WO2020063823A1

WO2020063823A1 - Anti-pd-1 antibodies and uses thereof

Info

Publication number: WO2020063823A1
Application number: PCT/CN2019/108417
Authority: WO
Inventors: Jing Li; Shou LI; Hong Li; Honghai GAO; Yuan Lin; Shali QI; Bing WAN; James Yan
Original assignee: Zai Lab (Shanghai) Co., Ltd.; Zlip Holding Limited
Priority date: 2018-09-27
Filing date: 2019-09-27
Publication date: 2020-04-02
Also published as: CN111253486B; CN111253486A

Abstract

Disclosed herein are anti-PD-1 antibodies and pharmaceutical compositions comprising an anti-PD-1 antibody for treating a disease or disorder.

Description

ANTI-PD-1 ANTIBODIES AND USES THEREOF

BACKGROUND OF THE DISCLOSURE

This application claims the benefit of PCT International Application No. PCT/CN2018/107872, filed September 27, 2018, which is incorporated by reference herein in its entirety. The immune system is capable of recognizing and eliminating tumor cells within a tumor microenvironment. Both innate and adaptive immunity act as a complementary network that recognize and remove these cells. Immunotherapy is a treatment method that modulates the immune system to recognize and subsequently eliminate the tumor cells.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are anti-PD-1 antibodies, pharmaceutical compositions thereof, and methods of their use.

Disclosed herein, in certain embodiments, is an anti-PD-1 antibody comprising three variable heavy chain complementarity determining regions (CDRs) and three variable light chain complementarity determining regions (CDRs) , wherein the anti-PD-1 antibody specifically binds to a different epitope on the extracellular domain of PD-1 compared to nivolumab and pembrolizumab, and wherein a binding affinity of the anti-PD-1 antibody is comparable to a binding affinity of nivolumab or pembrolizumab. In some embodiments, the VH CDR1 sequence consists of X ₁YX ₂MS; wherein X ₁ is S or T and X2 is G or T; the VH CDR2 sequence consists of X ₃ISX ₄GGX ₅DTYYPDX ₆VKG; wherein X ₃ is T or Y; X ₄ is G or F; X ₅ is R or G; and X ₆ is S or T; and the VH CDR3 sequence consists of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆; wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.

Disclosed herein, in certain embodiments, is an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , wherein: (a) the VH CDR1 sequence consists of X ₁YX ₂MS; wherein X ₁ is S or T and X2 is G or T; (b) the VH CDR2 sequence consists of X ₃ISX ₄GGX ₅DTYYPDX ₆VKG; wherein X ₃ is T or Y; X ₄ is G or F; X ₅ is R or G; and X ₆ is S or T; and (c) the VH CDR3 sequence consists of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆; wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.

In some embodiments, the VL CDR1 sequence consists of RASX ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈; wherein X ₁₇ is E or Q; X ₁₈ is S or D; X ₁₉ is V or I; X ₂₀ is D or S; X ₂₁ is S, N or D; X ₂₂ is Y or F; X ₂₃ is G or L; X ₂₄ is I or N; X ₂₅ is present or absence and if present, is S; X ₂₆ is present or absence and if present, is F; X ₂₇ is present or absence and if present, is M; X ₂₈ is present or absence and if present, is N; the VL CDR2 sequence consists of X ₂₉X ₃₀SX ₃₁X ₃₂X ₃₃S; wherein X ₂₉ is A or Y; X ₃₀ is A or T; X ₃₁ is N or R; X ₃₂ is Q or L; X ₃₃ is G or H; and the VL CDR3 sequence consists of QQX ₃₄X ₃₅X ₃₆X ₃₇PWT; wherein X ₃₄ is S or G; X ₃₅ is K or D; X ₃₆ is E or M; X ₃₇ is V or I.

In some embodiments, the VH CDR1 sequence is selected from SEQ ID NOs: 1, 4, or 7. In some embodiments, the VH CDR2 sequence is selected from SEQ ID NOs: 2, 5, or 8. In some embodiments, the VH CDR3 sequence is selected from SEQ ID NOs: 3, 6, or 9.

In some embodiments, the VL CDR1 sequence is selected from SEQ ID NOs: 19, 22, or 25. In some embodiments, the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, or 26. In some embodiments, the VL CDR3 sequence is selected from SEQ ID NOs: 21, 24, or 27.

In some embodiments, the VH CDR1 sequence is selected from SEQ ID NOs: 1, 4, or 7; the VH CDR2 sequence is selected from SEQ ID NOs: 2, 5, or 8; and the VH CDR3 sequence consists of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆; wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.

In some embodiments, the VH CDR1 sequence is selected from SEQ ID NOs: 1, 4, or 7; the VH CDR2 sequence is selected from SEQ ID NOs: 2, 5, or 8; and the VH CDR3 sequence is selected from SEQ ID NOs: 3, 6, or 9.

In some embodiments, the VL CDR1 sequence is selected from SEQ ID NOs: 19, 22, or 25; the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, or 26; and the VL CDR3 sequence consists of QQX ₃₄X ₃₅X ₃₆X ₃₇PWT; wherein X ₃₄ is S or G; X ₃₅ is K or D; X ₃₆ is E or M; X ₃₇ is V or I.

In some embodiments, the VL CDR1 sequence consists of RASX ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈; wherein X ₁₇ is E or Q; X ₁₈ is S or D; X ₁₉ is V or I; X ₂₀ is D or S; X ₂₁ is S, N or D; X ₂₂ is Y or F; X ₂₃ is G or L; X ₂₄ is I or N; X ₂₅ is present or absence and if present, is S; X ₂₆ is present or absence and if present, is F; X ₂₇ is present or absence and if present, is M; X ₂₈ is present or absence and if present, is N; the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, or 26; and the VL CDR3 sequence is selected from SEQ ID NOs: 21, 24, or 27.

In some embodiments, the VL CDR1 sequence is selected from SEQ ID NOs: 19, 22, or 25; the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, or 26; and the VL CDR3 sequence is selected from SEQ ID NOs: 21, 24, or 27.

In some embodiments, the VH CDR1 sequence is selected from SEQ ID NOs: 10, 13, or 16. In some embodiments, the VH CDR2 sequence is selected from SEQ ID NOs: 11, 14, or 17. In some embodiments, the VH CDR3 sequence is selected from SEQ ID NOs: 12, 15, or 18.

In some embodiments, the VL CDR1 sequence is selected from SEQ ID NOs: 28, 31, or 34. In some embodiments, the VL CDR2 sequence is selected from SEQ ID NOs: 29, 32, or 35. In some embodiments, the VL CDR3 sequence is selected from SEQ ID NOs: 30, 33, 36.

In some embodiments, the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 1-3 and three VL CDRs according to SEQ ID NOs: 19-21.

In some embodiments, the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 4-6 and three VL CDRs according to SEQ ID NOs: 22-24.

In some embodiments, the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 7-9 and three VL CDRs according to SEQ ID NOs: 25-27.

In some embodiments, the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 10-12 and three VL CDRs according to SEQ ID NOs: 28-30.

In some embodiments, the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 13-15 and three VL CDRs according to SEQ ID NOs: 31-33.

In some embodiments, the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 16-18 and three VL CDRs according to SEQ ID NOs: 34-36.

In some embodiments, the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 38 and the VL sequence according to SEQ ID NO: 40.

In some embodiments, the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 42 and the VL sequence according to SEQ ID NO: 44.

In some embodiments, the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 46 and the VL sequence according to SEQ ID NO: 48.

In some embodiments, the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 50 and the VL sequence according to SEQ ID NO: 52.

In some embodiments, the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 54 and the VL sequence according to SEQ ID NO: 56.

In some embodiments, the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 58 and the VL sequence according to SEQ ID NO: 60.

In some embodiments, the binding affinity of the anti-PD-1 antibody is higher than the binding affinity of nivolumab.

In some embodiments, the binding affinity of the anti-PD-1 antibody is higher than the binding affinity of pembrolizumab.

In some embodiments, the binding affinity of the anti-PD-1 antibody is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or higher than the binding affinity of nivolumab or pembrolizumab.

In some embodiments, the binding affinity of the anti-PD-1 antibody is about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or higher than the binding affinity of nivolumab or pembrolizumab.

In some embodiments, the ani-PD-1 antibody blocks interaction of PD-1 with PD-L1 and/or PD-L2. In some embodiments, the ani-PD-1 antibody has a >40%inhibition, >50%inhibition, or >60%inhibition at an antibody concentration range of about 5-7 nM. In some embodiments, the ani-PD-1 antibody has a >46%inhibition at an antibody concentration of 6.67nM.

In some embodiments, the anti-PD-1 antibody has a KD of less than 8e-9 M, less than 6e-9 M, less than 4e-9 M, less than 2.5e-9M, less than 2e-9 M, less than 1.5e-9 M, or less than 1.2e-9 M. In some embodiments, the anti-PD-1 antibody has a KD of about 2.43e-9 M. In some embodiments, the anti-PD-1 antibody has a KD of about 1.16e-9 M.

In some embodiments, the anti-PD-1 antibody has an IC ₅₀ similar to nivolumab and/or pembrolizumab.

In some embodiments, the anti-PD-1 antibody induces cytokine production. In some embodiments, the cytokine is IL-2 or INF-γ.

In some embodiments, the anti-PD-1 antibody comprises an IgG1 framework. In some embodiments, the anti-PD-1 antibody comprises an IgG4 framework. In some embodiments, the framework is a humanized IgG1 or IgG4 framework. In some embodiments, the IgG4 framework comprises a S228P mutation.

Disclosed herein, in certain embodiments, is an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , wherein the VH CDR1 sequence is selected from SEQ ID NOs: 1, 4, 7, 10, 13, or 16; the VH CDR2 sequence is selected from SEQ ID NOs: 2, 5, 8, 11, 14, or 17; and the VH CDR3 sequence is selected from SEQ ID NOs: 3, 6, 9, 12, 15, or 18. In some embodiments, the VL CDR1 sequence is selected from SEQ ID NOs: 19, 22, 25, 28, 31, or 34; the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, 26, 29, 32, or 35; and the VL CDR3 sequence is selected from SEQ ID NOs: 21, 24, 27, 30, 33, or 36.

Disclosed herein, in certain embodiments, is an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) according to SEQ ID NOs 1-3: and three variable light chain (VL) complementarity determining regions (CDRs) according to SEQ ID NOs: 19-21.

Disclosed herein, in certain embodiments, is an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) according to SEQ ID NOs: 4-6 and three variable light chain (VL) complementarity determining regions (CDRs) according to SEQ ID NOs: 22-24.

Disclosed herein, in certain embodiments, is an anti-PD-1 antibody comprising a variable heavy chain sequence and a variable light chain sequence pair selected from SEQ ID NOs: 38 and 40, 42 and 44, 46 and 48, 50 and 52, 54 and 56, 58 and 60.

Disclosed herein, in certain embodiments, is an anti-PD-1 antibody comprising a variable heavy chain sequence selected from SEQ ID NOs: 62, 64, and 66 and a variable light chain sequence selected from SEQ ID NOs: 68, 70, 72, and 74.

Disclosed herein, in certain embodiments, is an anti-PD-1 antibody comprising a variable heavy chain sequence selected from SEQ ID NOs: 76, 78, 80, and 82 and a variable light chain sequence selected from SEQ ID NOs: 84, 86, 88, 90, and 92.

Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising an anti-PD-1 antibody described above, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

Disclosed herein, in certain embodiments, is an isolated nucleic acid molecule encoding an anti-PD-1 antibody described above.

Disclosed herein, in certain embodiments, is a vector comprising a nucleic acid sequence encoding an anti-PD-1 antibody described above.

Disclosed herein, in certain embodiments, is a host cell producing an anti-PD-1 antibody described above.

Disclosed herein, in certain embodiments, is a method of treating a cancer in a subject in need thereof, comprising administering to the subject an anti-PD-1 antibody described above. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is bladder cancer, brain cancer, breast cancer, bladder cancer, bone cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, or uterine cancer. In some embodiments, the cancer is a lymphoma. In some embodiments, the cancer is Hodgkin’s lymphoma. In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the anti-PD-1 antibody and the additional therapeutic agent are administered sequentially. In some embodiments, the anti-PD-1 antibody is administered to the subject prior to administering the additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered to the subject prior to administering the anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody and the additional therapeutic agent are administered simultaneously. In some embodiments, the anti-PD-1 antibody and the additional therapeutic agent are formulated as separate dosage forms. In some embodiments, the subject is a human.

Disclosed herein, in certain embodiments, is a method of augmenting cytokine production in a subject having a cancer, comprising contacting cells of the subject with an anti-PD-1 antibody described above, thereby inducing an increase in the cytokine production relative to the cytokine production in cells of the same subject not contacted by the antibody. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is INF-γ. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is bladder cancer, brain cancer, breast cancer, bladder cancer, bone cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, or uterine cancer. In some embodiments, the cancer is a lymphoma. In some embodiments, the cancer is Hodgkin’s lymphoma. In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the anti-PD-1 antibody and the additional therapeutic agent are administered sequentially. In some embodiments, the anti-PD-1 antibody is administered to the subject prior to administering the additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered to the subject prior to administering the anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody and the additional therapeutic agent are administered simultaneously. In some embodiments, the anti- PD-1 antibody and the additional therapeutic agent are formulated as separate dosage forms. In some embodiments, the subject is a human.

Disclosed herein, in certain embodiments, is a method of inducing phagocytosis of a cell expressing PD-L1 and/or PD-L2, comprising blocking binding of the cell to a T cell expressing PD-1 with an anti-PD-1 antibody described above, thereby inducing phagocytosis of the cell. In some embodiments, the cell is a cancerous cell. In some embodiments, the cell is from a solid tumor. In some embodiments, the cell is from a hematologic malignancy. In some embodiments, the cell is from bladder cancer, brain cancer, breast cancer, bladder cancer, bone cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, or uterine cancer. In some embodiments, the cell is from a lymphoma. In some embodiments, the cell is from Hodgkin’s lymphoma. In some embodiments, the subject is a human.

Disclosed herein, in certain embodiments, is a kit comprising an anti-PD-1 antibody described above or a pharmaceutical composition described above.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity in the appended claims. The patent application file contains at least one drawing executed in color. Copies of this patent application with color drawing (s) will be provided by the Office upon request and payment of the necessary fee.

A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

FIG. 1 is a cartoon illustration of the programmed death 1 (PD-1) receptor and its ligands programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) , members of the CD28 and B7 families.

FIG. 2 shows human PD-1 specific antibody levels in the sera of immunized mice after 2 boosts using recombinant human PD-1 protein.

FIG. 3 shows full curve blocking activities of murine antibody hits in PD-1/PD-L1 binding assay.

FIG. 4A shows blocking of PD-1/PD-L2 binding by selected anti-PD-1 antibody hits in an ELISA assay.

FIG. 4B shows blocking of PD-1/PD-L2 binding by selected anti-PD-1 antibody hits in an ELISA assay.

FIG. 5 shows cross-reactivity to mouse PD-1 of murine anti-PD-1 antibodies.

FIG. 6 shows anti-PD-1 antibodies increased IL-2 production in a 2-dose DC/T MLR assay.

FIG. 7 shows anti-PD-1 antibodies increased IFN-γ production in a 2-dose DC/T MLR assay.

FIG. 8A shows increase of IL-2 production by anti-hPD-1 antibody hits in an immature DC/T MLR assay.

FIG. 8B shows increase of IFN-γ production by anti-hPD-1 antibody hits in an immature DC/T MLR assay.

FIG. 9 shows binding of chimeric anti-PD-1 antibodies to human PD-1 in an ELISA assay.

FIG. 10A shows FRET-based PD-1/PD-L1 binding/blocking assay for chimeric antibody hits.

FIG. 10B shows FRET-based PD-1/PD-L1 binding/blocking assay for chimeric antibody hits.

FIG. 11 shows blocking of PD-1/PD-L2 binding by chimeric anti-PD-1 antibodies in a protein-based ELISA assay.

FIG. 12A shows validation of chimeric antibody function in a first MLR assay.

FIG. 12B shows validation of chimeric antibody function in a second MLR assay.

FIG. 13A shows results of functional activities of selected chimeric PD-1 antibodies in Jurkat-NFAT luciferase reporter assay.

FIG. 13B shows results of functional activities of selected chimeric PD-1 antibodies in Jurkat-NFAT luciferase reporter assay.

FIG. 14A shows cell-based PD-1/PD-L1 binding blocking assay to test select PD-1 antibodies.

FIG. 14B shows cell-based PD-1/PD-L2 binding blocking assay to test select PD-1 antibodies.

FIG. 15A-Fig. 15D show inhibition of MC38 tumor growth in human PD-1 knock-in mice by treatment with anti-PD-1 antibodies. FIG. 15A: hIgG1isotype. FIG. 15B: Opdivo analogue. FIG. 15C: Xi31G1E10. FIG. 15D: Xi56B3C11.

FIG. 16A shows EC50s of 31G1E10 humanized PD-1 antibodies as determined by ELISA.

FIG. 16B shows EC50s of 56B3C11 humanized PD-1 antibodies as determined by ELISA.

FIG. 16C shows EC50s of 56B3C11 humanized PD-1 antibodies as determined by ELISA.

FIG. 17A and FIG 17B show PD-1/PD-L1 blocking activities of humanized PD-1 antibodies in two independent assays.

FIG. 18A-FIG. 18E show activities of select humanized PD-1 antibodies in PD-1/PD-L1 binding blocking in a luciferase reporter format

FIG. 19A shows activities of anti-PD-1 antibody variants in cell based PD-L1 binding blocking assay.

FIG. 19B shows activities of anti-PD-1 antibody variants in cell based PD-L2 binding blocking assay.

FIG. 20A and FIG. 20B show anti-PD-1 antibody variants increased IL-2 production in a DC/T MLR assay.

FIG. 20C and FIG. 20D show anti-PD-1 antibody variants increased IFN-γproduction in a DC/T MLR assay.

FIG. 21A and FIG. 21B show anti-PD-1 antibody variants increased IFN-γproduction in a CMV recall assay.

FIG. 22A shows in vivo tumor inhibition activity of pembrolizumab analogue in MC38 tumor model.

FIG. 22B shows in vivo tumor inhibition activity of 31G1E10-7 in MC38 tumor model.

FIG. 22C shows in vivo tumor inhibition activity of 31G1E10-12 in MC38 tumor model.

FIG. 22D shows in vivo tumor inhibition activity of 31G1E10-13 in MC38 tumor model.

FIG. 22E shows in vivo tumor inhibition activity of 31G1E10-18 in MC38 tumor model.

FIG. 23 shows freeze/thaw stability of select anti-PD-1 antibodies.

FIG. 24A shows accelerated stability of select anti-PD-1 antibodies at 4℃.

FIG. 24B shows accelerated stability of select anti-PD-1 antibodies at 25℃.

FIG. 24C shows accelerated stability of select anti-PD-1 antibodies at 40℃.

DETAILED DESCRIPTION OF THE DISCLOSURE

Programmed cell death 1, also known as PDCD1, is a type I transmembrane glycoprotein. It is an immune-receptor belonging to the CD28/CTLA-4 family that negatively regulates antigen receptor signaling by recruiting protein tyrosine phosphatase SHP-2 upon interacting with either of two ligands, PD-L1 or PD-L2 (see, e.g., Fig. 1) . PD-1 inhibits T-cell proliferation and production of related cytokines such as IL-1, IL-4, IL-10, and IFN-γ by suppressing activation and transduction of the PI3K/AKT pathway. In addition, co-ligation of PD-1 inhibits BCR-mediated signaling by dephosphorylating signal transducers. In some instances, PD-1 has been suggested to be involved in lymphocyte clonal selection and peripheral tolerance, and further contributes to the prevention of autoimmune diseases. Furthermore, PD-1 has been shown to be a regulator of virus-specific CD8+ T cell survival in HIV infection. As a cell surface molecule, PDCD1 regulates the adaptive immune response. Engagement of PD-1 by its ligands PD-L1 or PD-L2 transduces a signal that inhibits T-cell proliferation, cytokine production, and cytolytic function

Disclosed herein are antibodies that bind to PD-1 and inhibit the interaction of PD-1 to its ligands PD-L1 and/or PD-L2. In some instances, the antibodies described herein exhibits numerous desirable properties, such as for example, high affinity binding to PD-1 (e.g., to human PD-1) , enhances proliferation and expansion of tumor infiltrating lymphocytes, and decreases immune evasion of tumor cells.

Also disclosed herein, in certain embodiments, are pharmaceutical compositions that encompass an anti-PD-1 antibody, methods of use for the treatment of a cancer, and methods of inducing tumor cell kill activity.

Anti-PD-1 Antibodies

In certain embodiments, disclosed herein are anti-PD-1 antibodies. In some embodiments, the anti-PD-1 antibody binds to a different epitope on the extracellular domain of PD-1 compared to either nivolumab or pembrolizumab. In some instances, the anti-PD-1 antibody binds to a different epitope on the extracellular domain of PD-1 compared to nivolumab and pembrolizumab. In some cases, the binding affinity of the anti-PD-1 antibody is comparable to a binding affinity of either nivolumab or pembrolizumab. In additional cases, the anti-PD-1 antibody specifically binds to a different epitope on the extracellular domain of PD-1 compared to nivolumab and pembrolizumab, and a binding affinity of the anti-PD-1 antibody is comparable to a binding affinity of nivolumab or pembrolizumab.

In some embodiments, disclosed herein is anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , wherein:

a) the VH CDR1 sequence consists of X ₁YX ₂MS; wherein X ₁ is S or T and X2 is G or T;

b) the VH CDR2 sequence consists of X ₃ISX ₄GGX ₅DTYYPDX ₆VKG; wherein X ₃ is T or Y; X ₄ is G or F; X ₅ is R or G; and X ₆ is S or T; and

c) the VH CDR3 sequence consists of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆; wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.

In some embodiments, the VH CDR1 sequence is SYGMS, TYGMS, SYTMS, or TYTMS; the VH CDR2 sequence consists of X ₃ISX ₄GGX ₅DTYYPDX ₆VKG, wherein X ₃ is T or Y; X ₄ is G or F; X ₅ is R or G; and X ₆ is S or T; and the VH CDR3 sequence consists of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆, wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.

In some embodiments, the VH CDR1 sequence consists of X ₁YX ₂MS, wherein X ₁ is S or T and X2 is G or T; the VH CDR 2 sequence is TISGGGRDTYYPDSVKG, YISGGGRDTYYPDSVKG, TISFGGRDTYYPDSVKG, YISFGGRDTYYPDSVKG, TISGGGGDTYYPDSVKG, YISGGGGDTYYPDSVKG, YISFGGGDTYYPDSVKG, TISGGGRDTYYPDTVKG, YISFGGRDTYYPDTVKG, YISFGGGDTYYPDTVKG, TISFGGGDTYYPDTVKG, or TISGGGGDTYYPDTVKG; and the VH CDR3 sequence consists of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆, wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.

In some embodiments, the VH CDR1 sequence consists of X ₁YX ₂MS, wherein X ₁ is S or T and X2 is G or T; the VH CDR2 sequence consists of X ₃ISX ₄GGX ₅DTYYPDX ₆VKG, wherein X ₃ is T or Y; X ₄ is G or F; X ₅ is R or G; and X ₆ is S or T; and the VH CDR3 sequence is QRDSAWFAH, QGDSAWFAH, QRNSAWFAH, QRDYAWFAH, QRDSEWFAH, QRDSAGFAH, QRDSAWAAH, QRDSAWFPH, QRDSAWFAF, QRDSAWFAY, QGNSAWFAH, QGNYAWFAH, QGNSEWFAH, QGNSAGFAH, QGNSAWAAH, QGNSAWFPH, QGNSAWFAF, QGNSAWFAY, QGNYAWFAH, QGNYEWFAH, QGNYAGFAH, QGNYAWAAH, QGNYAWFPH, QGNYAWFAF, QGNYAWFAY, QGNYEWFAH, QGNYEGFAH, QGNYEWAAH, QGNYEWFPH, QGNYEWFAF, QGNYEWFAY, QGNYEGFAH, QGNYEGAAH, QGNYEGFPH, QGNYEGFAF, QGNYEGFAY, QGNYEGAAH, QGNYEGAPH, QGNYEGAPF, QGNYEGAPY, QRDSAWFAHA, QRDSAWFAHAY, QGNSAWFAHA, QGNSAWFAHAY, QGNYAWFAHA, QGNYAWFAHAY, QGNYEWFAHA, QGNYEWFAHAY, QGNYEGFAHA, QGNYEGFAHAY, QGNYEGAAHA, or QGNYEGAAHAY.

In some embodiments, the anti-PD-1 antibody comprises the VH CDR1 sequence selected from SEQ ID NOs: 1, 4, or 7; the VH CDR2 sequence selected from SEQ ID NOs: 2, 5, or 8; and the VH CDR3 sequence consisting of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆, wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VH CDR1 is SYGMS, SYTMS, TYGMS, SFYIH, SDYAWN, or NYWIE; the VH CDR2 is TISGGGRDTYYPDSVKG, YISFGGGDTYYPDTVKG, TISGGGRDTYYPDSVKG, WVYPGDTKYNEKFKG, YIIYSGSTSYNPSLKS, or NILPGTNNTNYNEKFKG; and the VH CDR3 is QRDSAWFAH, QGNYEGAPFAY, QRDSAWFAY, HNYDTMDY, NYGSSFYYFDY, or TFYGPFDY.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VH CDR1 is SYGMS, SYTMS, or TYGMS; the VH CDR2 is TISGGGRDTYYPDSVKG, YISFGGGDTYYPDTVKG, or TISGGGRDTYYPDSVKG; and the VH CDR3 is QRDSAWFAH, QGNYEGAPFAY, or QRDSAWFAY.

In some embodiments, an anti-PD-1 antibody comprises a VH CDR1, VH CDR2, and/or a VH CDR3 selected from Table 1.

Table 1.

In some embodiments, the anti-PD-1 antibody comprises:

a) the VL CDR1 sequence consists of RASX ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈; wherein X ₁₇ is E or Q; X ₁₈ is S or D; X ₁₉ is V or I; X ₂₀ is D or S; X ₂₁ is S, N or D; X ₂₂ is Y or F; X ₂₃ is G or L; X ₂₄ is I or N; X ₂₅ is present or absence and if present, is S; X ₂₆ is present or absence and if present, is F; X ₂₇ is present or absence and if present, is M; X ₂₈ is present or absence and if present, is N;

b) the VL CDR2 sequence consists of X ₂₉X ₃₀SX ₃₁X ₃₂X ₃₃S; wherein X ₂₉ is A or Y; X ₃₀ is A or T; X ₃₁ is N or R; X ₃₂ is Q or L; X ₃₃ is G or H; and

c) the VL CDR3 sequence consists of QQX ₃₄X ₃₅X ₃₆X ₃₇PWT; wherein X ₃₄ is S or G; X ₃₅ is K or D; X ₃₆ is E or M; X ₃₇ is V or I.

In some embodiments, the VL CDR1 is RASESVDSYGISFMN, RASQDISNFLN, RASESVDDYGISFMN, RASQEISGYLS, HASQGISSNIG, or KSSQSLLNSNSQKNYLA; the VL CDR2 sequence consists of X ₂₉X ₃₀SX ₃₁X ₃₂X ₃₃S, wherein X ₂₉ is A or Y; X ₃₀ is A or T; X ₃₁ is N or R; X ₃₂ is Q or L; X ₃₃ is G or H; and the VL CDR3 sequence consists of QQX ₃₄X ₃₅X ₃₆X ₃₇PWT, wherein X ₃₄ is S or G; X ₃₅ is K or D; X ₃₆ is E or M; X ₃₇ is V or I.

In some embodiments, the VL CDR1 sequence consists of RASX ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈, wherein X ₁₇ is E or Q; X ₁₈ is S or D; X ₁₉ is V or I; X ₂₀ is D or S; X ₂₁ is S, N or D; X ₂₂ is Y or F; X ₂₃ is G or L; X ₂₄ is I or N; X ₂₅ is present or absence and if present, is S; X ₂₆ is present or absence and if present, is F; X ₂₇ is present or absence and if present, is M; X ₂₈ is present or absence and if present, is N; the VL CDR2 sequence is AASNQGS, YTSRLHS, AASNQGS, AASTLDS, HGTNLED, or FTSTRES; and the VL CDR3 sequence consists of QQX ₃₄X ₃₅X ₃₆X ₃₇PWT, wherein X ₃₄ is S or G; X ₃₅ is K or D; X ₃₆ is E or M; X ₃₇ is V or I.

In some embodiments, the VL CDR1 sequence consists of RASX ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈, wherein X ₁₇ is E or Q; X ₁₈ is S or D; X ₁₉ is V or I; X ₂₀ is D or S; X ₂₁ is S, N or D; X ₂₂ is Y or F; X ₂₃ is G or L; X ₂₄ is I or N; X ₂₅ is present or absence and if present, is S; X ₂₆ is present or absence and if present, is F; X ₂₇ is present or absence and if present, is M; X ₂₈ is present or absence and if present, is N; the VL CDR2 sequence consists of X ₂₉X ₃₀SX ₃₁X ₃₂X ₃₃S, wherein X ₂₉ is A or Y; X ₃₀ is A or T; X ₃₁ is N or R; X ₃₂ is Q or L; X ₃₃ is G or H; and the VL CDR3 sequence is QQSKEVPWT, QQGDMIPWT, QQSKEVPWT, LQYASYPLT, VQYAQFPPT, or QQHYNTPYT.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VL CDR1 is RASESVDSYGISFMN, RASQDISNFLN, RASESVDDYGISFMN, RASQEISGYLS, HASQGISSNIG, or KSSQSLLNSNSQKNYLA; the VL CDR2 sequence is AASNQGS, YTSRLHS, AASNQGS, AASTLDS, HGTNLED, or FTSTRES; and the VL CDR3 sequence is QQSKEVPWT, QQGDMIPWT, QQSKEVPWT, LQYASYPLT, VQYAQFPPT, or QQHYNTPYT.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VL CDR1 is RASESVDSYGISFMN, RASQDISNFLN, or RASESVDDYGISFMN; the VL CDR2 sequence is AASNQGS, YTSRLHS, or AASNQGS; and the VL CDR3 sequence is QQSKEVPWT, QQGDMIPWT, or QQSKEVPWT.

In some embodiments, an anti-PD-1 antibody comprises a VL CDR1, VL CDR2, and/or a VL CDR3 selected from Table 2.

Table 2.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VH CDR1 is SEQ ID NO: 1, the VH CDR2 is SEQ ID NO: 2, and the VH CDR3 is SEQ ID NO: 3; and the VL CDR1 is 19, the VL CDR2 is SEQ ID NO: 20, and the VL CDR3 is SEQ ID NO: 21.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VH CDR1 is SEQ ID NO: 4, the VH CDR2 is SEQ ID NO: 5, and the VH CDR3 is SEQ ID NO: 6; and the VL CDR1 is 22, the VL CDR2 is SEQ ID NO: 23, and the VL CDR3 is SEQ ID NO: 24.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VH CDR1 is SEQ ID NO: 7, the VH CDR2 is SEQ ID NO: 8, and the VH CDR3 is SEQ ID NO: 9; and the VL CDR1 is 25, the VL CDR2 is SEQ ID NO: 26, and the VL CDR3 is SEQ ID NO: 27.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VH CDR1 is SEQ ID NO: 10, the VH CDR2 is SEQ ID NO: 11, and the VH CDR3 is SEQ ID NO: 12; and the VL CDR1 is 28, the VL CDR2 is SEQ ID NO: 29, and the VL CDR3 is SEQ ID NO: 30.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VH CDR1 is SEQ ID NO: 13, the VH CDR2 is SEQ ID NO: 14, and the VH CDR3 is SEQ ID NO: 15; and the VL CDR1 is 31, the VL CDR2 is SEQ ID NO: 32, and the VL CDR3 is SEQ ID NO: 33.

In some embodiments, an anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , in which the VH CDR1 is SEQ ID NO: 16, the VH CDR2 is SEQ ID NO: 17, and the VH CDR3 is SEQ ID NO: 18; and the VL CDR1 is 34, the VL CDR2 is SEQ ID NO: 35, and the VL CDR3 is SEQ ID NO: 36.

In some embodiments, the antibody comprises a humanized antibody, murine antibody, chimeric antibody, monoclonal antibody, monovalent antibody, bivalent antibody, or multi-valent antibody.

In some embodiments, the antibody comprises a full-length antibody. As used herein, the term “full-length” refers to an antibody comprising a Fab region and a Fc region, in which the Fc region comprises the CH2 and CH3 domains. In some instances, the antibody comprises a full-length humanized antibody. In other instances, the antibody comprises a full-length chimeric antibody. In additional instances, the antibody comprises a monovalent full-length antibody, a bivalent full-length antibody, or a multi-valent full-length antibody.

In some embodiments, the antibody comprises an antibody fragment. In some instances, the antibody fragment comprises a monovalent Fab’ , a divalent Fab ₂, F (ab) ' ₃ fragment, single-chain variable fragment (scFv) , bis-scFv, (scFv) ₂, diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein ( "dsFv" ) , single-domain antibody (sdantibody) , Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.

In some embodiments, an anti-PD-1 antibody comprises a VH and VL sequence as illustrated in Table 3.

Table 3

In some embodiments, an anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 38; or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 40. In some instances, the anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38; and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 40. In some instances, the anti-PD-1 antibody comprises SEQ ID NO: 38 and SEQ ID NO: 40.

In some embodiments, an anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 42; or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 44. In some instances, the anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 42; and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 44. In some instances, the anti-PD-1 antibody comprises SEQ ID NO: 42 and SEQ ID NO: 44.

In some embodiments, an anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 46; or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 48. In some instances, the anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 46; and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 48. In some instances, the anti-PD-1 antibody comprises SEQ ID NO: 46 and SEQ ID NO: 48.

In some embodiments, an anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 50; or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 52. In some instances, the anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 50; and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 52. In some instances, the anti-PD-1 antibody comprises SEQ ID NO: 50 and SEQ ID NO: 52.

In some embodiments, an anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 54; or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 56. In some instances, the anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 54; and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 56. In some instances, the anti-PD-1 antibody comprises SEQ ID NO: 54 and SEQ ID NO: 56.

In some embodiments, an anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 58; or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 60. In some instances, the anti-PD-1 antibody comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 58; and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 60. In some instances, the anti-PD-1 antibody comprises SEQ ID NO: 58 and SEQ ID NO: 60.

In some embodiments, an anti-PD-1 antibody comprises a VH and VL sequence as illustrated in Table 4.

Table 4

In some embodiments, an anti-PD-1 antibody comprises a VH and VL sequence as illustrated in Table 5.

Table 5

In some embodiments, the binding affinity of an anti-PD-1 antibody described above is higher than the binding affinity of nivolumab.

In some embodiments, the binding affinity of an anti-PD-1 antibody described above is higher than the binding affinity of pembrolizumab.

In some embodiments, the binding affinity of the anti-PD-1 antibody is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity is about 2-fold higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity is about 3-fold higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity is about 4-fold higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity is about 5-fold higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity is about 10-fold higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity is about 20-fold higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity is about 50-fold higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity is about 100-fold higher than the binding affinity of nivolumab or pembrolizumab.

In some embodiments, the binding affinity of the anti-PD-1 antibody is about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity of the anti-PD-1 antibody is about 10%or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity of the anti-PD-1 antibody is about 15%or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity of the anti-PD-1 antibody is about 20%or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity of the anti-PD-1 antibody is about 30%or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity of the anti-PD-1 antibody is about 40%or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity of the anti-PD-1 antibody is about 50%or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity of the anti-PD-1 antibody is about 100%or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity of the anti-PD-1 antibody is about 200%or higher than the binding affinity of nivolumab or pembrolizumab. In some instances, the binding affinity of the anti-PD-1 antibody is about 500%or higher than the binding affinity of nivolumab or pembrolizumab.

In some embodiments, an anti-PD-1 antibody described above has a >40%inhibition, >50%inhibition, or >60%inhibition at an antibody concentration range of about 5-7 nM. In some instances, the anti-PD-1 antibody has a >40%inhibition at an antibody concentration range of about 5-7 nM (e.g., about 5, 5.5, 6, 6.5, or 7 nM concentration) . In some instances, the anti-PD-1 antibody has a >50%inhibition at an antibody concentration range of about 5-7 nM (e.g., about 5, 5.5, 6, 6.5, or 7 nM concentration) . In some instances, the anti-PD-1 antibody has a >60%inhibition at an antibody concentration range of about 5-7 nM (e.g., about 5, 5.5, 6, 6.5, or 7 nM concentration) . In some cases, the ani-PD-1 antibody has a >46%inhibition at an antibody concentration of 6.67nM.

In some embodiments, an anti-PD-1 antibody described above has a KD of less than 8e-9 M, less than 6e-9 M, less than 4e-9 M, less than 2.5e-9M, less than 2e-9 M, less than 1.5e-9 M, or less than 1.2e-9 M. In some instances, the anti-PD-1 antibody has a KD of less than 8e-9 M. In some instances, the anti-PD-1 antibody has a KD of less than 6e-9 M. In some instances, the anti-PD-1 antibody has a KD of less than 4e-9 M. In some instances, the anti-PD-1 antibody has a KD of less than 2.5e-9M. In some instances, the anti-PD-1 antibody has a KD of less than 2e-9 M. In some instances, the anti-PD-1 antibody has a KD of less than 1.5e-9 M. In some instances, the anti-PD-1 antibody has a KD of less than 1.2e-9 M. In some cases, the anti-PD-1 antibody has a KD of about 2.43e-9 M. In some cases, the anti-PD-1 antibody has a KD of about 1.16e-9 M.

In some embodiments, an anti-PD-1 antibody described above has an IC ₅₀ similar to nivolumab and/or pembrolizumab.

In some embodiments, an anti-PD-1 antibody described above induces cytokine production. In some cases, the cytokine is IL-2 or INF-γ. In some cases, the cytokine is IL-2. In other cases, the cytokine is INF-γ.

In some embodiments, an anti-PD-1 antibody described above comprises an IgG1 framework. In some instances, the framework is a humanized IgG1.

In some embodiments, an anti-PD-1 antibody described above comprises an IgG4 framework. In some instances, the framework is a humanized IgG4 framework. In some cases, the IgG4 framework comprises a S228P mutation.

Methods of Use

Disclosed herein, in certain embodiments, is a method of treating a cancer by administering an anti-PD-1 antibody described above to a subject in need thereof. In some instances, the cancer is a solid tumor. In other instances, the cancer is a hematologic malignancy. In some cases, the cancer is bladder cancer, brain cancer, breast cancer, bladder cancer, bone cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, or uterine cancer. In some cases, the cancer is a lymphoma. In some cases, the cancer is Hodgkin’s lymphoma.

In some embodiments, the method further comprises administering an additional therapeutic agent. In some instances, the additional therapeutic agent comprises a first line cancer treatment. In some instances, the additional therapeutic agent comprises a chemotherapeutic agent or radiation.

In some embodiments, the subject has had surgery.

In some instances, the anti-PD-1 antibody and the additional therapeutic agent are administered sequentially. In some cases, the anti-PD-1 antibody is administered to the subject prior to administering the additional therapeutic agent. In other cases, the additional therapeutic agent is administered to the subject prior to administering the anti-PD-1 antibody.

In other instances, the anti-PD-1 antibody and the additional therapeutic agent are administered simultaneously.

In additional instances, the anti-PD-1 antibody and the additional therapeutic agent are formulated as separate dosage forms.

In some embodiments, also disclosed herein is a method of augmenting cytokine production in a subject having a cancer. In some cases, the cytokine is produced by a tumor-infiltrating lymphocyte (TIL) . In some instances, the cytokine comprises interleukin (IL) -2, IL-4, tumor necrosis factor-alpha (TNF-α) , transforming growth factor-beta 1 (TGF-β1) , IL-10, interferon-gamma (IFN-γ) , or granulocytemacrophage colony-stimulating factor (GM-CSF) . In some cases, the cytokine is IL-2. In some cases, the cytokine is IL-4. In some cases, the cytokine is TNF-α. In some cases, the cytokine is TGF-β1. In some cases, the cytokine is IL-10. In some cases, the cytokine is IFN-γ. In some cases, the cytokine is GM-CSF.

In some embodiments, additional disclosed herein is a method of inducing phagocytosis of cells expressing PD-L1 and/or PD-L2, which comprises blocking binding of the cell to a T cell expressing PD-1 with an anti-PD-1 antibody described above, thereby inducing phagocytosis of the cell. In some instances, the cell is a tumor cell. In some cases, the cell is from a solid tumor, e.g., from bladder cancer, brain cancer, breast cancer, bladder cancer, bone cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, or uterine cancer. In some cases, the cell is from a hematologic malignancy, e.g., from a lymphoma such as a Hodgkin’s lymphoma.

Antibody Production

In some embodiments, antibodies are raised by standard protocol by injecting a production animal with an antigenic composition. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. When utilizing an entire protein, or a larger section of the protein, antibodies may be raised by immunizing the production animal with the protein and a suitable adjuvant (e.g., Freund's , Freund's complete, oil-in-water emulsions, etc. ) . When a smaller peptide is utilized, it is advantageous to conjugate the peptide with a larger molecule to make an immunostimulatory conjugate. Commonly utilized conjugate proteins that are commercially available for such use include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) . In order to raise antibodies to particular epitopes, peptides derived from the full sequence may be utilized. Alternatively, in order to generate antibodies to relatively short peptide portions of the protein target, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as ovalbumin, BSA or KLH.

Polyclonal or monoclonal antigen binding units or antibodies can be produced from animals which have been genetically altered to produce human immunoglobulins. A transgenic animal can be produced by initially producing a “knock-out” animal which does not produce the animal's natural antibodies, and stably transforming the animal with a human antibody locus (e.g., by the use of a human artificial chromosome) . In such cases, only human antibodies are then made by the animal. Techniques for generating such animals, and deriving antibodies therefrom, are described in U.S. Pat. Nos. 6,162,963 and 6,150,584, incorporated fully herein by reference. Such antibodies can be referred to as human xenogenic antibodies.

Alternatively, antigen binding units can be produced from phage libraries containing human variable regions. See U.S. Pat. No. 6,174,708, incorporated fully herein by reference.

In some aspects of any of the embodiments disclosed herein, an antigen binding unit is produced by a hybridoma.

For monoclonal antigen binding units or monoclonal antibodies, hybridomas may be formed by isolating the stimulated immune cells, such as those from the spleen of the inoculated animal. These cells can then be fused to immortalized cells, such as myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The immortal cell line utilized can be selected to be deficient in enzymes necessary for the utilization of certain nutrients. Many such cell lines (such as myelomas) are known to those skilled in the art, and include, for example: thymidine kinase (TK) or hypoxanthine-guanine phosphoriboxyl transferase (HGPRT) . These deficiencies allow selection for fused cells according to their ability to grow on, for example, hypoxanthine aminopterinthymidine medium (HAT) .

In addition, the antigen binding unit may be produced by genetic engineering. Humanized, chimeric, or xenogeneic human antigen binding units, which produce less of an immune response when administered to humans, are of use in the present invention.

Antigen binding units disclosed herein can have a reduced propensity to induce an undesired immune response in humans, for example, anaphylactic shock, and can also exhibit a reduced propensity for priming an immune response which would prevent repeated dosage with the antibody therapeutic or imaging agent (e.g., the human-anti-murine-antibody “HAMA” response) . Such antigen binding units include, but are not limited to, humanized, chimeric, or xenogenic human antigen binding units.

Chimeric antigen binding units or chimeric antibodies can be made, for example, by recombinant means by combining the murine variable light and heavy chain regions (VK and VH) , obtained from a murine (or other animal-derived) hybridoma clone, with the human constant light and heavy chain regions, in order to produce an antibody with predominantly human domains. The production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Pat. No. 5,624,659, incorporated fully herein by reference) .

The term “humanized” as applies to a non-human (e.g. rodent or primate) antibodies are hybrid immunoglobulins, immunoglobulin chains or fragments thereof which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or primate having the desired specificity, affinity and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance and minimize immunogenicity when introduced into a human body. In some examples, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin.

Humanized antibodies can be engineered to contain human-like immunoglobulin domains, and incorporate only the complementarity-determining regions of the animal-derived antibody. This can be accomplished by carefully examining the sequence of the hyper-variable loops of the variable regions of a monoclonal antigen binding unit or monoclonal antibody, and fitting them to the structure of a human antigen binding unit or human antibody chains. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully herein by reference.

Methods for humanizing non-human antibodies are well known in the art. “Humanized” antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins. In some versions, the heavy (H) chain and light (L) chain constant (C) regions are replaced with human sequence. This can be a fusion polypeptide comprising a variable (V) region and a heterologous immunoglobulin C region. In some versions, the complementarity determining regions (CDRs) comprise non-human antibody sequences, while the V framework regions have also been converted to human sequences. See, for example, EP 0329400. In some versions, V regions are humanized by designing consensus sequences of human and mouse V regions, and converting residues outside the CDRs that are different between the consensus sequences.

In principle, a framework sequence from a humanized antibody can serve as the template for CDR grafting; however, it has been demonstrated that straight CDR replacement into such a framework can lead to significant loss of binding affinity to the antigen. Glaser et al. (1992) J. Immunol. 149: 2606; Tempest et al. (1992) Biotechnology 9: 266; and Shalaby et al. (1992) J. Exp. Med. 17: 217. The more homologous a human antibody (HuAb) is to the original murine antibody (muAb) , the less likely that the human framework will introduce distortions into the murine CDRs that could reduce affinity. Based on a sequence homology search against an antibody sequence database, the HuAb IC4 provides good framework homology to muM4TS. 22, although other highly homologous HuAbs would be suitable as well, especially kappa L chains from human subgroup I or H chains from human subgroup III. Kabat et al. (1987) . Various computer programs such as ENCAD (Levitt et al. (1983) J. Mol. Biol. 168: 595) are available to predict the ideal sequence for the V region. The invention thus encompasses HuAbs with different variable (V) regions. It is within the skill of one in the art to determine suitable V region sequences and to optimize these sequences. Methods for obtaining antibodies with reduced immunogenicity are also described in U.S. Pat. No. 5,270,202 and EP 699, 755.

Humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen (s) , is achieved.

A process for humanization of subject antigen binding units can be as follows. The best-fit germline acceptor heavy and light chain variable regions are selected based on homology, canonical structure and physical properties of the human antibody germlines for grafting. Computer modeling of mVH/VL versus grafted hVH/VL is performed and prototype humanized antibody sequence is generated. If modeling indicated a need for framework back-mutations, second variant with indicated FW changes is generated. DNA fragments encoding the selected germline frameworks and murine CDRs are synthesized. The synthesized DNA fragments are subcloned into IgG expression vectors and sequences are confirmed by DNA sequencing. The humanized antibodies are expressed in cells, such as 293F and the proteins are tested, for example in MDM phagocytosis assays and antigen binding assays. The humanized antigen binding units are compared with parental antigen binding units in antigen binding affinity, for example, by FACS on cells expressing the target antigen. If the affinity is greater than 2-fold lower than parental antigen binding unit, a second round of humanized variants can be generated and tested as described above.

As noted above, an antigen binding units can be either “monovalent” or “multivalent. ” Whereas the former has one binding site per antigen-binding unit, the latter contains multiple binding sites capable of binding to more than one antigen of the same or different kind. Depending on the number of binding sites, antigen binding units may be bivalent (having two antigen-binding sites) , trivalent (having three antigen-binding sites) , tetravalent (having four antigen-binding sites) , and so on.

Multivalent antigen binding units can be further classified on the basis of their binding specificities. A “monospecific” antigen binding unit is a molecule capable of binding to one or more antigens of the same kind. A “multispecific” antigen binding unit is a molecule having binding specificities for at least two different antigens. While such molecules normally will only bind two distinct antigens (i.e. bispecific antigen binding units) , antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein. This disclosure further provides multispecific antigen binding units. Multispecific antigen binding units are multivalent molecules capable of binding to at least two distinct antigens. Preferred multispecific antigen binding units are bispecific and trispecific molecules exhibiting binding specificities to two and three distinct antigens, respectively.

Polynucleotides and Vectors

In some embodiments, the present disclosure provides isolated nucleic acids encoding any of the antigen binding units disclosed herein. In another embodiment, the present disclosure provides vectors comprising a nucleic acid sequence encoding any antigen binding unit disclosed herein. In some embodiments, this invention provides isolated nucleic acids that encode a light-chain CDR and a heavy-chain CDR of an antigen binding unit disclosed herein.

The subject antigen binding units can be prepared by recombinant DNA technology, synthetic chemistry techniques, or a combination thereof. For instance, sequences encoding the desired components of the antigen binding units, including light chain CDRs and heavy chain CDRs are typically assembled cloned into an expression vector using standard molecular techniques know in the art. These sequences may be assembled from other vectors encoding the desired protein sequence, from PCR-generated fragments using respective template nucleic acids, or by assembly of synthetic oligonucleotides encoding the desired sequences. Expression systems can be created by transfecting a suitable cell with an expressing vector which comprises the antigen binding unit of interest.

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

Polynucleotides encoding antigen binding units can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant regions in place of the homologous non-human sequences. In that manner, chimeric antibodies are prepared that retain the binding specificity of the original antigen binding unit.

It is also understood that the polynucleotides embodied in the invention include those coding for functional equivalents and fragments thereof of the exemplified polypeptides. Functionally equivalent polypeptides include those that enhance, decrease or not significantly affect properties of the polypeptides encoded thereby. Functional equivalents may be polypeptides having conservative amino acid substitutions, analogs including fusions, and mutants.

Due to the degeneracy of the genetic code, there can be considerable variation in nucleotides of an antigen binding unit coding sequence, as well as sequences suitable for construction of the polynucleotide and vectors of the present invention. Sequence variants may have modified DNA or amino acid sequences, one or more substitutions, deletions, or additions, the net effect of which is to retain the desired antigen-binding activity. For instance, various substitutions can be made in the coding region that either do not alter the amino acids encoded or result in conservative changes. These substitutions are encompassed by the present invention. Conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. While conservative substitutions do effectively change one or more amino acid residues contained in the polypeptide to be produced, the substitutions are not expected to interfere with the antigen-binding activity of the resulting antigen binding units to be produced. Nucleotide substitutions that do not alter the amino acid residues encoded are useful for optimizing gene expression in different systems. Suitable substitutions are known to those of skill in the art and are made, for instance, to reflect preferred codon usage in the expression systems.

Where desired, the recombinant polynucleotides may comprise heterologous sequences that facilitate detection of the expression and purification of the gene product. Examples of such sequences are known in the art and include those encoding reporter proteins such as β-galactosidase, β-lactamase, chloramphenicol acetyltransferase (CAT) , luciferase, green fluorescent protein (GFP) and their derivatives. Other heterologous sequences that facilitate purification may code for epitopes such as Myc, HA (derived from influenza virus hemagglutinin) , His-6, FLAG, or the Fc portion of immunoglobulin, glutathione S-transferase (GST) , and maltose-binding protein (MBP) .

Polynucleotides disclosed herein can be conjugated to a variety of chemically functional moieties described above. Commonly employed moieties include labels capable of producing a detectable signal, signal peptides, agents that enhance immunologic reactivity, agents that facilitate coupling to a solid support, vaccine carriers, bioresponse modifiers, paramagnetic labels and drugs. The moieties can be covalently linked polynucleotide recombinantly or by other means known in the art.

Polynucleotides of the invention can comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, and polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.

Polynucleotides embodied in this invention can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequence data provided herein to obtain a desired polynucleotide by employing a DNA synthesizer or ordering from a commercial service.

Polynucleotides comprising a desired sequence can be inserted into a suitable vector which in turn can be introduced into a suitable host cell for replication and amplification. Accordingly, the invention encompasses a variety of vectors comprising one or more of the polynucleotides of the present invention. Also provided are selectable libraries of expression vectors comprising at least one vector encoding an antigen binding units disclosed herein.

Vectors of the present invention generally comprise transcriptional or translational control sequences required for expressing the antigen binding units. Suitable transcription or translational control sequences include but are not limited to replication origin, promoter, enhancer, repressor binding regions, transcription initiation sites, ribosome binding sites, translation initiation sites, and termination sites for transcription and translation.

The choice of promoters will largely depend on the host cells in which the vector is introduced. It is also possible, to utilize promoters normally associated with a desired light or heavy chain gene, provided that such control sequences are compatible with the host cell system. Cell-specific or tissue-specific promoters may also be used. A vast diversity of tissue specific promoters have been described and employed by artisans in the field. Exemplary promoters operative in selective animal cells include hepatocyte-specific promoters and cardiac muscle specific promoters. Depending on the choice of the recipient cell types, those skilled in the art will know of other suitable cell-specific or tissue-specific promoters applicable for the construction of the expression vectors of the present invention.

Using known molecular cloning or gene engineering techniques, appropriate transcriptional control sequences, enhancers, terminators, or any other genetic element known in the art can integrated in operative relationship, optionally additionally with intact selectable fusion genes to be expressed in accordance with the present invention. In addition to the above-described elements, the vectors may contain a selectable marker (for example, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector) , although such a marker gene can be carried on another polynucleotide sequence co-introduced into the host cell.

The polynucleotides and vectors of this invention have several specific uses. They are useful, for example, in expression systems for the production of antigen binding units. Such polynucleotides are useful as primers to effect amplification of desired polynucleotides. Furthermore, polynucleotides of this invention are also useful in pharmaceutical compositions including vaccines, diagnostics, and drugs.

The host cells of this invention can be used, inter alia, as repositories of the subject polynucleotides, vectors, or as vehicles for producing and screening desired antigen binding units based on their antigen binding specificities.

Accordingly, the invention provides a method of identifying an antigen binding unit that is immunoreactive with a desired antigen. Such a method can involve the following steps: (a) preparing a genetically diverse library of antigen binding units, wherein the library comprises at least one subject antigen binding unit; (b) contacting the library of antigen binding units with the desired antigen; (c) detecting a specific binding between antigen binding units and the antigen, thereby identifying the antigen binding unit that is immunoreactive with the desired antigen.

The ability of an antigen binding unit to specifically bind to a desired antigen can be tested by a variety of procedures well established in the art. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Gherardi et al. (1990) J. Immunol. Meth. 126: 61-68. Typically, antigen binding units exhibiting desired binding specificities can be detected directly by immunoassays, for example, by reacting labeled antigen binding units with the antigens that are immobilized on a solid support or substrate. In general, the substrate to which the antigen is adhered is fabricated with material exhibiting a low level of non-specific binding during immunoassay. An example solid support is made from one or more of the following types of materials: plastic polymers, glass, cellulose, nitrocellulose, semi-conducting material, and metal. In some examples, the substrate is petri dish, chromatography beads, magnetic beads, and the like.

For such solid-phase assays, the unreacted antigen binding units are removed by washing. In a liquid-phase assay, however, the unreacted antigen binding units are removed by some other separation technique, such as filtration or chromatography. After binding the antigen to the labeled antigen binding units, the amount of bound label is determined. A variation of this technique is a competitive assay, in which the antigen is bound to saturation with an original binding molecule. When a population of the subject antigen binding unit is introduced to the complex, only those that exhibit higher binding affinity will be able to compete, and thus remain bound to the antigen.

Alternatively, specific binding to a given antigen can be assessed by cell sorting, which involves presenting the desired antigen on the cells to be sorted, then labeling the target cells with antigen binding units that are coupled to detectable agents, followed by separating the labeled cells from the unlabeled ones in a cell sorter. A sophisticated cell separation method is fluorescence-activated cell sorting (FACS) . Cells traveling in single file in a fine stream are passed through a laser beam, and the fluorescence of each cell bound by the fluorescently labeled antigen binding unit is then measured.

Subsequent analysis of the eluted antigen binding units may involve protein sequencing for delineating the amino acid sequences of the light chains and heavy chains. Based on the deduced amino acid sequences, the cDNA encoding the antibody polypeptides can then be obtained by recombinant cloning methods including PCR, library screening, homology searches in existing nucleic acid databases, or any combination thereof. Commonly employed databases include but are not limited to GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS, and HTGS.

When a library of antigen binding unit is displayed on phage or bacterial particles, selection is preferably performed using affinity chromatography. The method typically proceeds with binding a library of phage antigen binding units to an antigen coated plates, column matrices, cells or to biotinylated antigen in solution followed by capture. The phages or bacteria bound to the solid phase are washed and then eluted by soluble hapten, acid or alkali. Alternatively, increasing concentrations of antigen can be used to dissociate the antigen binding units from the affinity matrix. For certain antigen binding units with extremely high affinity or avidity to the antigen, efficient elution may require high pH or mild reducing solution as described in WO 92/01047.

The efficiency of selection is likely to depend on a combination of several factors, including the kinetics of dissociation during washing, and whether multiple antigen binding units on a single phage or bacterium can simultaneously bind to antigens on a solid support. For example, antibodies with fast dissociation kinetics (and weak binding affinities) can be retained by use of short washes, multivalent display and a high coating density of antigen at the solid support. Conversely, the selection of antigen binding units with slow dissociation kinetics (and good binding affinities) can be favored by use of long washes, monovalent phages, and a low coating density of antigen.

Where desired, the library of antigen binding units can be pre-selected against an unrelated antigen to counter-select the undesired antigen binding units. The library may also be pre-selected against a related antigen in order to isolate, for example, anti-idiotypic antigen binding units.

Host Cells

In some embodiments, the present disclosure provides host cells expressing any one of the antigen binding units disclosed herein. A subject host cell typically comprises a nucleic acid encoding any one of the antigen binding units disclosed herein.

The invention provides host cells transfected with the polynucleotides, vectors, or a library of the vectors described above. The vectors can be introduced into a suitable prokaryotic or eukaryotic cell by any of a number of appropriate means, including electroporation, microprojectile bombardment; lipofection, infection (where the vector is coupled to an infectious agent) , transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances. The choice of the means for introducing vectors will often depend on features of the host cell.

For most animal cells, any of the above-mentioned methods is suitable for vector delivery. Preferred animal cells are vertebrate cells, preferably mammalian cells, capable of expressing exogenously introduced gene products in large quantity, e.g. at the milligram level. Non-limiting examples of preferred cells are NIH3T3 cells, COS, HeLa, and CHO cells.

Once introduced into a suitable host cell, expression of the antigen binding units can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of light chain CDRs or heavy chain CDRs, or the antigen binding unit can be detected and/or quantified by conventional hybridization assays (e.g. Northern blot analysis) , amplification procedures (e.g. RT-PCR) , SAGE (U.S. Pat. No. 5,695,937) , and array-based technologies (see e.g. U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934) , using probes complementary to any region of antigen binding unit polynucleotide.

Expression of the vector can also be determined by examining the antigen binding unit expressed. A variety of techniques are available in the art for protein analysis. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays) , “sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels) , western blot analysis, immunoprecipitation assays, immunoflourescent assays, and SDS-PAGE.

Preparation of Antigen-Binding Units

In some embodiments, the present disclosure provides methods of producing any antigen binding unit disclosed herein, wherein the method comprises culturing host cells expressing the antigen binding unit under conditions suitable for expressing the antigen binding unit, and isolating the antigen binding unit expressed by the host cell.

The expressed antigen binding units can be isolated using a variety of protein purification techniques known in the art. Generally, the antigen binding unit is isolated from culture media as secreted polypeptides, although they can be recovered from host cell lysates or bacterial periplasm, when directly produced without signal peptides. If the antigen binding units are membrane-bound, they can be solubilized by suitable detergent solutions commonly employed by artisans in the field. The recovered antigen binding units may be further purified by salt precipitation (e.g., with ammonium sulfate) , ion exchange chromatography (e.g. on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength) , gel filtration chromatography (including gel filtration HPLC) , and chromatography on tag-affinity column, or on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.

In addition, derivatized immunoglobulins with added chemical linkers, detectable moieties such as fluorescent dyes, enzymes, substrates, chemiluminescent moieties, specific binding moieties such as streptavidin, avidin, or biotin, or drug conjugates can be utilized in the methods and compositions of the present invention.

Additionally disclosed herein are antigen binding unites conjugated to a chemically functional moiety. Typically, the moiety is a label capable of producing a detectable signal. These conjugated antigen binding units are useful, for example, in detection systems such as quantitation of tumor burden, and imaging of metastatic foci and tumor imaging. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds substrate cofactors and inhibitors. See, for examples of patents teaching the use of such labels, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. The moieties can be covalently linked to antigen binding units, recombinantly linked, or conjugated to antigen binding units through a secondary reagent, such as a second antibody, protein A, or a biotin-avidin complex.

Other functional moieties include signal peptides, agents that enhance immunologic reactivity, agents that facilitate coupling to a solid support, vaccine carriers, bioresponse modifiers, paramagnetic labels and drugs. Signal peptides is a short amino acid sequence that directs a newly synthesized protein through a cellular membrane, usually the endoplasmic reticulum in eukaryotic cells, and either the inner membrane or both inner and outer membranes of bacteria. Signal peptides can be at the N-terminal portion of a polypeptide or the C-terminal portion of a polypeptide, and can be removed enzymatically between biosynthesis and secretion of the polypeptide from the cell. Such a peptide can be incorporated into an antigen binding units to allow secretion of the synthesized molecules.

Agents that enhance immunologic reactivity include, but are not limited to, bacterial superantigens. Agents that facilitate coupling to a solid support include, but are not limited to, biotin or avidin. Immunogen carriers include, but are not limited to, any physiologically acceptable buffers. Bioresponse modifiers include cytokines, particularly tumor necrosis factor (TNF) , interleukin-2, interleukin-4, granulocyte macrophage colony stimulating factor and γ-interferons.

Suitable drug moieties include antineoplastic agents. Non-limiting examples include radioisotopes, vinca alkaloids such as the vinblastine, vincristine and vindesine sulfates, adriamycin, bleomycin sulfate, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, lomustine, mechlororethamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbaze hydrochloride, streptozotocin, taxol, thioguanine, and uracil mustard.

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

Chemically functional moieties can be made recombinantly for instance by creating a fusion gene encoding the antigen binding unit and the functional moiety. Alternatively, the antigen binding unit can be chemically bonded to the moiety by any of a variety of well-established chemical procedures. For example, when the moiety is a protein, the linkage can be by way of heterobifunctional cross linkers, e.g., SPDP, carbodiimide glutaraldehyde, or the like. The moieties can be covalently linked, or conjugated, through a secondary reagent, such as a second antibody, protein A, or a biotin-avidin complex. Paramagnetic moieties and the conjugation thereof to antibodies are well-known in the art. See, e.g., Miltenyi et al. (1990) Cytometry 11: 231-238.

Pharmaceutical Compositions

In some embodiments, an anti-PD-1 antibody is further formulated as a pharmaceutical composition. In some instances, the pharmaceutical composition is formulated for administration to a subject by multiple administration routes, including but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular, intraarterial, intradermal, intraperitoneal, intravitreal, intracerebral, or intracerebroventricular) , oral, intranasal, buccal, rectal, or transdermal administration routes. In some instances, the pharmaceutical composition describe herein is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraarterial, intradermal, intraperitoneal, intravitreal, intracerebral, or intracerebroventricular) administration. In other instances, the pharmaceutical composition describe herein is formulated for oral administration. In still other instances, the pharmaceutical composition describe herein is formulated for intranasal administration.

In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations) , and mixed immediate and controlled release formulations.

In some instances, the pharmaceutical formulation includes multiparticulate formulations. In some instances, the pharmaceutical formulation includes nanoparticle formulations. Exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates) , nanofibers, nanohorns, nano-onions, nanorods, nanoropes and quantum dots. In some instances, a nanoparticle is a metal nanoparticle, e.g., a nanoparticle of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys or oxides thereof.

In some instances, a nanoparticle includes a core or a core and a shell, as in a core-shell nanoparticle. In some cases, a nanoparticle has at least one dimension of less than about 500nm, 400nm, 300nm, 200nm, or 100nm.

In some embodiments, the pharmaceutical compositions include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP) , cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995) ; Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins1999) .

In some instances, the pharmaceutical compositions further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

In some instances, the pharmaceutical compositions include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In some instances, the pharmaceutical compositions further include diluent which are used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds can include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as

dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar, such as

(Amstar) ; mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner’s sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

In some cases, the pharmaceutical compositions include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance. The term “disintegrate” include both the dissolution and dispersion of the dosage form when contacted with gastrointestinal fluid. Examples of disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or

or sodium starch glycolate such as

or

a cellulose such as a wood product, methylcrystalline cellulose, e.g.,

PH101,

PH102,

PH105,

P100,

Ming

and

methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose

cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as

HV (magnesium aluminum silicate) , a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

In some instances, the pharmaceutical compositions include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in the pharmaceutical compositions described herein for preventing, reducing or inhibiting adhesion or friction of materials. Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil

higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes,

boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as Carbowax ^TM, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid ^TM,

a starch such as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also function as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.

Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like.

Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630) , polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g.,

(BASF) , and the like. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactants are included to enhance physical stability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.

Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.

Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic applications. In some embodiments, the pharmaceutical composition is administered once per day, twice per day, three times per day or more. The pharmaceutical composition is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more. The pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the composition is given continuously; alternatively, the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday” ) . In some instances, the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday is from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder, or condition is retained.

In some embodiments, the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages is altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

In some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) . The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more of the compositions and methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container (s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

For example, the container (s) include an anti-PD-1 antibody as disclosed herein, host cells for producing one or more antibodies described herein, and/or vectors comprising nucleic acid molecules that encode the antibodies described herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.

A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a, ” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include” , “includes, ” and “included, ” is not limiting.

As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 μL” means “about 5 μL” and also “5 μL. ” Generally, the term “about” includes an amount that would be expected to be within experimental error, e.g., within 15%, 10%, or 5%.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As used herein, the terms “individual (s) ” , “subject (s) ” and “patient (s) ” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker) .

The terms “polypeptide” , “peptide” , and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear, cyclic, or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified, for example, via sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, ubiquitination, or any other manipulation, such as conjugation with a labeling component.

As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

A polypeptide or amino acid sequence “derived from” a designated protein refers to the origin of the polypeptide. Preferably, the polypeptide has an amino acid sequence that is essentially identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 10-20 amino acids, or at least 20-30 amino acids, or at least 30-50 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence. This terminology also includes a polypeptide expressed from a designated nucleic acid sequence.

EXAMPLES

These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1 Immunizations

For immunization with hPD-1 proteins, the protein was cross linked with immunoPlus (Genscript Inc. ) to enhance the cross reactivity to mPD-1 and then the adjuvant was mixed with protein at a 1: 1 v/v ratio, emulsified, and injected intraperitoneally.

Example 2 Serum titration by ELISA

hPD-1 protein was adsorbed onto 96-well, high protein binding plates (Costar) overnight at 4℃. Excess hPD-1 was removed by washing three times with PBS-Tween (0.1%v/v) and the wells were blocked with 1%w/v BSA (Sigma) in PBS for 1 hour at room temperature (RT) , plates were washed as described previously. Serial two-fold titrations of mouse serum were prepared, diluting samples in reagent diluent (0.1%w/v BSA/PBS) . 50μl/well of this titration was added to the coated ELISA plates. Plates were incubated at room temperature for at least 1 hour. Following incubation, plates were washed as before. Anti-mIg-HRP antibody (100ng/ml in reagent diluent; 50μl/well) was then added to the plates and incubated at RT for 1 hour. Unbound HRP-labelled anti-mouse Ig antibody was removed by washing as before. Plates were washed as before and 50μL of TMB (Sigma) was added to the plate. Then the reaction was stopped by adding 50μL of 1M sulfuric acid (Sinopharm Inc. ) . The optical density (OD) at 450nm was measured on a plate reader (Biotek) .

Example 3 Determination of serum titers by flow cytometry using Jurkat cells

Jurkat cells, suspended in FACS buffer (PBS + 1%w/v BSA + 0.1%w/v sodium azide) were distributed to a 96-well, u-bottom plate (Greiner) at a density of 10 ⁵ cells per well in 25 μl/well. A titration of mouse serum was prepared, diluting samples in FACS buffer. Twenty-five μL/well of this titration was then added to the cell plate. To determine the change in activity level due to immunization, serum from each animal prior to immunization was diluted to 1/100 in FACS buffer and 25μL/well added to the cells. Cells were incubated at 4 ℃ for 1 hour. Cells were washed twice with 150μL PBS, centrifuging after each wash step and aspirating supernatant (centrifuged at 300 xg for 3 min) . To detect antibody binding, AlexaFluor goat-anti-mouse IgG (Jackson ImmunoResearch) was diluted 1/500 in FACS buffer and 50 μL was added to the cells. Cells were incubated 1 hour at 4℃ in the dark, then washed twice with 150μL PBS as above, then resuspended in 100μl FACS buffer for analysis. AF647 signal intensity (geometric mean) was measured by flow cytometry using a BD FACS Array instrument.

Example 4 Murine tissue isolation and preparation

Mice were euthanized and sacrificed, spleens were excised from immunized mice, washed in 1x phosphate buffered saline (PBS) , and kept on ice until further processing. Tissues were prepared in buffer containing 1x PBS (Invitrogen) and 3%heat-inactivated foetal bovine serum (FBS) (Invitrogen) . Splenocytes were dispersed by mashing the tissue through a 45μm strainer (BD Falcon) and rinsing with 30 ml 3%FBS/PBS buffer before centrifugation at 700 g for 10 min at 4℃. To remove red blood cells, the pelleted splenocytes were resuspended in 4 ml of Red Blood Cell Lysis Buffer (Sigma) . After 4 min of incubation, the lysis reaction was stopped by addition of 3%FBS/1x PBS buffer. Cell clumps were filtered out with a 45 μm strainer. The remaining splenocytes were pelleted for further procedures.

Example 5 Hybridoma fusion

The isolated splenocytes were resuspended in 200μl of BSA fusion buffer, washed once and cell count was determined. SP2/0 cells were treated in the same way and washed twice with BSA fusion buffer. Splenocytes were fused at a ratio of 3: 1 with SP2/0 myeloma cells by electrofusion using a BTX ECM 2001 Electro Cell Manipulator (Harvard Apparatus) . Each fusion was left overnight in recovery medium (Dulbecco’s Modified Eagle’s Medium -high glucose (no phenol red, no L-G) containing OPI (Sigma) , L-Glutamax (Gibco) , 20%FBS (Gibco, batch-tested for hybridoma) and 2-mercaptoethanol) , then resuspended in complete DMEM medium with HAT, plated into 96-well plates, and cultured for another 7-10 days prior to screening.

Example 6 Primary screen: Binding to cells expressing human PD-1

Binding of secreted antibodies to cells expressing human PD-1 cells was determined using the BD FACSCalibur flow cytometer. Raji cells were seeded in round bottom 96 plates at 10 ⁵ cells per well in 50 μL F12+10%FBS (1.25x10 ⁵ cells/mL) and hybridoma cell culture supernatants were mixed with the cells. After washing 3 times with FACS buffer, cells were stained with AF647 labelled anti-mIgG antibody and fluorescence intensity was read by BD FACSCalibur for individual clones.

Example 7 Surface plasmon resonance (SPR)

SPR analysis was carried out using the Biacore T200 characterization system (GE Healthcare) . An anti-mouse capture surface was created on a CM5 biosensor chip by immobilising an anti-mouse antibody (GE Healthcare) by amine coupling. Antibodies in hybridoma supernatants were captured on this surface and human PD-1-his was used as the analyte at 256nM, 64nM, 16nM, 4nM, 1nM, and 0nM. For recombinant antibodies with a human Fc region, anti-human IgG (Jackson Immunoresearch) was immobilised on the biosensor surface by amine coupling. Human PD-1-his was used at 128nM, 32nM, 8nM, 2nM, and 0.5nM, and results were referenced to a non-binding control antibody.

Example 8 Sequence recovery of lead antibody candidates

Selected clones were used to prepare total RNA, which was used in an RT-PCR reaction to recover the heavy and light chain V-regions. Total RNA was extracted from hybridoma cells using TRIzol ^TM Reagent (Invitrogen) . The quantity and quality of the isolated RNA was analyzed spectrophotometrically. Murine IgG-specific reverse primers and mouse Ig-leader sequence-specific forward primer sets were used for the heavy chains. Murine kappa constant region specific reverse primers and murine kappa leader sequence specific forward primer sets were used for the kappa light chains. The RT-PCR products were separated by agarose gel electrophoresis with the DNA of the predicted size being gel purified and sequenced in the forward and reverse directions. Products were subcloned into a cloning vector and DNA of individual colonies submitted for sequencing. The DNA encoding the heavy chain variable regions of the selected lead antibodies was cloned into separate pREP4 expression plasmids (Invitrogen) in frame with the human IgG4P constant regions, the DNA encoding the light chain variable regions was cloned into pREP4 expression plasmids in frame with the human kappa constant regions using standard restriction enzyme digestion and ligation.

Hybridoma Generation

Example 9 Immunogens

PD-l protein was expressed from a DNA sequence encoding the extracellular domain (Met 1-Gln 167) of human PD-1 (NP_005009.2) with a C-terminal polyhistidine tag from Sinobiologicals (His-tag) . This protein was used for immunization.

Example 10 Immunization

Two mouse strains (Balb/c and C57/BL6) were used for immunization to generate anti-PD-1 monoclonal antibodies (Table 6) . Sera from serial boost or terminal blood samples were analyzed for the presence of specific antibodies (FIG. 2) . Serum titer data was used to select mice for hybridoma fusions. Details of the methods for titer determination are described in Examples 2 and 3.

Table 6 Details of animal immunization

Example 11 Hybridoma fusions

Single cell suspensions were prepared from spleens of the best responder animals and electro-fused with myeloma cells before seeding and culturing in 96-well plates. The hybridomas were then cultured in selection media for 7 days prior to screening of supernatants.

Hits screening and characterization

Example 12 Screening of primary hybridoma clones by FACS binding

Hybridoma supernatants were initially screened for PD-1 binding by flow cytometry using CHO-K1 cells expressing human PD-1 and CHO-K1 cells as control. The FACS was performed as follows: 2.5x10 ⁵ cells in 50μL per reaction was incubated with hybridoma supernatants and cells binding with primary antibodies were detected with secondary antibody (Goat-anti-mouse IgG (H&L) iFlour647) at 3 μg/ml. Nivolumab was set as positive control, and mouse IgG (GenScript) was set as negative control (3 μg/ml) .

About 75 FACS-positive primary clones were selected.

Example 13 Secondary FACS binding assay

The selected primary FACS binding clones were sub-cloned to obtain monoclonal cells for further testing by FACS binding. Some subclones retained the binding activities as compared with the parental clones, while some subclones lost the binding activities.

Example 14 Screening of blocking antibodies by HTRF assay

Based on the results of the secondary FACS binding assays, a panel of 23 unique positive binders was identified. CISBIO HTRF assay was performed in a 2-point dose point format to measure the blocking activities of these antibody hits in PD-1/PD-L1 binding. PD-1/PDL1 binding assay kit (63ADK000CPLPEB) , Human IgG control (R&D Systems) , and anti-PD-1 reference antibody, an analogue of nivolumab was used. The highest concentration of reference antibody was 200 nM and 3-fold serial dilution was made. 2 μl diluted antibodies were premixed with 4μl 10nM PD-L1-Euk and 4 μl 500 nM Tag-PD-1 in order. The mixture was incubated for 15 min at room temperature. Then 10 μl anti-Tag-XL665 was added and samples were incubated for 2 h. The plate was read with a Biotek Synergy Neo plate reader and the data were analyzed with Prism 5.0. Data were presented as relative inhibition percent I%, with the average positive control value as 100%, and average negative control value as 0%.

I%= (average PC value-sample value) / (average PC value-average NC value) *100%

The curves were generated using Graphpad Prism with nonlinear 4 parameter regression method. The top hits with at least 50%inhibition rate were listed in the Table 7.

Table 7 Blocking activities of murine anti-PD-1 antibodies in PD-1/PD-L1 binding by FRET assay

A total of 23 PD-1 antibodies were subjected to HTRF blocking assay at two dose points; 66.7 nM and 6.67 nM. The 2 dose points were chosen based on the pilot study with 9 reference antibodies. Antibodies 31G1E10, 56H6F7, 42F11B8, 31H7E10, 45E4E8, 33G5C7, 56B3C11, 42G2F11 and 6G12E8 were chosen for the full curve testing.

Example 15 Blocking activities of murine anti-PD-1 antibody hits in PD-1/PD-L1 binding by FRET assay

Full dose response blocking activity was measured for 10 selected anti-PD-1 hits using the HTRF assay. The method was described above, and antibody hits were prepared by 3-fold serial dilutions. Results are shown in FIG. 3

IC50s of murine anti-PD-1 antibodies for PD-1/PD-L1 binding are summarized in Table 8. Ref antibody Nivolumab has an IC50 value of 3.79 nM, with mini I%from 0.7153 to 12.74 and Max I%from 88.42 to 118.9.

Based on the full curve PD-1/PD-L1 binding blocking data, 31G1E10, 56H6F7, 42F11B8, 31H7E10, 45E4E8, 56B3C11 and 42G2F11have comparable IC50s to reference antibody (nivolumab analogue) . The additional antibodies displayed weaker PD-1/PD-L1 blocking activity as compared to the reference antibodies.

Example 16 Blocking activities of murine anti-PD-1 antibodies in PD-1/PD-L2 binding by ELISA

100μl/well 2μg/ml h-PD-L2-His (Sinobiological) was coated on 96-well plate at 4℃ overnight. After washing 3 times, 100 μl/well 1%BSA blocking solution was added and incubated for 1 hr at room temperature. 0.5μg/ml PD-1 protein (Sinobiological) with anti-PD-1 antibodies (diluted in 3-fold serial dilution from 33μg/mL) was mixed and pre-incubated for 0.5 hr at room temperature. 50 μl/well pre-incubated buffer was added and samples were incubated for 2 hr at room temperature. The plate was washed 3 times, and 50 μl/well 2nd antibody goat anti-hIgG-HRP (Fc specific, 1: 5000) (Abcam) was added to wells and incubated for 1 hour at room temperature. After washing 3 times, the plate was developed by adding 50μl TMB (Biopanda) for 1min and stopped by adding 50μl 1M HCL. The data was read by using Biotek SynergyNeo plate reader and analyzed by GraphPad Prism5.0 data analysis software. FIG. 4A and FIG. 4B show dose-dependent blocking activities of murine anti-PD-1 antibody hits in PD-1/PD-L2 binding .

The blocking activities of murine anti-PD-1 antibodies in PD-1/PD-L2 binding are summarized in Table 8. Of the antibodies tested, clone 31G1E10 has the lowest IC50 value. Blocking activities

Table 8 Summary of calculated IC50s (nM) of PD-1/PD-L2-blocking activities of murine antibody hits

Example 17 Epitope binning of exemplary anti-PD-1 antibodies

Epitope binning of exemplary anti-PD-1 antibodies were conducted using the Octet system with nivolumab and pembrolizumab analog antibodies as references. Nine neutralizing PD-1 antibodies and 2 reference antibodies were analyzed and grouped according to their competitive binding to reference antibodies. The ligand PD-1 was captured by Ni-NTA sensor and antibodies were flowed in assay buffer containing PBS pH 7.4, 0.1%BSA, 0.02%Tween after binding of reference antibodies. Epitope binning study showed that murine anti-PD-1 antibodies belongs to different groups as compared to reference antibodies such as nivolumab and pembrolizumab (Table 9) .

Table 9

Example 18 Cross-reactivity of anti-PD-1 hits with murine PD-1 protein

FACS analysis of CHO cells over-expressing murine PD-1was used for determining the cross-reactivity to murine PD-1 of antibody hits. CHO cells over-expressing human PD-1were set as control. As shown in FIG. 5, none of the hits bound to murine PD-1 as compared to their concentration dependent binding to human PD-1.

Example 19 Affinities of murine anti-PD-1 antibody hits

Six hits were selected for detailed kinetics study by Biacore characterization system based on results of binding and blocking assays described above. Each antibody was captured in flow cell FC2 around 100 RU respectively, using FC1 as the reference cell, followed by injection of antigen samples at varying concentrations. The signals with captured antibody subtracted from that without captured antibody were calculated with Biacore 8K evaluation software (Biacore) . The running buffer is HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05%surfactant P20) . Six antibodies were tested for affinities, with pembrolizumab analogue (CrownBio) and nivolumab analogue (Chempartners) as reference antibodies. The results are summarized in Table 10.

Table 10 Binding kinetics of 6 murine antibody hits as compared to reference antibodies

Clone. No.	ka (1/Ms)	kd (1/s)	KD (M)
31G1E10F12	1.71E+05	1.98E-04	1.16E-09
56H6F7D5	1.42E+05	7.05E-04	4.97E-09
42F11B8	1.10E+05	8.51E-04	7.71E-09
45E4B8E4	4.49E+05	6.89E-03	1.54E-08
56B3C11G9	1.22E+05	2.95E-04	2.43E-09
42G2F11C8	1.02E+05	6.76E-04	6.61E-09
Pembrolizumab	2.88E+06	4.74E-03	1.65E-09
Nivolumab	3.81E+05	1.52E-03	4.00E-09

Example 20 Function validation of anti-PD-1 antibodies by mature DC-T MLR assay

Monocytes were isolated by CD14 beads (human, Miltenyi Biotec) from PBMCs (ALLCELLS) , according to the protocols provided by the manufacturer. Monocytes were cultured with IL-4 (CrownBio, 35ng/ml) and GM-CSF (50ng/ml) for 7 days at 2x10 ⁶ cells per well (6-well plate) . On

day

7, 1 μg/ml LPS was added to the DC culture medium, DC cells were cultured with LPS for 24 hrs. After DC maturation, 20 μM Mitomycin C was added and incubated with DCs for 90 min. Meanwhile, human CD3+ T cells were purified from PBMCs by using a Pan T Cell Isolation Kit (Miltenyi Biotec) , 2×10 ⁵ purified CD3+ T cells and 4×10 ⁴ allogeneic mature dendritic cells were co-cultured in a total volume of 200 μl in the absence or presence of anti-PD-1 reference antibody or antibody hits for 5 days. Post 5-day culture, 50 μl culture supernatant was collected for evaluating levels of IL-2 (FIG. 6) and IFN-γ (FIG. 7) by using CISBIO kit.

In some instances, tested antibodies such as 31G1E10 have similar activities as compared to reference antibody.

Example 21 Additional function validation of anti-PD-1 antibodies by immature DC-T MLR assay

4x10 ⁴ immature DC cells from donor A5416 and 2x10 ⁵ CD3+ T cells from donor A5387 were isolated for MLR assay and cytokine determination. Increased levels of IL-2 (FIG. 8A) and IFN-γ (FIG. 8B) in the presence of anti-PD-1 murine antibody hits were observed. The response levels, however, were lower than those found in the mature DC-T MLR assay (FIG. 6, FIG. 7) .

Chimerization of murine anti-PD-1 antibodies

The V regions of six murine anti-PD-1 antibody hits, including 31G1E10, 56B3C11, 56H6F7, 42G3F11, 45E4E8, 42G2F11were chimerized with human IgG4 constant region with S228P mutation.

Example 22 Protein-based PD-1 binding activities of chimeric antibody hits.

Six chimeric antibodies were tested for their binding to PD-1 in a protein-based ELISA assay. PD-1 protein was coated at 100 μl/well, 1 μg/ml in 96-well plates. Chimeric antibody samples were diluted from 3 μg/ml, except 42G3F11 (from 9 μg/ml) and 45E4E8 (from 1 μg/ml) , in 3-fold series. Binding was detected by mouse anti-human IgG-Fc antibody (HRP, 1: 2, 500 dilution) . Titration curves were plotted (FIG. 9) using pembrolizumab and nivolumab as positive controls and IgG4 as a negative control.

Example 23 Activities of chimeric antibodies in blocking PD-1/PD-L1 binding

Six chimeric antibodies were tested for their abilities to block PD-1/PD-L1 binding in a FRET assay. Two separate experiments were performed, each with pembrolizumab and nivolumab analogue as positive controls. FIG. 10A and FIG. 10B show that the tested chimeric antibodies inhibited PD-1/PD-L1 binding in a concentration dependent manner. IC50s were comparable to that of pembrolizumab or nivolumab analogues.

Example 24 Activities of chimeric antibodies in blocking PD-1/PD-L2 binding

PD-1 protein prepared was coated on 96-well microplate, serially diluted antibody samples and bio-PD-L2 (Chempartner) solutions were then added, and Streptavidin-HRP (Sigma) was added for detection of bound PD-L2. FIG. 11 shows that the tested chimeric antibodies, except 42G2F11, inhibited PD-1/PD-L2 binding in a concentration dependent manner.

Example 25 Functional analysis of chimeric antibodies by MLR assay

DC/T MLR assays were performed to evaluate the chimeric murine antibodies with human IgG4 S228P mutation. Supernatants were collected for IFN-γ determination. In two assays, remarkable enhanced IFN-γ secretion was observed in the presence of chimeric anti-PD-1 antibodies as compared to isotype control (FIG. 12A and FIG. 12B) . Comparable effects to that of benchmarks were observed with antibodies 31G1E10, 56B3C11, and 56H6F7.

Example 26 Luciferase reporter assay to measure functional activities of chimeric anti-PD-1 antibodies

Two engineered cell lines, Jurkat-NFAT-PD-1-luciferase, and Hep3B-OS8-hPD-L1 were used to test the activation of PD-1 dependent downstream effects. Hep3B-OS8-PDL1 cells were plated in a 96-well plate (Perkin Elmer) one day before the assay. On the day of assay, 2x antibody solution was prepared in assay medium: 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, and 0nM. Medium was removed from pre-plated Hep3B-OS8-PDL1 cells. 50μl of antibody solution was added to the plate and incubated for 20-30min. Meanwhile, Jurkat (6C8) -NFAT-PD-1 cells were harvested using Assay Medium instead of Growth Medium. 50μl of Jurkat (6C8) -NFAT-PD-1 cells were added to the plate. 100μl of Assay Medium was added to Cell-Free Control wells. The assay plate was incubated in a humidified 37℃, 5%CO2 incubator for 6 hours. Luciferase activity (FIG. 13A and FIG. 13B) was measured by the ONE-Glo ^TM Luciferase Assay System (Promega) using a luminometer(ChemPartner) . Positive control was Keytruda analogue, 5.07 mg/ml, and negative control was hIgG4, 5.91mg/ml. EC50s calculated from the Jurkat-NFAT reporter assay are shown in Table 11.

Table 11.

Example 27 Activities of chimeric anti-PD-1 antibody leads in cell-based PD-L1 binding blocking assay

A FACS-based PD-L1 binding blocking assay was used to confirm the activities of top chimeric leads, xi31G1E10 and xi56B3C1. The plate was blocked with blocking buffer for 30 min. CHO-K1-PD-1-1F7 cells (Chempartner) were digested with TrypLE and stopped with culture medium. Cells were then added to the blocked plate and centrifuged before resuspending with diluted antibody solution. Biotin-labeled ligand solution was added to wells. The plate was shaken thoroughly, incubated at 4 ℃ for 2 hrs, then washed 3 times. Cells were suspended with secondary antibody (Streptavidin (SA) -Alexa488, Life Technologies) solution and incubated at 4℃ for 1h. Cells were washed and resuspended in blocking buffer. Keytruda analogue and Opdivo analogue were set as positive controls . hIgG4 was set as negative control . Ligand was Bio-PD-L1 (0.886 mg/ml) and Bio-PD-L2 (1.415 mg/ml) . FACS was performed on cells (FIG. 14A and FIG. 14B) .

Example 28 Inhibition of MC38 tumor growth in human PD-1 knock-in mice by chimeric antibody leads

MC38 tumor cells were maintained in vitro as a monolayer culture in DMEM plus 2 mM glutamate. Cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Each mouse was inoculated subcutaneously with MC38 cells (3 x 10 ⁵) for tumor development. Treatments were started on day 6 after tumor cell inoculation when the average tumor size reached approximately 70 mm ³. Mice were subjected to human IgG1 isotype, Opidvo analogue, Xi31G1E10, and Xi56B3C11 treatment respectively at 10 mpk thrice a week for three weeks (n=8/group) . Tumor size was measured and plotted for each mouse (FIG. 15A-15D) .

The data showed that TGI of reference antibody Nivolumab analogue was 94%, 84%for Xi31G1E10, and 101%for Xi56B3C11 respectively. Activities of these two antibodies were comparable to Nivolumab analogue.

Lead Optimization, characterization and selection

Example 29 Humanization design

Murine antibodies 31G1E10 and 56B3C11 were humanized by human germline structure matching, CDR grafting, and back-mutation. The combination of variants for humanized clone 31G1E10 is shown in Table 12.

Table 12

The combination of variants for humanized clone 56B3C11 is shown in Table 13.

Table 13

Example 30 Screening humanized variants by ELISA based PD-1 binding assay

The humanized variants generated from the above design were first screened by PD-1 binding assay. 1 μg/ml of PD-1 protein was coated on 96-well plates. Test antibodies were diluted from 10μg/ml in 3-fold serial dilution and 100 μL/well was added to the plate. The plate was sealed and incubated at room temperature for 1 hr. After washing 3 times with PBST (0.05%v/v) , 100 μL/well of 2nd antibody (anti-human IgG HRP, 1: 5000 diluted in 1%BSA/1 X PBS) was added and incubated for 1 hour before the plate was developed with TMB. Plate was read at OD450 on a BioTek reader and the data was analyzed with Graphpad to calculate the binding EC50s (FIG. 16A, 16B, and 16C) . Tables 14 and 15 show EC50s for the derivatives of each clone.

Table 14. EC50s of 31G1E10 derivatives

	EC50
Xi31G1E10	0.01398
31G1E10-1	0.01961
31G1E10-2	0.01168
31G1E10-3	0.02123

31G1E10-4	0.02703
31G1E10-5	0.01799
31G1E10-6	0.02208
31G1E10-7	0.01646
31G1E10-8	0.008458
31G1E10-9	0.02632
31G1E10-10	0.02231
31G1E10-11	0.01407
31G1E10-12	0.01624

Table 15. EC50s of 56B3C11 derivatives

	EC50		EC50
Xi56B3C11	0.07397	Xi56B3C11	0.07397
56B3C11-1	0.07722	56B3C11-11	0.1117
56B3C11-2	0.09484	56B3C11-12	0.1849
56B3C11-3	0.08802	56B3C11-13	0.1481
56B3C11-4	0.07979	56B3C11-14	0.1459
56B3C11-5	0.07815	56B3C11-15	0.1163
56B3C11-6	0.03044	56B3C11-16	0.1128
56B3C11-7	0.09073	56B3C11-17	0.06342
56B3C11-8	0.1047	56B3C11-18	0.06922
56B3C11-9	0.1061	56B3C11-19	0.07049
56B3C11-10	0.1172	56B3C11-20	0.05910

Example 31 Screening humanized variants by cell-based PD-1/PD-L1 blocking assay

A cell-based PD-1/PD-L1 blocking assay was performed as described earlier to select top clones from the humanized variants. Some clones were tested twice. The blocking assay results are shown in FIG. 17A and FIG. 17B.

Based on the IC50 calculation (Table 16) ,

derivative clones

4, 5, 7, 8, 10, 11, and 12 of 31G1E10, and

derivative clones

2, 3, 4, 5, 6, 13, and 18 of 56B3C11, were selected for further affinity measurement.

Table 16

Example 32 Affinity determination of humanized antibodies

For 31G1E10,

clones

4, 5, 7, 8, 10, 11, and 12, and for 56B3C11,

clones

2, 3, 4, 5, 6, 13, and 18, were further selected for Octet binding affinity studies.

Two sensor tips (AHC) from Fortebio were used to capture hFc-antibodies protein to the surface of the sensor, and added into antigen-containing wells, Wells with buffer only were set as negative controls. Dissociation processes were performed by putting sensors into kinetics buffer for 5 min at 30℃. The reference was subtracted from all curves. Regeneration condition: 5 seconds in Glycine (pH=1.5) and 5 seconds in PBS, repeating 5 times. Sample volume was 200 μL/well. Hydration solution volume was 200 μL/well. Flow rate was 1000 rpm/second; Biosensor hydration and sample plate equilibration for 10 min.

The Octet results showed (Table 17) that the humanized variants of Xi31G1E10 have lower off-rates as compared to the humanized variants of 56B3C11. The off-rates of humanized variants of 56B3C11 showed more variation.

Table 17

Antibody	KD (M)	kon (1/Ms)	kdis (1/s)
Keytruda analogue	7.51E-09	3.74E+05	2.81E-03
Opdivo analogue	1.11E-08	2.16E+05	2.41E-03
Xi 31G1E10	6.18E-09	2.07E+05	1.28E-03

31G1E10-4	7.53E-09	1.76E+05	1.32E-03
31G1E10-5	9.07E-09	2.03E+05	1.84E-03
31G1E10-7	9.97E-09	1.61E+05	1.60E-03
31G1E10-8	9.91E-09	1.47E+05	1.45E-03
31G1E10-10	6.28E-09	2.05E+05	1.29E-03
31G1E10-11	7.61E-09	2.37E+05	1.80E-03
31G1E10-12	8.25E-09	1.90E+05	1.57E-03
Xi 56B3C11	4.51E-09	1.55E+05	6.98E-04
56B3C11-2	9.78E-09	1.93E+05	1.89E-03
56B3C11-3	1.00E-08	1.96E+05	1.96E-03
56B3C11-4	9.64E-09	1.67E+05	1.61E-03
56B3C11-5	8.86E-09	1.83E+05	1.61E-03
56B3C11-6	1.03E-08	1.78E+05	1.83E-03
56B3C11-13	3.05E-09	1.82E+05	5.54E-04
56B3C11-18	8.20E-09	1.31E+05	1.08E-03

Example 33 Jurkat-NFAT Luciferase reporter assay for lead characterization

PD-1/PD-L1 functional blocking activity was measured in a quantitative luciferase reporter assay as described earlier. Nivolumab analogue and pembrolizumab analogue were set as references.

The calculated IC50 values are summarized in Table 18. Overall, these clones exhibited a similar activities among each other (FIG. 18A –18E) . For further characterization, clones with IC50s within 2-fold of the nivolumab or pembrolizumab analogue references were highlighted and subjected to further evaluation.

Table 18

Antibody	IC50
Xi 31G1E10	0.1398
31G1E10-4	0.3433
31G1E10-7	0.1795
31G1E10-10	0.2471
31G1E10-11	0.2036
31G1E10-12	0.1791
Xi 56B3C11	0.1766
56B3C11-2	0.3115
56B3C11-13	0.2114
56B3C11-15	0.2556
56B3C11-16	0.2579
56B3C11-18	0.2591

Pembrolizumab	0.1288
Nivolumab	0.08801
hIgG4	NA

Example 34 PD-1/PD-1 ligand binding blocking activities of humanized antibodies

Clones 31G1E10-7, 10, 11, 12, and Clones 56B3C11-13, -18 were tested using cell-based PD-1/PD-L1 (FIG. 19A) and PD-1/PD-L2 (FIG. 19B) binding blocking assays as described earlier. IC50 values calculated are shown in Table 19.

Table 19

Example 35 Characterization of humanized antibodies in DC/T MLR assay

PBMCs were isolated by gradient centrifugation and monocytes were cultured and i by adhesion to the culture dish. Monocytes were cultured in complete medium with GM-CSF and IL-4 for 6 days, medium was changed on day 3. DC cells were collected on day 5 and matured with the addition of 1 μg/ml LPS for 24 hours before treating with mitomycin. Meanwhile, human CD3+ T cells were purified from PBMCs using a Pan T-Cell Isolation Kit. 2×10 ⁵ purified CD3+_T cells and 4×10 ⁴ allogeneic mature dendritic cells were co-cultured in a total volume of 200 μl in the absence or presence or anti-PD-1 reference antibodies or humanized lead antibodies for 5 days. Culture supernatants were collected for evaluating the levels of cytokine IL-2 (FIG. 20 A and FIG. 20 B) three days and IFN-γ (FIG. 20 C and 20 D) 5 days after reaction was initiated respectively.

As shown in Table 20, the E50 values of IL-2 production are close to each other within both 31G1E10 variants and 56B3C11 variants. However, the INF-γ levels showed more variation and EC50 values were not calculated.

Table 20

Example 36 CMV antigen recall assay to evaluate humanized antibodies

Human mononuclear cells from PBMCs were separated by gradient centrifugation, and activated with 0.4 μg/ml CMV. Test antibodies were added to the culture and cells were incubated for 5 days. On day 5, culture supernatants were collected and IFN-γlevels were measured by ELISA (FIG. 21A and 21B) . Keytruda analogue was set as a positive control. IFN-γ levels were increased by all tested antibodies as compared to the control of CMV stimulation alone.

Example 37 In vivo anti-tumor efficacy of humanized antibodies in the MC38 syngeneic tumor model

A murine MC38 tumor growth inhibition model was established in hPD-1 knock-in mice. MC38 tumor cells were maintained in vitro as a monolayer culture in DMEM plus 2 mM glutamate. Cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Each mouse was inoculated subcutaneously with MC38 cells (3 x 10 ⁵) for tumor development. Treatments were started on day 6 after tumor cell inoculation when the average tumor size reached approximately 70 mm ³. Each group consisted of 8 tumor-bearing mice. The testing article was administrated to the mice according to the predetermined regimen as shown in the experimental design table (Table 21) .

Table 21

Tumor size was measured twice a week in two dimensions using a caliper. The tumor size was then used for calculations of T/C value. TGI was calculated for each group using the formula: TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100. Ti is the average tumor volume of a treatment group on a given day, T0 is the average tumor volume of the treatment group on the day of treatment start, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the vehicle group on the day of treatment start. Summary of statistical results, including mean and the standard error of the mean (SEM) , were provided for the tumor volume of each group at each time point (FIG. 22A-FIG. 22E) .

In vivo efficacy of both 31G1E10 and 56B3C11 were comparable to pembrolizumab in MC38 tumor model.

Example 38 Stability test with repeated freeze-thaw cycles

Repeated freeze/thaw cycles were performed to evaluate the top 4 humanized antibody variants. Antibodies were frozen and thawed for 8 cycles. Samples were analyzed by SEC-HPLC to determine the purity. Antibody concentrations were measured by Nanodrop to determine the recovery rate. As shown in FIG. 23, the repeated freeze/thaw studies showed that 31G1E10-12 was very stable at almost 100%monomer after repeated freeze/thaw cycles. The two 56B3C11 variants were also very stable, at 98%monomer throughout the study. In the case of 31G1E10-7, the protein showed some variation during the study. The recovery rate for all 4 proteins were consistently between 98%and 100%.

Example 39 Accelerated stability

Accelerated stability tests were performed to evaluate the selected lead antibodies. Antibodies were incubated at 4℃, 25℃, and 40℃ for 24 days. Samples were taken on day 0 (before test) ,

day

4, 8, 11, 15, and 24 and analyzed by SEC-HPLC to determine the purity. Antibody concentrations were measured by Nanodrop to determine the recovery rate.

Antibodies 31G1E10-12, 56B3C11-13, and 56B3C11-18 were shown to be very stable at all temperatures. Purity of these antibodies at 40℃ for 24 days were 99.2%, 97.7%, and 97.9% (FIG. 24C) , respectively. 31G1E10-7 had some fluctuations as compared to 31G1E10-12, but was also very stable in general. On day 24, the purity of 31G1E10-7 was 97.4% (4℃, FIG. 24A) , 98.6% (25℃, FIG. 24B) , and 97.1% (40℃, FIG. 24C) .

Example 40 Solubility test

Solubility tests were performed to evaluate the selected lead antibodies. Briefly, 31G1E10-7, -12, and 56B3C11-13, -18 antibodies were continually concentrated from 5 mg/ml to 11.4 mg/ml, 25mg/ml, 55 mg/ml, and up to 86 mg/ml. Visible aggregation was checked during the process and no aggregation was observed. Samples were analyzed by SEC-HPLC to determine the purity. Antibody concentrations were measured by Nanodrop to determine the recovery rate.

31G1E10-7 was concentrated from 4.1 mg/ml to 11.4 mg/ml, 25.2 mg/ml, 55 mg/ml, and 86 mg/ml. The monomer purity was 96.9%, 96.9%, 96.8%, and 96.8%respectively. Antibodies were recovered at 92.6% (11.4 mg/ml) , 91.1% (25.2 mg/ml) , 94.2%(55 mg/ml) , and 91% (86 mg/ml) .

31G1E10-12 was concentrated from 4.2 mg/ml to 10.2 mg/ml, 25.6 mg/ml, 55 mg/ml, and 72 mg/ml. The monomer purity was 100%for all samples. Antibodies were recovered at 88.2% (10.2 mg/ml) , 94.8% (25.6 mg/ml) , 91.1% (55 mg/ml) , and 84.2% (72mg/ml) .

56B3C11-13 was concentrated from 4.2 mg/ml to 10.2 mg/ml, 21.2 mg/ml, 45 mg/ml, and 76 mg/ml. The monomer purity was 98.8%for all samples. Antibodies were recovered at 88.3% (10.2 mg/ml) , 93.5% (21.2 mg/ml) , 91% (45 mg/ml) , and 91% (76mg/ml) .

56B3C11-18 was concentrated from 4.2 mg/ml to 10.6 mg/ml, 22.4 mg/ml, 43 mg/ml, and 84 mg/ml. The monomer purity was 98.2%for all samples. Antibodies were recovered at 85.3% (10.6 mg/ml) , 93.9% (22.4 mg/ml) , 88.5% (43 mg/ml) , and 99% (84mg/ml) .

Example 41 Thermostability measurement by DSC

Melting temperature (Tm) was assessed using Differential Scanning Calorimetry (DSC) . Scans were performed using an automated MicroCal VP-Capillary DSC equipped with a 96-well plate autosampler. Scans were run from 25-100 ℃ at the scan rate of 60 ℃/h. The sample data were analyzed using Origin 7.0 by subtraction of the reference data and the baseline as well as normalization to protein concentration. The unfolding transitions of each protein were fitted to calculate the transition temperatures (Tm) using the non-two-state unfolding model.

Melting temperatures were similar for 31G1E10 variants relative to those of 56B3C11 variants. Tm of 31G1E10-12 was slightly higher than that of 31G1E10-7.56B3C11 variants have similar Tms. 56B3C11-13 seemed to have slightly higher Tm than 56B3C11-18. All Tm are in normal range (Table 22) .

Table 22

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

An anti-PD-1 antibody comprising three variable heavy chain complementarity determining regions (CDRs) and three variable light chain complementarity determining regions (CDRs) , wherein the anti-PD-1 antibody specifically binds to a different epitope on the extracellular domain of PD-1 compared to nivolumab and pembrolizumab, and wherein a binding affinity of the anti-PD-1 antibody is comparable to a binding affinity of nivolumab or pembrolizumab.
The anti-PD-1 antibody of claim 1, wherein:

a) the VH CDR1 sequence consists of X ₁YX ₂MS;

wherein X ₁ is S or T and X2 is G or T;

b) the VH CDR2 sequence consists of X ₃ISX ₄GGX ₅DTYYPDX ₆VKG;

wherein X ₃ is T or Y; X ₄ is G or F; X ₅ is R or G; and X ₆ is S or T; and

c) the VH CDR3 sequence consists of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆;

wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.
An anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , wherein:

d) the VH CDR1 sequence consists of X ₁YX ₂MS;

wherein X ₁ is S or T and X2 is G or T;

e) the VH CDR2 sequence consists of X ₃ISX ₄GGX ₅DTYYPDX ₆VKG;

wherein X ₃ is T or Y; X ₄ is G or F; X ₅ is R or G; and X ₆ is S or T; and

f) the VH CDR3 sequence consists of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆;

wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.
The anti-PD-1 antibody of any one of the claims 1-3, wherein

a) the VL CDR1 sequence consists of

RASX ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈;

wherein X ₁₇ is E or Q; X ₁₈ is S or D; X ₁₉ is V or I; X ₂₀ is D or S; X ₂₁ is S, N or D; X ₂₂ is Y or F; X ₂₃ is G or L; X ₂₄ is I or N; X ₂₅ is present or absence and if present, is S; X ₂₆ is present or absence and if present, is F; X ₂₇ is present or absence and if present, is M; X ₂₈ is present or absence and if present, is N;

b) the VL CDR2 sequence consists of X ₂₉X ₃₀SX ₃₁X ₃₂X ₃₃S;

wherein X ₂₉ is A or Y; X ₃₀ is A or T; X ₃₁ is N or R; X ₃₂ is Q or L; X ₃₃ is G or H; and

c) the VL CDR3 sequence consists of QQX ₃₄X ₃₅X ₃₆X ₃₇PWT;

wherein X ₃₄ is S or G; X ₃₅ is K or D; X ₃₆ is E or M; X ₃₇ is V or I.
The anti-PD-1 antibody of any one of the claims 1-4, wherein the VH CDR1 sequence is selected from SEQ ID NOs: 1, 4, or 7.
The anti-PD-1 antibody of any one of the claims 1-5, wherein the VH CDR2 sequence is selected from SEQ ID NOs: 2, 5, or 8.
The anti-PD-1 antibody of any one of the claims 1-6, wherein the VH CDR3 sequence is selected from SEQ ID NOs: 3, 6, or 9.
The anti-PD-1 antibody of any one of the claims 1-7, wherein the VL CDR1 sequence is selected from SEQ ID NOs: 19, 22, or 25.
The anti-PD-1 antibody of any one of the claims 1-8, wherein the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, or 26.
The anti-PD-1 antibody of any one of the claims 1-9, wherein the VL CDR3 sequence is selected from SEQ ID NOs: 21, 24, or 27.
The anti-PD-1 antibody of any one of the claims 1-3, wherein:

a) the VH CDR1 sequence is selected from SEQ ID NOs: 1, 4, or 7;

b) the VH CDR2 sequence is selected from SEQ ID NOs: 2, 5, or 8; and

c) the VH CDR3 sequence consists of QX ₇X ₈X ₉X ₁₀X ₁₁X ₁₂X ₁₃X ₁₄X ₁₅X ₁₆;

wherein X ₇ is R or G; X ₈ is D or N; X ₉ is S or Y; X ₁₀ is A or E; X ₁₁ is W or G; X ₁₂ is F or A; X ₁₃ is A or P; X ₁₄ is H, F or Y; X ₁₅ is present or absence and if present, is A; and X ₁₆ is present or absence and if present, is Y.
The anti-PD-1 antibody of any one of the claims 1-3, wherein:

a) the VH CDR1 sequence is selected from SEQ ID NOs: 1, 4, or 7;

b) the VH CDR2 sequence is selected from SEQ ID NOs: 2, 5, or 8; and

c) the VH CDR3 sequence is selected from SEQ ID NOs: 3, 6, or 9.
The anti-PD-1 antibody of any one of the claims 1-3, 11, or 12, wherein

a) the VL CDR1 sequence is selected from SEQ ID NOs: 19, 22, or 25;

b) the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, or 26; and

c) the VL CDR3 sequence consists of QQX ₃₄X ₃₅X ₃₆X ₃₇PWT;

wherein X ₃₄ is S or G; X ₃₅ is K or D; X ₃₆ is E or M; X ₃₇ is V or I.
The anti-PD-1 antibody of any one of the claims 1-3, 11, 12, or 13, wherein

a) the VL CDR1 sequence consists of

RASX ₁₇X ₁₈X ₁₉X ₂₀X ₂₁X ₂₂X ₂₃X ₂₄X ₂₅X ₂₆X ₂₇X ₂₈;

wherein X ₁₇ is E or Q; X ₁₈ is S or D; X ₁₉ is V or I; X ₂₀ is D or S; X ₂₁ is S, N or D; X ₂₂ is Y or F; X ₂₃ is G or L; X ₂₄ is I or N; X ₂₅ is present or absence and if present, is S; X ₂₆ is present or absence and if present, is F; X ₂₇ is present or absence and if present, is M; X ₂₈ is present or absence and if present, is N;

b) the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, or 26; and

c) the VL CDR3 sequence is selected from SEQ ID NOs: 21, 24, or 27.
The anti-PD-1 antibody of any one of the claims 1-3 or 11-14, wherein

a) the VL CDR1 sequence is selected from SEQ ID NOs: 19, 22, or 25;

b) the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, or 26; and

c) the VL CDR3 sequence is selected from SEQ ID NOs: 21, 24, or 27.
The anti-PD-1 antibody of claim 1, wherein the VH CDR1 sequence is selected from SEQ ID NOs: 10, 13, or 16.
The anti-PD-1 antibody of claim 1, wherein the VH CDR2 sequence is selected from SEQ ID NOs: 11, 14, or 17.
The anti-PD-1 antibody of claim 1, wherein the VH CDR3 sequence is selected from SEQ ID NOs: 12, 15, or 18.
The anti-PD-1 antibody of claim 1, wherein the VL CDR1 sequence is selected from SEQ ID NOs: 28, 31, or 34.
The anti-PD-1 antibody of claim 1, wherein the VL CDR2 sequence is selected from SEQ ID NOs: 29, 32, or 35.
The anti-PD-1 antibody of claim 1, wherein the VL CDR3 sequence is selected from SEQ ID NOs: 30, 33, 36.
The anti-PD-1 antibody of any one of the claims 1-15, wherein the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 1-3 and three VL CDRs according to SEQ ID NOs: 19-21.
The anti-PD-1 antibody of any one of the claims 1-15, wherein the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 4-6 and three VL CDRs according to SEQ ID NOs: 22-24.
The anti-PD-1 antibody of any one of the claims 1-15, wherein the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 7-9 and three VL CDRs according to SEQ ID NOs: 25-27.
The anti-PD-1 antibody of claim 1, wherein the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 10-12 and three VL CDRs according to SEQ ID NOs: 28-30.
The anti-PD-1 antibody of claim 1, wherein the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 13-15 and three VL CDRs according to SEQ ID NOs: 31-33.
The anti-PD-1 antibody of claim 1, wherein the anti-PD-1 antibody comprises three VH CDRs according to SEQ ID NOs: 16-18 and three VL CDRs according to SEQ ID NOs: 34-36.
The anti-PD-1 antibody of any one of the claims 1-15 or 22, wherein the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 38 and the VL sequence according to SEQ ID NO: 40.
The anti-PD-1 antibody of any one of the claims 1 or 16-21, wherein the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 42 and the VL sequence according to SEQ ID NO: 44.
The anti-PD-1 antibody of any one of the claims 1 or 16-21, wherein the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 46 and the VL sequence according to SEQ ID NO: 48.
The anti-PD-1 antibody of any one of the claims 1 or 16-21, wherein the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 50 and the VL sequence according to SEQ ID NO: 52.
The anti-PD-1 antibody of any one of the claims 1-15 or 23, wherein the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 54 and the VL sequence according to SEQ ID NO: 56.
The anti-PD-1 antibody of any one of the claims 1-15 or 24, wherein the anti-PD-1 antibody comprises the VH sequence according to SEQ ID NO: 58 and the VL sequence according to SEQ ID NO: 60.
The anti-PD-1 antibody of any one of the claims 1-33, wherein the binding affinity of the anti-PD-1 antibody is higher than the binding affinity of nivolumab.
The anti-PD-1 antibody of any one of the claims 1-33, wherein the binding affinity of the anti-PD-1 antibody is higher than the binding affinity of pembrolizumab.
The anti-PD-1 antibody of any one of the claims 1-35, wherein the binding affinity of the anti-PD-1 antibody is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or higher than the binding affinity of nivolumab or pembrolizumab.
The anti-PD-1 antibody of any one of the claims 1-35, wherein the binding affinity of the anti-PD-1 antibody is about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or higher than the binding affinity of nivolumab or pembrolizumab.
The anti-PD-1 antibody of any one of the claims 1-37, wherein the ani-PD-1 antibody blocks interaction of PD-1 with PD-L1 and/or PD-L2.
The anti-PD-1 antibody of claim 38, wherein the ani-PD-1 antibody has a >40%inhibition, >50%inhibition, or >60%inhibition at an antibody concentration range of about 5-7 nM.
The anti-PD-1 antibody of claim 38, wherein the ani-PD-1 antibody has a >46%inhibition at an antibody concentration of 6.67nM.
The anti-PD-1 antibody of any one of the claims 1-40, wherein the anti-PD-1 antibody has a KD of less than 8e-9 M, less than 6e-9 M, less than 4e-9 M, less than 2.5e-9M, less than 2e-9 M, less than 1.5e-9 M, or less than 1.2e-9 M.
The anti-PD-1 antibody of any one of the claims 1-41, wherein the anti-PD-1 antibody has a KD of about 2.43e-9 M.
The anti-PD-1 antibody of any one of the claims 1-42, wherein the anti-PD-1 antibody has a KD of about 1.16e-9 M.
The anti-PD-1 antibody of any one of the claims 1-43, wherein the anti-PD-1 antibody has an IC ₅₀ similar to nivolumab and/or pembrolizumab.
The anti-PD-1 antibody of any one of the claims 1-44, wherein the anti-PD-1 antibody induces cytokine production.
The anti-PD-1 antibody of claim 45, wherein the cytokine is IL-2 or INF-γ.
The anti-PD-1 antibody of any one of the claims 1-46, wherein the anti-PD-1 antibody comprises an IgG1 framework.
The anti-PD-1 antibody of any one of the claims 1-46, wherein the anti-PD-1 antibody comprises an IgG4 framework.
The anti-PD-1 antibody of claim 47 or 48, wherein the framework is a humanized IgG1 or IgG4 framework.
The anti-PD-1 antibody of claim 48 or 49, wherein the IgG4 framework comprises a S228P mutation.
An anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) and three variable light chain (VL) complementarity determining regions (CDRs) , wherein the VH CDR1 sequence is selected from SEQ ID NOs: 1, 4, 7, 10, 13, or 16; the VH CDR2 sequence is selected from SEQ ID NOs: 2, 5, 8, 11, 14, or 17; and the VH CDR3 sequence is selected from SEQ ID NOs: 3, 6, 9, 12, 15, or 18.
The anti-PD-1 antibody of claim 51, wherein the VL CDR1 sequence is selected from SEQ ID NOs: 19, 22, 25, 28, 31, or 34; the VL CDR2 sequence is selected from SEQ ID NOs: 20, 23, 26, 29, 32, or 35; and the VL CDR3 sequence is selected from SEQ ID NOs: 21, 24, 27, 30, 33, or 36.
An anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) according to SEQ ID NOs 1-3: and three variable light chain (VL) complementarity determining regions (CDRs) according to SEQ ID NOs: 19-21.
An anti-PD-1 antibody comprising three variable heavy chain (VH) complementarity determining regions (CDRs) according to SEQ ID NOs: 4-6 and three variable light chain (VL) complementarity determining regions (CDRs) according to SEQ ID NOs: 22-24.
An anti-PD-1 antibody comprising a variable heavy chain sequence and a variable light chain sequence pair selected from SEQ ID NOs: 38 and 40, 42 and 44, 46 and 48, 50 and 52, 54 and 56, 58 and 60.
An anti-PD-1 antibody comprising a variable heavy chain sequence selected from SEQ ID NOs: 62, 64, and 66 and a variable light chain sequence selected from SEQ ID NOs: 68, 70, 72, and 74.
An anti-PD-1 antibody comprising a variable heavy chain sequence selected from SEQ ID NOs: 76, 78, 80, and 82 and a variable light chain sequence selected from SEQ ID NOs: 84, 86, 88, 90, and 92.
A pharmaceutical composition comprising an anti-PD-1 antibody of claims 1-57, and a pharmaceutically acceptable excipient.
The pharmaceutical composition of claim 58, wherein the pharmaceutical composition is formulated for systemic administration.
The pharmaceutical composition of claim 58 or 59, wherein the pharmaceutical composition is formulated for parenteral administration.
An isolated nucleic acid molecule encoding the antibody of any one of the claims 1-57.
A vector comprising a nucleic acid sequence encoding the antibody of any one of the claims 1-57.
A host cell producing an anti-PD-1 antibody of claims 1-57.
A method of treating a cancer in a subject in need thereof, comprising administering to the subject an anti-PD-1 antibody of claims 1-57.
A method of augmenting cytokine production in a subject having a cancer, comprising contacting cells of the subject with an anti-PD-1 antibody of claims 1-57, thereby inducing an increase in the cytokine production relative to the cytokine production in cells of the same subject not contacted by the antibody.
The method of claim 65, wherein the cytokine is IL-2.
The method of claim 65, wherein the cytokine is INF-γ.
The method of any one of the claims 64-67, wherein the cancer is a solid tumor.
The method of any one of the claims 64-67, wherein the cancer is a hematologic malignancy.
The method of any one of the claims 64-69, wherein the cancer is bladder cancer, brain cancer, breast cancer, bladder cancer, bone cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, or uterine cancer.
The method of any one of the claims 64-69, wherein the cancer is a lymphoma.
The method of any one of the claims 64-69, wherein the cancer is Hodgkin’s lymphoma.
The method of any one of the claims 64-72, further comprising administering an additional therapeutic agent.
The method of any one of the claims 64-73, wherein the anti-PD-1 antibody and the additional therapeutic agent are administered sequentially.
The method of claim 74, wherein the anti-PD-1 antibody is administered to the subject prior to administering the additional therapeutic agent.
The method of claim 74, wherein the additional therapeutic agent is administered to the subject prior to administering the anti-PD-1 antibody.
The method of any one of the claims 64-73, wherein the anti-PD-1 antibody and the additional therapeutic agent are administered simultaneously.
The method of any one of the claims 64-77, wherein the anti-PD-1 antibody and the additional therapeutic agent are formulated as separate dosage forms.
A method of inducing phagocytosis of a cell expressing PD-L1 and/or PD-L2, comprising blocking binding of the cell to a T cell expressing PD-1 with an anti-PD-1 antibody of claims 1-57, thereby inducing phagocytosis of the cell.
The method of claim 79, wherein the cell is a cancerous cell.
The method of claim 80, wherein the cell is from a solid tumor.
The method of claim 80, wherein the cell is from a hematologic malignancy.
The method of any one of the claims 80-82, wherein the cell is from bladder cancer, brain cancer, breast cancer, bladder cancer, bone cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, or uterine cancer.
The method of any one of the claims 80-82, wherein the cell is from a lymphoma.
The method of any one of the claims 80-82, wherein the cell is from Hodgkin’s lymphoma.
The method of any of the preceding claims, wherein the subject is a human.
A kit comprising an anti-PD-1 antibody of claims 1-57 or a pharmaceutical composition of claims 58-60.