Antibodies against programmed cell death protein 1, a PD-1 agonist

Abstract

Antibodies and antigen-binding fragments thereof that immunospecifically bind to PD-1, such as human or mouse PD-1, are agonists of PD-1, and are capable of inducing or promoting an immune response that activates immune cell proliferation or activity.Unlike most of the known anti-PD-1 and anti-PD-L1 antibodies that relieve inhibition of PD-1 function by physically blocking the interaction of PD-1 with an inactivating ligand, for example, by PD-L1, without wishing to be bound by theory, the disclosed antibodies and antigen-binding fragments thereof are currently best understood to immunospecifically bind to an epitope on PD-1 whereby PD-1 is directly activated.A method of treating cancer, comprising administering the antibody or antigen-binding fragment to a subject in need thereof.A polynucleotide encoding the antibody or antigen-binding fragment.A host cell comprising the polynucleotide.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 304,365, filed January 28, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates generally to antibodies that specifically bind to PD-1 and methods of their use. [Background technology]

[0003] The programmed death receptor protein 1 (PD-1) / programmed death receptor protein ligand 1 (PD-L1) pathway has shown promising clinical success as a target for cancer immunotherapy. Antibodies that inhibit this pathway by physically blocking the interaction between PD-1 and PD-L1 can enhance a patient's immune response against cancer cells. For example, pembrolizumab (Keytruda®, Merck & Co., Inc., Kenilworth, NJ) binds to a range of amino acids 64-89 of human PD-1 that overlaps with the portion of PD-1 that binds to PD-L1 (Zak et al., Cell Structure 25(8):1163-1174(2017)). Successful clinical trials with PD-1 monoclonal antibodies and other immune checkpoint inhibitors are opening new avenues in cancer immunology. However, because there are many cancer patients who do not respond to new immunotherapies, including anti-PD-1 inhibitor therapy, research into combination therapies and predictive biomarkers has become active (Iwai, Y. et al., Journal of Biomedical Science, 24: 26 (2017)).

[0004] It would therefore be desirable to provide compositions and methods for modulating PD-1 signaling. It would be particularly desirable to provide compositions and methods that are PD-1 agonists, i.e., that activate PD-1 independently of perturbing the PD-1 / PD-L1 interaction.

[0005] It would also be desirable to provide antibodies and antigen-binding fragments thereof that specifically bind to PD-1 and modulate PD-1 signaling. It would be particularly desirable to provide antibodies and antigen-binding fragments that are PD-1 agonists, i.e., that activate PD-1 independently of perturbing the PD-1 / PD-L1 interaction.

[0006] It would also be desirable to provide compositions and methods for treating cancer. It would also be desirable to provide compositions and methods for treating infectious diseases. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Zak et al.,Cell Structure 25(8):1163-1174(2017) [Non-Patent Document 2] Iwai, Y. et al., Journal of Biomedical Science, 24:26 (2017) Summary of the Invention [Means for solving the problem]

[0008] The present disclosure relates to antibodies and antigen-binding fragments thereof that immunospecifically bind to PD-1, preferably human or mouse PD-1, and are PD-1 agonists. Such antibodies can be used to induce or promote an immune response that activates immune cell proliferation or activity. In embodiments, the disclosed antibodies and antigen-binding fragments thereof specifically bind to PD-1 expressed on immune cells. In certain embodiments, the disclosed antibodies and antigen-binding fragments thereof specifically bind to a PD-1 epitope that is distant from the interaction site of PD-1 with PD-L1, e.g., a PD-1 epitope that does not bind to amino acid residues in the range of 64-89 of human PD-1. In certain embodiments, the antibodies or antigen-binding fragments do not compete with PD-L1 for binding to PD-1. Binding of the disclosed antibodies and antigen-binding fragments thereof to PD-1 on immune cells can transmit an activation signal into the immune cell, e.g., a signal that enhances or promotes activation of cytokine production and / or immune cell proliferation. Immune cells that express PD-1 include, but are not limited to, B cells and T cells, as well as bone marrow-derived cells (Riley, J., Immunol Rev., 229(1); 114-125 (2009)). In an embodiment, the immune cell is a T cell, preferably a CD8 + T cells.

[0009] The present disclosure also relates to a method of inducing, enhancing or promoting an immune response (e.g., an adaptive immune response) in a subject in need thereof by administering to the subject an effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof disclosed herein to induce, enhance or promote an immune response (e.g., an adaptive immune response) in the subject.

[0010] An antibody or antigen-binding fragment thereof with binding specificity for Programmed Death Receptor Protein 1 (PD-1) can comprise a light chain variable region and a heavy chain variable region, where the light chain variable region comprises CDR L1, CDR L2, and CDR L3 of Table 2 or Table 4, and the heavy chain variable region comprises CDR H1, CDR H2, and CDR H3 of Table 1 or Table 3.

[0011] In embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for programmed death receptor protein 1 (PD-1) can comprise a light chain variable region and a heavy chain variable region, wherein a) the light chain variable region is selected from the group consisting of QX 8 X 9 X 10 X 11 X 12 CDR L1, including X 13 X 14 CDR L2 containing S, and X 15 QX 16 X 17 X 18 X 19 PX 20 X 21 and a heavy chain variable region comprising CDR H1 comprising CDR H1 (SEQ ID NO: 394) comprising GYTFTTYG, CDR H2 comprising INTYSGVP (SEQ ID NO: 395), and CDR H3 comprising ARX 1 X 2 X 3 X 4 X 5 X 6 X 7 wherein X comprises a CDR H3 comprising 1 is selected from the group consisting of G and V; X 2 is selected from the group consisting of G, S, L, V, I, and E; X 3 is selected from the group consisting of R, G, P, W, S, H, and K; X 4 is selected from the group consisting of G, E, S, V, W, and K; X 5 is selected from the group consisting of I, F, S, G, D, and V; X 6 is selected from the group consisting of A, G, Y, and S; X 7 is selected from the group consisting of Y, H, and D; X 8 is selected from the group consisting of G, S, and D; X 9 is selected from the group consisting of I and L; X 10 is selected from the group consisting of S, V, G, and R; X 11 is selected from the group consisting of N, Y, T, S, and R; X 12 is selected from the group consisting of S, Y, D, and W; X13 is selected from the group consisting of A, K, Y, and D; X 14 is selected from the group consisting of A and V; X 15 is selected from the group consisting of Q, M, and L; X 16 is selected from the group consisting of S, G, and D; X 17 is selected from the group consisting of Y, T, N, and S; X 18 is selected from the group consisting of S, H, and D; X 19 is selected from the group consisting of T, W, and F; X 20 is selected from the group consisting of Y, P, R, and W; and X 21 is selected from the group consisting of T and F; or b) the light chain variable region is selected from the group consisting of QX 42 X 43 X 44 X 45 X 46 CDR L1, including X 47 CDR L2 containing AS, and X 48 X 49 X 50 X 51 X 52 X 53 X 54 and the heavy chain variable region comprises CDR L3 comprising GX 22 TFX 23 X 24 YX 25 CDR H1, IX containing (SEQ ID NO: 163) 26 X 27 X 28 X 29 X 30 X 31 CDR H2 containing T, and AX 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 FX 40 X 41 wherein X comprises a CDR H3 comprising 22 is selected from the group consisting of Y and Q; X 23 is selected from the group consisting of N, T, and G; X 24 is selected from the group consisting of S, I, and N; X 25is selected from the group consisting of W and L; X 26 is selected from the group consisting of H, L, R, and P; X 27 is selected from the group consisting of P and L; X 28 is selected from the group consisting of R, S, I, and N; X 29 is selected from the group consisting of G, D, N, and Y; X 30 is selected from the group consisting of I, S, G, and R; X 31 is selected from the group consisting of H, D, and Y; X 32 is selected from the group consisting of P and S; X 33 is selected from the group consisting of S, R, N, and Y; X 34 is selected from the group consisting of S, D, G, V, and F; X 35 is selected from the group consisting of S, N, G, I, D, and R; X 36 is selected from the group consisting of Y, N, C, L, S, and H; X 37 is selected from the group consisting of A, G, T, and E; X 38 is selected from the group consisting of W, C, S, R, D, and G; X 39 is selected from the group consisting of A, S, G, and D; X 40 is selected from the group consisting of A, S, and L; X 41 is selected from the group consisting of H, Y, and S; X 42 is selected from the group consisting of D, S, and G; X 43 is selected from the group consisting of V and I; X 44 is selected from the group consisting of S and G; X 45 is selected from the group consisting of T and S; X 46 is selected from the group consisting of A, S, D, and W; X 47 is selected from the group consisting of W, G, and D; X 48 is selected from the group consisting of Q and H; X 49 is selected from the group consisting of Q, K, and H; X 50 is selected from the group consisting of H, Y, F, R, D, and G; X 51 is selected from the group consisting of Y, N, Y, D, S, and G; X 52 is selected from the group consisting of R, S, V, T, and I; X 53is selected from the group consisting of S, A, F, T, Y, and W; and X 54 is selected from the group consisting of P, L, S, and T.

[0012] In embodiments, the antibody or antigen-binding fragment thereof comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-12; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-1; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-2; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-4; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-5; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-6 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-7; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-8; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-9; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-10; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-11; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-13; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-14 L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 4G9-15 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 4G9-16 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 4G9-17 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 4G9-18 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 4G9-19 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3;4G9-20 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-1 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-2 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-3 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-4 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-5 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3;5C2-6 CDR L1, CDR L2, CDR L3, CDR H1, CDR CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 5C2-7; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 5C2-8; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 5C2-9; or CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 5C2-10. In certain embodiments, the antibody or antigen-binding fragment comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-12; or CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-2. Antibodies 4G9-1 to 4G9-20 and 5C2-1 to C2-10 are described herein. In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-12;

[0013] In embodiments, the antibody or antigen-binding fragment comprises the light chain variable region and heavy chain variable region of 4G9-12; the light chain variable region and heavy chain variable region of 4G9-1; the light chain variable region and heavy chain variable region of 4G9-2; the light chain variable region and heavy chain variable region of 4G9-3; the light chain variable region and heavy chain variable region of 4G9-4; the light chain variable region and heavy chain variable region of 4G9-5; the light chain variable region and heavy chain variable region of 4G9-6 Variable regions; light and heavy chain variable regions of 4G9-7; light and heavy chain variable regions of 4G9-8; light and heavy chain variable regions of 4G9-9; light and heavy chain variable regions of 4G9-10; light and heavy chain variable regions of 4G9-11; light and heavy chain variable regions of 4G9-13; light and heavy chain variable regions of 4G9-14; light chain variable region of 4G9-15 and heavy chain variable regions; the light chain variable region and heavy chain variable region of 4G9-16; the light chain variable region and heavy chain variable region of 4G9-17; the light chain variable region and heavy chain variable region of 4G9-18; the light chain variable region and heavy chain variable region of 4G9-19; the light chain variable region and heavy chain variable region of 4G9-20; the light chain variable region and heavy chain variable region of 5C2-1; the light chain variable region and heavy chain variable region of 5C2-2; the light chain variable region and heavy chain variable region of 5C2-3 and heavy chain variable regions; the light chain variable region and heavy chain variable region of 5C2-4; the light chain variable region and heavy chain variable region of 5C2-5; the light chain variable region and heavy chain variable region of 5C2-6; the light chain variable region and heavy chain variable region of 5C2-7; the light chain variable region and heavy chain variable region of 5C2-8; the light chain variable region and heavy chain variable region of 5C2-9; or the light chain variable region and heavy chain variable region of 5C2-10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the light chain variable region and heavy chain variable region of 4G9-12. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the light chain variable region and heavy chain variable region of 4G9-2.

[0014] One embodiment provides an antibody or antigen-binding fragment thereof, wherein the CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 each independently have at least 75% identity, such as at least 85% identity, or at least 90% identity, with the corresponding CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of any one of antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10. In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 each independently have at least 75% identity, such as at least 85% identity, or at least 90% identity, with the corresponding CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-12.

[0015] One embodiment provides an antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region that are, respectively, independently, at least 75% identical, e.g., at least 85% identical, at least 90% identical, or at least 95% identical, to the corresponding light chain variable region and heavy chain variable region of any one of antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10. In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region that are, respectively, independently, at least 75% identical, e.g., at least 85% identical, at least 90% identical, or at least 95% identical, to the corresponding light chain variable region and heavy chain variable region of 4G9-12.

[0016] One embodiment provides an antibody or antigen-binding fragment thereof, wherein CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 each independently have three or fewer amino acid substitutions, such as two amino acid substitutions or one amino acid substitution, relative to the corresponding CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of any one of antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10.

[0017] An antibody or antigen-binding fragment thereof of the present disclosure can further comprise a light chain constant region or portion thereof, and / or a heavy chain constant region or portion thereof, for example, the heavy chain constant region can be an IgG constant region.

[0018] The antibodies or antigen-binding fragments thereof disclosed herein can increase the phosphorylated interleukin-2 inducible T cell kinase (pITK) level, the ITK / SHP2 ratio, or both the pITK level and the ITK / SHP2 ratio in cells by binding to PD-1 on the cells. By activating T cells, the antibodies or antigen-binding fragments thereof disclosed herein can induce T cell memory and / or inhibit T cell exhaustion. In one embodiment, the antibodies or antigen-binding fragments thereof induce central memory T cells (Tcm). In one embodiment, the antibodies or antigen-binding fragments thereof inhibit T cell exhaustion.

[0019] The present disclosure also relates to a polynucleotide(s) encoding the heavy or light chain, or both, of the above-mentioned antibodies or antigen-binding fragments thereof.

[0020] The present disclosure relates to a host cell comprising a polynucleotide(s). In an embodiment, the host cell is a mammalian cell (e.g., a human cell, a CHO cell, an immortalized human cell, an NS0 cell). In certain embodiments, the host cell may be a cell of a transgenic animal. In an embodiment, the transgenic animal is a mouse.

[0021] The present disclosure also relates to a pharmaceutical composition comprising the above-described antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier.

[0022] One embodiment provides an antibody or epitope-binding fragment thereof, or a fusion protein that immunospecifically binds to a PD-1 epitope that includes amino acids 96-110 of human PD-1 (hPD-1). In certain preferred embodiments, the antibody or antigen-binding fragment binds to a PD-1 epitope that includes amino acids 96-110 of hPD-1, which is expressed on the surface of immune cells. Without being bound to a particular theory or mechanism, it is believed that binding of this epitope of PD-1 by the antibody or antigen-binding fragment induces or enhances a signal through PD-1 that activates or stimulates the immune cell without perturbing the PD-1 / PD-L1 interaction. In an embodiment, the immune cell that is activated or stimulated is a T cell, e.g., a CD4 T cell.

[0023] In embodiments, the antibodies or antigen-binding fragments thereof may be human, murine, chimeric, humanized, monoclonal, bispecific, trispecific, or multispecific.

[0024] The present disclosure also relates to pharmaceutical compositions comprising one or more of the disclosed antibodies or antigen-binding fragments thereof. The pharmaceutical compositions may include a second therapeutic agent and / or a pharma- ceutical acceptable excipient. In embodiments, the second therapeutic agent may be a second antibody or antigen-binding fragment thereof that reduces inhibition of PD-1 by PD-L1.

[0025] The present disclosure also relates to methods for treating cancer comprising administering to a subject in need thereof an effective amount of an antibody, antigen-binding fragment, or pharmaceutical composition described herein. The present disclosure also relates to the antibody, antigen-binding fragment, or pharmaceutical composition disclosed herein for use as a medicament (e.g., particularly for treating cancer), and for use to treat cancer and / or for use in the manufacture of a medicament for treating cancer.

[0026] The disclosed therapeutic methods may further include administering one or more additional therapeutic agents to the subject. The one or more additional therapeutic agents may be an immunomodulatory agent. The immunomodulatory agent may be a checkpoint inhibitor (e.g., a checkpoint inhibitor that is a checkpoint inhibitor but does not inhibit the interaction of PD-1 with PD-L1 or PD-L2). For example, the checkpoint inhibitor may be an anti-LAG-3 antibody or an antigen-binding fragment thereof. As another example, the checkpoint inhibitor may be an anti-CTLA4 antibody or an antigen-binding fragment thereof. The immunomodulatory agent may be a T cell engager, including a bispecific T cell engager (BiTE), a bifunctional checkpoint inhibitory T cell engager (CiTE), a simultaneous multi-interacting T cell engager (SMITE), a trispecific killer engager (TriKE), a BiTE-expressing chimeric antigen receptor (CAR) T cell (CART.BiTE cell), and combinations thereof.

[0027] The one or more additional therapies can be cell therapy. For example, the cell therapy can be T cell therapy. The cell therapy can be tumor infiltrating lymphocyte (TIL) therapy. The cell therapy can be an engineered cell therapy, such as engineered TCR therapy, chimeric antigen receptor (CAR) therapy, or a combination thereof. In one embodiment, the cell therapy is chimeric antigen receptor T cell (CAR-T cell) therapy. In one embodiment, the cell therapy is engineered TCR T cell (TCR-T cell) therapy. For example, the method can include administering to the subject an effective combination comprising a therapeutically effective amount of (i) an antibody or antigen-binding fragment that is a PD-1 agonist as described herein, and (ii) a second antibody or antigen-binding fragment thereof that reduces inhibition of PD-1 by PD-L1. Without being bound by theory, the second antibody or antigen-binding fragment can bind to PD-1 or PD-L1 in a position that physically disrupts the interaction of PD-1 with PD-L1. In another embodiment, the method may include administering to the subject an effective combination comprising a therapeutically effective amount of (i) an antibody or antigen-binding fragment that is a PD-1 agonist as described herein, and (ii) an immune checkpoint inhibitor that does not bind to PD-1 or PD-L1, e.g., anti-CTLA4 or anti-LAG-3.

[0028] The present disclosure further relates to a method of inducing or enhancing central memory T cells, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment disclosed herein to a subject in need thereof.

[0029] The present disclosure further relates to a method of inhibiting T cell exhaustion comprising administering to a subject in need thereof a therapeutically effective amount of the antibodies or antigen-binding fragments disclosed herein. [Brief description of the drawings]

[0030] [Figure 1]Graphs showing human T cell activation from two donors, as determined by interferon gamma (IFNγ) levels, by antibodies 4G9-2 and 4G9-12 according to Example 3. A is a graph showing IFNγ levels following activation of human T cells from donor 1 with no agonistic anti-PD-1 antibody ("control"), with 4G9-2, or with 4G9-12 at antibody concentrations of 0 and 10 μg / mL. B is a graph showing IFNγ levels following activation of human T cells from donor 2 with no agonistic anti-PD-1 antibody ("control"), with 4G9-2, or with 4G9-12 at antibody concentrations of 0, 10, 20, and 50 μg / mL. For comparison, IFNγ levels without agonistic anti-PD-1 antibody treatment are shown as dashed lines.

[0031] [Diagram 2] FIG. 1 is a graph showing activation of human CD4+ T cells by antibodies 4G9-1 through 4G9-10 as determined by interferon gamma (IFNγ) levels according to Example 4, showing that 4G9-2 treatment significantly increased IFNγ (*p<0.05) compared to non-affinity matured antibody ("4G9 HC4-LC1"). The lower dotted line shows the average IFNγ levels in cells not treated with 4G9 lineage antibodies ("Untreated"). The upper dotted line shows the average IFNγ levels in cells treated with non-affinity matured antibody ("4G9 HC4-LC1").

[0032] [Figure 3A] 1 is a schematic diagram showing the experimental design to characterize memory and terminal effector T cell populations in response to agonistic antibody (4G9-12) treatment of human CD4+ T cells. [Figure 3B] Graphs showing the results of experiments characterizing memory T cell populations upon agonistic antibody (4G9-12) treatment of human CD4+ T cells. Graphs showing central memory T cells (CD45ROhigh / CD62Lhigh / CD45RAlow) as a percentage of CD4+ T cells following 4G9-12 treatment. [Figure 3C] Graphs showing the results of experiments characterizing terminal effector T cell populations in response to agonistic antibody (4G9-12) treatment of human CD4+ T cells. Graphs showing terminal effector T cells (CD45ROlow / CD62Llow / CD45RAhigh) as a percentage of CD4+ T cells following 4G9-12 treatment.

[0033] [Figure 4A] Figure 1 is a graph showing stimulation of CD4+ T cells by anti-PD-1 agonist antibodies, but not isotype control or PD-1 blocking antibodies, in the study described in Example 6. Figure 1 is a graph showing that treatment of donor CD4+ T cells with increasing concentrations of 4G9-2 or 4G9-12 antibodies activated T cells and induced IFNγ production, but treatment with a blocking anti-PD-1 antibody (PROMEGA Co., clone J1201, "Block-1") did not. [Figure 4B] Figure 1 is a graph showing stimulation of CD4+ T cells by anti-PD-1 agonist antibodies, but not isotype control or PD-1 blocking antibodies, in the study described in Example 6. Figure 2 is a graph showing that treatment of second donor CD4+ T cells with increasing concentrations of 4G9-2 or 4G9-12 antibodies activated T cells and induced IFNγ production, but not treatment with a blocking anti-PD-1 antibody (PROMEGA Co., clone J1201, "Block-1"). [Figure 4C] Figure 13 is a graph showing stimulation of CD4+ T cells by anti-PD-1 agonist antibodies, but not isotype control or PD-1 blocking antibodies, in the study described in Example 6. Figure 13 is a graph showing that treatment of CD4+ T cells from a third donor with increasing concentrations of 4G9-12 antibody activated T cells and induced IFNγ production, but not treatment with isotype control antibody ("Iso Ctrl"), blocking anti-PD-1 antibody (PROMEGA Co., clone J1201, "Block-1"), or pembrolizumab (blocking anti-PD-1 antibody, "Block-2").

[0034] [Diagram 5] Figure 13 is a graph showing that human CD4+ T cells treated with an anti-PD-1 agonist antibody (4G9-2) and both PD-L1 (e.g., PD-L1-IgG) and 4G9-2 increased T cell activation, as assessed by IFNγ release, compared to cells not treated with PD-L1 (e.g., PD-L1-IgG) or a blocking anti-PD-1 antibody ("untreated"). The results show that the anti-PD-1 agonist antibody dramatically increased T cell activation, as assessed by IFNγ release, even in the presence of PD-L1. Further experimental details are described in Example 7.

[0035] [Figure 6A] 1 is a histogram showing the results of a competitive binding experiment between PD-L1 and an agonistic anti-PD-1 antibody (4G9-12) as described in Example 8. A shows the dose-dependent binding of PD-L1 to PD-1 on the cell surface (PD-L1 fluorescence channel) at 1 μg / mL, 2 μg / mL, and 5 μg / mL of PD-L1. B shows that the binding of 4G9-12 (5 μg / ml) to cellular PD-1 is unaffected by varying PD-L1 concentrations. C shows that the dose-dependent binding of PD-L1 to PD1 on the cell surface is unaffected by the presence of 4G9-12 (5 μg / mL). [Figure 6B] Same as above. [Figure 6C] Same as above.

[0036] [Figure 7] FIG. 13 is a graph showing that intraperitoneal (ip) treatment with 4G9-12 (0.5 mg / kg or 1.0 mg / kg), and to a lesser extent, a blocking anti-PD-1 antibody (1.0 mg / kg, RMP1-14 clone), reduced mean tumor volume in a syngeneic mouse model of colorectal carcinoma compared to untreated mice ("Naive") in the study described in Example 9.

[0037] [Figure 8A] 1 is a graph showing the tumor volume for each mouse in a syngeneic mouse model of colorectal carcinoma, further described in Figure 7 and Example 9. FIG. 2 is a graph showing the tumor volume for each mouse in the untreated group. [Figure 8B] 1 is a graph showing the tumor volume of each mouse in an allogeneic mouse model of colorectal carcinoma, further described in FIG 7 and Example 9. FIG 1 is a graph showing the tumor volume of each mouse in the 4G9-12 antibody (1 mg / kg) group. [Figure 8C] 1 is a graph showing the tumor volume of each mouse in an allogeneic mouse model of colorectal carcinoma further described in FIG 7 and Example 9. FIG 1 is a graph showing the tumor volume of each mouse in the 4G9-12 antibody (0.5 mg / kg) group. [Figure 8D] Figure 13 is a graph showing the tumor volume for each mouse in a syngeneic mouse model of colorectal carcinoma, as further described in Figure 7 and Example 9. Figure 14 is a graph showing the tumor volume for each mouse in the blocking anti-PD-1 antibody (1 mg / kg, RMP1-14 clone) group.

[0038] [Figure 9] FIG. 13 is a graph showing that mean tumor volume is reduced in a colorectal carcinoma CT26 mouse model following intravenous (iv) treatment with 4G9-12 (0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, or 0.25 mg / kg), or a blocking anti-PD-1 antibody (1.0 mg / kg, RMP1-14 clone), compared to untreated mice ("Untreated"), as further described in Example 10.

[0039] [Figure 10A]9 and Example 10. A is a graph showing the tumor volume of each mouse in the colorectal carcinoma CT26 mouse model, which is further described in FIG. 9 and Example 10. A is a graph showing the tumor volume of each mouse in the untreated group. B is a graph showing the tumor volume of each mouse in the 4G9-12 antibody (0.01 mg / kg) group. C is a graph showing the tumor volume of each mouse in the 4G9-12 antibody (0.05 mg / kg) group. D is a graph showing the tumor volume of each mouse in the 4G9-12 antibody (0.1 mg / kg) group. E is a graph showing the tumor volume of each mouse in the 4G9-12 antibody (0.25 mg / kg) group. F is a graph showing the tumor volume of each mouse in the blocking anti-PD-1 antibody (1 mg / kg, RMP1-14 clone) group. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. [Figure 10F] Same as above.

[0040] [Figure 11A] FIG. 11 is a graph showing that in vivo treatment with 4G9-12 (an anti-PD-1 agonist antibody), but not treatment with a blocking anti-PD-1 antibody (RMP1-14), greatly increases the activation status of both CD4+ T cells and CD8+ T cells by ex vivo analysis compared to mice not treated with the antibody ("untreated"), as further described in Example 11. FIG. 12 is a graph showing that treatment with 4G9-12 (an anti-PD-1 agonist antibody), but not treatment with a blocking anti-PD-1 antibody (RMP1-14), greatly increases the activation status of CD4+ T cells using CD40L as a marker of activation status. FIG. 13 is a graph showing that treatment with 4G9-12 (an anti-PD-1 agonist antibody), but not treatment with a blocking anti-PD-1 antibody (RMP1-14), greatly increases the activation status of CD8+ T cells using CD25 as a marker of activation status. Statistical analysis consisted of one-way ANOVA with multiple comparison post-hoc tests. [Figure 11B] Same as above.

[0041] [Figure 12A] 1A-C are western blots and bar graphs showing that the intracellular ratio of ITK to SHP2 is increased following treatment of human CD4+ T cells with agonistic anti-PD-1 antibody 4G9-12, but not with blocking anti-PD-1 antibody (PROMEGA Co., "Control") or PD-L1 (e.g., PD-L1-IgG), as further described in Example 12. A is an image of a western blot for SHP2 and PD-1. B is an image of a western blot staining for PD-1 and ITK (interleukin-2 inducible T cell kinase). C is a bar graph of quantified ITK / SHP2 protein ratios. Values ​​were normalized to PD-1 protein levels. [Figure 12B] Same as above. [Figure 12C] Same as above.

[0042] [Figure 13] FIG. 1 is a graph showing that treatment with agonistic anti-PD-1 antibody 4G9-12 (25 μg / mL) but not PD-L1 (e.g., PD-L1-IgG), pembrolizumab ("Pembro"), or nivolumab ("Nivo") increases phosphorylated interleukin-2-inducible T cell kinase (pITK) in CD4+ T cells by normalized mean fluorescence intensity (MFI) obtained by flow cytometry compared to stimulated cells alone without additional treatment ("Stim"), as further described in Example 13. Data was normalized to the stimulation alone "Stim" group. The mean of the stimulation group is indicated by the dashed line. Statistical analysis consisted of one-way ANOVA with multiple comparison post-hoc testing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] definition As used herein, an antibody or antigen-binding fragment thereof is considered to "immunospecifically bind" to a target region or conformation of an antigen ("epitope") when the binding involves the antigen recognition site of the antibody or antigen-binding fragment. An antibody that immunospecifically binds to a particular epitope of a particular antigen may bind with lower affinity to other epitopes and / or antigens, but will not bind completely unrelated antigens, if the other antigens have any sequence or conformational similarity recognized by the antigen recognition site as determined, for example, by immunoassays, BIACORE® assays, or other assays known in the art. However, desirably, the antibody (and antigen-binding fragment thereof) does not cross-react with other antigens and / or epitopes. Antibodies and antigen-binding fragments may also bind other molecules, e.g., FcR receptors, in a non-immunospecific manner by binding regions, e.g., Fc regions, located in other regions / domains of the molecule that are not involved in the antigen recognition site.

[0044] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule that has at least one "variable region" antigen recognition site, including heavy and light chain variable regions. Antibodies also have at least one constant region, which does not contain the antigen recognition site.

[0045] The variable region includes a "hypervariable region" whose residues are responsible for antigen binding. The hypervariable region is made up of amino acid residues of the "complementarity determining regions" or "CDRs" (i.e., typically approximately residues 24-34 (CDR L1), 50-56 (CDR L2), and 89-97 (CDR L3) of the light chain variable region, and approximately residues 27-35 (CDR H1), 50-65 (CDR H2), and 95-102 (CDR H3) of the heavy chain variable region; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)), and / or residues of the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region; Chothia and Lesk; 1987, J. Mol. Biol. 196:901-917). "Framework Region" or "FR" residues are those variable region residues other than the hypervariable region residues as defined herein. Other well-known amino acid numbering conventions can be used to define the CDR and FR regions, such as the AHo scheme and the Chothia scheme.

[0046] The term antibody includes all types of antibodies derived from any species, such as monoclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (see, e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000; Cur. Plum, Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth, 231:25; International Publication Nos. WO 94 / 04678 and WO 94 / 25591; U.S. Pat. No. 0,305,079), single chain Fvs (scFvs) (see, e.g., Pluckthun in The Pharmacology of Monoclonal. Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, 1999; Schneider et al., 2002 ... Antibodies include, for example, antibodies that are capable of binding to a specific target molecule, such as ...

[0047] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that contain the antibody's complementarity determining regions ("CDRs") and, optionally, framework residues that comprise the antibody's "variable region" antigen recognition site, and that exhibit the ability to immunospecifically bind to an antigen. Such fragments include Fab', F(ab')2, Fv, single chain (ScFv), single variable region (dAb), etc., and mutants, naturally occurring variants thereof, as well as fusion proteins comprising the antibody's "variable region" antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or a receptor ligand, etc.).

[0048] As used herein, the term "fragment" refers to a peptide or polypeptide that comprises an amino acid sequence of at least 5 contiguous amino acid residues, e.g., at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0049] As used herein, the term "modulate" refers to the ability to change an effect, outcome, or activity (e.g., signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuating but not eliminating) or can completely eliminate such activity (e.g., neutralizing). Modulation can include internalization of the receptor after antibody binding, or reducing expression of the receptor on the target cell. Agonistic modulation can enhance or otherwise increase or enhance an activity (e.g., signal transduction). In yet further embodiments, such modulation can change the nature of the interaction between a ligand and its cognate receptor, so as to change the nature of the signal transduction induced. For example, a molecule, by binding to a ligand or receptor, can change the ability of such molecule to bind to other ligands or receptors, thereby changing their overall activity. Desirably, such modulation results in at least a 10% change in a measurable immune system activity, more desirably, at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, and even more desirably, at least a 100-fold change in such activity.

[0050] The term "substantially" used in the context of binding or exerting an effect is intended to indicate that the effect observed is physiologically or therapeutically relevant. Thus, for example, a molecule can substantially block the activity of a ligand or receptor if the degree of blockade is physiologically or therapeutically relevant (e.g., the degree is 60% or more complete, 70% or more complete, 75% or more complete, 80% or more complete, 85% or more complete, 90% or more complete, 95% or more complete, or 97% or more complete). Similarly, a molecule has substantially the same immunospecificity and / or properties as another molecule if such immunospecificity and properties are 60% or more identical, 70% or more identical, 75% or more identical, 80% or more identical, 85% or more identical, 90% or more identical, 95% or more identical, 97% or more identical, 98% or more identical, or 99% or more identical.

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

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

[0053] As used herein, the term "humanized antibody" refers to an immunoglobulin that includes a human framework region and one or more CDRs derived from a non-human (e.g., mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor." A constant region need not be present, but if present, it should be substantially identical to the constant region of a human immunoglobulin, 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 a natural human immunoglobulin sequence, except possibly for the CDRs. A humanized antibody is an antibody that includes a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody is not considered to encompass a typical chimeric antibody, for example, because the entire variable region of a chimeric antibody is non-human.

[0054] As used herein, "therapeutically effective amount" refers to an amount of a therapeutic agent that is sufficient to mediate clinically relevant elimination, reduction, or improvement of such symptoms under the conditions of administration. An effect is clinically relevant if its magnitude is sufficient to affect the health or prognosis of the recipient subject. A therapeutically effective amount may refer to an amount of a therapeutic agent that is sufficient to delay or minimize the onset of a disease, for example, to delay or minimize the spread of cancer. A therapeutically effective amount may also refer to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

[0055] As used herein, "immune cell" refers to any cell of hematopoietic origin, including, but not limited to, T cells, B cells, monocytes, dendritic cells, macrophages, and the like.

[0056] The terms "subject" and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans, pets (e.g., cats, dogs), farm and sport animals (e.g., cows, sheep, horses, camels), rodents such as mice and rats, and other laboratory animals, among others.

[0057] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited so that the sequences of the reference polypeptide and variant are similar overall and identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). The substituted or inserted amino acid residues may or may not be those encoded by the genetic code. A variant of a polypeptide may be a naturally occurring, such as an allelic variant, or a variant that is known not to occur naturally.

[0058] Modifications and changes can be made to the structure of the polypeptides of the present disclosure and still obtain molecules with similar properties to the polypeptides (e.g., conservative amino acid substitutions). For example, certain amino acids can be substituted for other amino acids in the sequence without significant loss of activity. Because it is the interacting ability and properties of a polypeptide that define its biological functional activity, certain amino acid sequence substitutions can be made to the polypeptide sequence to obtain polypeptides with similar properties.

[0059] When making such changes, the hydrophilicity index of an amino acid can be taken into consideration. The importance of the hydrophilicity amino acid index in conferring interactive biological function to a polypeptide is generally understood in the art. It is known that substitution of an amino acid with another amino acid having a similar hydrophilicity index or score can result in a polypeptide having similar biological activity. Each amino acid is assigned a hydrophilicity index based on its hydrophobicity and charge characteristics. These indicators 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); arginine (-4.5).

[0060] Without being bound by theory, the relative hydrophilicity index properties of amino acids determine the secondary structure of the resulting polypeptide, which in turn determines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. In general, it is known in the art that the replacement of an amino acid with another amino acid having a similar hydrophilicity index will result in a functionally equivalent polypeptide. In such alterations, the replacement of amino acids having hydrophilicity indices within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0061] Substitutions of similar amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functional equivalent polypeptide or peptide produced thereby is intended for use in immunological embodiments. The following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+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 (-4.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that substitution of an amino acid with another amino acid having a similar hydrophilicity value will still result in a biologically equivalent, and in particular an immunologically equivalent, polypeptide. Such alterations are preferably made with amino acids having hydrophilicity values ​​within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0062] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Representative substitutions taking into account the various aforementioned properties are well known to those of skill in the art and include the following (original residue→representative 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), (Trp→Tyr); (Tyr→Trp, Phe), and (Val→Ile, Leu). Thus, embodiments of the present disclosure contemplate functional or biological equivalents of such polypeptides. In particular, embodiments of the polypeptides may include variants having about 75% or more, e.g., 80%, 90%, or 95% or more sequence identity to the polypeptide of interest.

[0063] The term "percentage (%) of sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to those in a reference nucleic acid sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity. Alignment for determining the percentage of sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, SIM, or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment can be determined by known methods, including any algorithm designed to achieve maximum alignment over the entire length of the sequences to be compared.

[0064] For purposes herein, the percentage of sequence identity of a given nucleotide or amino acid sequence C to, with, or against a given nucleic acid sequence D (which can also be rephrased as a given sequence C having or containing a certain percentage of sequence identity to, with, or against a given sequence D) is calculated as follows: 100 * W / Z, where W is the number of nucleotides or amino acids that a sequence alignment program scores as a perfect match in its alignment of C and D, and Z is the total number of nucleotides or amino acids in D. It will be understood that if the length of sequence C is not equal to the length of sequence D, then the % sequence identity of C to D will not be equal to the % sequence identity of D to C.

[0065] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, and emulsions such as oil / water or water / oil emulsions, and various types of wetting agents.

[0066] composition Antibodies and antigen-binding fragments thereof that immunospecifically bind to PD-1 are provided. Contrary to the prevailing paradigm at the time of writing that anti-PD-1 antibodies act as "checkpoint inhibitors" that reduce the suppression of PD-1 activity by PD-L1 (Riley, J., Immunol Rev. 229(1):114-125 (2009)), the disclosed antibodies and antigen-binding fragments thereof immunospecifically bind to an epitope of PD-1 that is distant from the PD-L1 interaction site, are PD-1 agonists, and deliver activation signals to immune cells. The anti-PD-1 agonist antibodies and antigen-binding fragments disclosed herein not only inhibit T cell suppression, but also activate T cells. As shown and exemplified herein, the PD-1 agonists of the present disclosure can activate T cells, inhibit T cell exhaustion, and promote T cell memory in addition to inhibiting T cell suppression. In contrast, anti-PD-1 antibodies that block PD-L1 binding (e.g., non-agonistic PD-1 antibodies) act as checkpoint inhibitors. A recognized drawback of checkpoint inhibitors is that they can lead to T cell exhaustion and reduce T cell memory.

[0067] Programmed death receptor protein 1 (PD-1) The disclosed antibodies and antigen-binding fragments thereof immunospecifically bind to PD-1. For example, the antibodies and antigen-binding fragments thereof can bind to PD-1 having the amino acid sequence shown below.

[0068] Human PD-1 (UniProtKB Q15116) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 396)

[0069] Mouse PD-1 (UniProtKB Q02242) MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMVIGIMSALVGIPVLLLLAWALAVFCSTSMSEARGAGSKDDTLKEEPSAAPVPSVAYEELDFQGREKTPELPTACVHTEYATIVFTEGLGASAMGRRGSADGLQGPRPPRHEDGHCSWPL (SEQ ID NO: 397)

[0070] Preferably, the antibody or antigen-binding fragment thereof binds to PD-1 and is an agonist of PD-1. In certain embodiments, the antibodies and antigen-binding fragments of the present disclosure bind to an epitope of PD-1 that is not part of the structure of PD-1 that interacts with PD-L1. In certain embodiments, the antibodies and antigen-binding fragments immunospecifically bind to a PD-1 epitope comprising amino acids 96-110 of hPD-1 having the amino acid sequence TYLCGAISLAPKAQI (SEQ ID NO:398). This epitope is described in detail in US 11,021,540, which is incorporated herein by reference in its entirety. Without wishing to be bound by theory, it is believed that immunospecific binding of the antibodies or antigen-binding fragments of the present disclosure to a PD-1 epitope comprising amino acids 96-110 of hPD-1 activates signaling through PD-1.

[0071] antibody composition The disclosed anti-PD-1 antibodies or antigen-binding fragments thereof include any type of whole immunoglobulin (i.e., an antibody comprising two heavy chains and two light chains), fragments thereof, and proteins comprising at least the antigen-binding variable region of an antibody. In some embodiments, the disclosed antibodies comprise both an antibody light chain variable region and an antibody heavy chain variable region. In other embodiments, such molecules can further comprise one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The antibody can be selected from any type of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the constant region is a complement fixing constant region where it is desired that the antibody exhibit cytotoxic activity, typically of the IgG1 class. In other embodiments, where such cytotoxic activity is not desired, the constant region can be of the IgG2 or IgG4 class. The antibody can comprise a sequence of multiple classes or isotypes, and it is within the ordinary skill in the art to select a particular constant region to optimize desired effector functions.

[0072] Native heavy and light chain variable regions each contain four FW regions, which are largely in a β-sheet structure with three CDRs connecting them to form loops, which in some cases form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the FW regions and contribute, together with the CDRs of the other chain, to the formation of the antigen-binding site of antibodies.

[0073] Some embodiments provide antigen-binding fragments of anti-PD-1 antibodies, which may contain insertions, deletions, substitutions, or other selected modifications of particular regions or particular amino acid residues, whether or not linked to other sequences, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment (e.g., antigen-binding fragment).

[0074] Another embodiment provides a single chain antibody specific for PD-1. Methods for producing single chain antibodies are well known to those skilled in the art. Single chain antibodies can be produced by fusing the variable regions of heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single chain antibody variable fragments (scFvs) have been developed in which the C-terminus of one variable region is linked to the N-terminus of the other variable region via a peptide or linker of 15 to 25 amino acids without significantly destroying antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to be bound in the appropriate conformational orientation.

[0075] Another embodiment provides a bivalent single chain variable fragment (di-scFv) that can be designed by linking two scFvs. This can be done by creating a single peptide chain with two VH and two VL regions, resulting in a tandem scFv. scFvs can also be designed with a linker peptide that is too short (about 5 amino acids) for the two variable regions to fold together, which forces 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 scFvs, meaning that they have much higher affinity for the target. Even shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. These show even higher affinities than diabodies.

[0076] Another embodiment provides a monoclonal antibody specific for PD-1 that induces an activation signal in immune cells. Monoclonal antibodies can be obtained from a substantially homogeneous antibody population, i.e., the individual antibodies within the population are identical, except for naturally occurring mutations that may exist in a small subset of antibody molecules. Monoclonal antibodies include "chimeric" antibodies, and fragments of such antibodies, so long as they exhibit the desired activity. A chimeric antibody is one in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences of antibodies from a particular biological species or belonging to a particular antibody species or derivative, and the remaining portion of the chain(s) is identical or homologous to the corresponding sequences of antibodies from other biological species or belonging to other antibody classes or derivatives.

[0077] Chimeric and humanized antibodies Another embodiment provides chimeric anti-PD-1 antibodies and antigen-binding fragments thereof comprising one or more of the disclosed sequences, as well as functional variants thereof that bind to PD-1 and preferably transmit an activation signal to immune cells that express PD-1.

[0078] Methods for making chimeric antibodies are known in the art (see, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397). Chimeric antibodies comprising one or more CDRs from a non-human species and a framework region from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, such as CDR-grafting (EP 239,400; International Publication No. WO 91109967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), thinning or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969), and chain shuffling (U.S. Pat. No. 5,565,332).

[0079] The disclosed anti-PD-1 antibodies or antigen-binding fragments thereof can be human or humanized antibodies, or antigen-binding fragments thereof. Many non-human antibodies (e.g., antibodies derived from mice, rats, or rabbits) have natural antigenicity in humans and may cause undesirable immune responses when administered to humans. Therefore, using human or humanized antibodies in the present methods reduces the possibility that antibodies administered to humans will cause undesirable immune responses.

[0080] In the absence of endogenous immunoglobulin production, transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies upon immunization can be employed. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric or germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies upon antigen challenge. Such antibodies can also be produced using appropriate expression or display libraries, such as phage display.

[0081] Optionally, antibodies are made in other species and "humanized" for administration to humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies comprise a human immunoglobulin (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient antibody are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, rabbit, etc., having the desired specificity, affinity, capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are present neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FW regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0082] Methods for humanizing non-human antibodies are well known in the art and are described, for example, in European Patent Nos. EP 239,400, EP 592,106, and EP 519,596; International Publication Nos. WO 91 / 09967 and WO 93 / 17105; U.S. Patent Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991, Molecular immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-8.1:4; Roguska et al., 1994, PNAS 91:969-973; Tan et al. al.,2002,J.Immunol.169:1119-1125;Caldas et al.,2000,Protein Eng.13:353-360;Motet et al.,2000,Methods 20:267-79;Baca et al.,1997,J.Mot Chem.272:1067840684;Roguska et al.,1996,Protein Eng.9:895-904;Couto et al.,1995,Cancer Res.55(23 Supp):5973s-5977s;Couto Oat,1995,Cancer.Res.55:1717-22;Sandhu,1994,Gene 150:409-10;Pedersen et See, e.g., 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.

[0083] Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically introduced from an "import" variable region. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can essentially be performed by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. Thus, humanized forms of non-human antibodies (or fragments thereof) are chimeric antibodies or fragments in which substantially less of the human variable regions have been replaced with the corresponding sequences from a non-human species. In practice, humanized antibodies are typically human antibodies in which some of the CDR residues, and possibly some of the FW residues, have been replaced with residues from analogous sites in rodent antibodies.

[0084] The choice of human variable regions (both light and heavy chains) used to make a humanized antibody can be very important to reduce antigenicity. According to the "best fit" method, the sequence of the variable region of a rodent antibody is screened against the entire library of known human variable region sequences. The human sequence that is closest to the rodent sequence is then taken as the human framework (FW) of the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular derivative group of light or heavy chains. The same framework may be used for several different humanized antibodies.

[0085] It is further important that antibodies be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that are capable of graphically displaying probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FW residues can be selected and combined from the consensus and import sequences such that desired antibody properties, such as increased affinity for the target antigen(s), are achieved.

[0086] The present disclosure also relates to chimeric antigen receptors (CARs) comprising the light chain variable region and the heavy chain variable region of any of the antibodies or antigen-binding fragments disclosed herein. CARs are well-known transmembrane receptors that, when expressed in T cells, result in activation of the T cells by a desired antigen in an MHC-independent manner. Typically, CARs comprise an scFv with a desired binding specificity fused to a hinge region (e.g., IgG hinges of IgG1, IgG2 or IgG4 with or without and CH regions, CD28 hinge, CD8α hinge), a transmembrane region (e.g., CD28 transmembrane region and cytoplasmic tail), a CD3 zeta cytoplasmic region, and optionally one or more costimulatory regions (e.g., 4-1BB, OX40, CD28). In an embodiment, the CAR comprises the light chain variable region of any of the antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10, and the heavy chain variable region of any of the antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10.

[0087] A human antibody, humanized antibody, or chimeric antibody derivative can comprise substantially all of at least one, typically two, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the framework regions correspond to those of a human immunoglobulin consensus sequence. Such antibodies can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The constant region of such antibodies can be selected with regard to the proposed function of the antibody, in particular the effector functions that may be required. In some embodiments, the constant region of such antibodies can be or comprise a human IgA, IgD, IgE, IgG or IgM region. In certain embodiments, when the humanized antibody derivative is intended for therapeutic use and antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, human IgG constant regions are used, particularly those of the IgG1 and IgG3 isotypes. In alternative embodiments, IgG2 and IgG4 isotypes are used when the antibody is intended for therapeutic purposes and antibody effector function is not required. Fc constant regions comprising one or more amino acid modifications that alter antibody effector function, as disclosed in U.S. Patent Application Publication Nos. 2005 / 0037000 and 200510004514.

[0088] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences. For example, the donor CDR or consensus framework can be mutated by substituting, inserting, or deleting at least one residue (e.g., at least two residues, at least three residues, at least four residues, at least five residues, at least six residues, at least seven residues, at least eight residues) so that the CDR or framework residue at that site does not correspond to either the consensus or the donor antibody. In some embodiments, the CDR differs from the parental sequence by substitution, insertion, or deletion of one, two, three, or four residues. In some embodiments, the framework region differs from the parental sequence by substitution, insertion, or deletion of one, two, three, four, five, six, seven, or eight residues. In some embodiments, such mutations are not extensive. Usually, at least 75% of the humanized antibody residues correspond to those of the parental framework region (FW) and CDR sequences. For example, at least 80%, at least 90%, or at least 95%. In many cases, framework residues in framework regions are replaced with the corresponding residues of CDR-donor antibodies, for example to improve antigen binding. These framework replacements are identified by methods well known in the art, such as modeling the interaction between CDR and framework residues to identify framework residues important for antigen binding, and sequence comparison to identify unusual framework residues at specific positions. (See, for example, Queen et al., U.S. Patent No. 5,585,089; U.S. Publication 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).

[0089] In embodiments, the antibody or antigen-binding fragment is a human antibody or fragment, i.e., contains only sequences of human origin. Such human antibodies or antigen-binding fragments may be particularly desirable for therapeutic treatment of human subjects. Human antibodies can be produced by a variety of methods known in the art, such as phage display methods using antibody libraries derived from human immunoglobulin sequences (see U.S. Pat. Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO98 / 46645, WO94 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741). Such human antibodies can be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but that can express human immunoglobulin genes.

[0090] For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional by homologous recombination, either separately or simultaneously with the introduction of human immunoglobulin loci. In particular, J HHomozygous deletion of the region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are bred to generate homozygous offspring that express human antibodies. The transgenic mice can be immunized using conventional methods with a selected antigen, such as all or a portion of a polypeptide. Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For a review of this human antibody production technology, see Lonberg and Huszar 1995, Int. Rev. Immunol. 13:65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96433735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated by reference in their entireties.

[0091] DNA sequences encoding human receptor framework sequences include, but are not limited to, human germline VH regions VH1-18 and JH6, and FW regions of human germline VL regions VK-A26 and JK4. In certain embodiments, one or more CDRs can be inserted within the framework regions using routine recombinant DNA techniques. The framework regions can be naturally occurring framework regions or consensus framework regions and human framework regions. For a list of human framework regions, see, e.g., Chothia et al., 1998, "Structural Determinants In The Sequences Of Immunoglobulin Variable Domain," J. Mol. Biol. 278:457-479.

[0092] Fusion proteins In embodiments, the antibody or antigen-binding fragment may be a component of a fusion protein or fusion polypeptide, in which the antibody or antigen-binding fragment can bind to another polypeptide, such as a chimeric antigen receptor (CAR). The fusion polypeptide has a first fusion partner that includes all or a portion of one or more of the disclosed antibodies or antigen-binding fragments, which is covalently linked to a second polypeptide, either directly or via a linker peptide sequence that is covalently linked to the second polypeptide. The fusion protein may or may not include a region that functions to dimerize or multimerize two or more fusion proteins. The peptide / polypeptide linker region may be a separate region or may be included in one of the other regions of the fusion protein (the antibody / antigen-binding fragment or the second polypeptide). Similarly, the region that functions to dimerize or multimerize the fusion protein, if present, may be a separate region or may be included in one of the other regions of the fusion protein (the antibody / antigen-binding fragment, the second polypeptide, or the linker region). In embodiments, the dimerization / multimerization region and the peptide / polypeptide linker region are the same.

[0093] The fusion protein disclosed herein can be, for example, of Formula I: N-R1-R2-R3-C (I) where N represents the N-terminus of the fusion protein, C represents the C-terminus of the fusion protein, R1 can be all or a portion of one of the disclosed antibodies, antigen-binding fragments, or functional variants or fragments thereof, R2 is an optional peptide / polypeptide linker region, and R3 can be a second polypeptide. Alternatively, R3 can be all or a portion of one of the disclosed antibodies, antigen-binding fragments, or functional variants or fragments thereof, and R1 can be a second polypeptide.

[0094] Dimerization or multimerization can occur between two or more fusion proteins via a dimerization or multimerization region. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The dimer or multimer formed can be a homodimer / homomultimer or a heterodimer / heteromultimer.

[0095] Antibody sequence 4G9 and 5C2 sequences One embodiment of the present disclosure relates to affinity matured antibodies, or antigen-binding fragments thereof, derived from the humanized antibody isolated from hybridoma 4G9. In particular, the present embodiment provides antibodies, and antigen-binding fragments thereof, that contain sequences found in or related to sequences from 20 antibodies designated 4G9-1 through 4G9-20. The sequences of the heavy chain variable region (VH), CDR H1, CDR H2, and CDR H3 of each of these antibodies are shown in Table 1.

[0096] Antibodies 4G9-1, 4G9-2, 4G9-3, 4G9-4, 4G9-5, 4G9-6, 4G9-7, 4G9-8, 4G9-9 and 4G9-10 contain the same heavy and light chain CDRs as antibodies 4G9-11, 4G9-12, 4G9-13, 4G9-14, 4G9-15, 4G9-16, 4G9-17, 4G9-18, 4G9-19 and 4G9-20, respectively. Furthermore, the light chain variable regions of 4G9-1, 4G9-2, 4G9-3, 4G9-4, 4G9-5, 4G9-6, 4G9-7, 4G9-8, 4G9-9 and 4G9-10 are the same as the light chain variable regions of 4G9-11, 4G9-12, 4G9-13, 4G9-14, 4G9-15, 4G9-16, 4G9-17, 4G9-18, 4G9-19 and 4G9-20, respectively. However, the heavy chain variable regions of 4G9-1, 4G9-2, 4G9-3, 4G9-4, 4G9-5, 4G9-6, 4G9-7, 4G9-8, 4G9-9 and 4G9-10 differ from the heavy chain variable regions of 4G9-11, 4G9-12, 4G9-13, 4G9-14, 4G9-15, 4G9-16, 4G9-17, 4G9-18, 4G9-19 and 4G9-20, respectively, in that 4G9-11, 4G9-12, 4G9-13, 4G9-14, 4G9-15, 4G9-16, 4G9-17, 4G9-18, 4G9-19 and 4G9-20 each contain an N73D substitution. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0097] ARX 1 X 2 X 3 X 4 X 5 X 6 X7 In X 1 is selected from the group consisting of G and V; X 2 is selected from the group consisting of G, S, L, V, I, and E; X 3 is selected from the group consisting of R, G, P, W, S, H and K; X 4 is selected from the group consisting of G, E, S, V, W, and K; X 5 is selected from the group consisting of I, F, S, G, D, and V; X 6 is selected from the group consisting of A, G, Y, and S; and X 7 is selected from the group consisting of Y, H, and D.

[0098] The sequences of the light chain variable region (VK), CDR L1, CDR L2, and CDR L3 of each antibody are shown in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0099] QX 8 X 9 X 10 X 11 X 12 , X 13 X 14 S and X 15 QX 16 X 17 X 18 X 19 PX 20 X 21 In X 8 is selected from the group consisting of G, S, and D; X 9 is selected from the group consisting of I and L; X 10 is selected from the group consisting of S, V, G, and R; X 11 is selected from the group consisting of N, Y, T, S, and R; X12 is selected from the group consisting of S, Y, D, and W; X 13 is selected from the group consisting of A, K, Y, and D; X 14 is selected from the group consisting of A and V; X 15 is selected from the group consisting of Q, M, and L; X 16 is selected from the group consisting of S, G, and D; X 17 is selected from the group consisting of Y, T, N, and S; X 18 is selected from the group consisting of S, H, and D; X 19 is selected from the group consisting of T, W, and F; X 20 is selected from the group consisting of Y, P, R, and W; and X 21 is selected from the group consisting of T and F.

[0100] One embodiment of the present disclosure relates to affinity matured antibodies, or antigen-binding fragments thereof, derived from the humanized antibody isolated from hybridoma 5C2. In particular, the present embodiment provides antibodies, and antigen-binding fragments thereof, that contain sequences found in or related to sequences from 20 antibodies designated 5C2-1 to 5C2-10. The sequences of the heavy chain variable region (VH), CDR H1, CDR H2, and CDR H3 of each of these antibodies are shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3]

[0101] GX 22 TFX 23 X 24 YX 25 (SEQ ID NO: 163), IX 26 X 27 X 28 X 29 X 30 X 31 T, and AX 32 X 33X 34 X 35 X 36 X 37 X 38 X 39 FX 40 X 41 In X 22 is selected from the group consisting of Y and Q; X 23 is selected from the group consisting of N, T, and G; X 24 is selected from the group consisting of S, I, and N; X 25 is selected from the group consisting of W and L; X 26 is selected from the group consisting of H, L, R, and P; X 27 is selected from the group consisting of P and L; X 28 is selected from the group consisting of R, S, I, and N; X 29 is selected from the group consisting of G, D, N, and Y; X 30 is selected from the group consisting of I, S, G, and R; X 31 is selected from the group consisting of H, D, and Y; X 32 is selected from the group consisting of P and S; X 33 is selected from the group consisting of S, R, N, and Y; X 34 is selected from the group consisting of S, D, G, V, and F; X 35 is selected from the group consisting of S, N, G, I, D, and R; X 36 is selected from the group consisting of Y, N, C, L, S, and H; X 37 is selected from the group consisting of A, G, T, and E; X 38 is selected from the group consisting of W, C, S, R, D, and G; X 39 is selected from the group consisting of A, S, G, and D; X 40 is selected from the group consisting of A, S, and L; and X 41 is selected from the group consisting of H, Y, and S.

[0102] The sequences of the light chain variable region (VK), CDR L1, CDR L2, and CDR L3 of each antibody are shown in Table 4. [Table 4-1] [Table 4-2] [Table 4-3]

[0103] QX 42 X 43 X 44 X 45 X 46 , X 47 AS and X 48 X 49 X 50 X 51 X 52 X 53 X 54 In X 42 is selected from the group consisting of D, S, and G; X 43 is selected from the group consisting of V and I; X 44 is selected from the group consisting of S and G; X 45 is selected from the group consisting of T and S; X 46 is selected from the group consisting of A, S, D and W; X 47 is selected from the group consisting of W, G and D; X 48 is selected from the group consisting of Q and H; X 49 is selected from the group consisting of Q, K, and H; X 50 is selected from the group consisting of H, Y, F, R, D, and G; X 51 is selected from the group consisting of Y, N, Y, D, S, and G; X 52 is selected from the group consisting of R, S, V, T, and I; X 53 is selected from the group consisting of S, A, F, T, Y, and W; and X 54 is selected from the group consisting of P, L, S, and T.

[0104] In embodiments, an antibody or antigen-binding fragment thereof with binding specificity for Programmed Death Receptor Protein 1 (PD-1) comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises any CDR L1 in Table 2 or Table 4, and any CDR L2 in Table 2 or Table 4, and any CDR L3 in Table 2 or Table 4, and the heavy chain variable region comprises any CDR H1 in Table 1 or Table 3, any CDR H2 in Table 1 or Table 3, and any CDR H3 in Table 1 or Table 3.

[0105] In embodiments, an antibody or antigen-binding fragment thereof with binding specificity for Programmed Death Receptor Protein 1 (PD-1) comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR L1, CDR L2, and CDR L3 of any one of antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10, and the heavy chain variable region comprises CDR H1, CDR H2, and CDR H3 of any one of antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10.

[0106] In embodiments, the antibody or antigen-binding fragment thereof comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-1; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-2. H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-4; CDR L1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-5. CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-6;CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-7; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-8; CDR L1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-9. CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-10; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-11. H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-12;CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-13; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-14; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-15. CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3 each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-16; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3 each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-17. H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-18;CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-19; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 4G9-20; CDR L1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-1. CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-2; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-3. H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-4;CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-5; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-6; CDR L1, CDR H2, and CDR H3, each of which has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-7. CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-8; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-9. or CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identical to the amino acid sequence of the corresponding CDR of 5C2-10;

[0107] In embodiments, the antibody or antigen-binding fragment thereof is defined by CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each independently having at least 75% identity, such as at least 85% identity, or at least 90% identity, to the corresponding CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3 of any one of antibodies 4G9-1 to 4G9-20, and 5C2-1 to 5C2-10.

[0108] In embodiments, the antibody or antigen-binding fragment thereof comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-1; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-2; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-3. H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 4G9-4; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 4G9-5; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 4G9-6; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 4G9-7 L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3;CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 4G9-8; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 4G9-9; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 4G9-10; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 4G9-11 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3; each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-12; each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-13; each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-14; H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each independently having an amino acid sequence that is identical or contains one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-15;CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-16; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-17; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-18. H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-19; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 4G9-20; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 5C2-1; H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 5C2-2; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 5C2-3;CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 5C2-4; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 5C2-5; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 5C2-6; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the corresponding CDR sequence of 5C2-7. CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 5C2-8; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence identical to or containing one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 5C2-9;Alternatively, it comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has an amino acid sequence that is identical to or contains one or two amino acid substitutions, insertions, or deletions compared to the sequence of the corresponding CDR of 5C2-10. In the above embodiment, preferably, each CDR independently has the same amino acid sequence as the reference CDR, or contains one amino acid substitution, insertion, or deletion relative to the reference CDR. In the above embodiment, preferably, each of the one or two substitutions, insertions, or deletions is such that it is a conservative substitution;

[0109] In a particular embodiment, the antibody or antigen-binding fragment comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3 of 4G9-2. In another particular embodiment, the antibody or antigen-binding fragment comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3 of 4G9-12.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each independently having at most one deletion, insertion, or substitution compared to the CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3 of any one of antibodies 4G9-1 to 4G9-20, and 5C2-1 to 5C2-10.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3, each of which independently has at most two deletions, insertions, or substitutions compared to the CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3 of any one of antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10.

[0112] An embodiment provides an antibody or antigen-binding fragment thereof, wherein CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 each independently have three or fewer amino acid substitutions, for example two amino acid substitutions or one amino acid substitution, relative to the corresponding CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of any one of antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10.

[0113] In embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region that have at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-1, respectively; a light chain variable region and a heavy chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the corresponding light chain and heavy chain variable region sequences of 4G9-3, respectively; a light chain variable region and a heavy chain variable region having about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-4, respectively; a light chain variable region and a heavy chain variable region, each having about 98%, or at least about 99%, amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-5; a light chain variable region and a heavy chain variable region, each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-5;light chain and heavy chain variable regions, each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-6; a light chain variable region and a heavy chain variable region that have at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-8, respectively; a light chain variable region and a heavy chain variable region having an amino acid sequence identity of at least about 4%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% to the corresponding light chain and heavy chain variable region sequences of 4G9-9, respectively; or a light chain variable region and a heavy chain variable region that have at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-10, respectively;light chain and heavy chain variable regions, each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-11; a light chain variable region and a heavy chain variable region that have at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the corresponding light chain and heavy chain variable region sequences of 4G9-13, respectively; a light chain variable region and a heavy chain variable region having 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the corresponding light chain and heavy chain variable region sequences of 4G9-14, respectively; or at least about 99% amino acid sequence identity to the corresponding light and heavy chain variable region sequences of 4G9-15;light chain and heavy chain variable regions, each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-16; a light chain variable region and a heavy chain variable region that have at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the corresponding light chain and heavy chain variable region sequences of 4G9-18, respectively; a light chain variable region and a heavy chain variable region having 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the corresponding light chain and heavy chain variable region sequences of 4G9-19, respectively; or at least about 99% amino acid sequence identity to the corresponding light and heavy chain variable region sequences of 4G9-20;light chain and heavy chain variable regions each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 5C2-1; a light chain variable region and a heavy chain variable region that have at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 5C2-3, respectively; a light chain variable region and a heavy chain variable region having an amino acid sequence identity of at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% to the corresponding light chain and heavy chain variable region sequences of 5C2-4, respectively; or at least about 99% amino acid sequence identity to the corresponding light and heavy chain variable region sequences of 5C2-5;light chain and heavy chain variable regions each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 5C2-6; a light chain variable region and a heavy chain variable region that have at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the corresponding light chain and heavy chain variable region sequences of 5C2-8, respectively; a light chain variable region and a heavy chain variable region having at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity; a light chain variable region and a heavy chain variable region each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 5C2-9; or a light chain variable region and a heavy chain variable region each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 5C2-10; The variable regions include a light chain variable region and a heavy chain variable region having the same affinity.

[0114] For example, preferred antibodies or antigen-binding fragments include light chain and heavy chain variable regions that have at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-2, respectively; a light chain variable region and a heavy chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the corresponding light chain and heavy chain variable region sequences of 4G9-8, respectively; a light chain variable region and a heavy chain variable region having about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-9, respectively; a light chain variable region and a heavy chain variable region that have at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-12, respectively;a light chain variable region and a heavy chain variable region each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-14; a light chain variable region and a heavy chain variable region each having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the corresponding light chain and heavy chain variable region sequences of 4G9-18; a light chain variable region and a heavy chain variable region that have at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the corresponding light chain and heavy chain variable region sequences of 4G9-19; or a light chain variable region and a heavy chain variable region that have at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with the corresponding light chain and heavy chain variable region sequences of 5C2-8, respectively. Without being bound by theory, the exemplary antibodies or antigen-binding fragments of this section have desirable PD-1 agonist activity;

[0115] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the light chain variable region and the heavy chain variable region of 4G9-2. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the light chain variable region and the heavy chain variable region of 4G9-12.

[0116] In embodiments, the antibody or antigen-binding fragment has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85 a light chain variable region and a heavy chain variable region having at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions; each of which is at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, compared to the corresponding light chain and heavy chain variable region sequences of 4G9-2; light chain and heavy chain variable regions having at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions; each of which has at most 1, at most 2, at most 3, or more deletions, insertions, or substitutions compared to the corresponding light chain and heavy chain variable region sequences of 4G9-3; light chain variable regions and heavy chain variable regions with at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions;Each of the light and heavy chain variable region sequences has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85 a light chain variable region and a heavy chain variable region having at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, each of which is at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light chain and heavy chain variable region sequences of 4G9-5; at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light and heavy chain variable region sequences of 4G9-6; light chain variable regions and heavy chain variable regions with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions;Each of the light and heavy chain variable region sequences has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85 a light chain variable region and a heavy chain variable region having at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, each of which is at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light chain and heavy chain variable region sequences of 4G9-8; at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light and heavy chain variable region sequences of 4G9-9; light chain variable regions and heavy chain variable regions with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions;Each of the light and heavy chain variable region sequences has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85 a light chain variable region and a heavy chain variable region having at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, each of which is at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light chain and heavy chain variable region sequences of 4G9-11. at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light and heavy chain variable region sequences of 4G9-12; light chain variable regions and heavy chain variable regions with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions;Each of the light and heavy chain variable region sequences has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85 a light chain variable region and a heavy chain variable region having at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, each of which is at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light chain and heavy chain variable region sequences of 4G9-14. at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light and heavy chain variable region sequences of 4G9-15; light chain variable regions and heavy chain variable regions with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions;light chain and heavy chain variable regions, each having at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions compared to the corresponding light chain and heavy chain variable region sequences of 4G9-16; Each of the light and heavy chain variable region sequences has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85 a light chain variable region and a heavy chain variable region having at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, each of which is at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light chain and heavy chain variable region sequences of 4G9-18. at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light and heavy chain variable region sequences of 4G9-19; light chain variable regions and heavy chain variable regions with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions;Each of the light and heavy chain variable region sequences has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85 a light chain variable region and a heavy chain variable region having at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, each of which is at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light chain and heavy chain variable region sequences of 5C2-1; at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light and heavy chain variable region sequences of 5C2-2; light chain variable regions and heavy chain variable regions with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions;Each of the light and heavy chain variable region sequences has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85 a light chain variable region and a heavy chain variable region having at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, that are at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light chain and heavy chain variable region sequences of 5C2-4; at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light and heavy chain variable region sequences of 5C2-5; light chain variable regions and heavy chain variable regions with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions;Each of the light and heavy chain variable region sequences has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85 a light chain variable region and a heavy chain variable region having at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, that are at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light chain and heavy chain variable region sequences of 5C2-7; at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions, respectively, compared to the corresponding light and heavy chain variable region sequences of 5C2-8; light chain variable regions and heavy chain variable regions with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions;a light chain variable region and a heavy chain variable region, each of which has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions compared to the corresponding light chain and heavy chain variable region sequences of 5C2-9; or each comprising a light chain and a heavy chain variable region that have at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, or at most 24 deletions, insertions, or substitutions compared to the corresponding light chain and heavy chain variable region sequences of 5C2-10;

[0117] For example, preferred antibodies or antigen-binding fragments include light chain variable regions and heavy chain variable regions that have at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, or at most 18 deletions, insertions, or substitutions compared to the corresponding light and heavy chain variable region sequences of 4G9-2, respectively. or a light chain variable region and a heavy chain variable region that have at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, or at most 18 deletions, insertions, or substitutions compared to the corresponding light and heavy chain variable region sequences of 4G9-12.

[0118] An embodiment provides an antibody or antigen-binding fragment thereof, wherein the light chain variable region and the heavy chain variable region each independently have at least 75% identity, e.g., at least 85% identity, at least 90% identity, or at least 95% identity, to the corresponding light chain variable region and heavy chain variable region of any one of antibodies 4G9-1 to 4G9-20 and 5C2-1 to 5C2-10.

[0119] In some embodiments, an antibody or antigen-binding fragment thereof is defined by a light chain variable region and a heavy chain variable region that each independently have at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, or at most 18 deletions, insertions, or substitutions compared to the corresponding light chain and heavy chain variable region of any one of antibodies 4G9-1 to 4G9-20, and 5C2-1 to 5C2-10.

[0120] In embodiments, the antibody or antigen-binding fragment comprises the heavy and light chain amino acid sequences, or an antigen-binding portion of either sequence, of antibody 4G9-1, 4G9-2, 4G9-3, 4G9-4, 4G9-5, 4G9-6, 4G9-7, 4G9-8, 4G9-10, 5C2-1, 5C2-2, 5C2-3, 5C2-4, 5C2-5, 5C2-6, 5C2-7, 5C2-8, 5C2-9, or 5C2-10, including the sequences disclosed in Table 5 or Table 6. Tables 5 and 6 disclose the heavy and light chain sequences of 4G9-1 through 4G9-20 and 5C2-1 through 5C2-10 with or without a terminal lysine ("-K", heavy chain only), and with or without a signal peptide ("-SP"). [Table 5-1] [Table 5-2]

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

[0121] Polynucleotides encoding the 4G9 and 5C2 sequences In another aspect, the present invention relates to an isolated nucleic acid molecule (e.g., a polynucleotide) encoding an antibody or antigen-binding fragment of the anti-PD-1 agonist antibodies disclosed herein. The present disclosure provides a polynucleotide encoding an antibody or antigen-binding fragment comprising the amino acid sequence of antibodies 4G9-1 to 4G9-20 or 5C2-1 to 5C2-10 (e.g., SEQ ID NOs: 1 to 393, Tables 1 to 6). The polynucleotide can encode the entire antibody or antigen-binding fragment thereof, or a light chain, a heavy chain, a combination thereof, or a CDR thereof. In embodiments, the polynucleotide can encode the heavy and / or light chain of an antibody or antigen-binding fragment selected from those set forth in Tables 5 and 6 (e.g., SEQ ID NOs: 214 to 393). In embodiments, the polynucleotide may encode a heavy chain variable region (e.g., VH) and / or a light chain variable region (e.g., VK) selected from those presented in Tables 1-4 (e.g., SEQ ID NOs: 1-20, 83-102, 143-152, and 184-193). In embodiments, the polynucleotide may encode an antibody or antigen-binding fragment (e.g., a light chain and / or heavy chain variable region / domain) comprising a CDR sequence selected from those presented in Tables 1-4 (e.g., SEQ ID NOs: 21-82, 103-142, 153-183, 194-213). In embodiments, the polynucleotide encodes a heavy and / or light chain variable region of 4G9-12, e.g., SEQ ID NOs: 12 and 94. In such embodiments, the polynucleotide optionally further encodes a heavy chain constant region and / or a light chain constant region. In embodiments, the polynucleotide encodes the heavy and / or light chain variable regions of 4G9-2, e.g., SEQ ID NOs:2 and 84. In embodiments, the polynucleotide encodes an antibody or antigen-binding fragment that includes the CDR sequences of 4G9-12 (e.g., SEQ ID NOs:32, 53, and 74, and / or 114, KVS, and 134). In some embodiments, the polynucleotide encodes an antibody or antigen-binding fragment that includes the heavy and light chain variable regions of 4G9-12 (e.g., SEQ ID NOs:12 and 94).

[0122] The isolated polynucleotide molecule can be produced by standard techniques, including, but not limited to, general molecular cloning and chemical polynucleotide synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain isolated polynucleotides encoding mutant polypeptides. General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler ed. Cold Spring Harbor Laboratory Press, 1995. When RNA is used as a template, a complementary DNA (CDNA) strand can be synthesized using reverse transcriptase. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or amplification based on polynucleotide sequences can also be used to obtain isolated polynucleotides. See, for example, 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.

[0123] The isolated polynucleotides can be chemically synthesized (e.g., using phosphoramidite technology for automated 3' to 5' DNA synthesis) as a single polynucleotide molecule or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., more than 100 nucleotides) containing the desired sequence can be synthesized, each pair containing a short region of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pairs are annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in one double-stranded polynucleotide molecule per oligonucleotide pair, which can then be heated into the vector. The isolated polynucleotides can also be obtained by mutagenesis. Polynucleotides encoding proteins can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis by PER. See Short Protocols in Molecular Biology, Chapter 8, Ausubel et al. ed. Green Publishing Associates and John Wiley & Sons, 1992.

[0124] Those of skill in the art will recognize that due to the redundancy of the genetic code, any amino acid sequence may be encoded by multiple polynucleotides, and that, depending on the organism in which the polynucleotide is maintained, replicated, and / or transcribed, a polynucleotide sequence that encodes a particular amino acid sequence may be selected taking into account the codon bias of the organism.

[0125] The polynucleotide may be present in a vector, e.g., an expression vector. The polynucleotide may be introduced into a suitable host cell, such as a Chinese Hamster Ovary (CHO) cell or an NSO cell, for expression, and may be, for example, extrachromosomal or chromosomally inserted. Preferably, the host cell is a mammalian host cell, such as a CHO cell or an NSO cell, although bacterial, yeast, plant and other types of suitable cells may also be used.

[0126] In embodiments, the host cell may be part of a transgenic animal. Methods for producing transgenic animals that produce antibodies are known in the art. See, for example, A. Jakobovits, Curt Opin Biotechnol., 6(5):5.61-6 (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):113.4-43 (2007); Lonberg N. (2005) "Human antibodies from transgenic animals", Nat Biotechnol. 25(10):113.4-43 (2007). Biotechnol. 23(9):1117-25, and U.S. Patent Nos. 9,708,635; 9,686,970; 9,499,838; 9,445,581; 9;388,446; 8,835,712; 8,703,485; 8,232,449; 7,795,494; and 5,939,598.

[0127] Techniques for the manipulation of polynucleotides, vectors, expression vectors, host cells, and transgenic animals are well known in the art and need not be described in detail.

[0128] Pharmaceutical Compositions Pharmaceutical compositions comprising the disclosed antibodies and antigen-binding fragments thereof are provided. Pharmaceutical compositions typically include one or more pharma- ceutical acceptable excipients.

[0129] The pharmaceutical compositions disclosed herein are administered to a subject in a therapeutically effective amount. An "effective amount" or "therapeutically effective amount" is typically an amount or dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated, or otherwise provide the desired pharmacological and / or physiological effect. The exact dosage will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.

[0130] For the disclosed antibodies and antigen-binding fragments thereof, one of skill in the art would be able to ascertain an appropriate dosage, taking into account the treatment context, age, and general health of the recipient. The selected dosage will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. For the disclosed antibodies and antigen-binding fragments thereof, dosages of 0.001 to 20 mg per kg of body weight per day can generally be administered to a mammal. In the case of intravenous injection or infusion, dosages may generally be lower.

[0131] In certain embodiments, the antibodies and antigen-binding fragments thereof may be administered locally, for example, by direct injection into the site to be treated. Typically, injection results in an increased local concentration of the antibodies and / or antigen-binding fragments thereof, which may be higher than can be achieved by systemic administration. The antibodies and antigen-binding fragments thereof disclosed herein may be combined with a substrate, as described above, to help reduce passive diffusion of the polypeptide from the site to be treated, thereby resulting in an increased local concentration of the polypeptide composition.

[0132] Parenteral Formulations In some embodiments, compositions comprising the disclosed antibodies and antigen-binding fragments are administered in an aqueous solution by parenteral injection. Formulations may also be in the form of suspensions or emulsions. In general, pharmaceutical compositions comprise a therapeutically effective amount of an antibody or antigen-binding fragment thereof, and optionally, a pharma- ceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions may optionally include one or more of the following: diluents, sterile water, buffered saline of various buffer contents (e.g., Tris-HO, acetate, phosphate), pH, and ionic strength; and additives such as detergents and solubilizers (e.g., TWEEN® 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thiomersal, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.The formulation may be lyophilized and redissolved / resuspended immediately before use.The formulation may be sterilized, for example, by filtering, incorporating a sterilizing agent into the composition, irradiating the composition, or heating the composition.

[0133] Oral Formulations In some embodiments, the antibody composition is formulated for oral administration. Oral dosage forms of antibodies can resist proteolysis and deliver a larger fraction of immunoreactive antibodies locally in the digestive tract for the treatment of infections, or allow absorption of antibodies for the treatment or prevention of systemic conditions (Reilly, RM, et al. Clin Pharmacokinet., 32(4):313.M(1997); Victoria S Jasion and Bruce P Burnett, Nutr J.;14:22(2015); and Philippart, M. et al., Drug. Res(Stuttg)66(03):11.120(2016)).

[0134] Oral solid dosage forms are generally described in Chapter 89 of Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042). Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders; or granules, or particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, or incorporation of the material into liposomes. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antibody or antigen-binding fragment. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing. Co., Easton, Pa. 18042), pages 1435-1712, which are incorporated herein by reference.

[0135] The composition may be prepared in liquid form or in the form of a dry powder (e.g., lyophilized). Liposomal or proteinoid encapsulation may be used to formulate the composition. Liposomal encapsulation may be used, and liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). See also Marshall, K. Modern Pharmaceutics, Chapter 10, GS Banker and CTRhodes ed., 1979. In general, the formulation includes an antibody or antigen-binding fragment (or a chemical modification thereof) and an inactive ingredient that protects the antibody or antigen-binding fragment in the gastric environment and releases the biologically active substance in the intestine.

[0136] Antibodies and antigen-binding fragments thereof can be chemically modified so that oral administration of the derivatives is efficacious. In general, the chemical modifications contemplated are to attach at least one moiety to the moiety molecule itself, which allows for uptake into the bloodstream from the stomach or intestine, or directly into the intestinal mucosa. It is also desirable to increase the overall stability of the moiety(s) and increase their circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical applications. Other moieties which may be used include propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-trioxocane (see, e.g., Abuchowski and Davis. (1981) "Soluble Polymer-Enzyme Adducts", Enzymes as Drugs. Hocenberg and Roberts, O.S. (Wiley-Interscience: New York, NY) pp. 367-383; and Newmark, et al. (1982) J. App. Biochem. 4:185-189).

[0137] Another embodiment provides liquid dosage forms for oral administration, including pharma- ceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other ingredients, such as inert diluents; adjuvants, such as wetting agents, emulsifying and suspending agents; and sweetening, flavoring, and perfuming agents.

[0138] For oral formulations, the location of release may be the stomach, the small intestine (duodenum, jejunum, ileum), or the large intestine. In some embodiments, the release avoids the deleterious effects of the stomach environment, either by protecting the drug (or derivative) or by releasing the drug (or derivative) past the stomach environment, e.g., in the intestine. To ensure full gastric resistance, a coating that is impermeable to at least pH 5.0 is desirable. Examples of the more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP-55, polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™ cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, Shellac™. These coatings may be used as mixed films.

[0139] Controlled Delivery Polymeric Matrices The antibodies and antigen-binding fragments thereof disclosed herein can also be administered in controlled release formulations. The antibodies and antigen-binding fragments thereof can be incorporated into an inert matrix, such as a viscous material, that allows release by either diffusion or leaching mechanisms. Slowly degenerating matrices may also be incorporated into the formulation. Another form of controlled release is based on the Oros therapeutic system (Alza Corp.), in which the antibody or antigen-binding fragment thereof is encapsulated in a semipermeable membrane that allows water to enter and drug to be pushed out by osmotic effects through a single small opening.

[0140] Controlled release polymeric devices can be made to provide systemic, prolonged release after implantation or injection (microparticles) of the polymeric device (rods, cylinders, film disks). The matrix can be in the form of particulates, such as microspheres in which the drug is dispersed within a solid polymeric matrix, or microcapsules in which the core is a different material from the polymeric shell and the peptide is dispersed or suspended within the core, which may be liquid or solid in nature. Unless otherwise defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as thin slabs or films, from nanometers to centimeters, powders produced by milling or other standard techniques, or gels, such as hydrogels.

[0141] Either non-biodegradable or biodegradable matrices can be used to deliver the agonist anti-PD-1 antibodies, antigen-binding fragments, or nucleic acids encoding the agonist anti-PD-1 antibodies or antigen-binding fragments, although in some embodiments, biodegradable matrices are desired. These can be natural or synthetic polymers, although in some embodiments, synthetic polymers are desired for better characterization of the degradation and release characteristics. The polymer is selected based on the period over which release is desired. In some cases, a linear release may be most useful, while in other cases, a pulsed or "bulk release" may provide more effective results. The polymer may be a hydrogel (typically absorbing up to about 90% water by weight) and may optionally be crosslinked with multivalent ions or polymers.

[0142] The matrices can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed to manufacture microspheres for drug delivery, such as those described by Mathiowitz and Langer, J. Controlled Release, 5:13.22 (1987); Mathiowitz, et al., Reclaim Polymers, 6:275-283 (1987); and Mathiowitz et al., J. Appl. Polymer Sci., 35:755-774 (1988).

[0143] The devices can be formulated for localized release to treat the site of infusion or injection (usually at a much lower dose than would be required to treat the entire body), or for systemic delivery: they can be injected subcutaneously into muscle, fat, or swallowed.

[0144] Manufacturing method Methods for producing antibodies The disclosed antibodies can be produced in cell culture, phage, or in a variety of animals, including, but not limited to, cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. In embodiments, the various animals can be transgenic animals that have been genetically engineered to produce human or humanized antibodies. In embodiments, the antibodies are mammalian antibodies. Phage technology can be used to isolate the original antibody or to generate variants with altered specificity or affinity properties. Such techniques are common and well known in the art. In embodiments, the antibodies are produced by recombinant means well known in the art. For example, recombinant antibodies can be produced by transfecting a host cell with a vector that contains a DNA sequence encoding the antibody. One or more vectors can be used to transfect the host cell with DNA sequences expressing at least one VL region and one VH region. Exemplary descriptions of recombinant means of antibody generation and production include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principle And Practice (Academic Press, 1993); Current Protocols In Immunology (John Wiley & Sons, latest edition).

[0145] The disclosed antibodies and antigen-binding fragments can be modified by recombinant means to enhance the antibody's ability to mediate a desired function. In other words, the antibodies and antigen-binding fragments can be modified by substitution using recombinant means. Typically, the substitution is conservative. For example, at least one amino acid in the constant region of the antibody or antigen-binding fragment can be replaced with a different residue. See, for example, U.S. Pat. No. 5,624,821, U.S. Pat. No. 6,194,551, Application No. WO9958572; and Angal et al., Mol Immunol. 30:105-08 (1993). Amino acid modifications include amino acid deletions, additions, and substitutions. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. In many cases, the antibodies are labeled by covalent or non-covalent attachment of a substance that provides a detectable signal. A wide variety of labels and conjugation techniques are known and are widely reported in the scientific and patent literature. These antibodies can be screened for binding to proteins or polypeptides, see, e.g., Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

[0146] For example, suitable antibodies with desired biological activity can be identified using in vitro assays including, but not limited to, proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, trafficking, signal transduction, and in vivo assays such as tumor growth inhibition. The antibodies provided herein are also useful for diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for their ability to bind to a particular antigen without inhibiting the receptor binding activity or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays.

[0147] In another embodiment, monoclonal antibodies are provided, which are obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies, which are identical within the population, except for naturally occurring mutations that may exist in a small subset of antibody molecules. Monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody from a particular biological species or belonging to a particular antibody class or derivative, while the remaining portion of the chain(s) is identical or homologous to the corresponding sequence of an antibody from another biological species or belonging to another antibody class or derivative, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity.

[0148] Monoclonal antibodies can be made using any method that produces monoclonal antibodies. In the hybridoma method, a mouse or other suitable host animal is immunized, usually with an immunizing agent, to induce lymphocytes that produce, or have the capacity to produce, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0149] The disclosed antibodies may also be produced by recombinant DNA methods. DNA encoding the disclosed antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the minimal antibody heavy and light chains). Libraries of antibodies or active antibody fragments (e.g., antigen-binding fragments) can also be produced and screened using phage display technology.

[0150] Methods for producing antibodies using protein chemistry are also known in the art. One method for producing a protein constituting an antibody is to link two or more peptides or polypeptides by protein chemistry techniques. For example, a peptide or polypeptide can be chemically synthesized using currently available laboratory equipment using Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry. (Applied Biosystems, Inc. Foster City, CA). Those skilled in the art can readily appreciate that, for example, a peptide or polypeptide corresponding to an antibody can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthetic resin, but another fragment of an antibody can be synthesized and then cleaved from the resin, thereby exposing a functionally blocked terminal group on the other fragment. By a peptide condensation reaction, these two fragments can be covalently linked via a peptide bond at the carboxyl and amino termini, respectively, to form an antibody or fragment thereof. Alternatively, the peptides or polypeptides are independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides can be linked by similar peptide condensation reactions to form antibodies or antigen-binding fragments thereof.

[0151] For example, enzymatic ligation of cloned or synthetic peptide regions can join relatively short peptide fragments to create larger peptide fragments, polypeptides, or entire protein regions. Alternatively, native chemical ligation of synthetic peptides can be used to synthesize large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide-α-thioester with another unprotected peptide region containing an amino-terminal Cys residue to obtain a thioester-linked intermediate as the first 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.

[0152] How to use The disclosed antibodies and antigen-binding fragments thereof can be used to regulate immune responses in a subject in need thereof. One embodiment provides a method of activating immune cells, e.g., T cells, that express PD-1 by administering the disclosed antibodies and antigen-binding fragments thereof, optionally with a second therapeutic agent, to expand or enhance the biological activity of immune cells that express PD-1.

[0153] Stimulating the immune response Treatment strategies Methods of inducing or enhancing an immune response in a subject are provided. Typically, the methods include administering to a subject a therapeutically effective amount of one or more of the disclosed antibodies and antigen-binding fragments thereof to immunospecifically bind to PD-1 and induce, enhance or enhance a stimulatory or activating signal through PD-1 to activate immune cells. The immune response can be, for example, induction, enhancement or enhancement of T cell activation, secretion of cytokines by immune cells, proliferation of T cells. The disclosed antibodies or antigen-binding fragments thereof can be administered to a subject in need thereof in a therapeutically effective amount to overcome T cell exhaustion and / or T cell energetics. Overcoming I cell exhaustion or T cell anergy can be determined by measuring T cell function using known techniques.

[0154] The method can be used in vivo or ex vivo to induce, promote or enhance a stimulatory immune response.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof is administered directly to the subject. It is also possible to administer a nucleic acid encoding the antibody or antigen-binding fragment thereof to the subject. Preferably, the nucleic acid, once administered, can be expressed to produce an antibody or antigen-binding fragment that binds to PD-1. In some embodiments, the antibody or antigen-binding fragment thereof is contacted with cells (e.g., immune cells) ex vivo and the therapeutic cells are administered to the subject (e.g., adoptive transfer). The antibody or antigen-binding fragment thereof can enable a more robust immune response. The disclosed compositions are useful for stimulating or enhancing T cell-mediated immune responses that trigger an activation signal via PD-1 on immune cells.

[0156] Target patients Cancer Treatment The disclosed antibodies and compositions and methods thereof can be used to treat cancer. In general, the agents are used to stimulate or enhance an immune response against cancer in a subject by administering to the subject an amount of the disclosed antibodies or antigen-binding fragments thereof that induce, promote, or enhance an activation signal via PD-1. The methods can alleviate one or more symptoms of cancer. The methods can be used to reduce or inhibit tumor growth or cause tumor regression. For example, as disclosed and exemplified herein, the antibodies or antigen-binding fragments of the present disclosure can also have anti-tumor activity, induce central memory T cells (Tcm), and / or inhibit T cell exhaustion.

[0157] Immune cells activated by the disclosed antibodies or fragments thereof can kill cancer cells and reduce the tumor burden in a subject. The tumor can be benign or malignant, and if malignant, can be at any stage of disease progression. In yet other embodiments, the cancer is associated with a particular cancer antigen (e.g., pan-carcinoma antigen (KS1 / 4), ovarian carcinoma antigen (CA125), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2 / neu, etc.).

[0158] The methods and antibody compositions disclosed herein are useful for the treatment or prevention of a variety of cancers or other abnormal proliferative disorders, including, but not limited to, carcinomas, including those of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, and skin; squamous cell carcinoma; lymphoid hematopoietic malignancies, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkett's lymphoma; acute and chronic myeloma. hematopoietic tumors of myeloid lineage, including myeloid leukemia, myeloid leukemia, and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma.

[0159] Cancers caused by abnormalities in apoptosis can also be treated by the disclosed methods and compositions. Such cancers include, but are not limited to, follicular lymphoma, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In certain embodiments, malignant tumors or abnormal proliferation changes (such as transformation and dysplasia) or hyperproliferative disorders in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus are treated or prevented by the methods and compositions. In other specific embodiments, sarcoma, melanoma, or leukemia are treated or prevented by the methods and compositions.

[0160] Specific cancers and related disorders that may be treated or prevented by the methods and compositions disclosed herein include leukemias, including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroid leukemia, and myelodysplastic syndromes, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and the like; polycythemia vera; Hodgkin's or non-Hodgkin's lymphomas (e.g., diffuse anaplastic lymphoma kinase (ALK) negative, large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma (DLBCL), large B-cell lymphoma (DLBCL), and the like; Lymphomas, including, but not limited to, lymphoma kinase (ALX) positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase (ALK) positive, ALK+ anaplastic large cell lymphoma (ALCL), acute myeloid lymphoma (AML); multiple myeloma, including, but not limited to, smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of unknown severity; benign monoclonal gammopathy; heavy chain disease; bone sarcoma (bone sarcoma), osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma, and the like; glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-neural sarcoma, and the like; brain tumors, including but not limited to glioma, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pinocytoma, pinoblastoma, primary cerebral lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal gland cancer, including but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer, including but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer;pancreatic cancer, including but not limited to islet cell adenomas, gastrinomas, glucagonomas, VIPomas, somatostatinomas, and carcinoid or islet cell tumors; pituitary cancer, including but not limited to Cushing's disease, prolactinomas, acromegaly, and diabetes insipidus; ocular cancer, including but not limited to intraocular melanomas such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma; squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and vulvar cancer, including but not limited to Paget's disease; cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancer, including but not limited to ovarian epithelial carcinoma, borderline malignant tumors, germ cell tumors, and stromal tumors; esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; adenocarcinoma, fungus (polypoid) ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, gastric cancer, including but not limited to liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, including but not limited to adenocarcinoma; cholangiocarcinoma, including but not limited to papillary, nodular, and diffuse; lung cancer, including but not limited to non-small cell lung cancer, squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung carcinoma; germinal tumors, seminoma, anaplastic, classical (typical), spermatocytic, non-seminomatous epithelioma, embryonal carcinoma, teratocarcinoma, choriocarcinoma (yolk sac tumor). prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer; oral cancer, including but not limited to squamous cell carcinoma; basal cell carcinoma; salivary gland cancer, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentigo melanoma; kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or uterus); Wilms' tumor;and bladder cancer, including, but not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. Additionally, cancers include myosarcoma, osteosarcoma, endothelial sarcoma, lymphangioendothelial sarcoma, mesothelioma, synovium, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma (for reviews of such diseases, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia, and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America). In certain embodiments, agonistic anti-PD-1 antibodies or antigen-binding fragments (e.g., antibodies having the heavy and light chain CDRs of 4G9-12) are used to treat non-small cell lung cancer in a patient in need thereof. For example, the present invention relates to a method of treating non-small cell lung cancer comprising administering to a subject in need thereof a therapeutically effective amount of an agonistic anti-PD-1 antibody or antigen-binding fragment (e.g., an antibody having the heavy and light chain CDRs of 4G9-12);

[0161] The agonist anti-PD-1 antibodies and antigen-binding fragments disclosed herein can be used to treat cancers that are resistant or have acquired resistance to checkpoint inhibition therapy. Resistance to checkpoint inhibition therapy can be primary resistance (e.g., the cancer is refractory to initial and subsequent treatments with checkpoint inhibition therapy) or secondary resistance (e.g., the cancer was responsive to initial or initial treatment but is refractory to subsequent treatments with checkpoint inhibition therapy). Resistance to checkpoint inhibition therapy can occur, for example, by downregulation or lack of expression of PD-L1 in tumors (e.g., PD-L1 negative cancers).

[0162] In some preferred embodiments, the agonistic anti-PD-1 antibodies and antigen-binding fragments disclosed herein can be used to treat a subject having a cancer with secondary resistance to checkpoint inhibition therapy, such as PD-1 checkpoint inhibition therapy. Accordingly, the present disclosure relates to a method of treating a cancer with secondary resistance to checkpoint inhibition therapy (e.g., PD-1 checkpoint inhibition therapy), comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonistic antibody or antigen-binding fragment disclosed herein.

[0163] In some preferred embodiments, the agonistic anti-PD-1 antibodies and antigen-binding fragments disclosed herein can be used to treat a subject having a cancer that has primary resistance to PD-1 checkpoint inhibition. Accordingly, the present disclosure relates to a method of treating a cancer that has primary resistance to checkpoint inhibition therapy (e.g., PD-1 checkpoint inhibition therapy), comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonistic antibody or antigen-binding fragment disclosed herein.

[0164] In some preferred embodiments, the agonistic anti-PD-1 antibodies and antigen-binding fragments disclosed herein can be used to treat a subject having a PD-L1 negative tumor (e.g., a PD-L1 downregulated cancer). Accordingly, the present disclosure relates to a method of treating a cancer that is PD-L1 negative (e.g., a PD-L1 downregulated cancer), comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonistic antibody or antigen-binding fragment disclosed herein.

[0165] Treating infection The disclosed antibody compositions and methods can be used to treat infections and infectious diseases. The agents are used to stimulate or enhance a subject's immune response to an infection by administering to the subject an amount of one or more of the disclosed antibodies or antigen-binding fragments thereof that deliver an activating or stimulatory signal through PD-1. The methods can alleviate one or more symptoms of the infection.

[0166] Infection or disease can be caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that invade cells and are attacked by cytotoxic T lymphocytes.

[0167] Infection or disease can be acute or chronic. Acute infection is usually short-lasting infection. During acute microbial infection, immune cells begin to express immunomodulatory receptors. Thus, in some embodiments, the method comprises increasing the immune stimulatory response to acute infection.

[0168] The infection may be caused by, for example, but not limited to, Candida albicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or Mycobacterium.

[0169] In some embodiments, the disclosed antibody compositions are used to treat chronic infections, eg, infections in which T cell exhaustion has occurred and the infection remains in the host for extended periods of time.

[0170] Examples of infections to be treated include chronic infections caused by hepatitis viruses, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes viruses, Epstein-Barr virus, and human papillomavirus.

[0171] Because viral infections are primarily cleared by T cells, increased T cell activity is therapeutically useful when it is beneficial for an animal or human subject to clear an infecting virus more quickly or thoroughly. Thus, the disclosed compositions can be administered for the treatment of localized or systemic viral infections, including, but not limited to, immunodeficiencies (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), and the common cold (e.g., human rhinovirus), as well as other viral infections caused by, for example, HTLV, hepatitis virus, respiratory syncytial virus, vaccinia virus, rabies virus, and SARS-CoV2 virus. The molecules can be administered locally to treat viral skin diseases, such as herpes lesions, shingles, and genital warts. The molecules can also be administered systemically to treat systemic viral diseases, including, but not limited to, AIDS influenza, the common cold, covid-19, or encephalitis.

[0172] Representative infections that can be treated include infections caused by microorganisms, including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, B.Dellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium; Heliobacter, Hemophilus, Hemophilus influenza II (HIB), Hypomicrobium, Legionella, Leptospirosis, Listeria, Meningococcus A, B & C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Rhodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaete, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yersinia, Cryptococcus neoformans、Histoplasma capsulatum、Candida albicans、Candida tropicalis、Nocardia asteroides、Rickettsia ricketsii、Rickettsia typhi、Mycoplasma pneumoniae、Chlamydialpsittaci、Chlamydial trachomatis、Plasmodium falciparum、Trypanosoma brucei、Entamoeba histolytica、Toxoplasma gondii、Trichomonas vaginalis、and、Schistosoma mansoni。.

[0173] Other microorganisms that can be treated using the disclosed compositions and methods include bacteria such as Klebsiella, Serratia, Pasteurella; pathogens associated with cholera, tetanus, botulism, anthrax, plague, and Lyme disease; or fungal or parasitic pathogens such as Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus, Aspergillus (fumigatus, niger, etc.), Mucorales genera (mucor, absidia, rhizophus), Sporothrix (schenkii), Blastomyces (dermatitidis), Paracoccidioides (brasiliensis), Coccidioides (immitis), as well as Histoplasma (capsulatuma), Entamoeba, Histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondi, Sporothrix, Blastomyces, Paracoccidioides, Coccidioides, Histoplasma, Entamoeba, Histolytica, Balanadium, Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis, Plasmodium, Babesia, or Trypanosoma, etc.

[0174] Combination Therapy to Boost Immune Response The disclosed antibodies and antigen-binding fragments thereof, and compositions thereof, can be administered to a subject in need thereof alone or in combination with one or more additional therapeutic agents. In some embodiments, the antibodies and antigen-binding fragments thereof, and the additional therapeutic agent are administered separately but simultaneously. The antibodies and antigen-binding fragments thereof, and the additional therapeutic agent can also be administered as part of the same composition. In other embodiments, the antibodies and antigen-binding fragments thereof, and the second therapeutic agent are administered separately, at different times, but as part of the same treatment regimen. The additional therapeutic agent can be administered before, after, or alternating with the administration of the disclosed antibodies and antigen-binding fragments thereof.

[0175] The subject may be administered the first therapeutic agent 1, 2, 3, 4, 5, 6, or more hours or 1, 2, 3, 4, 5, 6, 7, or more days prior to administration of the second therapeutic agent. In 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. Antibodies and antigen-binding fragments thereof may be the first therapeutic agent or the second therapeutic agent.

[0176] The antibodies and antigen-binding fragments thereof, as well as the additional therapeutic agent, can be administered as part of a treatment regimen. For example, if a first therapeutic agent can be administered to a subject every 4 days, the second therapeutic agent can be administered on the first, second, third, fourth day, or a combination thereof. The first therapeutic agent or the second therapeutic agent can be administered repeatedly throughout the treatment regimen.

[0177] Exemplary additional therapeutic agents include, but are not limited to, cytokines (e.g., IL-2, IL-12), chemotherapeutic agents, radionuclides, other immunotherapeutic agents, enzymes, antibiotics, antiviral agents (particularly protease inhibitors alone or in combination with nucleosides for the treatment of HIV or Hepatitis B or C), antiparasitic agents, growth factors, growth inhibitory agents, hormones, hormone antagonists, antibodies and biologically active fragments thereof (including humanized antibodies, single chain antibodies, chimeric antibodies), antigen and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatory drugs, ligands that bind toll-like receptors and activate the innate immune system (including, but not limited to, CpG oligonucleotides), molecules that recruit and optimize the adaptive immune system (e.g., bispecific T cell engagers), and other molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells, and helper T cells, as well as other molecules that inactivate or downregulate suppressor T cells and regulatory T cells.

[0178] The additional therapeutic agent can be selected based on the condition, disorder, or disease to be treated. For example, the antibodies or antigen-binding fragments disclosed herein can be co-administered with one or more additional agents that function to enhance or promote the immune response, or to reduce or inhibit the immune response. The selection of an additional therapeutic agent other than the antibodies or antigen-binding fragments disclosed herein is a routine matter for the skilled artisan having the benefit of this disclosure.

[0179] Additional therapeutic agents suitable for use in combination with the anti-PD-1 agonist antibodies or antigen-binding fragments described herein include cell therapy. Suitable cell therapies can include, for example, dendritic cells, B cells, T cells (e.g., αβT cells, CD3+T cells, CD4+T cells, CD8+T cells, γδT cells, etc.), macrophages, NK cells, NKT cells, and combinations thereof. Suitable cell therapies include, but are not limited to, stem cell therapy, T cell therapy (e.g., tumor infiltrating lymphocytes (TIL)), or engineered cell therapy (e.g., chimeric antigen receptor (CAR) therapy, engineered TCR therapy). In some preferred embodiments, the agonist anti-PD-1 antibodies and / or antigen-binding fragments disclosed herein can be used in combination with cell therapy to treat a subject with cancer. Thus, the present disclosure relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonist antibody or antigen-binding fragment disclosed herein and a cell therapy. In some preferred embodiments, the agonistic anti-PD-1 antibodies and / or antigen-binding fragments disclosed herein can be used in combination with T cell therapy to treat a subject with cancer. Accordingly, the present disclosure relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonistic antibody or antigen-binding fragment disclosed herein and T cell therapy. In some preferred embodiments, the agonistic anti-PD-1 antibodies and / or antigen-binding fragments disclosed herein can be used in combination with tumor infiltrating lymphocytes (TILs) to treat a subject with cancer. Accordingly, the present disclosure relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonistic antibody or antigen-binding fragment disclosed herein and tumor infiltrating lymphocytes (TILs).In some preferred embodiments, the agonistic anti-PD-1 antibodies and / or antigen-binding fragments disclosed herein can be used in combination with engineered cell therapy (e.g., engineered TCR therapy, chimeric antigen receptor (CAR) therapy, or a combination thereof) to treat a subject with cancer. Accordingly, the present disclosure relates to a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonistic antibody or antigen-binding fragment disclosed herein and an engineered cell therapy (e.g., engineered TCR therapy, chimeric antigen receptor (CAR) therapy, or a combination thereof).

[0180] One example of a suitable engineered cell therapy is chimeric antigen receptor therapy. Chimeric antigen receptor therapy can be CAR-T cells (e.g., axicabtagene ciloleucel, brexucabtagene autoleucel, ciltacabtagene autoleucel, idecabtagene vicleucel, lisocabtagene maraleucel, tisagenlecleucel), CAR-NK cells, CAR-macrophages, and combinations thereof. In some aspects, the present disclosure relates to administering agonistic anti-PD-1 antibodies together with CAR-T cells to a subject in need thereof. In some preferred embodiments, the agonistic anti-PD-1 antibodies and antigen-binding fragments disclosed herein can be used in combination with CAR-T cells to treat cancer in a subject in need thereof. Thus, the present disclosure relates to a method of treating cancer, comprising administering a therapeutically effective amount of the agonistic anti-PD-1 antibodies and antigen-binding fragments disclosed herein in combination with CAR-T cells to a subject in need thereof.

[0181] Another example of a suitable engineered cell therapy is engineered T cell receptor therapy. Engineered T cell receptor therapy can include engineered TCRs, TCR subunits, TCR fragments, and combinations thereof. Suitable engineered T cell receptor therapies include, but are not limited to, engineered TCR T cells (TCR-T cells), engineered TCR natural killer cells (TCR-NK), engineered TCR macrophages (TCR-macrophages), and combinations thereof. For example, an agonist anti-PD-1 antibody or antigen-binding fragment thereof can be used to target CD8 T cells expressing an engineered (e.g., transduced) TCR. + As another example, an agonist anti-PD-1 antibody or antigen-binding fragment thereof can be combined with a CD4 T cell expressing an engineered (e.g., transduced) TCR. + In some preferred embodiments, the agonistic anti-PD-1 antibodies and antigen-binding fragments disclosed herein can be used in combination with engineered TCR T cells (TCR-T cells) to treat cancer in a subject in need thereof. Accordingly, the present disclosure relates to a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an agonistic anti-PD-1 antibody or antigen-binding fragment disclosed herein and engineered TCR T cells (TCR-T cells).

[0182] When the agonistic anti-PD-1 antibodies or antigen-binding fragments disclosed herein are combined with a cell therapy, the two therapeutic agents can be administered to a subject in need thereof substantially simultaneously (e.g., simultaneously), or the cell therapy can be administered before or after the PD-1 antibody or antigen-binding fragment thereof. The timing of administration of each therapeutic agent can vary, but typically they are administered so that their respective pharmacological activities overlap in the subject.

[0183] The engineered TCR or chimeric antigen receptor can be designed to bind any antigen. For example, the TCR or chimeric antigen receptor can specifically bind any of the following antigens: CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, FAP, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, Tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-Acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, legumain, HPV E6, E7, intestinal carboxylesterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, fibronectin EDB (EDB-FN), 5T4 carcinoembryonic antigen, and IGLL1.

[0184] Other therapeutic agents suitable for use in combination with anti-PD-1 agonist antibodies include immunomodulatory agents that target and alter a patient's immune response, for example to enhance immune effector responses against tumors. Examples of suitable immunomodulatory agents include immune stimulatory molecules, such as cytokines (e.g., IL-2, IL-12, etc.), adjuvants, T cell engagers, immune stimulatory antibodies (e.g., anti-CD3), and agents that block inhibitory immune pathways, such as immune checkpoint inhibitors (e.g., antibodies that bind to immune checkpoint proteins). Immune checkpoint inhibitors include antibodies and antigen-binding fragments that bind to immune checkpoint proteins, such as: PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (CD152) that binds to B7-1 (CD80) and B7-2 (CD86), LAG that binds to galectin 3, LSECtin, and FGL1. 3 (CD223); TIM3 (HAVCR2) that binds to Ceacam1 and galectin 9 ligand; TIGIT (VSTM3, WUCAM) that binds to CD112 and CD155; BTLA (CD272), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55) that binds to HVEM (TNFRSF14); CD134 (TNRFSR4, OX40) that binds to CD252 (OX-40L), etc. Numerous therapeutic agents, such as antibodies that bind to immune checkpoint proteins and inhibit their immunosuppressive activity, have been developed as antitumor drugs. Several such agents are currently on the market, including: the anti-PD-1 antibodies pembrolizumab, dostallimab, cemiplimab-rwlc, nivolumab, camrelizumab, tislelizumab, toripalimab, and sintilimab; the anti-PD-L1 antibodies avelumab, durvalumab, and atezolizumab; the anti-CTLA-4 antibody ipilimumab; and the anti-LAG-3 antibody relatlimab. In some aspects, the present disclosure relates to combinations of agonistic anti-PD-1 antibodies with anti-CTLA4 antibodies (e.g., ipilimumab).In some aspects, the disclosure relates to a combination of an agonist anti-PD-1 antibody with an anti-LAG-3 antibody (e.g., relatlimab). In some preferred embodiments, the agonist anti-PD-1 antibody and / or antigen-binding fragment disclosed herein can be used in combination with an immunomodulatory agent to treat a subject with cancer. Accordingly, the disclosure relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonist antibody or antigen-binding fragment disclosed herein and an immunomodulatory agent. In some preferred embodiments, the agonist anti-PD-1 antibody and / or antigen-binding fragment disclosed herein can be used in combination with an anti-CTLA4 antibody or antigen-binding fragment thereof to treat a subject with cancer. Accordingly, the disclosure relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonist antibody or antigen-binding fragment disclosed herein and an anti-CTLA4 antibody or antigen-binding fragment thereof. In some preferred embodiments, the agonistic anti-PD-1 antibodies and / or antigen-binding fragments disclosed herein can be used in combination with an anti-LAG-3 antibody or antigen-binding fragment thereof to treat a subject with cancer. Accordingly, the present disclosure relates to a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonistic antibody or antigen-binding fragment disclosed herein and an anti-LAG-3 antibody or antigen-binding fragment thereof.

[0185] In a preferred embodiment, the agonist anti-PD-1 antibody or antigen-binding fragment is administered in combination with a checkpoint inhibitor that does not bind to PD-L1, PD-L2, and / or PD-1. For example, an agonist anti-PD-1 antibody or antigen-binding fragment may be administered with an antibody that binds any of the following: CTLA-4 (CD152), which binds B7-1 (CD80) and B7-2 (CD86); LAG 3 (CD223), which binds galectin 3, LSECtin, and FGL1; TIM3 (HAVCR2), which binds Ceacam1 and galectin 9 ligand; TIGIT (VSTM3, WUCAM), which binds CD112 and CD155; BTLA (CD272), which binds HVEM (TNFRSF14), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55), which binds HVEM, or CD134 (TNRFSR4, OX40), which binds CD252 (OX-40L). In more specific examples, the checkpoint inhibitor is an anti-CTLA-4 antibody, such as ipilimumab, and / or an anti-LAG-3 antibody, such as relatlimab. In some preferred embodiments, the agonistic anti-PD-1 antibodies and / or antigen-binding fragments disclosed herein can be used in combination with a checkpoint inhibitor (e.g., a checkpoint inhibitor that does not inhibit the interaction of PD-1 with PD-L1 or PD-L2) to treat a subject with cancer. Thus, the present disclosure relates to a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonistic antibody or antigen-binding fragment disclosed herein and a checkpoint inhibitor (e.g., a checkpoint inhibitor that does not inhibit the interaction of PD-1 with PD-L1 or PD-L2).

[0186] In one particular embodiment, the antibodies and antigen-binding fragments thereof may be co-administered with a PD-1 antagonist, i.e., a molecule that inhibits the suppression of PD-1 by PD-L1. In some embodiments, the PD-1 antagonist binds directly to the PD-1 receptor without inducing inhibitory signaling and also binds to ligands of the PD-1 receptor to reduce or inhibit ligands that induce signaling through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and cause inhibitory signaling, fewer cells are attenuated by the negative signals delivered by PD-1 signaling and a more robust immune response can be achieved.

[0187] PD-1 signaling is believed to be driven by the binding of PD-1 ligands (such as 87-H1 and Er-DC) in close proximity to peptide antigens presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Nail. Acad. Sci; KA, 105:10275-10276 (2008)). Thus, proteins, antibodies, or small molecules that block co-ligation of PD-1 and TCR on the cell membrane are also useful PD-1 antagonists.

[0188] In some embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or prevents PD-1 receptor signaling by binding to a ligand of PD-1 or to PD-1 itself, particularly when such binding is not followed by co-ligation of PD-1 with the TCR, and thereby does not induce inhibitory signaling through the PD-1 receptor. Other PD-1 antagonists contemplated by the methods of the invention include antibodies that bind to PD-1 or a ligand of PD-1, as well as other antibodies.

[0189] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following U.S. Patent Nos. 7,332,582, 7,488,802, 7,521,051, 7,524,498, 7,563,869, 7,981,416, 8,088,905, 8,287,856, 8,580,247, 8,728,474, 8,779,105, 9,067,999, 9,073,994, 9,084,776, 9,205,148, 9,358,289, 9,387,247, 9,492,539, and 9,492,540, all of which are incorporated by reference in their entireties. See also Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0190] Exemplary anti-PD-L1 antibodies include, but are not limited to, those described in U.S. Patent Nos. 8,383,796, 9,102,725, 9,273,135, 9,393,301, and 9,580,507, all of which are specifically incorporated by reference herein in their entireties.

[0191] Exemplary anti-PD-L2 antibodies include, but are not limited to, those described in U.S. Pat. Nos. 7,411,051, 7,052,694, 7,390,888, 8188238, and 9255147, all of which are specifically incorporated by reference in their entireties.

[0192] Other suitable examples of immunomodulatory agents for combination with the agonist anti-PD-1 antibodies or antigen-binding fragments described herein include T cell engaging therapies (e.g., T cell engagers). T cell engagers are agents that direct the cytotoxic activity of T cells to target tumors. Suitable T cell engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bifunctional checkpoint inhibitory T cell engagers (CiTEs), simultaneous multiply interacting T cell engagers (SMITEs), trispecific killer engagers (TriKEs), BiTE-expressing chimeric antigen receptor (CAR) T cells (CART.BiTE cells), or combinations thereof. The present disclosure relates to compositions comprising agonist anti-PD-1 antibodies and bispecific T cell engagers, and methods of use thereof. In some preferred embodiments, the agonistic anti-PD-1 antibodies and / or antigen-binding fragments disclosed herein can be used in combination with T cell engagers (e.g., bispecific T cell engagers (BiTEs), bifunctional checkpoint inhibitory T cell engagers (CiTEs), simultaneous multiply interacting T cell engagers (SMITEs), trispecific killer engagers (TriKEs), BiTE-expressing chimeric antigen receptor (CAR) T cells (CART.BiTE cells), and combinations thereof) to treat a subject with cancer. Accordingly, the disclosure relates to a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a PD-1 agonist antibody or antigen-binding fragment disclosed herein, and a therapeutically effective amount of a T cell engager (e.g., bispecific T cell engagers (BiTEs), bifunctional checkpoint inhibitory T cell engagers (CiTEs), simultaneous multiply interacting T cell engagers (SMITEs), trispecific killer engagers (TriKEs), BiTE-expressing chimeric antigen receptor (CAR) T cells (CART.BiTE cells), and combinations thereof).

[0193] Additional therapeutic agents that can be used in embodiments include those disclosed in US Pat. No. 11,021,540.

[0194] equivalent It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present invention described herein will be obvious and may be made using suitable equivalents without departing from the scope of the present disclosure or embodiments. Although specific compositions and methods have been described in detail, the same will be more clearly understood by reference to the following examples, which are introduced for purposes of illustration only and are not intended to be limiting. EXAMPLES

[0195] Example 1: hPD-1-HIS binding of 4G9-1 to 4G9-10 Antibodies 4G9-1 to 4G9-10 shown in Tables 1 to 2 and 5 were subjected to a HIS binding assay against human PD-1 (hPd-1). This assay involved the following steps.

[0196] 100 μl of 10 μg / ml PD-1-Protein (Acro Biosystems Cat. No. HM5221) was coated on a Maxisorpplate overnight at 4°C. The next morning, wells were washed once with PBST and blocked with 200 μl / well Superblock / 4% BSA for 2 hours. Wells were washed 3X with 1X PBST. Then, 100 μl of 4G9-12 at 1-100 μg / ml in Superblock / 4% BSA was added for 2 hours at RT or overnight at 4°C. Wells were washed 3X with 1X PBST. 100 μl of biotinylated anti-human IgG (ThermoFisher Cat. No. A18827) was added to the wells and allowed to bind for 1 hour at RT, as suggested by the manufacturer. In some experiments, anti-human IgG-HRP was used directly (signal remains detectable).

[0197] Wells were washed 3 times with 1X PBST. 100μl of 1:5000 SA HRP was added to the wells and allowed to bind for 1 hour at RT. Wells were washed 3 times with 1X PBST followed by 3 times with 1X PBS. 100μl of TMB was added to the wells and development was stopped with 2N sulfuric acid. Optical density at 450nm (OD450) was measured.

[0198] The results are as shown in Table 7. [Table 7]

[0199] Example 2: hPD-1-HIS binding of 5C2-1 to 5C2-10 Antibodies 5C2-1 to 5C2-10 shown in Tables 3 to 4 and 6 were subjected to a HIS binding assay with human PD-1 (hPd-1). The optical density at 450 nm (OD450) was determined according to the procedure shown in Example 1.

[0200] The results are as shown in Table 8. [Table 8]

[0201] Example 3: Activation of human T cells with 4G9-2 and 4G9-12 Antibodies 4G9-2 and 4G9-12 were assayed for PD-1 agonist activity according to the following in vitro procedure. In summary, IFNγ levels in the supernatant were due to T cell activation, which was due to PD-1 agonist activity by the antibodies. As a control, an antibody against human immunoglobulin G1 (hIgG1) was used.

[0202] Materials used were human CD4+ / CD45+ / CD25- naive T cells (BioVit, Hicksville, NY), human IFNγ ELISA kit (Invitrogen, catalog no. KHC4021), LymphoOne T cell growth xeno-free medium (Takara, catalog no. WK552S), certified fetal bovine serum, Performance Plus (Gibco, catalog no. 16000-044), Dynabead human T activator CD3 / CD28 (Gibco, catalog no. 11132D), interleukin-2 (IL-2; PeproTech, catalog no. 200-02); round-bottom sterile 96-well plates; and a plate reader for ELISA.

[0203] The following procedure was followed, and all procedures up to the ELISA measurement of IFNγ concentration were carried out under sterile conditions.

[0204] Human naive CD4 T cells were thawed. The procedure was performed twice, using T cells from a different donor each time. The cells were washed twice with PBS and LymphoOne cell culture medium was added. The cells were counted and the concentration was adjusted to 1.5 million / mL in LymphoOne medium. IL-2 and anti-CD3 / anti-CD28 coated Dynabeads were added according to the manufacturer's suggestions. Cells (100 μL per well) were added to round-bottom 96-well plates and incubated for 48 h at 37 °C with 5% CO2. Antibodies were added at final concentrations of 10-50 μg / mL. The same volume (20 μL) of medium was added to untreated (0 μg / mL antibody) wells, and control antibodies were also used in the same volume and concentration. The cells were then incubated for 24 h at 37 °C with 5% CO2, and the supernatant was collected by centrifugation.

[0205] Supernatants were pre-diluted from 1:1 to 1:50 and harvested supernatants were subjected to ELISA to detect IFNγ according to the manufacturer's protocol. IFNγ concentrations determined by ELISA are shown in Figures 1A and 1B.

[0206] Figures 1A and 1B show that IFNγ production by T cells in the absence of antibody or at the tested concentrations of the control anti-hIgG1 antibody was essentially the same for each donor. Culturing cells with 4G9-2 or 4G9-12 at concentrations of 10-50 μg / mL increased IFNγ production by approximately 75-125% in donor 1 and approximately 25-65% in donor 2. The increase in IFNγ production for donor 2 showed a dose-dependent response for both antibodies. The results indicate that 4G9-2 and 4G9-12 functioned as PD-1 agonists and increased T cell activation compared to T cell activation in cells untreated with agonistic anti-PD-1 antibodies (dashed lines in Figures 1A and 1B).

[0207] Example 4: Activation of human T cells by affinity matured antibodies 4G9-1 to 4G9-10 The affinity matured antibodies 4G9-1 to 4G9-10 were assayed for PD-1 agonist activity essentially as described in Example 3. Briefly, human CD4 + T cells were isolated from donors and stimulated with anti-CD3 / anti-CD28 coated beads and IL-2 for 24 hours. After 24 hours, cells were treated with selected antibodies from 4G9-1 to 4G9-10, a control antibody for the original 4G9 CDR that was not affinity matured (4G9 HC4-LC1), or left untreated. After a further 48 hours of culture, supernatants were collected and tested for IFNγ production by ELISA. This experiment was performed in duplicate wells for each treatment. Data from a representative experiment are shown in Figure 2. IFNγ production was significantly ( * Treatment with 4G9-2 antibody induced the highest IFNγ production compared to all other anti-PD1 agonist antibodies tested in the study.

[0208] Example 5: Evaluation of T memory cell induction and T cell exhaustion in human T cells by 4G9-12 antibody Human CD4 + T cells (BIOIVT LLC) were stimulated with beads coated with anti-CD3 and anti-CD28 antibodies (DYNABEADS™ GIBCO (THERMO FISHER SCIENTIFIC INC.)) and IL-2 (100 U / mL, R&D SYSTEMS, INC.) essentially as described in Example 3. After 72 hours of stimulation, cells were treated with 4G9-12 anti-PD-1 agonist antibody (25 μg / mL) or left untreated (control). After 48 hours of treatment with anti-PD-1 agonist antibody, cells were harvested, washed, and stained with fluorescently labeled antibodies. Stained cells were analyzed by flow cytometry. Figure 3A is a schematic diagram of the experiment. Central memory T cells (Tcm) express CD45RO high / CD62 Lhigh / CD45RA lowExhausted T cells (e.g., terminal effector T cells (Tte)) express CD45RO low / CD62L low / CD45RA high Treatment with 4G9-12 increased CD4 + The Tcm population was significantly * p<0.05), and the Tte population was significantly ( * p<0.05) (see Figures 3B and 3C, respectively).

[0209] Example 6: Comparison of 4G9-12 antibody and blocking anti-PD-1 antibody in human T cells The aim of this study was to determine the expression of primary human CD4+ cells after treatment with different concentrations of 4G9-12 and a blocking anti-PD-1 antibody. + The aim of this study was to test the activation of T cells. 4G9-12-mediated T cell stimulation was performed using purified human CD4 + T cells were pre-stimulated with human anti-CD3 / anti-CD28 Dynabeads and IL-2 for 24 hours at 37° C., essentially as described in Example 3. After the pre-stimulation step, cells were exposed to 4G9-12, 4G9-2, isotype control (Iso Ctrl), or a blocking anti-PD-1 (PROMEGA Co, clone J1201 ("Block-1", Figures 4A, 4B, and 4C) or pembrolizumab ("Block-2", Figure 4C) antibody, which is not a PD-1 agonist) at various concentrations for 48 hours, and IFN-γ levels were measured using ELISA (see Figures 4A-4C). Blocking anti-PD-1 antibodies did not increase IFN-γ levels in supernatants from purified CD4 T cells when compared to levels observed in untreated cell supernatants (dashed lines in Figures 4A-4C), while 4G9-12 and 4G9-2 treatments resulted in a similar concentration-dependent increase in IFN-γ. This result indicates that T cell stimulation, as assessed by IFN-γ production, is induced by the agonistic anti-PD-1 antibodies disclosed herein, but not by antibodies that bind PD-1 and inhibit its interaction with PD-L1, such as J1201 and pembrolizumab.

[0210] Example 7: Functional assessment of agonist antibody activity in the presence of PD-L1 or PD-1 blocking antibodies The 4G9-2 antibody was tested for its ability to activate human CD4 T cells prestimulated (24 hours) with CD3 / CD28-coated Dynabeads and IL-2, essentially as described in Example 3, but in the absence or presence of plate-bound PD-L1 in the form of an Fc fusion protein (PD-L1-IgG). As shown in Figure 5, after 72 hours of exposure to 25 μg / mL of 4G9-2, there was a greater than three-fold increase in IFN-γ levels in the supernatants (measured using an ELISA from THERMO FISHER SCIENTIFIC Inc.) compared to untreated T cells or T cells exposed to a blocking anti-PD-1 antibody (PROMEGA Co., clone J1201 "Blocking Ab", 25 μg / mL), confirming that 4G9-2 activates T cells. Pretreatment of cells with plate-bound PD-L1 (PD-L1-IgG) inhibited IFN-γ production, whereas 4G9-2 treatment in the presence of plate-bound PD-L1 (PD-L1-IgG) resulted in T cell activation comparable to that achieved by 4G9-2 treatment alone, suggesting that agonistic antibodies such as 4G9-2 can activate T cells even in the presence of their natural ligand (PD-L1).

[0211] Example 8: Evaluation of anti-PD-1 agonist antibodies that can potentially compete with PD-L1 To evaluate whether anti-PD-1 agonist antibodies can bind to PD-1 in competition with PD-L1 or can bind to PD-1 simultaneously with PD-L1, we used Raji PD-1 +APC-labeled 4G9-12 was assessed at 5 μg / mL for its potential to competitively bind with V450-labeled human PD-L1-IgG (1, 2, or 5 μg / mL) in cells by flow cytometry. PD-L1 alone binds to PD-1, and higher binding was observed with increasing concentrations of PD-L1, as shown in Figure 6A (PDL1 fluorescence). As shown in Figure 6B (4G9-12 fluorescence) and Figure 6C (PD-L1 fluorescence), both 4G9-12 and PD-L1-IgG were able to bind to the target simultaneously, and the presence of increasing concentrations of PD-L1-IgG did not affect the binding of 4G9-12. These data also indicate that PD-L1-IgG maintains similar levels of binding in the presence of 4G9-12. These data support that PD-1 agonistic antibodies such as 4G9-12 do not compete with PD-L1 for binding to PD-1 and therefore their agonistic activity is not due to inhibition of PD-L1 binding.

[0212] Example 9: Evaluation of intraperitoneal administration of 4G9-12 antibody in a syngeneic colorectal carcinoma mouse tumor model The antitumor activity of 4G9-12 was evaluated in vivo using a syngeneic colorectal carcinoma (CRC) tumor model in BALB / c mice. Mice were subcutaneously (SC) injected with CT26 tumor cells (1×105 cells / mouse) on day 0, and 4G9-12 (0.5 or 1 mg / kg) or a non-PD-1 agonist commercial benchmark PD-1 blocking antibody (clone RMP1-14 (BioXCell, 1 mg / kg)) was intraperitoneally (IP) injected 10, 13, 16, and 20 days after tumor implantation (0, 3, 6, and 10 days after treatment). A group of tumor-implanted mice that did not receive antibody served as a negative control group. Tumor volume was monitored. Both doses of 4G9-12 showed significant antitumor activity compared to the untreated control group and the group treated with 1 mg / kg PD-1 blocking antibody. There was no significant difference between the 0.5 mg / kg and 1 mg / kg doses of 4G9-12, suggesting that at these doses, the antitumor activity reached a stable response (see Figure 7). Individual tumor volumes for each mouse are shown in Figures 8A-8D. This study demonstrated the antitumor activity of 4G9-12 in a mouse model of colorectal cancer and showed that the agonistic antibody had superior antitumor activity compared to the PD-1 blocking antibody.

[0213] Example 10: Evaluation of intravenous administration of 4G9-12 antibody in a syngeneic colorectal carcinoma mouse tumor model The antitumor activity of 4G9-12 was evaluated in vivo using a syngeneic colorectal carcinoma (CRC) tumor model in BALB / c mice. Mice were inoculated with CT26 tumor cells (1x10 5Cells / mouse) were subcutaneously injected, and 4G9-12 (0.01, 0.05, 0.1, or 0.25 mg / kg) or a commercially available benchmark PD-1 blocking antibody (clone RMP1-14, BioXCell; 1 mg / kg, non-agonistic antibody) was administered by intravenous (IV) injection 14, 18, 22, and 25 days after tumor implantation (days 0, 4, 8, and 11 after administration). A group of tumor-implanted mice that did not receive antibody served as a negative control group. Tumor volumes were monitored for all mice. All doses of 4G9-12 resulted in statistically significant inhibition of tumor growth compared to untreated controls, and doses of 4G9-12 at 0.1 mg / kg and below responded similarly to each other and to 1 mg / kg of anti-PD-1 blocking antibody (see Figure 9). Individual tumor volumes for each mouse are shown in Figures 10A-10F. The benchmark PD-1 blocking antibody (4-fold higher concentration) was less active compared to 0.25 mg / kg 4G9-12. This study demonstrated the anti-tumor activity of agonistic anti-PD-1 antibodies such as 4G9-12 administered intravenously in a mouse model of CRC. These data indicate that the agonistic anti-PD-1 antibody 4G9-12 enhances tumor growth inhibition compared to PD-1 blocking (e.g., non-agonistic) antibodies.

[0214] Example 11: Ex vivo evaluation of T cell activation by anti-PD-1 agonist antibodies in the CT26 tumor model The antitumor activity of 4G9-12 was evaluated in vivo using a CT26 tumor model in BALB / c mice. CT26 cells (1 × 10 5 Ten days after tumor implantation, when the tumors reached a diameter of 4-6 mm, mice were treated with a single dose of 4G9-12 (1 mg / kg), a single dose of a PD-1 blocking antibody (RMP1-14 clone, 1 mg / kg, non-agonist antibody), or no treatment. Three days after treatment, mice were euthanized and tumors were isolated. Tumors were treated and CD4 + and CD8 + The activation status of T cells was assessed by flow cytometry. + CD40L is a marker for T cell activation, and CD8 +CD25 was used as a marker of T cell activation. These data suggest that agonistic anti-PD-1 antibodies such as 4G9-12, but not blocking anti-PD-1 antibodies, inhibit tumor-infiltrating CD4 + and CD8 + In contrast to blocking anti-PD-1 antibodies, 4G9-12 inhibited tumor-infiltrating CD4 T cells after a single dose. + and CD8 + Induces T cell activation (see Figures 11A and 11B).

[0215] Example 12: Assessment of cell signaling and T cell activation To investigate whether the intracellular signals mediated by PD-1 differ when PD-1 is bound to 4G9-12, PD-L1 ligands, and agonist antibodies such as anti-PD-1 blocking antibodies, we investigated the intracellular signaling pathways of human CD4 + T cells were stimulated with anti-CD3 / anti-CD28 Dynabeads and IL-2, essentially as described in Example 3. After stimulation, cells were treated with either 25 μg / ml of 4G9-12, PD-L1-IgG (R&D SYSTEMS, INC.), or anti-human PD-1 (clone J1201, PROMEGA Co., non-agonistic antibody) for 30 minutes. After 30 minutes of treatment, cells were washed and lysed, and immunoprecipitation was performed with Protein A, followed by Western blot analysis of ITK, SHP2, and PD-1 (see Figures 12A and 12B). The ratio of ITK / SHP2 (normalized to the level of PD-1) was determined, and the ratio showed that 4G9-12 increased the ITK / SHP2 ratio by 3-fold compared to the anti-PD-1 blocking antibody. As expected, PD-L1-IgG treatment decreased the ITK / HP2 ratio (see Figure 12C). Of particular note, 4G9-12 treatment increased the ITK / HP2 ratio compared to a control anti-human PD-1 blocking antibody, indicating that agonistic antibodies such as 4G9-12 induce signaling through PD-1 that is distinct from PD-L1-induced signaling.

[0216] Example 13: Human CD4 +Flow cytometric analysis of cell signaling induced by blocking and agonistic anti-PD-1 antibodies in T cells Because ITK is a key molecule in the mechanism of action of agonistic PD-1 antibodies, flow cytometry was used to evaluate the effect of 4G9-12 on the phosphorylation of ITK in comparison to the blocking anti-PD-1 antibodies (e.g., non-agonistic antibodies) pembrolizumab ("Pembro") and nivolumab ("Nivo"). + T cells were stimulated with anti-CD3 / anti-CD28 Dynabeads and IL-2 for 24 hours substantially as described in Example 3, and then treated with 25 μg / mL of 4G9-12, PD-L1-IgG (R&D SYSTEMS, INC.), pembrolizumab, nivolumab, or left untreated ("Stim") for 24 hours. At the end of treatment, cells were washed, fixed, permeabilized, stained with Live / Dead and PE-labeled anti-phospho-ITK antibody, and prepared for flow cytometry. PD-L1-IgG treatment reduced the phosphorylation of ITK. In contrast to the blocking PD-1 antibodies, pembrolizumab and nivolumab, 4G9-12 significantly increased the levels of phospho-ITK compared to untreated ("Stim") cells (see FIG. 13). These results provide a basis for how treatment with agonistic antibodies such as 4G9-12 leads to T cell activation, as ITK is an activating molecule within the T cell receptor signaling pathway.

Claims

1. 1. An antibody or antigen-binding fragment thereof having binding specificity for Programmed Death Receptor Protein 1 (PD-1), comprising a light chain variable region and a heavy chain variable region, CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-12; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-1; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-4; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-5; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-6; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-7; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-8; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-9; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-10; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 5C2-1; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 5C2-2; 5C2-3 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-4 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-5 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-6 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-7 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; 5C2-8 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 5C2-9; or CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 5C2-10; The antibody or antigen-binding fragment thereof.

2. the antibody or antigen-binding fragment thereof comprising: CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-12; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-4; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-8; CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-9; or 5C2-8 CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3; The antibody or antigen-binding fragment thereof of claim 1 .

3. the antibody or antigen-binding fragment thereof comprising: CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3 of 4G9-12; The antibody or antigen-binding fragment thereof of claim 1 .

4. 1. An antibody or antigen-binding fragment thereof having binding specificity for Programmed Death Receptor Protein 1 (PD-1), comprising a light chain variable region and a heavy chain variable region, a) the light chain variable region is ８ X ９ X １０ X １１ X １２ CDR L1, X １３ X １４ CDR L2 containing S, and X １５ QX １６ X １７ X １８ X １９ P.X. ２０ X ２１ and a CDR L3 comprising: １ X ２ X ３ X ４ X ５ X ６ X ７ and a CDR H3 comprising: １ is selected from the group consisting of G and V; X ２ is selected from the group consisting of G, S, L, V, I, and E; X ３ is selected from the group consisting of R, G, P, W, S, H, and K; X ４ is selected from the group consisting of G, E, S, V, W, and K; X ５ is selected from the group consisting of I, F, S, G, D, and V; X ６ is selected from the group consisting of A, G, Y, and S; X ７ is selected from the group consisting of Y, H, and D; X ８ is selected from the group consisting of G, S, and D; X ９ is selected from the group consisting of I and L; X １０ is selected from the group consisting of S, V, G, and R; X １１ is selected from the group consisting of N, Y, T, S, and R; X １２ is selected from the group consisting of S, Y, D, and W; X １３ is selected from the group consisting of A, K, Y, and D; X １４ is selected from the group consisting of A and V; X １５ is selected from the group consisting of Q, M, and L; X １６ is selected from the group consisting of S, G, and D; X １７ is selected from the group consisting of Y, T, N, and S; X １８ is selected from the group consisting of S, H, and D; X １９ is selected from the group consisting of T, W, and F; X ２０ is selected from the group consisting of Y, P, R, and W; and X ２１ is selected from the group consisting of T and F; or b) the light chain variable region is ４２ X ４３ X ４４ X ４５ X ４６ CDR L1, X ４７ CDR L2 containing AS, and X ４８ X ４９ X ５０ X ５１ X ５２ X ５３ X ５４ and wherein the heavy chain variable region comprises a CDR L3 comprising ２２ TFX ２３ X ２４ YX ２５ CDR H1, IX comprising (SEQ ID NO: 163) ２６ X ２７ X ２８ X ２９ X ３０ X ３１ CDR H2 containing T, and AX ３２ X ３３ X ３４ X ３５ X ３６ X ３７ X ３８ X ３９ Forex ４０ X ４１ and a CDR H3 comprising: ２２ is selected from the group consisting of Y and Q; X ２３ is selected from the group consisting of N, T, and G; X ２４ is selected from the group consisting of S, I, and N; X ２５ is selected from the group consisting of W and L; X ２６ is selected from the group consisting of H, L, R, and P; X ２７ is selected from the group consisting of P and L; X ２８ is selected from the group consisting of R, S, I, and N; X ２９ is selected from the group consisting of G, D, N, and Y; X ３０ is selected from the group consisting of I, S, G, and R; X ３１ is selected from the group consisting of H, D, and Y; X ３２ is selected from the group consisting of P and S; X ３３ is selected from the group consisting of S, R, N, and Y; X ３４ is selected from the group consisting of S, D, G, V, and F; X ３５ is selected from the group consisting of S, N, G, I, D, and R; X ３６ is selected from the group consisting of Y, N, C, L, S, and H; X ３７ is selected from the group consisting of A, G, T, and E; X ３８ is selected from the group consisting of W, C, S, R, D, and G; X ３９ is selected from the group consisting of A, S, G, and D; X ４０ is selected from the group consisting of A, S, and L; X ４１ is selected from the group consisting of H, Y, and S; X ４２ is selected from the group consisting of D, S, and G; X ４３ is selected from the group consisting of V and I; X ４４ is selected from the group consisting of S and G; X ４５ is selected from the group consisting of T and S; X ４６ is selected from the group consisting of A, S, D, and W; X ４７ is selected from the group consisting of W, G, and D; X ４８ is selected from the group consisting of Q and H; X ４９ is selected from the group consisting of Q, K, and H; X ５０ is selected from the group consisting of H, Y, F, R, D, and G; X ５１ is selected from the group consisting of Y, N, Y, D, S, and G; X ５２ is selected from the group consisting of R, S, V, T, and I; X ５３ is selected from the group consisting of S, A, F, T, Y, and W; and X ５４ is selected from the group consisting of P, L, S, and T; The antibody or antigen-binding fragment thereof.

5. the antibody or antigen-binding fragment thereof comprising: the light chain variable region and the heavy chain variable region of 4G9-12; the light chain variable region and the heavy chain variable region of 4G9-1; the light chain variable region and the heavy chain variable region of 4G9-2; the light chain variable region and the heavy chain variable region of 4G9-3; the light chain variable region and the heavy chain variable region of 4G9-4; the light chain variable region and the heavy chain variable region of 4G9-5; the light chain variable region and the heavy chain variable region of 4G9-6; the light chain variable region and the heavy chain variable region of 4G9-7; the light chain variable region and the heavy chain variable region of 4G9-8; the light chain variable region and the heavy chain variable region of 4G9-9; the light chain variable region and the heavy chain variable region of 4G9-10; the light chain variable region and the heavy chain variable region of 4G9-11; the light chain variable region and the heavy chain variable region of 4G9-13; the light chain variable region and the heavy chain variable region of 4G9-14; the light chain variable region and the heavy chain variable region of 4G9-15; the light chain variable region and the heavy chain variable region of 4G9-16; the light chain variable region and the heavy chain variable region of 4G9-17; the light chain variable region and the heavy chain variable region of 4G9-18; the light chain variable region and the heavy chain variable region of 4G9-19; the light chain variable region and the heavy chain variable region of 4G9-20; the light chain variable region and the heavy chain variable region of 5C2-1; the light chain variable region and the heavy chain variable region of 5C2-2; the light chain variable region and the heavy chain variable region of 5C2-3; the light chain variable region and the heavy chain variable region of 5C2-4; the light chain variable region and the heavy chain variable region of 5C2-5; the light chain variable region and the heavy chain variable region of 5C2-6; the light chain variable region and the heavy chain variable region of 5C2-7; the light chain variable region and the heavy chain variable region of 5C2-8; the light chain variable region and the heavy chain variable region of 5C2-9; or the light chain variable region and the heavy chain variable region of 5C2-10; 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising:

6. the antibody or antigen-binding fragment thereof comprising: the light chain variable region and the heavy chain variable region of 4G9-12; the light chain variable region and the heavy chain variable region of 4G9-2; the light chain variable region and the heavy chain variable region of 4G9-4; the light chain variable region and the heavy chain variable region of 4G9-8; the light chain variable region and the heavy chain variable region of 4G9-9; the light chain variable region and the heavy chain variable region of 4G9-14; the light chain variable region and the heavy chain variable region of 4G9-18; the light chain variable region and the heavy chain variable region of 4G9-19; or the light chain variable region and the heavy chain variable region of 5C2-8; The antibody or antigen-binding fragment thereof of claim 1 or 4, comprising:

7. the antibody or antigen-binding fragment thereof comprising: the light chain variable region and the heavy chain variable region of 4G9-12; or the light chain variable region and the heavy chain variable region of 4G9-2; The antibody or antigen-binding fragment thereof of claim 6, comprising:

8. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, further comprising a light chain constant region and a heavy chain constant region.

9. The antibody or antigen-binding fragment of claim 8, wherein the heavy chain constant region is an IgG constant region.

10. The antibody or antigen-binding fragment of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment is a PD-1 agonist.

11. The antibody or antigen-binding fragment of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment does not compete with PD-L1 for binding to PD-1.

12. The antibody or antigen-binding fragment of any one of claims 1 to 11, wherein the antibody or antigen-binding fragment increases intracellular pITK levels, ITK / SHP2 ratio, or both pITK levels and ITK / SHP2 ratio by binding to PD-1 on the cell.

13. The antibody or antigen-binding fragment of any one of claims 1 to 12, which induces central memory T cells (Tcm).

14. The antibody or antigen-binding fragment of any one of claims 1 to 13, which inhibits T cell exhaustion.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 and a pharma- ceutically acceptable carrier.

16. 17. A method for treating cancer, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 14, or the pharmaceutical composition of claim 15, to a subject in need thereof.

17. 17. The method of claim 16, further comprising administering to the subject one or more additional therapeutic agents.

18. 18. The method of claim 17, wherein the one or more additional therapeutic agents is an immunomodulatory agent.

19. 19. The method of claim 18, wherein the immunomodulatory agent is a checkpoint inhibitor, with the proviso that the checkpoint inhibitor does not inhibit the interaction of PD-1 with PD-L1 or PD-L2.

20. 20. The method of claim 19, wherein the checkpoint inhibitor is an anti-CTLA4 antibody or an antigen-binding fragment thereof.

21. 20. The method of claim 19, wherein the checkpoint inhibitor is an anti-LAG-3 antibody or an antigen-binding fragment thereof.

22. 20. The method of claim 18, wherein the immunomodulatory agent is a T cell engager.

23. 23. The method of claim 22, wherein the T cell engager is selected from the group consisting of a bispecific T cell engager (BiTE), a bifunctional checkpoint inhibitory T cell engager (CiTE), a simultaneous multi-interacting T cell engager (SMITE), a trispecific killer engager (TriKE), a BiTE-expressing chimeric antigen receptor (CAR) T cell (CART.BiTE cell), and combinations thereof.

24. 20. The method of claim 17, wherein the one or more additional therapeutic agents is a cell therapy.

25. 25. The method of claim 24, wherein the cell therapy is T cell therapy.

26. 25. The method of claim 24, wherein the cell therapy is a tumor infiltrating lymphocyte (TIL).

27. 25. The method of claim 24, wherein the cell therapy is an engineered cell therapy selected from the group consisting of engineered TCR therapy, chimeric antigen receptor (CAR) therapy, or a combination thereof.

28. 25. The method of claim 24, wherein the cell therapy is a chimeric antigen receptor T cell (CAR-T cell).

29. 25. The method of claim 24, wherein the cell therapy is an engineered TCR T cell (TCR-T cell).

30. The antibody or antigen-binding fragment of any one of claims 1 to 14 for use in the treatment of cancer.

31. An antigen-binding fragment antibody according to any one of claims 1 to 14 for use in the manufacture of a medicament for treating cancer.

32. A method of treating cancer, comprising administering to a subject in need thereof an effective combination comprising a therapeutically effective amount of (i) the antibody or antigen-binding fragment of any one of claims 1 to 14, or the pharmaceutical composition of claim 15, and (ii) a second antibody or antigen-binding fragment thereof, which reduces inhibition of PD-1 by PD-L1.

33. A combination of an antibody or antigen-binding fragment of any one of claims 1 to 14 and a second antibody or antigen-binding fragment thereof that relieves the inhibition of PD-1 by PD-L1 for use in the treatment of cancer.

34. 15. A combination of an antibody of an antigen-binding fragment of any one of claims 1 to 14 and a second antibody or antigen-binding fragment thereof that reduces inhibition of PD-1 by PD-L1 for use in the manufacture of a medicament for treating cancer.

35. A method for inducing or enhancing central memory T cells, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 14.

36. 15. A method for inhibiting T cell exhaustion, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 14.

37. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 14.

38. A host cell comprising the polynucleotide of claim 37.

39. 40. The host cell of claim 38, wherein the host cell is a mammalian cell.

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