Antibody against programmed cell death protein 1

Abstract

Antibodies and antigen-binding fragments thereof are provided that immunospecifically bind to PD-1, preferably human or mouse PD-1, and induce or promote an immune response that activates immune cell proliferation and activity. Contrary to the existing paradigm that PD-1 only promotes suppressive immune responses, the disclosed antibodies and antigen-binding fragments thereof immunospecifically bind to PD-1 and deliver an activation signal to immune cells that activates rather than suppresses them. In one embodiment, the disclosed antibodies and antigen-binding fragments thereof specifically bind to PD-1 expressed on immune cells. Binding of the disclosed antibodies and antigen-binding fragments thereof to PD-1 on immune cells transduces an activation signal into the immune cell, e.g., a signal that enhances or promotes activation of cytokine production and / or immune cell proliferation. Immune cells that express PD-1 include, but are not limited to, B and T cells, and bone marrow-derived cells. In one embodiment, the immune cells are T cells, preferably CD8+ T cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 62 / 555,156, filed September 7, 2017, 62 / 624,843, filed February 1, 2018, and 62 / 657,323, filed April 13, 2018, all of which are incorporated by reference in their entirety.

[0002] Sequence Listing Reference The Sequence Listing, submitted on September 6, 2018 as a text file named "064466.071 sequence listing_ST25.txt," created on August 21, 2018, and having a size of 55.2 kilobytes, is hereby incorporated by reference pursuant to 37 CFR 1.52(e)(5).

[0003] Technical field of the invention The present invention relates generally to immunomodulation and to antibodies that specifically bind to PD-1 and methods of their use. [Background technology]

[0004] Background of the Invention The programmed death receptor protein (PD-1) / programmed death receptor protein ligand 1 (PD-L1) pathway has shown promising clinical success as a target for cancer immunotherapy. Current antibodies targeting either PD-1 or PD-L1 can block this interaction and enhance immune responses against cancer cells. Successful clinical trials using PD-1 monoclonal antibodies and other immune checkpoint inhibitors have paved new paths in cancer immunology. However, a large subset of cancer patients fails to respond to new immunotherapies, leading to intensified research into combination therapies and predictive biomarkers (Iwai, Y., et al., Journal of Biomedical Science, 24:26 (2017)).

[0005] Accordingly, it is an object of the present invention to provide compositions and methods for modulating PD-1 signaling.

[0006] Another object of the present invention is to provide antibodies and antigen-binding fragments thereof that specifically bind to PD-1 and modulate PD-1 signaling.

[0007] Another object of the present invention is to provide compositions and methods for treating cancer.

[0008] Another object of the present invention is to provide compositions and methods for treating infectious diseases. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Iwai, Y., et al., Journal of Biomedical Science, 24:26 (2017) Summary of the Invention

[0010] Antibodies and antigen-binding fragments thereof are provided that immunospecifically bind to PD-1, preferably human or mouse PD-1, and induce or promote an immune response that activates immune cell proliferation and activity. In one embodiment, the disclosed antibodies and antigen-binding fragments thereof specifically bind to PD-1 expressed on immune cells. Binding of the disclosed antibodies and antigen-binding fragments thereof to PD-1 on immune cells transduces an activation signal into the immune cell, e.g., a signal that enhances or promotes activation of cytokine production and / or immune cell proliferation. Immune cells that express PD-1 include, but are not limited to, B and T cells, and bone marrow-derived cells (Riley, J., Immunol Rev. 229(1):114-125(2009)). In one embodiment, the immune cell is a T cell, preferably a CD8+ T cell.

[0011] Another embodiment provides a method of stimulating, enhancing, or enhancing an adaptive immune response in a subject in need thereof by administering to the subject an effective amount of the disclosed anti-PD-1 antibodies or antigen-binding fragments thereof to elicit, enhance, or promote an adaptive immune response in the subject.

[0012] One embodiment provides an antibody or antigen-binding fragment thereof having heavy chain complementarity-determining regions (CDRs) having the amino acid sequences set forth in SEQ ID NOs:6, 7, and 8, and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs:12, 13, and 14, wherein the antibody or antigen-binding fragment thereof immunospecifically binds to PD-1.

[0013] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:4 or 5.

[0014] One embodiment provides an antibody or antigen-binding fragment thereof having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:10 or 11.

[0015] One embodiment provides an antibody, or any antigen-binding fragment thereof, having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:4 or 5, and a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:10 or 11.

[0016] One embodiment provides a transgenic animal engineered to express any one of the disclosed antibodies or antigen-binding fragments thereof. In one embodiment, the animal is a mouse.

[0017] One embodiment provides a nucleic acid encoding a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:4 or 5.

[0018] One embodiment provides a nucleic acid encoding a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:10 or 11.

[0019] One embodiment provides an antibody or antigen-binding fragment thereof having heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 24, 13, and 25.

[0020] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:16 or 17.

[0021] One embodiment provides an antibody or antigen-binding fragment thereof having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:22 or 23.

[0022] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 16 or 17 and a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 22 or 23.

[0023] One embodiment provides a nucleic acid encoding a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:16 or 17.

[0024] One embodiment provides a nucleic acid encoding a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:22 or 23.

[0025] One embodiment provides an antibody or antigen-binding fragment thereof having heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs:29, 30, and 31, and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs:35, 36, and 37.

[0026] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:27 or 28.

[0027] One embodiment provides an antibody or antigen-binding fragment thereof having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:33 or 34.

[0028] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:27 or 28 and a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:33 or 34.

[0029] One embodiment provides a nucleic acid encoding a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:27 or 28.

[0030] One embodiment provides a nucleic acid encoding a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:33 or 34.

[0031] One embodiment provides an antibody, or antigen-binding fragment thereof, containing three light chain CDRs having amino acid sequences selected from the group consisting of SEQ ID NO: 12, 13, 14, 24, 25, 35, 36, or 37.

[0032] Another embodiment provides an antibody, or antigen-binding fragment thereof, containing three heavy chain CDRs having amino acid sequences selected from the group consisting of SEQ ID NOs: 6, 7, 8, 18, 19, 20, 29, 30, or 31.

[0033] Another embodiment provides an antibody, or antigen-binding fragment thereof, containing three light chain CDRs having amino acid sequences selected from the group consisting of SEQ ID NO: 12, 13, 14, 24, 25, 35, 36, or 37, and three heavy chain CDRs having amino acid sequences selected from the group consisting of SEQ ID NO: 6, 7, 8, 18, 19, 20, 29, 30, or 31.

[0034] One embodiment provides an antibody or epitope-binding fragment thereof, or a fusion protein that immunospecifically binds to SEQ ID NO:38. In one embodiment, the antibody binds to SEQ ID NO:38 on PD-1. In one embodiment, the antibody binds to PD-1 expressed on the surface of an immune cell and induces or promotes a signal through PD-1 that activates or stimulates the immune cell. In one embodiment, the activated or stimulated immune cell is a T cell, e.g., a CD8 +T cells.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof is human, murine, chimeric, humanized, monoclonal, bispecific, trispecific, or multispecific.

[0036] One embodiment provides a pharmaceutical composition comprising one or more of the disclosed antibodies or antigen-binding fragments thereof. In some embodiments, the pharmaceutical composition comprises a second therapeutic agent and / or a pharmaceutically acceptable excipient. An exemplary second therapeutic agent includes cyclophosphamide.

[0037] One embodiment provides a method of eliciting, promoting, or enhancing an immune response in a subject in need thereof by administering to the subject an effective amount of one or more of the disclosed antibodies or antigen-binding fragments thereof to elicit, promote, or enhance the immune response in the subject.

[0038] One embodiment provides a method of treating cancer in a subject in need thereof by administering to the subject an effective amount of one or more of the disclosed antibodies or antigen-binding fragments thereof to treat the cancer in the subject.

[0039] One embodiment provides a method of reducing tumor burden in a subject in need thereof by administering to the subject an effective amount of one or more of the disclosed antibodies or antigen-binding fragments thereof to reduce tumor burden in the subject.

[0040] One embodiment provides a method of treating an infectious disease in a subject in need thereof by administering to the subject an effective amount of one or more of the disclosed antibodies or antigen-binding fragments thereof to treat the infectious disease in the subject. [The present invention 1001] heavy chain complementarity determining regions (CDRs) having the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8; light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14; An antibody or antigen-binding fragment thereof comprising: [The present invention 1002] 1001. An antibody or antigen-binding fragment thereof of the present invention, comprising a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4 or 5. [The present invention 1003] The antibody or antigen-binding fragment thereof of the present invention 1001 or 1002, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 10 or 11. [The present invention 1004] a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4 or 5; a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 10 or 11; An antibody or antigen-binding fragment thereof comprising: [The present invention 1005] A transgenic animal engineered to express any one of the antibodies or antigen-binding fragments thereof of the present inventions 1001 to 1004. [The present invention 1006] The transgenic animal of the present invention 1005, which is a rodent. [The present invention 1007] The transgenic animal of the present invention 1006, wherein the rodent is a mouse. [The present invention 1008] A nucleic acid encoding a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4 or 5. [The present invention 1009] A nucleic acid encoding a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 10 or 11. [The present invention 1010] heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20; light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 24, 13, and 25; An antibody or antigen-binding fragment thereof comprising: [The present invention 1011] 10. The antibody or antigen-binding fragment thereof of the present invention 1010, comprising a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 16 or 17. [The present invention 1012] The antibody or antigen-binding fragment thereof of the invention 1010 or 1011, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 22 or 23. [The present invention 1013] a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 16 or 17; a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 22 or 23; An antibody or antigen-binding fragment thereof comprising: [The present invention 1014] A transgenic animal engineered to express any one of the antibodies or antigen-binding fragments thereof of the present invention. [The present invention 1015] The transgenic animal of the present invention 1014, which is a rodent. [The present invention 1016] The transgenic animal of the present invention 1015, wherein the rodent is a mouse. [The present invention 1017] A nucleic acid encoding a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 16 or 17. [The present invention 1018] A nucleic acid encoding a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 22 or 23. [The present invention 1019] heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 29, 30, and 31; light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 35, 36, and 37; An antibody or antigen-binding fragment thereof comprising: [The present invention 1020] 1019. An antibody or antigen-binding fragment thereof of the present invention, comprising a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 27 or 28. [The present invention 1021] The antibody or antigen-binding fragment thereof of the invention 1019 or 1020, comprising a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or 34. [The present invention 1022] a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 27 or 28; a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or 34; An antibody or antigen-binding fragment thereof comprising: [The present invention 1023] A transgenic animal engineered to express any one of the antibodies of the present invention 1019 to 1022 or an antigen-binding fragment thereof. [The present invention 1024] A transgenic animal of the present invention 1023 that is a rodent. [The present invention 1025] The transgenic animal of the present invention 1024, wherein the rodent is a mouse. [The present invention 1026] A nucleic acid encoding a heavy chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 27 or 28. [The present invention 1027] A nucleic acid encoding a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or 34. [The present invention 1028] Any of 1001 to 1004, 1010 to 1013, or 1019 to 1022 antibodies or antigen-binding fragments thereof of the present invention, which are human, murine, chimeric, humanized, monoclonal, bispecific, trispecific, or multispecific. [The present invention 1029] An antibody or antigen-binding fragment thereof comprising three light chain CDRs having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 13, 14, 24, 25, 35, 36, or 37. [The present invention 1030] An antibody or antigen-binding fragment thereof comprising three heavy chain CDRs having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 7, 8, 18, 19, 20, 29, 30, or 31. [The present invention 1031] three light chain CDRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 24, 25, 35, 36, or 37; three heavy chain CDRs having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 18, 19, 20, 29, 30, or 31; An antibody or antigen-binding fragment thereof comprising: [The present invention 1032] An antibody or epitope-binding fragment thereof, or a fusion protein that immunospecifically binds to SEQ ID NO:38. [The present invention 1033] The antibody or epitope-binding fragment thereof or fusion protein of the present invention 1032, wherein the immune cell is a T cell. [The present invention 1034] The T cells are CD8 + The antibody or epitope-binding fragment thereof or fusion protein of the present invention 1033, which is a T cell. [This invention 1035] The antibody or epitope-binding fragment thereof, or fusion protein of the present invention 1032, wherein the antibody or antigen-binding fragment thereof, or fusion protein binds to PD-1. [The present invention 1036] An antibody or epitope-binding fragment thereof, or a fusion protein that immunospecifically binds to SEQ ID NO: 38 on PD-1 expressed on the surface of an immune cell, and induces or promotes a signal through PD-1 that activates or stimulates the immune cell. [This invention 1037] A pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof of the present invention 1001 to 1004, 1010 to 1013, 1019 to 1022, or 1029 to 1036. [The present invention 1038] The pharmaceutical composition of invention 1037, further comprising a second therapeutic agent. [This invention 1039] The pharmaceutical composition of any one of claims 1037 to 1038, further comprising a pharmaceutically acceptable excipient. [The present invention 1040] The pharmaceutical composition of claim 1038, wherein the second therapeutic agent comprises cyclophosphamide. [The present invention 1041] 1. A method for inducing, promoting, or enhancing an immune response in a subject in need thereof, comprising: The method comprises the step of administering to the subject an effective amount of any one of the antibodies or antigen-binding fragments thereof of 1001 to 1004, 1010 to 1013, 1019 to 1022, or 1029 to 1036 of the present invention, or any one of the pharmaceutical compositions of 1037 to 1040 of the present invention, to induce, promote, or enhance an immune response in the subject. [The present invention 1042] 1. A method for treating cancer in a subject in need thereof, comprising: The method comprises the step of administering to the subject an effective amount of any one of the antibodies or antigen-binding fragments thereof of 1001 to 1004, 1010 to 1013, 1019 to 1022, or 1029 to 1036 of the present invention, or any one of the pharmaceutical compositions of 1037 to 1040 of the present invention, to treat cancer in the subject. [This invention 1043] 1. A method for reducing tumor burden in a subject in need thereof, comprising: The method comprises the step of administering to the subject an effective amount of any one of the antibodies or antigen-binding fragments thereof of 1001 to 1004, 1010 to 1013, 1019 to 1022, or 1029 to 1036 of the present invention, or any one of the pharmaceutical compositions of 1037 to 1040 of the present invention, to reduce tumor burden in the subject. [This invention 1044] 1. A method for treating an infectious disease in a subject in need thereof, comprising: The method comprises the step of administering to the subject an effective amount of any one of the antibodies or antigen-binding fragments thereof of 1001 to 1004, 1010 to 1013, 1019 to 1022, or 1029 to 1036 of the present invention, or any one of the pharmaceutical compositions of 1037 to 1040 of the present invention, to treat the infection in the subject. [Brief explanation of the drawings]

[0041] [Figure 1] 1 is a graph showing the interaction kinetics between monoclonal antibody 4G9 and human PD-1 as a function of time. The graph shows traces from concentrations of human PD-1 at 0, 125, 250, 500, 500, and 1000 nM. [Figure 2]1 is a graph showing the interaction kinetics between monoclonal antibody 4G9 and mouse PD-1 as a function of time. The graph shows traces from concentrations of mouse PD-1 at 0, 62.5, 125, 500, 500, and 1000 nM. [Figure 3] 1 is a graph showing the interaction kinetics between monoclonal antibody 4C12 and human PD-1 as a function of time. The graph shows traces from concentrations of human PD-1 at 0, 125, 250, 500, 500, and 1000 nM. [Figure 4] 1 is a graph showing the interaction kinetics between monoclonal antibody 5C2 and human PD-1 as a function of time. The graph shows traces from concentrations of mouse PD-1 at 0 and 1000 nM. [Figure 5] 1 is a graph showing the interaction kinetics between monoclonal antibody 5C2 and mouse PD-1 as a function of time. The graph shows traces from concentrations of human PD-1 at 0, 62.5, 125, 250, 500, 500, and 1000 nM. [Figure 6A] Flow cytometry histograms of EL4 cells stained with an isotype control antibody or the commercial anti-PD-1 antibody J43. [Figure 6B] 10 is a flow cytometry histogram of EL4 cells stained with secondary antibody alone or antibodies 4G9, 5C2, and 4C12. [Figure 6C] 10 is a flow cytometry histogram of El4 cells stained with secondary antibody alone or antibody 4G9. [Figure 6D] 10 is a flow cytometry histogram of El4 cells stained with secondary antibody alone or antibody 5C2. [Figure 6E] 10 is a flow cytometry histogram of El4 cells stained with secondary antibody alone or antibody 4C12. [Figure 6F] 1 is a bar graph showing binding of various purified mouse PD-1 antibodies to EL4 cells. [Figure 7A]Figures 7A and 7B are bar graphs showing the concentration of IFNγ (Figure 7A) or IL-2 in supernatants from CD4 T cells treated with various antibodies. The X-axis is treatment group, and the Y-axis is concentration (ng / mL). [Figure 7B] See legend to Figure 7A. [Figure 7C] 1 is a bar graph showing IFNγ concentrations in supernatants from human CD4 T cells treated with 4G9 or 5C2 antibodies. The X-axis represents treatment group and the Y-axis represents concentration (ng / mL). [Figure 8] 1 is a bar graph showing the level of intracellular staining of pAKT in mouse CD4 T cells treated with various antibodies. The X-axis represents treatment group, and the Y-axis represents pAKT(S473) MFI. [Figure 9] 1 is a Western blot showing IgG heavy and light chains in various antibodies. [Figure 10] 1 is a bar graph showing the binding of 4G9 and 5C2 antibodies to human PD-1-Fc. The X-axis represents antibody concentration, and the Y-axis represents OD450. [Figure 11] 11A and 11B are flow cytometry histograms showing binding of 4G9 and 5C2 antibodies to PD-1 on CD4 T cells from PD-1 KO mice (FIG. 11A) or PD-1 WT mice (FIG. 11B). [Figure 12] 1 is a line graph showing pS6 expression in CD4 T cells treated with 4G9, 5C2, commercial Ab-1, commercial Ab-2, or untreated. The X-axis represents total protein concentration (μg / mL), and the Y-axis represents OD450. [Figure 13A] FIG. 1 is a schematic diagram showing the experimental design for the TC-1 tumor experiments. [Figure 13B] 1 is a line graph showing the mean tumor volume (cm3) over time (days) for TC-1 tumor-bearing mice treated with E7 Vax, 4G9, RMP 1-14, E7 Vax + RMP 1-14, E7 Vax + 4G9, or untreated. The X-axis represents time (days) and the Y-axis represents the mean tumor volume (cm3). [Figure 13C]1 is a line graph showing survival over time for TC-1 tumor-bearing mice treated with E7 Vax, 4G9, RMP 1-14, E7 Vax+RMP 1-14, E7 Vax+4G9, or untreated. [Figure 13D] A line graph showing the mean tumor volume (cm3) over time (days) for TC-1 tumor-bearing mice treated with E7 Vax, 4C12, 5C2, RMP 1-14, E7 Vax+RMP 1-14, E7 Vax+4C12, E7 Vax+5C2, or untreated. [Figure 14A] FIG. 1 is a schematic diagram of the experimental design for the TC-1 tumor experiments. [Figure 14B] 1 is a line graph showing the mean tumor volume (cm3) over time (days) for TC-1 tumor-bearing mice treated with E7 Vax, 4G9, RMP 1-14, J43, E7 Vax+4G9, E7 Vax+RMP 1-14, E7 Vax+J43, or untreated. The X-axis represents time (days) and the Y-axis represents the mean tumor volume (cm3). [Figure 14C] 1 is a line graph showing the survival rate over time of TC-1 tumor-bearing mice treated with E7 Vax, 4G9, 4C12, 5C2, RMP 1-14, J43, E7 Vax+4G9, E7 Vax+4C12, E7 Vax+5C2, E7 Vax+RMP 1-14, E7 Vax+J43, or untreated. The x-axis represents time (days), and the y-axis represents survival rate. DETAILED DESCRIPTION OF THE INVENTION

[0042] Detailed Description of the Invention I. Definition As used herein, a molecule is said to be capable of "immunospecifically binding" to a second molecule if such binding exhibits the specificity and affinity of the antibody for its cognate antigen. An antibody is said to be capable of immunospecifically binding to a target region or conformation ("epitope") of an antigen if such binding involves a recognition site on an immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind other antigens with lower affinity, but not to completely unrelated antigens, if the other antigens have some sequence or conformational similarity recognized by the antigen recognition site, as determined, for example, by immunoassay, BIACORE® assay, or other assays known in the art. However, preferably, antibodies (and their antigen-binding fragments) do not cross-react with other antigens. Antibodies may also bind other molecules in a non-immunospecific manner, such as FcR receptors, via binding domains in other regions / domains of the molecule that do not involve an antigen recognition site, such as the Fc region.

[0043] As used herein, a molecule is said to "physiologically specifically bind" to a second molecule if such binding exhibits the specificity and affinity of a receptor for its cognate binding ligand. A molecule may be capable of physiologically specifically binding to two or more other molecules.

[0044] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule having a "variable region" antigen recognition site. The term "variable region" is intended to distinguish such domains of an immunoglobulin from domains broadly shared by antibodies (such as the antibody Fc domain). The variable region includes "hypervariable regions" whose residues are responsible for antigen binding. The hypervariable regions comprise amino acid residues from the "complementarity-determining regions" or "CDRs" (i.e., typically approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and approximately residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or residues from the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light-chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.The term antibody includes monoclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (see, e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; U.S. Pat. No. 6,005,079), single-chain Fvs (scFv) (see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, 1999; New York, pp. 269-315 (1994)), single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id and anti-anti-Id antibodies against antibodies). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0045] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that contain the antibody's complementarity-determining regions ("CDRs") and, optionally, framework residues comprising the antibody's "variable region" antigen recognition site, and that exhibit the ability to immunospecifically bind to an antigen. Such fragments include Fab', F(ab'), Fv, single-chain (ScFv), and mutants, naturally occurring variants thereof, as well as fusion proteins comprising the antibody's "variable region" antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor, or a receptor ligand, etc.).

[0046] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0047] The term "binding molecule," as used herein, is intended to refer to a molecule that specifically interacts with and binds to a particular target. Targets can include biological molecules or small (chemical) molecules. Target molecules can define antigens or antigenic portions. Examples of binding molecules include, but are not limited to, antibodies (including monoclonal antibodies, bispecific antibodies, and antibody fragments), fusion proteins, and other antigen-binding molecules known to those skilled in the art.

[0048] As used herein, the term "modulate" refers to the ability to alter an effect, outcome, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuating rather than neutralizing) or can completely abolish (e.g., neutralize) such activity. Modulation can include receptor internalization after antibody binding or reducing receptor expression on the target cell. Agonistic modulation can enhance or otherwise increase or potentiate an activity (e.g., signal transduction). In still further embodiments, such modulation can alter the nature of the interaction between a ligand and its cognate receptor to alter the nature of the induced signal transduction. For example, a molecule, by binding to a ligand or receptor, can alter the ability of such molecules to bind to other ligands or receptors, thereby altering their overall activity. Preferably, such modulation will provide at least a 10% change in measurable immune system activity, more preferably at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or even more preferably at least a 100-fold change in such activity.

[0049] The term "substantially," when used in the context of binding or an effect exhibited, is intended to indicate that the observed effect is physiologically or therapeutically significant. Thus, for example, a molecule can substantially block the activity of a ligand or receptor if the degree of blockage is physiologically or therapeutically significant (e.g., if such degree is greater than 60% complete, greater than 70% complete, greater than 75% complete, greater than 80% complete, greater than 85% complete, greater than 90% complete, greater than 95% complete, or greater than 97% complete). Similarly, a molecule is said to have substantially the same immunospecificity and / or characteristics as another molecule if such immunospecificity and characteristics are greater than 60% identity, greater than 70% identity, greater than 75% identity, greater than 80% identity, greater than 85% identity, greater than 90% identity, greater than 95% identity, or greater than 97% identity).

[0050] As used herein, a "costimulatory" signal encompasses positive costimulatory signals (eg, signals that result in an enhancement of an activity) and negative costimulatory signals (eg, signals that result in an inhibition of an activity).

[0051] As used herein, the term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to the same target as a parent or reference antibody, but differs in amino acid sequence from the parent or reference antibody or antigen-binding fragment thereof by including substitutions, additions, deletions, or modifications of one, two, three, four, five, or more amino acid residues relative to the parent or reference antibody or antigen-binding fragment thereof. Preferably, such a derivative will have substantially the same immunospecificity and / or characteristics as the parent or reference antibody or antigen-binding fragment thereof, or the same immunospecificity and characteristics. The amino acid substitutions or additions of such derivatives can include naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" encompasses, for example, chimeric or humanized variants, as well as variants with altered CH1, hinge, CH2, CH3, or CH4 regions, to form, for example, antibodies with variant Fc regions exhibiting enhanced or diminished effector or binding characteristics.

[0052] As used herein, a "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.

[0053] As used herein, the term "humanized antibody" refers to an immunoglobulin containing a human framework region and one or more CDRs from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-99%, preferably about 95% or more identical. Thus, all portions of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding portions of native human immunoglobulin sequences. A humanized antibody is an antibody containing a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, since the entire variable region of a chimeric antibody is non-human.

[0054] As used herein, the term "endogenous concentration" refers to the level at which a molecule is naturally expressed (i.e., in the absence of an expression vector or recombinant promoter) by a cell (which may be a normal cell, a cancer cell, or an infected cell).

[0055] As used herein, the terms "treat," "treating," "treatment," and "therapeutic use" refer to the elimination, reduction, or amelioration of one or more symptoms of a disease or disorder exacerbated by an anti-PD-1 antibody or antigenic fragment thereof.

[0056] As used herein, "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to mediate clinically significant elimination, reduction, or improvement of such symptoms. An effect is clinically significant if its magnitude is sufficient to affect the health or prognosis of the recipient subject. A therapeutically effective amount can refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of disease, for example, to delay or minimize the spread of cancer. A therapeutically effective amount can also refer to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of disease.

[0057] As used herein, the term "prophylactic agent" refers to an agent that can be used to prevent a disorder or disease prior to the detection of any symptoms of such disorder or disease. A "prophylactically effective" amount is the amount of a prophylactic agent sufficient to mediate such protection. A prophylactically effective amount can also refer to the amount of a prophylactic agent that provides a prophylactic benefit in the prevention of disease.

[0058] As used herein, the term "cancer" refers to a neoplasm or tumor resulting from the abnormal, uncontrolled growth of cells. As used herein, cancer explicitly includes leukemia and lymphoma. The term "cancer" refers to a disease involving cells that have the potential to metastasize to distant sites and exhibit phenotypic traits different from those of non-cancerous cells, such as the formation of colonies in a three-dimensional substrate such as soft agar, or the formation of a tubular network or web-like matrix in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancerous cells do not form colonies in soft agar, but form distinct sphere-like structures in a three-dimensional basement membrane or extracellular matrix preparation.

[0059] As used herein, "immune cell" refers to any cell of hematopoietic origin, including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.

[0060] As used herein, "valency" refers to the number of available binding sites per molecule.

[0061] As used herein, the terms "immune," "immunological," or "immune" response refer to the development in a recipient patient of a beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) response to a peptide. Such a response can be an active response elicited by administration of an immunogen, or a passive response elicited by administration of antibodies or primed T cells. A cellular immune response is defined as a response to antigen-specific CD4 + T helper cells and / or CD8 +The presence of a cell-mediated immunological response is induced by the presentation of polypeptide epitopes in association with class I or class II MHC molecules to activate cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, and the activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 + The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0062] As used herein, an "immunogenic agent" or "immunogen" is capable of eliciting an immunological response against itself after administration to a mammal, optionally with an adjuvant.

[0063] As used herein, the terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans, rodents, e.g., mice and rats, and other laboratory animals.

[0064] As used herein, the term "polypeptide" refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. Polypeptides may be "exogenous," meaning they are "heterologous," i.e., foreign, to the host cell in which they are used, such as human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as amino acid residue sequences. The sequences are written from left to right in the direction from amino acid to carboxy-terminus. In accordance with standard nomenclature, amino acid residue sequences are referred to by either three-letter or one-letter codes as shown below. Alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0065] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be a naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0066] Modifications and changes can be made in the structure of the disclosed polypeptides to obtain molecules that still have similar characteristics to the polypeptides (e.g., conservative amino acid substitutions). For example, certain amino acids can be substituted for other amino acids in the sequence without significant loss of activity. Because the biological functional activity of a polypeptide is defined by the polypeptide's ability to interact and properties, certain amino acid sequence substitutions can be made in the polypeptide sequence to still obtain polypeptides with similar properties.

[0067] When making such changes, the hydropathic index of amino acids can be taken into consideration. The importance of the hydropathic amino acid index in providing interactive biological function to a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted with other amino acids having similar hydropathic indexes or scores, still resulting in polypeptides with similar biological activity. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), aspartate (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0068] The relative hydropathic characteristics of amino acids are believed to determine the secondary structure of the resulting polypeptide, which in turn defines the polypeptide's interactions with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted with another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, substitution of amino acids whose hydropathic index is within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0069] Similar amino acid substitutions based on hydrophilicity may also be made, particularly when the resulting biologically functional equivalent polypeptides or peptides are intended for use in immunological embodiments. The following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartate (+3.0±1), glutamate (+3.0±1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), proline (-0.5±1), threonine (-0.4), alanine (-0.5), histidine (-0.5), cysteine ​​(-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and particularly an immunologically equivalent, polypeptide. In such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0070] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various above characteristics are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg:Lys), (Asn:Gln, His), (Asp:Glu, Cys, Ser), (Gln:Asn), (Glu:Asp), (Gly:Ala), (His:Asn, Gln), (Ile:Leu, Val), (Leu:Ile, Val), (Lys:Arg), (Met:Leu, Tyr), (Ser:Thr), (Thr:Ser), (Tip:Tyr), (Tyr:Trp, Phe), and (Val:Ile, Leu). Thus, embodiments of the present disclosure contemplate functional or biological equivalents of such polypeptides. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of interest.

[0071] The term "sequence identity percentage (%)" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and optionally introducing gaps to achieve the maximum sequence identity percentage.The alignment for determining the sequence identity percentage can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequence to be compared, can be determined by known methods.

[0072] For purposes herein, the % sequence identity of a given nucleotide or amino acid sequence C to, with, or against a given nucleic acid sequence D (which may alternatively be expressed as a given sequence C having or containing a certain % sequence identity to, with, or against a given sequence D) is calculated as follows: Fraction W / Z×100 where W is the number of nucleotides or amino acids that a sequence alignment program will score as a perfect match in its alignment of C and D, and Z is the total number of nucleotides or amino acids in D. It will be understood that if the length of sequence C is not equal to the length of sequence D, then the % sequence identity of C to D will not be equal to the % sequence identity of D to C.

[0073] As used herein, the term "pharmaceutically acceptable carrier" includes standard pharmaceutical carriers such as phosphate buffered saline, water, and emulsions, such as oil / water or water / oil emulsions, as well as any of the various types of wetting agents.

[0074] As used herein, the terms "antigenic determinant" and "epitope" are used interchangeably and refer to the structure recognized by an antibody.

[0075] As used herein, a "conformational epitope" is an epitope that comprises a discontinuous section of the amino acid sequence of an antigen. Antibodies bind to conformational epitopes based on the 3D surface features, shape, or tertiary structure of the antigen.

[0076] As used herein, a "linear epitope" is an epitope formed by a contiguous sequence of amino acids from an antigen. A linear epitope typically contains about 5 to about 10 contiguous amino acid residues. An antibody binds to a linear epitope based on the primary sequence of the antigen.

[0077] As used herein, a "paratope," also called an "antigen-binding site," is the part of an antibody that recognizes and binds to an antigen.

[0078] II. Composition Antibodies and antigen-binding fragments thereof that immunospecifically bind to PD-1 are provided. Contrary to the existing paradigm that PD-1 only promotes suppressive immune responses (Riley, J., Immunol Rev. 229(1):114-125(2009)), the disclosed antibodies and antigen-binding fragments thereof immunospecifically bind to PD-1 and deliver an activation signal to immune cells that activates rather than suppresses them.

[0079] A. Programmed death receptor protein 1 (PD-1) The disclosed antibodies and antigen-binding fragments thereof immunospecifically bind to PD-1. The antibodies and antigen-binding fragments thereof can bind to PD-1, for example, having the amino acid sequences provided below.

[0080] The amino acid sequences of human PD-1 and mouse PD-1 are known in the art and include, for example:

[0081] Human PD-1 TIFF0007795860000001.tif41165 Accession: AJS10360, which is specifically incorporated herein by reference in its entirety.

[0082] Mouse PD-1 TIFF0007795860000002.tif42165, which is specifically incorporated herein by reference in its entirety.

[0083] B. Antibody composition The disclosed anti-PD-1 antibodies or antigen-binding fragments thereof include whole immunoglobulins of any class (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. In some embodiments, the disclosed antibodies contain both an antibody light chain and at least the variable domain of an antibody heavy chain. In other embodiments, such molecules can further comprise one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain (particularly the CH1 and hinge regions, or the CH1, hinge, and CH2 regions, or the CH1, hinge, CH2, and CH3 regions). The antibody can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the constant domain is a complement-fixing constant domain when it is desired that the antibody exhibit cytotoxic activity, and the class is typically IgG1. In other embodiments, when such cytotoxic activity is not desired, the constant domain can be of the IgG2 or IgG4 class. Antibodies can comprise sequences from more than one class or isotype, and selection of particular constant domains to optimize desired effector functions is within the skill of the art.

[0084] The variable domains vary in sequence among antibodies and are responsible for the binding and specificity of each particular antibody to its specific antigen. However, variability is not usually uniformly distributed throughout the variable domains of antibodies. It is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). Natural heavy-chain and light-chain variable domains each contain four FR regions that primarily adopt a beta-sheet structure connected by three CDRs, which form loops that connect, and in some cases form part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.

[0085] Some embodiments provide biologically active fragments of anti-PD-1 antibodies, which may contain insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not attached to other sequences, provided that the activity of the fragment is not significantly altered or diminished compared to the unmodified antibody or antibody fragment.

[0086] Another embodiment provides a single-chain antibody specific for PD-1. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be made by fusing heavy and light chain variable domains together using a short peptide linker, thereby reconstituting an antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs), in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain by a 15-25 amino acid peptide or linker, have been developed without significantly disrupting antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind together in their proper conformational orientation.

[0087] Another embodiment provides a bivalent single-chain variable fragment (di-scFv) that can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, resulting in a tandem scFv. ScFvs can also be designed with a linker peptide (approximately five amino acids) that is too short for the two variable regions to fold together, causing the scFvs to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning they also have much higher affinity for their targets. Even shorter linkers (one or two amino acids) result in the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit even higher affinity for their targets than diabodies.

[0088] Another embodiment provides a monoclonal antibody specific for PD-1 that induces an activation signal in immune cells. Monoclonal antibodies can be obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity.

[0089] 1. Chimeric and humanized antibodies Another embodiment provides chimeric anti-PD-1 antibodies and antigen-binding fragments thereof comprising one or more of the disclosed sequences, and functional variants thereof that bind to PD-1 and transmit an activation signal to immune cells that express PD-1 are also provided.

[0090] Methods for producing chimeric antibodies are known in the art (see, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397). Chimeric antibodies comprising one or more CDRs from a non-human species and a framework region from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400, International Publication No. WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106, EP 519,596, Padlan, 1991, Molecular Immunology 28(4 / 5):489-498, Studnicka et al., 1994, Protein Engineering 7:805, and Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969), and chain shuffling (U.S. Pat. No. 5,565,332).

[0091] The disclosed anti-PD-1 antibodies or antigen-binding fragments thereof can be human or humanized antibodies, or antigen-binding fragments thereof. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and therefore may induce undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods helps reduce the likelihood that antibodies administered to humans will induce undesirable immune responses.

[0092] Transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production after immunization can be used. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies after antigen challenge.

[0093] Optionally, antibodies are generated in other species and "humanized" for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies comprise human immunoglobulins (recipient antibodies) in which residues from the complementarity-determining regions (CDRs) of the recipient antibody are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will contain 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 consensus sequence. The humanized antibody optimally will also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0094] Methods for humanizing non-human antibodies are well known in the art and are described, for example, in European Patent Nos. EP 239,400, EP 592,106, and EP 519,596, International Publication Nos. WO 91 / 09967 and WO 93 / 17105, U.S. Pat. Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886, and 6,407,213, Padlan, 1991, Molecular Immunology 28(4 / 5):489-498, Studnicka et al., 1994, Protein Engineering 7(6):805-814, Roguska et al., 1994, PNAS 91:969-973, Tan et al.,2002, J.Immunol.169:1119-1125, Caldas et al.,2000, Protein Eng.13:353-360, Morea et al.,2000,Methods 20:267-79, Baca et al. al.,1997, J.Biol.Chem.272:10678-10684, Roguska et al.,1996, Protein Eng.9:895-904, Couto et al.,1995, Cancer Res.55(23 Supp):5973s-5977s, Couto et al.,1995,Cancer Res.55:1717-22, Sandhu, 1994, Gene 150:409-10; Pedersen et al., 1994, J. Mol. Biol. 235:959-973; Jones et al., 1986, Nature 321:522-525; Reichmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

[0095] Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which typically come from an "import" variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can essentially be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, humanized forms of non-human antibodies (or fragments thereof) are chimeric antibodies or fragments in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0096] The selection of both light and heavy human variable domains used to create a humanized antibody can be crucial to reducing antigenicity. According to the "best fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework (FR) for the humanized antibody. Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies.

[0097] It is further important that antibodies be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display the probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the potential role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this method, FR residues can be selected and combined from consensus and import sequences to achieve the desired antibody characteristic, e.g., increased affinity for the target antigen(s). In general, CDR residues are directly and most substantially involved in influencing antigen binding.

[0098] A human, humanized, or chimeric antibody derivative can comprise substantially all of at least one, typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (donor antibody) and all or substantially all of the framework regions are FR regions of a human immunoglobulin consensus sequence. Such antibodies can also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. The constant domain of such antibodies can be selected with regard to the proposed function of the antibody, particularly any effector functions that may be required. In some embodiments, the constant domain of such antibodies can be or comprise a human IgA, IgD, IgE, IgG, or IgM domain. In certain embodiments, human IgG constant domains, particularly of the IgG1 and IgG3 isotypes, are used when the humanized antibody derivative is intended for therapeutic use and antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, are required. In alternative embodiments, IgG2 and IgG4 isotypes are used for therapeutic purposes and when antibody effector function is not required. Fc constant domains comprising one or more amino acid modifications that alter antibody effector function, such as those disclosed in U.S. Patent Application Publication Nos. 2005 / 0037000 and 2005 / 0064514.

[0099] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences; for example, the donor CDR or consensus framework may be mutated by substitution, insertion, or deletion of at least one residue such that the CDR or framework residue at that site corresponds to neither the consensus nor the donor antibody. In some embodiments, such mutations are not extensive. Typically, at least 75%, more often 90%, or more than 95% of the humanized antibody residues will correspond to residues in the parental framework region (FR) and CDR sequences.Humanized antibodies can be produced by a variety of techniques, including CDR grafting (EP 239,400, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1995, Protein Engineering 7(6):805-814). al., 1994, Proc. Natl. Acad. Sci. 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, U.S. Pat. al.,2002, J.Immunol.169:1119-25, Caldas et al.,2000, Protein Eng.13:353-60, Morea et al.,2000, Methods 20:267-79, Baca et al.,1997, J.Biol.Chem.272:10678-84, Roguska et al. al., 1996, Protein Eng. 9:895-904, Couto et al., 1995, Cancer Res. 55(23 Supp):5973s-5977s, Couto et al., 1995, Cancer Res. 55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al., 1994, J. Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321:522-525, Riechmann et al., 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596.

[0100] In many cases, framework residues in framework regions will be replaced with corresponding residues from CDR donor antibody to change, for example, improve, antigen binding.These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction of CDR and framework residues to identify the framework residues that are important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions.(See, for example, Queen et al., U.S. Patent No. 5,585,089; U.S. Publication No. 2004 / 0049014 and U.S. Publication No. 2003 / 0229208; U.S. Patent No. 6,350,861; U.S. Patent No. 6,180,370; U.S. Patent No. 5,693,762; U.S. Patent No. 5,693,761; U.S. Patent No. 5,585,089 and U.S. Patent No. 5,530,101; and Riechmann et al., 1988, Nature 332:323).

[0101] Human, chimeric, or humanized derivatives of the disclosed mouse anti-human Siglec-15 antibodies can be used in in vivo methods in humans. Mouse antibodies or antibodies from other species can be advantageously used for many uses (e.g., in vitro or in situ detection assays, acute in vivo uses, etc.). Such human or humanized antibodies can include amino acid residue substitutions, deletions, or additions in one or more non-human CDRs. Humanized antibody derivatives can have substantially the same binding, stronger binding, or weaker binding compared to non-derivatized humanized antibodies. In certain embodiments, one, two, three, four, or five amino acid residues in the CDRs are substituted, deleted, or added (i.e., mutated). Fully human antibodies are particularly desirable for therapeutic treatment of human subjects.

[0102] Such human antibodies can be produced by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences (see U.S. Pat. Nos. 4,444,887 and 4,716,111, and International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741). Such human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.

[0103] For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, J HHomozygous deletion of the region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized using conventional methods with a selected antigen, e.g., all or a portion of a polypeptide. Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology (see, e.g., U.S. Patent No. 5,916,771). The human immunoglobulin transgenes harbored by the transgenic mice are rearranged during B cell differentiation and subsequently undergo switching and somatic mutation. Thus, such techniques can be used to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93, incorporated herein by reference in its entirety). For further discussion of this technology for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, for example, International Publication Nos. WO98 / 24893, WO96 / 34096, and WO96 / 33735, and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated herein by reference in their entireties. Additionally, companies can work to provide human antibodies against selected antigens using techniques similar to those described above.

[0104] DNA sequences encoding human acceptor framework sequences include, but are not limited to, FR segments from human germline VH segments VH1-18 and JH6, and human germline VL segments VK-A26 and JK4. In certain embodiments, one or more of the CDRs are inserted into the framework regions using conventional recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions, and human framework regions (e.g., see Chothia et al., 1998, "Structural Determinants In The Sequences Of Immunoglobulin Variable Domain," J. Mol. Biol. 278:457-479, for a list of human framework regions).

[0105] C. Antibody Sequence 1.4C12 heavy chain sequence One embodiment provides a murine monoclonal antibody or antigen-binding fragment thereof isolated from hybridoma 4C12.

[0106] Another embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000003.tif126164

[0107] The underlined sequences correspond to the complementarity determining regions (CDRs). The double underlined sequences correspond to the constant regions. The dashed underlined sequences correspond to the leader sequences.

[0108] The nucleic acid can be in a vector, e.g., an expression vector. The nucleic acid can be extrachromosomal or inserted into the chromosome of the host cell, e.g., a Chinese hamster ovary cell.

[0109] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000004.tif46160

[0110] Single underlining corresponds to the leader sequence, double underlining corresponds to the CDRs, and dashed underlining corresponds to the constant region.

[0111] Another embodiment provides an antibody or antigen-binding fragment thereof having a leader sequence-less heavy chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the following: TIFF0007795860000005.tif41167 Double underlines correspond to CDRs and dashed underlines correspond to constant regions.

[0112] The amino acid sequence of CDR1 of the 4C12 heavy chain is TIFF0007795860000006.tif5128.

[0113] The amino acid sequence of CDR2 of the 4C12 heavy chain is TIFF0007795860000007.tif6128.

[0114] The amino acid sequence of CDR3 of the 4C12 heavy chain is TIFF0007795860000008.tif5128.

[0115] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain according to SEQ ID NO:4 or 5.

[0116] Another embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:3.

[0117] One embodiment provides an antibody having three different CDRs selected from the group consisting of SEQ ID NOs: 6, 7, and 8.

[0118] 2.4C12 light chain sequence Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000009.tif66165

[0119] Dashed underlines represent leader sequences, single underlines represent CDRs, and double underlines represent constant regions.

[0120] The nucleic acid can be in a vector, e.g., an expression vector. The nucleic acid can be extrachromosomal or inserted into the chromosome of the host cell, e.g., a Chinese hamster ovary cell.

[0121] Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000010.tif24159 Underlines represent leader sequences. Double underlines represent CDRs. Dashed underlines represent constant regions.

[0122] Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain without a leader sequence, the light chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the following: TIFF0007795860000011.tif24165 Double underlines represent CDRs. Dashed underlines represent constant regions.

[0123] The amino acid sequence of CDR1 of the 4C12 light chain is The file is TIFF0007795860000012.tif5128.

[0124] The amino acid sequence of CDR2 of the 4C12 light chain is The file is TIFF0007795860000013.tif5128.

[0125] The amino acid sequence of the CDR3 of the 4C12 light chain is The file is TIFF0007795860000014.tif5128.

[0126] One embodiment provides an antibody or antigen-binding fragment thereof having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:10 or 11.

[0127] Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:9.

[0128] One embodiment provides an antibody having three different CDRs selected from the group consisting of SEQ ID NOs: 12, 13, and 14.

[0129] Another embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain having an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:4 or 5, and a light chain having an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:10 or 11, or a combination of such light and heavy chains.

[0130] Another embodiment provides an antibody or antigen-binding fragment thereof having three different heavy chain CDRs with amino acids selected from the group consisting of SEQ ID NOs:6, 7, and 8, and three different light chain CDRs with amino acids selected from the group consisting of SEQ ID NOs:12, 13, and 14.

[0131] 3.2B5 heavy chain sequence One embodiment provides a murine monoclonal antibody or antigen-binding fragment thereof isolated from hybridoma 2B5.

[0132] Another embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000015.tif163165

[0133] The underlined sequences correspond to the complementarity determining regions (CDRs). The double underlined sequences correspond to the constant regions. The dashed underlined sequences correspond to the leader sequences.

[0134] The nucleic acid can be in a vector, e.g., an expression vector. The nucleic acid can be extrachromosomal or inserted into the chromosome of the host cell, e.g., a Chinese hamster ovary cell.

[0135] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000016.tif54158

[0136] Single underlining corresponds to the leader sequence, double underlining corresponds to the CDRs, and dashed underlining corresponds to the constant region.

[0137] Another embodiment provides an antibody or antigen-binding fragment thereof having a leader sequence-less heavy chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the following: TIFF0007795860000017.tif54166 Double underlines correspond to CDRs and dashed underlines correspond to constant regions.

[0138] The amino acid sequence of CDR1 of the 2B5 heavy chain is TIFF0007795860000018.tif5128.

[0139] The amino acid sequence of CDR2 of the 2B5 heavy chain is The file is TIFF0007795860000019.tif5128.

[0140] The amino acid sequence of the CDR3 of the 2B5 heavy chain is The file is TIFF0007795860000020.tif5128.

[0141] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain according to SEQ ID NO:16 or 17.

[0142] Another embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:15.

[0143] One embodiment provides an antibody having three different CDRs selected from the group consisting of SEQ ID NOs: 18, 19, and 20.

[0144] 4.2B5 light chain sequence Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000021.tif66164

[0145] Dashed underlines represent leader sequences, single underlines represent CDRs, and double underlines represent constant regions.

[0146] The nucleic acid can be in a vector, e.g., an expression vector. The nucleic acid can be extrachromosomal or inserted into the chromosome of the host cell, e.g., a Chinese hamster ovary cell.

[0147] Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000022.tif23162 Underlines represent leader sequences. Double underlines represent CDRs. Dashed underlines represent constant regions.

[0148] Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain without a leader sequence, the light chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the following: TIFF0007795860000023.tif24165 Double underlines represent CDRs. Dashed underlines represent constant regions.

[0149] The amino acid sequence of CDR1 of the 2B5 light chain is The file is TIFF0007795860000024.tif5128.

[0150] The amino acid sequence of CDR2 of the 2B5 light chain is The file is TIFF0007795860000025.tif5128.

[0151] The amino acid sequence of the CDR3 of the 2B5 light chain is TIFF0007795860000026.tif5128.

[0152] One embodiment provides an antibody or antigen-binding fragment thereof having a light chain having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:22 or 23.

[0153] Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:21.

[0154] One embodiment provides an antibody having three different CDRs selected from the group consisting of SEQ ID NOs: 24, 13, and 25.

[0155] Another embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain having an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 16 or 17, and a light chain having an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 22 or 23, or a combination of such light and heavy chains.

[0156] Another embodiment provides an antibody or antigen-binding fragment thereof having three different heavy chain CDRs with amino acids selected from the group consisting of SEQ ID NOs: 18, 19, and 20, and three different light chain CDRs with amino acids selected from the group consisting of SEQ ID NOs: 24, 13, and 25.

[0157] 6.4G9 heavy chain sequence One embodiment provides a murine monoclonal antibody or antigen-binding fragment thereof isolated from hybridoma 4G9.

[0158] Another embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000027.tif132165

[0159] The underlined sequences correspond to the complementarity determining regions (CDRs). The double underlined sequences correspond to the constant regions. The dashed underlined sequences correspond to the leader sequences.

[0160] The nucleic acid can be in a vector, e.g., an expression vector. The nucleic acid can be extrachromosomal or inserted into the chromosome of the host cell, e.g., a Chinese hamster ovary cell.

[0161] One embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000028.tif47164

[0162] Single underlining corresponds to the leader sequence, double underlining corresponds to the CDRs, and dashed underlining corresponds to the constant region.

[0163] Another embodiment provides an antibody or antigen-binding fragment thereof having a leader sequence-less heavy chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the following: TIFF0007795860000029.tif48167 Double underlines correspond to CDRs and dashed underlines correspond to constant regions.

[0164] The amino acid sequence of CDR1 of the 4G9 heavy chain is The file is TIFF0007795860000030.tif5128.

[0165] The amino acid sequence of CDR2 of the 4G9 heavy chain is The file is TIFF0007795860000031.tif5128.

[0166] The amino acid sequence of the CDR3 of the 4G9 heavy chain is The file is TIFF0007795860000032.tif5128.

[0167] One embodiment provides a 4G9 antibody or antigen-binding fragment thereof having a heavy chain according to SEQ ID NO:27 or 28.

[0168] Another embodiment provides a 4G9 antibody or antigen-binding fragment thereof having a heavy chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:26.

[0169] One embodiment provides a 4G9 antibody having three different CDRs selected from the group consisting of SEQ ID NOs: 29, 30, and 31.

[0170] 7.4G9 light chain sequence Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000033.tif66164

[0171] Dashed underlines represent leader sequences, single underlines represent CDRs, and double underlines represent constant regions.

[0172] The nucleic acid can be in a vector, e.g., an expression vector. The nucleic acid can be extrachromosomal or inserted into the chromosome of the host cell, e.g., a Chinese hamster ovary cell.

[0173] Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to: TIFF0007795860000034.tif24166 Underlines represent leader sequences. Double underlines represent CDRs. Dashed underlines represent constant regions.

[0174] Another embodiment provides an antibody or antigen-binding fragment thereof having a light chain without a leader sequence, the light chain having amino acids with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the following: TIFF0007795860000035.tif24166 Double underlines represent CDRs. Dashed underlines represent constant regions.

[0175] The amino acid sequence of CDR1 of the 4G9 light chain is The file is TIFF0007795860000036.tif5128.

[0176] The amino acid sequence of CDR2 of the 4G9 light chain is The file is TIFF0007795860000037.tif5128.

[0177] The amino acid sequence of the CDR3 of the 4G9 light chain is TIFF0007795860000038.tif5128.

[0178] One embodiment provides a 4G9 antibody or antigen-binding fragment thereof having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:33 or 34.

[0179] Another embodiment provides a 4G9 antibody or antigen-binding fragment thereof having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:32.

[0180] One embodiment provides a 4G9 antibody having three different CDRs selected from the group consisting of SEQ ID NOs: 35, 36, and 37.

[0181] Another embodiment provides an antibody or antigen-binding fragment thereof having a heavy chain having an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:27 or 28, and a light chain having an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:33 or 34, or a combination of such light and heavy chains.

[0182] Another embodiment provides a 4G9 antibody or antigen-binding fragment thereof having three different heavy chain CDRs with amino acids selected from the group consisting of SEQ ID NOs: 29, 30, and 31, and three different light chain CDRs with amino acids selected from the group consisting of SEQ ID NOs: 35, 36, and 37.

[0183] D. PD-1 activating epitopes Epitope-specific PD-1 binding moieties are disclosed herein. In one embodiment, the disclosed binding moieties immunospecifically bind to PD-1 and activate PD-1-mediated signaling.

[0184] The disclosed PD-1 epitope is formed by amino acids 96-110 of SEQ ID NO:1 and has the following amino acid sequence: TIFF0007795860000039.tif5128

[0185] 1. Antibodies One embodiment provides an antibody or epitope-binding fragment thereof that immunospecifically binds to SEQ ID NO:38 on the surface of an immune cell and activates the immune cell. In one embodiment, the preferred immune cell is a T cell, more specifically a CD8 + In another embodiment, the antibody or epitope-binding fragment thereof immunospecifically binds to SEQ ID NO:38 of PD-1 on the surface of an immune cell and promotes or induces an activation signal through PD-1 to activate the immune cell.

[0186] The epitope-specific antibody can be a monoclonal antibody, a humanized antibody, a human antibody, a murine antibody, a chimeric antibody, or a fragment thereof. Exemplary antibodies and methods for their production are discussed below.

[0187] 2. Fusion Proteins In one embodiment, the epitope-specific PD-1-binding moiety is a fusion protein. All or a portion of one or more of the above-disclosed PD-1 epitope antibodies or epitope-binding fragments can be conjugated to another polypeptide to form a fusion protein. The fusion polypeptide has a first fusion partner comprising all or a portion of one or more of the disclosed PD-1 epitope antibodies or epitope-binding fragments fused to a second polypeptide, either directly or via a linker peptide sequence fused to the second polypeptide. The fusion protein optionally contains a domain that functions to dimerize or multimerize two or more fusion proteins. The peptide / polypeptide linker domain can be a separate domain or can be contained within one of the other domains of the fusion protein (the first polypeptide or the second polypeptide). Similarly, the domain that functions to dimerize or multimerize the fusion protein can be a separate domain or can be contained within one of the other domains of the fusion protein (the first polypeptide, the second polypeptide, or the peptide / polypeptide linker domain). In one embodiment, the dimerization / multimerization domain and the peptide / polypeptide linker domain are the same.

[0188] The fusion proteins disclosed herein are of Formula I: N-R1-R2-R3-C where "N" represents the N-terminus of the fusion protein, "C" represents the C-terminus of the fusion protein, "R1" is all or a portion of a disclosed PD-1 epitope antibody or epitope-binding fragment, or a functional variant or fragment thereof, "R2" is an optional peptide / polypeptide linker domain, and "R3" is a second polypeptide. Alternatively, R3 is all or a portion of a disclosed PD-1 epitope antibody or epitope-binding fragment, or a functional variant or fragment thereof, and R1 is a second polypeptide.

[0189] Dimerization or multimerization can occur between or within two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur through chemical crosslinking. The resulting dimers or multimers can be homodimers / homomultimers or heterodimers / heteromultimers.

[0190] 3. Aptamers In some embodiments, the epitope-specific binding moiety is an aptamer. An aptamer is a molecule that preferably interacts with a target molecule in a specific manner. In one embodiment, the aptamer binds to SEQ ID NO:38 on the surface of immune cells and promotes or induces an activation signal through PD-1 to activate the immune cell. Typically, aptamers are small nucleic acids ranging from 15 to 50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind to proteins, cells, small organic molecules, or peptides. Aptamers can bind to small molecules such as ATP and theophylline, as well as large molecules such as reverse transcriptase and thrombin. Aptamers can bind very tightly to target molecules, with a Kd of less than 10-12 M. Aptamers preferably bind to target molecules with a Kd of less than 10-6, 10-8, 10-10, or 10-12. Aptamers can bind to target molecules with very high specificity. For example, the aptamer has been isolated that has the binding affinity between target molecule and another molecule that differs only at a single position on the molecule that is more than 10,000 times different.Preferably, the Kd of aptamer for target molecule is at least 10,100,1000,10,000 or 100,000 times lower than the Kd of background binding molecules.For example, when comparing polypeptides, the background molecules are preferably different polypeptides.Representative examples of the method for making and using the aptamer that binds to various different target molecules are known in the art.

[0191] 4.Small molecules In some embodiments, the epitope-specific binding moiety can be a small molecule. The term "small molecule" generally refers to a small organic compound having a molecular weight greater than about 100 and less than about 2,500 daltons, preferably between 100 and 2,000 daltons, more preferably between about 100 and about 1,250 daltons, more preferably between about 100 and about 1,000 daltons, more preferably between about 100 and about 1,000 daltons, more preferably between about 100 and about 750 daltons, and more preferably between about 200 and about 500 daltons. Small molecule agonists of activating epitopes of PD-1 can be identified using conventional screening methods. In some embodiments, screening assays can involve random screening of large libraries of test compounds. Assays can involve determining PD-1-induced immune responses or T cell activation.

[0192] E. Pharmaceutical Compositions Pharmaceutical compositions comprising the disclosed antibodies and antigen-binding fragments thereof are provided. The pharmaceutical compositions containing the antibodies and antigen-binding fragments thereof may be for parenteral administration (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection).

[0193] In some in vivo approaches, the compositions disclosed herein are administered to subjects in therapeutically effective amounts.As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage that is sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated, or otherwise provide the desired pharmacological and / or physiological effect.The exact dosage will vary according to various factors, such as subject-dependent variables (e.g., age, immune system health, etc.), disease, and treatment being performed.

[0194] As further research is conducted on the disclosed antibodies and antigen-binding fragments thereof, information regarding appropriate dosage levels for treating various conditions in various patients will emerge, enabling those skilled in the art to ascertain appropriate administration, taking into account the recipient's treatment background, age, and general health. The selected dosage will depend on the desired therapeutic effect, route of administration, and desired duration of treatment. The disclosed antibodies and antigen-binding fragments thereof are generally administered to mammals at dosage levels of 0.001 to 20 mg / kg of body weight daily. Generally, for intravenous injection or infusion, dosages may be lower.

[0195] In certain embodiments, antibodies and antigen-binding fragments thereof are administered locally, for example, by injection directly into the site to be treated. Typically, injection results in an increased localized concentration of the immunomodulatory agent composition that is greater than that which can be achieved by systemic administration. The immunomodulatory agent composition may be combined with a matrix, as described above, to help increase the localized concentration of the polypeptide composition by reducing passive diffusion of the polypeptide outside the site to be treated.

[0196] 1. Formulations for parenteral administration In some embodiments, compositions containing the disclosed antibodies and antigen-binding fragments are administered in aqueous solution by parenteral injection. Formulations can also be in the form of suspensions or emulsions. Generally, pharmaceutical compositions are provided that contain an effective amount of an antibody or antigen-binding fragment thereof, and optionally contain pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions optionally contain one or more of the following: diluents, sterile water, buffered saline solutions of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; and additives, such as detergents and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimersol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol). Examples of non-aqueous solvent or vehicle are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.Preparation can be lyophilized and redissolved / resuspended immediately before use.Preparation can be sterilized by, for example, filtering through bacteria-retaining filter, by incorporating sterilizing agent into composition, by irradiating composition, or by heating composition.

[0197] 2. Formulations for oral administration In some embodiments, the antibody composition is formulated for oral delivery. Oral dosage forms of antibodies can resist proteolysis and deliver a greater proportion of immunoreactive antibodies locally in the gastrointestinal tract for the treatment of infections, or allow for the absorption of antibodies for the treatment or prevention of systemic conditions (Reilly, RM, et al. Clin Pharmacokinet., 32(4):313-23(1997); Victoria S Jasion and Bruce P Burnett, Nutr J.; 14:22(2015); and Philippart, M., et al., Drug Res (Stuttg) 66(03):113-120(2016)).

[0198] Oral solid dosage forms are generally described in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co., Easton, Pa. 18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules, or those in which the material is incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, or into liposomes. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed compositions. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, incorporated herein by reference. The compositions can be prepared in liquid form or in dry powder (e.g., lyophilized) form. Liposomal or proteinoid encapsulation can be used to formulate the composition. Liposomal encapsulation can be used, and liposomes can be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics, Edited by GS Banker and CT Rhodes, Chapter 10, 1979. Generally, the formulation will include a peptide (or a chemically modified form thereof) and an inactive ingredient that protects the peptide in the stomach environment and releases biologically active material in the intestine.

[0199] Antibodies and their antigen-binding fragments can be chemically modified to allow for effective oral delivery of the derivatives. Generally, the intended chemical modification is the attachment of at least one moiety to the component molecule itself, which allows for uptake into the bloodstream from the stomach or intestine, or direct uptake into the intestinal mucosa. Also desirable is an increase in the overall stability of the component(s) and an increase in circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical use. Other moieties that can be used include propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane, and poly-1,3,6-tioxocane [see, e.g., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs, Hocenberg and Roberts, eds. (Wiley-Interscience: New York, NY) pp. 367-383, and Newmark, et al. (1982) J. Appl. Biochem. 4:185-189].

[0200] Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain inert diluents; adjuvants such as wetting agents, emulsifying agents, and suspending agents; and other components including sweetening, flavoring, and perfuming agents.

[0201] For oral formulations, the location of release may be the stomach, the small intestine (duodenum, jejunum, or ileum), or the large intestine. In some embodiments, release will avoid the adverse effects of the gastric environment, either by protecting the drug (or derivative) or by releasing the drug (or derivative) beyond the gastric environment, such as in the intestine. To ensure complete gastric resistance, a coating impermeable to at least pH 5.0 is essential. Examples of more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™, cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings may also be used as mixed films.

[0202] 3. Controlled Delivery Polymer Matrices The antibodies and antigen-binding fragments thereof disclosed herein can also be administered in controlled-release formulations. The antibodies and antigen-binding fragments thereof can be incorporated into an inert matrix that allows release by either diffusion or leaching, for example, gum. Slowly degenerating matrices can also be incorporated into the formulation. Another form of controlled release is based on the Oros Therapeutic System (Alza Corp.), in which the antibodies or antigen-binding fragments thereof are encapsulated in a semipermeable membrane that allows water to enter and the drug to be pushed out through a single small opening by osmotic effect.

[0203] Controlled-release polymeric devices can be fabricated for systemic, prolonged release after implantation or injection (microparticles) of the polymeric device (rods, cylinders, films, discs). The matrix can be in the form of microparticles, such as microspheres, where the drug is dispersed within a solid polymer matrix or microcapsules, where the core has a different material from the polymer shell and the peptide is dispersed or suspended in the core, which can be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, polymers can be cast as thin slabs or films ranging from a few nanometers to 4 centimeters, powders produced by milling or other standard techniques, or gels, such as hydrogels.

[0204] Either non-biodegradable or biodegradable matrices can be used to deliver fusion polypeptides or nucleic acids encoding the fusion polypeptides, although biodegradable matrices are preferred in some embodiments. These can be natural or synthetic polymers, although synthetic polymers are preferred in some embodiments due to better characterization of their degradation and release profiles. The polymer is selected based on the desired period of release. In some cases, linear release may be most useful, while in others, pulsed or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically absorbing up to about 90% water by weight) and may optionally be crosslinked with multivalent ions or polymers.

[0205] The matrix can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art.Biodegradable microspheres can be prepared using any of the methods developed to make microspheres for drug delivery, as described, for example, by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0206] The devices can be formulated for local release to treat an area of ​​implantation or injection, which will typically deliver a much lower dose than for systemic treatment or systemic delivery. They can be implanted or injected subcutaneously into muscle, fat, or swallowed.

[0207] III. Manufacturing method A. Antibody Production Methods The disclosed antibodies can be produced in cell culture, phage, or various animals, including, but not limited to, cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. In one embodiment, the various animals can be transgenic animals genetically engineered to produce human or humanized antibodies. Thus, in one embodiment, the antibodies are mammalian antibodies. Phage technology can be used to isolate initial antibodies or to generate variants with modified specificity or affinity characteristics. Such techniques are routine and well known in the art. In one embodiment, antibodies are produced by recombinant means known in the art. For example, recombinant antibodies can be produced by transforming host cells with vectors containing DNA sequences encoding the antibodies. One or more vectors are used to transform DNA sequences that express at least one VL and one VH region into the host cell. Exemplary descriptions of recombinant means of antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997), Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000), Goding, Monoclonal Antibodies: Principles And Practice (Academic Press, 1993), and Current Protocols In Immunology (John Wiley & Sons, latest edition).

[0208] The disclosed antibodies can be modified by recombinant means to increase the antibody's effectiveness in mediating a desired function. Accordingly, it is within the scope of the present invention that antibodies can be modified by substitution using recombinant means. Typically, the substitutions will be conservative. For example, at least one amino acid in the antibody's constant region can be replaced with a different residue. See, for example, U.S. Pat. No. 5,624,821, U.S. Pat. No. 6,194,551, WO9958572, and Angal, et al., Mol. Immunol. 30:105-08 (1993). Amino acid modifications include amino acid deletions, additions, and substitutions. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Antibodies are often labeled by either covalent or noncovalent attachment to a substance that provides a detectable signal. A wide variety of labeling and conjugation techniques are known and are widely reported in both the scientific and patent literature. These antibodies can be screened for binding to proteins, polypeptides, see, e.g., Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0209] For example, suitable antibodies with desired biological activity can be identified using in vitro assays, including but not limited to, proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, and signal transduction, and in vivo assays, such as tumor growth inhibition. The antibodies provided herein can also be useful for diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for their ability to bind to specific antigens without inhibiting the antigen's receptor binding or biological activity. As neutralizing antibodies, they can be useful in competitive binding assays.

[0210] Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulins of any class (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The variable domain varies in sequence among antibodies and is used to determine the binding and specificity of each particular antibody for its specific antigen. However, variability is not usually uniformly distributed throughout the variable domain of an antibody. It is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called the framework (FR). Natural heavy and light chain variable domains each contain four FR regions that primarily adopt a beta-sheet structure connected by three CDRs, which form loops that connect, and in some cases form part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.

[0211] Also disclosed are biologically active antibody fragments, which may contain insertions, deletions, substitutions, or other selected modifications of particular regions or particular amino acid residues, whether or not attached to other sequences, provided that the activity of the fragment is not significantly altered or diminished compared to the unmodified antibody or antibody fragment.

[0212] The techniques can also be adapted to produce single-chain antibodies based on the disclosed antibodies and antigen-binding fragments thereof. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be made by fusing heavy and light chain variable domains together using a short peptide linker, thereby reconstituting an antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs), in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain by a 15-25 amino acid peptide or linker, have been developed without significantly disrupting antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind together in their proper conformational orientation.

[0213] One embodiment provides a bivalent single-chain variable fragment (di-scFv) that can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, resulting in a tandem scFv. ScFvs can also be designed with a linker peptide (approximately five amino acids) that is too short for the two variable regions to fold together, causing the scFvs to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning they also have much higher affinity for their targets. Even shorter linkers (one or two amino acids) result in the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit even higher affinity for their targets than diabodies.

[0214] Another embodiment provides monoclonal antibodies obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired antagonist activity.

[0215] Monoclonal antibodies can be produced using any procedure that produces monoclonal antibodies. In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0216] The disclosed antibodies can also be produced by recombinant DNA methods. DNA encoding the disclosed antibodies can be easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of mouse antibodies). Libraries of antibodies or active antibody fragments can also be produced and screened using phage display technology.

[0217] Methods for producing antibodies using protein chemistry are also known in the art. One method for producing proteins, including antibodies, is to link two or more peptides or polypeptides together using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry (Applied Biosystems, Inc., Foster City, CA). Those skilled in the art can readily understand that peptides or polypeptides corresponding to antibodies can be synthesized, for example, by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from a synthetic resin, while another fragment of the antibody can be synthesized and then cleaved from the resin, thereby exposing the terminal group that is functionally blocked on the other fragment. By a peptide condensation reaction, these two fragments can be covalently linked via peptide bonds at their carboxyl and amino termini to form an antibody or fragment thereof. Alternatively, peptides or polypeptides can be independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides can be linked to form antibodies or antigen-binding fragments thereof through similar peptide condensation reactions.

[0218] For example, enzymatic ligation of cloned or synthetic peptide segments allows relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or entire protein domains. Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct larger peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide-alpha-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to yield a thioester-linked intermediate as the initial covalent product. Without modification of reaction conditions, this intermediate undergoes a spontaneous and rapid intramolecular reaction to form a native peptide bond at the ligation site.

[0219] B. Methods for Producing Isolated Nucleic Acid Molecules One embodiment provides a nucleic acid encoding the disclosed antibody or antigen-binding fragment thereof. The nucleic acid can encode the entire antibody, or an antigen-binding fragment thereof, or a light chain, a heavy chain, a combination, or a CDR thereof.

[0220] Isolated nucleic acid molecules can be produced by standard techniques, including, but not limited to, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain isolated nucleic acids encoding mutant polypeptides. PCR is a technique in which a target nucleic acid is enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond is used to design oligonucleotide primers identical in sequence to opposite strands of the amplified template. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers are typically 14-40 nucleotides in length but can range from 10 nucleotides to several hundred nucleotides in length. General PCR techniques are described, for example, in *PCR Primer: A Laboratory Manual*, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When RNA is used as a template source, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplification can also be used to obtain isolated nucleic acids. See, e.g., Lewis (1992) Genetic Engineering News 12:1, Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878, and Weiss (1991) Science 254:1292-1293.

[0221] Isolated nucleic acids can be chemically synthesized either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3'→5' direction). For example, one or more pairs of long oligonucleotides (e.g., more than 100 nucleotides) containing the desired sequence can be synthesized, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) so that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single double-stranded nucleic acid molecule per oligonucleotide pair, which can then be ligated into a vector. Isolated nucleic acids can also be obtained by mutagenesis. Protein-encoding nucleic acids can be mutated using standard techniques, including PCR-mediated oligonucleotide-directed mutagenesis and / or site-specific mutagenesis. See Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al., 1992.

[0222] IV.How to use The disclosed antibodies and antigen-binding fragments thereof can be used to modulate immune responses in subjects in need thereof. One embodiment provides a method of activating PD-1-expressing immune cells, e.g., T cells, to expand and enhance the biological activity of PD-1-expressing immune cells by administering the disclosed antibodies and antigen-binding fragments thereof, optionally with a second therapeutic agent.

[0223] A. Stimulating the immune response 1. Treatment Strategy Methods for inducing or enhancing an immune response in a subject are provided. Typically, the methods include administering to the subject an effective amount of one or more of the disclosed antibodies and antigen-binding fragments thereof to immunospecifically bind to PD-1 and induce, promote, or enhance a stimulatory or activation signal through PD-1 to activate immune cells. The immune response can induce, promote, or enhance, for example, immune cells, T cell activation by T cell proliferation, and cytokine secretion. The disclosed antibodies or antigen-binding fragments thereof can be administered to a subject in need thereof in an amount effective to overcome T cell depletion and / or T cell anergy. Overcoming T cell depletion or T cell anergy can be determined by measuring T cell function using known techniques.

[0224] The methods can be used in vivo or ex vivo to induce, promote or enhance a stimulatory immune response.

[0225] In some embodiments, the antibody or antigen-binding fragment thereof, or a nucleic acid encoding the antibody or antigen-binding fragment thereof, is administered directly to a subject. In some embodiments, the antibody or antigen-binding fragment thereof is contacted with cells (e.g., immune cells) ex vivo, and the treated cells are administered to a subject (e.g., adoptive transfer). The antibody or antigen-binding fragment thereof can enable a more robust immune response. The disclosed compositions are useful for triggering an activation signal through PD-1 on immune cells and stimulating or enhancing an immune response involving T cells.

[0226] 2. Subjects to be treated Cancer treatment The disclosed antibodies and compositions and methods thereof can be used to treat cancer. Generally, these agents are used to stimulate or enhance an immune response against cancer in a subject by administering to the subject an amount of the disclosed antibodies or antigen-binding fragments thereof that induces, promotes, or enhances an activation signal through PD-1. The method can reduce one or more symptoms of cancer.

[0227] Immune cells activated by the disclosed antibodies or fragments thereof can kill cells and reduce tumor burden in a subject. The term "cancer cells" is intended to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor that is confined to a localized area. In other embodiments, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and spread to distant sites. In still other embodiments, cancer is associated with a specific cancer antigen (e.g., pan-cancer antigen (KS 1 / 4), ovarian cancer antigen (CA125), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[0228] The methods and antibody compositions disclosed herein are useful for the treatment or prevention of various cancers and other abnormal proliferative diseases, including (but not limited to): cancers including those of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, and skin; including squamous cell carcinoma; hematopoietic malignancies of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; acute and chronic myeloid leukemia. Hematopoietic tumors of myeloid lineage, including leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including malignant melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including malignant melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma.

[0229] Cancer caused by abnormal apoptosis can also be treated by the disclosed method and composition.Such cancers may include, but are not limited to, follicular lymphoma, cancer with p53 mutation, hormone-dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndrome.In certain embodiments, malignant tumors or abnormal proliferation changes (such as dysplasia and dysplasia), or hyperproliferative disorders are treated or prevented by the present method and composition in ovary, bladder, breast, colon, lung, skin, pancreas, or uterus.In other specific embodiments, sarcoma, malignant melanoma, or leukemia are treated or prevented by the present method and composition.

[0230] Specific cancers and related disorders that may be treated or prevented by the methods and compositions disclosed herein include, but are not limited to, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, and chronic leukemias, such as, but not limited to, myelodysplastic syndromes, chronic myeloid (granular) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; Hodgkin's or non-Hodgkin's lymphoma (e.g., diffuse anaplastic lymphoma kinase (ALK)-negative, large B-cell lymphoma (DLBCL)); diffuse leukemia ...leukemia), chronic myeloid (granular) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; Lymphomas, including but not limited to anaplastic lymphoma kinase (ALK)-positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK)-positive, ALK+ anaplastic large cell lymphoma (ALCL), acute myeloid lymphoma (AML); multiple myeloma, including but not limited to smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; osteosarcoma Bone and connective tissue sarcomas, such as, but not limited to, osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor of bone, osteofibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma, fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, non-glial tumors, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor brain tumors, such as, but not limited to, follicular carcinoma, pineoblastoma, and primary cerebral lymphoma; breast cancer, such as, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal gland cancer, including, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer;Pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumors, and carcinoid or islet cell tumors; pituitary cancer, including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancer, including but not limited to intraocular melanoma, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, and malignant melanoma; vulvar cancer, including but not limited to melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial cancer and uterine sarcoma; ovarian cancer, including but not limited to ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; ovarian cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, malignant melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma esophageal cancer, including but not limited to adenocarcinoma, fungus (polypoid), ulcerating, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, including but not limited to adenocarcinoma; bile duct cancer, including but not limited to papillary, nodular, and diffuse; non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer. lung cancer, including but not limited to; testicular cancer, including but not limited to germ cell tumor, seminoma, undifferentiated, classic (typical), spermatocyte, non-seminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor), prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer; oral cancer, including but not limited to squamous cell carcinoma; basal carcinoma; salivary gland cancer, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cyst; pharyngeal cancer, including but not limited to squamous cell carcinoma and verrucous carcinoma;Skin cancers, including but not limited to basal cell carcinoma, squamous cell carcinoma, and malignant melanoma, superficial spreading malignant melanoma, nodular malignant melanoma, lentigo maligna malignant melanoma, acral lentiginous malignant melanoma; kidney cancers, including but not limited to renal cell carcinoma, adenocarcinoma, adrenal nephroma, fibrosarcoma, transitional cell carcinoma (of the renal pelvis and / or ureter); Wilms' tumor; and bladder cancers, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myxosarcoma, osteosarcoma, endothelial sarcoma, lymphangioendothelial sarcoma, mesothelioma, synovium, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (for reviews of such diseases, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia, and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America);

[0231] b. Treating infections The disclosed antibody compositions and methods can be used to treat infectious diseases and infectious disorders. Generally, these agents are used to stimulate or enhance an immune response to an infectious disease in a subject by administering to the subject an amount of one or more of the disclosed antibodies or antigen-binding fragments thereof that sends an activating or stimulatory signal through PD-1. The method can reduce one or more symptoms of the infectious disease.

[0232] Infection or disease can be caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that enter cells, i.e., are attacked by cytotoxic T lymphocytes.

[0233] Infection or disease can be acute or chronic. Acute infection is typically a short-term infection. During acute microbial infection, immune cells begin to express immunomodulatory receptors. Thus, in some embodiments, the method comprises enhancing an immune stimulatory response to acute infection.

[0234] The infection may be caused by, for example, but not limited to, Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacterium.

[0235] In some embodiments, the disclosed antibody compositions are used to treat chronic infections, such as those that result in T cell depletion or T cell anergy, allowing the infection to persist in the host for extended periods of time.

[0236] Exemplary infections to be treated are chronic infections caused by hepatitis virus, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes virus, Epstein-Barr virus, or human papillomavirus.

[0237] Because viral infections are primarily eliminated by T cells, increased T cell activity is therapeutically useful in situations where more rapid and thorough elimination of infectious viral agents would be beneficial to animal or human subjects. Thus, the disclosed compositions can be administered to treat local or systemic viral infections, including, but not limited to, immunodeficiencies (e.g., HIV), papillomas (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), and the common cold (e.g., human rhinovirus), as well as other viral infections caused by, for example, HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus, and rabies virus. The molecules can be administered locally to treat viral skin diseases such as herpes zoster or shingles, or genital warts. The molecules can also be administered systemically to treat systemic viral diseases, including, but not limited to, AIDS, influenza, the common cold, or encephalitis.

[0238] The active ingredients include Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, and Bor relia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacte rium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus s, Hemophilus, Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A、B、およびC、Methanobacterium、Micrococcus、Myobacterium、Mycoplasma、Myxococcus、Neisseri a、Nitrobacter、Oscillatoria、Prochloron、Proteus、Pseudomonas、Phodospirillum、Rickettsi a、Salmonella、Shigella、Spirillum、Spirochaeta、Staphylococcus、Streptococcus、Streptomy ces、Sulfolobus、Thermoplasma、Thiobacillus、およびTreponema、Vibrio、Yersinia、Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum、Trypanosoma brucei、Entamoeba histolytica、Toxoplasma gondii、Trichomonas vaginalisおよびSchistosomaInfections caused by microorganisms, including but not limited to, Clostridium perfringens, Clostridium diffusum, Clostridium globulin ...

[0239] Other microorganisms that may be treated using the disclosed compositions and methods include bacteria such as Klebsiella, Serratia, Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis), and Histoplasma (capsulatuma), Entamoeba, histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia and fungal or parasitic pathogens such as Plasmodium, Babesia, or Trypanosoma, including but not limited to: Plasmodium lambia, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondi, Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia, or Trypanosoma.

[0240] V. Combination Therapies to Enhance Immune Responses The disclosed antibodies and antigen-binding fragments thereof and compositions thereof can be administered to a subject in need thereof alone or in combination with one or more additional therapeutic agents. In some embodiments, the antibodies and antigen-binding fragments thereof and the additional therapeutic agent are administered separately but simultaneously. The antibodies and antigen-binding fragments thereof and the additional therapeutic agent can also be administered as part of the same composition. In other embodiments, the antibodies and antigen-binding fragments thereof and the second therapeutic agent are administered separately and at different times but as part of the same treatment regimen. The additional therapeutic agent can be administered before, after, or alternating with the administration of the disclosed antibodies and antigen-binding fragments thereof.

[0241] The subject may be administered the first therapeutic agent 1, 2, 3, 4, 5, 6 hours or more, or 1, 2, 3, 4, 5, 6, 7 days or more before the administration of the second therapeutic agent. In some embodiments, the subject may be administered one or more doses of the first agent every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days before the first administration of the second agent. Antibodies and antigen-binding fragments thereof may be the first or second therapeutic agent.

[0242] Antibody and its antigen-binding fragment and additional therapeutic agent can be administered as part of a treatment plan.For example, if a first therapeutic agent can be administered to a subject every 4 days, a second therapeutic agent can be administered on the 1st, 2nd, 3rd, or 4th day, or a combination thereof.The first therapeutic agent or the second therapeutic agent can be repeatedly administered throughout the entire treatment plan.

[0243] Exemplary additional therapeutic agents include, but are not limited to, cytokines, chemotherapeutics, radionuclides, other immunotherapeutics, enzymes, antibiotics, antivirals (particularly protease inhibitors alone or in combination with nucleosides for the treatment of HIV or Hepatitis B or C), antiparasitic agents (helminths, protozoans), growth factors, growth inhibitory agents, hormones, hormone antagonists, antibodies and biologically active fragments thereof (including humanized, single chain, and chimeric antibodies), antigen and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatory drugs, ligands that bind toll-like receptors to activate the innate immune system (including, but not limited to, CpG oligonucleotides), molecules that mobilize and optimize the adaptive immune system, other molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells and helper T cells, and other molecules that inactivate or downregulate suppressor or regulatory T cells.

[0244] The additional therapeutic agent is selected based on the condition, disorder, or disease being treated. For example, an immunomodulatory agent can be co-administered with one or more additional agents that function to enhance or promote the immune response, or to reduce or inhibit the immune response.

[0245] A. Antibacterial agents In one embodiment, the antibodies and antigen-binding fragments thereof can be administered to a subject in combination with an antimicrobial agent, such as an antibiotic, antifungal, antiviral, antiparasitic, or essential oil. In another embodiment, the disclosed antibodies and antigen-binding fragments thereof can be used in the treatment and prevention of disease, as well as in a preventative or prophylactic role in situations of severe traumatic injury, such as large burns, open fractures, accidental amputations, or other wounds.

[0246] In some embodiments, subjects are administered antibodies and antigen-binding fragments thereof and / or antibacterial agents upon admission to prevent further bacterial, fungal, or viral complications. Antibiotics can target pathogens, and antibodies and antigen-binding fragments thereof can stimulate the immune system to provide an enhanced response to treat or prevent further infection or disease.

[0247] 1. Chemotherapy drugs Antibodies and antigen-binding fragments thereof may be combined with one or more chemotherapeutic agents and pro-apoptotic agents. Representative chemotherapeutic agents include amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, chrysamphase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, and ribozyme. Exemplary pro-apoptotic agents include, but are not limited to, posomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. Exemplary pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

[0248] 2. Other immunomodulators a.PD-1 antagonist In some embodiments, the antibodies and antigen-binding fragments thereof are co-administered with a PD-1 antagonist. Programmed death-1 (PD-1) is a member of the CD28 family of receptors that, when triggered on T cells, delivers a negative immune response. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell expansion and / or the strength and / or duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Patent Nos. 8,114,845, 8,609,089, and 8,709,416, which are specifically incorporated herein by reference in their entireties, and include compounds or agents that either bind to and block the ligand of PD-1, interfering with or inhibiting binding of the ligand to the PD-1 receptor, or that directly bind to and block the PD-1 receptor without triggering inhibitory signaling through the PD-1 receptor.

[0249] In some embodiments, the PD-1 receptor antagonist binds directly to the PD-1 receptor without causing inhibitory signaling and also binds to a ligand of the PD-1 receptor, reducing or inhibiting the ligand from triggering signaling through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and cause transmission of an inhibitory signal, fewer cells are compromised by the negative signals delivered by PD-1 signaling, and a more robust immune response may be achieved.

[0250] PD-1 signaling is thought to be triggered by binding of PD-1 ligands (such as B7-H1 or B7-DC) in close proximity to peptide antigens presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Thus, proteins, antibodies, or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

[0251] In some embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or interferes with PD-1 receptor signaling by binding to a ligand of PD-1 or to PD-1 itself, particularly when such binding is not followed by co-ligation of the TCR and PD-1, thereby not causing inhibitory signaling through the PD-1 receptor. Other PD-1 antagonists contemplated by the methods of the invention include antibodies that bind to PD-1 or a ligand of PD-1, as well as other antibodies.

[0252] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following U.S. Patent Nos. 7,332,582, 7,488,802, 7,521,051, 7,524,498, 7,563,869, 7,981,416, 8,088,905, 8,287,856, 8,580,247, 8,728,474, 8,779,105, 9,067,999, 9,073,994, 9,084,776, 9,205,148, 9,358,289, 9,387,247, 9,492,539, and 9,492,540, all of which are incorporated by reference in their entirety.

[0253] See also Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0254] Exemplary anti-PD-L1 antibodies include, but are not limited to, those described in the following U.S. Patent Nos. 8,383,796, 9,102,725, 9,273,135, 9,393,301, and 9,580,507, all of which are specifically incorporated by reference herein in their entirety.

[0255] Regarding anti-B7-DC (also referred to as anti-PD-L2) antibodies, see U.S. Patent Nos. 7,411,051, 7,052,694, 7,390,888, 8,188,238, and 9,255,147.

[0256] Other exemplary PD-1 receptor antagonists include, but are not limited to, PD-L2 polypeptides (including homologs and variants thereof), and active fragments of any of the above, and fusion proteins incorporating any of the above. In some embodiments, the fusion protein comprises a soluble portion of B7-DC linked to the Fc portion of an antibody, such as human IgG, and does not incorporate all or a portion of the transmembrane portion of human B7-DC.

[0257] The PD-1 antagonist can also be a fragment of mammalian PD-L1, e.g., from a mouse or a primate, such as a human, which binds to and blocks PD-1 but does not result in inhibitory signaling through PD-1. The fragment can also be part of a fusion protein, e.g., an Ig fusion protein.

[0258] Other useful polypeptide PD-1 antagonists include those that bind to the ligand of the PD-1 receptor. These include PD-1 receptor proteins, or soluble fragments thereof, that can bind to PD-1 ligands, such as PD-L1 or B7-DC, and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signaling. PD-L1 has also been shown to bind to the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include soluble ECD portions of the PD-1 protein that contain mutations, such as the A99L mutation, that increase binding to the natural ligand (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or its soluble fragments that can bind to PD-L1 ligands and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signaling, are also useful.

[0259] PD-1 and PD-L1 antisense nucleic acids, both DNA and RNA, and siRNA molecules are also called PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells and the production of T cell ligands such as PD-L1 and / or PD-L2. For example, siRNA (e.g., about 21 nucleotides long (specific to the gene encoding PD-1) or encoding a PD-1 ligand (oligonucleotides can be easily purchased commercially) can be complexed with a carrier such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8):2231-2244(2009)), and can be easily taken up by cells that express PD-1 and its ligand and reduce the expression of these receptors and ligands, thereby achieving a reduction in inhibitory signaling in T cells and thereby activating T cells.

[0260] b. CTLA4 antagonist Other molecules useful for mediating the effects of T cells in immune responses are also contemplated as additional therapeutic agents. In some embodiments, the molecule is a CTLA4 antagonist, such as an antagonist anti-CTLA4 antibody. An example of an anti-CTLA4 antibody contemplated for use in the methods of the present invention includes the antibody described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0261] Dosages for anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and can range, for example, from 0.1 to 100 mg / kg, with shorter ranges being 1 to 50 mg / kg or 10 to 20 mg / kg. Suitable doses for human subjects can be 5 to 15 mg / kg, with 10 mg / kg of antibody (e.g., human anti-PD-1 antibody) being a specific embodiment.

[0262] Specific examples of anti-CTLA4 antibodies useful in the methods of the present invention include the human anti-CTLA4 antibody ipilimumab, administered at a dose of, for example, about 10 mg / kg, and a human anti-CTLA4 antibody, administered at a dose of, for example, about 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[0263] In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand and prevent it from binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002)). Such small organic compounds can be administered alone or together with anti-CTLA4 antibodies to reduce inhibitory signaling in T cells.

[0264] 3. Enhancers In some embodiments, the additional therapeutic agent comprises a potentiating agent, which acts to increase the effectiveness of the immune response upregulator, optionally by more than one mechanism, although the precise mechanism of action is not critical to the broad practice of the invention.

[0265] In some embodiments, the potentiating agent is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan®, or Neosar®) is an oxazaphosphorine drug, and analogs include ifosfamide (IFO, Ifex), perfosfamide, trophosphamide (trofosfamide; Ixoten), and pharmaceutically acceptable salts, solvates, prodrugs, and metabolites thereof (U.S. Patent Application Publication No. 2007 / 0202077, incorporated in its entirety). Ifosfamide (MITOXANA®) is a structural analog of cyclophosphamide, and its mechanism of action is considered to be the same as or substantially similar to that of cyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) and trofosfamide are also alkylating agents structurally related to cyclophosphamide. For example, perfosfamide alkylates DNA, thereby inhibiting DNA replication and RNA and protein synthesis. New oxazaphosphorine derivatives have been designed and evaluated in an attempt to improve selectivity and response and reduce host toxicity (Liang J, Huang M, Duan W, Yu XQ, Zhou S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des. 2007;13(9):963-78. Review). These include mafosfamide (NSC 345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide mustard), S-(-)-bromophosfamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine), and NSC 613060 (aldophosphamide thiazolidinedione). Mahofosfamide is an oxazaphosphorine analogue, the chemically stable 4-thioethanesulfonate salt of 4-hydroxy-CPA. Glufosfamide is an IFO derivative in which isophosphoramide mustard, an alkylated metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule.Additional cyclophosphamide analogs are described in US Pat. No. 5,190,929, entitled "Cyclophosphamide analogs useful as anti-tumor agents," which is incorporated herein by reference in its entirety.

[0266] Although CTX itself is non-toxic, some of its metabolites are cytotoxic alkylating agents that induce DNA cross-linking and, at higher doses, strand breakage. Many cells are resistant to CTX because they express high levels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). Because lymphocytes (but not hematopoietic stem cells) express only low levels of ALDH, CTX targets proliferating lymphocytes, whereas cycling cells are most sensitive to DNA alkylating agents.

[0267] In one embodiment, low doses of CTX are used in combination with the disclosed antibodies and their antigen-binding fragments. Low doses of CTX (less than 200 mg / kg) can have immunostimulatory effects, including stimulating anti-tumor immune responses in human and mouse cancer models (Brode & Cooke Crit Rev. Immunol. 28:109-126 (2008)). These low doses are subtherapeutic and do not have direct anti-tumor activity. In contrast, high doses of CTX inhibit anti-tumor responses. Several mechanisms may explain the role of CTX in enhancing anti-tumor immune responses: (a) depletion of CD4+CD25+FoxP3+ Tregs (and specifically, proliferating Tregs, which may be particularly suppressive); (b) depletion of B lymphocytes; (c) induction of nitric oxide (NO), which leads to the suppression of tumor cell growth; and (d) mobilization and expansion of CD11b+Gr-1+MDSCs. These primary effects have many secondary effects; for example, following Treg depletion, macrophages produce more IFN-γ and less IL-10. CTX has also been shown to induce type I IFN expression and promote homeostatic proliferation of lymphocytes.

[0268] Treg depletion is most often cited as the mechanism by which CTX enhances antitumor immune responses. This conclusion is based in part on the results of adoptive transfer experiments. In the AB1-HA tumor model, CTX treatment on day 9 resulted in a 75% cure rate. Transfer of purified Tregs on day 12 almost completely inhibited the CTX response (van der Most et al. Cancer Immunol. Immunother. 58:1219-1228 (2009)). Similar results were seen in the HHD2 tumor model, where adoptive transfer of CD4+CD25+ Tregs after CTX pretreatment eliminated the therapeutic response to the vaccine (Taieb, JJ Immunol. 176:2722-2729 (2006)).

[0269] Numerous human clinical trials have demonstrated that low-dose CTX is a safe, well-tolerated, and effective agent for promoting anti-tumor immune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47:1-12 (1998)).

[0270] In one embodiment, the optimal dose of CTX for enhancing anti-tumor immune responses is one that reduces total T cell counts by reducing Treg levels below the normal range, but is sub-therapeutic (see Machiels et al. Cancer Res. 61:3689-3697 (2001)).

[0271] In some embodiments, CTX is 300 mg / m 2 In another embodiment, the average male (6 feet, 170 pounds (78 kg), 1.98 m) 2 300 mg / m2 body surface area 2In contrast, the dose of CTX is 8 mg / kg, or 624 mg of total protein. In mouse models of cancer, efficacy has been seen at doses ranging from 15 to 150 mg / kg, which corresponds to 0.45 to 4.5 mg of total protein in a 30 g mouse (Machiels et al. Cancer Res. 61:3689-3697 (2001); Hengst et al. Cancer Res. 41:2163-2167 (1981); Hengst Cancer Res. 40:2135-2141 (1980)).

[0272] For larger mammals such as primates, including human patients, such mg / m 2 Doses can be used, but unit doses administered at finite time intervals can also be used.Such unit doses can be administered daily for a finite period, and up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days, etc. are all specifically contemplated by the present invention.The same schedule can also be applied to other enhancing agents listed herein.

[0273] In other embodiments, the potentiating agent is an agent that reduces the activity and / or number of regulatory T lymphocytes (T-regs), such as sunitinib (SUTENT®), anti-TGFβ, or imatinib (GLEEVAC®). The listed treatment regimens may also include the administration of adjunctive agents.

[0274] Useful enhancers also include antimitotic agents such as paclitaxol, aromatase inhibitors (e.g., Letrozole), and angiogenesis inhibitors (VEGF inhibitors such as Avastin, VEGF-Trap) (see, e.g., Li et al., Vascular endothelial growth factor blockade reduces intratumoral regulatory T cells and enhances the efficacy of a GM-CSF-secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov 15;12(22):6808-16), anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18 antagonists.

[0275] VI. Transgenic Animals One embodiment provides a transgenic animal that produces an antibody or antigen-binding fragment thereof having heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, and light chain CDRs having amino acids according to SEQ ID NOs: 12, 13, and 14. In one embodiment, the transgenic animal is a rodent, e.g., a mouse.

[0276] Another embodiment provides a transgenic animal that produces an antibody or antigen-binding fragment thereof having heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, and light chain CDRs having amino acids selected from the group consisting of SEQ ID NOs: 24, 13, and 25. In one embodiment, the transgenic animal is a rodent, e.g., a mouse.

[0277] Another embodiment provides a transgenic animal that produces an antibody or antigen-binding fragment thereof having heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 29, 30, and 31, and light chain CDRs having amino acids according to SEQ ID NOs: 35, 36, and 37. In one embodiment, the transgenic animal is a rodent, e.g., a mouse.

[0278] Methods for producing transgenic animals that produce antibodies are known in the art. For example, see A. Jakobovits, Curr Opin Biotechnol., 6(5):561-6(1995) and Bruggemann, M., et al., Arch Immunol Ther Exp(Warsz)., 63(2):101-108(2015); Jakobovits, A., et al., "From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice," Nat Biotechnol. 25(10):1134-43 (2007); Lonberg N. (2005) "Human antibodies from transgenic animals," Nat Biotechnol. 23(9):1117-25, and U.S. Patent Nos. 9,708,635, 9,686,970, 9,499,838, 9,445,581, 9,388,446, 8,835,712, 8,703,485, 8,232,449, 7,795,494, and 5,939,598. [Example]

[0279] Example 1: Anti-PD-1 antibody production result The production of anti-PD-1 antibodies yielded clones 4G9, 4C12, and 5C2, which were selected for characterization.

[0280] Example 2: Interaction kinetics between anti-PD-1 antibodies and PD-1 material and method Antibodies from clones 4G9, 4C12, and 5C2 were characterized using a Biocore™ system available from GE. The analyte was mouse or human PD-1, and the ligand was an anti-PD-1 antibody. Analyte concentrations were 0, 62.5, 125, 250, 500, and 1000 nM, where indicated.

[0281] result Figure 1 and Table 1 show the interaction analysis of 4G9 with human PD-1. The equilibrium association constant (K A ) is 9.52 × 10 5 (1 / M). The equilibrium dissociation constant (K D ) is 1.05 × 10 -6 (M).

[0282] Table 1. Interaction analysis of 4G9 with human PD-1 TIFF0007795860000040.tif41170

[0283] Figure 2 and Table 2 show the interaction analysis of 4G9 with mouse PD-1. The equilibrium association constant (K A ) is 1.94 × 10 5 (1 / M). The equilibrium dissociation constant (K D ) is 5.15 × 10 -6 (M).

[0284] Table 2. Analysis of 4G9 interaction with mouse PD-1 TIFF0007795860000041.tif41170

[0285] Figure 3 and Table 3 show the interaction analysis of 4C12 with human PD-1. The equilibrium association constant (K A ) is 3.14 × 10 6 (1 / M). The equilibrium dissociation constant (K D ) is 3.19 × 10 -7 (M).

[0286] Table 3. Interaction analysis of 4C12 with human PD-1 TIFF0007795860000042.tif41170

[0287] Figure 4 and Table 4 show the interaction analysis of 5C2 with human PD-1. The equilibrium association constant (K A ) is 2.02 × 10 5 (1 / M). The equilibrium dissociation constant (K D ) is 4.95 × 10 -6 (M).

[0288] Table 4. Interaction analysis of 5C2 with human PD-1 TIFF0007795860000043.tif41170

[0289] Figure 5 and Table 5 show the interaction analysis of 5C2 with mouse PD-1. The equilibrium association constant (K A ) is 1.18 × 10 6 (1 / M). The equilibrium dissociation constant (K D ) is 8.50 x 10 -7 (M).

[0290] Table 5. Interaction analysis of 5C2 with mouse PD-1 TIFF0007795860000044.tif41170

[0291] Example 3: Binding of anti-PD-1 antibodies to EL4 cells material and method Murine EL4 cells, which constitutively express PD-1, were used in a fluorescence-activated cell sorter to assess the binding of 4G9, 5C2, and 4C12 to the cells.

[0292] result Figure 6A is a flow cytometry histogram showing the binding of a commercially available anti-PD-1 antibody to EL4 cells, whereas a control isotype antibody does not. Figure 6B is a flow cytometry histogram showing the binding of 4G9, 5C2, and 4C12 to EL4 cells, whereas a secondary antibody alone does not. Figure 6C is a flow cytometry histogram showing the binding of the 4G9 anti-PD-1 antibody to EL4 cells, whereas a secondary antibody alone does not. Figure 6D is a flow cytometry histogram showing the binding of the 5C2 anti-PD-1 antibody to EL4 cells, whereas a secondary antibody alone does not. Figure 6E is a flow cytometry histogram showing the binding of the 4C12 anti-PD-1 antibody to EL4 cells, whereas a secondary antibody alone does not.

[0293] Example 4: Agonistic activity of anti-PD-1 antibodies material and method Mouse CD4 T cells Purified mouse CD4 T cells were stimulated with anti-CD3 / anti-CD28 Abs for 48 hours, then anti-PD-1 Abs (10 μg / mL) were added and cultured with Protein A beads for an additional 48 hours. In some samples, anti-PD-L1 Abs were added to block the PD-1 / PD-L1 interaction and analyze the agonistic effects of the test Abs. IFNγ and IL-2 concentrations in the supernatants were detected using a CBA assay.

[0294] Human CD4 T cells Purified human CD4 T cells were stimulated with anti-CD3 / anti-CD28 Abs for 48 hours, then anti-PD-1 Abs (1 or 10 μg / mL) were added and cultured with protein A beads for another 48 hours. IFNγ concentrations in the supernatants were detected using the CBA assay.

[0295] result Figures 7A and 7B show that stimulated CD4 T cells treated with anti-PD-1 antibodies 5C2, 4C12, and 4G9 had higher concentrations of IFNγ and IL-2 in the supernatant compared to untreated cells or cells treated with anti-PD-L1 antibodies. Figure 7C shows that stimulated human CD4 T cells treated with anti-PD-1 antibodies 4G9 and 5C2 had higher concentrations of IFNγ supernatant compared to untreated cells or cells treated with an isotype control antibody.

[0296] Example 5: Hybridomas enhance Akt phosphorylation material and method Intracellular staining of pAKT(S473) was used as a marker of T cell activation.

[0297] result Hybridomas from mice immunized with peptide E enhanced phosphorylation of Akt (S473) in mouse CD4 T cells (FIG. 8).

[0298] Example 6: Characterization of three anti-PD-1 antibodies material and method Purified antibodies from hybridomas 5C2, 4C12, and 4G9 were characterized.

[0299] result The isotype of each of the three anti-PD-1 antibodies was determined. Both 5C2 and 4C12 were found to be of the IgG1 isotype, while 4G9 was found to be of the IgG2b isotype (Figure 9). Hybridoma sequencing showed 100% sequence identity to the 5C2 and 4C12 antibodies.

[0300] Example 7: 4G9 and 5C2 specifically bind to human PD-1 material and method: An ELISA assay was used to determine the binding of 4G9 and 5C2 to human PD-1-Fc.

[0301] result: Figure 10 shows the results of an ELISA assay to assess the binding of the 4G9 and 5C2 antibodies to human PD-1-Fc. Both 4G9 and 5C2 specifically bind to human PD-1. Figure 11A is a flow cytometry histogram showing that 4G9 and 5C2 do not bind to PD-1 KO CD4 T cells, but do bind to CD4 T cells from wild-type mice (Figure 11B).

[0302] Example 8: Signal Transduction material and method: Murine CD4 T cells were pre-stimulated for 48 hours to ensure PD-1 expression and then treated with purified 4G9 and 5C2 antibodies. The concentration of pS6 was determined using an ELISA kit (Cell Signaling Tech).

[0303] result: In contrast to blocking antibodies (RMP1-14 and J43), 4G9 and 5C2 activated T cells through the S6 pathway. Treatment of mouse CD4 T cells pre-stimulated for 48 hours (to ensure PD-1 expression) with purified 4G9 and 5C2 antibodies significantly increased pS6 within the linear range (ELISA kit, Cell Signaling Tech; *P<0.05, **P<0.01, ***P<0.001 compared with untreated). This phenomenon is due to direct activation rather than PD-1 / PD-L1 blockade, because treatment with commercially available Ab-1 (RMP1-14) and Ab-2 (J43), which block the PD-1 / PD-L1 interaction, did not result in an increase in pS6.

[0304] Example 8: In vivo efficacy evaluation material and method: Figure 13A is a schematic diagram of the TC-1 tumor model used in this experiment. Briefly, on day 0, TC-1 tumor cells were subcutaneously injected into mice. On days 10 (D10), 17 (D17), and 24 (D24) after tumor injection, the mice were treated with vaccine (E7+PADRE+Quil A). On days 10, 14, 17, 21, 24, and 28, the mice were treated with anti-PD-1 antibody.

[0305] result: The anti-PD-1 antibodies 4G9, 4C12, and 5C2 reduced tumor volume and increased survival when combined with the E7 vaccine (Figures 13B-13D).

[0306] Example 9: In vivo efficacy evaluation - anti-EpE antibodies material and method: Antibodies against epitope E were generated and tested in the TC-1 tumor model described above in Example 8 and in Figure 14A.

[0307] result: Mice treated with 4G9, an antibody directed against epitope E, showed significantly lower tumor volume and higher survival rates compared to traditional checkpoint inhibitors, the anti-PD1 blocking antibodies RMP1-14 and J43 (Figures 14B-14C).

Claims

1. a heavy chain complementarity determining region (CDR) 1 having the amino acid sequence set forth in SEQ ID NO:6; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:8; a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 12; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 14; An anti-PD-1 antibody or antigen-binding fragment thereof comprising:

2. An anti-PD-1 antibody or antigen-binding fragment thereof, comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) sequence of SEQ ID NO: 4 or 5, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) sequence of SEQ ID NO: 10 or 11.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4 or 5.

4. The antibody or antigen-binding fragment thereof of claim 1 or 3, comprising a light chain having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 10 or 11.

5. a heavy chain having at least 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4 or 5; a light chain having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 10 or 11; The antibody or antigen-binding fragment thereof of claim 1, comprising:

6. A non-human transgenic animal engineered to express the antibody or antigen-binding fragment thereof of any one of claims 1 to 5.

7. The transgenic animal of claim 6, which is a rodent.

8. The transgenic animal of claim 7 , wherein the rodent is a mouse.

9. A nucleic acid comprising a sequence encoding an anti-PD-1 antibody or antigen-binding fragment thereof, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:6, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:8, and having at least 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:4 or 5; Encoding the heavy chain, a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 14, and having at least 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 10 or 11; Encoding the light chain, Nucleic acid.

10. A nucleic acid comprising a sequence encoding the anti-PD-1 antibody or antigen-binding fragment thereof of claim 1 or 2.

11. A cell comprising a nucleic acid comprising a sequence encoding an anti-PD-1 antibody or antigen-binding fragment thereof, a nucleic acid comprising a sequence encoding a heavy chain comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:6, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:8; and A nucleic acid comprising a sequence encoding a light chain comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 12, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 13, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:

14. including, cells.

12. A cell comprising a nucleic acid comprising a sequence encoding an anti-PD-1 antibody or antigen-binding fragment thereof, a nucleic acid comprising a sequence encoding a heavy chain comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) sequence of SEQ ID NO: 4 or 5; and a nucleic acid comprising a sequence encoding a light chain comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within the light chain variable region (LCVR) sequence of SEQ ID NO: 10 or 11; including, cells.

13. a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:29; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 30; and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 31; a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 35; a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 36, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 37; An anti-PD-1 antibody or antigen-binding fragment thereof comprising:

14. An anti-PD-1 antibody or antigen-binding fragment thereof, comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) sequence of SEQ ID NO: 27 or 28, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) sequence of SEQ ID NO: 33 or 34.

15. The antibody or antigen-binding fragment thereof of claim 13, comprising a heavy chain having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 27 or 28.

16. 16. The antibody or antigen-binding fragment thereof of claim 13 or 15, comprising a light chain having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or 34.

17. a heavy chain having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 27 or 28; a light chain having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or 34; The antibody or antigen-binding fragment thereof of claim 13, comprising:

18. A non-human transgenic animal engineered to express the antibody or antigen-binding fragment thereof of any one of claims 13 to 17.

19. 19. The transgenic animal of claim 18, which is a rodent.

20. 20. The transgenic animal of claim 19, wherein the rodent is a mouse.

21. A nucleic acid comprising a sequence encoding an anti-PD-1 antibody or antigen-binding fragment thereof, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:29, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:30, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:31, and encoding a heavy chain having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:27 or 28; encoding a light chain comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 35, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 36, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 37, and having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or 34; Nucleic acid.

22. A nucleic acid comprising a sequence encoding the anti-PD-1 antibody or antigen-binding fragment thereof of claim 13 or 14.

23. A cell comprising a nucleic acid comprising a sequence encoding an anti-PD-1 antibody or antigen-binding fragment thereof, a nucleic acid comprising a sequence encoding a heavy chain comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:29, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:30, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:31; and A nucleic acid comprising a sequence encoding a light chain comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:35, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:36, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:

37. including, cells.

24. A cell comprising a nucleic acid comprising a sequence encoding an anti-PD-1 antibody or antigen-binding fragment thereof, a nucleic acid comprising a sequence encoding a heavy chain comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) sequence of SEQ ID NO: 27 or 28; and a nucleic acid comprising a sequence encoding a light chain comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within the light chain variable region (LCVR) sequence of SEQ ID NO: 33 or 34; including, cells.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or 13 to 17, which is a monoclonal antibody.

26. 18. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, or 13 to 17, which is a murine or chimeric antibody.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or 13 to 17, which is a humanized antibody.

28. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, or 13 to 17, which is bispecific, trispecific, or multispecific.

29. The antibody of any one of claims 1 to 5, 13 to 17, or 25 to 28, which is a whole immunoglobulin.

30. 30. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, 13 to 17, or 25 to 29, which is IgG, IgE, IgM, IgD, IgA, or IgY.

31. IgG １ , IgG ２ , IgG ３ , IgG ４ , IgA １ , or IgA ２ 30. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, 13 to 17, or 25 to 29,

32. Fab', F(ab') ２ 32. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, 13 to 17, 25 to 28, 30, or 31, which is an Fv, single chain (ScFv), di-scFv, diabody, or tribody.

33. 33. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, 13 to 17, or 25 to 32, which immunospecifically binds to the amino acid sequence set forth in SEQ ID NO: 38 on the surface of an immune cell.

34. 34. The antibody or antigen-binding fragment thereof of claim 33, wherein the immune cell is a T cell.

35. The T cell is CD8 ＋ The antibody or antigen-binding fragment thereof of claim 34, which is a T cell.

36. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, 13 to 17, or 25 to 32, which immunospecifically binds to the amino acid sequence set forth in SEQ ID NO: 38 on PD-1 expressed on the surface of immune cells, and induces or promotes a signal that activates or stimulates immune cells through PD-1.

37. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, 13 to 17, or 25 to 36.

38. 38. The pharmaceutical composition of claim 37, further comprising a second therapeutic agent.

39. 39. The pharmaceutical composition of claim 37 or 38, further comprising a pharmaceutically acceptable excipient.

40. 39. The pharmaceutical composition of claim 38, wherein the second therapeutic agent comprises cyclophosphamide.

41. 41. The pharmaceutical composition of any one of claims 37 to 40 for use in a method for inducing, promoting or enhancing an immune response in a subject in need thereof.

42. 41. The pharmaceutical composition of any one of claims 37 to 40 for use in a method for treating cancer in a subject in need thereof.

43. 41. The pharmaceutical composition of any one of claims 37 to 40 for use in a method for reducing tumor burden in a subject in need thereof.

44. 41. The pharmaceutical composition of any one of claims 37 to 40 for use in a method for treating an infection in a subject in need thereof.

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