Antibody against programmed cell death protein 1

Abstract

The present invention provides an antibody and its antigen-binding fragment that immune-specifically bind to PD-1, preferably human or mouse PD-1, and induce or promote an immune response that activates immune cell proliferation and activity. [Solution] The antibody and its antigen-binding fragment of the Disclosure bind immune-specifically to PD-1 and deliver an activation signal to immune cells that activates them rather than suppresses them. In one embodiment, the antibody and its antigen-binding fragment of the Disclosure bind specifically to PD-1 expressed on immune cells. The binding of the antibody and its antigen-binding fragment of the Disclosure to PD-1 on immune cells causes the transmission of an activation signal into the immune cells, for example, a signal that enhances or promotes the activation of cytokine production and / or immune cell proliferation. In one embodiment, the immune cells are T cells, preferably CD8+ T cells.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Patent Application No. 62 / 555,156 filed on 7 September 2017, No. 62 / 624,843 filed on 1 February 2018, and No. 62 / 657,323 filed on 13 April 2018, all of which are incorporated in their entirety by reference.

[0002] Sequence listing reference The sequence listing, created on 21 August 2018 and filed on 6 September 2018 as a text file named "064466.071 sequence listing_ST25.txt" with a size of 55.2 kilobytes, is incorporated herein by reference in accordance with 1.52(e)(5) of the U.S. Patent Act Enforcement Rules.

[0003] Technical field of inventions The present invention generally relates to immunomodulation, as well as antibodies that specifically bind to PD-1 and methods for using them. [Background technology]

[0004] Background of the Invention The programmed cell death receptor protein (PD-1) / programmed cell 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 the immune response against cancer cells. Successful clinical trials with PD-1 monoclonal antibodies and other immune checkpoint inhibitors are opening new avenues in cancer immunology. However, because a large subset of cancer patients do not respond to new immunotherapies, research into combination therapies and predictive biomarkers is being intensified (Iwai, Y., et al., Journal of Biomedical Science, 24:26 (2017) (Non-patent Literature 1)).

[0005] Therefore, one object of the present invention is to provide compositions and methods for regulating PD-1 signaling.

[0006] Another object of the present invention is to provide an antibody and its antigen-binding fragment that specifically bind to PD-1 and regulate 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) [Overview of the project]

[0010] The present invention provides an antibody and its antigen-binding fragment that immune-specifically 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 antibody and its antigen-binding fragment specifically bind to PD-1 expressed on immune cells. The binding of the disclosed antibody and its antigen-binding fragment to PD-1 on immune cells transmits an activation signal into the immune cell, for example, a signal that enhances or promotes the activation of cytokine production and / or immune cell proliferation. Immune cells expressing PD-1 include, but are not limited to, B and T cells, as well as 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, promoting, or enhancing an adaptive immune response in a subject that needs it by administering to the subject an effective amount of the disclosed anti-PD-1 antibody or an antigen-binding fragment thereof to induce, enhance, or promote an adaptive immune response in the subject.

[0012] One embodiment provides an antibody or an antigen-binding fragment thereof having a heavy-chain complementarity-determining region (CDR) having the amino acid sequences set forth in SEQ ID NO: 6, 7, and 8 and a light-chain CDR having the amino acid sequences set forth in SEQ ID NO: 12, 13, and 14, wherein the antibody or antigen-binding fragment thereof binds immunospecifically to PD-1.

[0013] One embodiment provides an antibody or an 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.

[0014] One embodiment provides an antibody or an 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 with 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 with SEQ ID NO:10 or 11.

[0019] One embodiment provides an antibody or an antigen-binding fragment thereof having a heavy chain CDR having the amino acid sequences set forth in SEQ ID NO:18, 19, and 20 and a light chain CDR having the amino acid sequences set forth in SEQ ID NO:24, 13, and 25.

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

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

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

[0025] One embodiment provides an antibody or its antigen-binding fragment, comprising a heavy chain CDR having the amino acid sequences described in SEQ ID NO: 29, 30, and 31, and a light chain CDR having the amino acid sequences described in SEQ ID NO: 35, 36, and 37.

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

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

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

[0031] One embodiment provides an antibody, or its antigen-binding fragment, 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 its antigen-binding fragment, containing 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.

[0033] Another embodiment provides an antibody or its antigen-binding fragment, comprising 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 its epitope-binding fragment or fusion protein that binds immunospecifically 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 immune cells and induces or promotes a signal through PD-1 that activates or stimulates the immune cells. In one embodiment, the activated or stimulated immune cells are T cells, for example, CD8 +These are T cells.

[0035] In some embodiments, the antibody or its antigen-binding fragment is human, mouse, chimeric, humanized, monoclonal, bispecific, tripspecific, or multispecific.

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

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

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

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

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

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

[0042] Detailed description of the invention I. Definition As used herein, a molecule is said to be capable of “immunospecific binding” to a second molecule if such binding exhibits the antibody’s specificity and affinity to its congener antigen. An antibody is said to be capable of immunospecific 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 to other antigens with lower affinity if the other antigen has some sequence or conformational similarity recognized by the antigen recognition site, as determined, for example, by immunological assays, BIACORE® assays, or other assays known in the art, but will not bind to completely unrelated antigens. However, preferably, the antibody (and its antigen-binding fragments) do not cross-react with other antigens. Antibodies may also bind to other molecules in a non-immunospecific manner, such as FcR receptors, by binding domains in other regions / domains of the molecule that do not involve an antigen-recognition site, such as an 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 the receptor to its homologous 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 immunoglobulin from domains widely shared by antibodies (such as the antibody Fc domain). The variable region includes a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region consists of amino acid residues from the “complementarity-determining region” or “CDR” (i.e., typically residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of This includes residues from Health, Bethesda, MD. (1991), and / or from the "hypervariable loop" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 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 variable domain residues other than the hypervariable region residues as defined herein.The term antibody can refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (see, for example, 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. WO94 / 04678 and WO94 / 25591; U.S. Patent No. 6,005,079), single-stranded Fvs (scFv) (see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New See York, pp. 269-315 (1994), which include single-chain antibodies, disulfide-linked Fvs (sdFv), intracellular antibodies, and anti-idiotype (anti-Id) antibodies (e.g., 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 framework residues including the antibody’s complementarity-determining region (“CDR”) and optionally the antibody’s “variable region” antigen-recognition site, and exhibit the ability to bind immunospecifically to an antigen. Such fragments include Fab’, F(ab’)2, Fv, single-stranded (ScFv), and their mutants, spontaneous variants, as well as fusion proteins containing the antibody’s “variable region” antigen-recognition site and heterologous proteins (e.g., toxins, antigen-recognition sites of different antigens, enzymes, receptors, or receptor ligands).

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

[0047] As used herein, the term “binding molecule” is intended to refer to a molecule that specifically interacts with and binds to a particular target. The target may include a biomolecule or a small (chemical) molecule. The target molecule may define an antigen or an antigenic moiety. 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., signaling). Such modulation may be operative or antagonistic. Antagonistic modulation may be partial (i.e., weaken rather than neutralize) or may completely neutralize such activity (e.g., neutralize). Modulation may include the internalization of a receptor after antibody binding or a reduction in receptor expression on target cells. Apological modulation may enhance or otherwise increase or enhance activity (e.g., signaling). In further embodiments, such modulation may also alter the nature of the interaction between a ligand and its homologous receptor in order to alter the nature of the induced signaling. For example, a molecule may, by binding to a ligand or receptor, alter the ability of such molecule to bind to other ligands or receptors, thereby altering their overall activity. Preferably, such modulation will provide a change of at least 10% of measurable immune system activity, more preferably at least 50% of such activity, or at least 2x, 5x, 10x, or even more preferably at least 100x of such activity.

[0049] The term "substantially" is intended to indicate that the observed effect is physiologically or therapeutically significant when used in the context of binding or the effect being shown. For example, a molecule can substantially block the activity of a ligand or receptor if the degree of blockade is physiologically or therapeutically significant (e.g., if such a degree is greater than 60% completeness, greater than 70% completeness, greater than 75% completeness, greater than 80% completeness, greater than 85% completeness, greater than 90% completeness, greater than 95% completeness, or greater than 97% completeness). 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, the term “co-stimulus” signal includes both positive co-stimulus signals (e.g., signals that result in enhancement of activity) and negative co-stimulus signals (e.g., signals that result in inhibition of activity).

[0051] As used herein, the term “derivative” refers to an antibody or its antigen-binding fragment that binds immunospecifically to the same target as the parent antibody or reference antibody, but whose amino acid sequence differs from that of the parent antibody or reference antibody or its antigen-binding fragment by including substitutions, additions, deletions, or modifications of one, two, three, four, five, or more amino acid residues to the parent antibody or reference antibody or its antigen-binding fragment. Preferably, such derivatives will have substantially the same immunospecificity and / or characteristics as the parent antibody or reference antibody or its antigen-binding fragment, or have the same immunospecificity and characteristics. Amino acid substitutions or additions to such derivatives may include spontaneously occurring (i.e., DNA-encoded) or non-spontaneously occurring amino acid residues. The term “derivative” includes, for example, chimeric or humanized variants, as well as variants having modified CH1, hinge, CH2, CH3, or CH4 regions, to form antibodies having variant Fc regions that exhibit enhanced or reduced effector or binding characteristics.

[0052] As used herein, "chimeric antibody" refers to a molecule in which different parts of the antibody are derived from different immunoglobulin molecules, such as an antibody 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 non-human (usually mouse or rat) immunoglobulins. The non-human immunoglobulin providing the CDRs is called the “donor,” and the human immunoglobulin providing the framework is called the “acceptor.” Constant regions are not required to be present, but if present, they should be substantially identical to the constant regions of human immunoglobulins, i.e., at least about 85–99%, preferably about 95% or more identical. Thus, all parts of a humanized immunoglobulin are substantially identical to the corresponding parts of the natural human immunoglobulin sequence, except in some cases the CDRs. A humanized antibody is an antibody containing humanized light chain and humanized heavy chain immunoglobulins. For example, a humanized antibody would not encompass a typical chimeric antibody, since, for example, the entire variable region of a chimeric antibody is non-human.

[0054] As used herein, the term “endogenous concentration” means the level at which a molecule is expressed naturally (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 “to treat,” “to treat,” “treatment,” and “therapeutic use” refer to the elimination, reduction, or improvement of one or more symptoms of a disease or disorder exacerbated by an anti-PD-1 antibody or its antigenic fragment.

[0056] As used herein, “therapeutic dose” means an amount of therapeutic agent sufficient to mediate a clinically meaningful elimination, reduction, or improvement of such a condition. An effect is clinically meaningful if its magnitude is sufficient to affect the health or prognosis of the recipient. A therapeutic dose may also mean an amount of therapeutic agent sufficient to delay or minimize the onset of a disease, for example, the spread of cancer. A therapeutic dose may also mean an amount of therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0057] As used herein, the term “preventive agent” refers to an agent that can be used to prevent a disorder or disease before any symptom of such disorder or disease is detected. “Preventive effective” dose is the amount of a preventive agent sufficient to mediate such protection. The preventive effective dose may also refer to the amount of a preventive agent that provides a preventive benefit in the prevention of disease.

[0058] As used herein, the term “cancer” refers to a neoplasm or tumor resulting from the abnormal and uncontrolled growth of cells. As used herein, cancer expressly includes leukemia and lymphoma. The term “cancer” refers to a disease involving cells that have metastasized distally and may exhibit phenotypic traits distinct from those of non-cancerous cells, such as colonization in three-dimensional substrates like soft agar, or formation of tubular networks or web-like matrices in three-dimensional basement membranes or extracellular matrix preparations. Non-cancerous cells do not colonize in soft agar and form distinct spherical structures in three-dimensional basement membranes or extracellular matrix preparations.

[0059] As used herein, “immune cells” 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, “valentity” refers to the number of available binding sites per molecule.

[0061] As used herein, the terms “immunological,” “immunological,” or “immune” response refer to the occurrence of a beneficial humoral (antibody-mediated) and / or cellular (antigen-specific T cell-mediated or their secretion-mediated) response to a peptide in a recipient patient. Such a response may be an active response induced by the administration of an immunogen, or a passive response induced by the administration of an antibody or T cells that have received initial antigen stimulation. A cellular immune response may involve antigen-specific CD4 + T helper cells and / or CD8 +The response is induced by the presentation of polypeptide epitopes associated with class I or class II MHC molecules to activate cytotoxic T cells. The response may also be accompanied by the activation or recruitment of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, neutrophils, or other components of innate immunity. The presence of a cell-mediated immunological response is indicated in proliferation assays (CD4 + This can be determined by T cell (T cell) or CTL (cytotoxic T lymphocyte) assays. 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 immunized syngenes and measuring their protective or therapeutic effect in a second target.

[0062] As used herein, “immunogenic agent” or “immunogen” may optionally be used with adjuvants to induce an immunological response to itself in mammals after administration.

[0063] As used herein, the terms “individual,” “host,” “subject,” and “patient” are interchangeable herein and refer to mammals, including but not limited to humans, rodents, such as 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 modifications (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and their fragments. Polypeptides can be “exogenous,” meaning they are “heterogeneous,” or different from, 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. Their sequences are described from left to right, in the direction from amino acid to carboxyl terminus. Following 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 is different from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in its amino acid sequence from another reference polypeptide. Generally, the differences are limited to such that the sequences of the reference polypeptide and the variant are closely similar overall and identical in many regions. A variant and a reference polypeptide may differ in their amino acid sequences by one or more modifications (e.g., substitutions, additions, and / or deletions). The substituted or inserted amino acid residues may or may not be encoded by the genetic code. A variant of a polypeptide may be a naturally occurring one, such as an allele variant, or it may be a variant that is not known to occur naturally.

[0066] Modifications and alterations can be made to the structure of the disclosed polypeptide to obtain a molecule that still possesses similar characteristics to the polypeptide (e.g., conserved amino acid substitutions). For example, a particular amino acid may be substituted in the sequence for another amino acid without apparent loss of activity. Since the interaction ability and properties of a polypeptide define its biological and functional activity, a particular amino acid sequence substitution can be made in the polypeptide sequence, and a polypeptide with similar properties can still be obtained.

[0067] When making such modifications, the hydroxyl index of amino acids may be taken into consideration. The importance of the hydroxyl amino acid index in conferring interactive biological functions to polypeptides is generally understood in the art. It is known that certain amino acids can be substituted with other amino acids having similar hydroxyl indices or scores, resulting in polypeptides that still possess similar biological activity. Each amino acid is assigned a hydroxyl index based on its hydrophobic and charge characteristics. The 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 hydroxyl characteristics of amino acids are thought 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 amino acids can be substituted with other amino acids having similar hydroxyl indices, and functionally equivalent polypeptides can still be obtained. In such modifications, substitutions of amino acids with hydroxyl indices of ±2 are preferred, those of ±1 are particularly preferred, and those of ±0.5 are even more particularly preferred.

[0069] Similar amino acid substitutions based on hydrophilicity may also be made, particularly when the resulting biologically equivalent polypeptide or peptide is intended for use in immunological embodiments. The following hydrophilic values ​​are 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 by substituting an amino acid with another having a similar hydrophilicity value, a polypeptide that is still biologically equivalent, and especially immunologically equivalent, can be obtained. In such a change, substitution of an amino acid with a hydrophilicity value of ±2 is preferred, ±1 is particularly preferred, and ±0.5 is even more preferred.

[0070] As outlined above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering various of the above characteristics are well known to those skilled 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). Accordingly, embodiments of the present disclosure are intended to provide functional or bioequivalents of the polypeptides described above. In particular, the polypeptide embodiments may include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with 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 a nucleotide or amino acid in a reference nucleic acid sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum sequence identity percentage. Alignment for the purpose of determining sequence identity percentage can be achieved in various ways within the scope of the skills 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 algorithm required to achieve the maximum alignment over the entire length of the sequences being compared, can be determined by known methods.

[0072] For the purposes of this specification, the sequence identity percentage of a given nucleotide or amino acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be expressed as a given sequence C having, or containing, a particular sequence identity percentage to, with, or against a given sequence D) is calculated as follows: Fraction W / Z×100 In the formula, W is the number of nucleotides or amino acids that are scored as a perfect match in the alignment of C and D by the sequence alignment program, 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, 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 any of the standard pharmaceutically acceptable carriers, such as phosphate-buffered salt solutions, water, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents.

[0074] As used herein, the terms “antigenic determinant” and “epitope” are used interchangeably and refer to structures recognized by antibodies.

[0075] As used herein, “structural epitope” is an epitope containing discontinuous sections of the amino acid sequence of an antigen. Antibodies bind to structural 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 continuous sequence of amino acids from an antigen. A linear epitope typically contains about 5 to about 10 consecutive amino acid residues. Antibodies bind to linear epitopes based on the primary sequence of the antigen.

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

[0078] II. Composition An antibody and its antigen-binding fragment that immune-specifically bind to PD-1 are provided. Contrary to the existing paradigm that PD-1 only promotes a suppressive immune response (Riley, J., Immunol Rev. 229(1): 114-125 (2009)), the disclosed antibody and its antigen-binding fragment immune-specifically bind to PD-1 and deliver an activation signal to immune cells that activates them rather than suppresses them.

[0079] A. Programmed Death Receptor Protein 1 (PD-1) The disclosed antibody and its antigen-binding fragment bind immunospecifically to PD-1. The antibody and its antigen-binding fragment can bind to PD-1 having, for example, the amino acid sequence provided below.

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

[0081] Human PD-1 TIFF2026049006000001.tif41165 accession: AJS10360, which is incorporated herein by reference in its entirety.

[0082] Mouse PD-1 This is TIFF2026049006000002.tif42165, which is incorporated in its entirety by reference herein.

[0083] B. Antibody composition The disclosed anti-PD-1 antibody or its antigen-binding fragment comprises a whole immunoglobulin of any class (i.e., an intact antibody), its fragment, and a synthetic protein containing at least the antigen-binding variable domain of the antibody. In some embodiments, the disclosed antibody contains both the antibody light chain and at least the variable domain of the antibody heavy chain. In other embodiments, such a molecule may further comprise one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain (in particular, the CH1 and hinge region, or the CH1, hinge, and CH2 region, or the CH1, hinge, CH2, and CH3 region). The antibody may 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-binding constant domain when it is desirable for the antibody to exhibit cytotoxic activity, and the class is typically IgG1. In other embodiments, when such cytotoxic activity is undesirable, the constant domain may be of the IgG2 or IgG4 class. Antibodies may contain sequences from two or more classes or isotypes, and the selection of specific constant domains to optimize desired effector function is within the scope of the skills of the art.

[0084] Variable domains have different sequences depending on the antibody and are used for the binding and specificity of each particular antibody to its specific antigen. However, variability is not usually uniformly distributed throughout the antibody's variable domains. 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 portion of the variable domain is called the framework (FR). The natural heavy and light chain variable domains each contain four FR regions that take the form of a beta-sheet structure, primarily 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 other chains, contribute to the formation of the antibody's antigen-binding site.

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

[0086] Another embodiment provides a single-chain antibody specific to PD-1. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be produced by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstituted 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 peptide or linker of 15-25 amino acids, 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 appropriate conformational orientation.

[0087] Another embodiment provides a divalent single-stranded variable fragment (di-scFv) that can be manipulated by linking two scFvs. This can be done by producing a single peptide chain having two VH and two VL regions, resulting in a tandem scFv. ScFvs can also be designed with a linker peptide (about 5 amino acids) that is too short for the two variable regions to fold together, causing the scFv to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40 times lower than the corresponding scFv, meaning they also have a much higher affinity for their targets. Even shorter linkers (1 or 2 amino acids) result in the formation of trimers (tribodies or tribodies). Tetrabodies have also been produced. They exhibit even higher affinity for their targets than diabodies.

[0088] Another embodiment provides a PD-1-specific monoclonal antibody that induces an activation signal in immune cells. The monoclonal antibody may be obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible spontaneous mutations that may exist within a small subset of the antibody molecule. The monoclonal antibody includes a “chimeric” antibody in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody of a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence in an antibody of a different species or belonging to a different antibody class or subclass, and in a fragment of such an antibody (insofar as they exhibit the desired antagonistic activity).

[0089] 1. Chimeric antibodies and humanized antibodies Another embodiment provides a chimeric anti-PD-1 antibody comprising one or more of the disclosed sequences and its antigen-binding fragment, as well as functional variants thereof that bind to PD-1 and transmit an activation signal to immune cells expressing PD-1.

[0090] Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, Bio Techniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patents 6,311,415, 5,807,715, 4,816,567, and 4,816,397. Chimeric antibodies containing one or more CDRs from non-human species and framework regions from human immunoglobulin molecules can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP239,400, International Publication No. WO91 / 09967, and U.S. Patents No. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP592,106, EP519,596, 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. Patent No. 5,565,332).

[0091] The disclosed anti-PD-1 antibody or its antigen-binding fragment may be a human or humanized antibody, or its antigen-binding fragment. Many non-human antibodies (e.g., those derived from mouse, rat, or rabbit) are naturally antigenic in humans and can therefore cause undesirable immune responses when administered to humans. Thus, the use of human or humanized antibodies in the method helps reduce the likelihood that an antibody administered to a human will cause an undesirable immune response.

[0092] Transgenic animals (e.g., mice) capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production after immunization can be used. For example, homozygous deletion of the antibody heavy chain linkage region (J(H)) gene in chimeric and germline mutant mice has been shown to result in complete inhibition of endogenous antibody production. Transplantation of a human germline immunoglobulin gene array into such germline mutant mice would result in the production of human antibodies after antigen challenge.

[0093] Optionally, antibodies are produced in other species and “humanized” for administration in humans. The humanized form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing minimal sequences derived from non-human immunoglobulin. A humanized antibody contains human immunoglobulin (recipient antibody) in which residues from the complementarity-determining region (CDR) of the recipient antibody are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues not found in either the recipient antibody or the imported CDR or framework sequence. Generally, a humanized antibody will contain at least one, typically two, substantially all of the variable domains, where all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the FR region is the FR region of a human immunoglobulin consensus sequence. The humanized antibody will also optimally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin.

[0094] Methods for humanizing non-human antibodies are well known in the art, for example, European Patent Nos. EP239,400, EP592,106, and EP519,596; International Publications WO91 / 09967 and WO93 / 17105; U.S. Patents 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 See 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, humanized antibodies have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are often called “import” residues, which typically derive from an “import” variable domain. Antibody humanization techniques generally involve the use of recombinant DNA techniques to manipulate the DNA sequences encoding one or more polypeptide chains of an antibody molecule. Humanization can essentially be carried out by using rodent CDRs or CDR sequences in place of the corresponding sequences in a human antibody. Thus, the humanized form of a non-human antibody (or fragment thereof) is a chimeric antibody or fragment in which a substantially fraction of the intact human variable domain is replaced by the corresponding sequence from a non-human species. In practice, a humanized antibody is typically a human antibody in which several CDR residues and possibly several FR residues are replaced by residues from similar sites in a rodent antibody.

[0096] The selection of both light and heavy human variable domains used in the production of humanized antibodies can be crucial for reducing antigenicity. According to the "best fit" method, the sequences of variable domains in rodent antibodies are screened against an entire library of known human variable domain sequences. The human sequences most closely resembling the rodent sequences are then accepted as the human framework (FR) for the humanized antibody. Alternatively, a specific framework is used, derived from the consensus sequences of all human antibodies in a particular subgroup of the light or heavy chain. The same framework may be used for several different humanized antibodies.

[0097] It is even more important that antibodies are humanized while retaining high affinity for the antigen and other desirable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parent sequence and various conceptual humanized products, using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are commonly available and well known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional structures of selected candidate immunoglobulin sequences. Investigation of these displays allows for the analysis of the possible roles of residues in the functionalization of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this method, FR residues can be selected and combined from consensus and import sequences so that the desired antibody characteristics, e.g., increased affinity for the target antigen(s), are achieved. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

[0098] Human, humanized, or chimeric antibody derivatives may contain substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin (donor antibody), and all or substantially all of the framework region is the FR region of the human immunoglobulin consensus sequence. Such antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin. The constant domains of such antibodies may be selected with respect to the proposed function of the antibody, particularly any effector function that may be required. In some embodiments, the constant domains of such antibodies may be or contain human IgA, IgD, IgE, IgG, or IgM domains. In certain embodiments, human IgG constant domains, particularly those of the IgG1 and IgG3 isotypes, are used when the humanized antibody derivative is intended for therapeutic use and antibody effector function, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC) activity, is required. In alternative embodiments, the 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 modify antibody effector function, such as those disclosed in U.S. Patent Application Publications 2005 / 0037000 and 2005 / 0064514.

[0099] The framework and CDR regions of a humanized antibody do not need to correspond precisely to the parent sequence. For example, the donor CDR or consensus framework can be mutated by the substitution, insertion, or deletion of at least one residue, so that the CDR or framework residue at that site does not correspond to either the consensus or donor antibody. In some embodiments, such mutations are not widespread. Typically, at least 75%, and in more cases 90%, or even more than 95%, of the humanized antibody residues will correspond to residues in the parent framework region (FR) and CDR sequence.Humanized antibodies are used for CDR grafting (European Patent No. EP239,400, International Publication No. WO91 / 09967, and U.S. Patents No. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurface formation (European Patent Nos. EP592,106 and EP519,596, Padlan, 1991, Molecular Immunology 28(4 / 5):489-498, Studnicka et al., 1994, Protein Engineering 7(6):805-814, and Roguska et al. 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 Techniques disclosed in 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 include, but are not limited to, various techniques known in the art that can be used to produce these products.

[0100] In many cases, framework residues within the framework region will be substituted with corresponding residues from the CDR donor antibody to alter, for example, improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify abnormal framework residues at specific locations. (See, for example, Queen et al., U.S. Patent No. 5,585,089, U.S. Publication Nos. 2004 / 0049014 and 2003 / 0229208, U.S. Patent Nos. 6,350,861, 6,180,370, 5,693,762, 5,693,761, 5,585,089 and 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 antibody may be used in in vivo methods in humans. Mouse antibodies or antibodies of other species may be advantageously used for many applications (e.g., in vitro or in-situ detection assays, acute in vivo use, etc.). Such human or humanized antibodies may include amino acid residue substitutions, deletions, or additions in one or more non-human CDRs. Humanized antibody derivatives may have substantially the same, stronger, or weaker binding compared to non-derivative humanized antibodies. In certain embodiments, one, two, three, four, or five amino acid residues of the CDR 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 a variety of methods known in the art, including phage presentation methods using antibody libraries derived from human immunoglobulin sequences (see U.S. Patents 4,444,887 and 4,716,111, and International Publications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741). Such human antibodies can be produced using transgenic mice that can express human immunoglobulin genes but cannot express functional endogenous immunoglobulins.

[0103] For example, the human heavy-chain and light-chain immunoglobulin gene complex can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy-chain and light-chain genes. The mouse heavy-chain and light-chain immunoglobulin genes can be rendered non-functional, either independently of or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, J HHomozygous deletion of the region prevents endogenous antibody production. Modified embryonic stem cells are proliferated and microinjected into blastocysts to produce chimeric mice. These chimeric mice are then mated to produce homozygous offspring that express human antibodies. Transgenic mice are immunized using conventional methods with selected antigens, e.g., all or part of a polypeptide. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques (see, e.g., U.S. 5,916,771). The human immunoglobulin transgenes in transgenic mice are rearranged during B cell differentiation and subsequently undergo switching and somatic mutation. Thus, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies using such techniques. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93 (the whole work is incorporated herein by reference)). For further consideration of this technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see, for example, International Publications WO98 / 24893, WO96 / 34096, and WO96 / 33735, and U.S. Patents 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 (these are incorporated herein in their entirety by reference). In addition, companies can work to provide human antibodies against selected antigens using technologies similar to those described above.

[0104] DNA sequences encoding human acceptor framework sequences include, but are not limited to, the 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 CDRs are inserted into the framework region using commonly used recombinant DNA techniques. The framework region may be a spontaneously generated or consensus framework region, or a human framework region (for example, 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 mouse monoclonal antibody isolated from hybridoma 4C12 or its antigen-binding fragment.

[0106] Another embodiment provides an antibody or its antigen-binding fragment having a heavy chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: TIFF2026049006000003.tif126164

[0107] Underlined sequences correspond to complementarity-determining regions (CDRs). Double-underlined sequences correspond to stationary regions. Dashed underlined sequences correspond to leader sequences.

[0108] Nucleic acids may be present in a vector, such as an expression vector. They may be extrachromosomal or inserted into the chromosomes of a host cell, such as a Chinese hamster ovary cell.

[0109] One embodiment provides an antibody or an antigen-binding fragment having a heavy chain having amino acids that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following. TIFF2026049006000004.tif46160

[0110] A single underline corresponds to the leader sequence. A double underline corresponds to the CDR, and a dashed underline corresponds to the steady-state region.

[0111] Another embodiment provides an antibody or antigen-binding fragment having a leader sequence-less heavy chain having amino acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: In TIFF2026049006000005.tif41167, double underlines correspond to CDR, and dashed underlines correspond to the steady-state area.

[0112] The amino acid sequence of the 4C12 heavy chain CDR1 is: The filename is TIFF2026049006000006.tif5128.

[0113] The amino acid sequence of the 4C12 heavy chain CDR2 is: The filename is TIFF2026049006000007.tif6128.

[0114] The amino acid sequence of the 4C12 heavy chain CDR3 is: The filename is TIFF2026049006000008.tif5128.

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

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

[0117] One embodiment provides an antibody having three different CDRs selected from the group consisting of SEQ ID NO: 6, 7, and 8.

[0118] 2.4C12 light chain sequence Another embodiment provides an antibody or its antigen-binding fragment having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: TIFF2026049006000009.tif66165

[0119] A dashed underline indicates the leader sequence. A single underline indicates the CDR (Critical Distribution Range). A double underline indicates the steady-state region.

[0120] Nucleic acids may be present in a vector, such as an expression vector. They may be extrachromosomal or inserted into the chromosomes of a host cell, such as a Chinese hamster ovary cell.

[0121] Another embodiment provides an antibody or an antigen-binding fragment having a light chain having amino acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: In TIFF2026049006000010.tif24159, underlines indicate leader sequences. Double underlines indicate CDRs. Dashed underlines indicate steady-state regions.

[0122] Another embodiment provides an antibody or antigen-binding fragment having a leader sequence-less light chain having amino acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: In TIFF2026049006000011.tif24165, double underlines indicate CDR (Critical Distance Reduction). Dashed underlines indicate steady-state regions.

[0123] The amino acid sequence of CDR1 in the 4C12 light chain is: The filename is TIFF2026049006000012.tif5128.

[0124] The amino acid sequence of CDR2 in the 4C12 light chain is: The filename is TIFF2026049006000013.tif5128.

[0125] The amino acid sequence of CDR3 in the 4C12 light chain is: The filename is TIFF2026049006000014.tif5128.

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

[0127] Another embodiment provides an antibody or its antigen-binding fragment having a light chain encoded by a nucleic acid having sequence identity of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% with 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 having a heavy chain having an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with 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 with SEQ ID NO: 10 or 11, or a combination thereof of the light chain and heavy chain.

[0130] Another embodiment provides an antibody or its antigen-binding fragment, comprising three different heavy chain CDRs having amino acids selected from the group consisting of SEQ ID NO: 6, 7, and 8, and three different light chain CDRs having amino acids selected from the group consisting of SEQ ID NO: 12, 13, and 14.

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

[0132] Another embodiment provides an antibody or its antigen-binding fragment having a heavy chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: TIFF2026049006000015.tif163165

[0133] Underlined sequences correspond to complementarity-determining regions (CDRs). Double-underlined sequences correspond to stationary regions. Dashed underlined sequences correspond to leader sequences.

[0134] Nucleic acids may be present in a vector, such as an expression vector. They may be extrachromosomal or inserted into the chromosomes of a host cell, such as a Chinese hamster ovary cell.

[0135] One embodiment provides an antibody or an antigen-binding fragment having a heavy chain having amino acids that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following. TIFF2026049006000016.tif54158

[0136] A single underline corresponds to the leader sequence. A double underline corresponds to the CDR, and a dashed underline corresponds to the steady-state region.

[0137] Another embodiment provides an antibody or antigen-binding fragment having a leader sequence-less heavy chain having amino acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: In TIFF2026049006000017.tif54166, double underlines correspond to CDR, and dashed underlines correspond to the steady-state area.

[0138] The amino acid sequence of CDR1 in the 2B5 heavy chain is: The filename is TIFF2026049006000018.tif5128.

[0139] The amino acid sequence of CDR2 in the 2B5 heavy chain is: The filename is TIFF2026049006000019.tif5128.

[0140] The amino acid sequence of CDR3 in the 2B5 heavy chain is: The filename is TIFF2026049006000020.tif5128.

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

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

[0143] One embodiment provides an antibody having three different CDRs selected from the group consisting of SEQ ID NO: 18, 19, and 20.

[0144] 4.2B5 Light Chain Sequence Another embodiment provides an antibody or its antigen-binding fragment having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: TIFF2026049006000021.tif66164

[0145] A dashed underline indicates the leader sequence. A single underline indicates the CDR (Critical Distribution Range). A double underline indicates the steady-state region.

[0146] Nucleic acids may be present in a vector, such as an expression vector. They may be extrachromosomal or inserted into the chromosomes of a host cell, such as a Chinese hamster ovary cell.

[0147] Another embodiment provides an antibody or an antigen-binding fragment having a light chain having amino acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: In TIFF2026049006000022.tif23162, underlines indicate leader sequences. Double underlines indicate CDRs. Dashed underlines indicate steady-state regions.

[0148] Another embodiment provides an antibody or antigen-binding fragment having a leader sequence-less light chain having amino acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: In TIFF2026049006000023.tif24165, double underlines indicate CDR (Critical Distance Reduction). Dashed underlines indicate steady-state regions.

[0149] The amino acid sequence of CDR1 in the 2B5 light chain is: The filename is TIFF2026049006000024.tif5128.

[0150] The amino acid sequence of CDR2 in the 2B5 light chain is: The filename is TIFF2026049006000025.tif5128.

[0151] The amino acid sequence of CDR3 in the 2B5 light chain is: The filename is TIFF2026049006000026.tif5128.

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

[0153] Another embodiment provides an antibody or its antigen-binding fragment having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with 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 having a heavy chain having an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with 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 with SEQ ID NO: 22 or 23, or a combination thereof of the light chain and heavy chain.

[0156] Another embodiment provides an antibody or its antigen-binding fragment, comprising three different heavy chain CDRs having amino acids selected from the group consisting of SEQ ID NO: 18, 19, and 20, and three different light chain CDRs having amino acids selected from the group consisting of SEQ ID NO: 24, 13, and 25.

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

[0158] Another embodiment provides an antibody or its antigen-binding fragment having a heavy chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: TIFF2026049006000027.tif132165

[0159] Underlined sequences correspond to complementarity-determining regions (CDRs). Double-underlined sequences correspond to stationary regions. Dashed underlined sequences correspond to leader sequences.

[0160] Nucleic acids may be present in a vector, such as an expression vector. They may be extrachromosomal or inserted into the chromosomes of a host cell, such as a Chinese hamster ovary cell.

[0161] One embodiment provides an antibody or an antigen-binding fragment having a heavy chain having amino acids that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following. TIFF2026049006000028.tif47164

[0162] A single underline corresponds to the leader sequence. A double underline corresponds to the CDR, and a dashed underline corresponds to the steady-state region.

[0163] Another embodiment provides an antibody or antigen-binding fragment having a leader sequence-less heavy chain having amino acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: In TIFF2026049006000029.tif48167, double underlines correspond to CDR, and dashed underlines correspond to the steady-state area.

[0164] The amino acid sequence of the 4G9 heavy chain CDR1 is: The filename is TIFF2026049006000030.tif5128.

[0165] The amino acid sequence of the 4G9 heavy chain CDR2 is: The filename is TIFF2026049006000031.tif5128.

[0166] The amino acid sequence of the 4G9 heavy chain CDR3 is: The filename is TIFF2026049006000032.tif5128.

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

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

[0169] One embodiment provides a 4G9 antibody having three different CDRs selected from the group consisting of SEQ ID NO: 29, 30, and 31.

[0170] 7.4G9 light chain sequence Another embodiment provides an antibody or its antigen-binding fragment having a light chain encoded by a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: TIFF2026049006000033.tif66164

[0171] A dashed underline indicates the leader sequence. A single underline indicates the CDR (Critical Distribution Range). A double underline indicates the steady-state region.

[0172] Nucleic acids may be present in a vector, such as an expression vector. They may be extrachromosomal or inserted into the chromosomes of a host cell, such as a Chinese hamster ovary cell.

[0173] Another embodiment provides an antibody or an antigen-binding fragment having a light chain having amino acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: In TIFF2026049006000034.tif24166, underlines indicate leader sequences. Double underlines indicate CDRs. Dashed underlines indicate steady-state regions.

[0174] Another embodiment provides an antibody or antigen-binding fragment having a leader sequence-less light chain having amino acids having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the following: In TIFF2026049006000035.tif24166, double underlines indicate CDR (Critical Distance Reduction). Dashed underlines indicate steady-state regions.

[0175] The amino acid sequence of CDR1 in the 4G9 light chain is: The filename is TIFF2026049006000036.tif5128.

[0176] The amino acid sequence of CDR2 in the 4G9 light chain is: The filename is TIFF2026049006000037.tif5128.

[0177] The amino acid sequence of CDR3 in the 4G9 light chain is: The filename is TIFF2026049006000038.tif5128.

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

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

[0180] One embodiment provides a 4G9 antibody having three different CDRs selected from the group consisting of SEQ ID NO: 35, 36, and 37.

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

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

[0183] D.PD-1 activated epitope 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. TIFF2026049006000039.tif5128

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

[0186] Epitope-specific antibodies may be monoclonal antibodies, humanized antibodies, human antibodies, mouse antibodies, chimeric antibodies, or fragments thereof. Exemplary antibodies and methods for their production are discussed below.

[0187] 2. Fusion protein In one embodiment, the epitope-specific PD-1 binding moiety is a fusion protein. One or more of the disclosed PD-1 epitope antibodies or epitope-binding fragments can be bound to another polypeptide to form a fusion protein. The fusion polypeptide has a first fusion partner comprising 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 domains that function to dimerize or polymerize two or more fusion proteins. The peptide / polypeptide linker domain may be a separate domain or may 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 polymerize the fusion protein may be a separate domain or may 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 identical.

[0188] The fusion protein disclosed herein is of the form of formula I below: N-R1-R2-R3-C In the formula, "N" represents the N-terminus of the fusion protein, "C" represents the C-terminus of the fusion protein, "R1" is all or part of the disclosed PD-1 epitope antibody or epitope-binding fragment, or a functional variant or fragment thereof, "R2" is an arbitrary peptide / polypeptide linker domain, and "R3" is a second polypeptide. Alternatively, R3 is all or part of the 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 dimerizing or multimerizing domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The resulting dimers or multimers may be homodimers / homopolymers or heterodimers / heteropolymers.

[0190] 3. Aptamer In some embodiments, the epitope-specific binding site is an aptamer. An aptamer is a molecule that preferably interacts with a target molecule in a specific manner. In one embodiment, an aptamer binds to SEQ ID NO:38 on the surface of immune cells and promotes or induces an activation signal via PD-1 to activate the immune cells. Typically, an aptamer is a small nucleic acid ranging from 15 to 50 nucleotides in length that folds 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 strongly to target molecules with Kd values ​​less than 10⁻¹² M. It is preferable that aptamers bind to target molecules with Kd values ​​less than 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹². Aptamers can bind to target molecules with very high specificity. For example, aptamers have been isolated that have a binding affinity difference of more than 10,000 times between a target molecule and another molecule that differs by only a single position on the molecule. It is preferable that the aptamer has a Kd for the target molecule that is at least 10, 100, 1,000, 10,000, or 100,000 times lower than the Kd for the background binding molecule. For example, when comparing polypeptides, it is preferable that the background molecule is a different polypeptide. Representative examples of methods for preparing and using aptamers that bind to various different target molecules are known in the art.

[0191] 4.Small molecules In some embodiments, the epitope-specific binding site may be a small molecule. The term “small molecule” generally refers to small organic compounds having a molecular weight greater than about 100 daltons and less than about 2,500 daltons, preferably 100 to 2,000, more preferably about 100 to 1,250, more preferably about 100 to 1,000, more preferably about 100 to 750, and more preferably about 200 to 500 daltons. Small molecule agonists of the PD-1 activation epitope may be identified using commonly used screening methods. In some embodiments, the screening assay may include random screening of a large library of test compounds. The assay may include determining a PD-1-induced immune response or T cell activation.

[0192] E. Pharmaceutical Compositions A pharmaceutical composition comprising a disclosed antibody and its antigen-binding fragment is provided. The pharmaceutical composition containing the antibody and its antigen-binding fragment 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 the subject in a therapeutically effective dose. As used herein, the terms “effective dose” or “therapeutically effective dose” mean a dose 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 dose will vary depending on various factors such as subject-dependent variables (e.g., age, immune system health), disease, and the treatment being administered.

[0194] As further research is conducted on the disclosed antibodies and their antigen-binding fragments, information will emerge regarding dosage levels suitable for treating various conditions in various patients, and those skilled in the art will be able to determine the appropriate dosage considering the recipient's treatment background, age, and general health status. The selected dosage depends on the desired therapeutic effect, route of administration, and desired duration of treatment. For the disclosed antibodies and their antigen-binding fragments, a dosage level of 0.001 to 20 mg / kg of body weight is generally administered to mammals daily. Generally, dosages may be lower for intravenous injection or infusion.

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

[0196] 1. Formulations for parenteral administration In some embodiments, compositions containing the disclosed antibody and antigen-binding fragment are administered by parenteral injection in an aqueous solution. The formulation may also be in the form of a suspension or emulsion. Generally, pharmaceutical compositions are provided that contain an effective amount of the antibody or its antigen-binding fragment and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may optionally include: diluents, sterile water, buffered salines of various buffering agent content (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; and additives, e.g., detergents and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thymelsol, benzyl alcohol) and bulking agents (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations can be lyophilized and redissolved / resuspended immediately before use. The formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, by incorporating a sterilizing agent into the composition, by irradiation of the composition, or by heating the composition.

[0197] 2. Formulations for oral administration In some embodiments, the antibody composition is formulated for oral delivery. The oral dosage form of the antibody may be resistant to proteolysis and be able to deliver a larger proportion of immunoreactive antibodies locally into the gastrointestinal tract for the treatment of infection, or it may allow for the absorption of the antibody for the treatment or prevention of a systemic condition (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, lozenges or troches, cachets, pellets, powders, or granules, or materials incorporated into particulate preparations of polymer compounds such as polylactic acid or polyglycolic acid, or into liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed product. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042), pages 1435-1712, incorporated herein by reference. Compositions may be prepared in liquid form or in dry powder form (e.g., lyophilized). Liposomes or proteinoid encapsulation may be used to formulate the composition. Liposome encapsulation may be used, and liposomes may 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 CTRhodes Chapter 10, 1979. Generally, the formulation will contain a peptide (or a chemically modified form thereof) and an inert component that protects the peptide in the gastric environment and releases the biologically active material in the intestine.

[0199] Antibodies and their antigen-binding fragments can be chemically modified to facilitate oral delivery of derivatives. Generally, the intended chemical modification involves attachment of at least one component molecule to the molecule itself, while the other component allows for uptake into the bloodstream from the stomach or intestines, or direct uptake into the intestinal mucosa. Also desirable is increased overall stability of the component(s) and increased circulating time in the body. PEGylation is an exemplary chemical modification for pharmaceutical use. Other parts that may be used include propylene glycol, ethylene glycol and propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane, and poly-1,3,6-thioxocan [see, for example, 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, liquids, suspensions, and syrups, which may contain inert diluents; auxiliary agents such as wetting agents, emulsifiers, and suspending agents; and other components including sweeteners, flavoring agents, and fragrances.

[0201] For oral formulations, the site of release may be the stomach, small intestine (duodenum, jejunum, or ileum), or large intestine. In some embodiments, release will avoid adverse effects on the gastric environment either by protection of the drug (or derivative) or by release of the drug (or derivative) beyond the gastric environment, such as into the intestines. To ensure complete gastric resistance, an impermeable coating to at least pH 5.0 is essential. Examples of more common inert components used as enteric coatings include 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 can be used as mixed films.

[0202] 3. Controlled Delivery Polymer Matrix The antibodies and antigen-binding fragments disclosed herein may also be administered in controlled-release formulations. The antibodies and antigen-binding fragments may be incorporated into an inert matrix that allows for release by either a diffusion or leaching mechanism, for example, by gum. Slow-denaturing matrices may also be incorporated into the formulations. Another form of controlled release is based on the Oros therapeutic system (Alza Corp.), where the antibody or antigen-binding fragment is encapsulated in a semipermeable membrane that allows water to be added to push the drug through a single small opening by osmotic effect.

[0203] Controlled-release polymer devices can be fabricated for long-term systemic release after implantation or injection (microparticles) of polymer devices (rods, cylinders, films, disks). The matrix can be in the form of microparticles, such as microspheres, where the drug is dispersed in a solid polymer matrix or microcapsules, the core has a different material from the polymer shell, and the peptide is dispersed or suspended in the core, which can be essentially liquid or solid. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer can be cast as thin slabs or films ranging from a few nanometers to 4 centimeters, as a powder produced by grinding or other standard techniques, or as a gel such as a hydrogel.

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

[0205] The matrix may be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Biodegradable microspheres may be prepared using any of the methods developed for producing 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 may be formulated for local release to treat areas of implantation or injection, which would typically deliver much lower doses than those for systemic treatment or systemic delivery. They may be implanted or injected subcutaneously, intramuscularly, or subcutaneously, or swallowed.

[0207] III. Manufacturing method A. Method for producing antibodies The disclosed antibodies may be produced in cell culture, phages, or in a variety of animals, including but not limited to cattle, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. In one embodiment, the animals may be transgenic animals genetically engineered to produce human or humanized antibodies. Thus, in one embodiment, the antibodies are mammalian antibodies. Phage techniques may be used to isolate initial antibodies or to generate variants with modified specificity or affinity characteristics. Such techniques are common and well known in the art. In one embodiment, the antibodies are produced by recombinant means known in the art. For example, recombinant antibodies may be produced by transforming host cells with a vector containing a DNA sequence encoding the antibody. One or more vectors are used to transform host cells with a DNA sequence expressing at least one VL and one VH region. Examples of recombinant methods for 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 so as to increase the greater effectiveness of the antibody in mediating the desired function. Thus, the ability of an antibody to be modified by substitution using recombinant means is within the scope of the invention. Typically, the substitution will be a conservative substitution. For example, at least one amino acid within the constant region of the antibody can be replaced with a different residue. See, e.g., U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO9958572, and Angal, et al., Mol. Immunol. 30:105-08 (1993). Modifications in amino acids include deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Often, antibodies are labeled by binding either covalently or non-covalently to a substance that provides a detectable signal. A wide variety of labeling and conjugation techniques are known and widely reported in both 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 having the 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, signal transduction, and in vivo assays such as inhibition of tumor growth. 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 specifically to an antigen without inhibiting receptor binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays.

[0210] Antibodies that can be used in the disclosed compositions and methods include intact antibodies of any class (i.e., whole antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domains of antibodies. The variable domains have different sequences depending on the antibody and are used for the binding and specificity of that particular antibody to its particular antigen. However, the variability is not usually evenly distributed throughout the variable domains of the antibody. It typically concentrates in three segments called complementarity-determining regions (CDRs) or hypervariable regions, both in the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called frameworks (FRs). The variable domains of the native heavy and light chains each contain four FR regions that mainly take on a beta-sheet structure connected by three CDRs, which form loops that connect the beta-sheet structure and in some cases form part of it. The CDRs in each chain are held very close together by the FR regions and contribute, together with the CDRs from the other chain, to the formation of the antigen-binding site of the antibody.

[0211] Fragments of antibodies with biological activity are also disclosed. The fragments include 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 modified or reduced compared to the unmodified antibody or antibody fragment.

[0212] The technique can also be adapted to the production of single-chain antibodies based on the disclosed antibody and its antigen-binding fragment. Methods for the production of single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be produced by fusing the variable domains of the heavy and light chains together using a short peptide linker, thereby reconstituted 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 peptide or linker of 15-25 amino acids, 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 appropriate conformational orientation.

[0213] One embodiment provides a divalent single-stranded variable fragment (di-scFv) that can be manipulated by linking two scFvs. This can be done by producing a single peptide chain having two VH and two VL regions, resulting in a tandem scFv. ScFvs can also be designed with a linker peptide (about 5 amino acids) that is too short for the two variable regions to fold together, causing the scFv to dimerize. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40 times lower than the corresponding scFv, meaning they also have a much higher affinity for their targets. Even shorter linkers (1 or 2 amino acids) result in the formation of trimers (tribodies or tribodies). Tetrabodies have also been produced. They exhibit even higher affinity for their targets than diabodies.

[0214] Another embodiment provides a monoclonal antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible spontaneous mutations that may exist within a small subset of antibody molecules. The monoclonal antibody includes a “chimeric” antibody in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody of a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence in an antibody of a different species or belonging to a different antibody class or subclass, and in a fragment of such an antibody (insofar as they exhibit the desired antagonistic activity).

[0215] Monoclonal antibodies can be produced using any procedure for producing monoclonal antibodies. In the hybridoma method, mice or other suitable host animals are 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. The DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the mouse antibody). Libraries of antibodies or active antibody fragments can also be generated and screened using phage presentation techniques.

[0217] Methods for producing antibodies using protein chemistry are also known in the art. One method for producing an antibody-containing protein is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry, using currently available laboratory equipment (Applied Biosystems, Inc., Foster City, CA). Those skilled in the art will readily understand that peptides or polypeptides corresponding to antibodies can be synthesized, for example, by standard chemical reactions. For example, a peptide or polypeptide may be synthesized and not cleaved from a synthetic resin, while the other fragment of the antibody may be synthesized and subsequently cleaved from the resin, thereby exposing a 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 terminals, respectively, to form an antibody or fragment thereof. Alternatively, peptides or polypeptides can be synthesized independently in vivo as described above. Once isolated, these independent peptides or polypeptides can be ligated together to form antibodies or their antigen-binding fragments via similar peptide condensation reactions.

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

[0219] B. Method for producing isolated nucleic acid molecules One embodiment provides a nucleic acid encoding an antibody or its antigen-binding fragment. The nucleic acid may encode the entire antibody, its antigen-binding fragment, its light chain, heavy chain, combination, or its CDR.

[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 terminal or beyond region of the region of interest can be used to design oligonucleotide primers whose sequence is identical to that of the opposite strand of the template being amplified. PCR can be used to amplify specific sequences from DNA and RNA, including sequences from total genomic DNA or total cellular RNA. Primers are typically 14–40 nucleotides long, but can range from 10 to several hundred nucleotides long. 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 substitution amplification, auto-persistent sequence replication, or nucleic acid sequence-based amplification can also be used to obtain isolated nucleic acids. For example, see 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 techniques for automated DNA synthesis in the 3'→5' direction). For example, one or more pairs of long oligonucleotides (e.g., over 100 nucleotides) containing the desired sequence may be synthesized, each pair containing a complementary short segment (e.g., about 15 nucleotides) such that a double helix is ​​formed when this oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides to yield 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-coding nucleic acids can be mutated using standard techniques, including oligonucleotide-specific mutagenesis and / or site-directed mutagenesis via PCR. 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 their antigen-binding fragments can be used to modulate the immune response in subjects that require it. One embodiment provides a method for activating PD-1-expressing immune cells, such as T cells, by administering the disclosed antibodies and their antigen fragments, optionally including a second therapeutic agent, thereby promoting the proliferation of PD-1-expressing immune cells and enhancing their biological activity.

[0223] A. Immune response stimulation 1. Treatment Strategy A method is provided for inducing or enhancing an immune response in a subject. Typically, the method comprises administering to a subject an effective amount of one or more of the disclosed antibodies and their antigen-binding fragments in order to immune-specifically bind to PD-1 and induce, promote, or enhance a stimulating or activating signal through PD-1 for activating immune cells. The immune response may induce, promote, or enhance, for example, immune cell activity, T cell activation by T cell proliferation, or cytokine secretion. The disclosed antibodies or their antigen-binding fragments may be administered to a subject in need in an effective amount to overcome T cell depletion and / or T cell anergy. Overcoming T cell depletion or T cell anergy may be determined by measuring T cell function using known techniques.

[0224] The method may be used in vivo or ex vivo to induce, promote, or enhance a stimulated immune response.

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

[0226] 2. Who will be treated? a. Cancer treatment The disclosed antibodies and their compositions, as well as the methods, may be used to treat cancer. Generally, these agents are used to stimulate or enhance the immune response to cancer in a subject by administering to the subject an amount of the disclosed antibody or its antigen-binding fragment that induces, promotes, or enhances the activation signal via PD-1. The methods may reduce one or more symptoms of cancer.

[0227] Immune cells activated by the disclosed antibodies or fragments can kill cells in a target and reduce the tumor burden. 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 localized. In other embodiments, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and spread distally. In yet another embodiment, cancer is associated with a specific cancer antigen (e.g., pan-cancer antigen (KS1 / 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 disorders, including (but not limited to): cancers of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, neck, thyroid, and skin; including squamous cell carcinoma; lymphoid hematopoietic malignancies, including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and Burkitt lymphoma; and acute and chronic myeloid Hematopoietic malignancies of the myeloid lineage, including leukemia and promyelocytic leukemia; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; other tumors, including malignant melanoma, seminomas, teratocarcinomas, neuroblastomas, and gliomas; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, gliomas, and schwannomas; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including malignant melanoma, xeroderma pigmentosum, keratosacral cell carcinoma, seminomas, follicular thyroid carcinoma, and teratocarcinomas.

[0229] Cancers caused by abnormal apoptosis can also be treated by the disclosed methods and compositions. Such cancers can include, but are not limited to, follicular lymphoma, cancers with p53 mutations, hormone-dependent tumors of the breast, prostate and ovary, as well as premalignant lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In certain embodiments, malignant tumors or abnormal proliferative changes (such as dysplasia and anaplasia), or hyperproliferative disorders, are treated or prevented by the methods and compositions in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, malignant melanoma, or leukemia are treated or prevented by the methods and compositions.

[0230] Certain cancers and related disorders that can be treated or prevented by the methods and compositions disclosed herein include, but are not limited to, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, and myelodysplastic syndromes, chronic myeloid (granular) leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and other chronic leukemias; polycythemia vera; Hodgkin or non-Hodgkin lymphoma (e.g., diffuse anaplastic lymphoma kinase (ALK) negative, large B-cell lymphoma (DLBCL)); and bima 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), etc., but not limited to these; smoldering multiple myeloma, nonsecretory myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma, etc., but not limited to these; Waldenström macroglobulinemia; monoclonal gammaglobulinemia of unknown significance; benign monoclonal gammaglobulinemia; heavy chain disease; osteosarcoma (bone Sarcomas include, but are not limited to, bone and connective tissue sarcomas such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor of bone, osteofibrosarcoma, chordoma, periosteosarcoma, soft tissue sarcoma, tubosarcoma (angiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma, etc.; glioma, astrocytoma, brainstem glioma, ependymoma, oligodendrocyte, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor. Brain tumors, including but not limited to cytocytoma, pineal blastoma, and primary brain lymphoma; breast cancers, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, ductal carcinoma, medullary breast carcinoma, mucinous breast carcinoma, tubular breast carcinoma, papillary breast carcinoma, Paget's disease, and inflammatory breast carcinoma; adrenal cancers, including but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancers, including but not limited to papillary or follicular thyroid cancer, medullary thyroid carcinoma, and anaplastic thyroid carcinoma;Pancreatic cancers include, but are not limited to, insulinoma, gastrinoma, glucagonoma, bipoma, somatostatin-secreting tumors, and carcinoid or islet cell tumors; pituitary cancers include, but are not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; ocular cancers include, but are not limited to, intraocular melanomas such as iris melanoma, choroidal melanoma, and ciliary melanoma, as well as retinoblastoma; vaginal cancers include, but are not limited to, squamous cell carcinoma, adenocarcinoma, and malignant melanoma; squamous cell carcinoma, malignant Vulvar cancers include, but are not limited to, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers include, but are not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers include, but are not limited to, endometrial cancer and uterine sarcoma; ovarian cancers include, but are not limited to, ovarian epithelial carcinoma, borderline tumors, germ cell tumors, and stromal tumors; squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, malignant melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma, but these Esophageal cancer, including but not limited to adenocarcinoma, mycoplasmic (polypoid), ulcerative, superficially spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; gastric cancer; 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, etc. Lung cancer, not limited to germ cell tumors, seminomas, undifferentiated, classical (typical), spermatocyte, non-seminoma, embryonic cancer, teratoma, choriocarcinoma (yolk sac tumor), testicular cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma, not limited to these; prostate cancer, not limited to these; penile cancer; oral cancer, not limited to squamous cell carcinoma; basal cancer; salivary gland cancer, not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic cancer; pharyngeal cancer, not limited to squamous cell carcinoma and verrucous cancer;Skin cancers, including but not limited to basal cell carcinoma, squamous cell carcinoma, and malignant melanoma, superficial melanoma, nodular melanoma, lentigo malignant melanoma, and acral lentiginous melanoma; renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, and transitional cell carcinoma (pelvis and / or ureter); kidney cancers, including but not limited to Wilms' tumor; and bladder cancers, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. In addition, cancers include myxosarcoma, osteosarcoma, endosarcoma, lymphangiosarcoma, mesothelioma, synoviomas, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary carcinoma (for a review of such diseases, see Fishman et al., 1985, Medicine, 2d Ed., JBLippincott 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. Treatment of infectious diseases The disclosed antibody compositions and methods may be used to treat infectious diseases and infectious illnesses. Generally, these agents are used to stimulate or enhance the immune response to infection in a subject by administering one or more of the disclosed antibodies or antigen-binding fragments to the subject in amounts that transmit an activation or stimulating signal through PD-1. The methods may reduce one or more symptoms of the infection.

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

[0233] Infections or diseases can be acute or chronic. Acute infections are typically short-lived infections. During an acute microbial infection, immune cells begin to express immunomodulatory receptors. Therefore, in some embodiments, the method involves enhancing the immune-stimulating response to an acute infection.

[0234] Infections can be caused by, for example, 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, causing the infection to persist in the host for an extended period.

[0236] Exemplary infections treated include chronic infections caused by hepatitis viruses, human immunodeficiency viruses (HIV), human T-lymphotropic viruses (HTLV), herpesviruses, Epstein-Barr virus, or human papillomavirus.

[0237] Since 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 factors is beneficial to animal or human subjects. Therefore, the disclosed compositions may be administered for the treatment of topical or systemic viral infections, including but not limited to immunodeficiency (e.g., HIV), papillomas (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza A virus), and the common cold (e.g., human rhinovirus), as well as other viral infections caused by, for example, HTLV, hepatitis viruses, respiratory syncytial viruses, vaccinia viruses, and rabies viruses. The molecules can be administered topically to treat viral skin diseases such as herpes lesions or herpes zoster, 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, such as Mansoni, are not limited to these, but are also not limited to these.

[0239] Other microorganisms that can be treated using the disclosed compositions and methods include bacteria such as Klebsiella, Serratia, and 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., and Giardia. Examples include fungi or parasitic pathogens such as lambia, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondi, etc.), Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Histolytica, Balantidium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia, or Trypanosoma.

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

[0241] The subject may be administered the first therapeutic agent 1, 2, 3, 4, 5, or 6 hours or more, or 1, 2, 3, 4, 5, 6, or 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 therapeutic agent every 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, or 48 days prior to the first administration of the second therapeutic agent. The antibody and its antigen-binding fragment may be the first or second therapeutic agent.

[0242] Antibodies and their antigen-binding fragments, as well as additional therapeutic agents, may be administered as part of a treatment plan. For example, if the first therapeutic agent may be administered to the subject every four days, the second therapeutic agent may be administered on day 1, day 2, day 3, or day 4, or a combination thereof. The first or second therapeutic agent may be administered repeatedly throughout the entire treatment plan.

[0243] Exemplary additional therapeutic agents include, but are not limited to, cytokines, chemotherapeutic agents, radionuclides, other immunotherapeutic agents, enzymes, antibiotics, antivirals (in particular protease inhibitors, either alone or in combination with nucleosides for the treatment of HIV or hepatitis B or C), antiparasitic agents (helmins, protozoa), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and their bioactive fragments (including humanized, single-stranded, and chimeric antibodies), antigens and vaccine preparations (including adjuvants), peptide drugs, anti-inflammatory agents, ligands that bind to 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 inhibitory or regulatory T cells.

[0244] Additional therapeutic agents are selected based on the condition, disorder, or disease being treated. For example, immunomodulators may be administered concurrently with one or more additional drugs 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 antibody and its antigen-binding fragment may be administered to a subject in combination with an antibiotic, antifungal agent, antiviral agent, antiparasitic agent, or antimicrobial agent such as an essential oil. In another embodiment, the disclosed antibody and its antigen-binding fragment may be used for the treatment and prevention of disease, as well as for a preventive or prophylactic role in situations of severe traumatic injury such as major burns, open fractures, accidental amputations, or other wounds.

[0246] In some embodiments, subjects are administered antibodies and their antigen-binding fragments, as well as / or antibacterial agents, upon hospitalization to prevent further bacterial, fungal, or viral complications. Antibiotics can target target pathogens, and antibodies and their antigen-binding fragments can stimulate the immune system to provide an enhanced response, thereby treating or preventing further infections or diseases.

[0247] 1. Chemotherapy drugs Antibodies and their antigen-binding fragments can be combined with one or more chemotherapeutic agents and apoptosis promoters. Representative chemotherapeutic agents include amsacrin, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, chrysanpastase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, and ri Posomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, larcitrexed, satoraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof are examples of, but are not limited to, these. Representative apoptosis promoters include, but are not limited to, fludarabine, taurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

[0248] 2. Other immunomodulators a. PD-1 Antagonist In some embodiments, an antibody and its antigen-binding fragment are administered co-administered with a PD-1 antagonist. Programmed death-1 (PD-1) is a member of the CD28 family of receptors that deliver a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell growth and / or the intensity and / or duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Patents 8,114,845, 8,609,089, and 8,709,416, which are incorporated herein by reference in whole, and include compounds or agents that either bind to a ligand of PD-1 and block it to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or directly bind to and block the PD-1 receptor without inducing inhibitory signaling through the PD-1 receptor.

[0249] In some embodiments, PD-1 receptor antagonists bind directly to the PD-1 receptor without inducing inhibitory signaling, and also bind to ligands of the PD-1 receptor to reduce or inhibit ligands from inducing signaling through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and induce the transmission of inhibitory signals, fewer cells may be attenuated by the negative signal delivered by PD-1 signaling, resulting in a more robust immune response.

[0250] PD-1 signaling is thought to be triggered by the binding of PD-1 ligands (such as B7-H1 or B7-DC) that are in close proximity to peptide antigens indicated by the major histocompatibility complex (MHC) (see, for example, Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Therefore, 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 intervenes in PD-1 receptor signaling by binding to a ligand of PD-1 or PD-1 itself, in particular when co-ligation of the TCR and PD-1 does not follow such binding, thereby preventing inhibitory signaling through the PD-1 receptor. Other PD-1 antagonists intended by the methods of the present invention include antibodies that bind to PD-1 or a ligand of PD-1, and other antibodies.

[0252] Suitable anti-PD-1 antibodies include, but are not limited to, those described in U.S. Patent Nos. 7,332582, 7488802, 7521051, 7524498, 7563869, 7981416, 8088905, 8287856, 8580247, 8728474, 8779105, 9067999, 9073994, 9084776, 9205148, 9358289, 9387247, 9492539, and 9492540, and all of them are incorporated in their entirety by reference.

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

[0254] Examples of anti-PD-L1 antibodies include, but are not limited to, those described in U.S. Patent Nos. 8,383796, 9102725, 9273135, 9393301, and 9580507, all of which are incorporated herein by reference in their entirety.

[0255] For information regarding anti-B7-DC (also known as anti-PD-L2) antibodies, please refer to U.S. Patent Nos. 7,411,051, 7,052,694, 7,390,888, 8188238, and 9255147.

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

[0257] PD-1 antagonists can also be fragments of mammalian PD-L1, for example, from primates such as mice or humans. These fragments bind to and block PD-1, but do not result in inhibitory signaling through PD-1. The fragments can also be part of fusion proteins, such as Ig fusion proteins.

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

[0259] PD-1 and PD-L1 antisense nucleic acids, both DNA and RNA, as well as siRNA molecules, are also called PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells, as well as the production of T cell ligands such as PD-L1 and / or PD-L2. For example, siRNA (e.g., approximately 21 nucleotides long (specific to the gene encoding PD-1)) or siRNA encoding a PD-1 ligand (oligonucleotides are readily available commercially) forms complexes with carriers such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8):2231-2244 (2009)) and is readily taken up by cells, thereby achieving a reduction in inhibitory signaling in T cells and thus activating T cells, while expressing PD-1 and its ligands and reducing the expression of these receptors and ligands.

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

[0261] Dosages of anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and may range, for example, from 0.1 to 100 mg / kg, with shorter ranges of 1 to 50 mg / kg or 10 to 20 mg / kg. Appropriate doses for human subjects may 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 method of the present invention include, for example, ipilimumab, a human anti-CTLA4 antibody administered at a dose of approximately 10 mg / kg, and, for example, a human anti-CTLA4 antibody administered at a dose of approximately 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 its binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002)). Such small organic compounds can be administered alone or in combination with an anti-CTLA4 antibody to reduce inhibitory signaling on T cells.

[0264] 3. Enhancers In some embodiments, the additional therapeutic agent includes an enhancer. The enhancer may act to enhance the efficacy of the immune response upregulator by two or more mechanisms, but the exact mechanism of action is not essential for the broad implementation of the invention.

[0265] In some embodiments, the enhancer is cyclophosphamide. Cyclophosphamide (CTX, Cytoxan®, or Neosar®) is an oxazaphosphorine agent, and analogues include ifosfamide (IFO, Ifex), perphosphamide, trophosphamide (Ixoten), and their pharmaceutically acceptable salts, solvates, prodrugs, and metabolites (all incorporated in U.S. Patent Application No. 2007 / 0202077). Ifosfamide (MITOXANA®) is a structural analogue of cyclophosphamide, and its mechanism of action is considered to be identical or substantially the same as that of cyclophosphamide. Perphosphamide (4-hydroperoxycyclophosphamide) and trophosphamide are also alkylating agents structurally related to cyclophosphamide. For example, perphosphamides alkylate DNA, thereby inhibiting DNA replication as well as RNA and protein synthesis. Novel 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 maphosphamide (NSC 345842), glucosphamide (D19575, beta-D-glucosylisophosphoramide mustard), S-(-)-bromophosphamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine), and NSC 613060 (aldophosphamide thiazolidine). Maphosphamide is an oxazaphosphorine analog, a chemically stable 4-thioethanesulfonate of 4-hydroxy-CPA. Gluphosphamide is an IFO derivative in which isophosphoramide mustard, an alkylated metabolite of IFO, is glycosidicated to a beta-D-glucose molecule.Additional cyclophosphamide analogs are described in U.S. Patent No. 5,190,929, entitled "Cyclophosphamide analogs useful as anti-tumor agents," which is incorporated herein by reference in its entirety.

[0266] While CTX itself is non-toxic, some of its metabolites are cytotoxic alkylating agents that induce DNA crosslinking, disrupting the chain at higher doses. Many cells are resistant to CTX because they express high levels of the detoxification enzyme aldehyde dehydrogenase (ALDH). Since lymphocytes (not hematopoietic stem cells) express only low levels of ALDH, CTX targets proliferating lymphocytes, and cycling cells are most sensitive to DNA alkylating agents.

[0267] In one embodiment, a low dose of CTX is used in combination with a disclosed antibody and its antigen-binding fragment. Low doses of CTX (less than 200 mg / kg) can have immunostimulatory effects, including stimulation of the antitumor immune response in human and mouse models of cancer (Brode & Cooke Crit Rev.Immunol. 28:109-126 (2008)). These low doses are subthermal and do not have direct antitumor activity. In contrast, high doses of CTX inhibit the antitumor response. Several mechanisms may explain the role of CTX in enhancing the antitumor immune response: (a) depletion of CD4+CD25+FoxP3+Treg (and specifically, proliferating Treg which may be particularly suppressive), (b) depletion of B lymphocytes, (c) induction of nitric oxide (NO) resulting in inhibition of tumor cell growth, and (d) mobilization and expansion of CD11b+Gr-1+MDSCs. These primary effects have many secondary effects; for example, Treg-depleted 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 a mechanism by which CTX enhances the anti-tumor immune response. 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. The 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 abolished the therapeutic response to the vaccine (Taieb, J. J. Immunol. 176:2722-2729 (2006)).

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

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

[0271] In some embodiments, CTX is used as an immune enhancer at a dose of 300 mg / m 2 Another embodiment, the average male (6 feet, 170 pounds (78 kg), having a body surface area of 1.98 m 2 300 mg / m 2In contrast, the dose of CTX is 8 mg / kg, or 624 mg of total protein. In mouse models of cancer, efficacy was observed 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 While doses may be used, unit doses administered at finite time intervals may also be used. Such unit doses may be administered daily over a finite period, 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., all of which are specifically intended by the present invention. The same plan may be applied to other enhancers listed herein.

[0273] In other embodiments, the enhancer 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 plans may also include the administration of adjuvants.

[0274] Useful enhancers include mitotic inhibitors, such as paclitaxol; aromatase inhibitors (e.g., letrozole); and angiogenesis inhibitors (VEGF inhibitors, e.g., avastin, VEGF-Trap) (see, for example, 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 its antigen-binding fragment, comprising a heavy chain CDR having the amino acid sequences described in SEQ ID NO: 6, 7, and 8, and a light chain CDR having the amino acids according to SEQ ID NO: 12, 13, and 14. In one embodiment, the transgenic animal is a rodent, such as a mouse.

[0276] Another embodiment provides a transgenic animal that produces an antibody or an antigen-binding fragment, comprising a heavy chain CDR having the amino acid sequences described in SEQ ID NO: 18, 19, and 20, and a light chain CDR having amino acids selected from the group consisting of SEQ ID NO: 24, 13, and 25. In one embodiment, the transgenic animal is a rodent, such as a mouse.

[0277] Another embodiment provides a transgenic animal that produces an antibody or its antigen-binding fragment, comprising a heavy chain CDR having the amino acid sequences described in SEQ ID NO: 29, 30, and 31, and a light chain CDR having the amino acids according to SEQ ID NO: 35, 36, and 37. In one embodiment, the transgenic animal is a rodent, such as 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), and Lonberg N. (2005) “Human antibodies from transgenic animals.” Nat See Biotechnol. 23(9):1117-25, and U.S. patents 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. [Examples]

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

[0280] Example 2: Interaction dynamics between anti-PD-1 antibody and PD-1 material and method Antibodies from clones 4G9, 4C12, and 5C2 were characterized using the Biocore® system available from GE. The analytes were mouse or human PD-1, and the ligands were anti-PD-1 antibodies. 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. Equilibrium association constant (K A ) is 9.52 × 10 5 The equilibrium dissociation constant was (1 / M). D ) is 1.05 × 10 -6 It was (M).

[0282] (Table 1) Analysis of the interaction between human PD-1 and 4G9 TIFF2026049006000040.tif41170

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

[0284] (Table 2) Analysis of the interaction between mouse PD-1 and 4G9 TIFF2026049006000041.tif41170

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

[0286] (Table 3) Analysis of the interaction between human PD-1 and 4C12 TIFF2026049006000042.tif41170

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

[0288] (Table 4) Analysis of the interaction between human PD-1 and 5C2 TIFF2026049006000043.tif41170

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

[0290] (Table 5) Analysis of the interaction between mouse PD-1 and 5C2 TIFF2026049006000044.tif41170

[0291] Example 3: Binding of anti-PD-1 antibody to EL4 cells material and method We evaluated the binding of 4G9, 5C2, and 4C12 to mouse EL4 cells constitutively expressing PD-1 using a fluorescence-activated cell sorter.

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

[0293] Example 4: Agonist activity of anti-PD-1 antibody material and method Mouse CD4 T cells Purified mouse CD4 T cells were stimulated with anti-CD3 / anti-CD28 Ab for 48 hours, then cultured with protein A beads for another 48 hours after adding anti-PD-1 Ab (10 ug / mL). In some samples, anti-PD-L1 Ab was added to block the PD-1 / PD-L1 interaction, and the agonist effect of test Ab was analyzed. IFNγ and IL-2 concentrations in the supernatant were detected using a CBA assay.

[0294] Human CD4 T cells Purified human CD4 T cells were stimulated with anti-CD3 / anti-CD28 absorb for 48 hours, then treated with anti-PD-1 absorb (1 or 10 ug / mL) and cultured with protein A beads for another 48 hours. IFNγ concentration in the supernatant was detected using a 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 in the supernatant compared to untreated cells or cells treated with isotype control antibodies.

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

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

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

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

[0300] Example 7: 4G9 and 5C2 specifically bind to human PD-1. material and method: The binding of 4G9 and 5C2 to human PD-1-Fc was determined using an ELISA assay.

[0301] result: Figure 10 shows the results of an ELISA assay to evaluate the binding of 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: Mouse CD4 T cells were pre-stimulated for 48 hours and then treated with purified 4G9 and 5C2 antibodies to ensure PD-1 expression. The pS6 concentration 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 via 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 resulted in a significant increase in pS6 within a linear range (ELISA kit, Cell Signaling Tech, *P<0.05, **P<0.01, ***P<0.001 compared to untreated cells). This phenomenon is due to direct activation rather than PD-1 / PD-L1 blockade, as treatment with commercially available Ab-1 (RMP1-14) and Ab-2 (J43), which block PD-1 / PD-L1 interactions, does not result in increased 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, TC-1 tumor cells were subcutaneously injected into mice on day 0. Mice were treated with a vaccine (E7+PADRE+Quil A) on days 10 (D10), 17 (D17), and 24 (D24) after tumor injection. Mice were treated with anti-PD-1 antibody on days 10, 14, 17, 21, 24, and 28.

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

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

[0307] result: Mice treated with 4G9, an antibody produced 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).

[0308] Sequence information SEQUENCE LISTING <110> Augusta University Research Institute, Inc. <120> Antibodies to Programmed Cell Death Protein 1 <150> US 62 / 555,156 <151> 2017-09-07 <150> US 62 / 624,843 <151> 2018-02-01 <150> US 62 / 657,323 <151> 2018-04-13 <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 288 <212> PRT <213> Homo sapiens <400> 1 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 2 <211> 288 <212> PRT <213> Mus musculus <400> 2 Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln 1 5 10 15 Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp 20 25 30 Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala 35 40 45 Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met 50 55 60 Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala 65 70 75 80 Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln 85 90 95 Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp 100 105 110 Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val 130 135 140 Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro 145 150 155 160 Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met Ser Ala 165 170 175 Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp Ala Leu Ala Val 180 185 190 Phe Cys Ser Thr Ser Met Ser Glu Ala Arg Gly Ala Gly Ser Lys Asp 195 200 205 Asp Thr Leu Lys Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val Ala 210 215 220 Tyr Glu Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro 225 230 235 240 Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly 245 250 255 Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln 260 265 270 Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 3 <211> 1389 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 3 atgagatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactcccag 60 gtccaactgc agcagcctgg ggctgaactg gtgaagcctg gggcttcagt gaaggtgtcc 120 tgcaaggctt ctggctacac cttcaccagc tactggatgc actgggtgaa gcagaggcct 180 ggccaaggcc ttgagtggat tggaaggatt catccttctg atagtgatac taactacaat 240 caaaagttca agggcaaggc cacattgact gtagacaaat cctccagcac agcctacatg 300 cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcacc ctatggtaac 360 tacgcctccg ggtttgctta ctggggccaa gggactctgg tcactgtctc tgcagccaaa 420 acgacacccc catctgtcta tccactggcc cctggatctg ctgcccaaac taactccatg 480 gtgaccctgg gatgcctggt caagggctat ttccctgagc cagtgacagt gacctggaac 540 tctggatccc tgtccagcgg tgtgcacacc ttcccagctg tcctgcagtc tgacctctac 600 actctgagca gctcagtgac tgtcccctcc agcacctggc ccagccagac cgtcacctgc 660 aacgttgccc acccggccag cagcaccaag gtggacaaga aaattgtgcc cagggattgt 720 ggttgtaagc cttgcatatg tacagtccca gaagtatcat ctgtcttcat cttcccccca 780 aagcccaagg atgtgctcac cattactctg actcctaagg tcacgtgtgt tgtggtagac 840 atcagcaagg atgatcccga ggtccagttc agctggtttg tagatgatgt ggaggtgcac 900 acagctcaga cgaaaccccg ggaggagcag atcaacagca ctttccgttc agtcagtgaa 960 cttcccatca tgcaccagga ctggctcaat ggcaaggagt tcaaatgcag ggtcaacagt 1020 gcagctttcc ctgcccccat cgagaaaacc atctccaaaa ccaaaggcag accgaaggct 1080 ccacaggtgt acaccattcc acctcccaag gagcagatgg ccaaggataa agtcagtctg 1140 acctgcatga taacaaactt cttccctgaa gacattactg tggagtggca gtggaatggg 1200 cagccagcgg agaactacaa gaacactcag cccatcatgg acacagatgg ctcttacttc 1260 gtctacagca agctcaatgt gcagaagagc aactgggagg caggaaatac tttcacctgc 1320 tctgtgttac atgagggcct gcacaaccac catactgaga agagcctctc ccactctcct 1380 ggtaaatga 1389 <210> 4 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Met Arg Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Arg Ile His Pro Ser Asp Ser Asp Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Pro Tyr Gly Asn Tyr Ala Ser Gly Phe Ala Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro 130 135 140 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 145 150 155 160 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 195 200 205 Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His 210 215 220 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 225 230 235 240 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 245 250 255 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 260 265 270 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 275 280 285 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 290 295 300 Lys Pro Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu 305 310 315 320 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 325 330 335 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 355 360 365 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 370 375 380 Thr Asn Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 385 390 395 400 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp 405 410 415 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 420 425 430 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 435 440 445 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450 455 460 <210> 5 <211> 443 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 5 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile His Pro Ser Asp Ser Asp Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Pro Tyr Gly Asn Tyr Ala Ser Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg 275 280 285 Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 6 Ser Tyr Trp Met His 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 Arg Ile His Pro Ser Asp Ser Asp Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Tyr Gly Asn Tyr Ala Ser Gly Phe Ala Tyr 1 5 10 <210> 9 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 9 atgggcatca agatggagtc acagattcag gcatttgtat tcgtgtttct ctggttgtct 60 ggtgttgacg gagacattgt gatgacccag tctcacaaat tcatgtccac atcagtagga 120 gacagggtca gcatcacctg caaggccagt caggatgtga gtactgctgt agcctggtat 180 caacaaaaac cagggcaatc tcctaaacta ctgatttact gggcatccac ccggcacact 240 ggagtccctg atcgcttcac aggcagtgga tctgggacag attatactct caccatcagc 300 agtgtgcagg ctgaagacct ggcactttat tactgtcagc aacattatag cactccgtgg 360 acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc 420 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480 aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540 aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600 agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag 717 <210> 10 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 10 Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe 1 5 10 15 Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His 20 25 30 Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys 35 40 45 Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr 65 70 75 80 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr 85 90 95 Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys 100 105 110 Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130 135 140 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145 150 155 160 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165 170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 195 200 205 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210 215 220 Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 <210> 11 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 12 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 13 Trp Ala Ser Thr Arg His Thr 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 14 Gln Gln His Tyr Ser Thr Pro Trp Thr 1 5 <210> 15 <211> 1773 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 15 atggaatgga gcagagtctt tatctttctc ctatcagtaa ctgcaggtgt tcactcccag 60 gtccagctgc agcagtctgg agctgagctg gtaaggcctg ggacttcagt gaaggtgtcc 120 tgcaaggctt ctggatacgc cttcactaat tacttgatag agtgggtaaa gcagaggcct 180 ggacagggcc ttgagtggat tggagtgatt aatcctggaa gtggtggtac taactacaat 240 gagaagttca agggcaaggc aacactgact gcagacaaat cctccagcac tgcctacatg 300 cagctcagca gcctgacatc tgaggactct gcggtctatt tctgtgcaag atcagctcag 360 gcccctgact actggggcca aggcaccact ctcacagtct cctcagagag tcagtccttc 420 ccaaatgtct tccccctcgt ctcctgcgag agccccctgt ctgataagaa tctggtggcc 480 atgggctgcc tggcccggga cttcctgccc agcaccattt ccttcacctg gaactaccag 540 aacaacactg aagtcatcca gggtatcaga accttcccaa cactgaggac agggggcaag 600 tacctagcca cctcgcaggt gttgctgtct cccaagagca tccttgaagg ttcagatgaa 660 tacctggtat gcaaaatcca ctacggaggc aaaaacaaag atctgcatgt gcccattcca 720 gctgtcgcag agatgaaccc caatgtaaat gtgttcgtcc caccacggga tggcttctct 780 ggccctgcac cacgcaagtc taaactcatc tgcgaggcca cgaacttcac tccaaaaccg 840 atcacagtat cctggctaaa ggatgggaag ctcgtggaat ctggcttcac cacagatccg 900 gtgaccatcg agaacaaagg atccacaccc caaacctaca aggtcataag cacacttacc 960 atctctgaaa tcgactggct gaacctgaat gtgtacacct gccgtgtgga tcacaggggt 1020 ctcaccttct tgaagaacgt gtcctccaca tgtgctgcca gtccctccac agacatccta 1080 accttcacca tccccccctc ctttgccgac atcttcctca gcaagtccgc taacctgacc 1140 tgtctggtct caaacctggc aacctatgaa accctgaata tctcctgggc ttctcaaagt 1200 ggtgaaccac tggaaaccaa aattaaaatc atggaaagcc atcccaatgg caccttcagt 1260 gctaagggtg tggctagtgt ttgtgtggaa gactggaata acaggaagga atttgtgtgt 1320 actgtgactc acagggatct gccttcacca cagaagaaat tcatctcaaa acccaatgag 1380 gtgcacaaac atccacctgc tgtgtacctg ctgccaccag ctcgtgagca actgaacctg 1440 agggagtcag ccacagtcac ctgcctggtg aagggcttct ctcctgcaga catcagtgtg 1500 cagtggcttc agagagggca actcttgccc caagagaagt atgtgaccag tgccccgatg 1560 ccagagcctg gggccccagg cttctacttt acccacagca tcctgactgt gacagaggag 1620 gaatggaact ccggagagac ctatacctgt gttgtaggcc acgaggccct gccacacctg 1680 gtgaccgaga ggaccgtgga caagtccact ggtaaaccca cactgtacaa tgtctccctg 1740 atcatgtctg acacaggcgg cacctgctat is 1773 <210> 16 <211> 590 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 16 Met Glu Trp Ser Arg Val Phe Ile Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30 Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe 35 40 45 Thr Asn Tyr Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Ser Ala Gln Ala Pro Asp Tyr Trp Gly Gln Gly 115 120 125 Thr Thr Leu Thr Val Ser Ser Glu Ser Gln Ser Phe Pro Asn Val Phe 130 135 140 Pro Leu Val Ser Cys Glu Ser Pro Leu Ser Asp Lys Asn Leu Val Ala 145 150 155 160 Met Gly Cys Leu Ala Arg Asp Phe Leu Pro Ser Thr Ile Ser Phe Thr 165 170 175 Trp Asn Tyr Gln Asn Asn Thr Glu Val Ile Gln Gly Ile Arg Thr Phe 180 185 190 Pro Thr Leu Arg Thr Gly Gly Lys Tyr Leu Ala Thr Ser Gln Val Leu 195 200 205 Leu Ser Pro Lys Ser Ile Leu Glu Gly Ser Asp Glu Tyr Leu Val Cys 210 215 220 Lys Ile His Tyr Gly Gly Lys Asn Lys Asp Leu His Val Pro Ile Pro 225 230 235 240 Ala Val Ala Glu Met Asn Pro Asn Val Asn Val Phe Val Pro Pro Arg 245 250 255 Asp Gly Phe Ser Gly Pro Ala Pro Arg Lys Ser Lys Leu Ile Cys Glu 260 265 270 Ala Thr Asn Phe Thr Pro Lys Pro Ile Thr Val Ser Trp Leu Lys Asp 275 280 285 Gly Lys Leu Val Glu Ser Gly Phe Thr Thr Asp Pro Val Thr Ile Glu 290 295 300 Asn Lys Gly Ser Thr Pro Gln Thr Tyr Lys Val Ile Ser Thr Leu Thr 305 310 315 320 Ile Ser Glu Ile Asp Trp Leu Asn Leu Asn Val Tyr Thr Cys Arg Val 325 330 335 Asp His Arg Gly Leu Thr Phe Leu Lys Asn Val Ser Ser Thr Cys Ala 340 345 350 Ala Ser Pro Ser Thr Asp Ile Leu Thr Phe Thr Ile Pro Pro Ser Phe 355 360 365 Ala Asp Ile Phe Leu Ser Lys Ser Ala Asn Leu Thr Cys Leu Val Ser 370 375 380 Asn Leu Ala Thr Tyr Glu Thr Leu Asn Ile Ser Trp Ala Ser Gln Ser 385 390 395 400 Gly Glu Pro Leu Glu Thr Lys Ile Lys Ile Met Glu Ser His Pro Asn 405 410 415 Gly Thr Phe Ser Ala Lys Gly Val Ala Ser Val Cys Val Glu Asp Trp 420 425 430 Asn Asn Arg Lys Glu Phe Val Cys Thr Val Thr His Arg Asp Leu Pro 435 440 445 Ser Pro Gln Lys Lys Phe Ile Ser Lys Pro Asn Glu Val His Lys His 450 455 460 Pro Pro Ala Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu 465 470 475 480 Arg Glu Ser Ala Thr Val Thr Cys Leu Val Lys Gly Phe Ser Pro Ala 485 490 495 Asp Ile Ser Val Gln Trp Leu Gln Arg Gly Gln Leu Leu Pro Gln Glu 500 505 510 Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gly Ala Pro Gly Phe 515 520 525 Tyr Phe Thr His Ser Ile Leu Thr Val Thr Glu Glu Glu Trp Asn Ser 530 535 540 Gly Glu Thr Tyr Thr Cys Val Val Gly His Glu Ala Leu Pro His Leu 545 550 555 560 Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr 565 570 575 Asn Val Ser Leu Ile Met Ser Asp Thr Gly Gly Thr Cys Tyr 580 585 590 <210> 17 <211> 571 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Ala Gln Ala Pro Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Glu Ser Gln Ser Phe Pro Asn Val Phe Pro Leu Val 115 120 125 Ser Cys Glu Ser Pro Leu Ser Asp Lys Asn Leu Val Ala Met Gly Cys 130 135 140 Leu Ala Arg Asp Phe Leu Pro Ser Thr Ile Ser Phe Thr Trp Asn Tyr 145 150 155 160 Gln Asn Asn Thr Glu Val Ile Gln Gly Ile Arg Thr Phe Pro Thr Leu 165 170 175 Arg Thr Gly Gly Lys Tyr Leu Ala Thr Ser Gln Val Leu Leu Ser Pro 180 185 190 Lys Ser Ile Leu Glu Gly Ser Asp Glu Tyr Leu Val Cys Lys Ile His 195 200 205 Tyr Gly Gly Lys Asn Lys Asp Leu His Val Pro Ile Pro Ala Val Ala 210 215 220 Glu Met Asn Pro Asn Val Asn Val Phe Val Pro Pro Arg Asp Gly Phe 225 230 235 240 Ser Gly Pro Ala Pro Arg Lys Ser Lys Leu Ile Cys Glu Ala Thr Asn 245 250 255 Phe Thr Pro Lys Pro Ile Thr Val Ser Trp Leu Lys Asp Gly Lys Leu 260 265 270 Val Glu Ser Gly Phe Thr Thr Asp Pro Val Thr Ile Glu Asn Lys Gly 275 280 285 Ser Thr Pro Gln Thr Tyr Lys Val Ile Ser Thr Leu Thr Ile Ser Glu 290 295 300 Ile Asp Trp Leu Asn Leu Asn Val Tyr Thr Cys Arg Val Asp His Arg 305 310 315 320 Gly Leu Thr Phe Leu Lys Asn Val Ser Ser Thr Cys Ala Ala Ser Pro 325 330 335 Ser Thr Asp Ile Leu Thr Phe Thr Ile Pro Pro Ser Phe Ala Asp Ile 340 345 350 Phe Leu Ser Lys Ser Ala Asn Leu Thr Cys Leu Val Ser Asn Leu Ala 355 360 365 Thr Tyr Glu Thr Leu Asn Ile Ser Trp Ala Ser Gln Ser Gly Glu Pro 370 375 380 Leu Glu Thr Lys Ile Lys Ile Met Glu Ser His Pro Asn Gly Thr Phe 385 390 395 400 Ser Ala Lys Gly Val Ala Ser Val Cys Val Glu Asp Trp Asn Asn Arg 405 410 415 Lys Glu Phe Val Cys Thr Val Thr His Arg Asp Leu Pro Ser Pro Gln 420 425 430 Lys Lys Phe Ile Ser Lys Pro Asn Glu Val His Lys His Pro Pro Ala 435 440 445 Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser 450 455 460 Ala Thr Val Thr Cys Leu Val Lys Gly Phe Ser Pro Ala Asp Ile Ser 465 470 475 480 Val Gln Trp Leu Gln Arg Gly Gln Leu Leu Pro Gln Glu Lys Tyr Val 485 490 495 Thr Ser Ala Pro Met Pro Glu Pro Gly Ala Pro Gly Phe Tyr Phe Thr 500 505 510 His Ser Ile Leu Thr Val Thr Glu Glu Glu Trp Asn Ser Gly Glu Thr 515 520 525 Tyr Thr Cys Val Val Gly His Glu Ala Leu Pro His Leu Val Thr Glu 530 535 540 Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser 545 550 555 560 Leu Ile Met Ser Asp Thr Gly Gly Thr Cys Tyr 565 570 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 18 Asn Tyr Leu Ile Glu 1 5 <210> 19 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 19 Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 20 Ser Ala Gln Ala Pro Asp Tyr 1 5 <210> 21 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 21 atgggcatca agatggagac acattctcag gtctttgtat acatgttgct gtggttgtct 60 ggtgttgaag gagacattgt gatgacccag tctcacaaat tcatgtccac atcagtagga 120 gacagggtca gcatcacctg caaggccagt caggatgtgg gtactgctgt agcctggtat 180 caacagaaac cagggcaatc tcctaaacta ctgatttact gggcatccac ccggcacact 240 ggagtccctg atcgcttcac aggcagtgga tctgggacag atttcactct caccattagc 300 aatgtgcagt ctgaagactt ggcagattat ttctgtcagc aatatagcag ctatccattc 360 acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgcacc aactgtatcc 420 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480 aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540 aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600 agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag 717 <210> 22 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 22 Met Gly Ile Lys Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu 1 5 10 15 Leu Trp Leu Ser Gly Val Glu Gly Asp Ile Val Met Thr Gln Ser His 20 25 30 Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys 35 40 45 Ala Ser Gln Asp Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr 65 70 75 80 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys 100 105 110 Gln Gln Tyr Ser Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130 135 140 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145 150 155 160 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165 170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 195 200 205 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210 215 220 Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 <210> 23 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 23 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 24 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 24 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 25 Gln Gln His Tyr Ser Thr Pro Trp Thr 1 5 <210> 26 <211> 1416 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 26 atgggttggc tgtggaactt gctattcctg atggcagctg cccaaagtgc ccaagcacag 60 atccagttgg tacagtctgg acctgagctg aagaagcctg gagagacagt caagatctcc 120 tgcaaggctt ctgggtatac cttcacaacc tatggaatga cctgggtgaa acaggctcca 180 ggaaagggtt taaagtggat gggctggata aacacctact ctggagtgcc aacatatgct 240 gatgacttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg 300 cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgcaag agggggacgg 360 gggtttgctt actggggcca agggactctg gtcactgtct ctgcagccaa aacaacaccc 420 ccatcagtct atccactggc ccctgggtgt ggagatacaa ctggttcctc tgtgactctg 480 ggatgcctgg tcaagggcta cttccctgag tcagtgactg tgacttggaa ctctggatcc 540 ctgtccagca gtgtgcacac cttcccagct ctcctgcagt ctggactcta cactatgagc 600 agctcagtga ctgtcccctc cagcacctgg ccaagtcaga ccgtcacctg cagcgttgct 660 cacccagcca gcagcaccac ggtggacaaa aaacttgagc ccagcgggcc catttcaaca 720 atcaacccct gtcctccatg caaggagtgt cacaaatgcc cagctcctaa cctcgagggt 780 ggaccatccg tcttcatctt ccctccaaat atcaaggatg tactcatgat ctccctgaca 840 cccaaggtca cgtgtgtggt ggtggatgtg agcgaggatg acccagacgt ccggatcagc 900 960 aacagtacta tccgggtggt cagtgccctc cccatccagc accaggactg gatgagtggc 1020 aaggagttca aatgcaaggt caacaacaaa gacctcccat cacccatcga gagaaccatc 1080 tcaaaaatta aagggctagt cagagctcca caagtataca tcttgccgcc accagcagag 1140 cagttgtcca ggaagatgt cagtctcact tgctgtcg tggcttca ccctggagac 1200 atcagtgtgg agtggaccag caatgggcat acagaggaga actahagga caccgcacca 1260 gtcctggact ctgacggttc ttactcata tacagcaagc tcgattaaa aacaagcaag 1320 tgggagaaaa cagattcctt ctcatgcaac gtgagacacg agggtctgaa aaattacac 1380 ctgaagaga ccatctcccg gtctccgggt aaatga 1416 <210> 27 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 27 Met Gly Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser 1 5 10 15 Only Gln Only Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 20 25 30 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Thr Tyr Gly Met Thr Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60 Lys Trp Met Gly Trp Ile Asn Thr Tyr Ser Gly Val Pro Thr Tyr Ala 65 70 75 80 Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90 95 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Gly Gly Arg Gly Phe Ala Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr 130 135 140 Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 145 150 155 160 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp 165 170 175 Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu 180 185 190 Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser 195 200 205 Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala Ser 210 215 220 Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr 225 230 235 240 Ile Asn Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro 245 250 255 Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys 260 265 270 Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val 275 280 285 Asp Val Ser Glu Asp Asp Pro Asp Val Arg Ile Ser Trp Phe Val Asn 290 295 300 Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr 305 310 315 320 Asn Ser Thr Ile Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp 325 330 335 Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu 340 345 350 Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg 355 360 365 Ala Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg 370 375 380 Lys Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp 385 390 395 400 Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys 405 410 415 Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser 420 425 430 Lys Leu Asp Ile Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser 435 440 445 Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr 450 455 460 Ile Ser Arg Ser Pro Gly Lys 465 470 <210> 28 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 28 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Gly Met Thr Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Arg Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala 115 120 125 Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu 130 135 140 Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly 145 150 155 160 Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly 165 170 175 Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro 180 185 190 Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr 195 200 205 Val Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro 210 215 220 Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu 225 230 235 240 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu 245 250 255 Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Glu Asp Asp Pro Asp Val Arg Ile Ser Trp Phe Val Asn Asn Val Glu 275 280 285 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 290 295 300 Ile Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 305 310 315 320 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro 325 330 335 Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln 340 345 350 Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val 355 360 365 Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val 370 375 380 Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asp 405 410 415 Ile Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val 420 425 430 Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg 435 440 445 Ser Pro Gly Lys 450 <210> 29 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 29 Thr Tyr Gly Met Thr 1 5 <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 30 Trp Ile Asn Thr Tyr Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 31 Gly Gly Arg Gly Phe Ala Tyr 1 5 <210> 32 <211> 705 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 32 atggtatcca cacctcagtt ccttgtattt ttgcttttct ggattccagc ctccagaggt 60 gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga aagagtcagt 120 ttctcctgca gggccagtca gagcattggc acaagcatac actggtatca gcaaagaaca 180 aatggttctc caaggcttct cataaagtat gcttctgagt ctatctctgg gatcccttcc 240 aggtttagtg gcagtggatc agggacagat tttactctta gcatcaacag tgtggagtct 300 gagatattg cagattatta ctgtcacaa agtatagct ggccgtacac gttcggaggg 360 gggaccaagc tggaaataaa acggctgat gctgcaccaa ctgtatccat cttcccacca 420 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gctcttgaa caactctac 480 cccaaagaca tcaatgtca gtggagatt gatggcagtg acgacaaaa tggcgtcctg 540 aacagttgga ctgatcagga cagcaagac agcacctaca gcatgagcag caccctcacg 600 ttgaccaagg acgagtatga acgacatac agctatacct gtgaggccac tcacagaca 660 tcaacttcac ccattgtcaa gagctcaac aggaatgagt gttag 705 <210> 33 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 33 Met Val Ser Thr Pro Gln Phe Leu Val Phe Leu Leu Phe Trp Ile Pro 1 5 10 15 The Arg Gly Asp Serpent Has Thr Gln Pro Server 20 25 30 Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn 100 105 110 Ser Trp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 130 135 140 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 145 150 155 160 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 165 170 175 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 195 200 205 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 210 215 220 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 <210> 34 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 34 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 35 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 35 Arg Ala Ser Gln Ser Ile Gly Thr Ser Ile His 1 5 10 <210> 36 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 36 Tyr Ala Ser Glu Ser Ile Ser 1 5 <210> 37 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 37 Gln Gln Ser Asn Ser Trp Pro Tyr Thr 1 5 <210> 38 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 38 Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile 1 5 10 15

Claims

1. A heavy chain complementarity determination region (CDR) 1 having an amino acid sequence NYLIE, Heavy chain CDR2 having the amino acid sequence VINPGSGGTNYNEKFKG, and A heavy chain CDR3 having the amino acid sequence SAQAPDY, Light chain CDR1 having the amino acid sequence KASQDVGTAVA, or KASQDVSTAVA, Light chain CDR2 having the amino acid sequence WASTRH, and Light chain CDR3 having the amino acid sequence QQYSSYPFT or QQHYSTPWT and An anti-PD-1 antibody or its antigen-binding fragment, including the above.

2. An anti-PD-1 antibody or its antigen-binding fragment comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) sequence of SEQ ID NO: 16 or 17, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) sequence of SEQ ID NO: 22 or 23.

3. The antibody or antigen-binding fragment according to claim 1, comprising a heavy chain having at least 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 16 or 17.

4. The antibody or antigen-binding fragment according to claim 1 or 3, comprising a light chain having at least 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 22 or 23.

5. A heavy chain having at least 90%, 95%, 99%, or 100% sequence identity with sequence number 16 or 17. A light chain having at least 90%, 95%, 99%, or 100% sequence identity with sequence number 22 or 23. The antibody or antigen-binding fragment according to claim 1, comprising:

6. A non-human transgenic animal manipulated to express an antibody or antigen-binding fragment according to any one of claims 1 to 5.

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

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

9. A nucleic acid comprising a sequence encoding an anti-PD-1 antibody or its antigen-binding fragment, A heavy chain CDR1 having the amino acid sequence NYLIE, a heavy chain CDR2 having the amino acid sequence VINPGSGGTNYNEKFKG, and a heavy chain CDR3 having the amino acid sequence SAQAPDY, and having at least 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 16 or 17. Codes the heavy chain, A light chain CDR1 having the amino acid sequence KASQDVGTAVA or KASQDVSTAVA, a light chain CDR2 having the amino acid sequence WASTRHT, and a light chain CDR3 having the amino acid sequence QQYSSYPFT or QQHYSTPWT, and having at least 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 22 or 23. Code the light chain Nucleic acid.

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

11. A cell comprising nucleic acid containing a sequence encoding an anti-PD-1 antibody or its antigen-binding fragment, A nucleic acid comprising a sequence encoding a heavy chain including heavy chain CDR1 having the amino acid sequence NYLIE, heavy chain CDR2 having the amino acid sequence VINPGSGGTNYNEKFKG, and heavy chain CDR3 having the amino acid sequence SAQAPDY, and A nucleic acid comprising a sequence encoding a light chain including light chain CDR1 having the amino acid sequence KASQDVGTAVA or KASQDVSTAVA, light chain CDR2 having the amino acid sequence WASTRH, and light chain CDR3 having the amino acid sequence QQYSSYPFT or QQHYSTPWT. Cells that include this.

12. A cell comprising nucleic acid containing a sequence encoding an anti-PD-1 antibody or its antigen-binding fragment, A nucleic acid comprising a sequence encoding a heavy chain containing three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) sequence of SEQ ID NO: 16 or 17, and A nucleic acid comprising a sequence encoding a light chain, including three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) sequence of SEQ ID NO: 22 or 23, Cells that include this.

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

14. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, which is a mouse or chimeric antibody.

15. A humanized antibody, the antibody according to any one of claims 1 to 5, or an antigen-binding fragment thereof.

16. An antibody or antigen-binding fragment according to any one of claims 1 to 5, which is bispecific, triplicate, or multispecific.

17. The antibody according to any one of claims 1 to 5 or 13 to 16, wherein the entire immunoglobulin is used.

18. An antibody or antigen-binding fragment according to any one of claims 1 to 5 or 13 to 16, wherein the antibody is IgG, IgE, IgM, IgD, IgA, or IgY.

19. IgG １ , IgG ２ , IgG ３ , IgG ４ IgA １ , or IgA ２ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or 13 to 16.

20. Fab', F(ab') ２ An antibody or antigen-binding fragment according to any one of claims 1 to 5, 13 to 16, 18, or 19, wherein the antibody or antigen-binding fragment is Fv, single-stranded (ScFv), di-scFv, diabody, or tribody.

21. An antibody or antigen-binding fragment according to any one of claims 1 to 5, 13 to 16, or 18 to 20, which is immunospecifically bound to the amino acid sequence described in SEQ ID NO: 38 on the surface of an immune cell.

22. The antibody or antigen-binding fragment according to claim 21, wherein the immune cell is a T cell.

23. The T cell is CD8 ＋ The antibody or antigen-binding fragment according to claim 22, which is a T cell.

24. An antibody or antigen-binding fragment according to any one of claims 1 to 5, 13 to 16, or 18 to 20, which immune-specifically binds to the amino acid sequence described 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.

25. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 5, 13 to 16, or 18 to 24.

26. The pharmaceutical composition according to claim 25, further comprising a second therapeutic agent.

27. The pharmaceutical composition according to claim 25 or 26, further comprising a pharmaceutically acceptable excipient.

28. The pharmaceutical composition according to claim 27, wherein the second therapeutic agent comprises cyclophosphamide.

29. A pharmaceutical composition according to any one of claims 25 to 28, for use in a method of inducing, promoting, or enhancing an immune response in a subject in need thereof.

30. A pharmaceutical composition according to any one of claims 25 to 28, for use in a method for treating cancer in a person in need thereof.

31. A pharmaceutical composition according to any one of claims 25 to 28, for use in a method for reducing tumor burden in a subject that requires it.

32. A pharmaceutical composition according to any one of claims 25 to 28, for use in a method for treating an infectious disease in a person in need thereof.

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