PD-1 agonist and methods of using the same

Abstract

To provide a PD-1-binding agent comprising an immunoglobulin heavy chain polypeptide and immunoglobulin light chain polypeptide, as well as a method for making the PD-1-binding agent, and a pharmaceutical composition comprising the PD-1-binding agent.SOLUTION: A anti-PD-1 binding agent comprises immunoglobulin heavy chain variable regions containing a specific sequence, or immunoglobulin heavy chain variable regions containing at least CDR1, CDR2, and CDR3 thereof, and immunoglobulin light chain variable regions containing a specific sequence different from the aforementioned sequence, or immunoglobulin light chain variable regions containing at least CDR1, CDR2, and CDR3 thereof. Also provided are a pharmaceutical composition comprising (a) the anti-PD-1 binding agent and (b) a pharmaceutically acceptable carrier, and the uses of the anti-PD-1 binding agent or the pharmaceutical composition to treat inflammatory or autoimmune disorder in mammals.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - References to Related Applications This patent application claims priority to U.S. Provisional Patent Application No. 62 / 857,699, filed on June 5, 2019; U.S. Provisional Patent Application No. 62 / 863,193, filed on June 18, 2019; and U.S. Provisional Patent Application No. 62 / 983,512, filed on February 28, 2020, the entire disclosures of which are incorporated herein by reference.

Background Art

[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 first identified by subtractive hybridization of an apoptotic mouse T - cell line (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 of PD-1, PD ligand 1 (PD-L1) and PD ligand 2 (PD-L2), have been identified, and both of these belong to the B7 protein superfamily (Greenwald et al. supra). PD-L1 is expressed in a variety of cell types including cells of the lung, heart, thymus, spleen, and kidney (see, for example, Freeman et al., J. Exp. Med., 192(7):1027-1034 (2000); and Yamazaki et al., J. Immunol., 169(10):5538-5545 (2002)). Expression of PD-L1 is upregulated on macrophages and dendritic cells (DC) in response to lipopolysaccharide (LPS) and GM-CSF treatment, and on T cells and B cells during 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, for example, 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 exhibits a more restricted expression pattern and is expressed primarily by antigen-presenting cells (e.g., dendritic cells and macrophages) and some tumor cell lines (see, for example, 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)). Clustering of PD-1 induced by PD-L1 has been found to induce recruitment of the SHP2 phosphatase, which preferentially dephosphorylates CD28 and suppresses T cell function (Hui et al., Science, 355:1428-1433 (2017)). Deficiency of PD-1 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 have been associated with a high incidence of progression of systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, and multiple sclerosis (see, 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 advances in inhibiting PD-1 activity for treating various types of cancer and for immune enhancement (e.g., for treating infectious diseases), there is a need for a high-affinity PD-1 binding agent (e.g., an antibody) that promotes negative signaling and functions as a PD-1 agonist by binding to PD-1. SUMMARY OF THE INVENTION

[0006] Brief Summary of the Invention The present invention provides an agonistic PD-1 binding agent. 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: CDR1 comprising SEQ ID NO: 7; CDR2 comprising SEQ ID NO: 8; and CDR3 comprising SEQ ID NO: 9; and 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.

[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] Furthermore, the present invention provides an isolated or purified nucleic acid sequence encoding the aforementioned immunoglobulin polypeptide, a vector comprising such a nucleic acid sequence, an isolated PD-1 binder comprising the aforementioned immunoglobulin polypeptide, a nucleic acid sequence encoding such a PD-1 binder, a vector comprising such a nucleic acid sequence, an isolated cell comprising such a vector, a composition comprising such a PD-1 binder or such a vector together with a pharmaceutically acceptable carrier, and an immune response by administering an effective amount of such a composition to a mammal to inhibit and provide a method for treating inflammatory or autoimmune disorders in mammals.

Brief Description of the Drawings

[0011] Brief description of several figures of the drawings

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Mode for Carrying Out the Invention

[0012] Detailed Description of the Invention The present invention provides a PD-1 binder. 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 has been 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, a transmembrane region, and an intracellular tail accompanied by a short extracellular stalk. The PD-1 intracellular tail functions to negatively regulate T cell receptor signaling by recruiting tyrosine phosphatase when phosphorylated (see, for example, Ishida et al. supra; and Blank et al. supra), and contains two phosphorylation sites located in the immunoreceptor tyrosine-based inhibitory motif and the immunoreceptor tyrosine-based switch motif.

[0013] In some embodiments, the PD-1 binder provided herein is an agonist, which means that the PD-1 binder binds to PD-1 but does not significantly inhibit the binding of PD-1 to its PD-1 ligand, thereby maintaining the ability of PD-1 to negatively regulate T cell receptor signaling. According to certain embodiments, the PD-1 binder provided herein can induce or stimulate the ability of PD-1 to negatively regulate T cell receptor signaling and suppress the immune response. In certain embodiments, a PD-1 binder that binds to PD-1 with an epitope that includes, consists essentially of, or consists of residues 33-41 (sequence: NPPTFSPAL) and / or 96-110 (sequence: RVTQLPNGRDFHMSV) of human PD1 is provided.

[0014] The PD-1 binder comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, each of which contains three complementarity-determining regions (CDRs) commonly referred to as CDR1, CDR2, or CDR3. The CDR regions are also designated in the nomenclature using "H" or "L" to indicate the heavy or light chain respectively, i.e., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3. 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 (e.g., Kabat, et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH (1991); Chothia, et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol., 196:901- 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) (see).

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

[0016] In certain embodiments, the PD-1 binder can include 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 binder includes an immunoglobulin heavy chain variable region that includes a CDR of any one of SEQ ID NOs: 24-33, where the CDR is as provided above or as determined by any of various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo). Optionally, the immunoglobulin heavy chain variable region that includes a CDR of any one 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 one of SEQ ID NOs: 24-33.

[0017] In addition to or alternatively to the Ig heavy chain variable region described above, the anti-PD-1 binding agent can comprise an 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 to SEQ ID NO: 34 or 35 (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). 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 various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo). Optionally, the 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 binder may include 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 the immunoglobulin heavy chain variable region of SEQ ID NO: 29; or an immunoglobulin heavy chain variable region including 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 as determined by any of various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo)); and 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 the immunoglobulin light chain variable region of SEQ ID NO: 35, or an immunoglobulin light chain variable region including 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 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 their CDRs 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 their CDRs 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 their CDRs as determined by Martin. 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 their CDRs as determined by IGMT. 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 their CDRs 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 where the sequences retain the heavy and light chain CDRs of SEQ ID NOs: 36 and 37, respectively, 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).

[0019] According to another embodiment, the PD-1 binder may be 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 the immunoglobulin heavy chain variable region of SEQ ID NO: 24; or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO: 24 (where 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 as determined by any of various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo)); and 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 the immunoglobulin light chain variable region of SEQ ID NO: 34, or an immunoglobulin light chain variable region comprising at least the CDRs of SEQ ID NO: 34 (where 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 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 their CDRs 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 their CDRs 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 their CDRs 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 their CDRs determined by IGMT. In some embodiments. In some cases, 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 their CDRs determined by AHo.

[0020] According to one embodiment, the PD-1 binder may include 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 the immunoglobulin heavy chain variable region of SEQ ID NO: 30; or an immunoglobulin heavy chain variable region including 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 as determined by any of various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo)); and 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 the immunoglobulin light chain variable region of SEQ ID NO: 35, or an immunoglobulin light chain variable region including 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 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 their CDRs 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 their CDRs 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 their CDRs 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 their CDRs 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 their CDRs as determined by AHo.

[0021] According to another aspect, the anti-PD-1 binding agent comprises an immunoglobulin heavy chain variable region comprising CDR1 comprising SEQ ID NO: 7; CDR2 comprising SEQ ID NO: 8; and CDR3 comprising SEQ ID NO: 9; and 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. In some embodiments, the heavy chain CDR1 comprises any one 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 is any one of the immunoglobulin heavy chain variable regions of SEQ ID NOs: 43-47 or 61-63, or at least 80%, 85%, or 90% sequence identity to any one of SEQ ID NOs: 43-47 or 61-63 (e.g., at least 80%, at least 81%, at least 82%, at least comprising 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). In some embodiments, the PD-1 binder comprises an immunoglobulin heavy chain variable region comprising any one of the CDRs of SEQ ID NOs: 43-47 or 61-63, wherein the CDR is as provided above or as determined by any of various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo). Optionally, the immunoglobulin heavy chain variable region comprising any one of the CDRs 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 above Ig heavy chain variable regions (e.g., SEQ ID NOs: 43-47 or 61-63), the anti-PD-1 binding agent can include an immunoglobulin light chain variable region of any of SEQ ID NOs: 48-50, 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: 48-50. In other embodiments, the PD-1 binding agent includes an immunoglobulin light chain variable region that includes a CDR of any of SEQ ID NOs: 48-50, where the CDR is as provided above or as determined by any of various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo). Optionally, the immunoglobulin light chain variable region that includes a CDR of any of SEQ ID NOs: 48-50 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 one of SEQ ID NOs: 48-50.

[0024] In certain embodiments, the anti-PD-1 binding agent is the 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, where the CDR regions are as provided above (e.g For example, as determined by either CDR1 - SEQ ID NO:57, CDR2 - SEQ ID NO:42, and CDR3 - SEQ ID NO:9) or any of various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo); and the immunoglobulin light chain variable region of SEQ ID NO:49, or an amino acid sequence having at least 80%, 85%, or 90% sequence identity to SEQ ID NO:49 (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:49 (where 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 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:47 and the light chain variable region of SEQ ID NO:49, or their CDRs as determined at least by Kabat. 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 their CDRs as determined at least by Chothia. 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 their CDRs as determined at least by Martin. 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 their CDRs as determined at least 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 their CDRs as determined by at least AHo. As a further example, the anti-PD-1 binding agent comprises an immunoglobulin heavy chain comprising SEQ ID NO: 51 and an immunoglobulin light chain comprising SEQ ID NO: 52, 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 SEQ ID NOs: 51 and 52, optionally where the sequence retains the heavy and light chain CDRs of SEQ ID NOs: 51 and 52 as determined according to any of the various known immunoglobulin numbering schemes (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo) as provided above.

[0025] In another embodiment, the anti-PD-1 binding agent is an amino acid sequence having the immunoglobulin heavy chain variable region of SEQ ID NO: 46, or 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, where 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 various known immunoglobulin numbering schemes (e.g., Kabat, Ch as determined by any of othia, Martin (Enhanced Chothia), IGMT, or AHo); and the immunoglobulin light chain variable region of SEQ ID NO: 50, or an amino acid sequence having at least 80%, 85%, or 90% sequence identity to SEQ ID NO: 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); or an immunoglobulin heavy chain variable region comprising at least the CDRs of SEQ ID NO: 50 (where 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 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: 46 and the light chain variable region of SEQ ID NO: 50, or at least their CDRs as determined by Kabat. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 46 and the light chain variable region of SEQ ID NO: 50, or at least their CDRs as determined by Chothia. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 46 and the light chain variable region of SEQ ID NO: 50, or at least their CDRs as determined by Martin. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 46 and the light chain variable region of SEQ ID NO: 50, or at least their CDRs as determined by IGMT. In some embodiments, the antibody comprises the heavy chain variable region of SEQ ID NO: 46 and the light chain variable region of SEQ ID NO: 50, or at least their CDRs as determined by AHo.

[0026] The "identity" of the sequences described herein can be determined by comparing the nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. 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 greater of the lengths of the sequence of interest or the reference sequence). Many mathematical algorithms are known for obtaining optimal alignments and calculating identity between two or more sequences, and are incorporated into many available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), the BLAST programs (e.g., BLAST 2.1, BL2SEQ, and their later versions), and the FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching). 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 substitution of one amino acid at a given position or residue in a polypeptide sequence with another amino acid at the same position or residue. An amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative, depending on whether the substitution is by an amino acid residue having similar properties to the residue being replaced. A functional way to define common properties 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 a group preferentially exchange with each other and thus are most similar to each other in their effect on the 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 "slightly polar aliphatic subgroup" consists of methionine, serine, threonine, and cysteine. The "aliphatic polar / charged subgroup" consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine. 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] 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, e.g., lysine in place of arginine such that a positive charge can be maintained and vice versa, glutamic acid in place of aspartic acid such that a negative charge can be maintained and vice versa, serine in place of threonine such that a free - OH can be maintained, glutamine in place of asparagine such that a free - NH2 can be maintained. "Semi - conservative mutations" include amino acid substitutions of amino acids that are within the same group listed herein but not within the same subgroup. For example, substitution of aspartic acid in place of asparagine, or substitution of asparagine in place of lysine, includes amino acids within the same group but different subgroups. "Non - conservative mutations" include amino acid substitutions between different groups, e.g., lysine in place of tryptophan, phenylalanine in place of serine, etc.

[0032] In some embodiments, the PD - 1 binder 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. An isolated PD - 1 binder can be any type of molecule or construct that includes at least the specific immunoglobulin heavy and light chain variable regions. That is, the PD - 1 binder can be, for example, an intact immunoglobulin or antibody as 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 means any molecule or construct that includes some portion of the immunoglobulin or antibody and binds to the target antigen. Such fragments generally include at least a portion of the heavy and light chain variable regions that contain the CDRs, and optionally can include a portion of the constant region, along with other elements (e.g., linkers, etc.) that are not normally part of an immunoglobulin or antibody. Examples of such "fragments" are (i) V L , V H , C LA Fab fragment, which is a monovalent fragment consisting of a CH1 domain, (ii) an F(ab’)2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region, (iii) an Fv fragment consisting of the V L and V H domains, (iv) a Fab’ fragment resulting from cleaving the disulfide bridge of the F(ab’)2 fragment using mild reducing conditions; (v) a diabody; (vi) a single-chain variable region (scFv), and (vii) a disulfide-stabilized Fv fragment (dsFv), including but not limited to these.

[0033] In some embodiments, the PD-1 binder comprises an immunoglobulin heavy chain constant region, such as a fragment crystallizable (Fc) region or a part thereof. The Fc region can be any Ig class / subclass (including variants thereof, which can be IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), IgM). In certain embodiments, the PD-1 binder comprises an Fc region that binds to an Fc receptor of an antigen-presenting cell (e.g., dendritic cell, macrophage, Langerhans cell, or B cell). The Fc receptor can be an Fcγ receptor (FcγR) such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b). In one embodiment, the PD-1 binder comprises an Fc region that binds to an FcγR such as IgG1. That is, in some embodiments, the PD-1 binder is a “whole” or “complete” Ig (i.e., an antibody). In additional embodiments, the PD-1 binder is an IgG antibody, particularly an IgG1 antibody.

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

[0035] The isolated PD-1 binder can be, or can be obtained from, a human antibody, a non-human antibody, or a chimeric antibody. By "chimeric" is meant an antibody or fragment thereof that comprises both human and non-human regions. Preferably, the isolated PD-1 binder is a humanized antibody. A "humanized" antibody is a monoclonal antibody that comprises at least one CDR obtained from or derived from a human antibody framework and a non-human antibody. Non-human antibodies include, for example, antibodies isolated from any non-human animal such as rodents (e.g., mice or rats). A humanized antibody can comprise one , two, or three CDRs. In a preferred embodiment of the invention, the CDRH3 of the PD-1 binder of the invention is obtained from or derived from a murine monoclonal antibody, while the remaining variable and constant regions of the PD-1 binder of the invention are obtained from or derived from a human monoclonal antibody.

[0036] Human antibodies, non-human antibodies, chimeric antibodies, or humanized antibodies can be obtained by any means including those via in vitro sources (e.g., hybridomas or cell lines that recombinantly produce antibodies) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and include, for example, 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 antibodies or chimeric antibodies can be generated 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 the endogenous antibody genes have been effectively replaced with human antibody genes include, but are not limited to, 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 generated using any suitable method known in the art, including, for example, transplantation of 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)) (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey (2009)). In one embodiment, humanized antibodies can be produced using, for example, the method described in U.S. Patent Application Publication No. 2011 / 0287485A1.

[0037] The PD-1 binder 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 the dissociation constant (K D) is represented as. The affinity of a binder 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 pM to about 1 nanomolar (nM), from about 1 nM to about 1 μM, or from about 1 μM to about 100 μM). In one embodiment, the PD-1 binder has a K D and can bind to the PD-1 protein having it. In another embodiment, the PD-1 binder has a K D and can bind to PD-1 having it that is less than or equal to 200 pM (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). In some embodiments, the PD-1 binder is cross-reactive with cynomolgus PD-1 and has an affinity within any of the foregoing ranges discussed with respect to human PD-1. The immunoglobulin affinity for the antigen or epitope can be measured using any assay recognized in the art. Such methods include, 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] A PD-1 binder binds to PD-1, but preferably does not completely inhibit the ability of PD-1 to negatively regulate the immune response, or in some cases, does not substantially inhibit the ability of PD-1 to negatively regulate the immune response, or even enhances the ability of PD-1 to negatively regulate the immune response. In some embodiments, the PD-1 binder does not completely block the binding between PD-1 and PD-L1, or preferably does not substantially reduce the binding between PD-1 and PD-L1. The evaluation of the degree to which a PD-1 binder inhibits PD-1 regulation of the immune response or PD-1 binding to PD-L1 can be carried out using an assay such as those described in the Examples or other assays known in the art. In some embodiments, the PD-1 binder 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, about 10% or less.

[0039] Methods of Use / Treatment The present invention provides a method for suppressing an immune response, particularly a T cell-mediated immune response, in a mammal by administering a PD-1 binder described herein to the mammal. The present invention further provides a 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 a method for treating any disease or injury 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 binder described herein to reduce or eliminate any symptoms of the disorder, or to prevent or inhibit the onset of such symptoms. Negative regulation of the immune system as used herein is synonymous with immunosuppression. In some cases, it will be understood that the PD-1 binder 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 can be an inflammatory or autoimmune disorder. Examples of inflammatory or autoimmune disorders include, for example, infectious diseases (viral, bacterial, fungal and parasitic), endotoxin shock associated with infectious diseases, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Behçet's disease, Alzheimer's disease, inflammatory bowel diseases including Crohn's disease and ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, psoriatic arthritis, vasculitis, antineutrophil cytoplasmic antibody-related (ANCA) vasculitis, surgical adhesions, stroke, type I diabetes, Lyme disease, arthritis, meningitis, autoimmune vasculitis, immune-mediated inflammatory disorders of the central and peripheral nervous systems such as multiple sclerosis, lupus (such as systemic lupus erythematosus and chronic discoid lupus erythematosus) and Guillain-Barré syndrome, atopic dermatitis, polymyositis, dermatomyositis, autoimmune liver disorders, fibrosing alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Ménière's disease, pemphigus, pemphigoid, primary biliary cholangitis, hepatitis, sarcoidosis, scleroderma (localized scleroderma , systemic scleroderma, and progressive systemic scleroderma), polyangiitis with granulomatosis, other autoimmune disorders, cholangitis, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, heart diseases including ischemic diseases such as myocardial infarction and atherosclerosis, arteritis nodosa (polyarteritis nodosa and microscopic polyangiitis), allergic granulomatous angiitis, hypersensitivity vasculitis, aortic inflammatory syndrome (Takayasu arteritis), temporal arteritis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochloridemia, Still's disease, Cogan's syndrome, RS3PE, rheumatoid polymyalgia, fibromyalgia syndrome, antiphospholipid antibody syndrome, eosinophilic myositis, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic 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, acquired epidermolysis bullosa, alopecia areata, vitiligo, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent fetal loss, or infertility associated with a lack of fetal-maternal tolerance.

[0041] In some embodiments, the disease or disorder is giant cell arteritis, polymyalgia rheumatica, primary Sjögren'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 be associated with, 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," etc. refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or the adverse symptoms caused by the disease. For this purpose, the methods of the invention include administering a "therapeutically effective amount" of a PD-1 binder. A "therapeutically effective amount" refers to an amount effective at the dosage and for the period required to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on factors such as the condition, age, sex, and body weight of the individual, as well as the ability of the PD-1 binder to elicit the desired response in the individual.

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

[0044] A PD-1 binder 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 present 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 can 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, and the like. Optionally, the composition can be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The composition can be produced according to conventional techniques described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0045] Typical dosages of the PD-1 binder can be in the range of, for example, 1 pg / kg to 20 mg / kg of the body weight of an animal or a human; however, dosages below or above this exemplary range are within the scope of the present invention. The parenteral dosage per day is from about 0.00001 μg / kg to about 20 mg / kg of the 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 the 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 a range defined by any two of the foregoing values), more preferably from about 1 μg / kg to 5 mg / kg of the 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 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). The therapeutic or prophylactic effect can be monitored by regular evaluation of the patient being treated. For repeated administration over several days, depending on the condition, the treatment can be repeated until the desired suppression of the disease symptoms occurs. However, other dosing regimens can be useful and are within the scope of the present invention. The desired dosage can be delivered by single bolus administration of the composition, multiple bolus administrations of the composition, or continuous infusion administration of the composition.

[0046] The PD-1 binder can be administered to mammals using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, intraoral, 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 mammals using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0047] Once administered to a mammal (e.g., a human), the biological activity of the PD-1 binder of the present invention can be measured by any suitable method known in the art. For example, the biological activity can be evaluated by determining the stability of a particular PD-1 binder. In one embodiment of the present invention, the PD-1 binder (e.g., an antibody) has an in vivo half-life 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 binder has an in vivo half-life 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 binder has an in vivo half-life 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 binder of the present invention can be administered alone or in combination with other active agents or drugs. For example, the PD-1 binder can be administered in combination with other agents for the treatment or prevention of the diseases disclosed herein. In this regard, the PD-1 binder can be used in combination with, for example, other monoclonal antibodies, viruses that kill diseases, gene therapy, and adoptive It can be used in combination with at least one other inhibitor of inflammatory or autoimmune disorders, including T cell transfer, and / or surgery. The PD-1 binder of the present invention described herein can also be used in combination with at least one other immunosuppressant, including, for example, methotrexate, corticosteroids, and other small molecule agents used to treat autoimmune and inflammatory diseases. When the method of the present invention treats an infectious disease, the PD-1 binder 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 (e.g., live attenuated vaccine, subunit vaccine, recombinant vector vaccine, and small molecule antiviral therapies (e.g., virus replication inhibitors and nucleoside analogs).

[0049] In addition to therapeutic use, the PD-1 binder described herein can be used in diagnostic or research applications. In this regard, the PD-1 binder can be used in methods for diagnosing cancer or infectious diseases. In a similar manner, the PD-1 binder can be used in assays that monitor PD-1 protein levels in subjects being tested for diseases or disorders associated with abnormal PD-1 expression. Research applications include, for example, methods that utilize a PD-1 binder and a label to detect PD-1 protein in a sample, such as in a human body fluid or in a cell or tissue extract. The PD-1 binder can be used with or without modifications such as covalent or non-covalent labels at a detectable moiety. For example, the detectable moiety can be a radioisotope (e.g.,3 H, 14 C, 32 P, 35 S, or 125 I), 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 moiety that can be a detectable part to which an antigen-binding agent (e.g., an antibody) is separately conjugated. Any method known in the art for separately conjugating an antigen-binding agent (e.g., an antibody) to a detectable part can 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] The PD-1 protein level can be measured using the PD-1 binding agent of the present invention by any suitable method known in the art. Such methods include, for example, radioimmunoassay (RIA) and FACS. The normal or standard expression value of the PD-1 protein can be demonstrated by using any suitable technique to combine a sample containing or suspected of containing a PD-1 polypeptide, under conditions suitable for forming an antigen-antibody complex, with a PD-1 specific antibody. The antibody is labeled directly or indirectly with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, moieties, fluorescent substances, luminescent substances, and radioactive substances (see, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)). Then, the amount of the PD-1 polypeptide expressed in the sample is compared with the standard value.

[0051] The PD-1 binder can be provided in a kit, i.e., a packaged combination of a predetermined amount of reagents having instructions for performing a diagnostic assay. If the PD-1 binder is labeled with an enzyme, the kit preferably includes the substrate and cofactors required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). Further, other additives such as stabilizers, buffers (e.g., blocking buffers or lysis buffers) can be included in the kit. The relative amounts of the various reagents can be varied to provide a concentration of the reagents in solution that substantially optimizes the sensitivity of the assay. The reagents can be provided as a dry powder (typically lyophilized) containing an excipient that provides a reagent solution having an appropriate concentration upon dissolution. Additives can be included in the kit.

[0052] Nucleic acids, cells, manufacturing methods The present invention also provides one or more isolated or purified nucleic acid sequences encoding a PD-1 binder 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 the heavy chain variable regions of SEQ ID NOs: 29 and 47, respectively, and SEQ ID NOs: 53 and 55 encoding 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 the light chain variable regions of SEQ ID NOs: 35 and 49, respectively, and SEQ ID NOs: 54 and 56 encoding the complete heavy 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 can be single-stranded or double-stranded and which can contain unnatural 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., including double- and single-stranded DNA, as well as double- and single-stranded RNA. The terms include, by way of equivalent, any analogs of RNA or DNA made from nucleotide analogs, and modified polynucleotides including, but not limited to, methylated and / or capped polynucleotides. Nucleic acids are typically linked through phosphodiester bonds to form nucleic acid sequences or polynucleotides, although many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, etc.).

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

[0055] In addition to the nucleic acid sequence encoding the immunoglobulin heavy and / or light chain, the vector may include expression control sequences such as a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), etc. that provide for the expression of the coding sequence in the host cell. 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] A number of promoters, including constitutive, inducible, and repressive promoters from a variety of different sources, are well known in the art. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeast, and bacteria, and suitable promoters from these sources are readily available or can be produced synthetically based on publicly available sequences from depositories such as the 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, the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone induction 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] As used herein, the term "enhancer" refers to, for example, a DNA sequence that 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 binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A number of 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 as well as other commercial or individual sources). Many polynucleotides, including promoters (such as the commonly used CMV promoter), also contain enhancer sequences. Enhancers can be located upstream, within, or downstream of the coding sequence.

[0058] Vectors can also contain a selectable marker gene. As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that enables cells expressing the nucleic acid sequence to be specifically selected in the presence of the corresponding selection agent. Suitable selectable marker genes are known in the art and include, for example, International Patent Application Publications WO1992 / 008796 and WO1994 / 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., 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 are described in U.S. Pat. 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 persists as an extrachromosomal segment of DNA within the host cell in the presence of 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 Epstein - Barr nuclear antigen 1 (EBNA1) and the Epstein - Barr virus (EBV) origin of replication (oriP). 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 the T antigen and the SV40 origin of replication instead of EBNA1 and oriP.

[0060] Other suitable vectors include integrative expression vectors that can be randomly integrated into the host cell's DNA or that can contain recombination sites that allow for specific recombination between the expression vector and the host cell's chromosome. Such integrative expression vectors can utilize the endogenous expression control sequences of the host cell's chromosome to effect the expression of the desired protein. Examples of vectors that integrate in a site-specific manner can be found, for example, in the components of the flp-in system from Invitrogen (Carlsbad, CA) (e.g., pcDNA™5 / FRT), or in cre-lox systems such as those found in the pExchange-6 core vector from Stratagene (La Jolla, CA). Examples of vectors that integrate randomly into the host cell's chromosome include, for example, pcDNA3.3 from ThermoFisher (Carlsbad, CA) (when introduced in the absence of the T antigen), 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] The nucleic acid sequence encoding the amino acid sequence of the present invention can be provided to cells (i.e., in cis) on the same vector. 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, the nucleic acid sequence encoding the amino acid sequence of the present invention can be provided to a population of cells (i.e., in trans) on separate vectors. Each of the nucleic acid sequences in each of the separate vectors can include the same or different expression control sequences. The separate vectors can be provided to the cells simultaneously.

[0063] The vector(s) containing the nucleic acid(s) encoding the amino acid sequence of the present invention can be introduced into a host cell that can express the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell. As such, the present invention provides an in vitro cell or cell line containing the vector of the present invention. The present invention also provides an in vitro cell or cell line that expresses an immunoglobulin heavy and / or light chain polypeptide or that expresses a PD-1 binder. Preferred host cells are those that can grow easily and reliably, have a moderately fast growth rate, have a well-characterized expression system, and can be transformed or transfected easily and efficiently.

[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 Escherichia coli), Pseudomonas, Streptomyces, Salmonella, and Erwinia. Particularly useful prokaryotic cells include various strains of E. coli (e.g., K12, HB101 (ATCC number 33694), DH5α, DH10, MC1061 (ATCC number 53338), and CC102).

[0065] In some embodiments, the vector is introduced into eukaryotic cells. 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, and many are described in American Available from the 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 number 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 number CRL1573), and 3T3 cells (e.g., ATCC number CCL92). Other suitable mammalian cell lines include simian COS - 1 (e.g., ATCC number CRL1650) and COS - 7 cell lines (e.g., ATCC number CRL1651), and CV - 1 cell line (e.g., ATCC number CCL70). Further exemplary mammalian host cells include mouse cell line NS0, which is a derivative of the mouse myeloma strain MOPC21 (e.g., Tysabri), and primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from in vitro culture of primary tissues, 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 suitable mammalian host cells and methods for cell transformation, culture, amplification, screening, and purification are known in the art.

[0068] In some embodiments, the mammalian cell is a human cell. For example, the mammalian cell can be a human lymphocyte or lymphocyte - derived cell line, such as a cell line 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] The nucleic acid sequence encoding the amino acid sequence of the present invention can be introduced into cells by any suitable technique such as "transfection", "transformation", or "transduction". In the present specification, "transfection", "transformation", or "transduction" as used 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, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after the growth of infectious particles in suitable packaging cells, many of which are commercially available.

[0070] Nucleic acids and cells can be used for any purpose, such as for the production of the PD-1 binding agents described herein. In this regard, the present invention provides a method for producing a PD-1 binding agent, which includes culturing cells containing nucleic acids encoding the heavy and / or light immunoglobulin polypeptides of the PD-1 binding agent. In other words, the method includes expressing in the cells nucleic acids encoding the immunoglobulin heavy and / or light chains of the PD-1 binding agent. 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 can further include 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 in no way be construed as limiting its scope.

Example

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

[0073] The 437M5-112 antibody is derived from single cell PCR on sorted PD-1 binding IgG switched B cells from immunized mouse spleen. The 3.7C6 antibody is derived from a mouse hybridoma generated by standard fusion techniques from spleen cells of immunized mice. The antibodies were humanized using standard techniques described herein. The final optimized antibodies were expressed in CHO cells. The antibody sequences are summarized in Tables 1A, 1B, and 1C, where the "H" and "L" chains refer to the heavy and light chains, respectively, and the CDRs are determined to contain amino acids according to both the Kabat and IMGT definitions (Table 1B) or according to Kabat or IMGT for specific antibodies (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 HEK293 cells stably transfected with the antibodies.

[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), and 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 (2 x 10 total per sample 5It was stained with each antibody at the indicated concentration for 40 minutes at 4°C with gentle shaking, centrifuged, and washed once. The cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes at room temperature, washed, and the antibody was detected with phycoerythrin (PE)-conjugated goat anti-human kappa (Southern Biotechnology, Birmingham, AL) for 15 minutes at 4°C with gentle shaking. The cells were washed, resuspended, and the fluorescence of the bound antibody was quantified on a BD FACSArray™ (BD Biosciences, San Jose, CA). The data were analyzed for the median fluorescence intensity (MFI) using FlowJo® analysis software (FlowJo, LLC). EC 50 values were determined in GraphPad Prism 5.0 (GraphPad Software) using a log (agonist) versus response-variable slope (4-parameter) curve fit. The results are shown in FIGS. 1 and 2, and the EC 50 values are described 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 bind to 2-day anti-CD3 / anti-CD28-activated human peripheral blood CD4 + T cells.

[0082] Primary human peripheral blood CD4 + T cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic bead separation (CD4 +Prepared using a T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) and activated with plastic-coated anti-CD3 and anti-CD28 in 6-well plates for 48 hours. Cells (1 x 10 per sample 5 ) were washed and stained with each antibody at the indicated concentration in a V-bottom 96-well plate with gentle shaking at 4°C for 30 minutes, 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, resuspended, and the fluorescence of the bound antibody 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 values are described 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 chicken egg lysozyme.

[0083]

Table 6

[0084] Example 3 This example records 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] A competition assay was performed to test for competition for PD-1 binding between the anti-PD-1 antibody and the PD-L1-Fc or PD-L2-Fc constructs. As shown in Figures 4-7, the antibodies tested showed moderate competition with PD-L1 (about 70% maximum inhibition) and strong competition with PD-L2; another antibody tested showed weak / minimal competition with PD-L1 (about 15% maximum inhibition) and moderate competition with PD-L2 (about 70% maximum inhibition).

[0086] CHO-K1 cells were stably transfected to express human PD-1, and high-expressing clones were selected. Cells were harvested by Accutase™ treatment (Innovative Cell Technologies, San Diego, CA) and placed in U-bottom 96-well plates (2x10 5 cells / well). For the test, the PD-L1 competing antibody was 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 a 10-minute incubation on ice, the antibody / DyL650-PD-L1-Fc mixture was added to the cells at 4°C for 30 minutes with gentle shaking. The cells were centrifuged, washed once, resuspended in buffer containing propidium iodide, and the bound PD-L1-Fc fluorescence was quantified on a BD FACSArray™ (BD Biosciences, San Jose, CA). The data were analyzed for PD-L1 geometric mean 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. The results are shown in Figures 4 and 5, and the resulting IC 50The values are described 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”) is the 437M5-112 anti-PD-1 antibody described in Example 1. APE12095 (“12095.03” and “12095.04”) is 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 recovered by Accutase™ treatment (Innovative Cell Technologies, San Dirgo, CA) and placed into U-bottom 96-well plates (2 x 10 5 cells / well). For testing, PD-L2 competing 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 a 10-minute incubation on ice, the antibody / DyL650-PD-L2-Fc mixture was added to the cells at 4°C for 30 minutes with gentle shaking. The cells were centrifuged, washed once, resuspended in buffer containing propidium iodide, and the bound PD-L2-Fc fluorescence was quantified on a BD FACSArray™ (BD Biosciences, San Jose, CA). The data were analyzed for PD-L2 geometric mean 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. The results are shown in Figures 6 and 7, and the resulting IC 50Values are described 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") is the 437M5 - 112 anti - PD - 1 antibody described in Example 1. APE12095 ("12095.03" and "12095.04") is 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) was conjugated with anti-CD3 (10 μg), anti-PD-1 or PD-L1-Fc (40 μg), and the negative control antibody-conjugated chicken egg lysozyme (50 μg) according to the manufacturer's instructions to a total of 100 μg of conjugated protein. The degree of bead coupling was quantified by flow cytometry. Primary human peripheral blood CD4 + T cells were prepared using magnetic bead separation of PBMC (CD4 + T Cell Isolation Kit, Miltenyi Biotec, Auburn, CA). Purified CD4 + T cells (1 x 10 5 cells / well) were incubated for 72 hours with different numbers of beads (bead:T cell ratios of 4:1, 2:1, or 1:1) in the presence of soluble anti-CD28 (eBioscience; shown at 250 ng / ml, 100 ng / ml, or 50 ng / ml). Secreted IFNγ in the culture supernatant was quantified by ELISA (R&D Systems, Minneapolis, MN). As shown in FIGS. 8A and 8B and summarized in Table 8, the anti-PD-1 antibodies (437M5-112 and 3.7C6) disclosed herein showed consistent inhibitory (agonist) activity comparable to PD-L1-Fc in the bead assay.

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

[0095]

Table 11

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

[0097] For plate-based agonist assays, 96-well plates were continuously coated overnight at 4° C. with anti-CD3 (0.3 μg / ml), the wells were aspirated and washed with PBS, and then subjected to a second coating overnight at 4° C. with various concentrations of anti-PD-1 antibodies or PD-L1-Fc shown in FIGS. 12-14. 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. The cells were harvested, washed and incubated for 48 hours in anti-CD3 / anti-PD-1 coated wells (1×10 5 cells / well) in the presence of human gamma globulin (100 μg / ml). Secreted IL-2 in the culture supernatants was quantified by ELISA (R&D Systems, Minneapolis, MN). Inhibition of IL-2 production by the PD-1 antibodies across 3 PBMC donors is shown in FIGS. 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 agonist 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 a U-bottom 96-well plate 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 tetramer PD-L1-Fc, the anti-PD-1 IgG1 antibodies described herein (3.7C6 APE12095; 437M5-112 APE12043), or control human IgG1. Secreted IFNγ in the culture supernatant was quantified by ELISA (R&D Systems, Minneapolis, MN). In this tetanus toxoid recall response whole blood assay, as shown in FIGS. 15A (positive and negative controls) and 15B (anti-PD-1 antibody), potent agonist antibody activity of the anti-PD-1 antibodies described herein was observed in the presence of blocking anti-PD-L1 / anti-PD-L2.

[0100] The IgG1 3.7C6 anti-PD-1 antibody was compared to the same antibody prepared as human IgG2 (FIGS. 15C and 15D). The anti-PD-1 IgG2 version of the antibody had the same activated T cell binding as anti-PD-1 IgG1 but did not show 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 decrease the immune response in whole blood in a concentration-dependent manner.

[0102] Human whole blood stimulated in vitro with an appropriate antigen will elicit a specific T cell recall immune response when the donor has previously experienced exposure to the antigen of interest. The immune response can be measured by IFN-γ and IL17A levels.

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

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

[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) showed binding kinetics by surface plasmon resonance (SPR) comparable to that of human and cynomolgus monkey PD-1. The tight binding kinetics approached the limit of the instrument. The SPR data was in good agreement with the equilibrium binding affinity of APE12095 determined by the kinetic exclusion assay (KinExA®). The final affinity measurements were KinExA® K D : 75 pM for human PD-1; and KinExA® K D : 450 pM for cynomolgus monkey PD-1.

[0107] 437M5-112 (APE12043.05 and APE12538.01) also showed binding kinetics by SPR comparable to that of human and cynomolgus monkey PD-1. The tight binding kinetics approached the limit of the instrument. The SPR data was in good agreement with the equilibrium binding affinity of APE12043 determined by KinExA®. The final affinity measurements were KinExA® K D : 51 pM for human PD-1; and KinExA® K D : 210 pM for cynomolgus monkey PD-1. The K for the anti-PD-1 antibodies disclosed herein by surface plasmon resonance and KinExA® D measurements are summarized in Table 9.

[0108]

Table 12

[0109] The binding affinity and thermal stability of the APE12537 antibody were compared with those of a similar antibody designated as "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 the positions in the sequence listing). The results provided in Table 10 indicate that these mutations increased the 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 the kinetic constants were globally fitted using a 1:1 binding model. Biotinylated human or cynomolgus monkey PD-1 extracellular domain monomer was captured at a concentration of 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 give a low response to prevent the affinity effect on the dissociation rate. The Tm measurement values 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 are effective in vivo in a xenogeneic NSG / Hu - PBMC graft - versus - host disease (GvHD) model.

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

[0114] The 3.7C6 PD-1 agonist antibody (APE12095) disclosed herein showed statistically significant efficacy against the isotype control in the time to 10% weight loss (Figure 16C). The 437M5-112 anti-PD-1 agonist antibody also showed statistically significant efficacy against the isotype control in the time to 10% weight loss (Figure 16D). The responses to both anti-PD-1 antibodies were bimodal, and the proportion of animals in each group survived completely in the study (Figures 16C and 16D).

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

[0116] The study design of the single-dose pharmacokinetics and tolerance study in cynomolgus monkeys is described in Table 11. The evaluations during the study were as follows: - Clinical pathology, pre-dose (2 times), days 2, 6, 22, and 35 (Charles River Laboratories (CRL)) - Blood FACS panel - major leukocyte populations and 20 T cell subsets, pre-dose (2 times), days 2, 6, 22, and 35 (CRL) - B, T, NK, monocytes - CD4, CD8, T central memory, T effector memory, PD-1 + , act ivated CD4 + and CD8 + , Treg - Receptor occupancy, days 4, 14, 28, 35 - PK sample analysis, anti-drug antibody analysis at pre-dose and day 35 - 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 hours and 24 hours, day 7 and day 35 - PK parameter analysis using Phoenix® WinNonlin® (Certara, USA).

[0117] Both PD-1 antibodies showed pharmacokinetic properties with good performance at detectable drug levels in all animals on 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 study are described in Tables 11 and 12.

[0118]

Table 14

[0119]

Table 15

[0120]

Table 16

[0121] As shown, all 6 animals administered with antibody APE12538.01 had measurable / low-titer anti-drug antibodies on day 36. Of the 6 animals administered with antibody APE12537.01 2 of the 6 animals had measurable / low-titer anti-drug antibodies on day 36. There were no significant changes in any of the cytokines to be evaluated. Furthermore, as shown in FIGS. 18A and 18B, sustained 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 antibody disclosed herein induced recruitment of the phosphatase SHP2 to the PD-1 cytoplasmic domain in Jurkat cells transfected with PD-1.

[0123] Antibody 3.7C6 (APE12890) or a human IgG1 isotype control antibody recognizing hen egg lysozyme and a fixed amount of anti-CD3 (UCHT1 clone; BioLegend, San Diego, CA) were conjugated to magnetic beads (Dynabeads™ M-280 Tosylated; conjugated to Invitrogen (trademark) / ThermoFisher Scientific. The anti-PD-1 antibody on the beads mimics the involvement of FcγR by the antibody on antigen-presenting cells. Stable human PD-1 transfected Jurkat cells were stimulated with the indicated beads for either 2 minutes or 10 minutes, the cells were lysed, and PD-1 was immunoprecipitated by the addition of 3.7C6 binding 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), after activation of PD-1 Jurkat cells with anti-CD3, the 3.7C6 antibody described herein, but not the isotype control antibody, induced recruitment of the phosphatase SHP2 to the PD-1 cytoplasmic domain, but did not induce recruitment of SHP1. 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. No recruitment of SHP was seen with soluble nivolumab. In combination with T cell activation and CD28 co-stimulation, antibody 3.7C6 also decreased phosphorylation of ZAP70 and LAT (data not shown). Antibody 3.7C6 had no effect on the signaling pathway 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 to which the 3.7C6 antibody (APE12537) disclosed herein binds was performed at Biomotif AB (Danderyd, Sweden) using recombinant human PD-1 monomer. The structure of PD-1 is publicly available through the Protein Data Bank (PDB) operated by the National Center for Biotechnology Information (Bethesda, MD) (see also accession 4ZqK (Zak, K. M. et al., 2015, Structure 23:2341-2348; and PDB accession 5GGR (Lee, J. Y. et al., 2016, Nat Commun., 7:13354)). One major peptide labeled "HDX mapped β-hairpin" in FIGS. 20A and 20B was protected from hydrogen-deuterium exchange by the 3.7C6 antibody. The "HDX mapped β-hairpin" is composed of amino acids 96-110 of PD-1, which have the sequence RVTQLPNGRDFHMSV. Another major peptide is amino acid 33 of PD-1 It is composed of ~41, and the amino acid has the sequence NPPTFSPAL. Figures 20A and 20B show a space-filling model (light gray) of the crystal structure of the human PD-L1 extracellular binding domain and a ribbon model (black) of the crystal structure of the docked human PD-1 extracellular domain (PD-1 and PD-L1 structures from NCBI PDB). The molecules are oriented with the membrane-proximal region of PD-1 at the lower left (Figure 20A) and rotated 90° to show the membrane-proximal region of PD-1 at the lower center (Figure 20B). Human PD-1 monomers containing different sets of mutations in defined surface regions were expressed, and the binding of the 3.7C6 antibody (APE12095) disclosed herein was evaluated by surface plasmon resonance. Mutations in the region labeled "PD-1 triple mutant" in Figures 20A and 20B completely abrogated the binding of the 3.7C6 antibody disclosed herein. Mutations at the top of the loop labeled "HDX mapped β hairpin" in Figures 20A and 20B had no effect on the binding of the 3.7C6 antibody disclosed herein. The combination of hydrogen-deuterium exchange and PD-1 mutation mapping demonstrated that the 3.7C6 PD-1 agonist antibody disclosed herein binds to the region depicted by the dotted circle in Figure 20B, which is on the opposite side of PD-1 from the PD-L1 binding site.

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

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

[0128] PBMCs were isolated from the blood of alopecia areata donors and cultured in plates in the presence of keratinocyte peptides antigen (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 (2×10 5 cells / well). After 5 days, the cells were washed and further incubated in ELISpot plates for 20 hours to detect the number of IFNγ-secreting cells. The results from each donor and treatment group were normalized against untreated wells to enable statistical comparison of data from 12 donors for the treatment and negative controls.

[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 control IgG1 isotype. 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 and compared to the control IgG1 isotype tetramer. Both the anti-PD-1 antibody 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 antibody disclosed herein inhibited the production of IFNγ in peripheral blood mononuclear cells (PBMCs) from alopecia areata donors stimulated with melanocyte peptide antigen.

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

[0132] PBMCs were isolated from the blood of alopecia areata donors and cultured in plates (2×105 cells / well) in the presence of melanocyte peptide antigen (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 by Meso Scale Discovery (Meso Scale Diagnostics, Rockville, MD). Results from each donor and treatment were normalized to untreated wells to enable statistical comparison of data from 12 donors for the treatments and negative controls. Additionally, after 5 days, cells were washed and further incubated for 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 enable statistical comparison of data from 12 donors for the treatments and negative controls. The results are shown in FIGS. 23A - 23D.

[0133] As shown in Figure 23A, the IgG1 3.7C6 anti-PD-1 antibody inhibited IFNγ production in a concentration-dependent manner compared to the control IgG1 isotype. As shown in Figure 23B, the positive control PD-L1-IgG1 Fc tetramer inhibited IFNγ production in a concentration-dependent manner compared to the IgG1 isotype tetramer and compared to the control IgG1 isotype tetramer. Both the anti-PD-1 antibody 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 antibody and the PD-L1-IgG1 Fc tetramer described herein significantly decreased 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 antibody disclosed herein is effective in vivo at a dose of 3 mg / kg in a xenogeneic NSG / Hu-PBMC graft-versus-host disease (GvHD) model.

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

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

[0137] There was no significant difference in survival between the 30 mg / kg of anti-PD-1 agonist IgG1 (3.7C6) and the 3 mg / kg dose group of anti-PD-1 agonist IgG1 (3.7C6). This suggests that efficacy in the GvHD model can be obtained at doses less than 3 mg / kg.

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

[0139] The use of the terms "a", "an", "the", and "at least one" and similar referents in the context of describing the present invention (particularly in the context of the following claims) should be construed to cover both the singular and the plural forms unless otherwise indicated herein or clearly contradicted by the context. The term "at least one" accompanied by a list of one or more items (e.g., "at least one of A and B") should be construed 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 the context. The terms "comprising", "having", "including", and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless specifically stated otherwise. The recitation of a range of values herein is merely intended to serve as a convenient method of referring individually to each separate value within the range, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any example, or exemplary language (e.g., "such as") provided herein is merely intended to better illuminate the invention and does not limit 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 the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend for the invention to be practiced 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. Further, any combination of the above elements in all possible variations is included in the invention as long as such combinations are not otherwise indicated herein or are not clearly precluded by context.

Claims

1. An immunoglobulin heavy chain variable region comprising SEQ ID NO: 29, or an immunoglobulin heavy chain variable region comprising at least CDR1, CDR2 and CDR3 thereof; and an immunoglobulin light chain variable region comprising SEQ ID NO: 35, or at least CDR1, CDR2 and CDR3 thereof; An anti-PD-1 binding agent comprising:

2. The immunoglobulin heavy chain variable region comprises at least CDR1, CDR2, and CDR3 of SEQ ID NO:29, as determined by the numbering according to Kabat, Chothia, Martin, IMGT, or AHo; and the immunoglobulin light chain variable region comprises at least CDR1, CDR2 and CDR3 of SEQ ID NO: 35, as determined by the numbering according to Kabat, Chothia, Martin, IMGT or AHo; 2. The anti-PD-1 binding agent of claim 1.

3. An immunoglobulin heavy chain variable region comprising the following CDRs as determined by Kabat numbering: CDR1: SEQ ID NO:76, CDR2: SEQ ID NO:77, CDR3: comprises SEQ ID NO:78, and The immunoglobulin light chain variable region comprises the following CDRs as determined by Kabat numbering: CDR1: SEQ ID NO:79, CDR2: SEQ ID NO: 80, CDR3: comprising SEQ ID NO: 81, 2. The anti-PD-1 binding agent of claim 1.

4. The immunoglobulin heavy chain variable region comprises the following CDRs as determined by IMGT numbering: CDR1: SEQ ID NO: 82, CDR2: SEQ ID NO:83, CDR3: comprises SEQ ID NO: 84, and The immunoglobulin light chain variable region comprises the following CDRs as determined by IMGT numbering: CDR1: SEQ ID NO: 85, CDR2: SEQ ID NO: 86, CDR3: comprising SEQ ID NO: 87, 2. The anti-PD-1 binding agent of claim 1.

5. The immunoglobulin heavy chain variable region comprises at least 90% sequence identity to SEQ ID NO:29; and the immunoglobulin light chain variable region comprises at least 90% sequence identity to SEQ ID NO:35; 2. The anti-PD-1 binding agent of claim 1.

6. An immunoglobulin heavy chain variable region of SEQ ID NO: 29, and comprising an immunoglobulin light chain variable region of SEQ ID NO: 35; 2. The anti-PD-1 binding agent of claim 1.

7. An immunoglobulin heavy chain comprising SEQ ID NO:36, and comprising an immunoglobulin light chain comprising SEQ ID NO: 37, 2. The anti-PD-1 binding agent of claim 1.

8. The anti-PD-1 binding agent of any one of claims 1 to 7, wherein the anti-PD-1 binding agent is an antibody, an antigen-binding antibody fragment, or a conjugate thereof.

9. The anti-PD-1 binding agent of any one of claims 1-6, wherein the anti-PD-1 binding agent is a F(ab')2, Fab', Fab, Fv, scFv, dsFv, or a single chain binding polypeptide.

10. The anti-PD-1 binding agent of any one of claims 1 to 6, wherein the anti-PD-1 binding agent comprises an IgG Fc region that binds to an Fc receptor on an antigen presenting cell.

11. The anti-PD-1 binding agent of any one of claims 1 to 7, wherein the anti-PD-1 binding agent comprises an IgG1 Fc region or other Fc region that binds to FcγR.

12. A pharmaceutical composition comprising: (a) an anti-PD-1 binding agent of any one of claims 1 to 11; and (b) a pharma- ceutically acceptable carrier.

13. (1-1) An immunoglobulin heavy chain comprising an immunoglobulin heavy chain variable region comprising SEQ ID NO: 29, or an immunoglobulin heavy chain variable region comprising at least CDR1, CDR2 and CDR3 thereof; (1-2) An immunoglobulin light chain comprising an immunoglobulin light chain variable region comprising SEQ ID NO: 35, or an immunoglobulin light chain variable region comprising at least CDR1, CDR2 and CDR3 thereof; or (2) The anti-PD-1 binding agent according to any one of claims 1 to 11, A nucleic acid encoding 14. A cell expressing an anti-PD-1 binding agent according to any one of claims 1 to 11.

15. A method for producing an anti-PD-1 binding agent according to any one of claims 1 to 11, said method comprising administering to a cell: (1) A nucleic acid encoding the anti-PD-1 binding agent of any one of claims 1 to 11; or (2-1) A nucleic acid encoding an immunoglobulin heavy chain comprising an immunoglobulin heavy chain variable region comprising SEQ ID NO: 29, or an immunoglobulin heavy chain variable region comprising at least CDR1, CDR2, and CDR3 thereof; and (2-2) A nucleic acid encoding an immunoglobulin light chain comprising an immunoglobulin light chain variable region comprising SEQ ID NO: 35, or an immunoglobulin light chain variable region comprising at least CDR1, CDR2 and CDR3 thereof; The method of claim 1, further comprising expressing 16. An anti-PD-1 binding agent according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12 for inhibiting an immune response in a mammal.

17. An anti-PD-1 binding agent according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12 for treating an inflammatory or autoimmune disorder in a mammal.

18. The anti-PD-1 binding agent or pharmaceutical composition of claim 17, 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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