PD-1 agonist and method of using the same

Abstract

To provide an anti-PD-1 binder, a pharmaceutical composition including the binder, and a method for treating inflammatory or autoimmune disorder.SOLUTION: Provided is an anti-PD-1 binder including an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In one embodiment, the immunoglobulin heavy chain variable region includes CDR1 including a specific sequence, CDR2 including a specific sequence, and CDR3 including a specific sequence, and the immunoglobulin light chain variable region includes CDR1 including a specific sequence, CDR2 including a specific sequence, and CDR3 including a specific sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 62 / 857,699, filed June 5, 2019; U.S. Provisional Patent Application No. 62 / 863,193, filed June 18, 2019; and U.S. Provisional Patent Application No. 62 / 983,512, filed February 28, 2020, the entire disclosures of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Programmed death 1 (PD-1), also known as programmed cell death 1, is a 268-amino acid type I transmembrane protein that was first identified by subtractive hybridization of a mouse T cell line undergoing apoptosis (Isida et al., Embo J., 11:3887-95 (1992)). PD-1 is a member of the CD28 / CTLA-4 family of T cell regulators and has been reported to be expressed on activated T cells, B cells, and myeloid cells (Greenwald et al., Annu. Rev. Immunol., 23:515-548 (2005); and Sharpe et al., Nat. Immunol., 8:239-245 (2007)).

[0003] Two ligands for PD-1, PD-ligand 1 (PD-L1) and PD-ligand 2 (PD-L2), have been identified, both of which belong to the B7 protein superfamily (Greenwald et al., supra). PD-L1 is expressed on a variety of cell types, including cells of the lung, heart, thymus, spleen, and kidney (see, e.g., Freeman et al., J. Exp. Med., 192(7):1027-1034 (2000); and Yamazaki et al., J. Immunol., 169(10):5538-5545 (2002)). PD-L1 expression is upregulated on macrophages and dendritic cells (DCs) in response to lipopolysaccharide (LPS) and GM-CSF treatment, and on T cells and B cells upon signaling via the T cell and B cell receptors. PD-L1 is also expressed in a variety of mouse and human tumor cell lines (see, e.g., Iwai et al., Proc. Natl. Acad. Sci. USA, 99(19):12293-12297(2002); and Blank et al., Cancer Res., 64(3):1140-1145(2004)). In contrast, PD-L2 shows a more restricted expression pattern, being expressed primarily by antigen-presenting cells (e.g., dendritic cells and macrophages) and some tumor cell lines (see, e.g., Latchman et al., Nat. Immunol., 2(3):261-238(2001)).

[0004] PD-1 negatively regulates T cell activation, and this inhibitory function is associated with immunoreceptor tyrosine-based switch motifs (ITSMs) in the cytoplasmic domain (see, e.g., Greenwald et al., supra; and Parry et al., Mol. Cell. Biol., 25:9543-9553 (2005)). PD-L1-induced PD-1 clustering has been found to induce recruitment of SHP2 phosphatase, which preferentially dephosphorylates CD28 to suppress T cell function (Hui et al., Science, 355:1428-1433 (2017)). PD-1 deficiency can lead to autoimmunity. For example, C57BL / 6 PD-1 knockout mice have been shown to develop a lupus-like syndrome (see, e.g., Nishimura et al., Immunity, 11:141-1151 (1999)). In humans, single nucleotide polymorphisms in the PD-1 gene are associated with a higher incidence of progression of systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, and multiple sclerosis (e.g., Nielsen et al., Tissue Antigens, 62(6):492-497 (2003); Bertsias et al., Arthritis Rheum., 60(1):207-218 (2009); Ni et al., Hum. Genet., 121(2):223-232 (2007); Tahoori et al., Clin. Exp. Rheumatol., 29(5):763-767 (2011); and Kroner et al., Ann. Neurol., 58(1):50-57 (2005).

[0005] Despite recent progress in inhibiting PD-1 activity to treat various types of cancer and for immune enhancement (e.g., to treat infectious diseases), there is a need for PD-1 binding agents (e.g., antibodies) that bind to PD-1 with high affinity, promote negative signaling, and function as PD-1 agonists. Summary of the Invention

[0006] Brief Summary of the Invention The present invention provides agonistic PD-1-binding agents. In one embodiment, the PD-1-binding agent comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises: CDR1 comprising SEQ ID NO:1; CDR2 comprising SEQ ID NO:2; and CDR3 comprising SEQ ID NO:3; and the immunoglobulin light chain variable region comprises CDR1 comprising SEQ ID NO:4; CDR2 comprising SEQ ID NO:5; and CDR3 comprising SEQ ID NO:6.

[0007] Also provided are anti-PD-1 binding agents comprising an immunoglobulin heavy chain variable region having at least 80%, 85%, or 90% sequence identity to any one of SEQ ID NOs: 24-33, or a heavy chain variable region comprising at least the CDR regions of SEQ ID NOs: 24-33, and / or an immunoglobulin light chain variable region having at least 80%, 85%, or 90% sequence identity to SEQ ID NO: 34 or 35, or a light chain variable region comprising at least the CDR regions of SEQ ID NO: 34 or 35.

[0008] In another aspect, the PD-1-binding agent comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region comprises: a CDR1 comprising SEQ ID NO:7; a CDR2 comprising SEQ ID NO:8; and a CDR3 comprising SEQ ID NO:9; and the immunoglobulin light chain variable region comprises a CDR1 comprising SEQ ID NO:10; a CDR2 comprising SEQ ID NO:11; and a CDR3 comprising SEQ ID NO:12.

[0009] Also provided are anti-PD-1 binding agents comprising an immunoglobulin heavy chain variable region having at least 80%, 85%, or 90% sequence identity to any one of SEQ ID NOs: 43-47 or 61-63, or a heavy chain variable region comprising at least their CDR regions, and / or an immunoglobulin light chain variable region having at least 80%, 85%, or 90% sequence identity to SEQ ID NOs: 48-50, or a light chain variable region comprising at least their CDR regions.

[0010] The present invention further provides isolated or purified nucleic acid sequences encoding the aforementioned immunoglobulin polypeptides, vectors comprising such nucleic acid sequences, isolated PD-1-binding agents comprising the aforementioned immunoglobulin polypeptides, nucleic acid sequences encoding such PD-1-binding agents, vectors comprising such nucleic acid sequences, isolated cells comprising such vectors, compositions comprising such PD-1-binding agents or such vectors together with a pharmaceutically acceptable carrier, and methods for inducing an immune response by administering an effective amount of such a composition to a mammal. and a method for treating inflammatory or autoimmune disorders in a mammal. [Brief explanation of the drawings]

[0011] A brief description of some of the figures in the drawing [Figure 1] FIG. 1 is a graph showing the results of anti-PD-1 antibody binding to HEK293 cells stably transfected with human PD-1. [Figure 2] FIG. 2 is a graph showing the results of anti-PD-1 antibody binding to HEK293 cells stably transfected with cynomolgus monkey PD-1. [Figure 3] FIG. 3 is a graph showing the results of anti-PD-1 antibody binding to anti-CD3 / anti-CD28 activated human peripheral blood CD4+ T cells over a 2-day period. [Figure 4] Figures 4-7 are graphs showing the results of testing anti-PD-1 antibodies to compete with either PD-L1-Fc or PD-L2-Fc for binding to PD-1 CHO-K1 cells. Figure 4 is a graph showing the results of a competition assay demonstrating the ability of anti-PD-1 antibodies to compete with PD-L1-Fc for binding to CHO-K1 cells stably transfected with human PD-1. [Figure 5] Figure 5 is a graph depicting the results of a competition assay demonstrating the ability of anti-PD-1 antibodies to compete with PD-L1-Fc for binding to CHO-K1 cells stably transfected with human PD-1. [Figure 6]Figure 6 is a graph depicting the results of a competition assay demonstrating the ability of anti-PD-1 antibodies to compete with PD-L2-Fc for binding to CHO-K1 cells stably transfected with human PD-1. [Figure 7] Figure 7 is a graph depicting the results of a competition assay demonstrating the ability of anti-PD-1 antibodies to compete with PD-L2-Fc for binding to CHO-K1 cells stably transfected with human PD-1. [Figure 8] Figure 8A is a graph showing the agonistic activity performance of anti-PD-1 antibodies in a bead-based CD4+ T cell agonist assay using a 2:1 bead-to-cell ratio, and Figure 8B is a graph showing the agonistic activity performance of anti-PD-1 antibodies in a bead-based CD4+ T cell agonist assay using a 1:1 bead-to-cell ratio. [Figure 9] Figure 9A is a graph showing the agonistic activity performance of anti-PD-1 antibodies in a bead-based CD4+ T cell agonist assay using a 4:1 bead-to-cell ratio, Figure 9B is a graph showing the agonistic activity performance of anti-PD-1 antibodies in a bead-based CD4+ T cell agonist assay using a 2:1 bead-to-cell ratio, and Figure 9C is a graph showing the agonistic activity performance of anti-PD-1 antibodies in a bead-based CD4+ T cell agonist assay using a 1:1 bead-to-cell ratio. [Figure 10A] FIG. 10A is a graph showing the mean % inhibition of IFNγ production across multiple donors in a bead-based CD4+ T cell agonist assay for anti-PD-1 antibodies. [Figure 10B] FIG. 10B is a chart providing a description of the anti-PD-1 antibodies, the % inhibition of IFNγ, and the number of donors included in FIG. 10A. [Figure 11A] FIG. 11A is a graph showing the mean % inhibition of IFNγ production across multiple donors in a bead-based CD4+ T cell agonist assay for anti-PD-1 antibodies. [Figure 11B]Figure 11B is a graph showing the mean % inhibition of IFNγ production across the same donors in a bead-based CD4+ T cell agonist assay for the reference PD-1 agonist, PD-L1-Fc. [Figure 11C] FIG. 11C is a chart providing the candidate antibodies, antibody description, % inhibition of IFNγ, and the number of donors included in FIGS. 11A and 11B. [Figure 12A] Figure 12A is a graph showing the agonist potency of anti-PD-1 antibodies in inhibiting IL-2 production in a plate-based human PBMC agonist assay (donor #747). [Figure 12B] Figure 12B is a graph showing the agonistic potency of anti-PD-1 antibodies and PD-L1-Fc in inhibiting IL-2 production in a plate-based human PBMC agonist assay (donor #500). [Figure 13A] Figure 13A is a graph showing the agonist potency of anti-PD-1 antibodies and PD-L1-Fc in inhibiting IL-2 production in a plate-based human PBMC agonist assay (frozen donor #500). [Figure 13B] Figure 13B is a graph showing the agonist potency of anti-PD-1 antibodies and PD-L1-Fc in inhibiting IL-2 production in a plate-based human PBMC agonist assay (frozen donor #500). [Figure 14A] Figure 14A is a graph showing the agonist potency of anti-PD-1 antibodies and PD-L1-Fc in inhibiting IL-2 production in a plate-based human PBMC agonist assay (frozen donor #1202). [Figure 14B] Figure 14B is a graph showing the agonist potency of anti-PD-1 antibodies and PD-L1-Fc in inhibiting IL-2 production in a plate-based human PBMC agonist assay (frozen donor #1202). [Figure 15A]Figure 15A is a graph showing the observed agonistic activity of PD-L1-Fc tetramer in a human whole blood tetanus recall assay, and the lack of agonistic activity of nivolumab in the presence of blocking anti-PD-L1 / anti-PD-L2. [Figure 15B] Figure 15B is a graph showing the observed agonistic activity of PD-1 agonist antibodies in a human whole blood tetanus recall assay in the presence of blocking anti-PD-L1 / anti-PD-L2. [Figure 15C] Figure 15C is a graph showing the effect of WT IgG1 anti-PD-1 agonist antibody on IFNγ in a human whole blood tetanus recall assay (closed triangle data points). [Figure 15D] Figure 15D is a graph showing the effect of IgG2 isotype anti-PD-1 antibodies on IFNγ in a human whole blood tetanus recall assay (open triangle data points). [Figure 16A] FIG. 16A is a schematic diagram of the xenogeneic NSG / Hu-PBMC mouse model for studying graft-versus-host disease as described in Example 8, according to an embodiment of the present invention. [Figure 16B] FIG. 16B is a schematic diagram showing the timeline, dosing schedule, and model groups of the NSG / Hu-PBMC graft-versus-host disease study described in Example 8, according to an embodiment of the present invention. [Figure 16C] Figure 16C is a graph showing the results of the time to >10% weight loss from the NSG / Hu-PBMC graft-versus-host disease study in Example 8 for anti-PD-1 antibodies. [Figure 16D] Figure 16D is a graph showing the results of the time to >10% body weight loss from the NSG / Hu-PBMC graft-versus-host disease study in Example 8 for anti-PD-1 antibodies. [Figure 17A] Figure 17A is a graph showing the pharmacokinetic profile of anti-PD-1 antibodies in cynomolgus monkeys following a single intravenous or subcutaneous dose of 10 mg / kg. [Figure 17B]Figure 17B is a graph showing the pharmacokinetic profiles of anti-PD-1 antibodies in cynomolgus monkeys following a single intravenous or subcutaneous dose of 10 mg / kg. [Figure 18A] Figure 18A is a graph showing CD3+ T cell PD-1 receptor occupancy in cynomolgus monkeys after a single 10 mg / kg intravenous or subcutaneous dose of anti-PD-1 antibody. [Figure 18B] Figure 18B is a graph showing CD3+ T cell PD-1 receptor occupancy in cynomolgus monkeys after a single intravenous or subcutaneous dose of 10 mg / kg of anti-PD-1 antibody. [Figure 19A] Figure 19A is an SDS-PAGE gel showing the results of immunoblotting of PD-1 immunoprecipitates with either anti-PD-1 (top), anti-SHP2 (middle), or anti-SHP1 (bottom). [Figure 19B] FIG. 19B is a graph showing densitometric quantification of the immunoblot shown in FIG. 19A. [Figure 20] Figure 20A shows a space-filling model of the crystal structure of the human PD-L1 extracellular binding domain (gray) docked with a ribbon model representation of the crystal structure of the human PD-1 extracellular domain (black). The molecule is oriented with the membrane-proximal region of PD-1 at the bottom left. Figure 20B shows a space-filling model of the crystal structure of the human PD-L1 extracellular binding domain (gray) docked with a ribbon model representation of the crystal structure of the human PD-1 extracellular domain (black). The molecule has been rotated 90° compared to the representation of the molecule shown in Figure 20A, with the membrane-proximal region of PD-1 at the bottom center. [Figure 21A] Figure 21A is a graph showing the effect of IgG1 3.7C6 anti-PD-1 antibody compared to the IgG1 isotype on secreted IFNγ in PBMCs from alopecia areata donors stimulated with keratinocyte antigens. [Figure 21B] Figure 21B is a graph showing the effect of PD-L1-IgG1 Fc tetramers compared to IgG1 isotype tetramers on secreted IFNγ in PBMCs from alopecia areata donors stimulated with keratinocyte antigens. [Figure 21C]Figure 21C is a graph showing the effect of IgG1 3.7C6 anti-PD-1 antibody on the number of IFNγ spot-forming cells (SFCs) in PBMCs isolated from alopecia areata donors stimulated with keratinocyte antigens. [Figure 21D] Figure 21D is a graph showing the effect of PD-L1 IgG1-Fc tetramer on the number of IFNγ spot-forming cells (SFCs) in PBMCs isolated from alopecia areata donors stimulated with keratinocyte antigens. [Figure 22] Figure 22A is a graph showing the effect of IgG1 3.7C6 anti-PD-1 antibody compared to the IgG1 isotype on secreted INFγ and IL-17A in a tetanus toxoid-specific antigen recall assay. Figure 22B is a graph showing the effect of IgG1 3.7C6 anti-PD-1 antibody compared to the IgG1 isotype on secreted IL-17A in a tetanus toxoid-specific antigen recall assay. [Figure 23A] Figure 23A is a graph showing the effect of IgG1 3.7C6 anti-PD-1 antibody compared to the igG1 isotype on secreted IFNγ in PBMCs from alopecia areata donors stimulated with melanocyte antigens. [Figure 23B] Figure 23B is a graph showing the effect of PD-L1-IgG1-Fc tetramers compared to IgG1 isotype tetramers on secreted IFNγ in PBMCs from alopecia areata donors stimulated with melanocyte antigens. [Figure 23C] Figure 23C is a graph showing the effect of IgG1 3.7C6 anti-PD-1 antibody on the number of IFNγ SFCs in PBMCs isolated from alopecia areata donors stimulated with melanocyte antigens. [Figure 23D] Figure 23D is a graph showing the effect of PD-L1 IgG1-Fc tetramer on the number of IFNγ SFCs in PBMCs isolated from alopecia areata donors stimulated with melanocyte antigens. [Figure 24A]FIG. 24A is a schematic diagram of the xenogeneic NSG / Hu-PBMC mouse model for studying graft-versus-host disease as described in Example 15, according to an embodiment of the present invention. [Figure 24B] FIG. 24B is a schematic diagram showing the timeline, dosing schedule, and model groups of the NSG / Hu-PBMC graft-versus-host disease study described in Example 15, according to an embodiment of the present invention. [Figure 24C] Figure 24C is a graph showing the time to death results of the NSG / Hu-PBMC graft-versus-host disease study in Example 15 for the anti-PD-1 agonist IgG1 antibody 3.7C6. [Figure 24D] FIG. 24D is a graph showing the results of percent body weight change from the start of the study for individual animals relative to the isotype control over the course of the study. [Figure 24E] Figure 24E is a graph showing the results of percent body weight change from the start of the study for individual animals for the anti-PD-1 agonist IgG1 antibody 3.7C6 at a dose of 30 mg / kg over the course of the study. [Figure 24F] Figure 24F is a graph showing the results of percent body weight change from the start of the study for individual animals for the anti-PD-1 agonist IgG1 antibody 3.7C6 at a dose of 10 mg / kg over the course of the study. [Figure 24G] Figure 24G is a graph showing the results of percent body weight change from the start of the study for individual animals for the anti-PD-1 agonist IgG1 antibody 3.7C6 at a dose of 3 mg / kg over the course of the study. [Figure 24H] FIG. 24H is a graph showing the results of percent body weight change from the start of the study for individual animals for the positive control CTLA-4-Ig over the course of the study. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description of the Invention The present invention provides PD-1 binding agents. As discussed above, programmed death 1 (PD-1) (also known as programmed cell death 1) is a 268-amino acid type I transmembrane protein (Ishida et al., supra). PD-1 is a member of the CD28 / CTLA-4 family of T cell regulators and is reported to be expressed on activated T cells, B cells, and myeloid cells (Greenwald et al., supra; and Sharpe et al., supra). PD-1 contains an extracellular IgV domain followed by a short extracellular stalk, a transmembrane region, and an intracellular tail. The PD-1 intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, which function to negatively regulate T cell receptor signaling by recruiting tyrosine phosphatases when phosphorylated (see, e.g., Ishida et al., supra; and Blank et al., supra).

[0013] In some embodiments, the PD-1-binding agents provided herein are agonists, meaning that they bind to PD-1 but do not significantly inhibit the binding of PD-1 to a PD-1 ligand, thereby maintaining the ability of PD-1 to negatively regulate T cell receptor signaling. According to certain embodiments, the PD-1-binding agents provided herein can induce or stimulate the ability of PD-1 to negatively regulate T cell receptor signaling and suppress an immune response. In certain embodiments, PD-1-binding agents are provided that bind to PD-1 at an epitope comprising, consisting essentially of, or consisting of residues 33-41 of human PD1 (sequence: NPPTFSPAL) and / or 96-110 of human PD-1 (sequence: RVTQLPNGRDFHMSV).

[0014] The PD-1-binding agent comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, each of which contains three complementarity-determining regions (CDRs), typically referred to as CDR1, CDR2, or CDR3. The CDR regions are also referred to as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, using "H" or "L" to denote heavy or light chain, respectively, in the nomenclature. The CDRs of a given Ig sequence can be determined by any of several conventional numbering schemes, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo (see, e.g., Kabat, et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH (1991); Chothia, et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol., 196:901-903). 917(1987);Al-Lazikani et al.,Standard Conformations for the Canonical Structures of Immunoglobulins,J. Mol. Biol.,273:927-948(1997);Abhinandan et al.,Analysis and Improvements to Kabat and Structurally Correct Numbering of Antibody Variable Domains,Mol. Immunol.,45:3832-3839(2008);Lefranc et al.,The IMGT unique numbering for immunoglobulins,T cell Receptors and Ig-like domains,The Immunologist,7:132-136(1999);Lefranc et al.,IMGT unique numbering for immunoglobulin and T cell receptor variable domains and I superfamily V-like domains,Dev. Comp. Immunol.,27:55-77(2003); and Honegger et al.,Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J. Mol. Biol. 309:657-670 (2001)).

[0015] According to one aspect of the invention, the immunoglobulin heavy chain variable region of the PD-1-binding agent comprises a CDR1 comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO:2, and a CDR3 comprising SEQ ID NO:3; and the immunoglobulin light chain variable region comprises a CDR1 comprising SEQ ID NO:4, a CDR2 comprising SEQ ID NO:5, and a CDR3 comprising SEQ ID NO:6. In some embodiments, the heavy chain CDR1 comprises any one of SEQ ID NOs:13-18. Additionally or alternatively, some embodiments of the heavy chain CDR3 comprise any one of SEQ ID NOs:19-21. Additionally, the light chain CDR1 may comprise SEQ ID NO:22 or 23.

[0016] In certain embodiments, the PD-1-binding agent may comprise an immunoglobulin heavy chain variable region of any one of SEQ ID NOs:24-33, or an amino acid sequence having at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to any one of SEQ ID NOs:24-33. In other embodiments, the PD-1-binding agent comprises an immunoglobulin heavy chain variable region comprising the CDRs of any of SEQ ID NOs:24-33, where the CDRs are as provided above or as determined by any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo). Optionally, an immunoglobulin heavy chain variable region comprising the CDRs of any of SEQ ID NOs: 24-33 also has an amino acid sequence having at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to any of SEQ ID NOs: 24-33.

[0017] In addition to, or alternatively, the Ig heavy chain variable regions described above, the anti-PD-1-binding agent can comprise the immunoglobulin light chain variable region of SEQ ID NO: 34 or 35, or an amino acid sequence having at least 80%, 85%, or 90% sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 34 or 35. In other embodiments, the PD-1-binding agent comprises an immunoglobulin light chain variable region comprising the CDRs of SEQ ID NO: 34 or 35, where the CDRs are as provided above or as determined by any of a variety of known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo). Optionally, an immunoglobulin light chain variable region comprising the CDRs of SEQ ID NO: 34 or 35 also has an amino acid sequence having at least 80%, 85%, or 90% sequence identity to SEQ ID NO: 34 or 35 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity).

[0018] According to one embodiment, the PD-1-binding agent comprises an immunoglobulin heavy chain variable region of SEQ ID NO:29, or an amino acid sequence having at least 80%, 85%, or 90% sequence identity thereto (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity); or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO:29, wherein the CDR regions are as provided above (e.g., CDR1—SEQ ID NO:15, CDR2—SEQ ID NO:2, and CDR3—SEQ ID NO:20) or are numbered according to various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced and an immunoglobulin light chain variable region of SEQ ID NO: 35 or at least 80%, 85%, or 90% sequence identity thereto (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least and / or an immunoglobulin light chain variable region comprising at least the CDRs of SEQ ID NO: 35 (wherein the CDR regions are as provided above (e.g., CDR1—SEQ ID NO: 23, CDR2—SEQ ID NO: 5, and CDR3—SEQ ID NO: 6) or as determined by any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo)).In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO:29 and the light chain variable region of SEQ ID NO:35, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO:29 and the light chain variable region of SEQ ID NO:35, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO:29 and the light chain variable region of SEQ ID NO:35, or at least the CDRs thereof as determined by Martin ... In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 35, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 35, or at least the CDRs thereof as determined by AHo. As a further example, the anti-PD-1 binding agent can comprise an immunoglobulin heavy chain comprising SEQ ID NO: 36 and an immunoglobulin light chain comprising SEQ ID NO: 37, or amino acid sequences having at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NOs: 36 and 37, respectively, optionally wherein the sequences retain the heavy and light chain CDRs of SEQ ID NOs: 36 and 37, respectively, wherein the CDRs are as provided above or are numbered according to various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo).

[0019] According to another embodiment, the PD-1-binding agent comprises an immunoglobulin heavy chain variable region of SEQ ID NO:24, or an amino acid sequence having at least 80%, 85%, or 90% sequence identity thereto (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto); or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO:24, wherein the CDR regions are as provided above (e.g., CDR1—SEQ ID NO:13, CDR2—SEQ ID NO:2, and CDR3—SEQ ID NO:19) or are numbered according to various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced and an immunoglobulin light chain variable region of SEQ ID NO: 34 or at least 80%, 85%, or 90% sequence identity thereto (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least or 100% sequence identity) or an immunoglobulin light chain variable region comprising at least the CDRs of SEQ ID NO: 34 (wherein the CDR regions are as provided above (e.g., CDR1—SEQ ID NO: 22, CDR2—SEQ ID NO: 5, and CDR3—SEQ ID NO: 6) or as determined by any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo)).In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO:24 and the light chain variable region of SEQ ID NO:34, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO:24 and the light chain variable region of SEQ ID NO:34, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO:24 and the light chain variable region of SEQ ID NO:34, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO:24 and the light chain variable region of SEQ ID NO:34, or at least the CDRs thereof as determined by IGMT ... For example, the antibody comprises the heavy chain variable region of SEQ ID NO: 24 and the light chain variable region of SEQ ID NO: 34, or at least the CDRs thereof as determined by AHo.

[0020] According to one embodiment, the PD-1-binding agent comprises an immunoglobulin heavy chain variable region of SEQ ID NO: 30, or an amino acid sequence having at least 80%, 85%, or 90% sequence identity thereto (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity); or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO: 30, wherein the CDR regions are as provided above (e.g., CDR1—SEQ ID NO: 15, CDR2—SEQ ID NO: 2, and CDR3—SEQ ID NO: 21) or are numbered according to various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced and an immunoglobulin light chain variable region of SEQ ID NO: 35 or at least 80%, 85%, or 90% sequence identity thereto (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least 1 and / or an immunoglobulin light chain variable region comprising at least the CDRs of SEQ ID NO: 35 (wherein the CDR regions are as provided above (e.g., CDR1—SEQ ID NO: 23, CDR2—SEQ ID NO: 5, and CDR3—SEQ ID NO: 6) or as determined by any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo)).In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 30 and the light chain variable region of SEQ ID NO: 35, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 30 and the light chain variable region of SEQ ID NO: 35, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 30 and the light chain variable region of SEQ ID NO: 35, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 30 and the light chain variable region of SEQ ID NO: 35, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 30 and the light chain variable region of SEQ ID NO: 35, or at least the CDRs thereof as determined by AHo.

[0021] According to another aspect, the anti-PD-1 binding agent comprises an immunoglobulin heavy chain variable region comprising a CDR1 comprising SEQ ID NO:7, a CDR2 comprising SEQ ID NO:8, and a CDR3 comprising SEQ ID NO:9; and an immunoglobulin light chain variable region comprising a CDR1 comprising SEQ ID NO:10, a CDR2 comprising SEQ ID NO:11, and a CDR3 comprising SEQ ID NO:12. In some embodiments, the heavy chain CDR1 comprises any of SEQ ID NOs:57-60. In some embodiments, the heavy chain CDR2 comprises any one of SEQ ID NOs:38-42.

[0022] In some embodiments, the PD-1-binding agent comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 43-47 or 61-63, or a PD-1-binding domain that shares at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 1109%, at least 1111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least In some embodiments, the PD-1-binding agent comprises an amino acid sequence having at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the PD-1 binding agent. In some embodiments, the PD-1-binding agent comprises an immunoglobulin heavy chain variable region comprising the CDRs of any of SEQ ID NOs: 43-47 or 61-63, wherein the CDRs are as provided above or as determined by any of a variety of known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo). Optionally, an immunoglobulin heavy chain variable region comprising the CDRs of any one of SEQ ID NOs: 43-47 or 61-63 also has at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to any one of SEQ ID NOs: 43-47 or 61-63.

[0023] In addition to, or alternatively to, the Ig heavy chain variable regions described above (e.g., SEQ ID NOS: 43-47 or 61-63), the anti-PD-1 binding agent can comprise an immunoglobulin light chain variable region of any of SEQ ID NOS: 48-50, or an amino acid sequence with at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to any one of SEQ ID NOS: 48-50. In other embodiments, the PD-1-binding agent comprises an immunoglobulin light chain variable region comprising the CDRs of any of SEQ ID NOs: 48-50, where the CDRs are as provided above or as determined by any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo). Optionally, an immunoglobulin light chain variable region comprising the CDRs of any of SEQ ID NOs: 48-50 also has an amino acid sequence having at least 80%, 85%, or 90% sequence identity to any of SEQ ID NOs: 48-50 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity).

[0024] In specific embodiments, the anti-PD-1 binding agent comprises an immunoglobulin heavy chain variable region of SEQ ID NO: 47, or an amino acid sequence having at least 80%, 85%, or 90% sequence identity to SEQ ID NO: 47 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity); or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO: 47 (wherein the CDR regions are as provided above (e.g., for example, CDR1—SEQ ID NO:57, CDR2—SEQ ID NO:42, and CDR3—SEQ ID NO:9) or as determined by any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo); and an immunoglobulin light chain variable region of SEQ ID NO:49, or at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 13 or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO:49 (wherein the CDR regions are as provided above (e.g., CDR1—SEQ ID NO:10, CDR2—SEQ ID NO:11, and CDR3—SEQ ID NO:12) or according to various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo). In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 47 and a light chain variable region of SEQ ID NO: 49, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 47 and a light chain variable region of SEQ ID NO: 49, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 47 and a light chain variable region of SEQ ID NO: 49, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 47 and a light chain variable region of SEQ ID NO: 49, or at least the CDRs thereof as determined by IGMT.In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 47 and the light chain variable region of SEQ ID NO: 49, or at least the CDRs thereof as determined by AHo. As a further example, the anti-PD-1 binding agent can comprise an immunoglobulin heavy chain comprising SEQ ID NO:51 and an immunoglobulin light chain comprising SEQ ID NO:52, or an amino acid sequence with at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NOs:51 and 52, optionally wherein the sequences are numbered according to any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced The heavy and light chain CDRs of SEQ ID NOs: 51 and 52 are retained as determined according to either the NIH (Immuno-Chothia), IGMT, or AHo).

[0025] In another embodiment, the anti-PD-1 binding agent comprises an immunoglobulin heavy chain variable region of SEQ ID NO:46, or an amino acid sequence having at least 80%, 85%, or 90% sequence identity to SEQ ID NO:46 (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity); or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO:46 (wherein the CDR regions are as provided above (e.g., CDR1—SEQ ID NO:57, CDR2—SEQ ID NO:41, and CDR3—SEQ ID NO:9) or are numbered according to various known immunoglobulin numbering schemes (e.g., Kabat, Ch and an immunoglobulin light chain variable region of SEQ ID NO: 50, or at least 80%, 85%, or 90% sequence identity (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least 14 or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO:50 (wherein the CDR regions are as provided above (e.g., CDR1—SEQ ID NO:10, CDR2—SEQ ID NO:11, and CDR3—SEQ ID NO:12) or as determined by any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo)). In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO:46 and a light chain variable region of SEQ ID NO:50, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO:46 and a light chain variable region of SEQ ID NO:50, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 46 and a light chain variable region of SEQ ID NO: 50, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 46 and a light chain variable region of SEQ ID NO: 50, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody comprises a heavy chain variable region of SEQ ID NO: 46 and a light chain variable region of SEQ ID NO: 50, or at least the CDRs thereof as determined by AHo.

[0026] The sequence "identity" described herein can be determined by comparing a nucleic acid or amino acid sequence of interest with a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., identical) between the sequence of interest and the reference sequence, divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). Many mathematical algorithms for obtaining optimal alignment and calculating the identity between two or more sequences are known and are incorporated into many available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for aligning nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and their later versions), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity search). Sequence alignment algorithms are also disclosed, for example, in Altschul et al., J. Molecular Biol., 215(3):403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7):951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0027] Variations in sequence identity can be achieved by the addition, substitution, or deletion of one or more amino acid residues. An amino acid "replacement" or "substitution" refers to the replacement of one amino acid at a given position or residue in a polypeptide sequence with another amino acid at the same position or residue. Amino acid replacements or substitutions can be conservative, semi-conservative, or non-conservative, depending on whether the substitution is with an amino acid residue with similar properties to the replaced residue. A practical method for defining common characteristics between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, groups of amino acids can be defined when the amino acids within the group preferentially exchange with each other and are therefore most similar to each other in their impact on overall protein structure (Schulz and Schirmer, supra).

[0028] Amino acids can be broadly grouped as "aromatic" or "aliphatic." Aromatic amino acids contain an aromatic ring. Examples of "aromatic" amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly grouped as "aliphatic." Examples of "aliphatic" amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine ​​(C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R or Arg).

[0029] Aliphatic amino acids can be subdivided into four subgroups: the "large aliphatic non-polar subgroup" consists of valine, leucine, and isoleucine; the "aliphatic slightly polar subgroup" consists of methionine, serine, threonine, and cysteine; the "aliphatic polar / charged subgroup" consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine; and the "small residue subgroup" consists of glycine and alanine. The group of charged / polar amino acids can be subdivided into three subgroups: the "positively charged subgroup" consisting of lysine and arginine, the "negatively charged subgroup" consisting of glutamic acid and aspartic acid, and the "polar subgroup" consisting of asparagine and glutamine.

[0030] The aromatic amino acids can be subdivided into two subgroups: the "nitrogen ring subgroup" consisting of histidine and tryptophan, and the "phenyl subgroup" consisting of phenylalanine and tyrosine.

[0031] Examples of conservative amino acid substitutions include substitutions of amino acids within the above subgroups, such as lysine for arginine and vice versa, so that a positive charge can be maintained, glutamic acid for aspartic acid and vice versa, so that a negative charge can be maintained, serine for threonine, so that a free -OH can be maintained, and glutamine for asparagine, so that a free -NH can be maintained. "Semi-conservative mutation" includes amino acid substitutions of amino acids within the same group listed herein but not within the same subgroup. For example, substitutions of aspartic acid for asparagine or asparagine for lysine include amino acids within the same group but from different subgroups. "Non-conservative mutations" include substitutions of amino acids between different groups, for example, lysine for tryptophan, phenylalanine for serine, etc.

[0032] In some embodiments, the PD-1-binding agent can comprise, consist essentially of, or consist of immunoglobulin heavy and light chain variable regions or the complete heavy and light chain polypeptides provided herein. The isolated PD-1-binding agent can be any type of molecule or construct comprising at least a particular immunoglobulin heavy and light chain variable region. That is, the PD-1-binding agent can be, for example, an entire immunoglobulin or antibody described herein, or an antigen-binding (PD-1-binding) immunoglobulin or antibody "fragment." The term "fragment," as used with respect to an antibody or immunoglobulin, refers to any molecule or construct that comprises some portion of an immunoglobulin or antibody and binds to a target antigen. Such fragments generally contain at least a portion of the heavy and light chain variable regions, including the CDRs, and may also include portions of the constant regions, optionally along with other elements not normally part of immunoglobulins or antibodies (e.g., linkers, etc.). Examples of such "fragments" include: (i) V L , V H , C L(ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a single-chain V of an antibody. L and V H (iv) Fab' fragments resulting from cleavage of the disulfide bridges of the F(ab')2 fragment using mild reducing conditions; (v) diabodies; (vi) single-chain variable regions (scFv), and (vii) disulfide-stabilized Fv fragments (dsFv).

[0033] In some embodiments, the PD-1-binding agent comprises an immunoglobulin heavy chain constant region, such as a fragment crystallizable (Fc) region or portion thereof. The Fc region can be any Ig class / subclass (including variants thereof, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), IgM. In certain embodiments, the PD-1-binding agent comprises an Fc region that binds to an Fc receptor on an antigen-presenting cell (e.g., a dendritic cell, macrophage, Langerhans cell, or B cell). Fc receptors include FcγRI (CD64), FcγRIIA (CD32), FcγRII (CD33), FcγRII (CD34), FcγRII (CD35), FcγRII (CD36), FcγRII (CD37), FcγRII (CD38), FcγRII (CD39 ... The PD-1-binding agent may be an Fcγ receptor (FcγR), such as FcγRIIIB (CD32), FcγRIIIA (CD16a), or FcγRIIIB (CD16b). In one embodiment, the PD-1-binding agent comprises an Fc region that binds to an FcγR, such as IgG1. Thus, in some embodiments, the PD-1-binding agent is a "whole" or "complete" Ig (i.e., antibody). In additional embodiments, the PD-1-binding agent is an IgG antibody, particularly an IgG1 antibody.

[0034] The isolated PD-1-binding agent may also be an antibody conjugate. In this regard, the isolated PD-1-binding agent may be a conjugate comprising a PD-1-binding agent (e.g., an anti-PD-1 antibody or antibody fragment) and another biologically active moiety. For example, the PD-1-binding agent may be conjugated to a peptide, a fluorescent molecule, or a chemotherapeutic agent, particularly an agent useful in suppressing an immune response.

[0035] The isolated PD-1-binding agent can be or can be derived from a human antibody, a non-human antibody, or a chimeric antibody. By "chimeric" is meant an antibody or fragment thereof that contains both human and non-human regions. Preferably, the isolated PD-1-binding agent is a humanized antibody. A "humanized" antibody is a monoclonal antibody that contains a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat). A humanized antibody is an antibody that contains at least one CDR obtained or derived from a non-human antibody. In a preferred embodiment of the invention, the CDRH3 of a PD-1-binding agent of the invention is obtained or derived from a murine monoclonal antibody, while the remaining variable and constant regions of the PD-1-binding agent of the invention are obtained or derived from a human monoclonal antibody.

[0036] Human, non-human, chimeric, or humanized antibodies can be obtained by any means, including via in vitro sources (e.g., hybridomas or cell lines that recombinantly produce antibodies) and in vivo sources (e.g., rodents). Methods for producing antibodies are known in the art and are described, for example, in Koehler and Milstein, Eur. J. Immunol., 5:511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al.(eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY(2001);Starkie et al., PLoS One, 11(3):e0152282 (2016)). In certain embodiments, human or chimeric antibodies can be produced using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes have been replaced with one or more human immunoglobulin genes. Examples of transgenic mice in which endogenous antibody genes have been effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSE™, Kirin TC MOUSE™, and Kyowa Kirin KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25 (2005) and Lonberg, Handb. Exp. Pharmacol., 181:69-97 (2008)). Humanized antibodies can be produced using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey (2009)), including, for example, grafting non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36(1):25-34 (2005); and Hou et al., J. Biochem., 144(1):115-120 (2008)). In one embodiment, humanized antibodies can be produced using, for example, the methods described in U.S. Patent Application Publication No. 2011 / 0287485A1.

[0037] The PD-1 binding agent can have any suitable affinity for human PD-1. The term "affinity" refers to the equilibrium constant for the reversible binding of two agents, and is expressed as the dissociation constant (K DThe affinity of a binding agent for a ligand, such as the affinity of an antibody for an epitope, can be, for example, from about 1 picomolar (pM) to about 100 micromolar (μM) (e.g., from about 1 picomolar (pM) to about 1 nanomolar (nM), from about 1 nM to about 1 micromolar (μM), or from about 1 μM to about 100 μM). In one embodiment, the PD-1-binding agent has a K of 1 nM or less (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values). D In another embodiment, the PD-1-binding agent can bind to a PD-1 protein having a K of 200 pM or less (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoing values). D In some embodiments, the PD-1-binding agent is cross-reactive with cynomolgus monkey PD-1 with an affinity within any of the ranges discussed above for human PD-1. Immunoglobulin affinity for an epitope can be measured using any art-recognized assay, including, for example, fluorescence-activated cell sorting (FACS), separable beads (e.g., magnetic beads), surface plasmon resonance (SPR), liquid-phase competition (KinExA®), antigen panning, and / or ELISA (see, e.g., Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY, 2001).

[0038] PD-1-binding agents bind to PD-1 but preferably do not completely inhibit the ability of PD-1 to negatively regulate an immune response, or in some cases do not substantially inhibit or even enhance the ability of PD-1 to negatively regulate an immune response. In some embodiments, the PD-1-binding agent does not completely block, or preferably does not substantially reduce, the binding between PD-1 and PD-L1. Assessment of the extent to which a PD-1-binding agent inhibits PD-1 regulation of an immune response or PD-1 binding to PD-L1 can be performed using assays such as those described in the Examples or other assays known in the art. In some embodiments, the PD-1-binding agent inhibits PD-1 binding to PD-L1 by about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, or about 10% or less.

[0039] Methods of Use / Treatment The present invention provides methods for suppressing immune responses, particularly T cell-mediated immune responses, in a mammal by administering to the mammal a PD-1-binding agent described herein. The present invention further provides methods for treating any disease or disorder in which a decrease in PD-1 activity (e.g., a decrease in PD-1 signaling through decreased PD-L1 binding, such as a decrease in negative regulation of the immune system) causes or contributes to the pathological effects of the disease, or any disease or disorder in which an increase in PD-1 activity (e.g., an increase in PD-1 signaling through PD-L1 binding, such as an increase in negative regulation of the immune system) would have a therapeutic effect, the method comprising administering to a mammal a PD-1-binding agent described herein to reduce or eliminate any symptoms of the disorder, or prevent or inhibit the onset of such symptoms. As used herein, negative regulation of the immune system is synonymous with immunosuppression. It will be appreciated that in some cases, the PD-1-binding agent can be administered prior to the onset of symptoms (e.g., prior to exposure to an antigen that elicits an immune response) to prevent, suppress, or reduce the severity of the immune response upon introduction of the antigen.

[0040] The disease or disorder may be an inflammatory or autoimmune disorder. Examples of inflammatory or autoimmune disorders include, for example, infectious diseases (viral, bacterial, fungal, and parasitic), endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Behcet's disease, Alzheimer's disease, inflammatory bowel disease including Crohn's disease and ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, Pyronidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, antineutrophil cytoplasmic antibody-associated (ANCA) vasculitis, surgical adhesions, stroke, type I diabetes, Lyme disease, and arthritis. Immune-mediated inflammatory disorders of the central and peripheral nervous system such as meningoencephalitis, autoimmune vasculitis, multiple sclerosis, lupus (such as systemic lupus erythematosus and chronic discoid lupus erythematosus) and Guillain-Barr syndrome, atopic dermatitis, polymyositis, dermatomyositis, autoimmune liver disease, fibrosing alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, pemphigoid, primary biliary cholangitis, hepatitis, sarcoidosis, and scleroderma (localized scleroderma). , systemic sclerosis, and progressive systemic sclerosis), sarcomatosis with polyangiitis, other autosomal dominant disorders, cholangitis, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, heart disease including ischemic diseases such as myocardial infarction and atherosclerosis, arteritis nodosa (polyarteritis nodosa and microscopic polyangiitis), allergic granulomatous vasculitis, hypersensitivity vasculitis, aortitis syndrome (Takayasu's arteritis), temporal arteritis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochloremia, Still's disease, Cogan's syndrome, RS3PE, polymyalgia rheumatica, fibromyalgia syndrome, antiphospholipid syndrome, eosinophilic myositis, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megakaryoblastic anemia, hemolytic anemia, autoimmune neutropenia, Hashimoto's thyroiditis, autoimmune adrenal insufficiency, primary hypothyroidism, idiopathic Addison's disease (chronic adrenal insufficiency), herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent fetal loss, or infertility related to lack of feto-maternal tolerance.

[0041] In some embodiments, the disease or disorder is giant cell arteritis, polymyalgia rheumatica, primary Sjogren's syndrome, TNF-resistant rheumatoid arthritis, alopecia areata, primary biliary cholangitis (PBC), graft-versus-host disease (GvHD), vitiligo, ANCA vasculitis, type 1 diabetes, or non-infectious uveitis.

[0042] An "immune response" can involve, for example, antibody production and / or activation of immune effector cells (e.g., T cells), production of inflammatory cytokines, or any of the indications or disorders described herein or otherwise known in the art. As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is curative, i.e., the effect partially or completely cures the disease and / or adverse symptoms resulting from the disease. To this end, the methods of the invention involve administering a "therapeutically effective amount" of a PD-1-binding agent. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the PD-1-binding agent to elicit a desired response in an individual.

[0043] Alternatively, the pharmacological and / or physiological effect can be prophylactic, i.e., the effect completely or partially prevents the disease or condition. In this regard, the methods of the invention involve administering a "prophylactically effective amount" of a PD-1-binding agent. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of disease onset).

[0044] The PD-1-binding agent can be part of a composition suitable for administration to a mammal. Preferably, the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition comprising a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and an amino acid sequence, antigen-binding agent, or vector of the invention. Any suitable carrier can be used within the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition can also include any other excipients used in the formulation of therapeutic molecules (e.g., proteins or antibodies), particularly parenteral formulations, including, for example, buffers, tonicity modifiers, stabilizers, surfactants, etc. The composition can optionally be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be produced according to conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0045] Typical doses of PD-1-binding agents can be, for example, in the range of 1 pg / kg to 20 mg / kg of animal or human body weight; however, doses below or above this exemplary range are within the scope of the invention. Daily parenteral dosages may range from about 0.00001 μg / kg to about 20 mg / kg of total body weight (e.g., about 0.001 μg / kg, about 0.1 μg / kg, about 1 μg / kg, about 5 μg / kg, about 10 μg / kg, about 100 μg / kg, about 500 μg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, or a range defined by any two of the foregoing values), preferably from about 0.1 μg / kg to about 10 mg / kg of total body weight (e.g., about 0.5 μg / kg, about 1 μg / kg, about 50 μg / kg, about 150 μg / kg, about 300 μg / kg, about 750 μg / kg, about 1.5 mg / kg, about 5 mg / kg, or or a range defined by any two of the foregoing values), more preferably about 1 μg / kg to 5 mg / kg of total body weight (e.g., about 3 μg / kg, about 15 μg / kg, about 75 μg / kg, about 300 μg / kg, about 900 μg / kg, about 2 mg / kg, about 4 mg / kg, or a range defined by any two of the foregoing values), and even more preferably about 0.5 to 15 mg / kg of body weight per day (e.g., about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 6 mg / kg, about 9 mg / kg, about 11 mg / kg, about 13 mg / kg, or a range defined by any two of the foregoing values). Therapeutic or prophylactic effectiveness can be monitored by periodic evaluation of the patient being treated. For repeated administrations over several days or longer, depending on the condition, treatment can be repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the present invention. The desired dosage can be delivered by administering a single bolus of the composition, multiple boluses of the composition, or a continuous infusion of the composition.

[0046] The PD-1 binding agent can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition is preferably suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection.

[0047] Once administered to a mammal (e.g., a human), the biological activity of the PD-1-binding agents of the invention can be measured by any suitable method known in the art. For example, biological activity can be assessed by determining the stability of a particular PD-1-binding agent. In one embodiment of the invention, the PD-1-binding agent (e.g., an antibody) has an in vivo half-life of between about 30 minutes and 45 days (e.g., about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 1 day, about 5 days, about 10 days, about 15 days, about 25 days, about 35 days, about 40 days, about 45 days, or a range defined by any two of the foregoing values). In another embodiment, the PD-1-binding agent has an in vivo half-life of between about 2 hours and 20 days (e.g., about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 2 days, about 3 days, about 7 days, about 12 days, about 14 days, about 17 days, about 19 days, or a range defined by any two of the foregoing values). In another embodiment, the PD-1-binding agent has an in vivo half-life of between about 10 days and about 40 days (e.g., about 10 days, about 13 days, about 16 days, about 18 days, about 20 days, about 23 days, about 26 days, about 29 days, about 30 days, about 33 days, about 37 days, about 38 days, about 39 days, about 40 days, or a range defined by any two of the foregoing values).

[0048] The PD-1-binding agents of the present invention can be administered alone or in combination with other active agents or drugs. For example, the PD-1-binding agents can be administered in combination with other agents for the treatment or prevention of the diseases disclosed herein. In this regard, the PD-1-binding agents can be administered in combination with, for example, other monoclonal antibodies, disease-killing viruses, gene therapies, and adoptive therapies. It can be used in combination with at least one other inflammatory or autoimmune disorder inhibitor, including T cell transfer, and / or surgery. The PD-1-binding agents of the invention described herein can also be used in combination with at least one other immunosuppressant, including, for example, methotrexate, corticosteroids, and other small molecule drugs used to treat autoimmune and inflammatory diseases. When the methods of the invention treat infectious diseases, the PD-1-binding agents can be administered in combination with at least one antibacterial agent or at least one antiviral agent. In this regard, the antibacterial agent can be any suitable antibiotic known in the art. The antiviral agent can be any suitable type of vaccine that specifically targets a particular virus, such as a live attenuated vaccine, a subunit vaccine, a recombinant vector vaccine, and a small molecule antiviral therapy (e.g., a viral replication inhibitor and a nucleoside analog).

[0049] In addition to therapeutic uses, the PD-1-binding agents described herein can be used in diagnostic or research applications. In this regard, the PD-1-binding agents can be used in methods for diagnosing cancer or infectious diseases. In a similar manner, the PD-1-binding agents can be used in assays to monitor PD-1 protein levels in subjects being tested for diseases or disorders associated with aberrant PD-1 expression. Research applications include, for example, methods that utilize the PD-1-binding agents and a label to detect PD-1 protein in a sample, e.g., in a human body fluid or in a cell or tissue extract. The PD-1-binding agents can be used with or without modification, such as covalent or non-covalent labeling with a detectable moiety. For example, the detectable moiety can be a radioisotope (e.g.,3 H, 14 C. 32 P, 35 S, or 125 The detectable moiety may be a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), an enzyme (e.g., alkaline phosphatase, beta-galactosidase, or horseradish peroxidase), or a prosthetic group. Any method known in the art for separately conjugating an antigen-binding agent (e.g., an antibody) to a detectable moiety may be used in the context of the present invention (see, e.g., Hunter et al., Nature, 194:495-496 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol. Meth., 40:219-230 (1981); and Nygren, J. Histochem. Cytochem., 30:407-412 (1982)).

[0050] PD-1 protein levels can be measured using the PD-1-binding agents of the invention by any suitable method known in the art. Such methods include, for example, radioimmunoassay (RIA) and FACS. Normal or standard expression levels of PD-1 protein can be established using any suitable technique, for example, by combining a sample containing or suspected of containing PD-1 polypeptide with a PD-1-specific antibody under conditions suitable for forming an antigen-antibody complex. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials (see, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)). The amount of PD-1 polypeptide expressed in the sample is then compared with the standard value.

[0051] The PD-1-binding agent can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. When the PD-1-binding agent is labeled with an enzyme, the kit desirably includes substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), etc., may be included. Additives may be included in the kit. The relative amounts of the various reagents may be varied to provide concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. The reagents may be provided as dry powders (typically lyophilized) that include excipients that, upon dissolution, provide a reagent solution having the appropriate concentration.

[0052] Nucleic acids, cells, manufacturing methods The present invention also provides one or more isolated or purified nucleic acid sequences encoding a PD-1-binding agent or its individual heavy or light chain immunoglobulin polypeptides. That is, in one embodiment, the nucleic acid encodes an immunoglobulin light chain variable region or a complete immunoglobulin light chain provided herein. In another embodiment, the nucleic acid encodes an immunoglobulin heavy chain variable region or a complete immunoglobulin light chain provided herein. In yet another embodiment, the nucleic acid encodes both an immunoglobulin light chain variable region or a complete immunoglobulin light chain and an immunoglobulin heavy chain variable region or a complete immunoglobulin heavy chain provided herein. Examples of nucleic acid sequences encoding immunoglobulin heavy chains are provided by SEQ ID NOS: 53 and 55, which encode the heavy chain variable regions of SEQ ID NOS: 29 and 47, respectively, and the complete heavy and light chains of SEQ ID NOS: 36 and 51, respectively. Examples of nucleic acid sequences encoding immunoglobulin light chains are provided by SEQ ID NOS: 54 and 56, which encode the light chain variable regions of SEQ ID NOS: 35 and 49, respectively, and the complete chains and light chains of SEQ ID NOS: 37 and 52, respectively.

[0053] The terms "nucleic acid" and "nucleic acid sequence" are intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which may be single-stranded or double-stranded and may contain non-natural or modified nucleotides. As used herein, the terms "nucleic acid" and "polynucleotide" refer to polymeric forms of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, i.e., double- and single-stranded DNA, and double- and single-stranded RNA. The terms also include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, as well as modified polynucleotides, such as, but not limited to, methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, although many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, etc.).

[0054] Nucleic acid can be part of a vector. Vector can be, for example, a plasmid, an episome, a cosmid, a viral vector (for example, retrovirus or adenovirus), or a phage. Suitable vectors and vector preparation methods are well known in the art (see, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0055] In addition to the nucleic acid sequences encoding immunoglobulin heavy and / or light chains, the vectors can include expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc., that provide for expression of the coding sequences in host cells. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press. , San Diego, Calif. (1990).

[0056] Numerous promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art. Representative promoter sources include, for example, viruses, mammals, insects, plants, yeast, and bacteria, and suitable promoters from these sources are readily available or can be synthetically produced based on publicly available sequences, for example, from depositories such as ATCC and other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, CA), the LACSWITCH™ system (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nuc. Acid. Res., 27:4324-4327 (1999); Nuc. Acid. Res., 28:e99 (2000); U.S. Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308:123-144(2005)).

[0057] The term "enhancer" as used herein refers to a DNA sequence that, for example, increases the transcription of a nucleic acid sequence to which it is operably linked. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. Numerous enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (e.g., from depositories such as ATCC and other commercial or individual sources). Many polynucleotides containing promoters (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, internal, or downstream of a coding sequence.

[0058] A vector may also contain a selectable marker gene. As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that allows for the specific selection of cells that express the nucleic acid sequence in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art, and are described, for example, in International Patent Applications WO 1992 / 008796 and WO 1994 / 028143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567-3570 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1-14 (1981); Santerre et al., Gene, 30:147-156 (1984); Kent et al. al.,Science,237:901-903(1987);Wigler et al.,Cell,11:223-232(1977);Szybalska & Szybalski,Proc. Natl. Acad. Sci. USA, 48:2026-2034 (1962); Lowy et al., Cell, 22:817-823 (1980); and U.S. Patent Nos. 5,122,464 and 5,770,359.

[0059] In some embodiments, the vector is an "episomal expression vector" or "episome" that can replicate in a host cell and persist as an extrachromosomal segment of DNA within the host cell under appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize the Epstein-Barr nuclear antigen 1 (EBNA1) and Epstein-Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA) and pBK-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of episomal vectors that use T antigen and the SV40 origin of replication instead of EBNA1 and oriP.

[0060] Other suitable vectors include integrative expression vectors, which may be randomly integrated into the DNA of a host cell or may contain recombination sites that allow for specific recombination between the expression vector and the host cell chromosome. Such integrative expression vectors may utilize endogenous expression control sequences of the host cell chromosome to effect expression of the desired protein. Examples of vectors that integrate in a site-specific manner include, for example, components of the flp-in system (e.g., pcDNA™5 / FRT) from Invitrogen (Carlsbad, CA) or the cre-lox system, such as can be found in the pExchange-6 core vector from Stratagene (La Jolla, CA). Examples of vectors that randomly integrate into the host cell chromosome include, for example, pcDNA3.3 (when introduced in the absence of T antigen) from ThermoFisher (Carlsbad, CA), UCOE from Millipore (Billerica, MA), and pCI or pFN10A(ACT)FLEXI™ from Promega (Madison, WI).

[0061] Viral vectors can also be used. Representative commercially available viral expression vectors include, but are not limited to, the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands), the lentivirus-based pLP1 from ThermoFisher (Carlsbad, CA), and the retroviral vector pFB-ERV plus pCFB-EGSH from Agilent (Stratagene, La Jolla, CA).

[0062] Nucleic acid sequences encoding the amino acid sequences of the present invention can be provided to cells on the same vector (i.e., in cis). A unidirectional promoter can be used to control the expression of each nucleic acid sequence. In another embodiment, a combination of bidirectional and unidirectional promoters can be used to control the expression of multiple nucleic acid sequences. Alternatively, nucleic acid sequences encoding the amino acid sequences of the present invention can be provided to a population of cells on separate vectors (i.e., in trans). Each of the nucleic acid sequences in each of the separate vectors can contain the same or different expression control sequences. The separate vectors can be provided to cells simultaneously.

[0063] Vector(s) containing nucleic acid(s) encoding the amino acid sequences of the invention can be introduced into host cells capable of expressing the encoded polypeptides, including any suitable prokaryotic or eukaryotic cell. As such, the invention provides in vitro cells or cell lines containing the vectors of the invention. The invention also provides in vitro cells or cell lines that express immunoglobulin heavy and / or light chain polypeptides or express PD-1-binding agents. Preferred host cells are those that can be grown easily and reliably, have reasonably fast growth rates, possess well-characterized expression systems, and can be easily and efficiently transformed or transfected.

[0064] Examples of suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Brevibacillus), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include various strains of E. coli (e.g., K12, HB101 (ATCC No. 33694), DH5α, DH10, MC1061 (ATCC No. 53338), and CC102).

[0065] In some embodiments, the vector is introduced into a eukaryotic cell. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhinosporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeast cells include, for example, Saccharomyces cerevisiae and Pichia pastoris.

[0066] Suitable insect cells are described, for example, in Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67:4566-4579 (1993). Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad, CA).

[0067] In some embodiments, mammalian cells are utilized in the present invention. Many suitable mammalian host cells are known in the art, many of which are available from American These cells are available from the American Type Culture Collection (ATCC, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (e.g., ATCC No. CCL61), CHO DHFR cells (e.g., Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (e.g., ATCC No. CRL1573), and 3T3 cells (e.g., ATCC No. CCL92). Other suitable mammalian cell lines are monkey COS-1 (e.g., ATCC No. CRL1650) and COS-7 cell lines (e.g., ATCC No. CRL1651), and CV-1 cell lines (e.g., ATCC No. CCL70). Further exemplary mammalian host cells include primate and rodent cell lines, including the mouse cell line NS0, a derivative of the mouse myeloma line MOPC21 (e.g., Tysabri), and transformed cell lines. Normal diploid cells, cell lines derived from in vitro culture of primary tissue, and primary explants are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, all of which are available from the ATCC. Methods for selecting appropriate mammalian host cells and for cell transformation, culture, amplification, screening, and purification are known in the art.

[0068] In some embodiments, the mammalian cells are human cells. For example, the mammalian cells can be human lymphocytes or lymphocyte-derived cell lines, such as cell lines of pre-B lymphocyte origin. Examples of human lymphocyte cell lines include, without limitation, RAMOS (e.g., CRL-1596), Daudi (e.g., CCL-213), EB-3 (e.g., CCL-85), Raji cells (e.g., CCL-86), and derivatives thereof.

[0069] Nucleic acid sequences encoding the amino acid sequences of the present invention can be introduced into cells by any suitable technique, such as "transfection," "transformation," or "transduction." As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art, such as calcium phosphate DNA coprecipitation (see, e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-assisted microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell. Biol., 7:2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after propagation of the infectious particles in appropriate packaging cells, many of which are commercially available.

[0070] The nucleic acids and cells can be used for any purpose, such as for producing the PD-1-binding agents described herein. In this regard, the present invention provides a method for producing a PD-1-binding agent, comprising culturing cells containing nucleic acids encoding heavy and / or light immunoglobulin polypeptides of the PD-1-binding agent. Stated another way, the method comprises expressing nucleic acids encoding the immunoglobulin heavy and / or light chains of the PD-1-binding agent in the cells. It will be understood that the immunoglobulin heavy and light chains can be expressed from a single nucleic acid in a given cell, or the immunoglobulin heavy and light chains can be expressed from separate nucleic acids in the same cell. The method may further comprise recovering and / or purifying the PD-1-binding agent from the cells or cell culture medium using known techniques.

[0071] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0072] The following examples describe specific anti-PD-1 antibody heavy and light chain polypeptide sequences according to embodiments of the invention. The antibodies used in these examples are as described below.

[0073] The 437M5-112 antibody was derived by single-cell PCR on sorted PD-1-binding IgG-switched B cells from the spleens of immunized mice. The 3.7C6 antibody was derived from a murine hybridoma generated by standard fusion techniques from spleen cells of immunized mice. The antibody was humanized using standard techniques described herein. The final optimized antibody was expressed in CHO cells. The antibody sequences are summarized in Tables 1A, 1B, and 1C, where "H" and "L" chains refer to heavy and light chains, respectively, and CDRs are determined to contain amino acids according to both Kabat and IMGT definitions (Table 1B) or, for specific antibodies, according to Kabat or IMGT (Table 1C).

[0074] [Table 1]

[0075] [Table 2]

[0076] [Table 3]

[0077] Example 1 This example demonstrates that the antibodies disclosed herein exhibit saturable binding to human and cynomolgus PD-1 expressed in stably transfected HEK293 cells.

[0078] HEK293 cells were stably transfected to express either human PD-1 or cynomolgus PD-1. Cells were harvested by Accutase™ treatment (Innovative Cell Technologies, San Diego, CA). Cells expressing cynomolgus PD-1 were treated with the lipophilic fluorescent dye Vybrant® DiD (ThermoFisher Scientific, Carlsbad, CA) and then mixed with an equal number of unlabeled HEK293 cells expressing human PD-1. Cells (a total of 2 x 10 per sample) were then transfected with cynomolgus PD-1. 5) were stained with the indicated concentrations of each antibody for 40 minutes at 4°C with gentle shaking, centrifuged, and washed once. Cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes at room temperature, washed, and antibodies were detected with phycoerythrin (PE)-conjugated goat anti-human kappa (Southern Biotechnology, Birmingham, AL) for 15 minutes at 4°C with gentle shaking. Cells were washed and resuspended, and the fluorescence of bound antibodies was quantified on a BD FACSArray™ (BD Biosciences, San Jose, CA). Data were analyzed for median fluorescence intensity (MFI) using FlowJo® analysis software (FlowJo, LLC). EC 50 The value is (agonist) vs. response—determined in GraphPad Prism 5.0 (GraphPad Software) using a variable slope (4 parameter) curve fit. The results are shown in Figures 1 and 2, and EC 50 The values ​​are listed in Table 2 (human) and Table 3 (cynomolgus monkey). APE06339 is a human IgG1 isotype control antibody specific for hen egg lysozyme. APE08145 is a reference anti-PD-1 antibody.

[0079] [Table 4]

[0080] [Table 5]

[0081] Example 2 This example demonstrates that the antibodies disclosed herein inhibit the proliferation of human peripheral blood CD4 + Demonstrate binding to T cells.

[0082] Primary human peripheral blood CD4 + T cells were isolated by magnetic bead separation of peripheral blood mononuclear cells (PBMCs) (CD4 +T cells (1 x 10 per sample) were prepared using a T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) and activated with anti-CD3 and anti-CD28 on plastic-coated plates for 48 hours in 6-well plates. 5 ) were washed and stained with the indicated concentrations of each antibody in a V-bottom 96-well plate for 30 minutes at 4°C with gentle shaking, centrifuged, and washed once. Cells were fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature, washed, and the antibody was detected with Alexa Fluor 647-conjugated F(ab')2 goat anti-human IgG Fc (Jackson ImmunoResearch, West Grove, PA) for 10 minutes at 4°C. Cells were washed and resuspended, and the fluorescence of bound antibodies was quantified on a BD FACSArray™ (BD Biosciences, San Jose, CA). Data were analyzed for geometric mean fluorescence intensity (MFI) using FlowJo® analysis software (FlowJo, LLC). EC 50 Values ​​were determined in GraphPad Prism 7.02 (GraphPad Software) using a log(agonist) vs. response-variable slope (4 parameter) curve fit. Results are shown in Figure 3 and EC 50 The values ​​are listed in Table 4. APE10787 is a human IgG1 positive control antibody specific for PD-1, and APE06339 is a human IgG1 isotype control antibody specific for hen egg lysozyme.

[0083] [Table 6]

[0084] Example 3 This example documents the extent to which the antibodies disclosed herein compete with PD-L1 and PD-L2 for binding to CHO-K1 cells transfected with human PD-1.

[0085] Competition assays were performed to examine the competition for PD-1 binding between anti-PD-1 antibodies and PD-L1-Fc or PD-L2-Fc constructs. As shown in Figures 4-7, the tested antibodies exhibited moderate competition with PD-L1 (approximately 70% of maximum inhibition) and strong competition with PD-L2; another tested antibody exhibited weak / minimal competition with PD-L1 (approximately 15% of maximum inhibition) and moderate competition with PD-L2 (approximately 70% of maximum inhibition).

[0086] CHO-K1 cells were stably transfected to express human PD-1, and high-level expressing clones were selected. Cells were harvested by Accutase™ treatment (Innovative Cell Technologies, San Diego, CA) and plated in a U-bottom 96-well plate (2 x 10 5 For testing, PD-L1 competitive antibodies were serially diluted and pre-mixed with DyLight 650 (DyL650)-labeled human PD-L1-mouse IgG1 Fc fusion protein (Abcam, Cambridge, MA) (10 nM final concentration DyL650-PD-L1-Fc and antibody concentrations shown in Figures 4 and 5). After 10 minutes of incubation on ice, The antibody / DyL650-PD-L1-Fc mixture was added to the cells for 30 minutes at 4°C with gentle shaking. Cells were centrifuged, washed once, and resuspended in buffer containing propidium iodide, and bound PD-L1-Fc fluorescence was quantified on a BD FACSArray™ (BD Biosciences, San Jose, CA). Data were analyzed for PD-L1 geometric median fluorescence intensity (MFI) using FlowJo® analysis software (FlowJo, LLC). IC 50 Values ​​were determined in GraphPad Prism 7.02 (GraphPad Software) using a log(agonist) vs. response-variable slope (4 parameter) curve fit. Results are shown in Figures 4 and 5, and the resulting IC 50The values ​​are listed in Tables 4-5. APE10787 ("10787") is a human IgG1 positive control antagonist antibody specific for PD-1, and APE06339 ("06339.08") is a human IgG1 isotype control antibody specific for hen egg lysozyme. APE08145 ("08145.05" and "08145.06") are reference antibodies. APE12043 ("12043.02" and "12043.03") are the 437M5-112 anti-PD-1 antibody described in Example 1. APE12095 ("12095.03" and "12095.04") are the 3.7C6 anti-PD-1 antibody described in Example 1.

[0087] CHO-K1 cell clones stably expressing high levels of human PD-1 were harvested by Accutase™ treatment (Innovative Cell Technologies, San Diego, CA) and plated in U-bottom 96-well plates (2 x 10 5 Cells were then placed into the wells (100 μg / well). For testing, PD-L2 competitive antibodies were serially diluted and pre-mixed with DyL650-labeled human PD-L2-mouse IgG1 Fc fusion protein (Abcam, Cambridge, MA) (10 nM final concentration DyL650-PD-L2-Fc and antibody concentrations shown in Figures 6 and 7). After 10 minutes of incubation on ice, the antibody / DyL650-PD-L2-Fc mixture was added to the cells for 30 minutes at 4°C with gentle shaking. Cells were centrifuged, washed once, and resuspended in buffer containing propidium iodide, and bound PD-L2-Fc fluorescence was quantified on a BD FACSArray™ (BD Biosciences, San Jose, CA). Data were analyzed for PD-L2 geometric median fluorescence intensity (MFI) using FlowJo® analysis software (FlowJo, LLC). IC 50 Values ​​were determined in GraphPad Prism 7.02 (GraphPad Software) using a log(agonist) vs. response-variable slope (4 parameter) curve fit. Results are shown in Figures 6 and 7, and the resulting IC 50The values ​​are listed in Tables 6-7. APE10787 ("10787") is a human IgG1 positive control antagonist antibody specific for PD-1, and APE06339 ("06339.08") is a human IgG1 isotype control antibody specific for hen egg lysozyme. APE08145 ("08145.05" and "08145.06") are reference antibodies. APE12043 ("12043.02" and "12043.03") are the 437M5-112 anti-PD-1 antibody described in Example 1. APE12095 ("12095.03" and "12095.04") are the 3.7C6 anti-PD-1 antibody described in Example 1.

[0088] [Table 7]

[0089] [Table 8]

[0090] [Table 9]

[0091] [Table 10]

[0092] Example 4 This example demonstrates that the antibodies disclosed herein exhibit consistent agonist activity in bead-based and plate-based agonist assays.

[0093] For the bead-based agonist assay, Dynabeads® M-280 Tosylactivated (Invitrogen-Life Technologies, Carlsbad, CA) were coupled with anti-CD3 (10 μg), anti-PD-1 or PD-L1-Fc (40 μg), and negative control antibody-conjugated hen's egg lysozyme (50 μg) according to the manufacturer's instructions, for a total of 100 μg of coupled protein. The degree of bead coupling was quantified by flow cytometry. Primary human peripheral blood CD4 + T cells were isolated from magnetic bead-isolated PBMCs (CD4 + Purified CD4 T cells were prepared using a T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA). + T cells (1x10 5 T cells (1000 ng / ml, 1000 ng / ml, or 50 ng / ml, as indicated) were incubated with different numbers of beads (4:1, 2:1, or 1:1 ratios of beads to T cells) as indicated for 72 hours in the presence of soluble anti-CD28 (eBioscience; 250 ng / ml, 100 ng / ml, or 50 ng / ml, as indicated). Secreted IFNγ in the culture supernatant was quantified by ELISA (R&D Systems, Minneapolis, MN). As shown in Figures 8A and 8B and summarized in Table 8, the anti-PD-1 antibodies disclosed herein (437M5-112 and 3.7C6) exhibited consistent inhibitory (agonistic) activity comparable to that of PD-L1-Fc in the bead assay.

[0094] As shown in Figures 9A-9C, the 3.7C6 variants APE12093 and APE12095 were the best agonists in the bead-based assay, with stronger inhibition compared to PD-L1-Fc. The 437M5-112 variants APE12043 and APE12044 had improved agonistic activity compared to the parent antibody APE11844.

[0095] [Table 11]

[0096] Inhibition of IFNγ production by the anti-PD-1 antibodies disclosed herein across donors tested in the bead-based agonist assay is shown in Figures 10A-10B and Figures 11A-11C.

[0097] For plate-based agonist assays, 96-well plates were sequentially coated with anti-CD3 (0.3 μg / ml) overnight at 4°C. The wells were aspirated and washed with PBS, then subjected to a second coating with various concentrations of anti-PD-1 antibodies or PD-L1-Fc as shown in Figures 12-14 overnight at 4°C. Fresh or frozen human PBMCs were cultured for 48 hours in the presence of phytohemagglutinin (PHA; 2 μg / ml), harvested, washed to remove PHA, and cultured overnight in the presence of IL-2. Cells were harvested, washed, and plated onto the anti-CD3 / anti-PD-1 coated wells (1 x 10 cells) in the presence of human gamma globulin (100 μg / ml). 5 The cells were incubated in a 500-well plate (1000 x 1000 cells / well) for 48 hours. Secreted IL-2 in the culture supernatant was quantified by ELISA (R&D Systems, Minneapolis, MN). Inhibition of IL-2 production by PD-1 antibodies across three PBMC donors is shown in Figures 12A-12B, 13A-13B, and 14A-14B. Inhibition of IL-2 production by the anti-PD-1 antibodies disclosed herein was comparable to that induced by PD-L1-Fc.

[0098] Example 5 This example demonstrates that the anti-PD-1 antibodies disclosed herein exhibited agonistic antibody activity in solution in the presence of blocking anti-PD-L1 / anti-PD-L2.

[0099] Whole blood from tetanus toxoid-immunized donors was diluted 1:3 and cultured for 4 days in U-bottom 96-well plates in the presence of tetanus toxoid (Astarte Biologics, Bothell, WA; 5 μg / ml), anti-PD-L1 + anti-PD-L2 (BioLegend, San Diego, CA; 2 μg / ml each), and the indicated concentrations of tetrameric PD-L1-Fc, anti-PD-1 IgG1 antibodies described herein (3.7C6 APE12095; 437M5-112 APE12043), or control human IgG1. Secreted IFNγ in the culture supernatants was quantified by ELISA (R&D Systems, Minneapolis, MN). In this tetanus toxoid recall response whole blood assay, potent agonistic antibody activity of the anti-PD-1 antibodies described herein was observed in the presence of blocking anti-PD-L1 / anti-PD-L2, as shown in Figure 15A (positive and negative controls) and 15B (anti-PD-1 antibodies).

[0100] The IgG1 3.7C6 anti-PD-1 antibody was compared to the same antibody prepared as a human IgG2 (Figures 15C and 15D). The anti-PD-1 IgG2 version of the antibody exhibited anti-PD-1 It had identical activating T cell binding as IgG1 but showed no agonist activity. The IgG2, IgG4, or IgG1 (L234A, L235A) isotypes of the antibody also lacked agonist activity, demonstrating the requirement for FcγR engagement / antibody clustering for functional agonist activity.

[0101] Example 6 This example demonstrates that the anti-PD-1 antibodies provided herein reduce immune responses in whole blood in a concentration-dependent manner.

[0102] Human whole blood stimulated in vitro with the appropriate antigen will elicit a specific T cell recall immune response, which can be measured by IFN-γ and IL17A levels, provided the donor has previously been exposed to the antigen of interest.

[0103] Healthy human donors (N=6) were pre-screened for in vitro recall responsiveness to tetanus toxoid, an antigen to which the donors were likely previously exposed during standard tetanus vaccination. Whole blood from the donors was cultured for 96 hours in the presence of tetanus toxoid and either anti-PD-1 3.7C6 antibody (APE12890) or an irrelevant human IgG1 isotype control. After 96 hours of culture, supernatants were assayed for the presence of cytokines IFN-γ and IL17A using a cytokine detection kit (Meso Scale Diagnostics, Rockville, MD). The results are shown in Figures 22A and 22B.

[0104] Although some donors responded more strongly than others, all donors responded to tetanus toxoid-specific stimulation by producing significant amounts of IFN-γ and IL-17A. As shown in Figures 22A and 22B, the 3.7C6 antibody reduced the secretion of both IFN-γ and IL-17A in a concentration-dependent manner compared to the IgG1 isotype control antibody. The median IC of 3.7C6 in the human whole blood tetanus toxoid recall assay was 0.01. 50 and average IC 50 ±SD were determined to be 0.053 nM and 0.091 ± 0.115 nM, respectively, for IFN-γ inhibition, and 0.097 nM and 0.119 ± 0.098 nM, respectively, for IL-17A inhibition.

[0105] Example 7 This example demonstrates the binding kinetics (affinity) and thermal stability of the antibodies disclosed herein.

[0106] 3.7C6 (APE12095.06 and APE12537.01) exhibited comparable binding kinetics by surface plasmon resonance (SPR) to human and cynomolgus monkey PD-1. The tight binding kinetics approached the limits of the instrument. The SPR data agree well with the equilibrium binding affinity for APE12095 determined by a kinetic exclusion assay (KinExA®). Final affinity measurements were compared with the KinExA® K for human PD-1. D : 75 pM; and KinExA® K for cynomolgus monkey PD-1 D :450pM.

[0107] 437M5-112 (APE12043.05 and APE12538.01) also showed binding kinetics by SPR that were very comparable to human and cynomolgus monkey PD-1. The tight binding kinetics approached the limits of the instrument. The SPR data agree well with the equilibrium binding affinity for APE12043 determined by KinExA®. Final affinity measurements were compared with KinExA® K for human PD-1. D : 51 pM; and KinExA® K for cynomolgus monkey PD-1 D The K for the anti-PD-1 antibodies disclosed herein by surface plasmon resonance and KinExA® was 210 pM. D A summary of the measurements is given in Table 9.

[0108] [Table 12]

[0109] The APE12537 antibody binding affinity and thermal stability were compared to those of a similar antibody designated "030-13263 / 030-13264." APE12537 differed from 030-13263 / 030-13264 by two mutations in the heavy chain: A52aI and D62Q according to Kabat numbering (A53I and D63Q using positions in the sequence listing). The results, presented in Table 10, show that these mutations increased binding affinity and thermal stability.

[0110] [Table 13]

[0111] K for screening by surface plasmon resonance (SPR) D Measurements were performed on a Biacore T200 (GE Healthcare Life Sciences, Pittsburgh, PA), and kinetic constants were globally fitted using a 1:1 binding model. Biotinylated human or cynomolgus monkey PD-1 extracellular domain monomers were captured at 1 nM on a Biacore Sensor chip SA (GE Healthcare Life Sciences, Pittsburgh, PA) using a carboxymethylated dextran surface pre-immobilized with streptavidin. The captured antigen level was targeted to yield a low response to prevent affinity effects on dissociation rates. T measurements were determined by fluorescence-based thermal shift and differential scanning calorimetry.

[0112] Example 8 This example demonstrates that the anti-PD-1 antibodies disclosed herein exhibit in vivo efficacy in a xenogeneic NSG / Hu-PBMC graft-versus-host disease (GvHD) model.

[0113] A xenogeneic NSG / Hu-PBMC GvHD model testing the efficacy of the anti-PD-1 antibodies disclosed herein was performed at The Jackson Laboratory JAX® In Vivo Pharmacology Services (Sacramento, CA). NOD-scid IL2rγ null (NSG) mice were irradiated with 1 Gy followed by 3x10 6 Intravenous injection of human PBMCs was performed. The antibody was administered intraperitoneally at 10 mg / kg twice weekly for 4 weeks, starting the day after PBMC injection, and the belatacept biosimilar was administered intraperitoneally at 75 μg / mouse three times weekly for 4 weeks. The dosing regimen and treatment groups in this study are shown in Figure 16B. Disease was monitored three times weekly for weight loss, death, and GvHD score measurements: weight loss, activity, fur texture, pallor, and posture. Animals showing more than 10% weight loss were monitored for disease daily, and animals showing more than 20% weight loss from starting weight were monitored for safety. He was put to death mercifully.

[0114] The 3.7C6 PD-1 agonist antibody (APE12095) disclosed herein demonstrated statistically significant efficacy versus the isotype control in time to 10% body weight loss (Figure 16C). The 437M5-112 anti-PD-1 agonist antibody disclosed herein also demonstrated statistically significant efficacy versus the isotype control in time to 10% body weight loss (Figure 16D). The response to both anti-PD-1 antibodies was bimodal, with a proportion of animals in each group surviving the full study (Figures 16C and 16D).

[0115] Example 9 This example demonstrates the study design of a single-dose pharmacokinetic and tolerability study in cynomolgus monkeys.

[0116] The study design for the single-dose pharmacokinetic and tolerability study in cynomolgus monkeys is described in Table 11. Evaluations during the study were as follows: Clinical pathology, pre-dose (twice), days 2, 6, 22, and 35 (Charles River Laboratories (CRL)) -Blood FACS panel - major leukocyte populations and 20 T cell subsets, pre-dose (twice), days 2, 6, 22, and 35 (CRL) - B, T, NK, monocytes - CD4, CD8, T central memory, T effector memory, PD-1 + , life sexualized CD4 + and CD8 + , Tregs Receptor occupancy, days 4, 14, 28, and 35 -PK sample analysis, pre-dose and day 35 anti-drug antibody analysis Serum cytokine analysis (17-plex: IL-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-17a, G-CSF, GM-CSF, IFNγ, MIP1β, MIP1α, TNF-α), pre-dose, 4 and 24 hours, day 7 and day 35 -PK parameter analysis using Phoenix® WinNonlin® (Certara, USA).

[0117] Both PD-1 antibodies demonstrated well-behaved pharmacokinetic profiles with detectable drug levels in all animals at day 28 (Figures 17A and 17B). The study was completed on day 35. The doses were well tolerated, and no adverse clinical signs or changes in clinical pathology were observed. The results of the studies are described in Tables 11 and 12.

[0118] [Table 14]

[0119] [Table 15]

[0120] [Table 16]

[0121] As shown, all 6 animals administered antibody APE12538.01 had measurable / low titer anti-drug antibodies on day 36. Two of the six animals had measurable / low titer anti-drug antibodies on day 36. There were no significant changes in any of the cytokines evaluated. Furthermore, as shown in Figures 18A and 18B, persistent receptor occupancy through day 14 was observed in all animals except #1001 (administered IV with 437M5-112); some occupancy was seen in most animals through day 28.

[0122] Example 10 This example demonstrates that the anti-PD-1 antibodies disclosed herein induced recruitment of the phosphatase SHP2 to the PD-1 cytoplasmic domain in PD-1-transfected Jurkat cells.

[0123] Antibody 3.7C6 (APE12890) or a human IgG1 isotype control antibody that recognizes hen egg lysozyme and a fixed amount of anti-CD3 (UCHT1 clone; BioLegend, San Diego, CA) were attached to magnetic beads (Dynabeads™ M-280 Anti-PD-1 antibodies on beads were coupled to 3.7C6-conjugated beads (Figure 19A, top), anti-SHP2 (Figure 19A, middle), or anti-SHP1 (Figure 19A, bottom). The anti-PD-1 antibodies on the beads mimic FcγR engagement by antibodies on antigen-presenting cells. Stably human PD-1-transfected Jurkat cells were stimulated with the indicated beads for either 2 or 10 minutes, the cells were lysed, and PD-1 was immunoprecipitated by the addition of 3.7C6-conjugated beads. The immunoprecipitates were analyzed by SDS-PAGE and then immunoblotted with either anti-PD-1 (Figure 19A, top), anti-SHP2 (Figure 19A, middle), or anti-SHP1 (Figure 19A, bottom). In this signaling assay, as shown in Figures 19A (immunoblot) and 19B (densitometric quantification of immunoblot), following PD-1 Jurkat cell activation with anti-CD3, the 3.7C6 antibody described herein, but not an isotype control antibody, induced recruitment of the phosphatase SHP2, but not SHP1, to the PD-1 cytoplasmic domain. In the presence of anti-CD3-coated beads, as shown in Figures 19A-B, the PD-1 antagonist antibody nivolumab (used at 100 nM in solution) did not induce recruitment of either SHP2 or SHP1 to PD-1. Soluble nivolumab did not induce any SHP recruitment. In combination with T cell activation and CD28 costimulation, antibody 3.7C6 also reduced the phosphorylation of ZAP70 and LAT (data not shown). Antibody 3.7C6 had no effect on signaling pathways in the absence of T cell activation.

[0124] Example 11 This example demonstrates that the epitope on human PD-1 to which the 3.7C6 antibody disclosed herein binds is on the opposite face of PD-1 from the PD-L1 binding site.

[0125] Hydrogen-deuterium exchange mapping of the peptide on PD-1 bound by the 3.7C6 antibody (APE12537) disclosed herein was performed at Biomotif AB (Danderyd, Sweden) using recombinant human PD-1 monomer. The structure of PD-1 was obtained from the National Center for Biotechnology Information (NIRS). The HDX-mapped β-hairpin of PD-1 is publicly available through the Protein Data Bank (PDB) hosted by the National Institute of Information (Bethesda, MD) under accession 4ZqK (see also Zak, KM et al., 2015, Structure 23:2341-2348; and PDB accession 5GGR (see also Lee, JY et al., 2016, Nat Commun., 7:13354). One major peptide, labeled "HDX-mapped β-hairpin" in Figures 20A and 20B, was protected from hydrogen-deuterium exchange by the 3.7C6 antibody. The "HDX-mapped β-hairpin" consists of amino acids 96-110 of PD-1, which have the sequence RVTQLPNGRDFHMSV. Another major peptide consists of amino acids 33-34 of PD-1. It is composed of ∼41 amino acids, which have the sequence NPPTFSPAL. Figures 20A and 20B show a ribbon model (black) of the crystal structure of the human PD-1 extracellular domain docked with a space-filling model (light gray) of the crystal structure of the human PD-L1 extracellular binding domain (PD-1 and PD-L1 structures from the NCBI PDB). The molecule is oriented with the membrane-proximal region of PD-1 at the bottom left (Figure 20A) and rotated 90° to show the membrane-proximal region of PD-1 at the bottom center (Figure 20B). Human PD-1 monomers containing different sets of mutations in defined surface regions were expressed, and binding of the 3.7C6 antibody (APE12095) disclosed herein was assessed by surface plasmon resonance. Mutations in the region labeled "PD-1 triple point mutant" in Figures 20A and 20B completely abolished binding of the 3.7C6 antibody disclosed herein. Mutations in the upper part of the loop labeled "HDX mapped β hairpin" in Figures 20A and 20B did not affect binding of the 3.7C6 antibody disclosed herein. A combination of hydrogen-deuterium exchange and PD-1 mutation mapping demonstrated that the 3.7C6 PD-1 agonist antibody disclosed herein exhibits binding to the region delineated by the dotted circle in Figure 20B, on the opposite face of PD-1 from the PD-L1 binding site.

[0126] Example 12 This example demonstrates that the anti-PD-1 antibodies disclosed herein inhibited IFNγ production in peripheral blood mononuclear cells (PBMCs) from alopecia areata donors stimulated with keratinocyte peptide antigens.

[0127] Alopecia areata is a type of hair loss mediated by the immune system. Hair loss occurs when the immune privilege of hair follicles is destroyed by keratinocyte and melanocyte antigen-specific T cells that produce IFNγ. T cells infiltrate the root sheath of hair follicles. Activated T cells produce excessive IFNγ. Major histocompatibility complex class I and II molecules are abnormally expressed, resulting in the subsequent destruction of hair follicle cells and hair loss.

[0128] PBMCs were isolated from the blood of alopecia areata donors and cultured in plates (2 × 10 cells) in the presence of keratinocyte peptide antigens (peptide antigen pools were as described by Wang et al., J Invest Dermatol. 2016 Aug;136(8):1617-1626) and the indicated concentrations of tetrameric PD-L1-IgG1 Fc, IgG1 3.7C6 anti-PD-1 antibody (APE12890), or control IgG1 isotype tetramer, or control IgG1 isotype. 5 After 5 days, cells were washed and incubated in ELISpot plates for an additional 20 hours to detect the number of IFNγ-secreting cells. Results from each donor and treatment group were normalized to untreated wells to allow statistical comparison of data from 12 donors for treatment and negative control.

[0129] As shown in Figure 21A, the IgG1 3.7C6 anti-PD-1 antibody inhibited IFNγ production in a concentration-dependent manner compared to the IgG1 isotype control. As shown in Figure 21B, the positive control PD-L1-IgG1 Fc tetramer inhibited IFNγ production in a concentration-dependent manner compared to the IgG1 isotype tetramer. Both the anti-PD-1 antibodies and the PD-L1-IgG1 Fc tetramer described herein significantly inhibited the number of IFNγ-secreting cells at concentrations of 1 nM or greater (p<0.001), as shown in Figures 21C and 21D.

[0130] Example 13 This example demonstrates that the anti-PD-1 antibodies disclosed herein inhibited IFNγ production in peripheral blood mononuclear cells (PBMCs) from alopecia areata donors stimulated with a melanocyte peptide antigen.

[0131] Alopecia areata is a type of hair loss mediated by the immune system. Hair loss and / or loss of hair pigmentation occurs when the immune privilege of hair follicles is disrupted by keratinocyte- and melanocyte-antigen-specific T cells that produce IFNγ. T cells infiltrate the root sheath of hair follicles. Activated T cells produce excessive IFNγ. Major histocompatibility complex class I and II molecules are abnormally expressed, resulting in the subsequent destruction of hair follicle cells and hair loss. A similar melanocyte-specific T cell response in the skin results in the destruction of melanocytes in vitiligo.

[0132] PBMCs were isolated from the blood of alopecia areata donors and cultured in plates (2 × 10 cells / well) in the presence of melanocyte peptide antigens (the peptide antigen pool was as described by Wang et al., J Invest Dermatol. 2016 Aug 136(8):1617-1626) and the indicated concentrations of tetrameric PD-L1-IgG1 Fc, IgG1 3.7C6 anti-PD-1 antibody (APE12890), or control IgG1 isotype tetramer or control IgG1 isotype. After 5 days, secreted IFNγ in the culture supernatant was quantified using Meso Scale Discovery (Meso Scale Diagnostics, Rockville, MD). Results from each donor and treatment were normalized to untreated wells to allow statistical comparison of data from 12 donors for treatment and negative control. After 5 days, cells were washed and incubated for an additional 20 hours in an ELISpot assay to detect the number of IFNγ-secreting cells. Results from each donor and treatment were normalized to untreated wells to allow statistical comparison of data from 12 donors for treatment and negative control. Results are shown in Figures 23A-23D.

[0133] As shown in Figure 23A, the IgG1 3.7C6 anti-PD-1 antibody concentration-dependently inhibited IFNγ production compared to the IgG1 isotype control. As shown in Figure 23B, the positive control PD-L1-IgG1 Fc tetramer concentration-dependently inhibited IFNγ production compared to the IgG1 isotype tetramer. Both the anti-PD-1 antibodies and the PD-L1-IgG1 Fc tetramer described herein significantly inhibited IFNγ production at concentrations of 100 nM or greater (p<0.001). As shown in Figures 23C and 23D, both the anti-PD-1 antibodies and the PD-L1-IgG1 Fc tetramer described herein significantly reduced the number of IFNγ-secreting cells at concentrations of 10 nM or greater (p<0.001).

[0134] Example 14 This example demonstrates that the anti-PD-1 antibodies disclosed herein exhibit in vivo efficacy in a xenogeneic NSG / Hu-PBMC graft-versus-host disease (GvHD) model at a dose of 3 mg / kg.

[0135] A xenogeneic NSG / Hu-PBMC GvHD model testing the efficacy of the anti-PD01 antibodies disclosed herein was performed at The Jackson Laboratory, Sacramento, CA. NOD-scid IL2rγ null (NSG) mice were irradiated with 1 Gy followed by 0.9x10 7 Intravenous injection of human PBMCs was performed. Antibodies were administered intraperitoneally at 30 mg / kg, 10 mg / kg, or 3 mg / kg twice weekly for 4 weeks, starting the day after PBMC injection. A fourth group received an irrelevant isotype control antibody at 30 mg / kg twice weekly, and a fifth group received CTLA-4-IgG, a known positive control for efficacy in the model, at 75 μg / mouse three times weekly. The dosing regimen and treatment groups in this study are shown in Figure 24B. Disease was monitored three times weekly by weight loss, death, and GvHD score measurements: weight loss, activity, fur texture, pallor, and posture. Patients exhibiting more than 10% weight loss were randomly assigned to receive the GvHD treatment. Animals were monitored daily for disease and animals showing weight loss of more than 20% from starting weight were euthanized.

[0136] The 3.7C6 PD-1 agonist antibody (APE12890) disclosed herein demonstrated a statistically significant benefit in survival versus the isotype control, increasing median survival time (Figure 24C). The percent of starting body weight for individual animals over the study period is shown in Figures 24D, 24E, 24F, 24G, and 24H for administration of 30 mg / kg isotype control IgG1, 30 mg / kg anti-PD-1 agonist IgG1 (3.7C6), 10 mg / kg anti-PD-1 agonist IgG1 (3.7C6), 3 mg / kg anti-PD-1 agonist IgG1 (3.7C6), and 75 μg / dose of CTLA-4-Ig (positive control), respectively.

[0137] There was no significant difference in survival between the anti-PD-1 agonist IgG1(3.7C6) 30 mg / kg and anti-PD-1 agonist IgG1(3.7C6) 3 mg / kg dose groups, suggesting that efficacy in the GvHD model may be achieved at doses less than 3 mg / kg.

[0138] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0139] Use of the terms "a," "an," "the," "at least one," and similar referents in the context of describing the present invention (particularly in the context of the claims below) should be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be interpreted to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be interpreted as open-ended terms (i.e., meaning "including, but not limited to"), unless specifically stated otherwise. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better clarify the invention and do not impose limitations on the scope of the invention unless otherwise claimed in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0140] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect that such variations will be utilized by those of ordinary skill in the art, and the inventors intend to practice the invention otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is also included. are encompassed by the present invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. 1. An anti-PD-1 binding agent comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, (a) the immunoglobulin heavy chain variable region comprises: CDR1 comprising SEQ ID NO: 1; CDR2 comprising SEQ ID NO:2; and CDR3 comprising SEQ ID NO:3 wherein the immunoglobulin light chain variable region comprises CDR1 comprising SEQ ID NO:4; CDR2 comprising SEQ ID NO:5; and CDR3 comprising SEQ ID NO:6 Includes; (b) the immunoglobulin heavy chain variable region comprises at least 80% sequence identity to any one of SEQ ID NOs: 24-33 and / or comprises at least the CDR regions of any one of SEQ ID NOs: 24-33, and the immunoglobulin light chain variable region comprises at least 80% sequence identity to SEQ ID NO: 34 or 35 and / or comprises at least the CDR regions of any one of SEQ ID NOs: 34 or 35; (c) the immunoglobulin heavy chain variable region comprises CDR1 comprising SEQ ID NO:7; CDR2 comprising SEQ ID NO:8; and CDR3 comprising SEQ ID NO:9 wherein the immunoglobulin light chain variable region comprises CDR1 comprising SEQ ID NO: 10; CDR2 comprising SEQ ID NO: 11; and CDR3 comprising SEQ ID NO: 12 Contains, or (d) the immunoglobulin heavy chain variable region comprises at least 80% sequence identity to, and / or comprises at least the CDR regions of, any one of SEQ ID NOs: 43-47 or 61-63, and the immunoglobulin light chain variable region comprises at least 80% sequence identity to, and / or comprises at least the CDR regions of, any one of SEQ ID NOs: 43-47 or 61-63, and

2. 2. The anti-PD-1 binding agent of claim 1, wherein the immunoglobulin heavy chain variable region comprises at least 90% sequence identity to any one of SEQ ID NOs:24-33, and / or comprises at least the CDR regions of any one of SEQ ID NOs:24-33.

3. 2. The anti-PD-1 binding agent of claim 1, comprising an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 24-33.

4. 4. The anti-PD-1 binding agent of any of claims 1-3, wherein the immunoglobulin light chain variable region comprises at least 90% sequence identity to SEQ ID NO: 34 or 35, and / or comprises at least the CDR regions of any one of SEQ ID NO: 34 or 35.

5. 4. The anti-PD-1 binding agent of any of claims 1-3, comprising the immunoglobulin light chain variable region of SEQ ID NO: 34 or 35.

6. Immunoglobulin heavy chain variable region CDR1 comprising SEQ ID NO: 1; CDR2 comprising SEQ ID NO:2; and CDR3 comprising SEQ ID NO:3 an immunoglobulin light chain variable region comprising CDR1 comprising SEQ ID NO:4; CDR2 comprising SEQ ID NO:5; and CDR3 comprising SEQ ID NO:6 2. The anti-PD-1 binding agent of claim 1, comprising:

7. 7. The anti-PD-1 binding agent of claim 6, wherein the immunoglobulin heavy chain variable region CDR1 comprises any one of SEQ ID NOs: 13-18.

8. 8. The anti-PD-1 binding agent of claim 6 or 7, wherein the immunoglobulin heavy chain variable region CDR3 comprises any one of SEQ ID NOs: 19-21.

9. 9. The anti-PD-1 binding agent of any one of claims 5 to 8, wherein the immunoglobulin light chain variable region CDR1 comprises SEQ ID NO: 22 or 23.

10. Immunoglobulin heavy chain variable region CDR1 comprising SEQ ID NO: 15; CDR2 comprising SEQ ID NO:2; and CDR3 comprising SEQ ID NO: 20 an immunoglobulin light chain variable region comprising CDR1 comprising SEQ ID NO:23; CDR2 comprising SEQ ID NO:5; and CDR3 comprising SEQ ID NO:6 or an immunoglobulin heavy chain variable region comprising CDR1 comprising SEQ ID NO: 15; CDR2 comprising SEQ ID NO:2; and CDR3 comprising SEQ ID NO: 21 an immunoglobulin light chain variable region comprising CDR1 comprising SEQ ID NO:23; CDR2 comprising SEQ ID NO:5; and CDR3 comprising SEQ ID NO:6 or an immunoglobulin heavy chain variable region comprising CDR1 comprising SEQ ID NO: 13; CDR2 comprising SEQ ID NO:2; and CDR3 comprising SEQ ID NO: 19 an immunoglobulin light chain variable region comprising CDR1 comprising SEQ ID NO:23; CDR2 comprising SEQ ID NO:5; and CDR3 comprising SEQ ID NO:6 5. The anti-PD-1 binding agent of claim 4, comprising:

11. the immunoglobulin heavy chain variable region comprises at least 90% sequence identity to SEQ ID NO:29, or comprises at least the CDR regions of SEQ ID NO:29, and the immunoglobulin light chain variable region comprises at least 90% sequence identity to SEQ ID NO:35, or comprises at least the CDR regions of SEQ ID NO:35; Alternatively, the immunoglobulin heavy chain variable region comprises at least 90% sequence identity to SEQ ID NO: 30, or comprises at least the CDR regions of SEQ ID NO: 30, and the immunoglobulin light chain variable region comprises at least 90% sequence identity to SEQ ID NO: 35, or comprises at least the CDR regions of SEQ ID NO:

30. Both contain the CDR regions of SEQ ID NO: 35; or the anti-PD-1 binding agent of claim 1, wherein the immunoglobulin heavy chain variable region comprises at least 90% sequence identity to SEQ ID NO:24, or comprises at least the CDR regions of SEQ ID NO:24, and the immunoglobulin light chain variable region comprises at least 90% sequence identity to SEQ ID NO:34, or comprises at least the CDR regions of SEQ ID NO:

34.

12. the immunoglobulin heavy chain variable region comprises SEQ ID NO:29 and the immunoglobulin light chain variable region comprises SEQ ID NO:35; or the immunoglobulin heavy chain variable region comprises SEQ ID NO: 30 and the immunoglobulin light chain variable region comprises SEQ ID NO: 35; Or, the anti-PD-1 binding agent of claim 11, wherein the immunoglobulin heavy chain variable region comprises SEQ ID NO:24 and the immunoglobulin light chain variable region comprises SEQ ID NO:

34.

13. 2. The anti-PD-1 binding agent of claim 1, wherein the immunoglobulin heavy chain variable region comprises at least 90% sequence identity to any one of SEQ ID NOs: 43-47 or 61-63, or comprises at least the CDR regions of any one of SEQ ID NOs: 43-47 or 61-63.

14. 14. The anti-PD-1 binding agent of claim 1 or 13, wherein the immunoglobulin light chain variable region comprises at least 90% sequence identity to SEQ ID NOs: 48-50, or comprises at least the CDR regions of any one of SEQ ID NOs: 48-50.

15. 15. The anti-PD-1 binding agent of claim 13 or 14, wherein the immunoglobulin heavy chain comprises any one of SEQ ID NOs: 43-47 or 61-63.

16. 16. The anti-PD-1 binding agent of any of claims 13-15, comprising an immunoglobulin light chain variable region of any one of SEQ ID NOs: 48-50.

17. Immunoglobulin heavy chain variable region CDR1 comprising SEQ ID NO:7; CDR2 comprising SEQ ID NO:8; and CDR3 comprising SEQ ID NO:9 an immunoglobulin light chain variable region comprising CDR1 comprising SEQ ID NO: 10; CDR2 comprising SEQ ID NO: 11; and CDR3 comprising SEQ ID NO: 12 2. The anti-PD-1 binding agent of claim 1, comprising:

18. 18. The anti-PD-1 binding agent of claim 17, wherein the immunoglobulin heavy chain variable region CDR1 comprises any one of SEQ ID NOs: 57-60.

19. 19. The anti-PD-1 binding agent of claim 17 or 18, wherein the immunoglobulin heavy chain variable region CDR2 comprises any one of SEQ ID NOs: 38-42.

20. Immunoglobulin heavy chain variable region CDR1 comprising SEQ ID NO:57; CDR2 comprising SEQ ID NO: 42; and CDR3 comprising SEQ ID NO:9 an immunoglobulin light chain variable region comprising CDR1 comprising SEQ ID NO: 10; CDR2 comprising SEQ ID NO: 11; and CDR3 comprising SEQ ID NO: 12 or an immunoglobulin heavy chain variable region comprising CDR1 comprising SEQ ID NO:57; CDR2 comprising SEQ ID NO: 41; and CDR3 comprising SEQ ID NO:9 an immunoglobulin light chain variable region comprising CDR1 comprising SEQ ID NO: 10; CDR2 comprising SEQ ID NO: 11; and CDR3 comprising SEQ ID NO: 12 18. The anti-PD-1 binding agent of claim 17, comprising:

21. the immunoglobulin heavy chain variable region comprises at least 90% sequence identity to SEQ ID NO:47 or comprises at least the CDR regions of SEQ ID NO:47; the immunoglobulin light chain variable region comprises at least 90% sequence identity to SEQ ID NO:49 or comprises at least the CDR regions of SEQ ID NO:49; or the immunoglobulin heavy chain variable region comprises at least 90% sequence identity to SEQ ID NO:46, or comprises at least the CDR regions of SEQ ID NO:46; 2. The anti-PD-1 binding agent of claim 1, wherein the immunoglobulin light chain variable region comprises at least 90% sequence identity to SEQ ID NO:50, or comprises at least the CDR regions of SEQ ID NO:

50.

22. comprising an immunoglobulin heavy chain variable region of SEQ ID NO: 47, and an immunoglobulin light chain variable region of SEQ ID NO: 49; Or the anti-PD-1 binding agent of claim 21, comprising an immunoglobulin heavy chain variable region of SEQ ID NO:46, and an immunoglobulin light chain variable region of SEQ ID NO:

50.

23. 23. The anti-PD-1 binding agent of any one of claims 1-22, wherein the anti-PD-1 binding agent is an antibody, an antibody conjugate, or an antigen-binding fragment thereof.

24. The anti-PD-1 binding agent is F(ab') ２ , Fab', Fab, Fv, scFv, dsFv, or single-chain binding polypeptide.

25. 25. The anti-PD-1 binding agent of any one of claims 1-24, wherein the anti-PD-1 binding agent comprises an IgG Fc region that binds to an Fc receptor on an antigen-presenting cell.

26. 26. The anti-PD-1 binding agent of any one of claims 1-25, wherein the anti-PD-1 binding agent comprises an IgG1 Fc region or other Fc region that binds to an FcγR.

27. 2. The anti-PD-1 binding agent of claim 1, comprising an immunoglobulin heavy chain comprising SEQ ID NO:36 and an immunoglobulin light chain comprising SEQ ID NO:

37.

28. 2. The anti-PD-1 binding agent of claim 1, comprising an immunoglobulin heavy chain comprising SEQ ID NO:51 and an immunoglobulin light chain comprising SEQ ID NO:

52.

29. 29. A pharmaceutical composition comprising: (a) the anti-PD-1 binding agent of any one of claims 1-28; and (b) a pharmaceutically acceptable carrier.

30. 30. A method of inhibiting an immune response in a mammal, comprising administering to the mammal the anti-PD-1 binding agent of any of claims 1-28 or the pharmaceutical composition of claim 29.

31. 30. A method of treating an inflammatory or autoimmune disorder in a mammal, comprising administering to a mammal having an inflammatory or autoimmune disorder an anti-PD-1 binding agent of any of claims 1-28 or a pharmaceutical composition of claim 29, thereby treating the disorder.

32. 32. The method of claim 31, wherein the inflammatory or autoimmune disorder is primary biliary cholangitis (PBC), graft-versus-host disease (GvHD), vitiligo, ANCA vasculitis, type 1 diabetes, or non-infectious uveitis.

33. 29. A nucleic acid encoding the immunoglobulin heavy chain and / or immunoglobulin light chain of the anti-PD-1 binding agent of any of claims 1-28, optionally in a vector.

34. A cell expressing the anti-PD-1 binding agent of any of claims 1 to 28.

35. 30. A method of producing the anti-PD-1-binding agent of any one of claims 1-28, the method comprising expressing in a cell a nucleic acid sequence encoding an immunoglobulin heavy chain and a nucleic acid sequence encoding an immunoglobulin light chain of the anti-PD-1-binding agent of any one of claims 1-28.

36. 30. The anti-PD-1 binding agent of any of claims 1-28, or the pharmaceutical composition of claim 29, for inhibiting an immune response in a mammal.

37. 30. The anti-PD-1 binding agent of any of claims 1-28, or the pharmaceutical composition of claim 29, for treating an inflammatory or autoimmune disorder in a mammal.

32. 38. The anti-PD-1 binding agent or pharmaceutical composition of claim 37, wherein the inflammatory or autoimmune disorder is primary biliary cholangitis (PBC), graft-versus-host disease (GvHD), vitiligo, ANCA vasculitis, type 1 diabetes, or non-infectious uveitis.

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