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, comprising administering a therapeutic agent comprising one or more antigen binding sites to cells involved in the autoimmune, allergic and / or inflammatory disease and depleting the cells.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 358,675, filed on July 6, 2022; and U.S. Provisional Patent Application No. 63 / 481,261, filed on January 24, 2023, the entire contents of each provisional patent application are hereby incorporated by reference for all purposes. Background Art

[0003] The immune system is tightly controlled 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 ends quickly under normal circumstances. However, in many chronic conditions, the inflammatory response persists and may lead to severe tissue and organ damage. Abnormal inflammatory responses have been shown to be associated with many chronic diseases, especially autoimmune diseases, in which immune cells act against self-proteins. Allergies are also caused by a dysregulated immune system that recognizes harmless non-self antigens. Mast cells and IgE antibodies take center stage in allergic reactions, while rogue T cells or B cells are the main players in autoimmunity.

[0004] There remains a need for therapeutic agents and treatments that fine-tune the activity of immune cells and the immune system, either alone or in combination, for autoimmune, allergic and inflammatory diseases. Summary of the invention

[0005] The present application provides methods and compositions for treating autoimmune, allergic and inflammatory diseases.

[0006] 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.

[0007] 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.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof has enhanced effector function. In some embodiments, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC).

[0009] In some embodiments, the antibody or its antigen binding fragment can deplete cells. In some embodiments, the cell is a cell expressing PD-1. 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 a B cell.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof is at least about 60%, about 75%, or about 90% afucosylated.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0012] (a)(i) a heavy chain variable (VH) region comprising:

[0013] A heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 2, or a sequence having 1 or 2 amino acid differences therefrom,

[0014] a HCDR2 having the amino acid sequence of SEQ ID NO: 3 or a sequence having 1 or 2 amino acid differences therefrom, and

[0015] A HCDR3 having the amino acid sequence of SEQ ID NO: 4, or a sequence having 1 or 2 amino acid differences therefrom; and

[0016] (ii) a light chain variable (VL) region comprising:

[0017] A light chain complementary determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 6, or a sequence having 1 or 2 amino acid differences therefrom,

[0018] LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence having 1 or 2 amino acid differences therefrom, and

[0019] LCDR3 having the amino acid sequence of SEQ ID NO: 8, or a sequence having 1 or 2 amino acid differences therefrom;

[0020] (b)(i) a VH region comprising:

[0021] A HCDR1 having the amino acid sequence of SEQ ID NO: 10 or a sequence having 1 or 2 amino acid differences therefrom,

[0022] a HCDR2 having the amino acid sequence of SEQ ID NO: 11 or a sequence having 1 or 2 amino acid differences therefrom, and

[0023] A HCDR3 having the amino acid sequence of SEQ ID NO: 12, or a sequence having 1 or 2 amino acid differences therefrom; and

[0024] (ii) a VL region comprising:

[0025] LCDR1 having the amino acid sequence of SEQ ID NO: 14 or a sequence having 1 or 2 amino acid differences therefrom,

[0026] LCDR2 having the amino acid sequence of SEQ ID NO: 15 or a sequence having 1 or 2 amino acid differences therefrom, and

[0027] LCDR3 having the amino acid sequence of SEQ ID NO: 16, or a sequence having 1 or 2 amino acid differences therefrom;

[0028] (c)(i) a VH region comprising:

[0029] A HCDR1 having the amino acid sequence of SEQ ID NO: 18 or a sequence having 1 or 2 amino acid differences therefrom,

[0030] a HCDR2 having the amino acid sequence of SEQ ID NO: 19 or a sequence having 1 or 2 amino acid differences therefrom, and

[0031] A HCDR3 having the amino acid sequence of SEQ ID NO: 20, or a sequence having 1 or 2 amino acid differences therefrom; and

[0032] (ii) a VL region comprising:

[0033] LCDR1 having the amino acid sequence of SEQ ID NO: 22 or a sequence having 1 or 2 amino acid differences therefrom,

[0034] LCDR2 having the amino acid sequence of SEQ ID NO: 23 or a sequence having 1 or 2 amino acid differences therefrom, and

[0035] LCDR3 having the amino acid sequence of SEQ ID NO: 24, or a sequence having 1 or 2 amino acid differences therefrom;

[0036] (d)(i) a VH region comprising:

[0037] A HCDR1 having the amino acid sequence of SEQ ID NO: 26 or a sequence having 1 or 2 amino acid differences therefrom,

[0038] a HCDR2 having the amino acid sequence of SEQ ID NO: 27 or a sequence having 1 or 2 amino acid differences therefrom, and

[0039] A HCDR3 having the amino acid sequence of SEQ ID NO: 28, or a sequence having 1 or 2 amino acid differences therefrom; and

[0040] (ii) a VL region comprising:

[0041] LCDR1 having the amino acid sequence of SEQ ID NO: 30 or a sequence having 1 or 2 amino acid differences therefrom,

[0042] LCDR2 having the amino acid sequence of SEQ ID NO: 31 or a sequence having 1 or 2 amino acid differences therefrom, and

[0043] LCDR3 having the amino acid sequence of SEQ ID NO: 32, or a sequence having 1 or 2 amino acid differences therefrom;

[0044] (e)(i) a VH region comprising:

[0045] A HCDR1 having the amino acid sequence of SEQ ID NO: 34 or a sequence having 1 or 2 amino acid differences therefrom,

[0046] a HCDR2 having the amino acid sequence of SEQ ID NO: 35 or a sequence having 1 or 2 amino acid differences therefrom, and

[0047] A HCDR3 having the amino acid sequence of SEQ ID NO: 36, or a sequence having 1 or 2 amino acid differences therefrom; and

[0048] (ii) a VL region comprising:

[0049] LCDR1 having the amino acid sequence of SEQ ID NO: 38 or a sequence having 1 or 2 amino acid differences therefrom,

[0050] LCDR2 having the amino acid sequence of SEQ ID NO: 39 or a sequence having 1 or 2 amino acid differences therefrom, and

[0051] LCDR3 having the amino acid sequence of SEQ ID NO: 40, or a sequence having 1 or 2 amino acid differences therefrom;

[0052] (f)(i) a VH region comprising:

[0053] A HCDR1 having the amino acid sequence of SEQ ID NO: 34 or a sequence having 1 or 2 amino acid differences therefrom,

[0054] a HCDR2 having the amino acid sequence of SEQ ID NO: 43 or a sequence having 1 or 2 amino acid differences therefrom, and

[0055] A HCDR3 having the amino acid sequence of SEQ ID NO: 36, or a sequence having 1 or 2 amino acid differences therefrom; and

[0056] (ii) a VL region comprising:

[0057] LCDR1 having the amino acid sequence of SEQ ID NO: 38 or a sequence having 1 or 2 amino acid differences therefrom,

[0058] LCDR2 having the amino acid sequence of SEQ ID NO: 39 or a sequence having 1 or 2 amino acid differences therefrom, and

[0059] LCDR3 having the amino acid sequence of SEQ ID NO: 40, or a sequence having 1 or 2 amino acid differences therefrom;

[0060] (g)(i) a VH region comprising:

[0061] A HCDR1 having the amino acid sequence of SEQ ID NO: 51 or a sequence having 1 or 2 amino acid differences therefrom,

[0062] a HCDR2 having the amino acid sequence of SEQ ID NO: 52, or a sequence having 1 or 2 amino acid differences therefrom, and

[0063] A HCDR3 having the amino acid sequence of SEQ ID NO: 53, or a sequence having 1 or 2 amino acid differences therefrom; and

[0064] (ii) a VL region comprising:

[0065] LCDR1 having the amino acid sequence of SEQ ID NO: 55 or a sequence having 1 or 2 amino acid differences therefrom,

[0066] LCDR2 having the amino acid sequence of SEQ ID NO: 56 or a sequence having 1 or 2 amino acid differences therefrom, and

[0067] LCDR3 having the amino acid sequence of SEQ ID NO: 57, or a sequence having 1 or 2 amino acid differences therefrom;

[0068] (h)(i) a VH region comprising:

[0069] A HCDR1 having the amino acid sequence of SEQ ID NO: 51 or a sequence having 1 or 2 amino acid differences therefrom,

[0070] a HCDR2 having the amino acid sequence of SEQ ID NO: 60 or a sequence having 1 or 2 amino acid differences therefrom, and

[0071] A HCDR3 having the amino acid sequence of SEQ ID NO: 53, or a sequence having 1 or 2 amino acid differences therefrom; and

[0072] (ii) a VL region comprising:

[0073] LCDR1 having the amino acid sequence of SEQ ID NO: 55 or a sequence having 1 or 2 amino acid differences therefrom,

[0074] LCDR2 having the amino acid sequence of SEQ ID NO: 56 or a sequence having 1 or 2 amino acid differences therefrom, and

[0075] LCDR3 having the amino acid sequence of SEQ ID NO: 57, or a sequence having 1 or 2 amino acid differences therefrom;

[0076] (i) (i) a VH region comprising:

[0077] A HCDR1 having the amino acid sequence of SEQ ID NO: 70 or a sequence having 1 or 2 amino acid differences therefrom,

[0078] a HCDR2 having the amino acid sequence of SEQ ID NO: 71 or a sequence having 1 or 2 amino acid differences therefrom, and

[0079] A HCDR3 having the amino acid sequence of SEQ ID NO: 72, or a sequence having 1 or 2 amino acid differences therefrom; and

[0080] (ii) a VL region comprising:

[0081] LCDR1 having the amino acid sequence of SEQ ID NO: 74 or a sequence having 1 or 2 amino acid differences therefrom,

[0082] LCDR2 having the amino acid sequence of SEQ ID NO: 75 or a sequence having 1 or 2 amino acid differences therefrom, and

[0083] LCDR3 having the amino acid sequence of SEQ ID NO: 76, or a sequence having 1 or 2 amino acid differences therefrom;

[0084] (j)(i) a VH region comprising:

[0085] A HCDR1 having the amino acid sequence of SEQ ID NO: 70 or a sequence having 1 or 2 amino acid differences therefrom,

[0086] a HCDR2 having the amino acid sequence of SEQ ID NO: 79 or a sequence having 1 or 2 amino acid differences therefrom, and

[0087] A HCDR3 having the amino acid sequence of SEQ ID NO: 72, or a sequence having 1 or 2 amino acid differences therefrom; and

[0088] (ii) a VL region comprising:

[0089] LCDR1 having the amino acid sequence of SEQ ID NO: 74 or a sequence having 1 or 2 amino acid differences therefrom,

[0090] LCDR2 having the amino acid sequence of SEQ ID NO: 75 or a sequence having 1 or 2 amino acid differences therefrom, and

[0091] LCDR3 having the amino acid sequence of SEQ ID NO: 76, or a sequence having 1 or 2 amino acid differences therefrom;

[0092] (k)(i) a VH region comprising:

[0093] A HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence having 1 or 2 amino acid differences therefrom,

[0094] a HCDR2 having the amino acid sequence of SEQ ID NO: 86 or a sequence having 1 or 2 amino acid differences therefrom, and

[0095] A HCDR3 having the amino acid sequence of SEQ ID NO: 87, or a sequence having 1 or 2 amino acid differences therefrom; and

[0096] (ii) a VL region comprising:

[0097] LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence having 1 or 2 amino acid differences therefrom,

[0098] LCDR2 having the amino acid sequence of SEQ ID NO: 90 or a sequence having 1 or 2 amino acid differences therefrom, and

[0099] LCDR3 having the amino acid sequence of SEQ ID NO: 91, or a sequence having 1 or 2 amino acid differences therefrom;

[0100] or

[0101] (l)(i) a VH region comprising:

[0102] A HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence having 1 or 2 amino acid differences therefrom,

[0103] a HCDR2 having the amino acid sequence of SEQ ID NO: 94 or a sequence having 1 or 2 amino acid differences therefrom, and

[0104] A HCDR3 having the amino acid sequence of SEQ ID NO: 87, or a sequence having 1 or 2 amino acid differences therefrom; and

[0105] (ii) a VL region comprising:

[0106] LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence having 1 or 2 amino acid differences therefrom,

[0107] LCDR2 having the amino acid sequence of SEQ ID NO: 90 or a sequence having 1 or 2 amino acid differences therefrom, and

[0108] A LCDR3 having the amino acid sequence of SEQ ID NO: 91, or a sequence having 1 or 2 amino acid differences therefrom.

[0109] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence that collectively differs by no more than two amino acid residues from:

[0110] (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;

[0111] (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;

[0112] (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;

[0113] (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;

[0114] (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;

[0115] (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;

[0116] (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;

[0117] (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;

[0118] (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;

[0119] (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;

[0120] (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;

[0121] or

[0122] (l) are 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.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0124] (a)(i) a VH region comprising:

[0125] A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2,

[0126] a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3, and

[0127] A CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4; and

[0128] (ii) a VL region comprising:

[0129] A CDR-L1 comprising the amino acid sequence of SEQ ID NO:6,

[0130] a CDR-L2 comprising the amino acid sequence of SEQ ID NO:7, and

[0131] a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8;

[0132] (b)(i) a VH region comprising:

[0133] A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and (ii) a VL region comprising:

[0134] a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16;

[0135] (c)(i) a VH region comprising:

[0136] A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and (ii) a VL region comprising:

[0137] a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:24;

[0138] (d)(i) a VH region comprising:

[0139] A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 28; and (ii) a VL region comprising:

[0140] a CDR-L1 comprising the amino acid sequence of SEQ ID NO:30, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:31, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:32;

[0141] (e)(i) a VH region comprising:

[0142] A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a VL region comprising:

[0143] a CDR-L1 comprising the amino acid sequence of SEQ ID NO:38, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:39, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:40;

[0144] (f)(i) a VH region comprising:

[0145] A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; and (ii) a VL region comprising:

[0146] a CDR-L1 comprising the amino acid sequence of SEQ ID NO:38, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:39, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:40;

[0147] (g)(i) a VH region comprising:

[0148] a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53; and (ii) a VL region comprising:

[0149] a CDR-L1 comprising the amino acid sequence of SEQ ID NO:55, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:57;

[0150] (h)(i) a VH region comprising:

[0151] A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53; and (ii) a VL region comprising:

[0152] a CDR-L1 comprising the amino acid sequence of SEQ ID NO:55, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:57;

[0153] (i) (i) a VH region comprising:

[0154] a CDR-H1 comprising the amino acid sequence of SEQ ID NO:70, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:71, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:72; and

[0155] (ii) a VL region comprising:

[0156] a CDR-L1 comprising the amino acid sequence of SEQ ID NO:74, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:75, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:76;

[0157] (j)(i) a VH region comprising:

[0158] a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72; and (ii) a VL region comprising:

[0159] a CDR-L1 comprising the amino acid sequence of SEQ ID NO:74, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:75, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:76;

[0160] (k)(i) a VH region comprising:

[0161] A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; and (ii) a VL region comprising:

[0162] a CDR-L1 comprising the amino acid sequence of SEQ ID NO:89, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:90, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:91;

[0163] or (l)(i) a VH region comprising:

[0164] A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 85, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 86, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; and (ii) a VL region comprising:

[0165] A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 89, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 90, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91. In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0166] (i) a VH region comprising:

[0167] a CDR-H1 comprising the amino acid sequence of SEQ ID NO:34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:43, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:36; and

[0168] (ii) a VL region comprising:

[0169] A CDR-L1 comprising the amino acid sequence of SEQ ID NO:38,

[0170] a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and

[0171] a CDR-L3 comprising the amino acid sequence of SEQ ID NO:40;

[0172] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0173] (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;

[0174] (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;

[0175] (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;

[0176] (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;

[0177] (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;

[0178] (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;

[0179] (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;

[0180] (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;

[0181] (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;

[0182] (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;

[0183] (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;

[0184] or

[0185] (l) a VH region comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with 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 with the amino acid sequence of SEQ ID NO:96 or 98.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0187] (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;

[0188] (b) a VH region having the amino acid sequence of SEQ ID NO: 9 and a VL region having the amino acid sequence of SEQ ID NO: 13;

[0189] (c) a VH region having the amino acid sequence of SEQ ID NO: 17 and a VL region having the amino acid sequence of SEQ ID NO: 21;

[0190] (d) a VH region having the amino acid sequence of SEQ ID NO: 25 and a VL region having the amino acid sequence of SEQ ID NO: 29;

[0191] (e) a VH region having the amino acid sequence of SEQ ID NO: 33 and a VL region having the amino acid sequence of SEQ ID NO: 37;

[0192] (f) 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;

[0193] (g) a VH region having the amino acid sequence of SEQ ID NO: 50 and a VL region having the amino acid sequence of SEQ ID NO: 54;

[0194] (h) a VH region having the amino acid sequence of SEQ ID NO: 56, 62, 64 or 66 and a VL region having the amino acid sequence of SEQ ID NO: 68;

[0195] (i) a VH region having the amino acid sequence of SEQ ID NO: 69 and a VL region having the amino acid sequence of SEQ ID NO: 73;

[0196] (j) a VH region having the amino acid sequence of SEQ ID NO: 78 and a VL region having the amino acid sequence of SEQ ID NO: 81 or 83;

[0197] (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;

[0198] or

[0199] (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.

[0200] In some embodiments, the antibody or antigen-binding fragment thereof comprises: 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.

[0201] In some embodiments, the antibody is a humanized antibody. In some embodiments, the antigen binding fragment is Fab, Fab', Fab2, F(ab')2, Fv, single-chain Fv (scFv) or diabody. In some embodiments, the antigen binding fragment is scFv.

[0202] In some embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region.

[0203] 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 with the amino acid sequence of SEQ ID NO: 100. In some embodiments, the antibody heavy chain constant region has an amino acid sequence of SEQ ID NO: 100.

[0204] 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 is a human IgG1 heavy chain constant region comprising one 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) S23 9D, 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, wherein 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, wherein 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, wherein the numbering of the constant region is according to the EU index.

[0205] In some embodiments, the antibody or antigen-binding fragment thereof is produced in a cell line that

[0206] (a) having a knockout of α-1,6-fucosyltransferase (Fut8); or

[0207] (b) Overexpression of β1,4-N-acetylglucosaminyltransferase III (GnT-III) and optionally overexpression of Golgi μ-mannosidase II (ManII).

[0208] 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 monkey PD-1.

[0209] In some embodiments, the antibody or antigen-binding fragment is capable of blocking the PD-1 / PD-L1 interaction.

[0210] 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.

[0211] In some embodiments, wherein 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.

[0212] In another aspect, a composition comprising an antibody or antigen-binding fragment thereof as described herein is provided. In some embodiments, the antibody or antigen-binding fragment comprises an Fc region and an N-glycoside-linked carbohydrate chain attached to the Fc region, optionally wherein less than 50% of the N-glycoside-linked carbohydrate chains contain fucose residues. In some embodiments, substantially no N-glycoside-linked carbohydrate chains contain fucose residues.

[0213] In another aspect, a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein is provided.

[0214] In another aspect, an expression vector comprising a polynucleotide described herein is provided.

[0215] In another aspect, a host cell comprising a polynucleotide or expression vector as described herein is provided. In some embodiments, the host cell is a mammalian or insect cell. In some embodiments, the host cell (a) comprises a Fut8 knockout; and / or (b) overexpresses GnT-III and optionally overexpresses ManII.

[0216] In another aspect, provided is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0217] In another aspect, a method of treating a disease or condition in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof as described herein. In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is an allergy. In some embodiments, the disease or condition is an inflammatory disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0218] Figure 1 Blockade of PD-1 / PD-L1 interaction by disclosed PD-1 antibodies is depicted. The percentage of blocking is plotted against a standard curve of a control PD-1 neutralizing antibody.

[0219] Figure 2The disclosed PD-1 antibodies were shown to inhibit IFN-γ secretion in mixed lymphocyte reactions (MLRs) elicited by co-culture of peripheral blood mononuclear cells (PBMCs) from different donors.

[0220] Figure 3 Depicted is the inhibition of activation of Jurkat reporter T cells incubated with Raji-APC cells modified to express the FcγRII receptor by PD-1 antibodies with a normal IgG1 Fc or a modified Fc containing mutations that increase binding to FcγRII.

[0221] Figure 4 Depicted is the inhibition of IL-2 secretion by Jurkat reporter T cells incubated with Raji-APC cells modified to express the FcγRII receptor by PD-1 antibodies with a normal IgG1 Fc or a modified Fc containing mutations that increase binding to FcγRII.

[0222] Figure 5A and Figure 5B The PD-1 antibody 3H4, which contains a modified Fc domain to increase binding to FcγRII, was shown to inhibit CD4( Figure 5A ) and CD8( Figure 5B )T cell proliferation.

[0223] Figure 6 It was shown that PD-1 antibody 3H4 containing an Fc domain modified to increase binding to FcγRII inhibited IFN-γ secretion in PBMCs compared to PD-1 antibody with normal IgG1 Fc and controls (isotype antibody control and no antibody control). DETAILED DESCRIPTION

[0224] definition

[0225] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context is inappropriate.

[0226] As used herein, when used in this article to refer to a value, the terms "about", "approximately" and "equivalent thereto" refer to a value similar to the reference value in the context of a reference value. Generally speaking, those skilled in the art who are familiar with the context will understand the relevant degree of variation covered by "about", "approximately" and "equivalent thereto" in this context. For example, in some embodiments, the terms "about", "approximately" and "equivalent thereto" can be covered in 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 value mentioned.

[0227] As used herein, "agonist" or "agonistic" when used in reference to an antigen binding site or a molecule containing an antigen binding site, means that the binding of the antigen binding site or molecule to its target results in stimulation or activation of the target, or enhances, increases, promotes, induces or prolongs one or more functions or biological activities of the target.

[0228] As used herein, the terms "antagonist," "antagonistic," "neutralizing," or "blocking" when used in reference to an antigen binding site or a molecule containing an antigen binding site, means that binding of the antigen binding site or molecule to its target results in inhibition of at least some biological activity of the target.

[0229] As used herein, "antibody" refers to a polypeptide whose amino acid sequence includes an immunoglobulin and a fragment thereof that specifically binds to a specified antigen, or a fragment thereof. The antibody 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). It will be appreciated by those of ordinary skill in the art that the characteristic sequence or portion of an antibody may 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). In addition, it will be appreciated by those of ordinary skill in the art that the characteristic sequence or portion of an antibody may include one or more polypeptide chains, and may include sequence elements found in the same polypeptide chain or in different polypeptide chains.

[0230] As used herein, "antibody mimics" refers to any molecule that can simulate the ability of an antibody to bind to an antigen, but is not limited to the antibody structure. Examples of antibody mimics include, but are not limited to, Affibodies, Affilins, Affimers, Afftins, Alphabodies, Anticalins, Avimers, Centyrins, DARPins, Fynomers, monobodies, nanobodies, and nanoCLAMPs.

[0231] An "antigen-binding fragment" of an antibody comprises a portion of an intact antibody that is still capable of binding antigen. In certain embodiments, papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. The Fab fragment consists of an entire light chain and the variable region domain (V H ) and the first constant domain of a heavy chain (C H1) composition. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site. In certain embodiments, treatment of an antibody with pepsin produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen binding activities and is still capable of cross-linking antigen. The Fab' fragment differs from the Fab fragment in that it has a single C H A few residues are added to the carboxyl terminus of the constant domain, including one or more cysteines from the antibody hinge region. Fab'-SH denotes Fab' in which the cysteine ​​residue(s) of the constant domain have 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.

[0232] As used herein, the term "antigen binding site" refers to any molecule or any part of a molecule that can bind to an antigen. In human antibodies, the antigen binding site is formed by amino acid residues of the N-terminal variable region ("V") of the heavy chain ("H") and the light chain ("L"). The three highly divergent segments within the heavy and light chain V regions are called "hypervariable regions", which are located between more conservative flanking segments called "framework regions" or "FRs". Therefore, the term "FR" refers to the amino acid sequence naturally occurring between and adjacent to the hypervariable regions of immunoglobulins. In human antibodies, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged 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 the bound antigen, and the three hypervariable regions of the heavy chain and the light chain are each called "complementarity determining region" or "CDR". In some animals, such as camels and cartilaginous fish, the antigen binding site may be formed by a single antibody chain, thereby providing a "single domain antibody". The antigen binding site may also be an antibody mimetic or present in an antibody mimetic.

[0233] CDR can be determined by the methods described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of Protein 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 according to these definitions usually include overlaps or subsets of amino acid residues when compared to each other. In certain embodiments, the term "CDR" is 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, Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of Protein of immunological interest. (1991). In certain embodiments, the heavy chain CDR and light chain CDR of an antibody are defined using different conventions. For example, in certain embodiments, the heavy chain CDR is defined according to MacCallum (supra) and the light chain CDR is defined according to Kabat (supra). CDRH1, CDRH2 and CDRH3 represent heavy chain CDRs, CDRL1, CDRL2 and CDRL3 represent light chain CDRs.

[0234] As used herein, the "binding affinity" of a molecule (e.g., antigen binding site) of the present disclosure to a given target (e.g., PD-1, PD-L1, or PD-L2) can be determined by a variety of methods known to those skilled in the art. Such methods include, but are not limited to, fluorescence titration, ELISA (enzyme-linked immunosorbent assay), calorimetry (e.g., isothermal titration calorimetry (ITC)), and surface plasmon resonance (SPR).

[0235] As used herein, "bispecific" refers to a molecule that is capable of specifically binding to at least two different targets. Typically, a bispecific molecule comprises two antigen binding sites, each site being specific for a different target. In some embodiments, a bispecific molecule is capable of binding to two targets simultaneously.

[0236] As used herein, "detectable affinity" refers to the ability to bind a given target with a certain affinity constant, usually measured by K. D or EC 50 The affinity constant is about 10 at most. -5 M or lower (lower K D or EC 50 Lower affinities are no longer measurable by common methods such as ELISA (enzyme-linked immunosorbent assay) and are therefore of secondary significance.

[0237] As used herein, the term "epitope" is an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule, which is called a paratope and consists of the six complementary determining regions of an antibody. A single antigen can have more than one epitope. Epitopes may be conformational or linear. Conformational epitopes consist of spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes consist of adjacent amino acid residues in a polypeptide chain.

[0238] The "Fc" fragment contains the carboxyl-terminal portions of two heavy chains linked together by disulfide bonds. The effector functions of an antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcR) on certain cell types.

[0239] "Fragments" with respect to antigens (e.g., PD-1, PD-L1, or PD-L2) refer to N-terminal and / or C-terminal truncations or protein domains of the antigen. In some embodiments, fragments of the antigen retain the ability of the full-length antigen to be recognized and / or bound by the antigen-binding fragments of the present disclosure.

[0240] As used herein, "fucosylation" or "fucosylated" refers to the presence of fucose residues within oligosaccharides attached to the antibody peptide backbone. Specifically, a fucosylated antibody comprises α(l,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue of one or two N-linked oligosaccharides attached to the Fc region of the antibody. "Non-fucosylated," "afucosylated," or "fucose-deficient" antibodies refer to glycosylated antibody variants comprising an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, the non-fucosylated or fucose-deficient antibodies have reduced fucose relative to the amount of fucose on the same antibody produced in a cell line. In some embodiments, antibodies with reduced fucose or lack of fucose have improved ADCC function.

[0241] The "degree of fucosylation" is the percentage of fucosylated oligosaccharides relative to all oligosaccharides, which can be identified by methods known in the art, such as by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in an N-glycosidase F-treated antibody composition. In a composition of a "fully fucosylated antibody", substantially all oligosaccharides contain fucose residues, i.e., are fucosylated. In some embodiments, a composition of a fully fucosylated antibody has a degree of fucosylation of at least about 90%. In contrast, in a composition of a "completely non-fucosylated antibody", substantially no oligosaccharides are fucosylated. In some embodiments, a composition of a completely non-fucosylated antibody has a degree of fucosylation of less than about 10%. In a composition of a "partially fucosylated antibody", only a portion of the oligosaccharides contain fucose. The individual antibodies in such compositions may not contain fucose residues in the N-linked oligosaccharides in the Fc region, contain fucose residues in one N-linked oligosaccharide in the Fc region, or contain fucose residues in two N-linked oligosaccharides in the Fc region, provided that the composition does not contain substantially all single antibodies lacking fucose residues in the N-linked oligosaccharides in the Fc region, nor does it contain substantially all single antibodies containing fucose residues in both N-linked oligosaccharides in the Fc region. In one embodiment, the 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 and methods and cell lines for producing antibodies with altered, reduced or eliminated fucosylation are known in the art (see, e.g., WO 2023 / 044390A1).

[0242] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, each antibody constituting the population is identical, except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies have a high degree of specificity (for a single antigenic site). In addition, compared with conventional (polyclonal) antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on the antigen. The modifier "monoclonal" indicates the characteristics of the antibody obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the antibody to be produced by any ad hoc method. For example, monoclonal antibodies can be made by a hybridoma method, or can be made by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibody" can also be separated from a phage antibody library.

[0243] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use, either in vivo or ex vivo.

[0244] 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, after administration to a subject, can provide a compound described herein or its active metabolite or residue. As known to those skilled in the art, the "salts" of the compounds described herein can be derived from inorganic or organic acids and bases.

[0245] 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, p-toluenesulfonic 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 pharmaceutically acceptable in themselves, may be used to prepare salts useful as intermediates to obtain the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0246] Exemplary bases include, but are not limited to, alkali metal (eg, sodium) hydroxides, alkaline earth metal (eg, magnesium) hydroxides, ammonia, and compounds of formula NW4 + Compounds (wherein W is C 1-4 Alkyl) etc.

[0247] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and the like. Other examples of salts include anions of the compounds described herein with suitable cations such as Na + NH4 + and NW4 + (W is C 1-4 Alkyl) and other compounds.

[0248] For therapeutic use, salts of the compounds described herein are considered pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0249] As used herein, the phrase "reference level" generally refers to a level that is considered "normal" for comparison purposes, such as the level of an appropriate control. For example, in the context of antibody effector function enhancement, a "reference level" may refer to the effector function level of an antibody comprising a wild-type constant region (Fc). As another example, in the context of target binding, a "reference level" may refer to the binding affinity level of a natural ligand of a target (e.g., as measured by K D or EC 50 The reference level may be determined contemporaneously, or may be predetermined, e.g. known or inferred from past observations.

[0250] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., rodents, apes, equines, bovines, porcines, canines, felines, etc.), and more preferably include humans.

[0251] As used herein, the phrases "therapeutically effective amount" and "effective amount" are used interchangeably and refer to an amount effective to achieve the desired therapeutic outcome at the necessary dosage and for the necessary period of time. The therapeutically effective amount may vary depending on a variety of factors, such as the type of disease (e.g., cancer), the disease state, the age, sex, and / or weight of the individual, and the ability of the immunoconjugate (or its pharmaceutical composition) to elicit the desired response in the individual. An effective amount may also be an amount in which any toxic or deleterious effects of the immunoconjugate or its pharmaceutical composition are offset by the therapeutically beneficial effects.

[0252] As used herein, "treating" a condition or "treatment" of the condition (e.g., a condition described herein, such as cancer) is a method of obtaining a beneficial or desired result (e.g., a clinical result). Beneficial or desired results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions; reduction in the extent of a disease, disorder, or condition; stabilization (i.e., not worsening) of the state of a disease, disorder, or condition; prevention of the spread of a disease, disorder, or condition (e.g., primary cancer and / or secondary metastasis); delay or slowing of the progression of a disease, disorder, or condition; improvement or palliation of a disease, disorder, or condition; and partial or complete and detectable or undetectable remission.

[0253] Antigen binding site

[0254] In one aspect, the present application provides an antigen binding site capable of binding to a given antigen (e.g., 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.

[0255] In some embodiments, the antigen binding site is capable of binding 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 B cell. Examples of such antigens include, but are not limited to, 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, C D20, CD22, CD28, CD38, CD39, CD72, CD73, CD79A, CD79B, glycoprotein 130 (gp130), BCMA, BAFF receptor (BAFF-R), TACI, integrin α4, integrin β7, FCGR2B, ICOS ligand, CD138, SLAMF7, LILR family members, fibroblast activation protein α (FAP), DLK-1, CD26, TE-7, CD29, PDGRFα, TGFβ 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.

[0256] In some embodiments, the antigen binding site provided is present as an antibody mimetic (e.g., an Affibody, Affilin, Affimer, afftin, Alphabody, Anticalin, avimer, Centyrin, DARPin, Fynomer, monobody, nanobody, or nanoCLAMP).

[0257] In some embodiments, 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 exists as a single chain variable fragment (scFv).

[0258] In some embodiments, the antigen binding site provided is capable of binding to PD-1. In some embodiments, the antigen binding site exists as an antibody. In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site that is capable of binding to PD-1 and exists as an antibody described herein, wherein such antigen binding site comprises 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 that is capable of binding to PD-1 and exists as an antibody described herein.

[0259] In some embodiments, an antigen binding site capable of binding to PD-1 is provided that 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.

[0260] In some embodiments, an antigen binding site capable of binding to PD-1 is provided comprising VH and VL, wherein VH comprises CDR-H1, CDR-H2 and CDR-H3, and 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 of VH or VL described in Table 5. The CDRs of 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 of which is independently selected from those described in Table 5.

[0261] In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to PD-1 as described herein, wherein such antigen binding site has a CDR sequence in which each CDR differs from the CDR sequence described in Table 5 by no more than two amino acid residues (e.g., two or one amino acid residue). In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to PD-1 as described herein, wherein such antigen binding site has a set of six CDRs, whose sequences generally differ from a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 5 by no more than two amino acid residues (e.g., two or one amino acid residue). In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to PD-1 as described herein, wherein such antigen binding site has a set of six CDRs, whose sequences generally differ from an anti-PD-1 sequence having a set of VH and VL selected from Table 5 by no more than two amino acid residues (e.g., two or one amino acid residue).

[0262] In some embodiments, an antigen binding site capable of binding to PD-1 is provided comprising a VH sequence as described in Table 5 and a VL sequence as described in Table 5. In some embodiments, an antigen binding site is provided, which is an antigen binding site described herein that is capable of binding to PD-1, such as an anti-PD-1 variant having a set of VH and VL selected from Table 5, wherein such antigen binding site has (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 the VH described 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 the VL described in Table 5.

[0263] In some embodiments, an antigen binding site capable of binding to PD-1 is provided, comprising a heavy chain sequence as described in Table 5 and a light chain sequence as described in Table 5. In some embodiments, an antigen binding site is provided, which is an antigen binding site capable of binding to PD-1 as described herein, such as a variant of anti-PD-1 having a group of heavy chains and light chains selected from Table 5, wherein such antigen binding site has (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 described 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 described in Table 5.

[0264] In certain embodiments, the antigen binding sites described herein are derived from 1-17. For example, in certain embodiments, the antigen binding sites described herein comprise a VH comprising an amino acid sequence that is 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 that is 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, the antigen binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 having VH and VL sequences of SEQ ID NOs: 1 and 5, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, VH comprises CDR-H1, CDR-H2 and CDR-H3, comprising amino acid sequences of SEQ ID NOs: 2, 3 and 4, respectively. In certain embodiments, VL comprises CDR-L1, CDR-L2 and CDR-L3, comprising amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively. In certain embodiments, the antigen binding site comprises (a) VH comprising CDR1, CDR2 and CDR3 comprising amino acid sequences of SEQ ID NOs: 2, 3 and 4, respectively; and (b) VL comprising CDR1, CDR2 and CDR3 comprising amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively.

[0265] In certain embodiments, the antigen binding sites described herein are derived from 1-28. For example, in certain embodiments, the antigen binding sites described herein comprise a VH comprising an amino acid sequence that is 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 that is 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 having VH and VL sequences of SEQ ID NOs: 9 and 13, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, 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, 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.

[0266] In certain embodiments, the antigen binding sites described herein are derived from 1-33. For example, in certain embodiments, the antigen binding sites described herein comprise a VH comprising an amino acid sequence that is 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 that is 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 having VH and VL sequences of SEQ ID NOs: 17 and 21, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, 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, 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.

[0267] In certain embodiments, the antigen binding sites described herein are derived from 1-35. For example, in certain embodiments, the antigen binding sites described herein comprise a VH comprising an amino acid sequence that is 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 that is 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 having VH and VL sequences of SEQ ID NOs: 25 and 29, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, 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, 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.

[0268] In certain embodiments, the antigen binding sites described herein are derived from murine 3H4. For example, in certain embodiments, the antigen binding sites described herein comprise a VH comprising an amino acid sequence that is 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 that is 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 having 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, VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NOs: 34, 35, and 36, respectively. In certain embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3 comprising 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.

[0269] In certain embodiments, the antigen binding sites described herein are derived from humanized 3H4. For example, in certain embodiments, the antigen binding sites described herein comprise a VH comprising an amino acid sequence that is 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 / or a VL comprising an amino acid sequence that is 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: 47 or 49. In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO: 42. In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO: 44. In certain embodiments, 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 having a VH sequence of SEQ ID NO: 42 or 45, which is determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art; and / or CDR-L1, CDR-L2 and CDR-L3 having a VL sequence of SEQ ID NO: 47 or 49, which is determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, the antigen binding site comprises (a) VH comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 34, 43 and 36, respectively; and (b) VL comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 38, 39 and 40, respectively.

[0270] In certain embodiments, the antigen binding sites described herein are derived from murine 2.3A9. For example, in certain embodiments, the antigen binding sites described herein comprise a VH comprising an amino acid sequence that is 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 that is 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 having VH and VL sequences of SEQ ID NOs: 50 and 54, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, 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, 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.

[0271] 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 an 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, VH comprises the amino acid sequence of SEQ ID NO: 59. In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO: 62. In certain embodiments, 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 having a VH sequence of SEQ ID NO: 59, 62, 64, or 66, which is determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art; and / or CDR-L1, CDR-L2, and CDR-L3 having a VL sequence of SEQ ID NO: 68, which is determined according to 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.

[0272] In certain embodiments, the antigen binding sites described herein are derived from murine 20B3.1. For example, in certain embodiments, the antigen binding sites described herein comprise a VH comprising an amino acid sequence that is 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 that is 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 having VH and VL sequences of SEQ ID NOs: 69 and 73, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, 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, 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.

[0273] In certain embodiments, the antigen binding sites described herein are derived from humanized 20B3.1. For example, in certain embodiments, the antigen binding sites described herein comprise 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, VH comprises the amino acid sequence of SEQ ID NO: 78. In certain embodiments, VL comprises the amino acid sequence of SEQ ID NO: 81. In certain embodiments, 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 having a VH sequence of SEQ ID NO: 78, which is determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art; and / or CDR-L1, CDR-L2 and CDR-L3 having a VL sequence of SEQ ID NO: 81 or 83, which is determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, the antigen binding site comprises (a) VH comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 70, 79 and 72, respectively; and (b) VL comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 74, 75 and 76, respectively.

[0274] In certain embodiments, the antigen binding sites described herein are derived from murine 1B8. For example, in certain embodiments, the antigen binding sites described herein comprise a VH comprising an amino acid sequence that is 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 that is 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 having VH and VL sequences of SEQ ID NOs: 84 and 88, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, 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, 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.

[0275] In certain embodiments, the antigen binding sites described herein are derived from humanized 1B8. For example, in certain embodiments, the antigen binding sites described herein comprise 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 / 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: 96 or 98. In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO: 93. In certain embodiments, VL comprises the amino acid sequence of SEQ ID NO: 96. In certain embodiments, 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 having a VH sequence of SEQ ID NO: 93, which is determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art; and / or CDR-L1, CDR-L2 and CDR-L3 having a VL sequence of SEQ ID NO: 96 or 98, which is determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, the antigen binding site comprises (a) VH comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 86, 94 and 87, respectively; and (b) VL comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 89, 90 and 91, respectively.

[0276] In each of the above embodiments, it is contemplated herein that the VH and / or VL sequences that bind to PD-1 may contain 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 VH and / or VL without affecting their ability to bind to PD-1. For example, it is contemplated herein that the VH and VL sequences that bind to PD-1 may contain cysteine ​​heterodimerization mutations, thereby facilitating the formation of disulfide bridges between VH and VL to form scFv.

[0277] In some embodiments, the antigen binding sites disclosed herein bind to human PD-1 or its extracellular region. In some embodiments, the antigen binding sites disclosed herein bind to PD-1 presented on the surface of a membrane (e.g., the plasma membrane of a cell).

[0278] In some embodiments, the antigen binding sites disclosed herein bind to cynomolgus monkey PD-1. In some embodiments, the antigen binding sites disclosed herein bind to cynomolgus monkey PD-1 with an affinity comparable to that of human PD-1.

[0279] In some embodiments, the antigen binding sites disclosed herein do not significantly bind to other PD-1 family members. In some embodiments, the antigen binding sites disclosed herein do not significantly bind to ICOS, CD28, or CTLA-4.

[0280] In some embodiments, the antigen binding sites disclosed herein are capable of inhibiting the binding of PD-L1 to PD-1. In some embodiments, the antigen binding sites disclosed herein are capable of agonizing PD-1.

[0281] In some embodiments, the antigen binding sites disclosed herein can reduce IL-2 secretion. In some embodiments, the antigen binding sites disclosed herein can inhibit (partially or completely) T cell activation. In some embodiments, the antigen binding sites disclosed herein can inhibit (partially or completely) T cell proliferation (e.g., CD4+ and / or CD8+ T cells).

[0282] In some embodiments, the antigen binding sites disclosed herein are capable of reducing inflammatory cytokine secretion. In some embodiments, the antigen binding sites disclosed herein are capable of reducing IFN-γ secretion.

[0283] Molecules containing antigen binding sites

[0284] Also provided herein are molecules containing disclosed antigen binding sites.Such molecules can be, but are not limited to, antibodies or their antigen binding fragments, antibody fragments, nanobodies, antibody mimetics, etc.In some embodiments, the molecules containing disclosed antigen binding sites are therapeutic agents, i.e., the molecules confer therapeutic benefits.Therefore, in some embodiments, therapeutic agents containing disclosed antigen binding sites are provided.In some embodiments, therapeutic agents can induce complement dependent cytotoxicity (CDC).In some embodiments, therapeutic agents can induce antibody dependent cell-mediated cytotoxicity (ADCC).In some embodiments, therapeutic agents can induce antibody dependent cellular phagocytosis (ADCP).In some embodiments, therapeutic agents can deplete immune cells (e.g., T cells, B cells, mast cells, macrophages, NK cells, DC, monocytes, neutrophils, fibroblasts or epithelial cells).

[0285] In some embodiments, the therapeutic agent is an antibody or an 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 its antigen-binding fragment is multispecific, such as bispecific. In some embodiments, the antibody is a non-fucosylated antibody (non-fucosylated antibody). In some embodiments, the antibody is at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1% fucosylated. In some embodiments, the antibody is afucosylated antibody. 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% afucosylated.

[0286] In some embodiments, the antibody or antigen-binding fragment thereof can bind to 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, C D19, CD20, CD22, CD28, CD38, CD39, CD72, CD73, CD79A, CD79B, glycoprotein 130 (gp130), BCMA, BAFF receptor (BAFF-R), TACI, integrin α4, integrin β7, FCGR2B, ICOS ligand, CD138, SLAMF7, LILR family members, fibroblast activation protein α (FAP), DLK-1, CD26, TE-7, CD29, PDGRFα, TGFβ receptor, MAS516, CD13, or a combination thereof. In some embodiments, the antibody or its antigen-binding fragment can bind to PD-1, PD-L1 or PD-L2. In some embodiments, the antibody or its antigen-binding fragment can bind to PD-1. In some embodiments, the antibody or its antigen-binding fragment can excite PD-1.

[0287] In some embodiments, therapeutic agents comprising two or more antigen binding sites are provided. In some embodiments, the therapeutic agent is bispecific.

[0288] In some embodiments, the therapeutic agent comprises a first antigen binding site that can bind to a marker of activated T cells, activated B cells, activated fibroblasts, or activated epithelial cells. In some embodiments, the first antigen binding site can bind to PD-1, PD-L1, PD-L2, CTLA-4, BTLA-4, or KLRG1. In some embodiments, the first antigen binding site can bind to PD-1, PD-L1, PD-L2, BCMA, or CD38. In some embodiments, the first antigen binding site can bind to FAP or PDGRFα.

[0289] In some embodiments, the therapeutic agent comprises a second antigen binding site that binds to a marker of activated T cells, activated B cells, activated fibroblasts, or activated epithelial cells. In some embodiments, the marker is directed to the same type of activated cells as the marker bound by the first antigen binding site. In some embodiments, the marker is directed to different types of activated cells as the marker bound by the first antigen binding site.

[0290] 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, PDGRFα, TGFβ receptor, MAS516 and CD13.

[0291] Various formats and uses of bispecific molecules are known in the art.The bispecific molecules according to the invention are not limited to any particular bispecific format or method of generating it.

[0292] In some embodiments, the bispecific molecule can be a bispecific antibody or a fragment or derivative thereof, including but not limited to: (i) a single antibody with two arms, each arm comprising a different antigen binding site; (ii) a bispecific scFv, for example, two scFvs connected via a peptide linker; (iii) a dual variable domain antibody (DVD-Ig), in which each light chain and heavy chain contains two variable domains connected in series by a short peptide bond; (iv) a bispecific (Fab′)2 fragment; (v) a diabody; (vi) a tandem diabody (TandAb), a fusion of two diabodies; and (vii) a "dock and lock (DNL)-Fab3", a trivalent bispecific consisting of two identical Fab fragments connected to different Fab fragments. Binding proteins; (viii) IgG-like molecules with engineered Fc to enforce heterodimerization; (ix) recombinant IgG-like dual-targeting molecules, in which each side of the molecule contains Fab fragments or partial Fab fragments 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 partial Fab fragment; (xi) Fc fusion molecules; (v) Fab fusion molecules; and (vi) antibodies and heavy chain antibodies (e.g., domain antibodies, nanobodies) based on scFv and diabodies, 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 with a heavy chain constant domain, Fc region or a portion thereof.

[0293] Examples of IgG-like molecules with engineered Fc include, but are not limited to, triomabs, knobs-into-holes (kih) molecules (e.g., kih IgG with a common light chain), CrossMAbs, orthoFab IgG molecules, electrostatic matching molecules, LUZ-Y molecules, DIG-body and PIG-body molecules, chain exchange engineered domain bodies (SEEDbody) molecules, Biclonics 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 The example of IgG fusion molecules includes but is not limited to DVD-Ig molecules and IgG-scFv. The example of Fc fusion molecules includes but is not limited to scFv / Fc fusions, SCORPION molecules and Fc-DART molecules. The example of Fab fusion bispecific antibodies includes but is not limited to F(ab)2 molecules, DNL molecules and Fab-Fv molecules. The examples of antibodies and domain antibodies based on scFv and double antibodies include but are not limited to bispecific T cell engagers (BiTE) molecules, tandem double antibody molecules (TandAb), dual affinity retargeting technology (DART) molecules, single-chain double antibody molecules, TCR-like antibodies (AIT, ReceptorLogics), human serum albumin scFv fusions, COMBODY molecules, dual-targeted nano antibodies and dual-targeted heavy chain domain antibodies only.

[0294] In some embodiments, the molecule containing the disclosed antigen binding site also comprises an antibody constant region or a fragment or variant thereof. In some embodiments, the antibody constant region can be, for example, a heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; in particular, selected from, for example, a (for example, human) heavy chain constant region of IgG1, IgG2, IgG3 and IgG4. In certain embodiments, the antibody constant region or a fragment or variant thereof has an amino acid sequence consistent with, for example, a heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE, preferably, for example, a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 at least 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). In certain embodiments, the antibody constant region can be a light chain constant region selected from, for example, a (e.g., human) light chain constant region of κ or λ. The constant region can be changed, for example, mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of the following: 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 can fix complement. In other embodiments, the antibody will 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 mutant that does not support binding to Fc receptors, for example, it has a mutagenic or missing Fc receptor binding region.

[0295] In some embodiments, the constant region comprises one or more mutations that enhance effector function.

[0296] 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 a heavy chain constant region of IgG1 comprising one or more of the following mutations, numbered based on 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) S23 9D 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; (u) E233D, G237D, H268D, P271G and A330R, or a combination thereof. In certain embodiments, the constant region comprises an IgG1 heavy chain constant region comprising S239D and H268D mutations, numbered based on the EU index. In certain embodiments, the constant region comprises an IgG1 heavy chain constant region comprising E233D, G237D, H268D, P271G, and A330R mutations, numbered based on the EU index.

[0297] In certain embodiments, the constant region comprises a heavy chain constant region of IgG1 comprising one or more of the following mutations, numbered based on the EU index: (a) 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.

[0298] In some embodiments, the constant region comprises one or more mutations that reduce effector function. For example, in certain embodiments, the constant region comprises a heavy chain constant region of IgG1, which comprises one or more of the following mutations, based on EU index numbering: (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 P33 1S; (g) N297A; (h) N297G; (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 based on 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 a heavy chain constant region of IgG4 comprising one or more of the following mutations, numbered based on 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.

[0299] In some embodiments, the antigen binding site is connected to an antibody constant region or a fragment or variant thereof. In certain embodiments, the antigen binding site is connected to an IgG constant region, including hinge, CH2 and CH3 domains (with or without a CH1 domain).

[0300] In some embodiments, the molecules containing the disclosed antigen binding sites further comprise an afucosylated Fc region. In some embodiments, the Fc region is an afucosylated IgG1 Fc region.

[0301] In some embodiments, the bispecific molecule can be a fusion protein containing one or more antibody mimetics. In some embodiments, the bispecific molecule can be a fusion protein containing one or more antibody mimetics and one or more antibodies or antigen-binding fragments thereof.

[0302] Amino acid sequence modification

[0303] Amino acid sequence modifications of the antigen binding sites disclosed herein and molecules containing the antigen binding sites (e.g., antibodies or antigen binding fragments thereof) 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 in the nucleic acid sequence encoding the antigen binding site or a molecule containing the antigen binding site, or by peptide synthesis. Such modifications may include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions may be performed, as long as the antigen binding site or a molecule containing the antigen binding site retains the desired properties and / or functions. In some embodiments, amino acid changes are introduced to alter post-translational processes, such as changing the number or position of glycosylation sites.

[0304] A useful method for identifying certain residues or regions as preferred mutagenesis positions is called "alanine scanning mutagenesis". In this method, a residue or a 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. Then, those amino acid positions that show functional sensitivity to substitution are improved by introducing additional or other variants at or for the substitution site. Therefore, although the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself does not need to be predetermined. For example, in order to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis can be performed at the target codon or region, and the expressed variants can be screened to obtain the desired activity.

[0305] Examples of amino acid sequence insertions include, but are not limited to, amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include, but are not limited to, N-terminal methionyl residues.

[0306] In some embodiments, the antigen binding site or a molecule containing the antigen binding site is fused at one terminus to another polypeptide, such as a cytotoxic polypeptide, an enzyme, or a polypeptide that increases the serum half-life of the antibody or antigen binding fragment.

[0307] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue replaced by a different residue in the amino acid sequence of the molecule. The most interesting sites for substitution mutagenesis are usually hypervariable regions, but framework region changes are also contemplated. Examples of conservative substitutions are shown under the heading "preferred substitutions" in Table 1. More substantial changes under the heading "exemplary substitutions" in Table 1, or changes further described below with reference to amino acid classes, can be introduced and the resulting antibodies or antigen-binding fragments screened.

[0308] Table 1. Exemplary amino acid substitutions

[0309]

[0310]

[0311] Substantial modification of the biological properties of antibodies can be achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are generally divided into several categories based on common side chain properties:

[0312] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0313] (2) Neutral hydrophilic: Cys, Ser, Thr;

[0314] (3) Acidic: Asp, Glu;

[0315] (4) Basic: Asn, Gln, His, Lys, Arg;

[0316] (5) Residues that affect chain orientation: Gly, Pro; and

[0317] (6) Aromatic: Trp, Tyr, Phe.

[0318] Non-conservative substitutions may entail exchanging a member of one of these classes for another class.

[0319] Additionally or alternatively, cysteine ​​residues that are not involved in maintaining the proper conformation of the antibody or antigen-binding fragment may be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0320] In some embodiments, substitution variants comprise substitutions within one or more hypervariable region residues of a parent antibody (e.g., a human antibody). Typically, variants with improved biological properties relative to the parent antibody from which they were generated are selected for further development.

[0321] The method for producing such substitution variants relates to affinity maturation using phage display. In one example of such a method, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The resulting antibody variants are displayed in a monovalent manner, such as from filamentous phage particles fused to the M13 gene III product packaged within each particle. The phage display variants are then screened for biological activity (e.g., binding affinity).

[0322] In order 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 in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody or antigen-binding fragment and the antigen. Such contact residues and adjacent residues are candidates for substitution according to the technology described in detail herein. Once such variants are generated, the variant group is screened, and antibodies with excellent properties in one or more related assays can be selected for further development.

[0323] In some embodiments, the original glycosylation pattern of the parent antibody is altered. Such alterations may include deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites not present in the antibody.

[0324] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of a 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 recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any 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 may also be used.

[0325] Addition of glycosylation sites to the antibody can be achieved by altering the amino acid sequence of the antibody or antigen-binding fragment so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0326] Nucleic acid molecules encoding amino acid sequence variants of antibodies or antigen-binding fragments can be prepared by a variety of methods known in the art. These methods include, but are 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 variants or non-variants of earlier preparations of antibodies or their antigen-binding fragments.

[0327] In some embodiments, modifications that increase serum half-life are used. For example, a salvage receptor binding epitope can be incorporated into a molecule containing a disclosed antigen binding site, as described, for example, in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0328] Methods for preparing antigen binding sites and molecules containing antigen binding sites

[0329] The above proteins can be prepared using recombinant DNA techniques well known to those skilled in the art.

[0330] For example, one or more nucleic acid sequences encoding a protein containing a disclosed antigen binding site can be cloned into one or more expression vectors; the expression vector can be stably transfected into a host cell capable of expressing the gene. After transfection, individual 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 amplification and maintenance of expression of proteins containing 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-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography.

[0331] Thus, also provided herein are isolated nucleic acids encoding antigen binding sites and molecules (eg, proteins) containing the antigen binding sites, vectors and host cells comprising the nucleic acids, and recombinant techniques for production.

[0332] In certain embodiments, one or more isolated nucleic acids are provided, which comprise sequences encoding the immunoglobulin heavy chain and / or immunoglobulin light chain variable regions of any antibody disclosed herein, and one or more expression vectors, which express the immunoglobulin heavy chain and / or immunoglobulin light chain variable regions of any antibody disclosed herein. A host cell comprising one or more of the aforementioned expression vectors and / or isolated nucleic acids is also provided.

[0333] In some embodiments, the antigen binding sites and molecules (eg, proteins) containing the antigen binding sites of the present disclosure can be fused to another agent, eg, another therapeutic agent. The construction of fusion proteins is within the ordinary skill in the art.

[0334] Monoclonal antibodies

[0335] Monoclonal antibodies may be made using the hybridoma method first described, or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0336] In the hybridoma method, mice or other suitable host animals, such as hamsters, are immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively or in addition, lymphocytes can be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent (e.g., polyethylene glycol) to form hybridoma cells.

[0337] The hybridoma cells thus prepared are seeded and grown in an appropriate culture medium that preferably contains 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 hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0338] The example of suitable fusion partner includes but is not limited to myeloma cell, and the myeloma cell effectively fuses, supports the stable high-level production of antibody by the selected antibody-producing cell, and is sensitive to the selective culture medium of the unfused parental cell. The example of suitable myeloma cell line includes but is not limited to mouse myeloma cell line, such as those derived from MOPC-21 and MPC-11 mouse tumors, which can be obtained from Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives, such as X63-Ag8-653 cells that can be obtained from American Type Culture Collection (American Type Culture Collection, Rockville, Md. USA). Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies are also described.

[0339] According to the hybridoma method, the culture medium in which the hybridoma cells are grown is then assayed for production of monoclonal antibodies directed against the antigen. For example, the binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0340] The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis.

[0341] Once hybridoma cells producing antibodies with desired specificity, affinity and / or activity have been identified, 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 culture media. In addition, hybridoma cells can be grown in vivo in animals as ascites tumors, for example, by injecting cells intraperitoneally into mice.

[0342] The antibody can be purified by conventional antibody purification procedures, such as affinity chromatography (e.g., using protein A or protein G-Sepharose ) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc., to appropriately separate the monoclonal antibodies secreted by the subclones from the culture medium, ascites or serum.

[0343] DNA encoding monoclonal antibodies can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). Hybridoma cells can be used as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into a host cell that does not originally produce antibody protein, such as an Escherichia coli cell, ape COS cell, Chinese hamster ovary (CHO) cell, or myeloma cell, to obtain the synthesis of monoclonal antibodies in recombinant host cells.

[0344] In certain embodiments, monoclonal antibodies or antibody fragments are isolated from antibody phage libraries. Human antibodies with high affinity (nM range) can be produced, for example, by chain shuffling. Combinatorial infection and in vivo recombination can provide strategies for building very large phage libraries. These techniques are feasible alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0345] The DNA encoding the antibody can be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy and light chain constant domain (CH and CL) sequences for the homologous murine sequences (see, e.g., U.S. Pat. No. 4,816,567), or by fusing the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide).

[0346] Human antibodies and phage display methods

[0347] Human antibodies can be produced by methods known in the art, including methods described herein. For example, transgenic animals (e.g., mice) can be produced that are capable of producing a complete human antibody library in the absence of endogenous immunoglobulin production after immunization. For example, homozygous deletions of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice have been described, resulting in complete inhibition of endogenous antibody production. Transferring human germline immunoglobulin gene arrays to such germline mutant mice will result in the production of human antibodies after antigen attack. See, e.g., U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852.

[0348] Alternatively, phage display technology can be used to produce human antibodies and antibody fragments from immunoglobulin variable (V) domain gene libraries of unimmunized donors in vitro. According to this technology, the antibody V domain gene frame is cloned into the major or minor coat protein gene of filamentous phage (such as M13 or fd), and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain single-stranded DNA copies of phage genomes, the selection based on the functional properties of antibodies also results in the selection of genes encoding antibodies that show those properties. Therefore, phage simulates some properties of B cells. Phage display can be carried out in various forms. Several sources of V gene segments can be used for phage display, such as random combinatorial libraries from V genes, such as libraries derived from immune mouse spleens. V gene libraries from unimmunized human donors can be constructed, and antibodies for antigens (including self-antigens) of different arrays can be separated substantially according to the method described in the art. See, for example, U.S. Patent No. 5,565,332 and No. 5,573,905.

[0349] Human antibodies can also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0350] Competition assays for determining whether an antibody binds to the same epitope as a disclosed antibody or competes for binding with a disclosed antibody 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.

[0351] Typically, competitive assays involve the use of antigens, test antibodies, and reference antibodies that are bound to a solid surface or expressed on a cell surface. The reference antibody is labeled, while the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of the labeled reference antibody bound to a solid surface or cell 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., competitive antibodies) include antibodies that bind to the same epitope or similar (e.g., overlapping) epitopes as the reference antibody, and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody to occur sterically hindered.

[0352] Competition assays can be performed in two orientations to ensure that the presence of the label does not interfere or otherwise inhibit binding. For example, in the first orientation, the reference antibody is labeled and the test antibody is not labeled, and in the second orientation, the test antibody is labeled and the reference antibody is not labeled.

[0353] 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, e.g., by at least 50%, 75%, 90%, 95%, or 99%, as measured in a competitive binding assay.

[0354] If substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody, then it can be determined that the two antibodies bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody, then it can be determined that the two antibodies bind to overlapping epitopes.

[0355] The antibodies disclosed herein can be further optimized (e.g., affinity maturation) to improve biochemical characteristics (including affinity and / or specificity), improve biophysical properties (including aggregation, stability, precipitation and / or non-specific interactions), and / or reduce immunogenicity. Affinity maturation procedures are within the ordinary skill in the art. For example, diversity can be introduced into immunoglobulin heavy chains and / or immunoglobulin light chains by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis and / or site-specific mutagenesis.

[0356] In certain embodiments, the isolated human antibody contains one or more somatic mutations. In these cases, the antibody can be modified to a human germline sequence to optimize the antibody (e.g., by a process known as germlining).

[0357] Typically, 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 was derived. For example, in certain embodiments, an optimized antibody has a higher affinity for an antigen when compared to a parent antibody.

[0358] Pharmaceutical composition

[0359] In certain embodiments, provided molecules containing 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 carrier" refers to any of a variety of physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof.

[0360] In some embodiments, the pharmaceutical composition comprises one or more tonicity agents or stabilizers. Non-limiting examples of such tonicity agents or stabilizers include sugars (e.g., sucrose), polyols (e.g., mannitol or sorbitol), and sodium chloride.

[0361] 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 ions or chelators (e.g., ethylenediaminetetraacetic acid (EDTA)).

[0362] In some embodiments, the pharmaceutical composition comprises one or more auxiliary substances, such as wetting or emulsifying agents, preservatives (eg, benzyl alcohol), or buffers, which can enhance the shelf life and / or effectiveness of the immunoconjugates disclosed herein.

[0363] Pharmaceutical compositions can be provided in any of a variety of forms. These include, for example, liquid, semisolid 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 some forms may depend on the intended mode of administration and therapeutic application.

[0364] In some embodiments, the pharmaceutical composition is in the form of an injectable or infusible solution.

[0365] Pharmaceutical compositions are usually sterile and stable under manufacturing, transportation and storage conditions. Pharmaceutical compositions can be formulated into, for example, solutions, microemulsions, dispersions, liposomes or other ordered structures. In some embodiments, pharmaceutical compositions are formulated into structures that are particularly suitable for high drug concentrations. For example, sterile injectable solutions can be prepared by incorporating therapeutic agents (e.g., immunoconjugates) in the desired amount into a suitable solvent having one or a combination of the components listed herein, optionally followed by sterilization (e.g., filtration sterilization). Typically, dispersions can be prepared by incorporating immunoconjugates into a sterile vehicle containing a basic dispersion medium and other components (e.g., those other components mentioned herein). In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods include vacuum drying and freeze drying, to produce, for example, powders of immunoconjugates and any other desired components from their previously sterile filtered solutions.

[0366] The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining a certain particle size (for example, in the case of a dispersion) and / or by the use of a surfactant. Prolonged absorption of an injectable composition can be achieved, for example, by including in the composition an agent that delays absorption (e.g., monostearate and / or gelatin).

[0367] Treatment

[0368] The present application provides methods for treating diseases or conditions using therapeutic agents (e.g., proteins, such as antibodies or antigen-binding fragments thereof) or pharmaceutical compositions described herein. The methods of treatment disclosed herein generally include administering to a subject in need thereof (e.g., a human subject) a therapeutically effective amount of a disclosed therapeutic agent or pharmaceutical composition containing an antigen-binding site.

[0369] 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 diseases (e.g., primary sclerosing cholangitis and autoimmune pancreatitis), Guillain-Barré 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 thyroiditis, autoimmune adrenal insufficiency, primary hypothyroidism, idiopathic Addison's disease (chronic adrenal insufficiency), type I 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 formulation of the present invention can also be used to prevent or treat graft-versus-host disease (GVHD).

[0370] 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 sensitivities, Alzheimer's disease, asthma, atherosclerotic and occlusive diseases, optionally atherosclerosis, ischemic heart disease, myocardial infarction, stroke, peripheral vascular disease or restenosis in vascular stents, autoimmune diseases, bronchitis, cancer, carditis, cataracts, celiac disease, chronic pain, chronic prostatitis, cirrhosis, colitis, connective tissue diseases, optionally systemic lupus erythematosus, systemic sclerosis, polymyositis, dermatomyositis or Sjögren's syndrome, corneal diseases, Crohn's disease, crystalline arthropathy, optionally gout, pseudogout, calcium pyrophosphate deposition disease, dementia, dermatitis, diabetes, dry eyes, eczema, edema, emphysema, fibromyalgia, gastroenteritis, gingivitis, glomerulonephritis, heart disease, hepatitis, hypertension, hypersensitivity reactions, inflammatory bowel disease, inflammatory disorders (including consequences of trauma or ischemia), insulin resistance, Anti-, Interstitial Cystitis, Iridocyclitis, Iritis, Joint Pain, Arthritis, Rheumatoid Arthritis, Lyme Disease, Metabolic Syndrome (Syndrome X), Multiple Sclerosis, Myositis, Nephritis, Obesity, Eye Disease (Including Uveitis), Osteopenia, Osteoporosis, Parkinson's Disease, Pelvic Inflammatory Disease, Periodontal Disease, Polyarteritis, Polychondritis, Polymyalgia Rheumatica, Psoriasis, Reperfusion Injury, Rheumatoid Arthritis, Rheumatic Diseases, Optional Rheumatoid Arthritis, Osteoarthritis or psoriatic arthritis, rheumatoid arthritis, sarcoidosis, scleroderma, sinusitis, Sjögren's syndrome, spastic colon, spondyloarthropathies, optionally ankylosing spondylitis, reactive arthritis or Reiter's syndrome, systemic candidiasis, tendinitis, transplant rejection, vaginitis, vascular disease including atherosclerotic vascular disease, vasculitis, optionally polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome or vasculitis.

[0371] The therapeutically effective amount can be administered via a single dose or via multiple doses (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten doses). When administered via multiple doses, any of a variety of suitable treatment regimens can be used, including regular administration (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.).

[0372] The dosage regimen (e.g., the amount of each therapeutic agent, the relative duration of therapy, etc.) that is effective in the treatment method may depend on the severity of the disease or disorder and the weight and general condition of the subject. For example, the therapeutically effective amount of a particular composition comprising a therapeutic agent applied to a mammal (e.g., a human) may be determined by a person of ordinary skill in the art taking into account individual differences in age, weight, and condition of the mammal. The therapeutically effective amount and / or the optimal amount may also be determined empirically by a person skilled in the art.

[0373] The molecules containing the antigen binding site or pharmaceutical compositions thereof can be administered by any of a variety of suitable routes, including but not limited to systemic routes, such as parenteral (eg, intravenous or subcutaneous) or enteral routes.

[0374] In many embodiments, administering a therapeutic agent or its pharmaceutical composition containing an antigen binding site according to the provided method results in depletion of cells involved in the disease to be treated. In some embodiments, the cell expresses an antigen that binds to the antigen binding site contained in the therapeutic agent. In some embodiments, the cell is an immune cell, such as 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 cell is an activated cell, such as an antigen-activated T cell or B cell.

[0375] List of Exemplary Embodiments

[0376] The present invention is further described by the following non-limiting exemplary embodiments:

[0377] Embodiment 1. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutic agent comprising one or more antigen binding sites,

[0378] wherein the antigen binding site is capable of binding to an antigen selected from PD-1, PD-L1 or PD-L2 on cells involved in the autoimmune disease,

[0379] and wherein administration of said therapeutic agent comprising one or more antigen binding sites results in depletion of said cells.

[0380] Embodiment 2. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutic agent comprising one or more antigen binding sites,

[0381] wherein the antigen binding site is capable of binding to an antigen selected from the following on cells involved in the autoimmune disease: 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, CD73, CD79A, CD79B, glycoprotein 130 (gp130), BCMA, BAFF receptor (BAFF-R), TACI, integrin α4, integrin β7, FCGR2B, ICOS ligand, CD138, SLAMF7, LILR family members, fibroblast activation protein α (FAP), DLK-1, CD26, TE-7, CD29, PDGRFα, TGFβ receptor, MAS516, CD13, or a combination thereof,

[0382] and wherein administration of said therapeutic agent comprising one or more antigen binding sites results in depletion of said cells.

[0383] Embodiment 3. A method of treating an allergic or inflammatory disease in a subject in need thereof, the method comprising administering to the subject a therapeutic agent comprising one or more antigen binding sites,

[0384] wherein the antigen binding site is capable of binding to an antigen selected from PD-1, PD-L1 or PD-L2 on cells involved in the allergic or inflammatory disease,

[0385] and wherein administration of said therapeutic agent comprising one or more antigen binding sites results in depletion of said cells.

[0386] Embodiment 4. A method of treating an allergic or inflammatory disease in a subject in need thereof, the method comprising administering to the subject a therapeutic agent comprising one or more antigen binding sites,

[0387] The antigen binding site is capable of binding to an antigen selected from the following on cells involved in the allergic or inflammatory disease: 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, CD73, CD79A, CD79B, glycoprotein 130 (gp130), BCMA, BAFF receptor, TACI, integrin α4, integrin β7, FCGR2B, ICOS ligand, CD138, SLAMF7 and LILR family members, fibroblast activation protein alpha (FAP), DLK-1, CD26, TE-7, CD29, PDGRFα, TGFβ receptor, MAS516, CD13, or a combination thereof,

[0388] and wherein administration of said therapeutic agent comprising one or more antigen binding sites results in depletion of said cells.

[0389] Embodiment 5. The method of any one of embodiments 1-4, wherein the cell expresses a marker 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, C D22, CD28, CD38, CD39, CD72, CD73, CD79A, CD79B, gp130, BCMA, BAFF receptor, TACI, integrin α4, integrin β7, FCGR2B, ICOS ligand, CD138, SLAMF7, LILR family members, FAP, DLK-1, CD26, TE-7, CD29, PDGRFα, TGFβ receptor, MAS516, CD13 or a combination thereof.

[0390] Embodiment 6. The method of any one of embodiments 1-5, wherein the cell is an immune cell.

[0391] Embodiment 7. The method of any one of embodiments 1-5, 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.

[0392] Embodiment 8. The method of embodiment 6 or 7, wherein the cell is activated.

[0393] Embodiment 9. The method of any one of embodiments 6-8, wherein the cell is an antigen-activated T cell or B cell.

[0394] Embodiment 10. The method of any one of embodiments 1-9, wherein the antigen binding site is present as a single chain variable fragment (scFv).

[0395] Embodiment 11. The method of any one of embodiments 1-10, wherein the antigen binding site is present as an antibody mimetic (e.g., an affinity body, an affilin, an affimer, an afftin, an alphabody, an anticalin, an avimer, a centyrin, a DARPin, a fynomer, a monoclonal antibody, a nanobody, or a nanoCLAMP).

[0396] Embodiment 12. The method of any one of embodiments 1-11, wherein the therapeutic agent is an antibody or antigen-binding fragment thereof comprising one or more antigen-binding sites.

[0397] Embodiment 13. The method of embodiment 12, wherein the antibody or antigen-binding fragment thereof is Fab, F(ab')2, Fab', scFv, Fv fragment, Fd fragment or diabody.

[0398] 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.

[0399] Embodiment 15. The method of embodiments 1-11, wherein the therapeutic agent is a bispecific antibody.

[0400] Embodiment 16. The method of embodiment 15, wherein the bispecific antibody comprises a first antigen binding site and a second antigen binding site, wherein

[0401] The first antigen binding site is capable of binding to PD-1, PD-L1, PD-L2, CTLA-4, BTLA or KLRG1, and

[0402] The second antigen binding site is capable of binding to a marker of activated T cells (e.g., TIM-3 or LAG-3).

[0403] Embodiment 17. The method of embodiment 15, wherein the bispecific antibody comprises a first antigen binding site and a second antigen binding site, wherein

[0404] The first antigen binding site is capable of binding to PD-1, PD-L1, PD-L2, BCMA or CD38, and

[0405] The second antigen binding site is capable of binding a marker of activated B cells (eg, CD19, CD20, BAFF-R or TACI).

[0406] Embodiment 18. The method of embodiment 15, wherein the bispecific antibody comprises a first antigen binding site and a second antigen binding site, wherein

[0407] The first antigen binding site is capable of binding to FAP or PDGRFα, and

[0408] The second antigen binding site is capable of binding to a marker of activated fibroblasts (eg, FAP, DLK-1, CD26, TE-7, CD29, PDGRFα, TGFβ receptor, MAS516 or CD13).

[0409] Embodiment 19. The method of any one of embodiments 1-18, wherein the therapeutic agent is an antibody or antigen-binding fragment thereof comprising an antibody heavy chain constant region.

[0410] Embodiment 20. The method of embodiment 20, wherein the antibody heavy chain constant region is a human IgG heavy chain constant region.

[0411] Embodiment 21. The method of any one of embodiments 1-20, wherein the therapeutic agent is an antibody or antigen-binding fragment thereof conjugated or fused to a label or a second therapeutic agent.

[0412] Embodiment 22. The method of embodiment 21, wherein the second therapeutic agent is a cytotoxic small molecule or an immunotoxin.

[0413] Embodiment 23. The method of any one of embodiments 1-22, wherein the therapeutic agent is capable of inducing complement dependent cytotoxicity (CDC).

[0414] Embodiment 24. The method of any one of embodiments 1-23, wherein the therapeutic agent is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).

[0415] Embodiment 25. The method of any one of embodiments 1-24, wherein the therapeutic agent is capable of inducing antibody-dependent cellular phagocytosis (ADCP).

[0416] Embodiment 26. The method of any one of embodiments 1-25, wherein the therapeutic agent is afucosylated.

[0417] Embodiment 27. The method of Embodiment 25, wherein the therapeutic agent is at least about 75% afucosylated.

[0418] Embodiment 28. The method of embodiment 26 or 27, wherein the therapeutic agent is capable of depleting immune cells.

[0419] Embodiment 29. The method of any one of embodiments 1-28, wherein the therapeutic agent is capable of agonizing PD-1.

[0420] Embodiment 30. The method of any one of embodiments 1-29, wherein the therapeutic agent comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-91, or a fragment thereof.

[0421] Embodiment 31. The method of any one of embodiments 1-29, wherein the therapeutic agent comprises at least one amino acid sequence selected from any one of SEQ ID NOs: 1-91, or a fragment thereof.

[0422] Embodiment 32. The method of any one of embodiments 1-29, wherein the therapeutic agent comprises at least two amino acid sequences selected from any one of SEQ ID NOs: 1-91, or a fragment thereof.

[0423] Embodiment 33. The method of any one of embodiments 1-29, wherein the therapeutic agent comprises at least one CDR of a sequence selected from any one of the following: SEQ ID NO: 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.

[0424] Embodiment 34. The method of any one of embodiments 1-29, wherein the therapeutic agent comprises at least two CDRs from a sequence selected from any one of the following: SEQ ID NO: 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.

[0425] Embodiment 35. The method of any one of embodiments 1-29, wherein the therapeutic agent comprises at least three CDRs from a sequence selected from any one of the following: SEQ ID NO: 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.

[0426] Embodiment 36. The method of any one of embodiments 1-29, wherein the therapeutic agent comprises

[0427] (a) the complementarity determining region 1 (CDR1), CDR2 and / or CDR3 of the heavy chain variable region (VH) shown in any one of SEQ ID NO: 1, 9, 17, 25, 33, 50, 69 and 84; and / or

[0428] (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.

[0429] Embodiment 37. The method of any one of embodiments 1-29, wherein the therapeutic agent comprises

[0430] (i)(a) VH comprising the following complementarity determining regions:

[0431] CDR-H1, comprising the amino acid sequence: SGGSIRSTRWWS (SEQ ID NO: 2);

[0432] CDR-H2, comprising the amino acid sequence: EIYHSGSTNYNPSLKS (SEQ ID NO: 3); and

[0433] CDR-H3, comprising the amino acid sequence: QDYGDSGDWYFDL (SEQ ID NO: 4); and / or

[0434] (b) VL comprising the following complementarity determining regions:

[0435] CDR-L1, comprising the amino acid sequence: TRSSGSIASNSVQ (SEQ ID NO: 6);

[0436] CDR-L2, comprising the amino acid sequence: EDNQRPS (SEQ ID NO: 7); and

[0437] CDR-L3, comprising the amino acid sequence: QSSDSSAVV (SEQ ID NO: 8); or

[0438] (ii) (a) a VH comprising the following complementarity determining regions:

[0439] CDR-H1, comprising the amino acid sequence: SYGIS (SEQ ID NO: 10);

[0440] CDR-H2, comprising the amino acid sequence: WISAYNGNTNYAQKLQ (SEQ ID NO: 11); and

[0441] CDR-H3 comprising the amino acid sequence: DADYSSGSGY (SEQ ID NO: 12); and / or

[0442] (b) VL comprising the following complementarity determining regions:

[0443] CDR-L1, comprising the amino acid sequence: SGDALPKQYAY (SEQ ID NO: 14);

[0444] CDR-L2, comprising the amino acid sequence: KDTERPS (SEQ ID NO: 15); and

[0445] CDR-L3, comprising the amino acid sequence: QSADNSITYRV (SEQ ID NO: 16); or

[0446] (iii)(a) VH comprising the following complementarity determining regions:

[0447] CDR-H1, comprising the amino acid sequence: SYYIH (SEQ ID NO: 18);

[0448] CDR-H2, comprising the amino acid sequence: IINPRGATISYAQKFQG (SEQ ID NO: 19); and

[0449] CDR-H3 comprising the amino acid sequence: AGIYGFDFDY (SEQ ID NO: 20); and / or

[0450] (b) VL comprising the following complementarity determining regions:

[0451] CDR-L1, comprising the amino acid sequence: TGTSNDVGGYNYVS (SEQ ID NO: 22);

[0452] CDR-L2, comprising the amino acid sequence: DVTNRPS (SEQ ID NO: 23); and

[0453] CDR-L3, comprising the amino acid sequence: SSYTIVTNFEVL (SEQ ID NO: 24); or

[0454] (iv)(a) a VH comprising the following complementarity determining regions:

[0455] CDR-H1, comprising the amino acid sequence: SGAYYWS (SEQ ID NO: 26);

[0456] CDR-H2, comprising the amino acid sequence: YIYYNGNTYYNPSLRS (SEQ ID NO: 27); and

[0457] CDR-H3 comprising the amino acid sequence: ASDYVWGGYRYMDAFDI (SEQ ID NO: 28); and / or

[0458] (b) VL comprising the following complementarity determining regions:

[0459] CDR-L1, comprising the amino acid sequence: SGSNSNIGSNSVN (SEQ ID NO: 30);

[0460] CDR-L2, comprising the amino acid sequence: GNNQRPS (SEQ ID NO: 31); and

[0461] CDR-L3, comprising the amino acid sequence: AAWDDSLNGP (SEQ ID NO: 32); or

[0462] (v)(a) VH comprising the following complementarity determining regions:

[0463] CDR-H1, comprising the amino acid sequence: DYYIN (SEQ ID NO: 34);

[0464] CDR-H2, comprising the amino acid sequence: WIYPGSVNTKYNEKFRG (SEQ ID NO: 35); and

[0465] CDR-H3 comprising the amino acid sequence: YSNWFFDV (SEQ ID NO: 36); and / or

[0466] (b) VL comprising the following complementarity determining regions:

[0467] CDR-L1, comprising the amino acid sequence: RASQEISGYLS (SEQ ID NO: 38);

[0468] CDR-L2, comprising the amino acid sequence: AASTLDS (SEQ ID NO: 39); and

[0469] CDR-L3, comprising the amino acid sequence: LQYASYPYT (SEQ ID NO: 40); or

[0470] (vi)(a) a VH comprising the following complementarity determining regions:

[0471] CDR-H1, comprising the amino acid sequence: DHAIH (SEQ ID NO: 51);

[0472] CDR-H2, comprising the amino acid sequence: YISPGYGDIEYNEKFKG (SEQ ID NO: 52); and

[0473] CDR-H3 comprising the amino acid sequence: TTGY (SEQ ID NO: 53); and / or

[0474] (b) VL comprising the following complementarity determining regions:

[0475] CDR-L1, comprising the amino acid sequence: KSSQSLLNSDGKTYLN (SEQ ID NO: 55);

[0476] CDR-L2, comprising the amino acid sequence: LVSELDS (SEQ ID NO: 56); and

[0477] CDR-L3, comprising the amino acid sequence: WQGTHFPH (SEQ ID NO: 57); or

[0478] (vii)(a) a VH comprising the following complementarity determining regions:

[0479] CDR-H1, comprising the amino acid sequence: SFGMS (SEQ ID NO: 70);

[0480] CDR-H2, comprising the amino acid sequence: TISGGGSNTYYPDSVKG (SEQ ID NO: 71); and

[0481] CDR-H3 comprising the amino acid sequence: IYDVAWFAY (SEQ ID NO: 72); and / or

[0482] (b) VL comprising the following complementarity determining regions:

[0483] CDR-L1, comprising the amino acid sequence: RASESVDNSGISFMN (SEQ ID NO: 74);

[0484] CDR-L2 comprises the amino acid sequence: AASNQGS (SEQ ID NO: 75); and CDR-L3 comprises the amino acid sequence: QQSKEVPWT (SEQ ID NO: 76); or

[0485] (viii)(a) a VH comprising the following complementarity determining regions:

[0486] CDR-H1, comprising the amino acid sequence: SYAMS (SEQ ID NO: 85);

[0487] CDR-H2, comprising the amino acid sequence: TITGGGRNTYYPDSVKG (SEQ ID NO: 86); and

[0488] CDR-H3 comprising the amino acid sequence: QGYDGYTWFAY (SEQ ID NO: 87); and / or

[0489] (b) VL comprising the following complementarity determining regions:

[0490] CDR-L1, comprising the amino acid sequence: RASESVDNSGISFMN (SEQ ID NO: 89);

[0491] CDR-L2, comprising the amino acid sequence: AASNPGS (SEQ ID NO:90); and

[0492] CDR-L3, comprising the amino acid sequence: QQSKEVPWT (SEQ ID NO:91).

[0493] Embodiment 38. The method of any one of embodiments 1-29 and 36-37, wherein the therapeutic agent comprises

[0494] (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

[0495] (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

[0496] (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

[0497] (iv) a VH comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 25; and / or a VL comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 29; or

[0498] (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

[0499] (vi) a VH comprising an amino acid sequence at least 85% identical to the VH of the HC as shown in any one of SEQ ID NOs: 41 or 44; and / or a VL comprising an amino acid sequence at least 85% identical to the VL of the LC as shown in any one of SEQ ID NOs: 46 or 48; or

[0500] (vii) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 50; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 55; or

[0501] (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

[0502] (ix) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 69; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 73; or

[0503] (x) a VH comprising an amino acid sequence at least 85% identical to the VH of the HC set forth in SEQ ID NO: 77; and / or a VL comprising an amino acid sequence at least 85% identical to the VL of the LC set forth in any one of SEQ ID NOs: 80 or 82; or

[0504] (xi) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 84; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 88; or

[0505] (xii) a VH comprising an amino acid sequence at least 85% identical to the VH of the HC shown in SEQ ID NO:92; and / or a VL comprising an amino acid sequence at least 85% identical to the VL of the LC shown in any one of SEQ ID NOs:95 or 97.

[0506] Embodiment 39. The method of any one of embodiments 1-29 and 36-37, wherein the therapeutic agent comprises

[0507] (i) a HC comprising CDR-H1, CDR-H2 and CDR-H3 of the HC shown in any one of SEQ ID NO: 41 or 44 and having at least 85% amino acid sequence identity thereto; and / or

[0508] An LC comprising CDR-L1, CDR-L2 and CDR-L3 of the LC shown in any one of SEQ ID NO: 46 or 48 and having at least 85% amino acid sequence identity thereto; or

[0509] (ii) a HC comprising CDR-H1, CDR-H2 and CDR-H3 of a HC shown in any one of SEQ ID NOs: 58, 61, 63 and 65 and having at least 85% amino acid sequence identity thereto; and / or

[0510] An LC comprising CDR-L1, CDR-L2 and CDR-L3 of the LC shown in SEQ ID NO:67 and having at least 85% amino acid sequence identity with SEQ ID NO:67; or

[0511] (iii) a HC comprising CDR-H1, CDR-H2 and CDR-H3 of the HC shown in SEQ ID NO: 77 and having at least 85% amino acid sequence identity with SEQ ID NO: 77; and / or

[0512] An LC comprising CDR-L1, CDR-L2 and CDR-L3 of the LC shown in any one of SEQ ID NO: 80 or 82 and having at least 85% amino acid sequence identity thereto; or

[0513] (iv) a HC comprising CDR-H1, CDR-H2 and CDR-H3 of the HC shown in SEQ ID NO: 92 and having at least 85% amino acid sequence identity with SEQ ID NO: 92; and / or

[0514] An LC comprising CDR-L1, CDR-L2 and CDR-L3 of the LC shown in any one of SEQ ID NO: 95 or 97 and having at least 85% amino acid sequence identity therewith.

[0515] 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%.

[0516] Embodiment 41. The antigen binding site of any one of embodiments 1-29 and 36-40, wherein the antigen binding site comprises

[0517] (i) a VH comprising the amino acid sequence of SEQ ID NO: 1; and / or

[0518] A VL comprising the amino acid sequence of SEQ ID NO: 5; or

[0519] (ii) a VH comprising the amino acid sequence of SEQ ID NO: 9; and / or

[0520] A VL comprising the amino acid sequence of SEQ ID NO: 13; or

[0521] (iii) a VH comprising the amino acid sequence of SEQ ID NO: 17; and / or

[0522] A VL comprising the amino acid sequence of SEQ ID NO: 21; or

[0523] (iv) a VH comprising the amino acid sequence of SEQ ID NO: 25; and / or

[0524] A VL comprising the amino acid sequence of SEQ ID NO: 29; or

[0525] (v) a VH comprising the amino acid sequence of SEQ ID NO: 33; and / or

[0526] A VL comprising the amino acid sequence of SEQ ID NO: 37; or

[0527] (vi) the VH of the HC shown in any one of SEQ ID NO: 41 or 44; and / or

[0528] The VL of the LC shown in any one of SEQ ID NO:46 or 48; or

[0529] (vii) a VH comprising the amino acid sequence of SEQ ID NO: 50; and / or

[0530] A VL comprising the amino acid sequence of SEQ ID NO: 55; or

[0531] (viii) the VH of the HC shown in any one of SEQ ID NOs: 58, 61, 63 and 65; and / or

[0532] The VL of the LC shown in SEQ ID NO:67; or

[0533] (ix) a VH comprising the amino acid sequence of SEQ ID NO: 69; and / or

[0534] A VL comprising the amino acid sequence of SEQ ID NO: 73; or

[0535] (x) the VH of the HC shown in SEQ ID NO: 77; and / or

[0536] The VL of the LC shown in any one of SEQ ID NO: 80 or 82; or

[0537] (xi) a VH comprising the amino acid sequence of SEQ ID NO: 84; and / or

[0538] A VL comprising the amino acid sequence of SEQ ID NO: 88; or

[0539] (xii) the VH of the HC shown in SEQ ID NO:92; and / or

[0540] VL of the LC shown in any one of SEQ ID NO:95 or 97.

[0541] Embodiment 42. The method of embodiment 12, wherein the antibody or antigen-binding fragment thereof is afucosylated.

[0542] Embodiment 43. The method of embodiment 42, wherein the antibody or antigen-binding fragment thereof is at least about 75% afucosylated.

[0543] Example

[0544] Example 1. Antibody Generation

[0545] Human antibodies targeted to antigens on cells involved in autoimmune, allergic, or inflammatory diseases are produced by methods known in the art.

[0546] In some embodiments, transgenic mice that cannot express functional endogenous immunoglobulins but express human immunoglobulin genes are used. Specifically, human heavy chain and light chain immunoglobulin gene complexes are introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions are introduced into mouse embryonic stem cells. The introduction of human immunoglobulin loci by homologous recombination occurs alone or simultaneously with the non-functionalization of mouse heavy chain and light chain immunoglobulin genes. Specifically, homozygous deletions in the JH region prevent endogenous antibody production. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then cultivated to produce homozygous offspring expressing human antibodies. With selected antigens, such as all or part of an antigen (e.g., PD-1, PD-L1, or PD-L2), transgenic mice are immunized in a normal manner. Conventional hybridoma technology can be used to obtain monoclonal antibodies against antigens from immunized transgenic mice. The human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation, followed by class switching and somatic mutations.

[0547] In some other embodiments, antibodies are generated by screening yeast display libraries.

[0548] Example 2. Binding of Antibodies to PD-1

[0549] Targeted binding of PD-1 antibodies to PD-1 expressed on activated T cells was assessed by flow cytometry.

[0550] T cells were activated by anti-CD3, anti-CD28 and anti-W6 / 32 treatment. Activation resulted in increased expression of PD-1 on the surface of T cells. After 3 days of activation, the cells were resuspended in FACS blocking buffer (PBS + 10% normal human serum) for 30 minutes. After washing, the cells were incubated with PD-1 test antibodies in FACS buffer (PBS + 0.5% bovine serum albumin + 0.09% sodium azide) and incubated at 4 ° C for 30 minutes. The cells were then washed and incubated with PE-labeled anti-human secondary antibodies at 4 ° C for 30 minutes in the dark. After washing, BD FACSSymphony A3 instrument (BD Biosciences) was used for cell fluorescence analysis. Cells were sorted by scattering properties, and cells were separated for single cell analysis using side scatter width to side scatter area. The binding of antibodies to cells was quantified by measuring the fluorescence emission of 561nm excitation wavelength using a 586 / 15nm filter. Data analysis was performed using FlowJo software. All tested PD-1 antibodies were able to bind to T cells expressing PD-1 (see Table 2).

[0551] Table 2. Binding of PD-1 antibodies to PD-1 expressed on T cells

[0552] Antibody Binds to PD-1 on activated T cells 3H4 + 2.3A9 + 20B3.1 + 1B8 + 1-17 + 1-28 + 1-33 + 1-35 + 1.A10 + 1G7 + 7G3 +

[0553] Example 3. Analysis of cross-reactivity and nonspecific binding of PD-1 antibodies by ELISA

[0554] An ELISA-based assay was used to evaluate cross-reactivity and nonspecific binding of PD-1 antibodies to human PD-1, cynomolgus monkey PD-1, and PD-1 family members.

[0555] The His-tagged extracellular domains of human PD-1, cynomolgus PD-1, and PD-1 family members ICOS, CD28, and CTLA-4 extracellular domains were fixed on 96-well ELISA plates. The plates were washed with PBST (phosphate-buffered saline + 0.05% Tween 20) and blocked with 1% BSA in PBS for 1 hour. After washing with PBST, the test PD-1 antibody was added to the coated plate and incubated at room temperature for 2 hours. The plates were washed and incubated with HRP-conjugated goat anti-human IgG secondary antibodies at room temperature for 1 hour. After washing, antibody binding was detected colorimetrically by adding 3,3',5,5'-tetramethylbenzidine (TMB), incubated at room temperature for 10-20 minutes, and then 2.5N sulfuric acid stop solution was added. The absorbance at 450nm was measured on a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism.

[0556] Exemplary results are depicted in Table 3. All PD-1 antibodies tested were able to bind to human and cynomolgus PD-1, but did not cross-react with PD-1 family members ICOS, CD28, or CTLA-4.

[0557] Table 3. Cross-reactivity of PD-1 antibodies

[0558]

[0559] Example 4. Analysis of the blocking effect of PD-1 antibody on PD-1 / PD-L1 by ELISA

[0560] An ELISA-based assay was used to assess the ability of the disclosed PD-1 antibodies to block the PD-1 / PD-L1 interaction.

[0561] In brief, human PD-L1 was fixed on a 96-well ELISA plate. The plate was washed and blocked with blocking buffer for 1.5 hours at 37°C. The plate was washed and the standard curve of the test PD-1 antibody, isotype control or PD-1 neutralizing antibody was 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 last wash, antibody binding was detected colorimetrically by adding substrate solution and incubating for 20 minutes under dark conditions, followed by adding stop solution. The absorbance at 450nm was measured on a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism.

[0562] The percentage of blocking for the tested PD-1 antibodies was determined by interpolating the values ​​obtained for each sample with the standard control curve (see Figure 1), and are summarized in Table 4. The tested antibodies showed a range of activities 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, while PD-1 antibodies 1-17 and 2.3A9 had no blocking effect.

[0563] Table 4. Blockade of PD-1 / PD-L1 interaction by PD-1 antibodies

[0564] Antibody Blocking percentage 1G7 97 7G3 96 20B3.1 94 1.8A10 92 1B8 90 1-28 87 1-33 68 1-35 58 3H4 46 1-17 7 2.3A9 0

[0565] Example 5. Inhibition of T cells in mixed lymphocyte reaction by PD-1 antibody

[0566] The ability of the disclosed PD-1 antibodies to agonize PD-1 and inhibit T cells in a mixed lymphocyte reaction (MLR) was evaluated.

[0567] 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 allogeneic stimulation because allogeneic T cells from one donor produce IFN-γ upon recognition of foreign MHC from the other donor.

[0568] PD-1 antibody or isotype control was added to the PBMC co-culture. PBMC alone (without antibody) was used as a control. The plates were incubated for 72 hours and then the IFN-γ levels present in the supernatant were measured by ELISA assay.

[0569] In brief, the IFN-γ capture antibody was fixed on a 96-well ELISA plate. The plate was washed with PBST and blocked with 1% BSA in PBS for 1 hour. After washing with PBST, the supernatant and IFN-γ standard curve were added to the coated plate and incubated at room temperature for 2 hours. The plate was washed and incubated at room temperature for 2 hours with the detection antibody conjugated with HRP. After washing, antibody binding was detected by colorimetry by adding TMB, and incubated at room temperature for 10-20 minutes, and then 2.5N sulfuric acid stop solution was added. The absorbance at 450nm was measured on a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism.

[0570] Exemplary results are 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, and some of them showed stronger inhibitory effects on IFN-γ secretion compared with the PD-1 agonist antibody rosnilimab.

[0571] Example 6. Inhibition of T cell activation by effector function modified PD-1 antibodies

[0572] The ability of effector function modified PD-1 antibodies to inhibit T cell activation was evaluated. A commercially available assay was used, comprising an antigen presenting cell line (APC) 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.

[0573] Raji-APC cells modified to express the FcγRII receptor were incubated with PD-1 antibodies with normal human IgG1 Fc or effector-enhanced IgG1 Fc, isotype controls, or culture medium at 37°C, 5% CO2 for 30 minutes. The effector-enhanced IgG1 Fc contained mutations S239D / H268D ("X3" mutation) or E233D / G237D / H268D / P271G / A330R ("V12" mutation) to increase binding to FcγRII. After incubation, Jurkat-Lucia TCR-hPD-1 cells were added to the culture, and the plates were incubated for another 5.5 hours at 37°C, 5% CO2. The cells were centrifuged at 400g for 5 minutes, and the supernatant was collected for further analysis.

[0574] The supernatant was transferred to a 96-well white plate. 50 μL of QUANTI-Luc4 reagent was added to the plate and luminescence was immediately read using a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism and the relative inhibition percentage was calculated compared to the signal obtained from the control (isotype control or untreated with antibody).

[0575] Exemplary results are in Figure 3 As shown in . X3 or V12 mutations enhance the ability of some PD-1 antibodies (eg, 3H4) to inhibit Jurkat cell activation compared to PD-1 antibodies with normal IgG1 Fc and isotype controls.

[0576] The 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 presented in Figure 4 As shown in , 3H4 antibodies containing X3 or V12 mutations significantly inhibited IL-2 secretion compared to 3H4 with normal IgG1 Fc and controls (isotype control or no antibody treatment).

[0577] Example 7. Inhibition of T cell proliferation by effector function modified PD-1 antibodies

[0578] The ability of effector function-modified PD-1 antibodies to inhibit T cell proliferation was evaluated.

[0579] PBMCs were stained with CellTrace Violet (CTV) dye at 37°C for 15 minutes. After washing twice in cell growth medium, PBMCs were plated on 96-well plates coated with anti-CD3 and anti-CD28, which were added to all wells to stimulate T cells. PD-1 antibodies, isotype controls, or PD-L1 with normal human IgG1 Fc or IgG1 Fc with enhanced effects containing X3 mutations were added to the corresponding cells, and the plates were incubated at 37°C, 5% CO2 for 72 hours. The supernatant was collected for ELISA analysis. The remaining cells on the plate were washed, resuspended in FACS blocking buffer on ice for 15 minutes, and incubated with APC-labeled anti-CD4 and BV785-labeled anti-CD8 antibodies. After washing, cell fluorescence analysis was performed using a BD FACS Symphony A3 instrument (BD Biosciences). The CTV fluorescence intensity of CD4 and CD8 T cell subsets was measured. Data analysis was performed using FlowJo software, and the percentage of proliferating cells for each T cell subtype was quantified.

[0580] Exemplary results are in Figure 5A and Figure 5B The PD-1 antibodies tested (including the X3 mutation that increases binding to FcγRII) inhibited CD4 and CD8 T cell proliferation.

[0581] Example 8 Inhibition of IFN-γ Secretion by Effector Function Modified PD-1 Antibodies The ability of effector function modified PD-1 antibodies to inhibit IFN-γ secretion was evaluated.

[0582] Supernatants of 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.

[0583] Exemplary results are in Figure 6 The tested PD-1 antibodies (including the X3 mutation that increases binding to FcγRII) significantly inhibited IFN-γ secretion more than the control and PD-1 antibodies (agonists or antagonists) with normal IgG1 Fc.

[0584] Sequence Appendix

[0585] In some sequences, the variable regions are shown in bold and the CDRs are underlined.

[0586] Table 5. Sequences of exemplary antigen binding sites capable of binding to PD-1

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597] Table 6. Other sequences

[0598]

[0599]

[0600]

[0601]

[0602] Incorporated by Reference

[0603] Unless otherwise indicated to the contrary, the entire disclosure of each patent document and scientific article cited herein is incorporated by reference for all purposes.

[0604] Equivalent solutions / other implementation plans

[0605] 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. Such equivalents are intended to be encompassed by the appended claims.

Claims

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

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

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

4. The antibody or antigen-binding fragment thereof of claims 1-3, wherein the antibody or antigen-binding fragment thereof has enhanced effector function.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC).

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

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

8. The antibody or antigen-binding fragment thereof according to 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 according to 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 cell is activated.

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

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

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

15. The antibody or antigen-binding fragment thereof of claim 13, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence that generally differs by no more than two amino acid residues from the following sequence: (a) are 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) are 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.

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

91.

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

40.

18. The antibody or antigen-binding fragment thereof according to any one of claims 14 to 17, comprising (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 with 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 with the amino acid sequence of SEQ ID NO:96 or 98.

19. The antibody or antigen-binding fragment thereof of claim 18, comprising: (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 the amino acid sequence of SEQ ID NO: 9 and a VL region having the amino acid sequence of SEQ ID NO: 13; (c) a VH region having the amino acid sequence of SEQ ID NO: 17 and a VL region having the amino acid sequence of SEQ ID NO: 21; (d) a VH region having the amino acid sequence of SEQ ID NO: 25 and a VL region having the amino acid sequence of SEQ ID NO: 29; (e) a VH region having the amino acid sequence of SEQ ID NO: 33 and a VL region having the amino acid sequence of SEQ ID NO: 37; (f) 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; (g) a VH region having the amino acid sequence of SEQ ID NO: 50 and a VL region having the amino acid sequence of SEQ ID NO: 54; (h) a VH region having the amino acid sequence of SEQ ID NO: 56, 62, 64 or 66 and a VL region having the amino acid sequence of SEQ ID NO: 68; (i) a VH region having the amino acid sequence of SEQ ID NO: 69 and a VL region having the amino acid sequence of SEQ ID NO: 73; (j) a VH region having the amino acid sequence of SEQ ID NO: 78 and a VL region having the 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.

20. The antibody or antigen-binding fragment thereof according to claim 19, comprising 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.

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

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

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

24. The antibody or antigen-binding fragment thereof of any one of claims 1-23, wherein the antibody or antigen-binding fragment thereof comprises 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. 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. The antibody or antigen-binding fragment thereof according to 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 or antigen-binding fragment thereof of claim 29, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region comprising one 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, wherein the constant regions are numbered according to the EU index.

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, wherein the numbering of the constant region is according to the EU index.

32. 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, wherein the numbering of the constant region is according to the EU index.

33. The antibody or antigen-binding fragment thereof of any one of claims 1 to 32, wherein the antibody or antigen-binding fragment thereof is produced in a cell line, the cell line (a) having a knockout of α-1,6-fucosyltransferase (Fut8); or (b) Overexpression of β1,4-N-acetylglucosaminyltransferase III (GnT-III) and optionally overexpression of Golgi μ-mannosidase II (ManII).

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 1-35, 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 1-36, 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 1-37, wherein the antibody or antigen-binding fragment is capable of inhibiting T cell proliferation.

39. 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.

40. The antibody or antigen-binding fragment thereof of any one of claims 1-39, wherein the antibody or antigen-binding fragment thereof is conjugated to an agent.

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 as described in any one of claims 1-42.

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

45. The composition of claim 44, wherein substantially none of the N-glycosidically 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. An expression vector comprising the polynucleotide of claim 46.

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

49. The host cell of claim 48, wherein the host cell is a mammalian or insect cell.

50. The host cell of claim 48 or 49, wherein the host cell (a) comprises a Fut8 knockout; and / or (b) overexpresses GnT-III and optionally overexpresses ManII.

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. A method of treating a disease or condition in a subject in need thereof, the method 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. The method of claim 52, wherein the disease or disorder is an autoimmune disease.

54. The method of claim 52, wherein the disease or condition is allergy.

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

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