Methods and compositions for treating autoimmune, allergic, and inflammatory diseases

Abstract

Disclosed herein are methods of treating an autoimmune, allergic, and / or inflammatory disease in a subject in need thereof, the methods comprising administering a therapeutic agent comprising one or more antigen binding sites to and depleting cells involved in the autoimmune, allergic, and / or inflammatory disease.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 358,675, filed July 6, 2022, and U.S. Provisional Patent Application No. 63 / 481,261, filed January 24, 2023, the entire contents of each of which are incorporated herein by reference for all purposes. [Background technology]

[0002] background The immune system is tightly regulated by various immune cells and co-stimulatory and co-inhibitory ligands and receptors. Inflammation is a normal physiological defense against pathogen infection and tissue damage and is rapidly resolved under normal circumstances. However, in many chronic conditions, the inflammatory response persists and can lead to significant tissue and organ damage. Abnormal inflammatory responses have been shown to be associated with many chronic diseases, particularly autoimmune diseases, in which immune cells act on self-proteins. Allergies also result from dysregulation of the immune system's ability to recognize harmless non-self antigens. While mast cells and IgE antibodies are central to allergic responses, rogue T cells or B cells are key actors in autoimmunity.

[0003] There remains a need for therapeutic agents and methods that can be used alone or in combination to fine-tune the activity of immune cells and the immune system for autoimmune, allergic, and inflammatory diseases. Summary of the Invention

[0004] overview The present application provides methods and compositions for treating autoimmune, allergic, and inflammatory diseases.

[0005] In one aspect, an antibody or antigen-binding fragment thereof capable of binding to PD-1 is provided, wherein the antibody or antigen-binding fragment thereof (a) is a PD-1 agonist, (b) has enhanced binding to an Fc receptor, and / or (c) is non-fucosylated.

[0006] In some embodiments, the Fc receptor is an inhibitory Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In some embodiments, the Fc receptor is FcγRI. In some embodiments, the Fc receptor is FcγRII. In some embodiments, the Fc receptor is FcγRIII.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof has enhanced effector functions, ie, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0008] In some embodiments, the antibody or antigen-binding fragment thereof is capable of depleting a cell. In some embodiments, the cell is a PD-1-expressing cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell, a B cell, a macrophage, a natural killer (NK) cell, a dendritic cell (DC), a monocyte, a neutrophil, a fibroblast, or an epithelial cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is activated. In some embodiments, the immune cell is an antigen-activated T cell or B cell.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof is at least about 60%, about 75%, or about 90% non-fucosylated.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof (a) (i) a heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 2 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 3 or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable (VH) region comprising: (ii) a light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 6 or a sequence which differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 8 or a sequence that differs therefrom by one or two amino acids. a light chain variable (VL) region comprising (b) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 10 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 11 or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 12 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 14 or a sequence that differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 15 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 16 or a sequence that differs therefrom by one or two amino acids. a VL region comprising (c) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 18 or a sequence that differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 19 or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 20 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 22 or a sequence that differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 23 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 24 or a sequence that differs therefrom by one or two amino acids. a VL region comprising (d) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 26 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 27 or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 28 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 30 or a sequence that differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 31 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 32, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (e) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 34 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 35 or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 36 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 38 or a sequence that differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 39 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 40 or a sequence that differs therefrom by one or two amino acids. a VL region comprising (f) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 34 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 43 or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 36 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 38 or a sequence that differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 39 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 40 or a sequence that differs therefrom by one or two amino acids. a VL region comprising (g) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 51 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 52, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 53 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 55 or a sequence that differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 56 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 57 or a sequence that differs therefrom by one or two amino acids. a VL region comprising (h) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 51 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 60, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 53 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 55 or a sequence that differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 56 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 57 or a sequence that differs therefrom by one or two amino acids. a VL region comprising (i) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 70 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 71 or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 72 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 74 or a sequence which differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 75 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 76 or a sequence that differs therefrom by one or two amino acids. a VL region comprising (j) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 70 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 79 or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 72 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 74 or a sequence which differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 75 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 76 or a sequence that differs therefrom by one or two amino acids. a VL region comprising (k) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 86, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 87 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence that differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 90 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 91 or a sequence that differs therefrom by one or two amino acids. a VL region comprising or (l) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence which differs therefrom by one or two amino acids; an HCDR2 having the amino acid sequence of SEQ ID NO: 94 or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 87 or a sequence that differs therefrom by one or two amino acids. and a VH region comprising (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence that differs therefrom by one or two amino acids; LCDR2 having the amino acid sequence of SEQ ID NO: 90 or a sequence which differs therefrom by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 91 or a sequence that differs therefrom by one or two amino acids. VL region containing Includes.

[0011] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are (a) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; (b) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; (c) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; (d) SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively; (e) SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; (f) SEQ ID NO: 34, SEQ ID NO: 43, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; (g) SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively; (h) SEQ ID NO: 51, SEQ ID NO: 60, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively; (i) SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively; (j) SEQ ID NO: 70, SEQ ID NO: 79, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively; (k) SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, respectively; or (l) SEQ ID NO: 85, SEQ ID NO: 94, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, respectively. and an amino acid sequence that differs from the sequence of by a total of no more than two amino acid residues.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8 a VL region comprising (b) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16 a VL region comprising (c) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 a VL region comprising (d) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32 a VL region comprising (e) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 a VL region comprising (f) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 a VL region comprising (g) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 a VL region comprising (h) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 a VL region comprising (i) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 71, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 74; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 76 a VL region comprising (j) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 74; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 76 a VL region comprising (k) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 89; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 90, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91 a VL region comprising or (l) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 89; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 90, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91 VL region containing Includes.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 and a VH region comprising (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 VL region containing Includes.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof (a) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5; (b) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 13; (c) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 17, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21; (d) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 29; (e) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 33, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 37; (f) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 42 or 45, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 47 or 49; (g) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; (h) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 56, 62, 64, or 66, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 68; (i) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 69, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 73; (j) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 78, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 81 or 83; (k) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 88; or (l) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 93, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 96 or 98. Includes.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof (a) a VH region having the amino acid sequence of SEQ ID NO: 1 and a VL region having the amino acid sequence of SEQ ID NO: 5; (b) a VH region having an amino acid sequence of SEQ ID NO: 9 and a VL region having an amino acid sequence of SEQ ID NO: 13; (c) a VH region having an amino acid sequence of SEQ ID NO: 17, and a VL region having an amino acid sequence of SEQ ID NO: 21; (d) a VH region having an amino acid sequence of SEQ ID NO: 25, and a VL region having an amino acid sequence of SEQ ID NO: 29; (e) a VH region having an amino acid sequence of SEQ ID NO: 33, and a VL region having an amino acid sequence of SEQ ID NO: 37; (f) a VH region having an amino acid sequence of SEQ ID NO: 42 or 45, and a VL region having an amino acid sequence of SEQ ID NO: 47 or 49; (g) a VH region having an amino acid sequence of SEQ ID NO: 50, and a VL region having an amino acid sequence of SEQ ID NO: 54; (h) a VH region having an amino acid sequence of SEQ ID NO: 56, 62, 64, or 66, and a VL region having an amino acid sequence of SEQ ID NO: 68; (i) a VH region having an amino acid sequence of SEQ ID NO: 69, and a VL region having an amino acid sequence of SEQ ID NO: 73; (j) a VH region having an amino acid sequence of SEQ ID NO: 78 and a VL region having an amino acid sequence of SEQ ID NO: 81 or 83; (k) a VH region having an amino acid sequence of SEQ ID NO: 84, and a VL region having an amino acid sequence of SEQ ID NO: 88; or (l) a VH region having the amino acid sequence of SEQ ID NO: 93 and a VL region having the amino acid sequence of SEQ ID NO: 96 or 98 Includes.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region having the amino acid sequence of SEQ ID NO: 42 or 45, and a VL region having the amino acid sequence of SEQ ID NO: 47 or 49.

[0017] In some embodiments, the antibody is a humanized antibody. In some embodiments, the antigen-binding fragment is a Fab, Fab', Fab2, F(ab')2, Fv, single-chain Fv (scFv), or diabody. In some embodiments, the antigen-binding fragment is an scFv.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region.

[0019] In some embodiments, the antibody heavy chain constant region is a human IgG heavy chain constant region. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region. In some embodiments, the antibody heavy chain constant region comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the antibody heavy chain constant region has the amino acid sequence of SEQ ID NO: 100.

[0020] In some embodiments, the antibody heavy chain constant region comprises one or more mutations that enhance effector function. In some embodiments, the antibody heavy chain constant region comprises one or more of the following mutations: (a) E233D, (b) G236D, (c) G237D, (d) P238D, (e) S239D, (f) S267E, (g) H268D, (h) P271G, (i) L328Y, L328F, or L328E, (j) A330R, (k) I332E, (l) 236D and H268D, (m) S239D and H268D, (n) S239D, H268D, L328Y, and I332E, (o (i) a human IgG1 heavy chain constant region comprising one or a combination of the following mutations: (a) P238D and L328E, (b) G237D, P271G, and A330R, (c) G237D, H268D, P271G, and A330R, (d) S267E and L328F, (e) S239D and S267E, (f) G236D and S276E, or (g) E233D, G237D, H268D, P271G, and A330R, where the numbering of the constant region is according to the EU index. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region comprising the following mutations: S239D and H268D, where the numbering of the constant region is according to the EU index. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region comprising the following mutations: E233D, G237D, H268D, P271G, and A330R, where the numbering of the constant region is according to the EU index.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof (a) a cell line with an alpha-1,6-fucosyltransferase (Fut8) knockout, or (b) Cell lines overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III) and optionally overexpressing Golgi μ-mannosidase II (ManII) It is produced in

[0022] In some embodiments, the antibody or antigen-binding fragment is capable of binding to human PD-1. In some embodiments, the antibody or antigen-binding fragment is capable of binding to cynomolgus PD-1.

[0023] In some embodiments, the antibody or antigen-binding fragment is capable of blocking the PD-1 / PD-L1 interaction.

[0024] In some embodiments, the antibody or antigen-binding fragment can inhibit T cell activation. In some embodiments, the antibody or antigen-binding fragment can inhibit T cell proliferation. In some embodiments, the antibody or antigen-binding fragment can inhibit IFN-γ secretion.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an agent. In some embodiments, the agent is a cytotoxic agent or a label. In some embodiments, the agent is a therapeutic agent.

[0026] In another aspect, a composition is provided comprising an antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment comprises an Fc region and N-glycoside-linked carbohydrate chains linked to the Fc region, and optionally, less than 50% of the N-glycoside-linked carbohydrate chains comprise a fucose residue. In some embodiments, substantially none of the N-glycoside-linked carbohydrate chains comprise a fucose residue.

[0027] In another aspect, there is provided a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein.

[0028] In another aspect, there is provided an expression vector comprising a polynucleotide described herein.

[0029] In another aspect, a host cell is provided that comprises the polynucleotide or expression vector described herein. In some embodiments, the host cell is a mammalian cell or an insect cell. In some embodiments, the host cell (a) comprises a Fut8 knockout and / or (b) overexpresses GnT-III and optionally ManII.

[0030] In another aspect, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0031] In another aspect, methods of treating a disease or disorder in a subject in need thereof are provided, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof described herein. In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is an allergy. In some embodiments, the disease or disorder is an inflammatory disease. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 shows blocking of the PD-1 / PD-L1 interaction by the disclosed PD-1 antibodies. Percent blocking is plotted against a standard curve of a control PD-1 neutralizing antibody. [Figure 2] We show that the disclosed PD-1 antibodies suppressed IFN-γ secretion in co-cultures of peripheral blood mononuclear cells (PBMCs) from different donors and triggered a mixed lymphocyte reaction (MLR). [Figure 3]Shown is the inhibition of activation of Jurkat reporter T cells incubated with Raji-APC cells modified to express the FcγRII receptor by a PD-1 antibody containing a normal IgG1 Fc or a modified Fc containing mutations to increase binding to FcγRII. [Figure 4] 1 shows inhibition of IL-2 secretion in Jurkat reporter T cells incubated with Raji-APC cells modified to express the FcγRII receptor by a PD-1 antibody containing a normal IgG1 Fc or a modified Fc containing mutations to increase binding to FcγRII. [Figure 5A] Figures 5A and 5B show that the PD-1 antibody 3H4, which contains modifications in the Fc domain to increase binding to FcγRII, inhibited CD4 (Figure 5A) and CD8 (Figure 5B) T cell proliferation. [Figure 5B] See legend to Figure 5A. [Figure 6] Figure 1 shows that PD-1 antibody 3H4, which contains modifications in the Fc domain to increase binding to FcγRII, inhibited IFN-γ secretion from PBMCs compared to a PD-1 antibody with a normal IgG1 Fc and controls (isotype antibody control and no antibody control). DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description definition As used herein, the terms "a" and "an" mean "one or more" and include pluralities unless the context is inappropriate.

[0034] As used herein, the terms "about," "approximately," and "comparable to," when used herein with respect to a value, refer to a value similar to the referenced value in the context of the referenced value. Generally, a person of ordinary skill in the art familiar with the context will understand the associated degree of variation encompassed by "about," "approximately," or "comparable to" in that context. For example, in some embodiments, the terms "about," "approximately," and "comparable to" can encompass values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0035] As used herein, "agonist" or "agonistic," when used in reference to an antigen-binding site or a molecule containing an antigen-binding site, refers to the fact that binding of the antigen-binding site or molecule to its target results in stimulation or activation of the target, or in enhancing, increasing, promoting, inducing, or prolonging one or more functions or biological activities of the target.

[0036] As used herein, the terms "antagonist," "agonistic," "neutralizing," or "blocking," when used in reference to an antigen-binding site or a molecule containing an antigen-binding site, refer to the fact that binding of the antigen-binding site or molecule to its target results in at least partial inhibition of the biological activity of the target.

[0037] As used herein, "antibody" refers to a polypeptide, or fragment thereof, whose amino acid sequence includes immunoglobulins and fragments thereof that specifically bind to a designated antigen. Antibodies according to the present invention can be of any type (e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4). Those skilled in the art will understand that a characteristic sequence or portion of an antibody can include amino acids found in one or more regions of the antibody (e.g., variable region, hypervariable region, constant region, heavy chain, light chain, and combinations thereof). Furthermore, those skilled in the art will understand that a characteristic sequence or portion of an antibody can include one or more polypeptide chains and can include sequence elements found in the same or different polypeptide chains.

[0038] As used herein, "antibody mimetic" refers to any molecule that can mimic the ability of an antibody to bind to an antigen, but is not limited to an antibody structure. Examples of antibody mimetics include, but are not limited to, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, centirins, DARPins, finomers, monobodies, nanobodies, and nanoCLAMPs.

[0039] An "antigen-binding fragment" of an antibody comprises a portion of an intact antibody that is still capable of antigen binding. In certain embodiments, papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting the ability to readily crystallize. The Fab fragment contains the variable region domain (V) of the heavy chain. H ) along with the entire light chain and the first constant domain of one heavy chain (C H1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. In certain embodiments, pepsin treatment of an antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. Fab' fragments contain one or more cysteines from the antibody hinge region, and the C H F(ab')2 antibody fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of one domain. Fab'-SH refers to Fab' in which the cysteine residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0040] As used herein, the term "antigen-binding site" refers to any molecule or any portion of a molecule capable of antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent extensions within the V regions of the heavy and light chains are called "hypervariable regions" and are inserted between more conserved side chain extensions known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In human antibodies, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs." In certain animals, such as camels and cartilaginous fish, the antigen-binding site can be formed by a single antibody chain, providing a "single domain antibody," and the antigen-binding site can be or be present in an antibody mimetic.

[0041] CDRs can be determined by the methods described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996). CDRs determined based on these definitions typically include overlapping or subsets of amino acid residues when compared with each other. In certain embodiments, the term "CDR" refers to a CDR as defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A., Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" refers to a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991). In certain embodiments, the heavy chain CDRs and light chain CDRs of an antibody are defined using different rules. For example, in certain embodiments, the heavy chain CDRs are defined according to MacCallum (supra) and the light chain CDRs are defined according to Kabat (supra), with CDRH1, CDRH2, and CDRH3 representing the heavy chain CDRs and CDRL1, CDRL2, and CDRL3 representing the light chain CDRs.

[0042] As used herein, the "binding affinity" of a molecule (e.g., an antigen-binding site) of the present disclosure for a given target (e.g., PD-1, PD-L1, or PD-L2) can be determined by a number of methods known to those of skill in the art, including, but not limited to, fluorescence titration, ELISA (enzyme-linked immunosorbent assay), calorimetry, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR).

[0043] As used herein, "bispecific" refers to a molecule that can specifically bind to at least two different targets. Typically, a bispecific molecule comprises two antigen binding sites, each of which is specific to a different target. In some embodiments, a bispecific molecule can simultaneously bind to two targets.

[0044] As used herein, "detectable affinity" typically refers to a D or EC 50 up to about 10, as measured by -5 It refers to the ability to bind to a given target with an affinity constant less than or equal to M (K D or EC 50 (Lower values represent better binding capacity.) Even lower affinities, which can no longer be measured by common methods such as ELISA (enzyme-linked immunosorbent assay), are of secondary importance.

[0045] As used herein, the term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope and composed of the six complementarity-determining regions of the antibody. A single antigen may have multiple epitopes. Epitopes can be conformational or linear. Conformational epitopes are composed of spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are composed of adjacent amino acid residues within a polypeptide chain.

[0046] The "Fc" fragment contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of the antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0047] A "fragment," with respect to an antigen (e.g., PD-1, PD-L1, or PD-L2), refers to an N-terminal and / or C-terminal truncation of the antigen or a domain of the protein. In some embodiments, a fragment of an antigen retains the ability of the full-length antigen to be recognized and / or bound by an antigen-binding site of the present disclosure.

[0048] As used herein, "fucosylation" or "fucosylated" refers to the presence of fucose residues in the oligosaccharides attached to the peptide backbone of an antibody. Specifically, a fucosylated antibody contains α(1,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue of one or both of the N-linked oligosaccharides attached to the antibody Fc region. "Afucosylated," "defucosylated," or "fucose-deficient" antibodies refer to glycosylated antibody variants comprising an Fc region, in which the carbohydrate structures attached to the Fc region have reduced fucose or lack fucose. In some embodiments, afucosylated or fucose-deficient antibodies have reduced fucose compared to the amount of fucose in the same antibody produced in a cell line. In some embodiments, antibodies with reduced fucose or lacking fucose have improved ADCC function.

[0049] "Degree of fucosylation" refers to the percentage of fucosylated oligosaccharides relative to total oligosaccharides, and can be determined by methods known in the art, for example, in N-glycosidase F-treated antibody compositions assessed by matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF MS). In a "fully fucosylated antibody" composition, essentially all of the oligosaccharides contain a fucose residue, i.e., are fucosylated. In some embodiments, a fully fucosylated antibody composition has a degree of fucosylation of at least about 90%. In contrast, in a "fully non-fucosylated antibody" composition, essentially none of the oligosaccharides are fucosylated. In some embodiments, a fully non-fucosylated antibody composition has a degree of fucosylation of less than about 10%. In a "partially fucosylated antibody" composition, only a portion of the oligosaccharides contain fucose. Individual antibodies in such compositions can contain none, one, or both fucose residues in the N-linked oligosaccharides in the Fc region, provided that the composition contains essentially all individual antibodies that contain fucose residues in both the Fc region and the N-linked oligosaccharides in the Fc region, or essentially all individual antibodies that lack fucose residues in the N-linked oligosaccharides. In one embodiment, a composition of partially fucosylated antibodies has a degree of fucosylation of about 10% to about 80% (e.g., about 50% to about 80%, about 60% to about 80%, or about 70% to about 80%). Assays for measuring the degree of fucosylation, as well as methods and cell lines for producing antibodies with altered, reduced, or ablated fucosylation, are known in the art (see, e.g., WO 2023 / 044390 A1).

[0050] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. For example, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced using hybridoma technology or by recombinant DNA technology (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries.

[0051] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that inherently renders the composition suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0052] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound described herein, which, upon administration to a subject, is capable of providing a compound described herein, or an active metabolite or residue thereof. As known to those skilled in the art, "salts" of the compounds described herein can be derived from inorganic or organic acids and bases.

[0053] Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Other acids, such as oxalic acid, while not themselves pharmaceutically acceptable, can be used in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0054] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and bases of formula NW4 + (Wherein W is C 1-4 Examples of compounds include:

[0055] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include, for example, Na + , NH4 + , and NW4 + (Wherein W is C 1-4 The anions of the compounds described herein include those compounded with a suitable cation, such as an alkyl group.

[0056] For therapeutic uses, the salts of the compounds described herein are contemplated as pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0057] As used herein, the phrase "baseline level" generally refers to a level considered "normal" for comparison purposes, e.g., the level of an appropriate control. For example, in the context of antibody effector function enhancement, "baseline level" may refer to the level of effector function of an antibody comprising a wild-type constant (Fc) region. As another example, in the context of target binding, "baseline level" may refer to the level of binding affinity (e.g., K D or EC 50 The reference level may be determined contemporaneously or may be predetermined, e.g., known or inferred from past observations.

[0058] As used herein, the terms "subject" and "patient" refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, etc.), and more preferably, humans.

[0059] As used herein, the phrases "therapeutically effective amount" and "effective amount" are used interchangeably and refer to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic effect. A therapeutically effective amount may vary depending on factors such as the type of disease (e.g., cancer), the state of the disease, the age, sex, and / or weight of the individual, and the ability of the immunoconjugate (or pharmaceutical composition thereof) to elicit a desired response in the individual. An effective amount may also be one in which any toxic or detrimental effects of the immunoconjugate or pharmaceutical composition thereof are outweighed by the therapeutically beneficial effects.

[0060] As used herein, "treating" a condition (e.g., a condition described herein, such as cancer) or "treatment" of a condition is an approach for obtaining a beneficial or desired result, e.g., a clinical outcome. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable; a reduction in the severity of a disease, disorder, or condition; a stable (i.e., non-worsening) state of a disease, disorder, or condition (e.g., of the primary cancer and / or secondary metastases); a delay or slowing of the progression of a disease, disorder, or condition; an improvement or palliation of a disease, disorder, or condition; and remission (whether partial or complete).

[0061] antigen binding site In one aspect, the present application provides antigen-binding sites capable of binding to a given antigen, for example, an antigen on a cell involved in an immune disease. In some embodiments, the antigen-binding site is capable of binding to an epitope within the antigen.

[0062] In some embodiments, the antigen-binding site can bind to an antigen on a cell involved in an autoimmune, allergic, or inflammatory disease. In some embodiments, the cell is an immune cell, such as a T cell, a B cell, a mast cell, a macrophage, a natural killer (NK) cell, a dendritic cell (DC), a monocyte, a neutrophil, a fibroblast, or an epithelial cell. In some embodiments, the cell is an activated cell, such as an antigen-activated T cell or a B cell. Examples of such antigens include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA-4, C-type lectins (CLECs), Siglecs, tumor necrosis factor (TNF) receptor superfamily members, 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), CD40 ligand (CD40L), CD30 ligand (CD30L), CD70 ligand (CD70L), CD27 ligand (CD27L), TIM-3, LAG-3, BTLA, KLRG1, 2B4, CD244, CD19, CD20, and CD22. , CD28, CD38, CD39, CD72, CD73, CD79A, CD79B, glycoprotein 130 (gp130), BCMA, BAFF receptor (BAFF-R), TACI, integrin alpha 4, integrin beta 7, FCGR2B, ICOS ligand, CD138, SLAMF7, LILR family members, fibroblast activation protein alpha (FAP), DLK-1, CD26, TE-7, CD29, PDGRF alpha, TGF beta receptor, MAS516, CD13, or a combination thereof. In some embodiments, the antigen is PD-1, PD-L1, or PD-L2. In some embodiments, the antigen is PD-1.

[0063] In some embodiments, the provided antigen binding site is present as an antibody mimetic, e.g., an affibody, affilin, affimer, aftin, alphabody, anticalin, avimer, centirin, DARPin, finomer, monobody, nanobody, or nanoCLAMP.

[0064] In some aspects, the antigen-binding site provided comprises a heavy chain variable domain and / or a light chain variable domain. In some embodiments, the antigen-binding site comprises a heavy chain variable domain comprising CDR-H1, CDR-H2, and CDR-H3, and / or a light chain variable domain comprising CDR-L1, CDR-L2, and CDR-L3. In some embodiments, the antigen-binding site is present as a single-chain variable fragment (scFv).

[0065] In some embodiments, the antigen-binding site provided is capable of binding to PD-1. In some embodiments, the antigen-binding site is present as an antibody. In some embodiments, variants of antigen-binding sites capable of binding to PD-1 and present as antibodies described herein are provided, wherein such antigen-binding sites comprise an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to the amino acid sequence contained in the antigen-binding site capable of binding to PD-1 and present as an antibody described herein.

[0066] In some embodiments, the provided antigen-binding site capable of binding to PD-1 comprises a heavy chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and / or a light chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3.

[0067] In some embodiments, provided antigen-binding sites capable of binding to PD-1 comprise a VH and a VL, wherein the VH comprises CDR-H1, CDR-H2, and CDR-H3, and the VL comprises CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are each independently selected from those for the VH or VL listed in Table 5. The CDRs for VH and VL are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from those listed in Table 5.

[0068] In some embodiments, antigen-binding sites are provided that are variants of antigen-binding sites capable of binding to PD-1 described herein, wherein such antigen-binding sites have CDR sequences that differ by no more than two amino acid residues per CDR (e.g., two or one amino acid residue) from the CDR sequences set forth in Table 5. In some embodiments, antigen-binding sites are provided that are variants of antigen-binding sites capable of binding to PD-1 described herein, wherein such antigen-binding sites have a set of six CDRs whose sequences differ by a total of no more than two amino acid residues (e.g., two or more amino acid residues) from the set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 5. In some embodiments, antigen-binding sites are provided that are variants of the antigen-binding sites capable of binding to PD-1 described herein, where such antigen-binding sites have a set of six CDRs whose sequences differ by a total of no more than two amino acid residues (e.g., two or one amino acid residue) from those of an anti-PD-1 having a set of VH and VL selected from Table 5.

[0069] In some embodiments, the provided antigen binding site capable of binding to PD-1 comprises a VH sequence set forth in Table 5 and a VL sequence set forth in Table 5. In some embodiments, antigen-binding sites are provided that are variants of antigen-binding sites capable of binding to PD-1 described herein, such as anti-PD-1 antibodies having a VH and VL set selected from Table 5, wherein such antigen-binding sites have (1) a VH comprising an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to the amino acid sequence of a VH set forth in Table 5, and (2) a light chain domain comprising an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to the amino acid sequence of a VL set forth in Table 5.

[0070] In some embodiments, provided antigen-binding sites capable of binding to PD-1 comprise a heavy chain sequence set forth in Table 5 and a light chain sequence set forth in Table 5. In some embodiments, provided are antigen-binding sites that are variants of antigen-binding sites capable of binding to PD-1 described herein, such as anti-PD-1 antibodies having a set of heavy and light chains selected from Table 5, wherein such antigen-binding sites have (1) a heavy chain comprising an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to the amino acid sequence of the heavy chain set forth in Table 5, and (2) a light chain comprising an amino acid sequence that is at least 85%, at least 87.5%, 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%, or at least 99% identical to the amino acid sequence of the light chain set forth in Table 5.

[0071] In certain embodiments, the antigen-binding site described herein is derived from 1-17. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 1, and a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO:5. In certain embodiments, an antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 1 and 5, respectively, as determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, a VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively. In certain embodiments, a VL comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively. In certain embodiments, an antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively.

[0072] In certain embodiments, the antigen-binding site described herein is derived from 1-28. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 9, and a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO: 13. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 9 and 13, respectively, as determined under Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively.

[0073] In certain embodiments, the antigen-binding site described herein is derived from 1-33. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 17, and a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO: 21. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 17 and 21, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively.

[0074] In certain embodiments, the antigen-binding site described herein is derived from 1-35. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO:25, and a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO:29. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 25 and 29, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively.

[0075] In certain embodiments, the antigen-binding site described herein is derived from 3H4. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 33, and a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO: 37. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 33 and 37, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 34, 35, and 36, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 38, 39, and 40, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 34, 35, and 36, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 38, 39, and 40, respectively.

[0076] In certain embodiments, the antigen-binding site described herein is derived from humanized 3H4. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 42 or 45, and a VL comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 47 or 49. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 44. In certain embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 47. In certain embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 49. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, and CDR-H3 of the VH sequence of SEQ ID NO: 42 or 45, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art, and / or CDR-L1, CDR-L2, and CDR-L3 of the VL sequence of SEQ ID NO: 47 or 49, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 34, 43, and 36, respectively, and (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 38, 39, and 40, respectively.

[0077] In certain embodiments, the antigen-binding site described herein is derived from murine 2.3A9. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 50, and a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO: 54. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 50 and 54, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 51, 52, and 53, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 55, 56, and 57, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 51, 52, and 53, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 55, 56, and 57, respectively.

[0078] In certain embodiments, the antigen-binding site described herein is derived from humanized 2.3A9. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 59, 62, 64, or 66, and / or a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO: 68. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 59. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 62. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 64. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 66. In certain embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 68. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, and CDR-H3 of the VH sequence of SEQ ID NO: 59, 62, 64, or 66, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art, and / or CDR-L1, CDR-L2, and CDR-L3 of the VL sequence of SEQ ID NO: 68, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 51, 60, and 53, respectively, and (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 55, 56, and 57, respectively.

[0079] In certain embodiments, the antigen-binding site described herein is derived from murine 20B3.1. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 69, and a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO: 73. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 69 and 73, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 70, 71, and 72, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 74, 75, and 76, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 70, 71, and 72, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 74, 75, and 76, respectively.

[0080] In certain embodiments, the antigen-binding site described herein is derived from humanized 20B3.1. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 78, and / or a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO: 81 or 83. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 78. In certain embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 81. In certain embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 83. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, and CDR-H3 of the VH sequence of SEQ ID NO: 78, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art, and / or CDR-L1, CDR-L2, and CDR-L3 of the VL sequence of SEQ ID NO: 81 or 83, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 70, 79, and 72, respectively, and (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 74, 75, and 76, respectively.

[0081] In certain embodiments, the antigen-binding site described herein is derived from mouse 1B8. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 84, and a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO: 88. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 84 and 88, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 85, 86, and 87, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 89, 90, and 91, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 85, 86, and 87, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 89, 90, and 91, respectively.

[0082] In certain embodiments, the antigen-binding site described herein is derived from humanized 1B8. For example, in certain embodiments, the antigen-binding site described herein comprises a VH comprising an amino acid sequence at least 90% (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%) identical to the amino acid sequence of SEQ ID NO: 93, and a VL comprising an amino acid sequence at least 90% (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%) identical to SEQ ID NO: 96 or 98. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 98. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, and CDR-H3 of the VH sequence of SEQ ID NO: 93, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art, and / or CDR-L1, CDR-L2, and CDR-L3 of the VL sequence of SEQ ID NO: 96 or 98, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOs: 86, 94, and 87, respectively, and (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 89, 90, and 91, respectively.

[0083] In each of the foregoing embodiments, the PD-1-binding VH and / or VL sequences can include amino acid changes (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the VH and / or VL that do not affect their ability to bind PD-1. For example, it is contemplated herein that the PD-1-binding VH and VL sequences can include cysteine heterodimerization mutations, which promote the formation of disulfide bridges between the VH and VL to form scFvs.

[0084] In some embodiments, the antigen-binding sites disclosed herein bind to human PD-1 or its extracellular domain. In some embodiments, the antigen-binding sites disclosed herein bind to PD-1 displayed on the surface of a membrane (e.g., the plasma membrane of a cell).

[0085] In some embodiments, the antigen-binding sites disclosed herein bind to PD-1. In some embodiments, the antigen-binding sites disclosed herein bind to cynomolgus PD-1 with an affinity similar to that which binds to human PD-1.

[0086] In some embodiments, the antigen-binding sites disclosed herein do not significantly bind to other PD-1 family members, hi some embodiments, the antigen-binding sites disclosed herein do not significantly bind to ICOS, CD28, or CTLA-4.

[0087] In some embodiments, the antigen-binding sites disclosed herein are capable of inhibiting the binding of PD-L1 to PD-1, hi some embodiments, the antigen-binding sites disclosed herein are capable of agonizing PD-1.

[0088] In some embodiments, the antigen-binding sites disclosed herein are capable of reducing IL-2 secretion. In some embodiments, the antigen-binding sites disclosed herein are capable of inhibiting (partially or fully) T cell activation. In some embodiments, the antigen-binding sites disclosed herein are capable of inhibiting (partially or fully) T cell proliferation (e.g., proliferation of CD4+ and / or CD8+ T cells).

[0089] In some embodiments, the antigen binding sites disclosed herein are capable of reducing inflammatory cytokine secretion, hi some embodiments, the antigen binding sites disclosed herein are capable of reducing IFN-γ secretion.

[0090] Molecules containing antigen-binding sites Also provided herein are molecules comprising the disclosed antigen-binding sites. Such molecules can be, but are not limited to, antibodies or antigen-binding fragments thereof, antibody fragments, nanobodies, antibody mimetics, etc. In some embodiments, molecules comprising the disclosed antigen-binding sites are therapeutic agents, i.e., the molecules provide a therapeutic benefit. Thus, in some embodiments, therapeutic agents comprising the disclosed antigen-binding sites are provided. In some embodiments, the therapeutic agent is capable of inducing complement-dependent cytotoxicity (CDC). In some embodiments, the therapeutic agent is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the therapeutic agent is capable of inducing antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the therapeutic agent is capable of depleting immune cells (e.g., T cells, B cells, mast cells, macrophages, NK cells, DCs, monocytes, neutrophils, fibroblasts, or epithelial cells).

[0091] In some embodiments, the therapeutic agent is an antibody or antigen-binding fragment thereof. In some embodiments, the term "antigen-binding fragment" includes, but is not limited to, Fab, scFab (single-chain Fab), F(ab')2, Fab', single-chain Fv (scFv), Fv fragment, or diabody (dimer of scFv). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody or antigen-binding fragment thereof is multispecific, e.g., bispecific. In some embodiments, the antibody is afucosylated (defucosylated). In some embodiments, the antibody is up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, or up to about 1% fucosylated. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% defucosylated.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, C-type lectin (CLEC), Siglec, tumor necrosis factor (TNF) receptor superfamily members, 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), CD40 ligand (CD40L), CD30 ligand (CD30L), CD70 ligand (CD70L), CD27 ligand (CD27L), TIM-3, LAG-3, BTLA, KLRG1, 2B4, CD244, CD19, CD20, CD22, The antibody or antigen-binding fragment thereof may bind to CD28, CD38, CD39, CD72, CD73, CD79A, CD79B, glycoprotein 130 (gp130), BCMA, BAFF receptor (BAFF-R), TACI, integrin alpha 4, integrin beta 7, FCGR2B, ICOS ligand, CD138, SLAMF7, LILR family members, fibroblast activation protein alpha (FAP), DLK-1, CD26, TE-7, CD29, PDGRF alpha, TGF beta receptor, MAS516, CD13, or a combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof may bind to PD-1, PD-L1, or PD-L2. In some embodiments, the antibody or antigen-binding fragment thereof may bind to PD-1. In some embodiments, the antibody or antigen-binding fragment thereof may agonize PD-1.

[0093] In some embodiments, a therapeutic agent is provided that comprises two or more antigen-binding sites, hi some embodiments, the therapeutic agent is bispecific.

[0094] In some embodiments, the Therapeutic Agent comprises a first antigen-binding site capable of binding to a marker for activated T cells, activated B cells, activated fibroblasts, or activated epithelial cells. In some embodiments, the first antigen-binding site is capable of binding to PD-1, PD-L1, PD-L2, CTLA-4, BTLA-4, or KLRG1. In some embodiments, the first antigen-binding site is capable of binding to PD-1, PD-L1, PD-L2, BCMA, or CD38. In some embodiments, the first antigen-binding site is capable of binding to FAP or PDGRF alpha.

[0095] In some embodiments, the therapeutic agent comprises a second antigen-binding site capable of binding to a marker for activated T cells, activated B cells, activated fibroblasts, or activated epithelial cells. In some embodiments, the marker is for the same type of activated cell to which the marker bound by the first antigen-binding site binds. In some embodiments, the marker is for a different type of activated cell to which the marker bound by the first antigen-binding site binds.

[0096] In some embodiments, markers of activated T cells include, but are not limited to, TIM-3 and LAG-3. In some embodiments, markers of activated B cells include, but are not limited to, CD19, CD20, BAFF-R, and TACI. In some embodiments, markers of activated fibroblasts include, but are not limited to, FAP, DLK-1, CD26, TE-7, CD29, PDGRF alpha, TGF beta receptor, MAS516, and CD13.

[0097] Various formats and uses of bispecific molecules are known in the art. Bispecific molecules according to the invention are not limited to any particular bispecific format or method of production.

[0098] In some embodiments, bispecific molecules include, but are not limited to, (i) a single antibody having two arms each containing a different antigen-binding site; (ii) a bispecific scFv, e.g., via two scFvs linked by a peptide linker; (iii) a dual variable domain antibody (DVD-Ig), where each light and heavy chain contains two variable domains in tandem via a short peptide bond; (iv) a bispecific (Fab')2 fragment; (v) a diabody; (vi) a tandem diabody (TandAb), which is a fusion of two diabodies; and (vii) a "dock-and-lock (DNL)-Fab3," a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; and (viii) a bispecific antibody that is forced to heterodimerize. (ix) recombinant IgG-like dual targeting molecules, in which both sides of the molecule each comprise an Fab fragment or portion of an Fab fragment of at least two different antibodies; (x) IgG fusion molecules, in which a full-length IgG antibody is fused to an additional Fab fragment or portion of an Fab fragment; (xi) Fc fusion molecules; (v) Fab fusion molecules; and (vi) bispecific antibodies or fragments or derivatives thereof comprising scFv- and diabody-based antibodies and heavy chain antibodies (e.g., domain antibodies, nanobodies) in which different scFvs, diabodies, or heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to a heavy chain constant domain, Fc region, or portion thereof.

[0099] Examples of IgG-like molecules with engineered Fc include, but are not limited to, triomabs, knobs-into-holes (kih) molecules (e.g., kih IgGs with a common light chain), CrossMAbs, orthoFab IgG molecules, electrostatically matched molecules, LUZ-Y molecules, DIG-body and PIG-body molecules, chain-exchange engineered domain body (SEEDbody) molecules, bichronix molecules, FcΔAdp molecules, bispecific IgG1 and IgG2 molecules, Azymetric scaffold molecules, and DuoBody molecules. Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, dual targeting (DT)-Ig molecules, two-in-one antibodies, mAbs, 2 Examples of IgG fusion molecules include, but are not limited to, DVD-Ig molecules and IgG-scFv. Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusions, SCORPION molecules, and Fc-DART molecules. Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 molecules, DNL molecules, and Fab-Fv molecules. Examples of scFv- and diabody-based antibodies and domain antibodies include, but are not limited to, bispecific T cell engager (BiTE) molecules, tandem diabody molecules (TandAb), dual affinity retargeting technology (DART) molecules, single-chain diabody molecules, TCR-like antibodies (AIT, ReceptorLogics), human serum albumin scfv fusions, COMBODY molecules, dual-targeting nanobodies, and dual-targeting heavy chain-only domain antibodies.

[0100] In some embodiments, the disclosed molecules comprising an antigen-binding site further comprise an antibody constant region, or a fragment or variant thereof. In some embodiments, the antibody constant region may be, for example, the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, and is particularly selected from, for example, the heavy chain constant regions of (e.g., human) IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody constant region, or a fragment or variant thereof, has an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to, for example, the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, or IgE, preferably, for example, the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the antibody constant region may be, for example, a light chain constant region selected from (e.g., human) kappa or lambda light chain constant regions. The constant region may be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and / or may fix complement. In another embodiment, the antibody does not recruit effector cells or fix complement. In another embodiment, the antibody has reduced or no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment, or other variant that does not support binding to Fc receptors, e.g., it has a mutated or deleted Fc receptor binding region.

[0101] In some embodiments, the constant region comprises one or more mutations that enhance effector function.

[0102] In some embodiments, the constant region comprises one or more mutations that enhance binding to FcγRII (CD32). For example, in certain embodiments, the constant region comprises one or more of the following mutations, numbered according to the EU index: (a) E233D, (b) G236D, (c) G237D, (d) P238D, (e) S239D, (f) S267E, (g) H268D, (h) P271G, (i) L328Y, L328F, or L328E, (j) A330R, (k) I332E, (l) G236D and H268D, (m) S239D and H268D, (n) S239D, H268D, (p ... In certain embodiments, the constant region comprises an IgG1 heavy chain constant region comprising one of the following mutations: (a) S239D and H268D, (b) S239D and S267E, (c) S239D and S267E, (d) S239D and S267E, (e) S239D, S267E, (f) S239D, S267E, (g) S239D, S267E, (h) S239D, S267E, (i) S239D, S267E, (j) S239D, S267E, (j) S239D, S267E, (k ... In certain embodiments, the constant region comprises the heavy chain constant region of IgG1 comprising the following mutations, as numbered according to the EU index: E233D, G237D, H268D, P271G, and A330R.

[0103] For example, in certain embodiments, the constant region comprises an IgG1 heavy chain constant region comprising one or more of the following mutations, numbered according to the EU index: F243L, R292P, Y300L, V305I, and / or P396L; (b) S239D and / or I332E; (c) S239D, I332E, and / or A330L; (d) S298A, E333A, and / or K334A; (e) G236A, S239D, and / or I332E; (f) K326W and / or E333S; (g) S267E, H268F, and / or S324T; or (h) E345R, E430G, and / or S440Y.

[0104] In some embodiments, the constant region comprises one or more mutations that reduce effector function. For example, in certain embodiments, the constant region comprises the following mutations, numbered according to the EU index: (a) L234A and / or L235A, (b) A327G, A330S, and / or P331S, (c) E233P, L234V, L235A, and / or G236del, (d) E233P, L234V, and / or L235A, (e) E233P, L234V, L235A, G236del, A327G, A330S, and / or P331S, (f) E233P, L234V, L235A, A327G, A330S, and / or P331S, (g) N297A, (h) N297 (i) N297Q, (j) L242C, N297C, and / or K334C, (k) A287C, N297G, and / or L306C, (l) R292C, N297G, and / or V302C, (m) N297G, V323C, and / or I332C, (n) V259C, N297G, and / or L306C, (o) L234F, L235Q, K322Q, M252Y, S254T, and / or T256E, (p) L234A, L235A, and / or P329G, or (q) L234A, L235Q, and K322Q. In certain embodiments, the constant region comprises a heavy chain constant region of IgG2 comprising one or more of the following mutations, numbered according to the EU index: (a) A330S and / or P331S; (b) V234A, G237A, P238S, H268A, V309L, A330S, and / or P331S; or (c) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, and / or T256E. In certain embodiments, the constant region comprises an IgG4 heavy chain constant region comprising one or more of the following mutations, numbered according to the EU index: (a) E233P, F234V, L235A, and / or G236del; (b) E233P, F234V, and / or L235A; (c) S228P and / or L235E; or (d) S228P and / or L235A.

[0105] In some embodiments, the antigen-binding site is linked to an antibody constant region or a fragment or variant thereof, hi certain embodiments, the antigen-binding site is linked to an IgG constant region comprising a hinge, CH2, and CH3 domain, with or without a CH1 domain.

[0106] In some embodiments, the molecules comprising the disclosed antigen-binding sites further comprise a non-fucosylated Fc region, hi some embodiments, the Fc region is a non-fucosylated IgG1 Fc region.

[0107] In some embodiments, a bispecific molecule can be a fusion protein comprising one or more antibody mimetics. In some embodiments, a bispecific molecule can be a fusion protein comprising one or more antibody mimetics and one or more antibodies or antigen-binding fragments thereof.

[0108] Amino acid sequence modifications Amino acid sequence modification(s) of the antigen-binding sites and molecules comprising antigen-binding sites (e.g., antibodies or antigen-binding fragments thereof) disclosed herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody or antigen-binding fragment. Amino acid sequence variants can be prepared, for example, by introducing appropriate nucleotide changes into the nucleic acid sequence encoding the antigen-binding site or molecule comprising the antigen-binding site, or by peptide synthesis. Such modifications can include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions can be made, provided that the antigen-binding site or molecule comprising the antigen-binding site retains the desired properties and / or function. In some embodiments, amino acid changes are introduced to alter post-translational processing, for example, changing the number or position of glycosylation sites.

[0109] A useful method for identifying specific residues or regions that are preferred sites for mutagenesis is called "alanine scanning mutagenesis." In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Amino acid locations that demonstrate functional sensitivity to the substitution are then refined by introducing additional or other variants at or in place of the substitution site. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis can be performed at the target codon or region, and the expressed variants can be screened for the desired activity.

[0110] Examples of amino acid sequence insertions include, but are not limited to, amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes, but is not limited to, an N-terminal methionyl residue.

[0111] In some embodiments, the antigen-binding site or a molecule comprising an antigen-binding site is fused at one end to another polypeptide, e.g., a cytotoxic polypeptide, an enzyme, or a polypeptide that increases the serum half-life of the antibody or antigen-binding fragment.

[0112] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the amino acid sequence of the molecule replaced with a different residue. The most interesting sites for substitution mutagenesis are typically hypervariable regions, although framework region alterations are also contemplated. Examples of conservative substitutions are shown in Table 1 under the heading of "preferred substitutions." More substantial changes may be introduced under the heading of "exemplary substitutions" in Table 1, or as further described below for amino acid classes, and the resulting antibodies or antigen-binding fragments are screened.

[0113] [Table 1]

[0114] Substantial modification of the biological properties of antibodies can be achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the region of the substitution, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are typically classified based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, (3) Acidic: Asp, Glu, (4) Basic: Asn, Gln, His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe.

[0115] Non-conservative substitutions may involve exchanging a member of one of these classes for another class.

[0116] Additionally or alternatively, cysteine residues not involved in maintaining the proper conformation of the antibody or antigen-binding fragment may generally be substituted with serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to an antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0117] In some embodiments, substitutional variants contain substitutions within one or more hypervariable region residues of the parent antibody (e.g., a human antibody). Generally, the resulting variant(s) that have improved biological properties relative to the parent antibody from which they are generated are selected for further development.

[0118] A method for generating such substitutional variants involves affinity maturation using phage display. In an example of such a method, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner, e.g., from filamentous phage particles, as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity).

[0119] To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody or antigen-binding fragment and the antigen. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, a panel of variants can be screened, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0120] In some embodiments, the original glycosylation pattern of the parent antibody is altered. Such alteration(s) can include deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody.

[0121] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0122] Addition of glycosylation sites to an antibody can be accomplished by altering the amino acid sequence of the antibody or antigen-binding fragment such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0123] Nucleic acid molecules encoding amino acid sequence variants of an antibody or antigen-binding fragment can be prepared by a variety of methods known in the art, including, but not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared variant or non-variant versions of an antibody or antigen-binding fragment thereof.

[0124] In some embodiments, modifications that increase serum half-life are used. For example, salvage receptor binding epitopes can be incorporated into molecules comprising the disclosed antigen-binding sites, for example, as described in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that causes an increase in the in vivo serum half-life of the IgG molecule.

[0125] Methods for producing antigen-binding sites and molecules containing antigen-binding sites The proteins herein above can be produced using recombinant DNA techniques well known to those skilled in the art.

[0126] For example, one or more nucleic acid sequences encoding proteins comprising the disclosed antigen-binding sites can be cloned into one or more expression vectors. The expression vectors can be stably transfected into host cells capable of expressing the gene(s). After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix. Clones can be cultured under conditions suitable for bioreactor scale-up and can maintain expression of proteins comprising the antigen-binding sites disclosed herein. Proteins can be isolated and purified using methods known in the art, such as centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0127] Accordingly, also provided herein are isolated nucleic acids encoding the antigen-binding sites and molecules (e.g., proteins) comprising the antigen-binding sites, vectors and host cells comprising the nucleic acids, and recombinant techniques for production.

[0128] In certain embodiments, provided are one or more isolated nucleic acids comprising sequences encoding the variable regions of the immunoglobulin heavy and / or immunoglobulin light chains of any of the antibodies disclosed herein, as well as one or more expression vectors expressing the variable regions of the immunoglobulin heavy and / or immunoglobulin light chains of any of the antibodies disclosed herein. Similarly, provided are host cells comprising one or more of the foregoing expression vectors and / or isolated nucleic acids.

[0129] In some embodiments, the antigen-binding sites and molecules (e.g., proteins) comprising the antigen-binding sites of the present disclosure may be fused to another agent, e.g., another therapeutic agent. Construction of fusion proteins is within the knowledge of one of ordinary skill in the art.

[0130] Monoclonal antibodies Monoclonal antibodies may initially be made using hybridoma methods or may be made by recombinant DNA methods (US Pat. No. 4,816,567).

[0131] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, or additionally, lymphocytes can be immunized in vitro. After immunization, the lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells.

[0132] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as the fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0133] Examples of suitable fusion partners include, but are not limited to, myeloma cells that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parent cells. Examples of suitable myeloma cell lines include, but are not limited to, mouse myeloma lines, such as those derived from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, Calif., USA), and SP-2 and derivatives, such as X63-Ag8-653 cells available from the American Type Culture Collection (Rockville, Md., USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies.

[0134] According to the hybridoma method, the culture medium in which the hybridoma cells are growing is then assayed for production of monoclonal antibodies directed against the antigen. For example, the binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0135] The binding affinity of the monoclonal antibody can, for example, be determined by Scatchard analysis.

[0136] Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned, for example, by limiting dilution procedures, and grown by standard methods. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by intraperitoneal injection of the cells into mice.

[0137] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification techniques such as, for example, affinity chromatography (e.g., using Protein A or Protein G-Sepharose®) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, or dialysis.

[0138] DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a murine antibody). Hybridoma cells can serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, resulting in the synthesis of the monoclonal antibody in the recombinant host cells.

[0139] In certain embodiments, monoclonal antibodies or antibody fragments are isolated from antibody phage libraries. High-affinity (nM range) human antibodies can be produced, for example, by chain shuffling. Combinatorial infection and in vivo recombination can provide strategies for constructing very large phage libraries. These techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0140] DNA encoding an antibody can be modified, for example, by substituting human heavy and light chain constant domain (CH and CL) sequences for the homologous murine sequences (e.g., U.S. Pat. No. 4,816,567), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide) to produce chimeric or fusion antibody polypeptides.

[0141] Human antibodies and phage display methods Human antibodies can be produced by methods known in the art, including those described herein. For example, it is possible to generate transgenic animals (e.g., mice) that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transplantation of such germline mutant mice with a human germline immunoglobulin gene array results in the production of human antibodies upon antigen challenge. See, e.g., U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807, and WO 97 / 17852.

[0142] Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in frame into either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats. Several sources of V gene segments can be used for phage display, for example, from random combinatorial libraries of V genes, such as libraries derived from the spleens of immunized mice. V gene repertoires from unimmunized human donors can be constructed, and antibodies against a wide variety of antigens (including self-antigens) can be essentially isolated following methods described in the art. See, for example, U.S. Patent Nos. 5,565,332 and 5,573,905.

[0143] Human antibodies may also be generated by in vitro activated B cells (see, eg, US Pat. Nos. 5,567,610 and 5,229,275).

[0144] Competition assays for determining whether an antibody binds to the same epitope as the disclosed antibodies or competes for binding with the disclosed antibodies are known in the art. Exemplary competition assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance (e.g., BIAcore analysis), biolayer interferometry, and flow cytometry.

[0145] Typically, a competitive assay involves the use of an antigen bound to a solid surface or expressed on a cell surface, a test antibody, and a reference antibody. The reference antibody is labeled, and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess (e.g., 1x, 5x, 10x, 20x, or 100x). Antibodies identified by competitive assays (e.g., competing antibodies) include antibodies that bind to the same or similar (e.g., overlapping) epitopes as the reference antibody, and antibodies that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody to create steric hindrance.

[0146] The competition assay can be performed in both directions to ensure that the presence of the label does not interfere with or otherwise inhibit binding, e.g., in one direction, the reference antibody is labeled and the test antibody is unlabeled, and in the second direction, the test antibody is labeled and the reference antibody is unlabeled.

[0147] A test antibody competes with a reference antibody for specific binding to an antigen if an excess of one antibody (e.g., 1x, 5x, 10x, 20x, or 100x) inhibits binding of the other antibody by, e.g., at least 50%, 75%, 90%, 95%, or 99%, as measured in a competitive binding assay.

[0148] Two antibodies can be determined to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies can be determined to bind overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0149] The antibodies disclosed herein may be further optimized (e.g., affinity matured) to improve biochemical properties, including affinity and / or specificity, and biophysical properties, including aggregation, stability, precipitation, and / or nonspecific interactions, and / or to reduce immunogenicity. Affinity maturation procedures are within the knowledge of those skilled in the art. For example, diversity can be introduced into the immunoglobulin heavy chain and / or immunoglobulin light chain by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis, and / or site-directed mutagenesis.

[0150] In certain embodiments, the isolated human antibody contains one or more somatic mutations. In these cases, the antibody can be modified to human germline sequences to optimize the antibody (e.g., by a process called germlining).

[0151] Generally, an optimized antibody has at least the same, or substantially the same, affinity for an antigen as the non-optimized (or parent) antibody from which it is derived. For example, in certain embodiments, an optimized antibody has a higher affinity for an antigen when compared to the parent antibody.

[0152] Pharmaceutical Compositions In certain embodiments, the provided molecules comprising the disclosed antigen-binding sites are incorporated into pharmaceutical compositions suitable for administration to a subject together with one or more pharmaceutically acceptable carriers. As used herein, "pharmaceutically acceptable carriers" refers to any of a variety of physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof.

[0153] In some embodiments, the pharmaceutical composition comprises one or more tonicity adjusting or stabilizing agents, non-limiting examples of which include sugars (e.g., sucrose), polyalcohols (e.g., mannitol or sorbitol), and sodium chloride.

[0154] In some embodiments, the pharmaceutical composition comprises one or more bulking agents and / or lyoprotectants (e.g., mannitol or glycine), buffers (e.g., phosphate, acetate, or histidine buffers), surfactants (e.g., polysorbates), antioxidants (e.g., methionine), and / or metal ion or chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)).

[0155] In some embodiments, a pharmaceutical composition contains one or more auxiliary substances, such as wetting or emulsifying agents, preservatives (e.g., benzyl alcohol), or buffers, which may enhance the shelf life and / or effectiveness of the immunoconjugates disclosed herein.

[0156] Pharmaceutical compositions can be provided in any of a variety of forms, including, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The suitability of a particular form may depend on the intended mode of administration and therapeutic application.

[0157] In some embodiments, the pharmaceutical composition is in the form of an injectable or infusible solution.

[0158] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture, transportation, and storage. Pharmaceutical compositions can be formulated, for example, as solutions, microemulsions, dispersions, liposomes, or other ordered structures. In some embodiments, pharmaceutical compositions are formulated as structures particularly suitable for high drug concentrations. For example, sterile injectable solutions can be prepared by incorporating a therapeutic agent (e.g., immunoconjugate) in a desired amount in an appropriate solvent with one or a combination of ingredients listed herein, optionally followed by sterilization (e.g., filter sterilization). Generally, dispersions can be prepared by incorporating the immunoconjugate into a sterile vehicle containing a basic dispersion medium and other ingredient(s), such as the additional ingredients mentioned herein. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum drying and freeze-drying to obtain a powder of the immunoconjugate and any additional desired ingredient(s), for example, from a previously sterile-filtered solution thereof.

[0159] The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a certain particle size (e.g., in the case of dispersions), and / or by the use of surfactants. Prolonged absorption of injectable compositions can be brought about, for example, by including in the composition an agent which delays absorption (e.g., monostearate salts and / or gelatin).

[0160] Treatment method The present application provides methods for treating diseases or disorders using the therapeutic agents (e.g., proteins such as antibodies or antigen-binding fragments thereof) or pharmaceutical compositions described herein. The therapeutic methods disclosed herein generally include administering a therapeutically effective amount of a disclosed therapeutic agent or pharmaceutical composition thereof that comprises an antigen-binding site to a subject (e.g., a human subject) in need thereof.

[0161] In some embodiments, the disease or disorder is an autoimmune disease. Examples of autoimmune diseases include, but are not limited to, Behcet's disease, systemic lupus erythematosus, multiple sclerosis (systemic sclerosis and progressive systemic sclerosis), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa and microscopic polyangiitis), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, Wegener's granulomatosis, mixed connective tissue disease, Sjögren's syndrome, adult-onset Still's disease, allergic granulomatous vasculitis, hypersensitivity vasculitis, Cogan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia syndrome, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related disorders (e.g., primary sclerosing cholangitis and autoimmune pancreatitis), and Guillain-Barré syndrome. syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cirrhosis, aortitis syndrome, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (hyperthyroidism), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, idiopathic Addison's disease (chronic adrenal insufficiency), type 1 diabetes mellitus, chronic discoid lupus erythematosus, localized scleroderma, psoriasis, psoriatic arthritis, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent miscarriage, and inflammatory bowel disease (ulcerative colitis and Crohn's disease). The formulations of the present invention may also be applied to the prevention or treatment of graft-versus-host disease (GVHD).

[0162] In some embodiments, the disease or disorder is an allergic or inflammatory disease. Examples of allergic and inflammatory diseases include, but are not limited to, acid reflux / heartburn, acne, acne vulgaris, allergies and hypersensitivity disorders, Alzheimer's disease, asthma, atherosclerosis and vascular occlusive disease, optionally atherosclerosis, ischemic heart disease, myocardial infarction, stroke, peripheral vascular disease, or stent restenosis, autoimmune diseases, bronchitis, cancer, carditis, cataracts, celiac disease, chronic pain, chronic prostatitis, and cirrhosis of the liver. , colitis, connective tissue disease, optionally systemic lupus erythematosus, systemic sclerosis, polymyositis, dermatomyositis, or Sjogren's syndrome, corneal disease, Crohn's disease, crystalline arthritis, optionally gout, pseudogout, calcium pyrophosphate crystal deposition disease, dementia, dermatitis, diabetes, dry eye, eczema, edema, emphysema, fibromyalgia, gastroenteritis, gingivitis, glomerulonephritis, heart disease, hepatitis, hypertension, hypersensitivity, inflammatory bowel disease, including as a result of trauma or ischemia Inflammatory conditions, insulin resistance, interstitial cystitis, iridocyclitis, iritis, joint pain, arthritis, rheumatoid arthritis, Lyme disease, metabolic syndrome (syndrome X), multiple sclerosis, myositis, nephritis, obesity, eye diseases including uveitis, osteopenia, osteoporosis, Parkinson's disease, pelvic inflammatory disease, periodontal disease, polyarteritis, polychondritis, polymyalgia rheumatica, psoriasis, reperfusion injury, rheumatoid arthritis, rheumatoid arthritis, any rheumatic disease. osteoarthritis, or psoriatic arthritis, rheumatoid arthritis, sarcoidosis, scleroderma, sinusitis, Sjogren's syndrome, spastic colon, spondyloarthritis, optionally ankylosing spondylitis, reactive arthritis, or Reiter's syndrome, systemic candidiasis, tendinitis, transplant rejection, vaginitis, vascular diseases including atherosclerotic vascular disease, vasculitis, optionally polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, or vasculitis.

[0163] A therapeutically effective amount can be administered via a single dose or via multiple doses (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 doses). When administered via multiple doses, any of a variety of suitable treatment regimens may be used, including administration at regular intervals (e.g., once every other day, once every three days, once every four days, once every five days, three times a week, twice a week, once a week, once every two weeks, once every three weeks, etc.).

[0164] The effective dosage regimen in a treatment method (e.g., the amount of each therapeutic agent, the relative timing of treatment, etc.) may depend on the severity of the disease or condition, as well as the weight and general condition of the subject. For example, the therapeutically effective amount of a particular composition containing a therapeutic agent to be applied to a mammal (e.g., a human) can be determined by one skilled in the art, taking into account individual differences in the age, weight, and condition of the mammal. The therapeutically effective amount and / or optimal amount can also be determined empirically by one skilled in the art.

[0165] A molecule comprising an antigen-binding site or a pharmaceutical composition thereof may be administered by any of a variety of suitable routes, including, but not limited to, systemic routes such as parenteral (e.g., intravenous or subcutaneous) or enteral routes.

[0166] In many embodiments, administering a therapeutic agent or pharmaceutical composition thereof comprising an antigen-binding site according to the provided methods results in the depletion of cells involved in the disease being treated. In some embodiments, the cells express an antigen to which the antigen-binding site contained in the therapeutic agent binds. In some embodiments, the cells are immune cells, such as T cells, B cells, macrophages, natural killer (NK) cells, dendritic cells (DCs), monocytes, neutrophils, fibroblasts, or epithelial cells. In some embodiments, the cells are activated cells, such as antigen-activated T cells or B cells.

[0167] List of Exemplary Embodiments The present invention is further illustrated by the following non-limiting exemplary embodiments. Embodiment 1. A method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutic agent comprising one or more antigen-binding sites; the antigen-binding site is capable of binding to an antigen on a cell involved in the autoimmune disease selected from PD-1, PD-L1, or PD-L2; The method, wherein said administering said therapeutic agent comprising one or more antigen binding sites results in depletion of said cells. Embodiment 2. A method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutic agent comprising one or more antigen-binding sites; The antigen-binding site is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, C-type lectin (CLEC), Siglec, tumor necrosis factor (TNF) receptor superfamily members, 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), CD40 ligand (CD40L), CD30 ligand (CD30L), CD70 ligand (CD70L), CD27 ligand (CD27L), TIM-3, LAG-3, BTLA, KLRG1, 2B4, CD244, CD19, CD20, CD22, CD28, CD38, CD39, CD72, C D73, CD79A, CD79B, glycoprotein 130 (gp130), BCMA, BAFF receptor (BAFF-R), TACI, integrin alpha 4, integrin beta 7, FCGR2B, ICOS ligand, CD138, SLAMF7, LILR family members, fibroblast activation protein alpha (FAP), DLK-1, CD26, TE-7, CD29, PDGRF alpha, TGF beta receptor, MAS516, CD13, or a combination thereof, The method, wherein said administering said therapeutic agent comprising one or more antigen binding sites results in depletion of said cells. Embodiment 3. A method of treating an allergic or inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutic agent comprising one or more antigen-binding sites; the antigen-binding site is capable of binding to an antigen on a cell involved in the allergic or inflammatory disease selected from PD-1, PD-L1, or PD-L2; The method, wherein said administering said therapeutic agent comprising one or more antigen binding sites results in depletion of said cells. Embodiment 4. A method of treating an allergic or inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutic agent comprising one or more antigen-binding sites; The antigen-binding site is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, C-type lectin (CLEC), Siglec, tumor necrosis factor (TNF) receptor superfamily members, 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), CD40 ligand (CD40L), CD30 ligand (CD30L), CD70 ligand (CD70L), CD27 ligand (CD27L), TIM-3, LAG-3, BTLA, KLRG1, 2B4, CD244, CD19, CD20, CD22, CD28, CD38, CD39, CD72, CD 73, CD79A, CD79B, glycoprotein 130 (gp130), BCMA, BAFF receptor, TACI, integrin alpha 4, integrin beta 7, FCGR2B, ICOS ligand, CD138, SLAMF7, and LILR family members, fibroblast activation protein alpha (FAP), DLK-1, CD26, TE-7, CD29, PDGRF alpha, TGF beta receptor, MAS516, CD13, or a combination thereof, on cells involved in said allergic or inflammatory disease; The method, wherein said administering said therapeutic agent comprising one or more antigen binding sites results in depletion of said cells. Embodiment 5. The cells are selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CLEC, Siglec, TNF receptor superfamily members, 4-1BBL, OX40L, CD40L, CD30L, CD70L, CD27L, TIM-3, LAG-3, BTLA, KLRG1, 2B4, CD244, CD19, CD20, CD22, CD28, CD38, CD39, CD72, CD73, CD79A, CD79B, gp1 30, BCMA, BAFF receptor, TACI, integrin alpha 4, integrin beta 7, FCGR2B, ICOS ligand, CD138, SLAMF7, LILR family members, FAP, DLK-1, CD26, TE-7, CD29, PDGRF alpha, TGF beta receptor, MAS516, CD13, or a combination thereof. Embodiment 6. The method of any one of embodiments 1 to 5, wherein the cell is an immune cell. Embodiment 7. The method of any one of embodiments 1 to 5, wherein the cell is a T cell, a B cell, a macrophage, a natural killer (NK) cell, a dendritic cell (DC), a monocyte, a neutrophil, a fibroblast, or an epithelial cell. Embodiment 8 The method of embodiment 6 or 7, wherein the cells are activated. Embodiment 9. The method of any one of embodiments 6 to 8, wherein the cells are antigen-activated T cells or B cells. Embodiment 10. The method of any one of embodiments 1 to 9, wherein the antigen-binding site is present as a single-chain fragment variable (scFv). Embodiment 11. The method of any one of embodiments 1 to 10, wherein the antigen-binding site is present as an antibody mimetic (e.g., an affibody, affilin, affimer, aftin, alphabody, anticalin, avimer, centirin, DARPin, finomer, monobody, or nanoCLAMP). Embodiment 12. The method of any one of embodiments 1 to 11, wherein the therapeutic agent is an antibody or antigen-binding fragment thereof comprising the one or more antigen-binding sites. Embodiment 13. The method of embodiment 12, wherein the antibody or antigen-binding fragment thereof is a Fab, F(ab')2, Fab', scFv, Fv fragment, Fd fragment, or diabody. Embodiment 14 The method of embodiment 12, wherein the antibody or antigen-binding fragment thereof is a humanized antibody, a human antibody, a multispecific antibody, or a bispecific antibody, or a fragment thereof. Embodiment 15. The method of any one of embodiments 1 to 11, wherein the therapeutic agent is a bispecific antibody. Embodiment 16. The bispecific antibody comprises a first antigen-binding site and a second antigen-binding site; the first antigen-binding site is capable of binding to PD-1, PD-L1, PD-L2, CTLA-4, BTLA, or KLRG1; 16. The method of embodiment 15, wherein the second antigen-binding site is capable of binding to a marker of activated T cells (e.g., TIM-3 or LAG-3). Embodiment 17. The bispecific antibody comprises a first antigen-binding site and a second antigen-binding site; the first antigen-binding site is capable of binding to PD-1, PD-L1, PD-L2, BCMA, or CD38; 16. The method of embodiment 15, wherein the second antigen-binding site is capable of binding to a marker of activated B cells (e.g., CD19, CD20, BAFF-R, or TACI). Embodiment 18. The bispecific antibody comprises a first antigen-binding site and a second antigen-binding site; the first antigen-binding site is capable of binding to FAP or PDGRF alpha; 16. The method of embodiment 15, wherein the second antigen-binding site is capable of binding to a marker for activated fibroblasts (e.g., FAP, DLK-1, CD26, TE-7, CD29, PDGRF alpha, TGF beta receptor, MAS516, or CD13). Embodiment 19. The method of any one of embodiments 1 to 18, wherein the therapeutic agent is an antibody or antigen-binding fragment thereof comprising an antibody heavy chain constant region. Embodiment 20. The method of embodiment 20, wherein the antibody heavy chain constant region is a human IgG heavy chain constant region. Embodiment 21. The method of any one of embodiments 1 to 20, wherein the therapeutic agent is an antibody or antigen-binding fragment thereof conjugated or fused to a label or a second therapeutic agent. Embodiment 22. The method of embodiment 21, wherein the second therapeutic agent is a cytotoxic small molecule or an immunotoxin. Embodiment 23. The method of any one of embodiments 1 to 22, wherein the therapeutic agent is capable of inducing complement-dependent cytotoxicity (CDC). Embodiment 24. The method of any one of embodiments 1 to 23, wherein the therapeutic agent is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC). Embodiment 25. The method of any one of embodiments 1 to 24, wherein the therapeutic agent is capable of inducing antibody-dependent cellular phagocytosis (ADCP). Embodiment 26. The method of any one of embodiments 1 to 25, wherein the therapeutic agent is defucosylated. Embodiment 27. The method of embodiment 25, wherein the therapeutic agent is at least about 75% defucosylated. Embodiment 28 The method of embodiment 26 or 27, wherein the therapeutic agent is capable of depleting immune cells. Embodiment 29. The method of any one of embodiments 1 to 28, wherein the therapeutic agent is capable of agonizing PD-1. Embodiment 30. The method of any one of embodiments 1 to 29, wherein the therapeutic agent comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 91, or a fragment thereof. Embodiment 31. The method of any one of embodiments 1 to 29, wherein the therapeutic agent comprises at least one amino acid sequence selected from any one of SEQ ID NOs: 1 to 91, or a fragment thereof. Embodiment 32. The method of any one of embodiments 1 to 29, wherein the therapeutic agent comprises at least two amino acid sequences selected from any one of SEQ ID NOs: 1 to 91, or fragments thereof. Embodiment 33. The method of any one of embodiments 1 to 29, wherein the therapeutic agent comprises at least one CDR found in a sequence selected from any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 44, 46, 48, 50, 54, 58, 61, 63, 65, 67, 69, 73, 77, 80, 82, 84, 88, 92, 95, and 97. Embodiment 34. The method of any one of embodiments 1 to 29, wherein the therapeutic agent comprises at least two CDRs from a sequence selected from any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 44, 46, 48, 50, 54, 58, 61, 63, 65, 67, 69, 73, 77, 80, 82, 84, 88, 92, 95, and 97. Embodiment 35. The method of any one of embodiments 1 to 29, wherein the therapeutic agent comprises at least three CDRs from a sequence selected from any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 44, 46, 48, 50, 54, 58, 61, 63, 65, 67, 69, 73, 77, 80, 82, 84, 88, 92, 95, and 97. Embodiment 36. The therapeutic agent is: (a) complementarity-determining region 1 (CDR1), CDR2, and / or CDR3 of the heavy chain variable region (VH) set forth in any one of SEQ ID NOs: 1, 9, 17, 25, 33, 50, 69, and 84, and / or (b) CDR1, CDR2, and / or CDR3 of the light chain variable region (VL) shown in any one of SEQ ID NOs: 5, 13, 21, 29, 37, 55, 73, and 88 30. The method of any one of embodiments 1 to 29, comprising: Embodiment 37. The therapeutic agent is: (i) (a) Complementarity-determining region: CDR-H1 comprising the amino acid sequence: SGGSIRSTRWWS (SEQ ID NO: 2); CDR-H2 comprising the amino acid sequence: EIYHSGSTNYNPSLKS (SEQ ID NO: 3), and CDR-H3 containing the amino acid sequence: QDYGDSGDWYFDL (SEQ ID NO: 4) and / or (b) Complementarity-Determining Region: CDR-L1 comprising the amino acid sequence: TRSSGSIASNSVQ (SEQ ID NO: 6); CDR-L2 comprising the amino acid sequence: EDNQRPS (SEQ ID NO: 7), and CDR-L3 containing the amino acid sequence: QSSDSSAVV (SEQ ID NO: 8) VL containing, or (ii) (a) Complementarity-determining region: CDR-H1 containing the amino acid sequence: SYGIS (SEQ ID NO: 10), CDR-H2 comprising the amino acid sequence: WISAYNGNTNYAQKLQ (SEQ ID NO: 11), and CDR-H3 containing the amino acid sequence: DADYSSGSGY (SEQ ID NO: 12) and / or (b) Complementarity-Determining Region: CDR-L1 comprising the amino acid sequence: SGDALPKQYAY (SEQ ID NO: 14); CDR-L2 comprising the amino acid sequence: KDTERPS (SEQ ID NO: 15), and CDR-L3 containing the amino acid sequence: QSADNSITYRV (SEQ ID NO: 16) VL containing, or (iii) (a) Complementarity-determining region: CDR-H1 comprising the amino acid sequence: SYYIH (SEQ ID NO: 18); CDR-H2 comprising the amino acid sequence: IINPRGATISYAQKFQG (SEQ ID NO: 19), and CDR-H3 containing the amino acid sequence: AGIYGFDFDY (SEQ ID NO: 20) and / or (b) Complementarity-Determining Region: CDR-L1 comprising the amino acid sequence: TGTSNDVGGYNYVS (SEQ ID NO: 22); CDR-L2 comprising the amino acid sequence: DVTNRPS (SEQ ID NO: 23), and CDR-L3 containing the amino acid sequence: SSYTIVTNFEVL (SEQ ID NO: 24) VL containing, or (iv) (a) Complementarity-determining region: CDR-H1 comprising the amino acid sequence: SGAYYWS (SEQ ID NO: 26); CDR-H2 comprising the amino acid sequence: YIYYNGNTYYNPSLRS (SEQ ID NO: 27), and CDR-H3 containing the amino acid sequence: ASDYVWGGYRYMDAFDI (SEQ ID NO: 28) and / or (b) Complementarity-Determining Region: CDR-L1 comprising the amino acid sequence: SGSNSNIGSNSVN (SEQ ID NO: 30), CDR-L2 comprising the amino acid sequence: GNNQRPS (SEQ ID NO: 31), and CDR-L3 containing the amino acid sequence: AAWDDSLNGP (SEQ ID NO: 32) VL containing, or (v) (a) Complementarity-determining region: CDR-H1 comprising the amino acid sequence: DYYIN (SEQ ID NO: 34); CDR-H2 comprising the amino acid sequence: WIYPGSVNTKYNEKFRG (SEQ ID NO: 35), and CDR-H3 containing the amino acid sequence: YSNWFFDV (SEQ ID NO: 36) and / or (b) Complementarity-Determining Region: CDR-L1 comprising the amino acid sequence: RASQEISGYLS (SEQ ID NO: 38); CDR-L2 comprising the amino acid sequence: AASTLDS (SEQ ID NO: 39), and CDR-L3 containing the amino acid sequence: LQYASYPYT (SEQ ID NO: 40) VL containing, or (vi) (a) Complementarity-determining region: CDR-H1 comprising the amino acid sequence: DHAIH (SEQ ID NO: 51), CDR-H2 comprising the amino acid sequence: YISPGYGDIEYNEKFKG (SEQ ID NO: 52), and CDR-H3 containing the amino acid sequence: TTGY (SEQ ID NO: 53) and / or (b) Complementarity-Determining Region: CDR-L1 comprising the amino acid sequence: KSSQSLLNSDGKTYLN (SEQ ID NO: 55); CDR-L2 comprising the amino acid sequence: LVSELDS (SEQ ID NO: 56), and CDR-L3 containing the amino acid sequence: WQGTHFPH (SEQ ID NO: 57) VL containing, or (vii) (a) Complementarity-determining region: CDR-H1 comprising the amino acid sequence: SFGMS (SEQ ID NO: 70); CDR-H2 comprising the amino acid sequence: TISGGGSNTYYPDSVKG (SEQ ID NO: 71), and CDR-H3 containing the amino acid sequence: IYDVAWFAY (SEQ ID NO: 72) and / or (b) Complementarity-Determining Region: CDR-L1 comprising the amino acid sequence: RASESVDNSGISFMN (SEQ ID NO: 74), CDR-L2 comprising the amino acid sequence: AASNQGS (SEQ ID NO: 75), and CDR-L3 containing the amino acid sequence: QQSKEVPWT (SEQ ID NO: 76) VL containing, or (viii) (a) Complementarity-determining region: CDR-H1 comprising the amino acid sequence: SYAMS (SEQ ID NO: 85), CDR-H2 comprising the amino acid sequence: TITGGGRNTYYPDSVKG (SEQ ID NO: 86), and CDR-H3 containing the amino acid sequence: QGYDGYTWFAY (SEQ ID NO: 87) and / or (b) Complementarity-Determining Region: CDR-L1 comprising the amino acid sequence: RASESVDNSGISFMN (SEQ ID NO: 89); CDR-L2 comprising the amino acid sequence: AASNPGS (SEQ ID NO: 90), and CDR-L3 containing the amino acid sequence: QQSKEVPWT (SEQ ID NO: 91) VL containing 30. The method of any one of embodiments 1 to 29, comprising: Embodiment 38. The therapeutic agent is: (i) a VH comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 1, and / or a VL comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 5; or (ii) a VH comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 9, and / or a VL comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 13; or (iii) a VH comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 17, and / or a VL comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 21; or (iv) a VH comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 25, and / or a VL comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 29, or (v) a VH comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 33, and / or a VL comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 37; or (vi) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC set forth in any one of SEQ ID NOs: 41 or 44, and / or a VL comprising an amino acid sequence having at least 85% identity to the VL of the LC set forth in any one of SEQ ID NOs: 46 or 48; or (vii) a VH comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 50, and / or a VL comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 55; or (viii) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC set forth in any one of SEQ ID NOs: 58, 61, 63, and 65, and / or a VL comprising an amino acid sequence having at least 85% identity to the VL of the LC set forth in SEQ ID NO: 67; or (ix) a VH comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 69, and / or a VL comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 73; or (x) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC set forth in SEQ ID NO: 77, and / or a VL comprising an amino acid sequence having at least 85% identity to the VL of the LC set forth in any one of SEQ ID NOs: 80 or 82; or (xi) a VH comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 84, and / or a VL comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 88; or (xii) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC set forth in SEQ ID NO: 92, and / or a VL comprising an amino acid sequence having at least 85% identity to the VL of the LC set forth in any one of SEQ ID NOs: 95 or 97. 38. The method of any one of embodiments 1 to 29 and 36 to 37, comprising: Embodiment 39. The therapeutic agent is: (i) a HC comprising CDR-H1, CDR-H2, and CDR-H3 of the HC set forth in any one of SEQ ID NOs: 41 or 44 and having at least 85% amino acid sequence identity thereto; and / or an LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC set forth in any one of SEQ ID NOs: 46 or 48, and having at least 85% amino acid sequence thereto; or (ii) a HC comprising CDR-H1, CDR-H2, and CDR-H3 of a HC set forth in any one of SEQ ID NOs: 58, 61, 63, and 65, and having at least 85% amino acid sequence thereto; and / or an LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC set forth in SEQ ID NO: 67 and having at least 85% amino acid sequence identity to SEQ ID NO: 67; or (iii) a HC comprising CDR-H1, CDR-H2, and CDR-H3 of the HC set forth in SEQ ID NO: 77 and having at least 85% amino acid sequence identity to SEQ ID NO: 77; and / or an LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC set forth in any one of SEQ ID NOs: 80 or 82, and having at least 85% amino acid sequence thereto; or (iv) a HC comprising CDR-H1, CDR-H2, and CDR-H3 of the HC set forth in SEQ ID NO: 92 and having at least 85% amino acid sequence identity to SEQ ID NO: 92; and / or LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC set forth in any one of SEQ ID NOs: 95 and 97, and having at least 85% amino acid sequence thereto. 38. The method of any one of embodiments 1 to 29 and 36 to 37, comprising: Embodiment 40. The antigen-binding site of embodiment 38 or 39, wherein the sequence identity is at least 90%, 95%, 96%, 97%, 98%, or 99%. Embodiment 41. The antigen-binding site comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 1, and / or a VL comprising the amino acid sequence of SEQ ID NO: 5, or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 9, and / or a VL comprising the amino acid sequence of SEQ ID NO: 13, or (iii) a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 21; or (iv) a VH comprising the amino acid sequence of SEQ ID NO: 25, and / or a VL comprising the amino acid sequence of SEQ ID NO: 29, or (v) a VH comprising the amino acid sequence of SEQ ID NO: 33, and / or a VL comprising the amino acid sequence of SEQ ID NO: 37, or (vi) the VH of the HC set forth in any one of SEQ ID NOs: 41 or 44, and / or the VL of the LC set forth in any one of SEQ ID NOs: 46 or 48, or (vii) a VH comprising the amino acid sequence of SEQ ID NO: 50, and / or a VL comprising the amino acid sequence of SEQ ID NO: 55; or (viii) the VH of the HC set forth in any one of SEQ ID NOs: 58, 61, 63, and 65; and / or the VL of LC set forth in SEQ ID NO: 67, or (ix) a VH comprising the amino acid sequence of SEQ ID NO: 69, and / or a VL comprising the amino acid sequence of SEQ ID NO: 73, or (x) the VH of the HC set forth in SEQ ID NO: 77, and / or the VL of the LC set forth in any one of SEQ ID NOs: 80 or 82, or (xi) a VH comprising the amino acid sequence of SEQ ID NO: 84, and / or a VL comprising the amino acid sequence of SEQ ID NO: 88; or (xii) the VH of HC set forth in SEQ ID NO: 92, and / or The VL of LC set forth in any one of SEQ ID NOs: 95 or 97 41. The antigen-binding site according to any one of embodiments 1 to 29 and 36 to 40, comprising: Embodiment 42 The method of embodiment 12, wherein the antibody or antigen-binding fragment thereof is defucosylated. Embodiment 43. The method of embodiment 42, wherein the antibody or antigen-binding fragment thereof is at least about 75% defucosylated. [Example]

[0168] Example 1. Antibody production Human antibodies that target antigens on cells involved in autoimmune, allergic, or inflammatory diseases are produced by methods known in the art.

[0169] In some embodiments, transgenic mice are used that are incapable of expressing functional endogenous immunoglobulins but express human immunoglobulin genes. In particular, human heavy and light chain immunoglobulin gene complexes are introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions are introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes are made non-functional separately from or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, such as all or part of an antigen (e.g., PD-1, PD-L1, or PD-L2). Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.

[0170] In some other embodiments, the antibodies are produced by screening a yeast display library.

[0171] Example 2. Antibody Binding to PD-1 Target binding of PD-1 antibodies to PD-1 expressed on activated T cells was assessed by flow cytometry.

[0172] T cells were activated with anti-CD3, anti-CD28, and anti-W6 / 32 treatment. Activation increased PD-1 expression on the T cell surface. After 3 days of activation, cells were resuspended in FACS blocking buffer (PBS + 10% normal human serum) for 30 minutes. After washing, cells were incubated with PD-1 test antibodies in FACS buffer (PBS + 0.5% bovine serum albumin + 0.09% sodium azide) for 30 minutes at 4°C. Cells were then washed and incubated with PE-conjugated anti-human secondary antibodies in the dark for 30 minutes at 4°C. After washing, cytofluorometric analysis was performed using a BD FACSSymphony A3 instrument (BD Biosciences). Cells were sorted by scatter characteristics and separated for single-cell analysis using side scatter width versus side scatter area. Antibody binding to cells was quantified by measuring fluorescence emission at an excitation wavelength of 561 nm using a 586 / 15 nm filter. Data analysis was performed using FlowJo software. All c-Kit antibodies tested were able to bind to PD-1 expressing T cells (see Table 2).

[0173] [Table 2]

[0174] Example 3. Cross-reactivity and non-specific binding of PD-1 antibodies analyzed by ELISA An ELISA-based assay was used to assess the cross-reactivity and nonspecific binding of PD-1 antibodies to human PD-1, cynomolgus monkey PD-1, and PD-1 family members.

[0175] His-tagged extracellular domains of human PD-1, cynomolgus monkey PD-1, and the PD-1 family members ICOS, CD28, and CTLA-4 were immobilized on a 96-well ELISA plate. The plate was washed with PBST (phosphate-buffered saline + 0.05% Tween 20) and blocked for 1 hour with PBS containing 1% BSA. After washing with PBST, the test PD-1 antibody was added to the coated plate and incubated for 2 hours at room temperature. The plate was washed and incubated with an HRP-conjugated goat anti-human IgG secondary antibody for 1 hour at room temperature. After washing, antibody binding was detected colorimetrically by adding 3,3',5,5'-tetramethylbenzidine (TMB) and incubating for 10–20 minutes at room temperature, followed by the addition of a 2.5N sulfuric acid stop solution. Absorbance at 450 nm was measured using a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism.

[0176] Exemplary results are shown in Table 3. All PD-1 antibodies tested were able to bind to both human and cynomolgus monkey PD-1, but did not cross-react with the PD-1 family members ICOS, CD28, or CTLA-4.

[0177] [Table 3]

[0178] Example 4. PD-1 / PD-L1 Blockade by PD-1 Antibodies Analyzed by ELISA The ability of the disclosed PD-1 antibodies to block PD-1 / PD-L1 interactions was assessed using an ELISA-based assay.

[0179] Briefly, human PD-L1 was immobilized on a 96-well ELISA plate. The plate was washed and blocked with blocking buffer at 37°C for 1.5 hours. The plate was washed, and test PD-1 antibodies, isotype controls, or a standard curve of PD-1 neutralizing antibodies were added to the coated plate and incubated at 37°C for 1 hour. The plate was washed and incubated with PD-1 biotin for 1 hour at 37°C. After washing, streptavidin-HRP was added to the plate, and the plate was incubated at 37°C for 1 hour. After the final wash, antibody binding was detected colorimetrically by adding sublate solution and incubating for 20 minutes protected from light, followed by the addition of stop solution. Absorbance at 450 nm was measured using a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism.

[0180] The percent blocking of the PD-1 antibodies tested was determined by interpolating the values obtained from each sample against the standard control curve (see Figure 1) and is summarized in Table 4. The antibodies tested showed a wide range of activity in blocking the PD-1 / PD-L1 interaction. For example, PD-1 antibodies such as 1G7 and 7G3 almost completely blocked the PD-1 / PD-L1 interaction, whereas PD-1 antibodies 1-17 and 2.3A9 did not.

[0181] [Table 4]

[0182] Example 5. T cell suppression in mixed lymphocyte reaction by PD-1 antibody The ability of the disclosed PD-1 antibodies to agonize PD-1 and suppress T cells in a mixed lymphocyte reaction (MLR) was evaluated.

[0183] Peripheral blood mononuclear cells (PBMCs) from two different donors were co-cultured in 96-well plates. Co-culture of PBMCs from different donors results in allostimulation, as allogeneic T cells from one donor produce IFN-γ after recognizing foreign MHC from the other donor.

[0184] PD-1 antibody or an isotype control was added to the PBMC co-cultures. PBMCs alone (no antibody) were used as a control. Plates were incubated for 72 hours, and the levels of IFN-γ present in the supernatants were measured by ELISA assay.

[0185] Briefly, IFN-γ capture antibody was immobilized on a 96-well ELISA plate. The plate was washed with PBST and blocked for 1 hour with 1% BSA in PBS. After washing with PBST, supernatants and an IFN-γ standard curve were added to the coated plate and incubated for 2 hours at room temperature. The plate was washed and incubated with an HRP-conjugated detection antibody for 2 hours at room temperature. After washing, antibody binding was detected colorimetrically by adding TMB and incubating for 10–20 minutes at room temperature, followed by the addition of a 2.5N sulfuric acid stop solution. Absorbance at 450 nm was measured using a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism.

[0186] Exemplary results are shown in Figure 2. PD-1 antibodies 1-28, 1-33, 1-35, 1-17, 20B3.1, 1B8, 3H4, 2.3A9, 1.8A10, 1G7, and 7G3 inhibited IFN-γ secretion, in part demonstrating enhanced inhibition of IFN-γ secretion compared to the PD-1 agonist antibody rosnilimab.

[0187] Example 6. Inhibition of T cell activation by effector function-modifying PD-1 antibodies The ability of effector function-modifying PD-1 antibodies to inhibit T cell activation was assessed using a commercially available assay that includes antigen-presenting cell lines (APCs) and a human Jurkat reporter T cell line expressing a specific T cell receptor (TCR) and a lucia luciferase reporter gene. In this assay, activation of Jurkat cells results in NFAT-mediated luminescence.

[0188] Raji-APC cells modified to express the FcγRII receptor were incubated with PD-1 antibody along with normal human IgG1 Fc or effector-enhanced IgG1 Fc, isotype control, or medium for 30 minutes at 37°C and 5% CO2. The effector-enhanced IgG1 Fc contained the mutations S239D / H268D ("X3" mutation) or E233D / G237D / H268D / P271G / A330R ("V12" mutation), which increase binding to FcγRII. After incubation, Jurkat-Lucia TCR-hPD-1 cells were added to the cultures, and the plates were incubated for an additional 5.5 hours at 37°C and 5% CO2. The cells were centrifuged at 400g for 5 minutes, and the supernatant was collected for further analysis.

[0189] The supernatant was transferred to a 96-well white plate. 50 μL of QUANTI-Luc 4 reagent was added to the plate, and luminescence was immediately read using a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism to calculate the relative percent inhibition compared to the signal obtained from controls (isotype control or no antibody treatment).

[0190] Exemplary results are shown in Figure 3. The X3 or V12 mutations enhanced the ability of several PD-1 antibodies (such as 3H4) to inhibit Jurkat cell activation compared to PD-1 antibodies with normal IgG1 Fc and isotype controls.

[0191] Supernatants were also analyzed for IL-2 levels by ELISA assay as described in Example 5, except that the IL-2 capture antibody was immobilized on a 96-well ELISA plate. Exemplary results are shown in Figure 4. 3H4 antibodies containing the X3 or V12 mutations significantly inhibited IL-2 secretion compared to 3H4 with normal IgG1 Fc and controls (isotype control or no antibody treatment).

[0192] Example 7. Inhibition of T cell activation by effector function-modifying PD-1 antibodies The ability of effector function-modifying PD-1 antibodies to inhibit T cell activation was evaluated.

[0193] PBMCs were stained with CellTrace Violet (CTV) dye for 15 minutes at 37°C. After two washes in cell growth medium, PBMCs were seeded onto 96-well plates coated with anti-CD3 and anti-CD28, and added to all wells to stimulate T cells. PD-1 antibodies containing normal or effector-enhanced human IgG1 Fc with an X3 mutation, an isotype control, or PD-L1 were added to each well, and the plates were incubated at 37°C and 5% CO2 for 72 hours. Supernatants were collected for ELISA analysis. The remaining cells on the plates were washed, resuspended in FACS blocking buffer, and incubated with APC-labeled anti-CD4 and BV785-labeled anti-CD8 antibodies for 15 minutes on ice. After washing, cytofluorometric analysis was performed using a BD FACS Symphony A3 instrument (BD Biosciences). CTV fluorescence intensity was measured for CD4 and CD8 T cell subsets. Data analysis was performed using FlowJo software to quantify the percentage of proliferating cells for each T cell subtype.

[0194] Exemplary results are shown in Figures 5A and 5B. Tested PD-1 antibodies containing X3 mutations that increase binding to FcγRII inhibited proliferation of CD4 and CD8 T cells.

[0195] Example 8. Inhibition of IFN-γ secretion by effector function-modifying PD-1 antibodies The ability of effector function-modifying PD-1 antibodies to inhibit IFN-γ secretion was evaluated.

[0196] Supernatants from stimulated PBMCs stained with CTV and incubated with PD-1 antibody, isotype control, or PD-L1 were analyzed for IFN-γ secretion by ELISA as described in Example 5.

[0197] Exemplary results are shown in Figure 6. Tested PD-1 antibodies containing X3 mutations that increase binding to FcγRII inhibited IFN-γ secretion more significantly than controls and PD-1 antibodies (agonist or antagonist) with normal IgG1 Fc.

[0198] Sequence Annex In some sequences, variable regions are shown in bold and CDRs are underlined.

[0199] [Table 5] TIFF2025525438000007.tif231164TIFF2025525438000008.tif233164TIFF2025525438000009.tif233164TIFF20255254380 00010.tif232164TIFF2025525438000011.tif231164TIFF2025525438000012.tif233164TIFF2025525438000013.tif109164

[0200] [Table 6] TIFF2025525438000015.tif232164TIFF2025525438000016.tif34164

[0201] Incorporation by Reference Unless otherwise stated, the entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0202] Equivalents / Alternative Embodiments Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. An antibody or antigen-binding fragment thereof capable of binding to PD-1, (a) a PD-1 agonist; (b) has enhanced binding to Fc receptors, and / or (c) nonfucosylated The antibody or antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the Fc receptor is an inhibitory Fc receptor (e.g., FcγRII).

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the Fc receptor is an activating Fc receptor (e.g., FcγRI or FcγRIII).

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which has an enhanced effector function.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the effector functions are antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which is capable of depleting cells.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the cell is a PD-1-expressing cell.

8. The antibody or antigen-binding fragment thereof of claim 6 or 7, wherein the cell is an immune cell.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the immune cell is a T cell, a B cell, a macrophage, a natural killer (NK) cell, a dendritic cell (DC), a monocyte, a neutrophil, a fibroblast, or an epithelial cell.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the immune cell is a T cell or a B cell.

11. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 10, wherein the immune cells are activated.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein the immune cell is an antigen-activated T cell or B cell.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which is at least about 60%, about 75%, or about 90% non-fucosylated.

14. below: (a) (i) a heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO:2, or a sequence which differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 3 or a sequence which differs therefrom by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence that differs therefrom by one or two amino acids. a heavy chain variable (VH) region comprising: (ii) a light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 6, or a sequence which differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 8 or a sequence that differs therefrom by one or two amino acids. a light chain variable (VL) region comprising: (b) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 10 or a sequence that differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 11 or a sequence which differs therefrom by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 12, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 14 or a sequence that differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 15, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 16, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (c) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 18 or a sequence which differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 19 or a sequence which differs therefrom by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 20, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 22 or a sequence that differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 23, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 24, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (d) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 26 or a sequence which differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 27, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 28, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 30, or a sequence which differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 31, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 32, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (e) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 34 or a sequence which differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 35, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 36, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 38 or a sequence which differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 39, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 40, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (f) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 34 or a sequence which differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 43, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 36, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 38 or a sequence which differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 39, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 40, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (g) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 51, or a sequence which differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 52, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 53, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 55 or a sequence which differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 56, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 57, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (h) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 51, or a sequence which differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 60, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 53, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 55 or a sequence which differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 56, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 57, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (i) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 70, or a sequence which differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 71, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 72, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 74, or a sequence which differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 75, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 76, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (j) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 70, or a sequence which differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 79, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 72, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 74, or a sequence which differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 75, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 76, or a sequence that differs therefrom by one or two amino acids. a VL region comprising (k) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence that differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 86, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 87, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence that differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 90, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 91, or a sequence that differs therefrom by one or two amino acids. a VL region comprising or (l) (i) HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence that differs therefrom by one or two amino acids; HCDR2 having the amino acid sequence of SEQ ID NO: 94, or a sequence which differs therefrom by one or two amino acids; and HCDR3 having the amino acid sequence of SEQ ID NO: 87, or a sequence that differs therefrom by one or two amino acids. and a VH region comprising: (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence that differs therefrom by one or two amino acids; an LCDR2 having the amino acid sequence of SEQ ID NO: 90, or a sequence which differs therefrom by one or two amino acids; and LCDR3 having the amino acid sequence of SEQ ID NO: 91, or a sequence that differs therefrom by one or two amino acids. A VL region comprising The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, comprising:

14. the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 are (a) SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; (b) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; (c) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; (d) SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively; (e) SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; (f) SEQ ID NO: 34, SEQ ID NO: 43, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; (g) SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively; (h) SEQ ID NO: 51, SEQ ID NO: 60, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively; (i) SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively; (j) SEQ ID NO: 70, SEQ ID NO: 79, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively; (k) SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, respectively; or (l) SEQ ID NO: 85, SEQ ID NO: 94, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, respectively.

14. The antibody or antigen-binding fragment thereof of claim 13, comprising an amino acid sequence that differs from the sequence of by a total of no more than two amino acid residues.

16. (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:8 a VL region comprising (b) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16 a VL region comprising (c) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 a VL region comprising (d) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32 a VL region comprising (e) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 a VL region comprising (f) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 a VL region comprising (g) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 a VL region comprising (h) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 a VL region comprising (i) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 71, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 74; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 76 a VL region comprising (j) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 74; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 76 a VL region comprising (k) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 89; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 90, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91 a VL region comprising or (l) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 89; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 90, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91 A VL region comprising The antibody or antigen-binding fragment thereof of claim 15, comprising:

17. (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 and a VH region comprising: (ii) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 40 A VL region comprising The antibody or antigen-binding fragment thereof of claim 16, comprising:

18. below: (a) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5; (b) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 13; (c) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 17, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21; (d) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 29; (e) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 33, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 37; (f) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 42 or 45, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 47 or 49; (g) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; (h) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 56, 62, 64, or 66, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 68; (i) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 69, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 73; (j) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 78, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 81 or 83; (k) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 88; or (l) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 93, and a VL region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 96 or 98. The antibody or antigen-binding fragment thereof according to any one of claims 14 to 17, comprising:

19. (a) a VH region having the amino acid sequence of SEQ ID NO: 1 and a VL region having the amino acid sequence of SEQ ID NO: 5; (b) a VH region having an amino acid sequence of SEQ ID NO: 9, and a VL region having an amino acid sequence of SEQ ID NO: 13; (c) a VH region having an amino acid sequence of SEQ ID NO: 17, and a VL region having an amino acid sequence of SEQ ID NO: 21; (d) a VH region having an amino acid sequence of SEQ ID NO: 25, and a VL region having an amino acid sequence of SEQ ID NO: 29; (e) a VH region having an amino acid sequence of SEQ ID NO: 33, and a VL region having an amino acid sequence of SEQ ID NO: 37; (f) a VH region having an amino acid sequence of SEQ ID NO: 42 or 45, and a VL region having an amino acid sequence of SEQ ID NO: 47 or 49; (g) a VH region having an amino acid sequence of SEQ ID NO: 50, and a VL region having an amino acid sequence of SEQ ID NO: 54; (h) a VH region having an amino acid sequence of SEQ ID NO: 56, 62, 64, or 66, and a VL region having an amino acid sequence of SEQ ID NO: 68; (i) a VH region having an amino acid sequence of SEQ ID NO: 69, and a VL region having an amino acid sequence of SEQ ID NO: 73; (j) a VH region having an amino acid sequence of SEQ ID NO: 78, and a VL region having an amino acid sequence of SEQ ID NO: 81 or 83; (k) a VH region having the amino acid sequence of SEQ ID NO: 84 and a VL region having the amino acid sequence of SEQ ID NO: 88; or (l) a VH region having the amino acid sequence of SEQ ID NO: 93, and a VL region having the amino acid sequence of SEQ ID NO: 96 or 98; 19. The antibody or antigen-binding fragment thereof of claim 18, comprising:

20. A VH region having the amino acid sequence of SEQ ID NO: 42 or 45, and a VL region having the amino acid sequence of SEQ ID NO: 47 or 49.

20. The antibody or antigen-binding fragment thereof of claim 19, comprising:

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, wherein the antibody is a humanized antibody.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antigen-binding fragment is a Fab, a Fab', a Fab2, a F(ab')2, an Fv, a single-chain Fv (scFv), or a diabody.

23. The antibody or antigen-binding fragment thereof of claim 22, wherein the antigen-binding fragment is an scFv.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, comprising an antibody heavy chain constant region.

25. The antibody or antigen-binding fragment thereof of claim 24, wherein the antibody heavy chain constant region is a human IgG heavy chain constant region.

26. The antibody or antigen-binding fragment thereof of claim 25, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region.

27. 27. The antibody or antigen-binding fragment thereof of claim 26, wherein the antibody heavy chain constant region comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:

100.

28. 28. The antibody or antigen-binding fragment thereof of claim 27, wherein the antibody heavy chain constant region has the amino acid sequence of SEQ ID NO:

100.

29. The antibody or antigen-binding fragment thereof of claim 25 or 26, wherein the antibody heavy chain constant region comprises one or more mutations that enhance effector function.

30. the antibody heavy chain constant region comprising one or more of the following mutations: (a) E233D, (b) G236D, (c) G237D, (d) P238D, (e) S239D, (f) S267E, (g) H268D, (h) P271G, (i) L328Y, L328F, or L328E, (j) A330R; (k) I332E, (l) G236D and H268D, (m) S239D and H268D, (n) S239D, H268D, L328Y, and I332E; (o) P238D and L328E, (p) G237D, P271G, and A330R, (q) G237D, H268D, P271G, and A330R; (r) S267E and L328F, (s) S239D and S267E, (t) G236D and S276E, or (u) E233D, G237D, H268D, P271G, and A330R or a combination thereof; The numbering of the constant region is according to the EU index:

30. The antibody or antigen-binding fragment thereof of claim 29.

31. The antibody or antigen-binding fragment thereof of claim 30, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region comprising the following mutations: S239D and H268D, and the numbering of the constant region is according to the EU index.

32. 31. The antibody or antigen-binding fragment thereof of claim 30, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region comprising the following mutations: E233D, G237D, H268D, P271G, and A330R, and the numbering of the constant region is according to the EU index.

33. (a) a cell line with an alpha-1,6-fucosyltransferase (Fut8) knockout, or (b) Cell lines overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III) and optionally overexpressing Golgi μ-mannosidase II (ManII) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 32, produced in

34. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 33, wherein the antibody or antigen-binding fragment is capable of binding to human PD-1.

35. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, wherein the antibody or antigen-binding fragment is capable of binding to cynomolgus monkey PD-1.

36. The antibody or antigen-binding fragment thereof of any one of claims 1 to 35, wherein the antibody or antigen-binding fragment is capable of blocking PD-1 / PD-L1 interaction.

37. The antibody or antigen-binding fragment thereof of any one of claims 1 to 36, wherein the antibody or antigen-binding fragment is capable of inhibiting T cell activation.

38. The antibody or antigen-binding fragment thereof of any one of claims 1 to 37, wherein the antibody or antigen-binding fragment is capable of inhibiting T-cell proliferation.

38. The antibody or antigen-binding fragment thereof of any one of claims 1 to 38, wherein the antibody or antigen-binding fragment is capable of inhibiting IFN-γ secretion.

39. The antibody or antigen-binding fragment thereof of any one of claims 1 to 39, conjugated to a drug.

41. The antibody or antigen-binding fragment thereof of claim 40, wherein the agent is a cytotoxic agent or a label.

42. The antibody or antigen-binding fragment thereof of claim 40, wherein the agent is a therapeutic agent.

43. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 42.

44. 44. The composition of claim 43, wherein the antibody or antigen-binding fragment comprises an Fc region and N-glycoside-linked carbohydrate chains linked to the Fc region, and optionally, less than 50% of the N-glycoside-linked carbohydrate chains comprise a fucose residue.

45. 45. The composition of claim 44, wherein substantially none of the N-glycoside-linked carbohydrate chains contain a fucose residue.

46. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 42.

47. 47. An expression vector comprising the polynucleotide of claim 46.

48. 48. A host cell comprising the polynucleotide of claim 46 or the expression vector of claim 47.

49. 49. The host cell of claim 48, which is a mammalian cell or an insect cell.

50. (a) comprises a Fut8 knockout; and / or (b) overexpressing GnT-III and optionally ManII; 50. A host cell according to claim 48 or 49.

51. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 42 and a pharmaceutically acceptable carrier.

52. 52. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-42, the composition of any one of claims 43-45, or the pharmaceutical composition of claim 51.

53. 53. The method of claim 52, wherein the disease or disorder is an autoimmune disease.

54. 53. The method of claim 52, wherein the disease or disorder is an allergy.

55. 53. The method of claim 52, wherein the disease or disorder is an inflammatory disease.

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