test biological sample from an individual. For example, the test biological sample may be exposed to an anti-PD-L1 antibody or antigen-binding fragment thereof that binds to and detects expressed PD-L1 protein. Alternatively, PD-L1 may also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, etc. In some embodiments, the test sample is derived from cancer cells or tissues, or tumor-infiltrating immune cells. In one embodiment, the presence or upregulation of the level of PD-L1 in the test biological sample indicates likely responsiveness. The term "upregulation" as used herein refers to an overall increase of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or more in the protein level of PD-L1 in a test sample detected using an antibody or antigen-binding fragment provided herein compared to the protein level of PD-L1 in a reference sample detected using the same antibody. A reference sample may be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample was obtained. For example, a reference sample may be a non-diseased sample adjacent or near the test sample (e.g., a tumor).

[0153] The antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic procedures or treatments. For example, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with chemotherapy, radiation therapy, surgery (e.g., tumor removal) for cancer treatment, one or more antiemetic or other treatments for complications from chemotherapy, or any other therapeutic agent for use in treating cancer or any medical disorder mediated by PD-1. In some of these embodiments, the antibodies or antigen-binding fragments disclosed herein administered in combination with one or more additional therapeutic agents are The antibody or antigen-binding fragment may be administered simultaneously with one or more additional therapeutic agents, and in some of these embodiments, the antibody or antigen-binding fragment and the additional therapeutic agent may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered "in combination" with another therapeutic agent does not necessarily have to be administered simultaneously with that agent or in the same composition as that agent. When the term "in combination" is used in the present invention, an antibody or antigen-binding fragment administered before or after another agent is considered to be administered "in combination" with that agent, even if the antibody or antigen-binding fragment and the second agent are administered by different routes. When possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein will be administered according to the schedule set forth in the product information sheet for that additional therapeutic agent, or according to protocols described in Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002) or well known in the art.

[0154] In one embodiment, the therapeutic agent can induce or enhance an immune response against cancer. For example, tumor vaccines may be used to induce an immune response against certain tumors or cancers. Cytokine therapy may also be used to enhance tumor antigen presentation to the immune system. Examples of cytokine therapy include, but are not limited to, interferons such as interferon-α, -β, and -γ; colony stimulating factors such as macrophage-CSF, granulocyte-macrophage CSF, and granulocyte-CSF; interleukins such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12; tumor necrosis factors such as TNF-α and TNF-β. Agents that inactivate immunosuppressive targets may also be used, such as TGF-beta inhibitors, IL-10 inhibitors, and Fas ligand inhibitors. Another group of agents includes agents that activate immune reactivity to tumor or cancer cells, for example, those that enhance T cell activation (e.g., agonists of T cell costimulatory molecules such as CTLA-4, ICOS, and OX-40), and those that enhance dendritic cell function and antigen presentation.

[0155] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments that fall within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive ability and without departing from the scope of the invention. It will be understood that many modifications can be made to the procedures described herein while still remaining within the scope of the invention. It is the intention of the inventors that such modifications are included within the scope of the invention. EXAMPLES

[0156] Example 1: Preparation of antibody hybridomas

[0157] 1.1 Preparation of immunogens: DNA encoding PD-1 and PD-L1ECD or full-length was synthesized and inserted into the expression vector pcDNA3.3. Plasmid DNA was maxi-prepped and the sequence of the inserted DNA was confirmed by sequencing. Fusion proteins PD-1ECD and PD-L1ECD containing various tags, including human Fc, mouse Fc, and His tags, were obtained by transfecting CHO-S cells or HEK293 cells with the human PD-1ECD gene. After 5 days, the supernatants harvested from the cultures of the transiently transfected cells were used for protein purification. The fusion proteins were purified and quantified for use in immunization and screening.

[0158] 1.2 Establishment of stable cell lines. To obtain tools for antibody screening and antibody evaluation, we generated PD-1 and PD-L1 transfected cell lines. Briefly, CHO-K1, 293F or Ba / F3 cells were transfected with pCND3.3 expression vectors containing full-length PD-1 or PD-L1 using Lipofectamine 2000 transfection kit according to the manufacturer's protocol. 48-72 hours after transfection, the transfected cells were cultured in medium containing blasticidin or G418 for selection. Over time, this selects for cells that have stably integrated the PD-1 or PD-L1 gene into their genomic DNA. Meanwhile, the cells were examined for expression of the genes of interest, PD-1 and PD-L1. Once expression was confirmed, single clones of interest were picked out by limiting dilution and scaled up to large volumes. The established monoclonal cell lines were then maintained in medium containing lower doses of the antibiotic blasticidin or G418. 1.3 Generation of antibody hybridomas

[0159] 1.3.1 Immunization and cell fusion: 8-10 week old OMT rats (obtained from Open Monoclonal Technology, Inc., Palo Alto, US) were immunized in the footpad with 10μg human PD-1ECD in TiterMax for the first boost, and repeated immunization with PD-1ECD protein in aluminum every 3 days. The rats were bled every 2 weeks to collect serum, and antibody titers were measured by ELISA or FACS assay. When the antibody titers reached a sufficient level, the rats were given a final boost without adjuvant (add 100μl of 1XPBS instead), and cell fusion was performed as follows: immunization B lymphocytes isolated from lymph nodes of immunized OMT rats were combined with myeloma cells (1:1 ratio). The cell mixture was washed and suspended in 5-10 ml of ECF solution. ECF solution was added to bring the concentration to 2x10 6 After electrical cell fusion, the cell suspension in the fusion chamber was immediately transferred to a sterile tube containing a larger volume of medium. After >24 h incubation at 37°C, the cell suspension was mixed and dispensed (0.5x10 6 cells / plate). Cells were incubated at 37°C, 5% CO 2 When the number of clones was large enough, 100 μl of the supernatant was transferred from the 96-well plate to the antibody screening assay.

[0160] 1.3.2 Initial and confirmatory screening of hybridoma supernatants: ELISA assay was used as the initial screening method to test the binding of hybridoma supernatants to PD-1 protein. Briefly, plates (Nunc) were coated with 1 μg / ml of soluble human PD-1 extracellular domain protein overnight at 4°C. After blocking and washing, hybridoma supernatants were transferred to the coated plates and incubated for 1 h at room temperature. The plates were then washed and then incubated with secondary antibodies goat anti-rat IgG1 HRP (Bentyl) and goat anti-rat IgG2b HRP (Bentyl) for 45 min. After washing, TMB substrate was added and the interaction was stopped by 2 M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Devices). To confirm the native binding of PD-1 antibodies to the conformational PD-1 molecule expressed on the cell membrane, FACS analysis was performed on the PD-1 transfected CHO-S cell line. 1x10 CHO-S cells expressing human PD-1 6 The hybridoma cells were transferred into a 96-well U-bottom plate (BD) at a density of 1000 cells / ml. Hybridoma supernatants were then transferred to the plate and incubated at 4°C for 1 hour. After washing with 1XPBS / 1%BSA, secondary antibody goat anti-rat FITC (JacksonImmunoresearchLab) was added and incubated with the cells for 1 hour at 4°C in the dark. Cells were then washed and resuspended in 1XPBS / 1%BSA or fixed with 4% paraformaldehyde and analyzed by flow cytometry (BD). Antibody binding to the parental CHO-S cell line was performed using the same method. Figure 1 shows the binding of anti-human PD-1 antibodies to PD-1-expressing CHO cells. CHO cells transfected with full-length human PD-1 were stained with an antibody against human PD-1 from a rat hybridoma, followed by secondary antibody staining with FITC-conjugated goat anti-rat IgGFc and analyzed by FACS. The data show that the antibody specifically binds to PD-1 expressed on CHO cells.

[0161] To test the binding affinity of the antibody to native PD-1 expressed on human CD4+ T cells, human CD4+ T cells were generated from PBMCs cultured for 3 days in the presence of IL-2 and OKT3 and stained with an antibody against human PD-1. The binding of the antibody to PD-1 on T cells was analyzed by FACS. As shown in Figure 3, the FACS analysis showed that the antibody specifically bound to native PD-1 expressed on CD4+ T cells.

[0162] Testing the inhibitory activity of antibodies was used as a confirmatory screen to select the antibody candidate hits. Selected antibodies were tested by FACS analysis for their ability to inhibit the binding of the ligand PD-L1 to PD-1-transfected CHO-S cells. CHO-S cells expressing human PD-1 were cultured at 1x10 6 Cells were then transferred into a 96-well U-bottom plate (BD) at a density of 1000 cells / ml. Antibodies were serially diluted in wash buffer (1XPBS / 1%BSA) and incubated with the cells for 1 hour at 4°C. After washing, human Fc fused human PD-L1 protein was added and incubated for 1 hour at 4°C. Secondary antibody goat anti-human IgGFcFITC antibody (no cross-reactivity to rat IgGFc, Jackson Immunoresearch Lab) was incubated with the cells for 1 hour at 4°C in the dark. Cells were then washed and resuspended in 1XPBS / 1%BSA or fixed with 4% paraformaldehyde and analyzed by flow cytometry (BD).

[0163] 1.3.3 Subcloning of hybridomas: After specific binding and specific inhibition were confirmed through initial and confirmatory screening, the positive hybridoma cell lines could be used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted in cloning medium to 5 cells / well, 1 cell / well and 0.5 cells / well. They were seeded in 96-well plates at 200 μl / well, one plate at 5 cells / well, one plate at 1 cell / well and four plates at 0.5 cells / well. All plates were incubated at 37°C and 5% CO2 All cell lines are incubated until tested in the ELISA assay.

[0164] Example 2: Sequencing of antibody hybridoma cells and characterization of fully human antibodies 2.1 Sequencing of antibody hybridoma cells: RNA was isolated from monoclonal hybridoma cells using Trizol reagent. The VH and VL of PD-1 antibody were amplified according to the following protocol: Briefly, RNA was first reverse transcribed into cDNA using reverse transcriptase as described below. Reaction system (20 μl): 10x RT buffer 2.0μl 25x dNTP mix (100mM) 0.8μl 10x RT random primer / oligo dT / specific primer 2.0 μl MultiScribe™ Reverse Transcriptase 1.0 μl RNase inhibitor 1.0 μl 2 μg RNA Nuclease Free H 2 O up to 20.0 μl

[0165] Reaction conditions [Table A]

[0166] The resulting cDNA is used as a template for subsequent PCR amplification using primers specific for the gene of interest. The PCR reaction was carried out as follows: [ka]

[0167] 10 μl of the PCR reaction is ligated with the pMD18-T vector. 10 μl of the ligation product is transformed into Top10 competent cells, and the mixture is transferred onto pre-warmed 2-YT+Cab plates according to standard protocols and incubated overnight. Positive clones are checked by PCR using M13-48 and M13-47 primers, followed by sequencing.

[0168] 2.2 Construction of a fully human antibody molecule: The VH and VL of PD-1 antibody were amplified as described above. The PCR reaction was purified using a PCR cleanup kit, and the VL and pCI vector were digested with restriction enzymes PmeI and BssHII at 37°C for 2 hours. The reaction was run on 1% agarose and gel extracted using a kit according to the manufacturer's instructions. Ligation of the digested VL and pCI vector was performed as follows: [ka]

[0169] The mixture was incubated at 16°C for 30 min. 10 μl of the reaction was used for transformation and clonal expansion. The confirmed clone was used to extract DNA of plasmid pCI-VL. The pCI-VL vector and VH fragment were then digested with XbaI and SalI, and the purified and digested VH and vector were ligated with T4 DNA ligase at 16°C for 30 min. After the inserted VL and VH sequences were confirmed by sequencing, the expression vector containing the whole IgG of the fully human PD-1 antibody was used for transient transfection and stable cell line development.

[0170] Example 3: Characterization of fully human antibodies 3.1 Binding affinity of PD-1 antibodies to cell surface PD-1 molecules tested by flow cytometry (FACS): Antibody binding affinity to cell surface PD-1 was performed by FACS analysis. 5x10 CHO-S cells expressing human PD-1 were 5Cells were transferred into 96-well U-bottom plates (BD) at a density of 1000 cells / ml. Antibodies to be tested were serially diluted 1:2 in wash buffer (1XPBS / 1%BSA) and incubated with the cells for 1 hour at 4°C. Secondary antibody goat anti-human IgGFcFITC (3.0 moles FITC per mole IgG (JacksonImmunoresearch Lab)) was added and incubated for 1 hour at 4°C in the dark. Cells were then washed once, resuspended in 1XPBS / 1%BSA, and analyzed by flow cytometry (BD). Fluorescence intensity was converted to the bound molecule / cell ratio based on the Quantum™ MESF kit (Bangs Laboratories, Inc.) quantification beads. KD was calculated using GraphPad Prism 5. Figure 2 shows the binding of fully human PD-1 antibodies (i.e., 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb) to CHO cells expressing PD-1. Fully human antibodies against human PD-1 were used to stain PD-1 transfected CHO cells, and the fully human PD-1 antibodies had an EC of approximately 2 nmol / L. 50 FACS analysis shows that it specifically binds to PD-1.

[0171] 1.103.11-v2hAb was generated by mutating a single amino acid Asn93 (Kabat numbering) on ​​the original antibody 1.103.11hAb-VH(20975-VL) to a serine residue to reduce the risk of glycosylation at the CDR residues. Although Asn93 is located in the light chain CDR3, the antibody-antigen complex model generated from computational docking suggested that Asn93 does not directly contact any residue on the antigen of human PD-1. Much of the binding function of the light chain CDR3 appears to be provided by the adjacent residue Tyr91, which interacts with several residues on the PD-1 FG loop. Cell-based functional assays of 1.103.11-v2hAb confirmed that the mutation did not affect any binding ability (see experimental results below and Figures 15 and 16).

[0172] The binding affinity of 1.103.11-v2hAb to human PD-1 was measured by FACS and ELIS assays. Figure 16 shows the binding of 1.103.11-v2hAb to CHO cells expressing PD-1 in various buffers, and the binding was also measured when the antibody was in formulation buffer or 1xPBS (pH 7.4) and CHO-S cells expressing human PD-1 were incubated at 2x10 5 The antibodies were tested under the same conditions as the FACS assay described above, except that they were transferred into 96-well U-bottom plates (BD) at a density of 1.103.11 cells / ml. Results were comparable to those of 1.103.11hAb. "1.103.11-v2hAb in buffer" refers to the antibody in formulation buffer, and "1.103.11-v2hAb in PBS" refers to the antibody in 1xPBS, pH 7.4. The antibody bound to cell surface PD-1 on CHO cells in both solutions, with no significant difference in affinity to human PD-1 between the two conditions (EC50 was approximately 2.52 nmol / L for 1xPBS and approximately 3.12 nmol / L for formulation buffer).

[0173] Figure 15 shows the binding of antibody 1.103.11-v2hAb in various solutions to PD-1 protein as measured by ELISA. Following the same ELISA protocol as described above, the incubation time of 1.103.11-v2hAb was 2 hours and the incubation time of the secondary antibody goat anti-human IgGFcHRP (1:5000, Abcam) was 1 hour. "1.103.11-v2hAb in buffer" refers to the antibody in the formulation buffer and "1.103.11-v2hAb in PBS" refers to the antibody in 1xPBS (pH 7.4). Binding affinity to human PD-1 was demonstrated in both conditions.

[0174] CHO cells expressing human PD-1 were incubated with different concentrations of antibodies against PD-1, and then human PD-L1 tagged with mouse Fc was added to the cells. Binding of human PD-L1 to PD-1 expressing cells was detected using FITC-conjugated goat anti-mouse IgG, followed by FACS analysis. As shown in Figure 4, antibodies against PD-1 inhibited the binding of PD-L1 to PD-1 transfected CHO cells. 1.103.11-v2 hAb was also tested for inhibition of PD-L1 binding to PD-1 transfected CHO cells, and the results were comparable to 1.103.11 hAb.

[0175] 3.2 Overall kinetic binding affinity tested by surface plasmon resonance (SPR): Antibodies were characterized for affinity and binding kinetics to PD-1 by SPR assay using ProteOnXPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized on a GLM sensor chip (Bio-Rad) through amine coupling. Purified antibodies were flowed over the sensor chip and captured by Protein A. The chip was rotated 90° and washed with running buffer (1XPBS / 0.01% Tween20, Bio-Rad) until the baseline was stable. Five concentrations of human PD-1 and running buffer were flowed over the antibody flow cell at a flow rate of 100 μL / min during a 240 s association phase followed by a 600 s dissociation phase. The chip was resuspended in pH 1.7H after each run. 3 PO 4 The association and dissociation curves were fitted to a 1:1 Langmuir binding model using ProteOn software.

[0176] Using surface plasmon resonance, the affinities of antibodies to PD-1 to recombinant human PD-1 ranged from 3.76E-9 to 1.76E-10 ml / L, as shown in Figure 7. The affinity of 1.103.11-v2 hAb is expected to be similar to that of 1.103.11 hAb.

[0177] 3.3 Screening for orthologs (cross-species) and homologs (cross-family):

[0178] 3.3.1 Cross-reactivity to cynomolgus monkey PD-1 and mouse PD-1: Cross-reactivity was measured by ELISA. Plates (Nunc) were coated with 1 μg / ml cynomolgus monkey PD-1 (SinoBiological) and mouse PD-1 (SinoBiological) overnight at 4°C. After blocking and washing, 1 μg / ml of antibody was added to the plate and incubated for 1 h at room temperature. Plates were then washed and then incubated with secondary antibodies goat anti-rat IgG1 HRP (Bentyl) and goat anti-rat IgG2 HRP (Bentyl). The plate was incubated with IgG2b HRP (Bentyl) for 45 min. After washing, TMB substrate was added and the interaction was stopped with 2 M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Devices).

[0179] Cross-species cross-reactivity studies indicate that the antibodies against PD-1 bind to monkey PD-1 in cynomolgus monkeys but not to mouse PD-1 (Figure 6). 1.103.11-v2hAb is predicted to perform comparable to 1.103.11hAb in the same experiments.

[0180] 3.3.2 Cross-reactivity to PD-1 family members CD28, CTLA4 and ICOS: To examine the cross-family binding activity of fully human antibodies, cell lines expressing PD-1, CD-28, CTLA4 or ICOS were stained with the antibodies, followed by secondary antibody staining with FITC-conjugated goat anti-human IgGFc. PD-1 expressing cells were used as a positive control. The corresponding parental cell lines were used as negative controls. Stained cells were analyzed using BD Biosciences FACSCantoII and FlowJo Version software.

[0181] Figure 5 shows that PD-1, CD28 transfected CHO cells and CTLA4 transfected 293F cells were stained with antibodies against PD-1 and analyzed by FACS. The results show that the PD-1 antibody specifically binds to PD-1, but not to the PD-1 family members CD28 and CTLA4. 1.103.11-v2hAb is expected to show comparable results to 1.103.11hAb in the same experiment.

[0182] 3.4 Epitope Bin Testing 3.4.1 The binding epitope of the PD-1 antibody was binned against reference antibodies A and B by SPR assay using ProteOnXPR36 (Bio-Rad). Reference antibodies A and B were immobilized on a GLC sensor chip (Bio-Rad) through amine coupling. Human PD-1 solution was flowed over the antibody immobilized channel and captured by the reference antibody. The chip was then rotated 90° and washed with running buffer until the baseline was stable. The selected antibody was flowed over the sensor chip.

[0183] 3.4.2 The binding epitope of PD-1 antibody was binned against reference antibodies A and B by FACS. CHO cells expressing human PD-1 on the cell surface were incubated with reference antibodies A and B at a concentration of 10 μg / ml for 1 hour. Cells were washed and PD-1 antibody of the present disclosure was added and incubated for 1 hour. Secondary antibody, anti-rat IgG-FITC, was added and incubated for 1 hour at 4°C. Cells were then washed once, resuspended in 1XPBS / 1%BSA and analyzed by flow cytometry (BD).

[0184] The SPR assay and FACS results for the bin studies indicated that the epitopes on human PD-1 bound by the fully human PD-1 antibodies (i.e., 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15 hAb, and 1.153.7 hAb) were distinct from the existing PD-1 antibodies (i.e., reference antibodies A and B). It is expected that 1.103.11-v2hAb will show comparable results to 1.103.11hAb in the same experiment.

[0185] 3.5 In vitro function of PD-1 antibodies tested by cell-based assays

[0186] 3.5.1 Effect of human PD-1 antibodies on T cell proliferation. Allogeneic reactions were used to test the effect of PD-1 antibodies on T lymphocyte proliferation. Primary MLRs stimulated with dendritic cells (DCs) were performed in 96-well U-bottom tissue culture plates in 200 μl of RPMI 1640 containing 10% FCS and antibiotics. DCs were 1×10 5 Total CD4 of allogeneic + DCs were mixed with T cells at DC:T cell ratios ranging from 1:10 to 1:100. Cultures were also performed in the presence or absence of neutralizing monoclonal antibodies:human PD-1 antibodies and reference antibodies A and B, used at 10 μg / ml. The assay was incubated for 5 days, with the final 16 hours at 4°C. 3 H]thymidine was added at 1 uCi / well. 3 H]thymidine incorporation was measured by scintillation counting, and proliferation responses were calculated as the average of triplicate wells [ 3 H thymidine incorporation was expressed as counts per minute. Counts attributable to DC alone were always below 1000 cpm. Results shown are representative of at least five experiments performed.

[0187] The human dendritic cells (DCs) and CD4 + T cells, CD8 +T cells and total cells were generated from PBMCs by the following procedure: Human monocytes were purified from PBMCs by negative selection using a human monocyte enrichment cocktail kit according to the manufacturer's instructions (StemCellMeylan). Briefly, PBMCs were isolated from the blood of healthy donors using a Ficoll-Paque gradient. Cells were washed twice with PBS and then diluted to 1X10 in isolation buffer. 8 The cells were resuspended at 2x10 cells / ml and incubated with monocyte enrichment antibody cocktail for 30 minutes at 4°C. The cells were washed and then incubated with magnetic colloids for 30 minutes at 4°C. Unlabeled monocytes were collected by passing through a MACS column. To generate iDCs, monocytes were diluted at 2x10 6 iDCs were cultured at a concentration of 1000 cells / ml in RPMI 1640 medium containing 10% FCS and antibiotics with GM-CSF (PeproTech, Rocky Hill, NJ; 800 U / ml) and IL-4 (PeproTech; 500 U / ml). Half of the medium was replaced with medium containing GM-CSF and IL-4 every other day. Mature DCs were generated by stimulating iDCs with LPS (026:B6; Sigma-Aldrich, St. Louis, MO; 1 μg / ml) for an additional 24 h on day 5. CD4 + T cells, CD8 + T cells and total T cells were prepared by purifying PBMCs with human CD4 + T cells, CD8 + T cells and enriched mixtures of total T cells were purified by negative selection by incubation with magnetic colloids.

[0188] Human CD4 + T cells were stimulated with allogeneic DCs in the presence or absence of PD-1 antibodies 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb. + T cell proliferation is 3H] thymidine incorporation. Figure 10 shows that 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.139.15 hAb, and 1.153.7 hAb enhanced T cell proliferation in a concentration-dependent manner. 1.103.11-v2 hAb is expected to show comparable results to 1.103.11 hAb in the same experiment.

[0189] 3.5.2 Effect of human PD-1 antibody on cytokine IFNγ secretion in vitro: To directly examine the effect of human PD-1 antibody inhibition on cytokine IFNγ production, we performed experiments on IFNγ production in allogeneic MLR. + T cells were cultured according to the manufacturer's instructions using CD4 + DCs were purified from PBMCs by negative selection using a T cell enrichment cocktail kit. Immature DCs were generated from monocytes by culturing them in GM-CSF and IL-4 for 5 days, and mature DCs were differentiated by overnight stimulation with 1 μg / ml LPS. CD4 +T cells were mixed with iDCs / mDCs at T cell:DC ratios of 10:1 to 100:1. Cultures were performed in the presence or absence of human PD-1 antibody and reference antibody. After 5 days, the supernatant from each culture was collected for cytokine IFNγ measurement. The level of IFNγ in the supernatant was measured by ELISA assay. Briefly, Maxisorp plates were coated with anti-human IFN-gamma monoclonal antibody (0.75 μg / ml; i.e., 1 / 1360 dilution) diluted in coating buffer at 50 μl / well (i.e., 3.7 μl of antibody in 5 ml of coating buffer for a full 96-well plate) and incubated overnight at 4°C. Residual protein binding capacity was blocked by adding 200 μl / well of blocking buffer for 2 hours. A dilution series of recombinant IFN-gamma is prepared in complete medium to serve as standards, 2-fold dilutions from 8000 pg / ml to 125 pg / ml, plus complete medium alone. Plates are washed and standards and test supernatants are added (100 μl / well) and incubated for 2-4 hours. Biotinylated anti-IFN-gamma monoclonal antibody (1 / 1333) in blocking buffer is added, followed by Extra-Avidin Peroxidase. The reaction is initiated by the addition of TMB substrate and stopped with 2M HCl. Absorbance is measured at 450 nm.

[0190] Figure 9 shows human CD4 + Figure 1 shows that cells were stimulated with allogeneic DCs in the presence or absence of antibodies 1.7.3hAb, 1.49.9hAb, 1.103.11hAb, 1.139.15 hAb, and 1.153.7 hAb. IFNγ levels were measured by ELISA. Results showed that the fully human PD-1 antibodies increased IFNγ secretion in a dose-dependent manner. 1.103.11-v2hAb is expected to show comparable results to 1.103.11hAb in the same experiment.

[0191] 3.5.3 Effect of human PD-1 antibody on in vitro production of interleukin 2 (IL-2): CD4 +T cells were mixed with iDCs / mDCs at T cell:DC ratios ranging from 10:1 to 100:1. Cultures were performed in the presence or absence of human PD-1 antibody and reference antibodies. After 5 days, the supernatants from each culture were collected for cytokine measurements. The levels of IL-2 in the supernatants were measured by ELISA assay.

[0192] Figure 8 shows human CD4 + Figure 1 shows that T cells were stimulated with allogeneic DCs in the presence or absence of lead or control antibodies. IL-2 levels were measured by ELISA. Results showed that antibodies against PD-1 increased IL-2 secretion in a dose-dependent manner. 1.103.11-v2hAb is expected to show comparable results to 1.103.11hAb in the same experiment.

[0193] 3.5.4 Effect of human PD-1 antibodies on cell proliferation and cytokine production in autoantigen-specific immune responses: In this assay, T cells and DCs were derived from the same donor. + T cells were purified from PBMCs and cultured in the presence of CMVpp65 peptide and low doses of IL2 (20 U / ml), while simultaneously, DCs were generated by culturing monocytes derived from the PBMCs of the same donor in GM-CSF and IL-4. After 5 days, CD4 + T cells were co-cultured with DCs pulsed with pp65 peptide in the presence or absence of human PD-1 antibody and reference antibody (as a control). On day 5, 100 μl of supernatant was removed from each culture for measurement of cytokines IFNγ and IL-2. The levels of IFNγ and IL-2 production were detected by ELISA assay. The proliferation of T cells specific for DCs pulsed with CMVpp65 peptide was [ 3 H]thymidine incorporation was examined.

[0194] As shown in Figure 11, PD-1 antibody inhibited the proliferation of CMV pp65 peptide-loaded autologous DCs. + -CD4 +It was found that 1.103.11-v2hAb enhanced T cell proliferation in a concentration-dependent manner. It is expected that 1.103.11-v2hAb will perform similarly to 1.103.11hAb in the same experiments.

[0195] 3.5.5 Effect of human PD-1 antibodies on the suppressive function of regulatory T cells (Treg): Regulatory T cells (Treg), a subpopulation of T cells, are key immune regulators and play a key role in maintaining self-tolerance. Elevated numbers of regulatory T cells have been found in patients with several cancers and are associated with a worse prognosis, and thus, the effect of CD4 + CD25 + Regulatory T cells are associated with tumors. To directly examine the effect of human PD-1 antibodies on the immune suppressive response, we performed experiments on regulatory T cells. CD4 + CD25 + and CD4 + CD25 - T cells were isolated using specific anti-CD25 microbeads (MiltenyiBiotec, Auburn, CA) and positive or negative selection, respectively. First, PBMCs were transfected with human CD4 + Negative selection by incubation with a T cell enrichment mixture and magnetic colloids to identify CD4 + T cells were purified. CD4 + The T cells were then resuspended in MACS buffer, incubated with CD25+ microbeads on ice for 30 min, washed, and loaded onto the column. + CD25 - T cells were collected from the flow-through and washed before use. + CD25 + The cells were then harvested from the column and washed before use. Regulatory T cells are CD4 + CD25 -T cells and DCs (1:1 ratio of regulatory T cells:Teff) were cultured in the presence or absence of human PD-1 antibody at a concentration of 10 μg / ml. Either no antibody or an isotype antibody was used as negative control. Culture supernatants were removed on day 5 for cytokine detection by ELISA, and cell proliferation was [ 3 H]thymidine was added at a concentration of 1uCi / well and incubated for an additional 18 hours. 3 [H]thymidine incorporation was measured by scintillation counting. As shown in Figure 12, the PD-1 antibody inhibited the suppressive function of regulatory T cells and restored proliferation and IFNγ secretion of responding T cells. It is expected that 1.103.11-v2 hAb will show comparable results to 1.103.11 hAb in the same experiment.

[0196] 3.6 ADCC / CDC Assay: Healthy PD-1 + To minimize unwanted toxicity to cells, the selected anti-PD-1 fully human antibodies were confirmed to have no ADCC and CDC functions.

[0197] 3.6.1 ADCC: Activated T cells expressing high levels of cell surface PD-1 were used as target cells and pre-incubated with various concentrations of fully human antibodies for 30 min in 96-well plates, followed by the addition of IL-2-activated PBMCs (used as a source of natural killer (NK) cells, i.e. effector cells) at an effector / target ratio of 50:1. Plates were incubated in 5% CO 2 The plates were incubated at 37°C for 6 hours in an incubator. Lysis of target cells was determined by a Cytotoxicity Detection Kit (Roche). Optical density was measured by a Molecular Devices SpectraMaxM5e plate reader. The results showed that the fully human antibodies against PD-1 tested did not mediate ADCC (Figure 13). 1.103.11-v2hAb is expected to show comparable results to 1.103.11hAb in the same experiment.

[0198] 3.6.2CDC: Target cells (activated T cells), diluted human serum complement (Quidel-A112) and various concentrations of fully human PD-1 antibody were mixed in a 96-well plate. The plate was incubated in 5% CO 2 Incubated for 4 hours at 37°C in an incubator. Lysis of target cells was determined by CellTiterglo (Promega-G7573). Rituxan (Roche) and human lymphocyte cell line Raji (CD20 positive) were used as positive controls. Data showed that PD-1 antibodies did not mediate CDC (Figure 14). 1.103.11-v2 hAb was comparable to 1.103.11 hAb in the same experiment. It is predicted to show positive results.

[0199] Example 4: Epitope mapping of fully human antibodies To determine the epitope differences between the antibody of the invention 1.103.11 hAb provided herein and the known hPD-1 antibody, KEYTRUDA, alanine scanning experiments on hPD-1 and evaluation of the effect on antibody binding were performed using 1.103.11 hAb, KEYTRUDA and 11.148.10 (a control hPD-1 antibody that binds to an epitope that does not overlap with the epitope of 1.103.11 hAb or that of KEYTRUDA).

[0200] Alanine residues in hPD-1 were mutated to glycine codons, and all other residues were mutated to alanine codons. Point amino acid substitutions were made for each residue in the extracellular domain (ECD) of hPD-1 using two consecutive PCR steps. The pcDNA3.3-hPD-1_ECD.His plasmid encoding human PD-1 and a C-terminal His tag was used as a template, and a set of mutagenesis primers was used in the first-step PCR using the QuikChange lightning multi-site-directed mutagenesis kit (Agilent Technologies, Palo Alto, Ca). DpnI endonuclease was used to digest the parental template after the mutant strand synthesis reaction. In the second-step PCR, a linear DNA expression cassette consisting of the CMV promoter, the extracellular domain (ECD) of PD-1, a His tag, and herpes simplex virus thymidine kinase (TK) polyadenylation was amplified and transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD).

[0201] Monoclonal antibodies 1.103.11 hAb, Keytruda and 11.148.10 hAb were coated in plates for ELISA binding assay. After interacting with the supernatant containing the quantified PD-1 variants, HRP-conjugated anti-His antibody (Rockland, Cat#200-303-382) was added as detection antibody. The absorbance was normalized according to the average of the control variants. After setting an additional cutoff value (<0.55) for the fold change of binding, the final determined epitope residues were identified.

[0202] The top 30 point-substitution hPD-1 mutants that significantly reduced antibody binding were shown in Table 6. Examination of the locations of all these residues on the hPD-1 crystal structure (PDB codes 3RRQ and 4ZQK) revealed that several amino acids (e.g., Val144, Leu142, Val110, Met108, Cys123, etc.) were completely buried in the protein and unlikely to come into direct contact with any antibody. The observed reduction in binding was most likely caused by instability or even disruption of the hPD-1 structure after alanine substitution. To avoid misinterpreting these data as epitope hotspots, we utilized a control antibody, 11.148.10 hAb, which binds to a completely different location on the antigen but is expected to respond to disruption of the hPD-1 structure if it indeed occurs. Mutants that affect both antibodies were treated as false hotspots and removed from the list. After setting an additional cutoff value (≤0.55) for the binding fold change, the final determined epitope residues were listed in Table 3. These were nine positions in 1.103.11 hAb, five positions in Keytruda, and ten residues in the control antibody 11.148.10 hAb.

[0203] [Table 2]

[0204] [Table 3]

[0205] Comparing the epitope residues of 1.103.11 hAb and KEYTRUDA in Table 3 revealed only two overlapping hotspot residues. The rest appeared to be quite different, which indicated that the two antibodies may adopt quite different mechanisms for hPD-1 binding and hPD-L1 inhibition. Reading the residue numbers in Table 3 is not straightforward to interpret the mechanisms. Therefore, for better visualization and comparison, all the data in Table 3, together with the hPD-L1 binding site, were mapped onto the hPD-1 crystal structure (Figure 17).

[0206] As shown in Figure 17, the hotspot residues responsible for hPD-L1 binding were all clustered in the center of C, F and G strands (Figure 17A). The two tested antibodies, 1.103.11hAb and KEYTRUDA, have distinctly different epitopes, even though both are functional in binding to hPD-1 and inhibiting hPD-L1 (Figure 17B shows for 1.103.11hAb, and Figure 17C shows for KEYTRUDA). The epitope of KEYTRUDA is mainly contributed by residues on the C'D loop (corresponding to the C'' strand of mPD-1), which did not intersect with the PD-L1 binding site at all. This suggested that the hPD-L1 inhibitory function of KEYTRUDA was more dependent on the steric hindrance effect provided by the size of the antibody. In contrast, our lead antibody 1.103.11hAb is composed of hotspots distributed across multiple locations and has direct overlap with the hPD-L1 binding site (Figure 17A, 17B). The 1.103.11hAb of the present invention inhibited hPD-L1 by outcompeting hPD-L1 in reacting with the common binding site. The 1.103.11hAb is therefore predicted to be more functional in further development.

[0207] However, antibody 11.148.10 hAb bound to a completely different site (Figure 17D) from the two functional antibodies, confirming that this antibody itself is a good control antibody to measure the functionality of hPD-1 when making alanine substitutions.

[0208] While the present disclosure has been particularly shown and described with reference to specific embodiments, some of which are preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the disclosure disclosed herein.

Claims

[Claim 1] A composition as described in the specification.

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