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

Abstract

Disclosed herein are compositions and methods for treating autoimmune, allergic, and / or inflammatory diseases in a subject in need thereof.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 619,442, filed January 10, 2024, the entire contents of which are incorporated herein by reference for all purposes. Background Technology

[0003] The immune system is tightly controlled by various immune cells, as well as co-stimulatory and co-inhibitory ligands and receptors. Inflammation is a normal physiological defense against pathogen infection and tissue damage, and it usually resolves quickly. However, in many chronic diseases, the inflammatory response persists and can 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 their own proteins. Allergies are also caused by a dysregulation of the immune system, leading to the recognition of harmless non-self antigens. Mast cells and IgE antibodies play a central role in allergic reactions, while rogue T cells or B cells are the main participants in autoimmunity.

[0004] There is still a need for therapeutic agents and treatments that fine-tune the activity of immune cells and the immune system, which can be used alone or in combination for autoimmune, allergic and inflammatory diseases. Summary of the Invention

[0005] This 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 comprises 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, an HCDR2 having the amino acid sequence of SEQ ID NO: 98, and an HCDR3 having the amino acid sequence of SEQ ID NO: 4.

[0007] In some embodiments, the antibody or antigen-binding fragment further includes a light chain variable (VL) region, which comprises: (a) Light chain complementarity-determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence of SEQ ID NO: 8; or (b) LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 103, and LCDR3 having the amino acid sequence of SEQ ID NO: 104; or (c) LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 7 and LCDR3 having the amino acid sequence of SEQ ID NO: 109.

[0008] On the other hand, an antibody or antigen-binding fragment thereof capable of binding to PD-1 is provided, wherein the antibody or antigen-binding fragment thereof comprises a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 12-19, 97, and 100; and / or a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 23-26, 102, 106, and 108.

[0009] In some embodiments, the antibody or antigen-binding fragment comprises: a VH region comprising an amino acid sequence selected from SEQ ID NO: 12-19, 97 and 100; and a VL region comprising an amino acid sequence selected from SEQ ID NO: 23-26, 102, 106 and 108.

[0010] On the other hand, an antibody or antigen-binding fragment thereof capable of binding to PD-1 is provided, wherein the antibody or antigen-binding fragment comprises: (a) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 26; or (b) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 18, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 21; or (c) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 18, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 26; or (d) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 19, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 26; or (e) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 15, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or (f) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 15, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 25; or (g) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 16, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or (h) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 16, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 25; or (i) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 17, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or (j) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 17, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 25. (k) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 97, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 102; or (l) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 100, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 106; or (m) includes a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 97, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 108.

[0011] In some implementations, the antibody or antigen-binding fragment comprises: (a) The VH region containing the amino acid sequence of SEQ ID NO: 9 and the VL region containing the amino acid sequence of SEQ ID NO: 26; or (b) The VH region containing the amino acid sequence of SEQ ID NO: 18 and the VL region containing the amino acid sequence of SEQ ID NO: 21; or (c) The VH region containing the amino acid sequence of SEQ ID NO: 18 and the VL region containing the amino acid sequence of SEQ ID NO: 26; or (d) The VH region containing the amino acid sequence of SEQ ID NO: 19 and the VL region containing the amino acid sequence of SEQ ID NO: 26; or (e) The VH region containing the amino acid sequence of SEQ ID NO: 15 and the VL region containing the amino acid sequence of SEQ ID NO: 24; or (f) The VH region containing the amino acid sequence of SEQ ID NO: 15 and the VL region containing the amino acid sequence of SEQ ID NO: 25; or (g) A VH region containing the amino acid sequence of SEQ ID NO: 16 and a VL region containing the amino acid sequence of SEQ ID NO: 24; or (h) The VH region containing the amino acid sequence of SEQ ID NO: 16 and the VL region containing the amino acid sequence of SEQ ID NO: 25; or (i) The VH region containing the amino acid sequence of SEQ ID NO: 17 and the VL region containing the amino acid sequence of SEQ ID NO: 24; or (j) The VH region containing the amino acid sequence of SEQ ID NO: 17 and the VL region containing the amino acid sequence of SEQ ID NO: 25; (k) A VH region containing the amino acid sequence of SEQ ID NO: 97 and a VL region containing the amino acid sequence of SEQ ID NO: 102; or (l) A VH region containing the amino acid sequence of SEQ ID NO: 100 and a VL region containing the amino acid sequence of SEQ ID NO: 106; or (m) The VH region containing the amino acid sequence of SEQ ID NO: 97 and the VL region containing the amino acid sequence of SEQ ID NO: 108.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 16, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing the amino acid sequence of SEQ ID NO: 16 and a VL region containing the amino acid sequence of SEQ ID NO: 24.

[0013] In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 17, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing the amino acid sequence of SEQ ID NO: 17 and a VL region containing the amino acid sequence of SEQ ID NO: 25.

[0014] In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 97, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 102. In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing the amino acid sequence of SEQ ID NO: 97 and a VL region containing the amino acid sequence of SEQ ID NO: 102.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 100, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 106. In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing the amino acid sequence of SEQ ID NO: 100 and a VL region containing the amino acid sequence of SEQ ID NO: 106.

[0016] In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 97, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 108. In some embodiments, the antibody or antigen-binding fragment comprises: a VH region containing the amino acid sequence of SEQ ID NO: 97 and a VL region containing the amino acid sequence of SEQ ID NO: 108.

[0017] 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 a biantibody. In some embodiments, the antigen-binding fragment is scFv.

[0018] In some implementations, the antibody or its antigen-binding fragment contains a constant region of the antibody heavy chain.

[0019] In some embodiments, the antibody heavy chain constant region is the human IgG heavy chain constant region. In some embodiments, the antibody heavy chain constant region is the 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: 111. In some embodiments, the antibody heavy chain constant region has the amino acid sequence of SEQ ID NO: 111.

[0020] In some embodiments, the antibody heavy chain constant region contains one or more mutations that enhance effector function. In some embodiments, the antibody heavy chain constant region contains one or more mutations that enhance binding to inhibitory Fc receptors (e.g., FcγRII).

[0021] In some implementations, the antibody heavy chain constant region is the human IgG1 heavy chain constant region containing one of the following mutations: (a) E233D; (b) G236D or G236A; (c) G237D; (d) P238D; (e) S239D; (f) S267E; (g) H268D or H268F; (h) P271G; (i) S324T; (j) L328Y, L328F, or L328E; (k) A330R; (l) I332E; (m) E345K, E345Y, E345Q, or E345R; (n) E430G, E430S, E430F, or E430T; (o) S440Y; (p) G236D and H268D; (q) S239D and H268D; (r) S239D, H268D, L328Y and I332E; (s) P238D and L328E; (t) G237D, P271G and A330R; (u) G237D, H268D, P271G and A330R; (v) S267E and L328F; (w) S239D and S267E; (x) G236D and S276E; (y) E233D, G237D, H268D, P271G and A330R; (z) S239D, H268D and E430G; or (aa) S239D, H268D and E345K, or combinations thereof, wherein the constant regions are numbered according to the EU index. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing one of the following mutations: (a) G236D or G236A; (b) S239D; (c) H268D or H268F; (d) L328Y; (e) S324T; (f) I332E; (g) E345K, E345Y, E345Q, or E345R; (h) E430G, E430S, E430F, or E430T; (i) S440Y; (j) S239D and H268D; or combinations thereof, wherein the constant region is numbered according to the EU index. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing the following mutations: S239D and H268D, and optionally E430G or E345K, wherein the constant region is numbered according to the EU index. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing the following mutations: S239D, H268D, and E430G, wherein the constant regions are numbered according to the EU index. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing the following mutations: S239D, H268D, and E345K, wherein the constant regions are numbered according to the EU index.In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing the following mutations: E233D, G237D, H268D, P271G, and A330R, wherein the constant regions are numbered according to the EU index. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing the following mutations: S267E and L328F, wherein the constant regions are numbered according to the EU index. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing the following mutations: E430G or E345K, wherein the constant regions are numbered according to the EU index. In some embodiments, the antibody heavy chain constant region contains 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: 125. In some embodiments, the antibody heavy chain constant region has the amino acid sequence of SEQ ID NO: 125.

[0022] On the other hand, antibodies or antigen-binding fragments thereof capable of binding to PD-1 are provided, wherein... The antibody or its antigen-binding fragment comprises a human IgG1 heavy chain constant region containing a mutation selected from E345K, E345Y, E345Q, E345R, E430G, E430S, E430F, E430T, and S440Y, wherein the constant region is numbered according to the EU index; and The antibody or its antigen-binding fragment contains (a) (i) Heavy chain variable (VH) region, which contains: Heavy chain complementarity-determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids. HCDR2 having the amino acid sequence of SEQ ID NO: 3 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) A light chain variable (VL) region, comprising: The light chain complementarity-determining region 1 (LCDR1) has the amino acid sequence of SEQ ID NO: 6 or a sequence that differs from it by one or two amino acids. LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 8 or a sequence that differs from it by one or two amino acids; (b) (i) The heavy chain variable (VH) region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 28 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 29 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 30 or a sequence differing from it by one or two amino acids; and (ii) A light chain variable (VL) region, comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 32 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 33 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 34 or a sequence that differs from it by one or two amino acids; (c) (i) The VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 36 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 37 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 38 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 40 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 41 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 42 or a sequence that differs from it by one or two amino acids; (d) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 44 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 45 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 46 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 48 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 49 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 50 or a sequence that differs from it by one or two amino acids; (e) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 52 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 53 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 54 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 56 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 57 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 58 or a sequence that differs from it by one or two amino acids; (f) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 10 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 8 or a sequence that differs from it by one or two amino acids; (g) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 60 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 61 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 62 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 64 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 65 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 66 or a sequence that differs from it by one or two amino acids; (h) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 60 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 68 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 62 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 64 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 65 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 66 or a sequence that differs from it by one or two amino acids; (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 73 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 74 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 75 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 77 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 78 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 79 or a sequence that differs from it by one or two amino acids; (j) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 73 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 81 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 75 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 77 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 78 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 79 or a sequence that differs from it by one or two amino acids; (k) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 86 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 87 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 90 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 91 or a sequence that differs from it by one or two amino acids; (l) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 93 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 87 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 90 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 91 or a sequence that differs from it by one or two amino acids; (m) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 98 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 103 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 104 or a sequence that differs from it by one or two amino acids; (n) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 98 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 8 or a sequence that differs from it by one or two amino acids; or (o) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 98 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 109 or a sequence that differs from it by one or two amino acids.

[0023] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise amino acid sequences that differ from the following sequences by no more than two amino acid residues overall: (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) are SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 respectively; (c) are SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 respectively; (d) are SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (e) are SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 respectively; (f) are SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively; (g) are SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66 respectively; (h) are SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66 respectively; (i) are SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively; (j) are SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively; (k) are 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; (l) are SEQ ID NO: 85, SEQ ID NO: 93, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, respectively; (m) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 103 and SEQ ID NO: 104 respectively; (n) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or (o) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 109, respectively.

[0024] In some implementations, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following amino acid sequences: (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) are SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 respectively; (c) are SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 respectively; (d) are SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (e) are SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 respectively; (f) are SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively; (g) are SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66 respectively; (h) are SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66 respectively; (i) are SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively; (j) are SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively; (k) are 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; (l) are SEQ ID NO: 85, SEQ ID NO: 93, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, respectively; (m) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 103 and SEQ ID NO: 104 respectively; (n) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or (o) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 109, respectively.

[0025] In some implementations, the antibody or its antigen-binding fragment comprises: (a) 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: 1, 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: 5; (b) 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: 27, 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: 31; (c) 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: 35, 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: 39; (d) 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: 43, 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: 47; (e) 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: 51, 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: 55; (f) 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: 9 or 11, 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: 20 or 21; (g) 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: 59, 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: 63; (h) 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: 67, 128, 69, or 60, 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: 71; (i) 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: 72, 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: 76; (j) 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: 80, 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: 82 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 with 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 with the amino acid sequence of SEQ ID NO: 88; (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: 92, 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: 94 or 95; (m) 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: 97, 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: 102 or 108; or (n) 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: 100, 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: 106.

[0026] In some implementations, the antibody or its antigen-binding fragment comprises: (a) The VH region having the amino acid sequence of SEQ ID NO: 1 and the VL region having the amino acid sequence of SEQ ID NO: 27; (b) The VH region having the amino acid sequence of SEQ ID NO: 1 and the VL region having the amino acid sequence of SEQ ID NO: 31; (c) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 35 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 39; (d) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 43 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 47; (e) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 51 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 55; (f) The VH region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 9 or 11 and the VL region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 20 or 21; (g) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 59 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 63; (h) The VH region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 67, 128, 69 or 60 and the VL region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 71; (i) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 72 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 76; (j) The VH region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 80 and the VL region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 82 or 83; (k) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 84 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 88; (l) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 92 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 94 or 95; (m) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 97 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 102 or 108; or (m) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 100 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 106.

[0027] In some implementations, the constant region of the IgG1 heavy chain contains E430G or E345K, wherein the constant region is numbered according to the EU index.

[0028] In some implementations, the IgG1 heavy chain constant region also includes one of the following mutations: (a) E233D; (b) G236D or G236A; (c) G237D; (d) P238D; (e) S239D; (f) S267E; (g) H268D or H268F; (h) P271G; (i) S324T; (j) L328Y, L328F, or L328E; (k) A330R; (l) I332E; (m) G236D and H268D; (n) S239D and H268D; (o) S239D, H268D, L328Y, and I332E; (p) P238D and L328E; (q) G237D, P271G, and A330R; (r) G237D, H268D, P271G, and A330R; (s) S267E and L328F; (t) S239D and S267E; (u) G236D and S276E; or (v) E233D, G237D, H268D, P271G, and A330R, or combinations thereof, wherein the constant regions are numbered according to the EU index. In some embodiments, the IgG1 heavy chain constant region contains the following mutations: S239D and H268D, wherein the constant regions are numbered according to the EU index. In some embodiments, the IgG1 heavy chain constant region contains the following mutations: E233D, G237D, H268D, P271G, and A330R, wherein the constant regions are numbered according to the EU index. In some embodiments, the IgG1 heavy chain constant region contains the following mutations: S267E and L328F, wherein the constant regions are numbered according to the EU index. In some embodiments, the IgG1 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: 125. In some embodiments, the IgG1 heavy chain constant region has the amino acid sequence of SEQ ID NO: 125.

[0029] In some embodiments, the antibody or its antigen-binding fragment is a PD-1 agonist. In some embodiments, the antibody or its antigen-binding fragment is capable of binding to human PD-1. In some embodiments, the antibody or its antigen-binding fragment is capable of binding to cynomolgus monkey PD-1. In some embodiments, the antibody or antigen-binding fragment is capable of blocking PD-1 / PD-L1 interaction.

[0030] In some embodiments, the antibody or its antigen-binding fragment has enhanced binding to the Fc receptor. In some embodiments, the Fc receptor is an inhibitory Fc receptor (e.g., FcγRII). In some embodiments, the Fc receptor is an activating Fc receptor (e.g., FcγRI or FcγRIII).

[0031] In some embodiments, the antibody or its antigen-binding fragment has enhanced effector function. In some embodiments, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function is antibody-dependent phagocytosis (ADCP). In some embodiments, the effector function is complement-dependent cytotoxicity (CDC).

[0032] In some embodiments, the antibody or its antigen-binding fragment can deplete cells. In some embodiments, the cells are PD-1-expressing cells. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells, B cells, macrophages, natural killer (NK) cells, dendritic cells (DCs), monocytes, neutrophils, fibroblasts, or epithelial cells. In some embodiments, the immune cells are T cells or B cells. In some embodiments, the immune cells are activated. In some embodiments, the immune cells are antigen-activated T cells or B cells.

[0033] In some embodiments, at least about 60%, about 75%, or about 90% of the antibody or its antigen-binding fragment is unfucosylated. In some embodiments, the antibody or its antigen-binding fragment is generated in a cell line, said cell line... (a) Possessing α-1,6-fucosyltransferase (Fut8) knockout; or (b) Overexpression of β1,4-N-acetylglucosamine transferase III (GnT-III) and optionally overexpression of Golgi μ-mannosidase II (ManII).

[0034] In some implementations, the aforementioned antibody or its antigen-binding fragment can inhibit T cell activation.

[0035] In some implementations, the aforementioned antibody or its antigen-binding fragment can inhibit T cell proliferation.

[0036] In some implementations, the antibody or its antigen-binding fragment can inhibit IFN-γ secretion.

[0037] In some embodiments, the antibody or its antigen-binding fragment 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.

[0038] On the other hand, compositions comprising the antibody or antigen-binding fragment thereof described herein are provided. In some embodiments, the antibody or antigen-binding fragment comprises an Fc region and an N-glycoside-linked carbohydrate chain linked to the Fc region, optionally wherein less than 50% of the N-glycoside-linked carbohydrate chain contains fucose residues. In some embodiments, substantially no N-glycoside-linked carbohydrate chain contains fucose residues.

[0039] On the other hand, a polynucleotide encoding the antibody or its antigen 5 binding fragment described herein is provided.

[0040] On the other hand, expression vectors containing the polynucleotides described herein are provided.

[0041] On the other hand, a host cell comprising the polynucleotide or expression vector described herein is provided. In some embodiments, the host cell is a mammalian or insect cell. In some embodiments, the host cell (a) comprises Fut8 knockout; and / or (b) overexpresses GnT-III and optionally overexpresses ManII.

[0042] On the other hand, pharmaceutical compositions comprising the antibody or its antigen-binding fragment described herein and a pharmaceutically acceptable carrier are provided.

[0043] On the other hand, methods for treating a disease or condition in a subject in need are provided, the methods comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof 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. Attached Figure Description

[0044] Figure 1 The activation of the Fcγ receptor FcγRIIIA by the publicly disclosed PD-1 antibody was described.

[0045] Figure 2 The inhibition of activation of Jurkat reporter T cells by PD-1 antibodies with wild-type IgG1 Fc or modified Fc containing mutations that increase binding to FcγRII and optionally induce antibody hexamer formation was described.

[0046] Figure 3A and Figure 3B This shows the effect in Raji cells ( Figure 3A ) and CHO-K1 cells ( Figure 3BComplement-dependent cytotoxicity of PD-1 antibodies containing wild-type IgG1 Fc or modified Fc with mutations that increase binding to FcγRII and optionally induce antibody hexamer formation.

[0047] Figure 4A and Figure 4B High levels of PD-1 antibody expression were shown in PD-1 antibodies with wild-type IgG1 Fc or modified Fc containing mutations that increase binding to FcγRII and optionally induce antibody hexamer formation. Figure 4A ) and moderate ( Figure 4B The depletion of CD8 T cells with PD-1 on the cell surface.

[0048] Figure 5 The binding of humanized PD-1 antibody to PD-1 on the cell surface was depicted.

[0049] Figure 6 The cross-reactivity of PD-1 antibody to human and cynomolgus monkey PD-1 was described.

[0050] Figures 7A-7C This demonstrates that PD-1 antibodies can interact with the Fcγ receptor CD16a ( Figure 7A CD32a Figure 7B ) and CD64 Figure 7C ) Activate signal transduction.

[0051] Figure 8 The inhibition of activation of Jurkat reporter T cells by a PD-1 antibody containing a mutant Fc that increases binding to FcγRII and induces antibody hexamer formation was described.

[0052] Figure 9 The antibody-dependent phagocytosis of PD-1 positive target cells induced by PD-1 antibody was described.

[0053] Figure 10 This study depicts antibody-dependent cell-mediated cytotoxicity mediated by natural killer cells in PD-1-positive target cells induced by PD-1 antibodies.

[0054] Figure 11 This study demonstrated that PD-1 antibody inhibited IFN-γ secretion from PBMCs after re-exposure to tetanus toxoid antigen. Detailed Implementation

[0055] definition Unless the context is inappropriate, the terms “a” and “an” as used herein mean “one or more” and include plurals.

[0056] As used herein, when referring to values, the terms “about,” “approximately,” and “equivalent to” refer to values ​​similar to the reference value in the context of the reference value. Generally, those skilled in the art who are familiar with the context will understand the extent of variation covered by “about,” “approximately,” and “equivalent to” in that context. For example, in some embodiments, the terms “about,” “approximately,” and “equivalent to” may cover a range of values ​​of 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 mentioned value.

[0057] As used herein, "agonist" or "stimulatory" when referring 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 the stimulation or activation of the target, or enhances, increases, promotes, induces or prolongs one or more functions or biological activities of the target.

[0058] As used herein, the terms “antagonist,” “antagonistic,” “neutralizing,” or “blocking,” when referring to an antigen-binding site or a molecule containing an antigen-binding site, mean that the binding of the antigen-binding site or molecule to its target results in the inhibition of at least some of the biological activity of the target.

[0059] As used herein, "antibody" refers to a polypeptide, or a fragment thereof, whose amino acid sequence comprises an immunoglobulin and a fragment thereof that specifically binds to a specified antigen. Antibodies according to the invention can be of any type (e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4). Those skilled in the art will understand that the characteristic sequence or portion of an antibody may include amino acids found in one or more regions of the antibody (e.g., variable regions, hypervariable regions, constant regions, heavy chains, light chains, and combinations thereof). Furthermore, those skilled in the art will understand 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.

[0060] As used herein, “antibody mimic” means any molecule capable of mimicking the ability of an antibody to bind to an antigen, but is not limited to antibody structures. Examples of antibody mimics include, but are not limited to, affibody, affilin, affimer, afftin, alphabody, anticalin, avimer, centyrin, DARPin, fynomer, monobody, nanobody, and nanoCLAMP.

[0061] An antibody's "antigen-binding fragment" contains a portion of the complete antibody that is still capable of binding antigens. In some embodiments, papain digestion of the antibody produces two identical antigen-binding fragments, called the "Fab" fragment, and a residual "Fc" fragment, the names reflecting its ease of crystallization. The Fab fragment consists of the entire light chain and a variable region (V) domain of the heavy chain. H ) and the first constant structural domain of a heavy chain (C H 1) Composition. Each Fab fragment is monovalent in terms of antigen binding, meaning it has a single antigen-binding site. In some embodiments, treating the antibody with pepsin produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking the antigen. The Fab' fragment differs from the Fab fragment in that it has a C... H Several residues are added to the carboxyl terminus of the 1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH indicates a Fab' where the (multiple) cysteine ​​residues in the constant domain have free thiol groups. The F(ab')2 antibody fragment is initially generated as a Fab' fragment pair with hinge cysteine ​​residues between them. Other chemical conjugations of antibody fragments are also known.

[0062] As used herein, the term "antigen binding site" refers to any molecule or any part of a molecule capable of binding 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"). Three highly divergent segments within the V region of the heavy and light chains are called "hypervariable regions," which lie between more conserved flanking segments called "frame regions" or "FRs." Thus, the term "FR" refers to the naturally occurring amino acid sequence between and adjacent to the hypervariable regions of an immunoglobulin. In human antibodies, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged opposite each other in three-dimensional space to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface that binds the antigen, and the three hypervariable regions of the heavy and light chains are called "complementarity-determining regions" or "CDRs." In some animals, such as camels and cartilaginous fish, the antigen binding site may be formed by a single antibody chain, thus providing a "single-domain antibody." The antigen binding site can also be an antibody mimic or be present in an antibody mimic.

[0063] CDRs can be determined using the methods described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977), 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 typically include overlaps or subsets of amino acid residues when compared with each other. In some embodiments, the term "CDR" is the CDR as defined in 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 some embodiments, the term "CDR" is the CDR as defined in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of Protein of immunological interest. (1991). In some embodiments, the heavy chain CDR and light chain CDR of the antibody are defined using different conventions. For example, in some embodiments, the heavy chain CDR is defined according to MacCallum (above), and the light chain CDR is defined according to Kabat (above). CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, while CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0064] As used herein, the “binding affinity” of the molecules disclosed herein (e.g., antigen-binding sites) for 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).

[0065] As used herein, "bispecific" refers to a molecule's ability to specifically bind to at least two different targets. Typically, a bispecific molecule contains two antigen-binding sites, each specific for a different target. In some embodiments, a bispecific molecule can bind to two targets simultaneously.

[0066] As used in this article, "detectable affinity" refers to the ability to bind to a given target with a certain affinity constant, usually through K... D or EC 50 Measurements show that the affinity constant is at most about 10. -5 M or lower (lower K) D or EC 50 The value reflects a better binding affinity. Lower affinity cannot be measured by commonly used methods such as ELISA (enzyme-linked immunosorbent assay) and is therefore of secondary significance.

[0067] 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. This antigen-binding site is called the complementation site and is composed of the six complementation determinants of the antibody. A single antigen may have more than one epitope. Epitopes can be conformational or linear. Conformational epitopes consist of spatially aligned amino acids from different segments of a linear polypeptide chain. Linear epitopes consist of adjacent amino acid residues in the polypeptide chain.

[0068] The “Fc” fragment contains the carboxyl-terminal portions of two heavy chains linked together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by the Fc receptor (FcR) in certain cell types.

[0069] The term "fragment" for an antigen (e.g., PD-1, PD-L1, or PD-L2) refers to a truncated N-terminus and / or C-terminus of the antigen, or a protein domain. In some embodiments, the fragment of the antigen retains the ability of the full-length antigen to be recognized and / or bound by the antigen-binding fragment of this disclosure.

[0070] As used herein, "fucosylated" or "fucosylated" refers to the presence of fucose residues within the oligosaccharide attached to the antibody peptide backbone. Specifically, fucosylated antibodies contain α(1,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue of one or two N-linked oligosaccharides attached to the antibody's Fc region. "Non-fucosylated," "unfucosylated," or "fucose-deficient" antibodies refer to glycosylated antibody variants containing an Fc region in which the carbohydrate structure attached to the Fc region has reduced or no fucose. In some embodiments, non-fucosylated or fucose-deficient antibodies have reduced fucose content relative to the same antibody produced in the cell line. In some embodiments, antibodies with reduced or no fucose exhibit improved ADCC function.

[0071] "Fucosylation degree" is the percentage of fucosylated oligosaccharides relative to all oligosaccharides, which can be identified by methods known in the art, such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) in antibody compositions treated with N-glycosidase F. In a "fully fucosylated antibody" composition, substantially all oligosaccharides contain fucose residues, i.e., are fucosylated. In some embodiments, the fully fucosylated antibody composition has at least about 90% fucosylation degree. Conversely, in a "fully unfucosylated antibody" composition, substantially no oligosaccharides are fucosylated. In some embodiments, the fully unfucosylated antibody composition has less than about 10% fucosylation degree. In a "partially fucosylated antibody" composition, only a portion of the oligosaccharides contain fucose. Individual antibodies in such compositions may be absent from 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 both N-linked oligosaccharides in the Fc region, provided that the composition does not contain individual antibodies lacking fucose residues in substantially all N-linked oligosaccharides in the Fc region, nor does it contain individual antibodies containing fucose residues in substantially all two N-linked oligosaccharides in the Fc region. In one embodiment, the composition of partially fucosylated antibodies has a fucosylation degree 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 generating antibodies with altered, reduced, or eliminated fucosylation are known in the art (see, for example, WO 2023 / 044390 A1).

[0072] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, for example, the individual antibodies constituting that population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific (targeting a single antigenic site). Furthermore, each monoclonal antibody targets a single determinant on an antigen, unlike conventional (polyclonal) antibody products which typically comprise different antibodies targeting different determinants (epitopes). The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be produced by hybridoma methods or by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries.

[0073] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier, thereby making the composition particularly suitable for diagnostic or therapeutic use in vivo or in vitro.

[0074] As used herein, the term "pharmaceutically acceptable salt" means any pharmaceutically acceptable salt (e.g., acid or base) of the compound described in this application that, upon administration to a subject, provides the compound described in this application or its active metabolites or residues. As is known to those skilled in the art, the "salt" of the compound described in this application may be derived from inorganic or organic acids and bases.

[0075] 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, although not pharmaceutically acceptable on their own, can be used to prepare salts that can be used as intermediates to obtain the compounds described in this application and their pharmaceutically acceptable acid addition salts.

[0076] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and the formula NW4. + Compounds (where W is C) 1-4 Alkyl groups, etc.

[0077] Exemplary salts include, but are not limited to: acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, disaccharides, dodecyl sulfates, ethanesulfonates, fumarates, glucono-heptahydrates, glycerophosphates, hemisulfates, heptahydrates, hexanoates, hydrochlorides, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, oxalates, dihydroxynaphthalate, pectates, persulfates, phenylpropionates, picrates, neopentanoates, propionates, succinates, tartrates, thiocyanates, toluenesulfonates, undecanoates, etc. Other examples of salts include anions of the compounds described in this application with suitable cations such as Na+. + NH4 + and NW4 + (where W is C) 1-4 Alkyl groups and other complexes.

[0078] For therapeutic use, salts of the compounds described in this application are considered pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be used, for example, to prepare or purify pharmaceutically acceptable compounds.

[0079] As used herein, the phrase “reference level” generally refers to a level considered “normal” for comparative purposes, such as the level of an appropriate control. For example, in the context of enhanced antibody effector function, “reference level” could refer to the effector function level of an antibody containing the wild-type constant region (Fc). As another example, in the context of target binding, “reference level” could refer to the binding affinity level of the target’s natural ligand (e.g., by K…). D or EC 50 (Value definition). The reference level can be determined simultaneously or predetermined, for example, if it is known or inferred from past observations.

[0080] 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, suidae, canines, felines, etc.), and more preferably include humans.

[0081] As used herein, the phrases “therapeutic effective amount” and “effective amount” are used interchangeably and refer to the amount that effectively achieves the desired therapeutic outcome at the necessary dose and for the required period of time. Therapeutic effective amounts can vary due to a variety of factors, such as disease type (e.g., cancer), disease state, age, sex, and / or individual weight, and the ability of the immunoconjugate (or its pharmaceutical composition) to elicit the desired response in an individual. Effective amounts can also be the amount at which any toxic or adverse effects of the immunoconjugate or its pharmaceutical composition are offset by the beneficial therapeutic effects.

[0082] As used herein, “treating” a disease or “treatment” of a disease (e.g., the disease described herein, such as cancer) is a method of achieving a beneficial or desired outcome (e.g., clinical outcome). Beneficial or desired outcomes may include, but are not limited to, reducing or improving one or more symptoms or conditions; reducing the severity of a disease, condition, or disorder; stabilizing (i.e., not worsening) the state of a disease, condition, or disorder; preventing the spread of a disease, condition, or disorder (e.g., primary cancer and / or secondary metastasis); delaying or slowing the progression of a disease, condition, or disorder; improving or reducing a disease, condition, or disorder; and partial or complete remission, whether detectable or undetectable.

[0083] antigen binding site In one aspect, this application provides antigen-binding sites 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.

[0084] In some embodiments, the antigen-binding site is capable of binding to antigens on cells involved in autoimmune, allergic, or inflammatory diseases. In some embodiments, the cells are immune cells, such as T cells, B cells, mast cells, macrophages, natural killer (NK) cells, dendritic cells (DCs), monocytes, neutrophils, fibroblasts, or epithelial cells. In some embodiments, the cells are activated cells, such as antigen-activated T cells or B cells. Examples of such antigens include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA-4, C-type lectin (CLEC), Siglec, members of the tumor necrosis factor (TNF) receptor superfamily, 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, and 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 combinations thereof. In some embodiments, the antigen is PD-1, PD-L1, or PD-L2. In some embodiments, the antigen is PD-1.

[0085] In some implementations, the provided antigen-binding site exists as an antibody mimic (e.g., affinity, affilin, affimer, afftin, alphabody, anticalin, avimer, centyrin, DARPin, fynomer, monoclonal antibody, nanobody, or nanoCLAMP).

[0086] In some embodiments, the provided antigen binding site 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 including CDR-H1, CDR-H2, and CDR-H3, and / or a light chain variable domain including CDR-L1, CDR-L2, and CDR-L3. In some embodiments, the antigen binding site is present as a single-stranded variable fragment (scFv).

[0087] In some embodiments, the provided antigen-binding site is capable of binding PD-1. In some embodiments, the antigen-binding site is present as an antibody. In some embodiments, an antigen-binding site is provided that is a variant of an antigen-binding site capable of binding PD-1 and present as an antibody as described herein, wherein such an antigen-binding site comprises an amino acid sequence having 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% identity with the amino acid sequence contained in the antigen-binding site capable of binding PD-1 and present as an antibody as described herein.

[0088] In some embodiments, the provided antigen-binding site capable of binding to PD-1 includes 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.

[0089] In some embodiments, the provided antigen-binding sites capable of binding to PD-1 comprise 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 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 some embodiments, the antigen-binding sites comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from those described in Table 5.

[0090] In some embodiments, an antigen binding site is provided, which is a variant of the PD-1-binding antigen binding site described herein, wherein such an antigen binding site has a CDR sequence that differs from the CDR sequence described in Table 5 by no more than two amino acid residues (e.g., two or one amino acid residues). In some embodiments, an antigen binding site is provided, which is a variant of the PD-1-binding antigen binding site described herein, wherein such an antigen binding site has a set of six CDRs whose sequences generally differ from the 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 residues). In some embodiments, an antigen binding site is provided, which is a variant of the PD-1-binding antigen binding site described herein, wherein such an antigen binding site has a set of six CDRs whose sequences generally differ from the sequences of anti-PD-1 having the set of VH and VL selected from Table 5 by no more than two amino acid residues (e.g., two or one amino acid residues).

[0091] In some embodiments, the provided antigen-binding site capable of binding PD-1 comprises 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 that is an antigen-binding site capable of binding PD-1 as described herein, for example, 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 having 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% identity with the amino acid sequence of the VH described in Table 5; and (2) a light chain domain comprising an amino acid sequence having 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% identity with the amino acid sequence of the VL described in Table 5.

[0092] In some embodiments, the antigen-binding site described herein is derived from mouse 3H4. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 1; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antigen binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the VH and VL sequences of SEQ ID NO: 1 and 5, respectively, determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art. In some embodiments, VH comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO: 2, 3, and 4, respectively. In some embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO: 6, 7, and 8, respectively. In some embodiments, the antigen binding site comprises: (a) VH, which comprises CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequences of SEQ ID NO: 2, 3 and 4, respectively; and (b) VL, which comprises CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequences of SEQ ID NO: 6, 7 and 8, respectively.

[0093] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4. For example, in some embodiments, the antigen-binding site described herein comprises: VH, wherein the VH comprises an amino acid sequence having 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%) identity with an amino acid sequence selected from SEQ ID NO: 9, 11-19, 97, and 100; and / or VL, wherein the VL comprises an amino acid sequence having 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%) identity with an amino acid sequence selected from SEQ ID NO: 20-26, 102, 106, and 108. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 102.In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 106. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the antigen binding site comprises CDR-H1, CDR-H2, and CDR-H3 of the VH sequence selected from SEQ ID NO: 9, 11-19, 97, and 100, 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 of the VL sequence selected from SEQ ID NO: 20-26, 102, 106, and 108, determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0094] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-2. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 9; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 21.

[0095] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-4. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 11; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 21.

[0096] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-5. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 9; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 26.

[0097] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-6. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 18; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 21.

[0098] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-7. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 18; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 26.

[0099] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-8. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 19; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 26.

[0100] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-V4L3. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 15; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 24.

[0101] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-V4L4. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 15; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 25.

[0102] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-V5L3. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 16; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 24.

[0103] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-V5L4. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 16; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 25.

[0104] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-V6L3. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 17; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 24.

[0105] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-V6L4. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 17; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 25.

[0106] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-879. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 97; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 102.

[0107] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-892. For example, in some embodiments, the antigen-binding site described herein comprises: VH, wherein the VH comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 100; and VL, wherein the VL comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 106.

[0108] In some embodiments, the antigen-binding site described herein is derived from humanized 3H4-887. For example, in some embodiments, the antigen-binding site described herein comprises: VH, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 97; and VL, which comprises an amino acid sequence having 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%) identity with the amino acid sequence of SEQ ID NO: 108.

[0109] In each of the above embodiments, it is envisioned that the VH and / or VL sequences binding to PD-1 may contain amino acid alterations (e.g., substitutions, deletions, or additions of at least 1, 2, 3, 4, 5, or 10 amino acids) in the frame regions of VH and / or VL without affecting their ability to bind to PD-1. For example, it is envisioned that the VH and VL sequences binding to PD-1 may contain cysteine ​​heterodimerization mutations, thereby facilitating the formation of disulfide bridges between VH and VL to form scFv.

[0110] In some embodiments, the antigen-binding sites disclosed herein bind human PD-1 or its extracellular region. In some embodiments, the antigen-binding sites disclosed herein bind PD-1 present on a membrane (e.g., the plasma membrane of a cell).

[0111] 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 for binding to human PD-1.

[0112] 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.

[0113] In some embodiments, the antigen-binding sites disclosed herein can inhibit the binding of PD-L1 to PD-1. In some embodiments, the antigen-binding sites disclosed herein can activate PD-1.

[0114] 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).

[0115] In some embodiments, the antigen binding sites disclosed herein can reduce the secretion of inflammatory cytokines. In some embodiments, the antigen binding sites disclosed herein can reduce IFN-γ secretion.

[0116] Molecules containing antigen-binding sites This document also provides molecules containing publicly disclosed antigen-binding sites. Such molecules may be, but are not limited to, antibodies or antigen-binding fragments thereof, antibody fragments, nanobodies, antibody mimics, etc. In some embodiments, the molecule containing the publicly disclosed antigen-binding site is a therapeutic agent, i.e., the molecule confers a therapeutic benefit. Therefore, in some embodiments, therapeutic agents containing publicly disclosed antigen-binding sites are provided. In some embodiments, the therapeutic agent is capable of inducing complement-dependent cytotoxicity (CDC). In some embodiments, the therapeutic agent is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the therapeutic agent is capable of inducing antibody-dependent phagocytosis (ADCP). In some embodiments, the therapeutic agent is capable of depleting immune cells (e.g., T cells, B cells, mast cells, macrophages, NK cells, dendritic cells, monocytes, neutrophils, fibroblasts, or epithelial cells).

[0117] 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 fragments, or biantibodies (dimers 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 (unfucosylated antibody). In some embodiments, the antibody is fucosylated by up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, or up to about 1%. In some embodiments, the antibody is a fucosylated 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% fucosylated.

[0118] In some implementations, the antibody or its antigen-binding fragment 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, CD19, CD20, CD22, CD28, CD38, CD39, CD72, CD73, CD79A, CD79B, and 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 combinations 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 activate PD-1.

[0119] In some embodiments, a therapeutic agent comprising two or more antigen-binding sites is provided. In some embodiments, the therapeutic agent is bispecific.

[0120] Various forms and uses of bispecific molecules are known in the art. The bispecific molecule according to the invention is not limited to any particular bispecific form or method of producing it.

[0121] In some embodiments, the bispecific molecule may be a bispecific antibody or a fragment or derivative thereof, including but not limited to: (i) a single antibody having two arms, each arm containing a different antigen-binding site; (ii) a bispecific scFv, for example, two scFvs linked via a peptide linker; (iii) a bivariate domain antibody (DVD-Ig) wherein each light chain and heavy chain contains two variable domains tandemly linked by short peptide bonds; (iv) a bispecific (Fab′)2 fragment; (v) a biantibody; (vi) a tandem biantibody (TandAb), a fusion of two biantibodies; and (vii) a "docking and locking (DNL)-Fab3", a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (viii) an IgG-like molecule having an engineered Fc to force heterodimerization; (ix) a recombinant IgG-like dual-targeting molecule wherein each side of the molecule contains Fab fragments or partial Fab fragments of at least two different antibodies; (x) an IgG fusion molecule wherein a full-length IgG antibody is fused to an additional Fab fragment or partial Fab fragment; (xi) an Fc fusion molecule; (v) Fab fusion molecules; and (vi) antibodies and heavy chain antibodies (e.g., domain antibodies, nanobodies) based on scFv and biantibodies, wherein different scFv, biantibodies 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.

[0122] Examples of IgG-like molecules with engineered Fc include, but are not limited to, triomab, knot-into-hole (kih) molecules (e.g., kih IgG with a common light chain), CrossMAb, orthoFab IgG molecules, electrostatically matched 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, dual-antibodies, and mAbs. 2Examples of IgG fusion molecules include, but are not limited to, DVD-Ig molecules and IgG-scFv. Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusions, SCORPION molecules, and Fc-DART molecules. Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 molecules, DNL molecules, and Fab-Fv molecules. Examples of antibodies and domain-specific antibodies based on scFv and bispecific antibodies include, but are not limited to, bispecific T-cell conjugates (BiTE) molecules, tandem bispecific antibodies (TandAb), dual-affinity retargeting technology (DART) molecules, single-chain bispecific antibodies, TCR-like antibodies (AIT, Receptor Logics), human serum albumin scfv fusions, COMBODY molecules, dual-targeting nanobodies, and dual-targeting heavy chain domain-only antibodies.

[0123] In some embodiments, the molecule containing the disclosed antigen-binding site further comprises an antibody constant region or a fragment or variant thereof. In some embodiments, the antibody constant region may be a heavy chain constant region of, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, and IgG4 (e.g., human). In some embodiments, the antibody constant region or a fragment or variant thereof has a heavy chain constant region that is, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, or IgE, preferably, an amino acid sequence of, for example, human IgG1, IgG2, IgG3, or IgG4 that has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity. In some embodiments, the antibody constant region may be a light chain constant region selected from, for example, κ or λ (e.g., human). The constant region can be altered, for example, by mutation, to modify the properties of the antibody (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, 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 does not recruit effector cells or fix complement. In yet another embodiment, the antibody has a reduced or non-existent ability to bind to the Fc receptor. For example, it is an isotype or subtype, fragment, or other mutant that does not support binding to the Fc receptor, for example, it has a mutagenic or missing Fc receptor-binding region.

[0124] In some implementations, the constant region contains one or more mutations that enhance the effect.

[0125] In some implementations, the constant region contains one or more mutations that enhance binding to FcγRII (CD32). For example, in some implementations, the constant region comprises the heavy chain constant region of IgG1 containing one or more of the following mutations, based on EU index numbers: (a) E233D or G236A; (b) G236D; (c) G237D; (d) P238D; (e) S239D; (f) S267E; (g) H268D; (h) P271G; (i) S324T; (j) L328Y, L328F, or L328E; (k) A330R; (l) I332E; (m) E345K, E345Y, E345Q, or E345R; (n) E430G, E430S, E430F, or E430T; (o) S440Y; (p) G236D and H268D; (q) S239D and H268D; (r) S239D, H268D, L328Y and I332E; (s) P238D and L328E; (t) G237D, P271G and A330R; (u) G237D, H268D, P271G and A330R; (v) S267E and L328F; (w) S239D and S267E; (x) G236D and S276E; (y) E233D, G237D, H268D, P271G and A330R, or combinations thereof. In some implementations, the constant region comprises the heavy chain constant region of IgG1 containing one or more of the following mutations, based on EU index numbers: (a) G236D or G236A; (b) S239D; (c) H268D or H268F; (d) L328Y; (e) S324T; (f) I332E; (g) E345K, E345Y, E345Q, or E345R; (h) E430G, E430S, E430F, or E430T; (i) S440Y, or combinations thereof. In some embodiments, the constant region comprises a heavy chain constant region of IgG1 containing one or more of the following mutations, based on EU index numbers: (a) G236D or G236A; (b) S239D; (c) H268D or H268F; (d) S324T; (e) L328Y; (f) I332E; (g) E345K, E345Y, E345Q, or E345R; (h) E430G, E430S, E430F, or E430T; (i) S440Y, or combinations thereof. In some embodiments, the constant region comprises an IgG1 heavy chain constant region containing S239D and H268D mutations, based on EU index numbers. In some embodiments, the constant region comprises an IgG1 heavy chain constant region containing S239D, H268D, and E430G mutations, based on EU index numbers.In some embodiments, the constant region includes an IgG1 heavy chain constant region containing S239D, H268D, and E345K mutations, based on EU index numbers. In some embodiments, the constant region includes an IgG1 heavy chain constant region containing E233D, G237D, H268D, P271G, and A330R mutations, based on EU index numbers. In some embodiments, the constant region includes an IgG1 heavy chain constant region containing E430G or E345K mutations, based on EU index numbers. In some embodiments, the constant region includes an IgG1 heavy chain constant region containing S267E and L328F mutations, based on EU index numbers.

[0126] In some implementations, the constant region comprises the heavy chain constant region of IgG1 containing one or more of the following mutations, based on EU index numbers: (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.

[0127] In some implementations, the constant region contains one or more mutations that reduce the effector function. For example, in some embodiments, the constant region comprises the heavy chain constant region of IgG1 containing one or more of the following mutations, based on EU index numbers: (a) L234A and / or L235A; (b) A327G, A330S, and / or P331S; (c) E233P, L234V, L235A, and / or G236del; (d) E233P, L234V, and / or L235A; (e) E233P, L234V, L235A, G236del, A327G, A330S, and / or P331S; (f) E233P, L234V, L235A, A327G, A330S, and / or P331S; (g) N297A; (h) N297G; (i) N297Q; (j) (k) L242C, N297C and / or K334C; (l) A287C, N297G and / or L306C; (m) R292C, N297G and / or V302C; (n) 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 some implementations, the constant region comprises the heavy chain constant region of IgG2 containing one or more of the following mutations, based on EU index numbers: (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 some implementations, the constant region comprises the heavy chain constant region of IgG4 containing one or more of the following mutations, based on EU index numbers: (a) E233P, F234V, L235A and / or G236del; (b) E233P, F234V and / or L235A; (c) S228P and / or L235E; or (d) S228P and / or L235A.

[0128] In some embodiments, the antigen-binding site is linked to the antibody constant region or a fragment or variant thereof. In some embodiments, the antigen-binding site is linked to the IgG constant region, including the hinge, CH2, and CH3 domains (with or without the CH1 domain).

[0129] In some embodiments, the molecule containing the disclosed antigen-binding site also includes a non-fucosylated Fc region. In some embodiments, the Fc region is a non-fucosylated IgG1 Fc region.

[0130] In some embodiments, the bispecific molecule may be a fusion protein containing one or more antibody mimics. In some embodiments, the bispecific molecule may be a fusion protein containing one or more antibody mimics and one or more antibodies or antigen-binding fragments thereof.

[0131] amino acid sequence modification Amino acid sequence modifications of antigen-binding sites and molecules containing antigen-binding sites (e.g., antibodies or antigen-binding fragments thereof) disclosed herein are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of antibodies or antigen-binding fragments. Amino acid sequence variants can be prepared, for example, by introducing appropriate nucleotide changes into the nucleic acid sequence encoding the antigen-binding site or a molecule containing the antigen-binding site, or through 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 made, as long as the antigen-binding site or the molecule containing the antigen-binding site retains the desired properties and / or function. In some embodiments, amino acid changes are introduced to alter post-translational processes, such as changing the number or position of glycosylation sites.

[0132] A useful method for identifying certain residues or regions as preferred mutagenic sites is called "alanine scan 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 a neutral or negatively charged amino acid (preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions that exhibit functional sensitivity to the substitution are then modified by introducing additional or different variants at or to the substitution site. Therefore, while the sites where amino acid sequence variations are introduced are predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scans or random mutagenesis can be performed on the target codon or region, and expressed variants can be screened to obtain the desired activity.

[0133] 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 intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include, but are not limited to, N-terminal methionine residues.

[0134] In some implementations, the antigen-binding site or a molecule containing the antigen-binding site is fused at one end to another polypeptide, such as a cytotoxic polypeptide, an enzyme, or a polypeptide that increases the serum half-life of an antibody or antigen-binding fragment.

[0135] Another type of variant is the amino acid substitution variant. These variants have at least one amino acid residue in the molecule's amino acid sequence replaced by a different residue. The sites of most interest in substitution mutagenesis are usually hypervariable regions, but framework region alterations are also considered. Examples of conserved substitutions are shown under the heading "Preferred Substitutions" in Table 1. More substantial variations can be introduced under the heading "Exemplary Substitutions" in Table 1, or variations further described below with reference to the amino acid categories, and the resulting antibody or antigen-binding fragments can be screened.

[0136] Table 1. Exemplary amino acid substitutions

[0137] Substantial modifications to antibody biological properties can be achieved through substitutions that significantly differ in their role in maintaining (a) the structure of the polypeptide backbone in the substituted region, such as sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the volume of the side chains. Naturally occurring residues are typically categorized based on common side chain properties: (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral and hydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Alkaline: Asn, Gln, His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Fang ethnic group: Trp, Tyr, Phe.

[0138] Non-conservative replacement may require replacing members of one of these categories with members of another category.

[0139] Alternatively, cysteine ​​residues that do not participate in maintaining the proper conformation of the antibody or antigen-binding fragment can be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent undesirable cross-linking. Conversely, cysteine ​​bonds can be added to antibodies to improve their stability (especially when the antibody is an antibody fragment such as an Fv fragment).

[0140] In some embodiments, the substitution variant comprises a substitution within one or more hypervariable residues of the parent antibody (e.g., a human antibody). Typically, resulting variants that have improved biological properties relative to the parent antibody that produced them are selected for further development.

[0141] The method for generating such substitution variants involves phage display affinity maturation. In one instance of this method, several hypervariable sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The resulting antibody variants are displayed monovalently, for example, from filamentous phage particles fused to the M13 gene III product packaged within each particle. The biological activity (e.g., binding affinity) of the phage display variants is then screened.

[0142] To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Optionally or additionally, analyzing the crystal structure of the antigen-antibody complex to identify contact sites between the antibody or antigen-binding fragment and the antigen may be beneficial. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the variant set is screened, and antibodies exhibiting superior properties in one or more relevant assays can be selected for further development.

[0143] In some implementations, the original glycosylation pattern of the parent antibody is altered. Such alterations may include the deletion of one or more carbohydrate moieties found in the antibody, and / or the addition of one or more glycosylation sites not present in the antibody.

[0144] Antibody glycosylation is typically N-linked or O-linked. N-linking refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in the 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 hydroxy amino acid (most commonly serine or threonine), although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0145] Glycosylation sites can be added to antibodies by altering the amino acid sequence of the antibody or antigen-binding fragment to include one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by adding or substituting one or more serine or threonine residues into the original antibody sequence (for O-linked glycosylation sites).

[0146] 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, preparation by isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of variant or non-variant forms of antibodies or their antigen-binding fragments prepared earlier.

[0147] In some embodiments, modifications that increase serum half-life are used. For example, a salvage receptor-binding epitope may be incorporated into a molecule containing a disclosed antigen-binding site, as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor-binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0148] Methods for preparing antigen-binding sites and molecules containing antigen-binding sites The proteins described above can be prepared using recombinant DNA techniques well known to those skilled in the art.

[0149] 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; said expression vectors can be stably transfected into host cells 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 scale-up and maintenance of expression of proteins containing the disclosed antigen-binding sites. Proteins can be isolated and purified using methods known in the art, such as centrifugation, deep filtration, cell lysis, homogenization, freeze-thaw cycles, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0150] Therefore, this article also provides isolated nucleic acids encoding antigen-binding sites and molecules (e.g., proteins) containing antigen-binding sites, vectors and host cells containing said nucleic acids, and recombinant technologies for production.

[0151] In some embodiments, one or more isolated nucleic acids are provided, comprising sequences encoding variable regions of the immunoglobulin heavy chain and / or immunoglobulin light chain of any antibody disclosed herein, and one or more expression vectors expressing the variable regions of the immunoglobulin heavy chain and / or immunoglobulin light chain of any antibody disclosed herein. Host cells comprising one or more of the aforementioned expression vectors and / or isolated nucleic acids are also provided.

[0152] In some embodiments, the antigen-binding site of this disclosure and the molecule containing the antigen-binding site (e.g., a protein) can be fused with another agent, such as another therapeutic agent. The construction of the fusion protein is within the scope of ordinary techniques in the art.

[0153] Monoclonal antibodies Monoclonal antibodies can be prepared using the hybridoma method described first, or by a recombinant DNA method (US Patent No. 4,816,567).

[0154] In the hybridoma approach, mice or other suitable host animals, such as hamsters, are immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to proteins used for immunization. Optionally or additionally, lymphocytes can be immunized in vitro. Following immunization, the lymphocytes are isolated and then fused with a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells.

[0155] The hybridoma cells thus prepared are seeded into a suitable culture medium and grown therein, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells (also known as fusion couples). For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0156] Examples of suitable fusion couples include, but are not limited to, myeloma cells that fuse efficiently, support stable high levels of antibody production from selected antibody-producing cells, and are sensitive to selective media containing unfused parental cells. Examples of suitable myeloma cell lines include, but are not limited to, mouse myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and SP-2 and its derivatives, such as X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md., USA. Human myeloma and mouse-human heterologous myeloma cell lines for the production of human monoclonal antibodies are also described.

[0157] According to the hybridoma method, the production of monoclonal antibodies against the antigen in the culture medium in which the hybridoma cells are grown is then measured. For example, the binding specificity of the monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0158] The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis.

[0159] Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned, for example, through a limiting dilution procedure, and grown using standard methods. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 media. Furthermore, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by intraperitoneal injection of cells into mice.

[0160] Subclonal secreted monoclonal antibodies are appropriately separated from culture medium, ascites, or serum using 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.

[0161] DNA encoding monoclonal antibodies can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies. Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells that do not normally produce antibody proteins, such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain monoclonal antibody synthesis in recombinant host cells.

[0162] In some implementations, monoclonal antibodies or antibody fragments are isolated from an antibody phage library. High-affinity (nM range) human antibodies can be generated, for example, through chain shuffling. Combined infection and in vivo recombination can provide strategies for constructing very large phage libraries. These techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques used for isolating monoclonal antibodies.

[0163] DNA encoding antibodies can be modified to produce chimeric or fusion antibody peptides, for example, by replacing homologous mouse sequences with human heavy and light chain constant domain (CH and CL) sequences (see, for example, U.S. Patent No. 4,816,567), or by fusing immunoglobulin-coding sequences with all or part of the coding sequence of a non-immunoglobulin peptide (heterologous peptide).

[0164] Human antibody and phage display methods Human antibodies can be generated by methods known in the art, including those described herein. For example, transgenic animals (e.g., mice) can be generated that, upon immunization, produce a complete library of human antibodies in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice has been described as resulting in complete suppression of endogenous antibody production. Transferring a human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, for example, U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852.

[0165] Optionally, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene libraries from unimmunized donors. According to this technology, antibody V domain gene frames are cloned into the major or minor coat protein genes of filamentous phages (e.g., M13 or fd) and displayed as functional antibody fragments on the surface of phage particles. Because the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also leads to the selection of genes encoding antibodies that exhibit those properties. Thus, the phage mimics some properties of B cells. Phage display can be performed in various forms. Several sources of V gene segments can be used for phage display, such as randomly combined libraries from the V gene, for example, libraries derived from the spleen of immunized mice. V gene libraries from unimmunized human donors can be constructed, and antibodies against different arrays of antigens (including self-antigens) can be isolated substantially according to the methods described in the art. See, for example, U.S. Patent Nos. 5,565,332 and 5,573,905.

[0166] Human antibodies can also be produced by activated B cells in vitro (see, for example, U.S. Patent Nos. 5,567,610 and 5,229,275).

[0167] Competitive assays for determining whether an antibody binds to the same epitope as the disclosed antibody or competes for binding with the disclosed antibody are known in the art. Exemplary competitive assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance (e.g., BIAcore analysis), biolayer interferometry, and flow cytometry.

[0168] Typically, competitive assays involve the use of an antigen that binds to a solid surface or is expressed on a cell surface, a test antibody, and a reference antibody. The reference antibody is labeled, while the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody that binds to the 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 or similar (e.g., overlapping) epitopes as the reference antibody, as well as antibodies that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody to cause steric hindrance.

[0169] Competitive assays can be performed in two directions to ensure that the presence of the label does not interfere with or otherwise inhibit binding. For example, in the first direction, the reference antibody is labeled and the test antibody is unlabeled, and in the second direction, the test antibody is labeled and the reference antibody is unlabeled.

[0170] If an excess of one antibody (e.g., 1x, 5x, 10x, 20x, or 100x) inhibits the binding of another antibody, for example, by at least 50%, 75%, 90%, 95%, or 99%, as measured in a competitive binding assay, then the test antibody competes with the reference antibody for specific binding to the antigen.

[0171] If substantially all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody, then the same epitope can be identified for the binding of both antibodies. If only a subset of amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody, then overlapping epitopes for the binding of both antibodies can be identified.

[0172] 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 nonspecific interactions), and / or reduce immunogenicity. Affinity maturation procedures are within the scope of common art. For example, diversity can be introduced into immunoglobulin heavy chains and / or immunoglobulin light chains through DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis, and / or site-specific mutagenesis.

[0173] In some implementations, the isolated human antibody contains one or more somatic mutations. In these cases, the antibody can be modified with a human germline sequence to optimize the antibody (e.g., through a process called germination).

[0174] Typically, optimized antibodies have at least the same or substantially the same affinity for the antigen as the non-optimized (or parental) antibody from which it is derived. For example, in some embodiments, the optimized antibody has a higher affinity for the antigen compared to the parent antibody.

[0175] Pharmaceutical Composition In some embodiments, the provided molecule containing the disclosed antigen-binding site is incorporated together with one or more pharmaceutically acceptable carriers into a pharmaceutical composition suitable for administration to a subject. As used herein, a "pharmaceutically acceptable carrier" means any of a variety of physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, and combinations thereof.

[0176] In some embodiments, the pharmaceutical composition comprises one or more tonic agents or stabilizers. Non-limiting examples of such tonic agents or stabilizers include sugars (e.g., sucrose), polyols (e.g., mannitol or sorbitol), and sodium chloride.

[0177] In some embodiments, the pharmaceutical composition comprises one or more extenders and / or lyophilization protectants (e.g., mannitol or glycine), buffers (e.g., phosphate, acetate, or histidine buffers), surfactants (e.g., polysorbate), antioxidants (e.g., methionine), and / or metal ions or chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)).

[0178] In some embodiments, the pharmaceutical composition comprises one or more excipients, such as wetting agents or emulsifiers, preservatives (e.g., benzyl alcohol), or buffers, which may enhance the shelf life and / or efficacy of the immunoconjugates disclosed herein.

[0179] Pharmaceutical compositions can be provided in any of a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The suitability of certain forms may depend on the intended mode of administration and therapeutic application.

[0180] In some embodiments, the pharmaceutical composition is in the form of an injectable or infusionable solution.

[0181] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture, transport, and storage. 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 particularly suitable for high drug concentrations. For example, a sterile injectable solution can be prepared by incorporating a therapeutic agent (e.g., an immunoconjugate) in a 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, an immunoconjugate can be prepared by incorporating the immunoconjugate into a sterile medium containing a base dispersion medium and other components (e.g., those additional components mentioned herein). In the case of sterile powders used to prepare sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying to produce, for example, powders of the immunoconjugate and any other desired components from a previously sterile filtered solution.

[0182] Appropriate fluidity of the solution can be maintained, for example, by using coatings such as lecithin, by maintaining certain particle sizes (e.g., in the case of dispersions), and / or by using surfactants. Extended absorption of the injectable composition can be achieved, for example, by including an agent that delays absorption (e.g., monostearate and / or gelatin) in the composition.

[0183] Treatment This application provides methods for treating a disease or condition using the therapeutic agents (e.g., proteins, such as antibodies or antigen-binding fragments thereof) or pharmaceutical compositions described herein. The treatment methods disclosed herein generally involve administering a therapeutically effective amount of the disclosed therapeutic agent containing an antigen-binding site or a pharmaceutical composition thereof to a subject in need (e.g., a human subject).

[0184] In some implementations, the disease or condition is an autoimmune disease. Examples of autoimmune diseases include, but are not limited to, Behcet's disease, systemic lupus erythematosus, multiple sclerosis (systemic scleroderma and progressive systemic scleroderma), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa and microscopic polyangiitis), aortitis syndrome (high-stress 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, etc. Autoimmune hepatitis, primary biliary cirrhosis, aortitis syndrome, Goodpass 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, chronic discoid lupus erythematosus, localized scleroderma, psoriasis, psoriatic arthritis, pemphigus, bullous pemphigoid, herpes gestationis, linear IgA bullous dermatitis, acquired epidermolysis bullosa, alopecia areata, vitiligo, Harada disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent miscarriage, and inflammatory bowel disease (ulcerative colitis and Crohn's disease). The prescription of this invention can also be used for the prevention or treatment of graft-versus-host disease (GVHD).

[0185] In some implementations, the disease or condition 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, atherosclerosis and occlusive vascular disease, optional atherosclerosis, ischemic heart disease, myocardial infarction, stroke, peripheral vascular disease or stent restenosis, autoimmune diseases, bronchitis, cancer, carditis, cataracts, celiac disease, chronic pain, chronic prostatitis, cirrhosis, colitis, connective tissue diseases, optional systemic lupus erythematosus, systemic sclerosis, polymyositis, dermatomyositis or Sjögren's syndrome, corneal diseases, Crohn's disease, crystalline arthritis, optional gout, pseudogout, calcium pyrophosphate deposition disease, dementia, dermatitis, diabetes, dry eye syndrome, eczema, edema, emphysema, fibromyalgia, gastroenteritis, gingivitis, glomerulonephritis, heart disease, hepatitis, hypertension, hypersensitivity reactions, inflammatory bowel disease, inflammatory disorders (including the consequences of trauma or ischemia), insulin resistance. Anti-inflammatory drugs, interstitial cystitis, iridocyclitis, iritis, arthritis, arthritis, rheumatoid arthritis, Lyme disease, metabolic syndrome (syndrome x), multiple sclerosis, myositis, nephritis, obesity, eye diseases (including uveitis), osteoporosis, osteoporosis, Parkinson's disease, pelvic inflammatory disease, periodontitis, 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, spondyloarthritis, optional ankylosing spondylitis, reactive arthritis or Wright syndrome, systemic candidiasis, tendinitis, transplant rejection, vaginitis, vascular diseases including atherosclerotic vascular disease, vasculitis, optional polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, or vasculitis.

[0186] The therapeutically effective dose 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 periodic administration (e.g., every other day, every three days, every four days, every five days, three times a week, twice a week, once a week, once every two weeks, once every three weeks, etc.).

[0187] Effective dosage regimens in treatment methods (e.g., the amount of each therapeutic agent, the relative duration of treatment, etc.) may depend on the severity of the disease or ailment and the subject's weight and general condition. For example, the therapeutically effective amount of a particular composition containing a therapeutic agent applied to mammals (e.g., humans) can be determined by a person skilled in the art, taking into account individual differences in the mammal's age, weight, and condition. The therapeutically effective amount and / or optimal amount can also be determined empirically by a person skilled in the art.

[0188] Molecules containing antigen-binding sites or pharmaceutical compositions thereof may be administered via any of a variety of suitable routes, including but not limited to systemic routes such as parenteral (e.g., intravenous or subcutaneous) or enteral routes.

[0189] In many embodiments, administration of a therapeutic agent or pharmaceutical composition thereof containing an antigen-binding site, according to the provided method, results in the depletion of cells involved in the disease to be treated. In some embodiments, the cells express an antigen that binds to the antigen-binding site contained in the therapeutic agent. In some embodiments, the cells are immune cells, such as T cells, B cells, macrophages, natural killer (NK) cells, dendritic cells (DCs), monocytes, neutrophils, fibroblasts, or epithelial cells. In some embodiments, the cells are activated cells, such as antigen-activated T cells or B cells.

[0190] Example Example 1. Activation of FcγRIIIA The ability of publicly disclosed PD-1 antibodies to activate the Fcγ receptor was evaluated using engineered Jurkat reporter T cell lines expressing a specific Fcγ receptor and the Lucia luciferase reporter gene. Activation of Jurkat cells resulted in NFAT-mediated luminescence.

[0191] In summary, PD-1-expressing cells were incubated with Jurkat-Lucia reporter cells expressing FcγRIIIA and wild-type human IgG1 Fc or containing IgG1 Fc with a mutant S239D / H268D (“X3” mutation) that increases binding to FcγRIIB for 6 hours at 37°C and 5% CO2. Jurkat-Lucia TCR-hPD-1 cells were added to the culture, and the plates were incubated for another 5.5 hours at 37°C and 5% CO2. After incubation, 20 μL of supernatant was transferred to 96-well white plates. 50 μL of QUANTI-Luc 4 reagent was added to the plates, and luminescence was immediately read using a plate reader (PerkinElmer). The fold change in activation was calculated by comparing the luminescence intensity of each sample in RLUs (relative light units) to the isotype control.

[0192] Exemplary results in Figure 1 As shown in the figure, humanized anti-PD-1 3H4 with or without the X3 mutation can activate FcγRIIIA, and humanized antibodies 3H4-2, 3H4-4, 3H4-5, 3H4-7 and 3H4-8 containing the X3 mutation showed the greatest fold increase in activation.

[0193] Table 2. Antibody Information

[0194] Example 2. Inhibition of T cell activation by PD-1 antibody The ability of the disclosed PD-1 antibody to inhibit T cell activation was evaluated using a commercially available assay comprising an antigen-presenting cell line (APC) and a human Jurkat reporter T cell line expressing a specific T cell receptor (TCR) and the Lucia luciferase reporter gene. In this assay, activation of Jurkat cells resulted in NFAT-mediated luminescence. Additional Jurkat reporter cell-based assays were performed as described in Example 4 to evaluate the ability of the PD-1 antibody to inhibit T cell activation.

[0195] Raji-APC cells modified to express the FcγRII receptor were incubated with an allotype control or PD-1 antibody at 37°C and 5% CO2 for 30 minutes. The antibodies tested included wild-type IgG1 Fc, IgG1 Fc carrying the X3 mutation, or IgG1 Fc carrying the X3 mutation and the E430G or E345K mutation (“HexaBody” mutation) that induces antibody hexamer formation. After incubation, Jurkat-Lucia TCR-hPD-1 cells were added to the culture, and the plates were incubated at 37°C and 5% CO2 for another 5.5 hours. Cells were centrifuged at 400 xg for 5 minutes, and the supernatant was transferred to 96-well white plates. 50 μL of QUANTI-Luc 4 reagent was added to the plate, and the luminescence was immediately read using a PerkinElmer plate reader. Data were analyzed using GraphPadPrism, and the relative inhibition percentage was calculated compared to the signal obtained from the allotype control.

[0196] Exemplary results in Figure 2 As shown in the figure, compared with the isotype control, 3H4 containing the X3 mutation and humanized 3H4-2, 3H4-4, 3H4-5, 3H4-6, 3H4-7, and 3H4-8 significantly inhibited Jurkat cell activation. Furthermore, 3H4 containing the X3 mutation and the HexaBody mutation also inhibited Jurkat cell activation.

[0197] Example 3. Complement-dependent cytotoxicity of PD-1 antibodies The complement-dependent cytotoxicity (CDC) of the exemplary PD-1 antibodies disclosed herein was evaluated in Raji cells or CHO-K1 cells.

[0198] For Raji cell-based assays, PD-1 positive Raji cells were incubated for 30 minutes at 37°C with PD-1 antibody 3H4, 3H4 cells with the X3 mutation (“3H4 X3”), and 3H4 cells with both X3 and HexaBody mutations (“3H4 X3 Hex”) in 96-well clear-bottomed white-walled plates. After incubation, fresh human serum isolated from whole blood was added, and the plates were incubated at 37°C and 5% CO2 for 4 hours. Subsequently, CellTiter-Glo was added to each well, and the plates were incubated in the dark on a benchtop shaker for 2 minutes. Efficacy was measured on a plate reader (PerkinElmer). The percentage of cytotoxicity was calculated by subtracting the background RLU from all samples and then applying the formula 100% × (1 – RLU of the sample) / (RLU of the antibody-free control).

[0199] For CHO-K1 cell-based assays, PD-1-positive CHO-K1 cells were incubated with serially diluted PD-1 antibody 3H4X3 HexaBody in clear-bottomed white-walled plates at room temperature for 30 minutes. After incubation, normal human serum complement was added, and the plates were incubated at 37°C and 5% CO2 for 4 hours. Subsequently, CellCounting-Lite 2.0 luminescent cell viability assay working solution was added to each well, and the plates were incubated at room temperature for 10 minutes. Luminescence was measured on a plate reader, and data were analyzed using Microsoft Office Excel 2016 and GraphPad Prism. Concentrations were plotted as a function of relative luminescence, and curve fitting was performed using a four-parameter nonlinear regression algorithm. The percentage of cytotoxicity was calculated by subtracting background RLU from all samples and then applying the formula 100% x (1 – RLU of the sample) / (RLU of the antibody-free control).

[0200] The result is Figure 3A and Figure 3B As shown in the figure, 3H4X3 Hex exhibits significant CDC activity, EC 100%. 50 It is 4.092 nM.

[0201] Example 4. Exhaustion of CD8 T cells induced by PD-1 antibody Assess the ability of an exemplary PD-1 antibody to deplete CD8 T cells.

[0202] Fresh whole blood was collected into heparinized vacuum blood collection tubes. PD-1 antibody or allotype control antibody (100 nM) was incubated with whole blood in polypropylene tubes at 37°C and 5% CO2 for 24 hours. After incubation, cells were stained with fluorescently labeled antibodies against PD-1, CD3, CD4, CD8, and CD45 at room temperature for 30 minutes. Cells were lysed by adding lysis buffer at room temperature for 15 minutes, centrifuged at 400 × g for 4 minutes, and washed twice with cell staining buffer. After washing, cell fluorescence analysis was performed using a BD FACSSymphony A3 (BD ​​Biosciences). Cells were sorted by scattering properties, and cells were separated for single-cell analysis using the side-scatter width to the side-scatter area. Cell-bound antibodies were quantified by measuring excited fluorescence emission and used to quantify the relative proportion of CD8 T cells with high and moderate PD-1 expression compared to the entire CD8 T cell population. Data analysis was performed using FlowJo software, and the percentage of CD8 T cells with high and moderate PD-1 expression remaining under each condition was normalized by comparison with isotype controls.

[0203] The result is Figure 4A and Figure 4B As shown in the figure, PD-1 antibodies 3H4 X3 and 3H4 X3 Hex have been shown to mediate the depletion of CD8 T cells with high and moderate levels of PD-1 expression on the cell surface.

[0204] Example 5. Binding of antibody to PD-1 The binding of humanized PD-1 antibody to human PD-1 was assessed using biolayer interferometry (BLI).

[0205] The experiments were performed on an Octet BLI instrument. The test antibody was loaded onto an anti-human IgG Fc capture (AHC) sensor, and the sensor was immersed in serially diluted His-tagged PD-1. Baselines were established after loading and before each association. Data were used only once for analysis. Kinetic constants were calculated using a single-value (1:1) curve fitting model.

[0206] Exemplary results are depicted in Table 2. All tested PD-1 antibodies were able to bind to human PD-1.

[0207] Table 3. Binding of PD-1 antibody to human PD-1

[0208] The binding of humanized PD-1 antibody to human PD-1 was assessed by flow cytometry. PD-1-positive Jurkat cells were suspended in FACS blocking buffer (PBS + 10% normal human serum) for 30 min. After washing, cells were incubated with the test PD-1 antibody in FACS buffer (PBS + 0.5% bovine serum albumin + 0.09% sodium azide) at 4°C for 30 min, washed, and incubated with PE-labeled anti-human secondary antibody (Invitrogen). Cells were then incubated in the dark at 4°C for 30 min. After washing, cell fluorescence analysis was performed using a BD FACS Symphony A3 (BD ​​Biosciences). Cells were sorted by scattering properties, and cells were separated for single-cell analysis using the side-scatter width to the side-scatter area. The binding of the antibody to cells was quantified by measuring fluorescence emission at a 561 nm excitation wavelength using a 586 / 15 nm filter. Data analysis was performed using FlowJo software.

[0209] Figure 5 Exemplary results are described in the paper. All tested PD-1 antibodies were able to bind to PD-1 on the cell surface.

[0210] Example 6. Cross-reactivity of PD-1 antibodies Surface plasmon resonance (SPR) combined with kinetic assays were used to evaluate the cross-reactivity of PD-1 antibodies to human PD-1 and cynomolgus monkey PD-1.

[0211] In short, the test antibody was loaded onto the AHC sensor, and the sensor was immersed in eleven-fold, three-fold serial dilutions of His-tagged PD-1. Kinetic assays were performed on a Biacore T200 instrument using multi-cycle kinetics at an analytical temperature of 25°C. Kinetic constants were calculated using a monovalent (1:1) curve fitting model.

[0212] Exemplary results are depicted in Table 3. The tested PD-1 antibody AHF29879_R was able to bind to human and cynomolgus monkey PD-1.

[0213] Table 4. Cross-reactivity of PD-1 antibodies

[0214] The cross-reactivity of PD-1 antibodies to human PD-1 and cynomolgus monkey PD-1 was also analyzed using an ELISA-based assay. His-tagged extracellular domains of human and cynomolgus monkey PD-1 were immobilized on 96-well ELISA plates (Nunc Maxisorp, Thermo Fisher) and blocked with 1% BSA in PBS for 1 hour. After washing with PBST (phosphate-buffered saline + 0.05% Tween 20), serially diluted test PD-1 antibodies were added to the coated plates and incubated at room temperature for 2 hours. The plates were then washed and incubated with HRP-conjugated goat anti-human IgG secondary antibody (Jackson Immunoresearch) at room temperature for 1 hour. After washing, antibody binding was detected colorimetrically by adding 3,3',5,5'-tetramethylbenzidine (TMB) and incubated at room temperature for 10–20 minutes, followed by the addition of 2.5N sulfuric acid stop solution. Absorbance was measured at 450 nm using a plate reader (PerkinElmer). Use GraphPad Prism to analyze data.

[0215] Figure 6 Exemplary results are depicted in the paper. The tested PD-1 antibody h3H4-879 was able to bind to human and cynomolgus monkey PD-1.

[0216] Example 7. PD-1 antibody signal transduction via Fcγ receptor The ability of effector-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 the Lucia luciferase reporter gene. In this assay, activation of Jurkat cells resulted in NFAT-mediated luminescence.

[0217] Target cells expressing PD-1 were incubated in multi-well plates with humanized PD-1 antibodies or allotype controls containing effector-enhanced IgG1 Fc, as well as Jurkat-Lucia reporter cell lines modified to express Fcγ receptors CD16a (FcγRIIIa), CD32a (FcγRIIa), or CD64 (FcγRI) for 6 hours at 37°C and 5% CO2. The effector-enhanced IgG1 Fc contained the mutant S239D / H268D (“X3” mutation) and the E430G mutation that induces antibody hexamer formation (collectively, the “X3 Hex” mutation). After incubation, 20 mL of supernatant from each well was transferred to a 96-well white-walled plate, and 50 mL of Quanti-Luc4 reagent (Invivogen) was added. Luminescence was measured on a PerkinElmer plate reader. The fold change in activation was calculated by comparing the RLU values ​​from each sample to the allotype control.

[0218] Exemplary results in Figures 7A-7C As shown in the figure, all tested PD-1 antibodies with the X3 mutation were able to signal via Fcγ receptors CD16a, CD32a, and CD64.

[0219] Example 8. Inhibition of T cell activation by PD-1 antibody The Jurkat reporter gene assay was used to assess the ability of effector-modified PD-1 antibodies to inhibit T cell activation.

[0220] Raji-APC cells (Invivogen) modified to express the FcγRIIb receptor were incubated with a test PD-1 antibody or allotype control containing IgG1 Fc with an X3 Hex mutation that increases binding to FcγRIIb for 30 min at 37°C and 5% CO2. Raji-PD-L1 cells were used as a positive control for agonist activity. After incubation, Jurkat-Lucia TCR-hPD-1 cells (Invivogen) were added to the culture, and the plates were incubated for another 5.5 h at 37°C and 5% CO2. The cells were centrifuged at 400 x g for 5 min, and 20 mL of the supernatant was transferred to a 96-well white plate. Activation of Jurkat cells was measured via a bioluminescent signal generated by Lucia luciferase. 50 mL of QUANTI-Luc 4 reagent (Invivogen) was added to the plate, and the luminescence (RLU) was read immediately using a PerkinElmer plate reader. Data were analyzed using a GraphPad Prism.

[0221] Exemplary results in Figure 8As shown in the figure, compared with the isotype control, all tested PD-1 antibodies significantly inhibited the activation of Jurkat cells.

[0222] Example 9. Phagocytosis induced by PD-1 antibody The Jurkat reporter gene assay was used to assess the ability of PD-1 antibodies to induce phagocytosis.

[0223] Raji PD-1 positive target cells were labeled with Cell Tracker Deep Red and added to the wells of a 96-well plate. The labeled target cells were incubated with increasing concentrations of the test PD-1 antibody or an allotype control at 37°C for 30 minutes. After incubation, PKH26-stained human monocyte-derived macrophages were added to the culture at a 5:1 effector-to-target cell ratio. The plates were incubated for 4 hours, and images were captured using a Cytation 5 cell imaging multimodal reader (Agilent). The number of cells undergoing phagocytosis was counted using Gen5 cell counting software.

[0224] Exemplary results in Figure 9 As shown in the figure. All tests of PD-1 demonstrated effective antibody-dependent phagocytosis of PD-1-positive target cells.

[0225] Example 10. Cytotoxicity induced by PD-1 antibody The killing ability of PD-1 antibodies was evaluated using a natural killer (NK) cell-mediated cytotoxicity assay.

[0226] CD4 T cells and autologous NK cells were isolated from PBMCs using the StemCell Technologies Isolation Kit. T cells were activated by treatment with anti-CD3 and anti-CD28, which increased PD-1 expression on the cell surface. NK cells were incubated overnight with human IL-15, and their purity was checked by flow cytometry. Activated T cells were incubated in 96-well plates at 37°C and 5% CO2 for 40 minutes with increasing concentrations of test PD-1 antibody or allotype control. NK cells were added to the culture, and the plates were incubated overnight (18–24 hours) at 37°C and 5% CO2. After overnight incubation, Triton X was added to the control wells and incubated for 15 minutes to induce total cell lysis. The plates were centrifuged at 250 xg for 10 minutes. 100 mL of supernatant from each well was transferred to an optically clear 96-well plate. 100 mL of freshly prepared LDH reaction mixture was added to each well, and the plates were incubated for up to 30 minutes for color development. Absorbance was measured at 492 nm using a PerkinElmer plate reader, with 650 nm used as a reference. Data were analyzed using a GraphPad Prism. The value obtained from the reference wavelength was subtracted from the value at 492 nM. The percentage of cytotoxicity was determined by comparing the sample OD value with the OD value of a Triton X well representing 100% cytotoxicity.

[0227] Exemplary results in Figure 10 As shown in the figure. All PD-1 tests demonstrated effective antibody-dependent cell-mediated cytotoxicity in PD-1-positive target cells.

[0228] Example 11. Inhibition of IFN-γ secretion by PD-1 antibody To assess the ability of PD-1 antibody to inhibit IFN-γ secretion.

[0229] PBMCs from recently vaccinated tetanus donors were incubated with tetanus toxoid and test PD-1 antibodies or allotype controls at 37°C and 5% CO2 for 4 days. IFN-γ levels in the culture supernatant were measured using a commercial IFN-γ ELISA kit (R&D Systems). IFN-γ capture antibodies were immobilized 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 supernatant and IFN-γ standard curve were added to the coated plates and incubated at room temperature for 2 hours. The plates were washed and incubated with HRP-conjugated detection antibodies at room temperature for 2 hours. After washing, antibody binding was detected colorimetrically by adding 3,3',5,5'-tetramethylbenzidine (TMB) and incubated at room temperature for 10–20 minutes, followed by the addition of 2.5N sulfuric acid stop solution. The absorbance at 450 nm was measured using a PerkinElmer plate reader. The data were analyzed using a GraphPad Prism.

[0230] Exemplary results in Figure 11 As shown in the figure, PD-1 significantly inhibited IFN-γ secretion in all tests.

[0231] Sequence Appendix In some sequences, variable regions are shown in bold, and CDRs are shown underlined.

[0232] Table 5. Sequences of exemplary antigen-binding sites capable of binding to PD-1

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240] Table 6. Other sequences

[0241]

[0242]

[0243] By incorporating references Unless otherwise stated, the full disclosure of each patent document and scientific article cited herein is incorporated by reference for all purposes.

[0244] Equivalent solution / Other implementation solutions Those skilled in the art will recognize or be able to determine many equivalent solutions to the specific embodiments described herein using only conventional experiments. Such equivalent solutions are intended to be covered 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 comprises a heavy chain variable (VH) region, comprising: The heavy chain complementarity-determining region 1 (HCDR1) has the amino acid sequence of SEQ ID NO:

2. HCDR2 having the amino acid sequence of SEQ ID NO: 98, and HCDR3 having the amino acid sequence of SEQ ID NO:

4.

2. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment thereof further comprises a light chain variable (VL) region, which includes: (a) Light chain complementarity-determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 6, LCDR2, having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence of SEQ ID NO: 8; or (b) LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2, having the amino acid sequence of SEQ ID NO: 103, and LCDR3 having the amino acid sequence of SEQ ID NO: 104; or (c) LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2, having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence SEQ ID NO:

109.

3. An antibody or antigen-binding fragment thereof capable of binding to PD-1, wherein the antibody or antigen-binding fragment thereof comprises... A heavy chain variable (VH) region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 12-19, 97, and 100; and / or The light chain variable (VL) region contains an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequences of SEQ ID NO: 23-26, 102, 106 and 108.

4. The antibody or its antigen-binding fragment as described in claim 3, wherein... The VH region contains an amino acid sequence selected from SEQ ID NO: 12-19, 97, and 100; and The VL region contains an amino acid sequence selected from SEQ ID NO: 23-26, 102, 106, and 108.

5. An antibody or antigen-binding fragment thereof capable of binding to PD-1, wherein the antibody or antigen-binding fragment thereof comprises... (a) A heavy chain variable (VH) region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9, and a light chain variable (VL) region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 26; or (b) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 18, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 21; or (c) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 18, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 26; or (d) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 19, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 26; or (e) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 15, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or (f) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 15, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 25; or (g) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 16, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or (h) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 16, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 25; or (i) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 17, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or (j) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 17, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 25; or (k) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 97, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 102; or (l) A VH region comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 100, and a VL region comprising an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 106; or (m) includes a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 97, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:

108.

6. The antibody or antigen-binding fragment thereof as described in claim 5, wherein... (a) The VH region contains the amino acid sequence of SEQ ID NO: 9, and the VL region contains the amino acid sequence of SEQ ID NO: 26; or (b) The VH region contains the amino acid sequence of SEQ ID NO: 18, and the VL region contains the amino acid sequence of SEQ ID NO: 21; or (c) The VH region contains the amino acid sequence of SEQ ID NO: 18, and the VL region contains the amino acid sequence of SEQ ID NO: 26; or (d) The VH region contains the amino acid sequence of SEQ ID NO: 19, and the VL region contains the amino acid sequence of SEQ ID NO: 26; or (e) The VH region contains the amino acid sequence of SEQ ID NO: 15, and the VL region contains the amino acid sequence of SEQ ID NO: 24; or (f) The VH region contains the amino acid sequence of SEQ ID NO: 15, and the VL region contains the amino acid sequence of SEQ ID NO: 25; or (g) The VH region contains the amino acid sequence of SEQ ID NO: 16, and the VL region contains the amino acid sequence of SEQ ID NO: 24; or (h) The VH region contains the amino acid sequence of SEQ ID NO: 16, and the VL region contains the amino acid sequence of SEQ ID NO: 25; or (i) The VH region contains the amino acid sequence of SEQ ID NO: 17, and the VL region contains the amino acid sequence of SEQ ID NO: 24; or (j) The VH region contains the amino acid sequence of SEQ ID NO: 17, and the VL region contains the amino acid sequence of SEQ ID NO: 25; or (k) The VH region contains the amino acid sequence of SEQ ID NO: 97, and the VL region contains the amino acid sequence of SEQ ID NO: 102; or (l) The VH region contains the amino acid sequence of SEQ ID NO: 100, and the VL region contains the amino acid sequence of SEQ ID NO: 106; or (m) The VH region contains the amino acid sequence of SEQ ID NO: 97, and the VL region contains the amino acid sequence of SEQ ID NO:

108.

7. The antibody or antigen-binding fragment thereof as claimed in claim 3 or 5, wherein the VH region comprises an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 16, and the VL region comprises an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:

24.

8. The antibody or antigen-binding fragment thereof as claimed in claim 7, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 16, and the VL region comprises the amino acid sequence of SEQ ID NO:

24.

9. The antibody or antigen-binding fragment thereof as claimed in claim 3 or 5, wherein the VH region comprises an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 17, and the VL region comprises an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:

25.

10. The antibody or antigen-binding fragment thereof as claimed in claim 9, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 17, and the VL region comprises the amino acid sequence of SEQ ID NO:

25.

11. The antibody or antigen-binding fragment thereof as claimed in claim 1 or 5, wherein the antibody or antigen-binding fragment thereof comprises a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 97, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:

102.

12. The antibody or antigen-binding fragment thereof as claimed in claim 11, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 97, and the VL region comprises the amino acid sequence of SEQ ID NO:

102.

13. The antibody or antigen-binding fragment thereof as claimed in claim 1 or 5, wherein the antibody or antigen-binding fragment thereof comprises a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 100, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:

106.

14. The antibody or antigen-binding fragment thereof as claimed in claim 13, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 100, and the VL region comprises the amino acid sequence of SEQ ID NO:

106.

15. The antibody or antigen-binding fragment thereof as claimed in claim 1 or 5, wherein the antibody or antigen-binding fragment thereof comprises a VH region containing an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 97, and a VL region containing an amino acid sequence having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:

108.

16. The antibody or antigen-binding fragment thereof as claimed in claim 9, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 97, and the VL region comprises the amino acid sequence of SEQ ID NO:

108.

17. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 16, wherein the antibody is a humanized antibody.

18. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 17, wherein the antigen-binding fragment is Fab, Fab', Fab2, F(ab')2, Fv, single-chain Fv (scFv) or a biantibody.

19. The antibody or antigen-binding fragment thereof as claimed in claim 18, wherein the antigen-binding fragment is scFv.

20. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 19, wherein the antibody or antigen-binding fragment thereof comprises a constant region of the antibody heavy chain.

21. The antibody or antigen-binding fragment thereof as claimed in claim 20, wherein the antibody heavy chain constant region is the human IgG heavy chain constant region.

22. The antibody or antigen-binding fragment thereof as claimed in claim 21, wherein the antibody heavy chain constant region is the human IgG1 heavy chain constant region.

23. The antibody or antigen-binding fragment thereof as claimed in claim 22, wherein the constant region of the antibody heavy chain 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:

111.

24. The antibody or antigen-binding fragment thereof as claimed in claim 22, wherein the constant region of the antibody heavy chain has the amino acid sequence of SEQ ID NO:

111.

25. The antibody or antigen-binding fragment thereof as claimed in claim 21 or 22, wherein the constant region of the antibody heavy chain contains one or more mutations that enhance effector function.

26. The antibody or antigen-binding fragment thereof as claimed in claim 21, 22 or 25, wherein the constant region of the antibody heavy chain contains one or more mutations that enhance binding to an inhibitory Fc receptor (e.g., FcγRII).

27. The antibody or antigen-binding fragment thereof as claimed in claim 25 or 26, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing one of the following mutations: (a) E233D; (b) G236D or G236A; (c) G237D; (d) P238D; (e) S239D; (f) S267E; (g) H268D or H268F; (h) P271G; (i) S324T; (j) L328Y, L328F or L328E; (k) A330R; (l) I332E; (m) E345K, E345Y, E345Q or E345R; (n) E430G, E430S, E430F or E430T; (o) S440Y; (p) G236D and H268D; (q) S239D and H268D; (r) S239D, H268D, L328Y and I332E; (s) P238D and L328E; (t) G237D, P271G and A330R; (u) G237D, H268D, P271G and A330R; (v) S267E and L328F; (w) S239D and S267E; (x) G236D and S276E; (y) E233D, G237D, H268D, P271G and A330R; (z) S239D, H268D, and E430G; or (aa) S239D, H268D and E345K, Or a combination thereof, wherein the constant regions are numbered according to the EU index.

28. The antibody or antigen-binding fragment thereof as claimed in claim 27, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing one of the following mutations: (a) G236D or G236A; (b) S239D; (c) H268D or H268F; (d) L328Y; (e) S324T; (f) I332E; (g) E345K, E345Y, E345Q or E345R; (h) E430G, E430S, E430F or E430T; (i) S440Y; (j) S239D and H268D; Or a combination thereof, wherein the numbering of the constant region is in accordance with the EU index.

29. The antibody or antigen-binding fragment thereof as claimed in any one of claims 25 to 28, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region comprising the following mutations: S239D and H268D, and optionally E430G or E345K, wherein the numbering of the constant region is in accordance with the EU index.

30. The antibody or antigen-binding fragment thereof as claimed in claim 29, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region comprising the following mutations: S239D, H268D, and E430G, wherein the numbering of the constant region is in accordance with the EU index.

31. The antibody or antigen-binding fragment thereof as claimed in any one of claims 25 to 28, 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 in accordance with the EU index.

32. The antibody or antigen-binding fragment thereof as claimed in any one of claims 25 to 28, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing the following mutations: S267E and L328F, wherein the numbering of the constant region is in accordance with the EU index.

33. The antibody or antigen-binding fragment thereof as claimed in any one of claims 25 to 32, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region containing the following mutations: E430G or E345K, wherein the numbering of the constant region is in accordance with the EU index.

34. The antibody or antigen-binding fragment thereof as claimed in claim 25 or 26, wherein the constant region of the antibody heavy chain 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:

125.

35. The antibody or antigen-binding fragment thereof as claimed in claim 34, wherein the constant region of the antibody heavy chain has the amino acid sequence of SEQ ID NO:

125.

36. An antibody or an antigen-binding fragment thereof capable of binding to PD-1, wherein: The antibody or its antigen-binding fragment comprises a human IgG1 heavy chain constant region containing a mutation selected from E345K, E345Y, E345Q, E345R, E430G, E430S, E430F, E430T, and S440Y, wherein the constant region is numbered according to the EU index; and The antibody or its antigen-binding fragment contains (a) (i) Heavy chain variable (VH) region, which contains: Heavy chain complementarity-determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids. HCDR2 having the amino acid sequence of SEQ ID NO: 3 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) A light chain variable (VL) region, comprising: The light chain complementarity-determining region 1 (LCDR1) has the amino acid sequence of SEQ ID NO: 6 or a sequence that differs from it by one or two amino acids. LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 8 or a sequence that differs from it by one or two amino acids; (b) (i) The heavy chain variable (VH) region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 28 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 29 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 30 or a sequence differing from it by one or two amino acids; and (ii) A light chain variable (VL) region, comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 32 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 33 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 34 or a sequence that differs from it by one or two amino acids; (c) (i) The VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 36 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 37 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 38 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 40 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 41 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 42 or a sequence that differs from it by one or two amino acids; (d) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 44 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 45 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 46 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 48 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 49 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 50 or a sequence that differs from it by one or two amino acids; (e) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 52 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 53 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 54 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 56 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 57 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 58 or a sequence that differs from it by one or two amino acids; (f) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 10 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 8 or a sequence that differs from it by one or two amino acids; (g) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 60 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 61 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 62 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 64 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 65 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 66 or a sequence that differs from it by one or two amino acids; (h) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 60 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 68 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 62 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 64 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 65 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 66 or a sequence that differs from it by one or two amino acids; (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 73 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 74 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 75 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 77 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 78 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 79 or a sequence that differs from it by one or two amino acids; (j) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 73 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 81 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 75 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 77 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 78 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 79 or a sequence that differs from it by one or two amino acids; (k) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 86 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 87 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 90 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 91 or a sequence that differs from it by one or two amino acids; (l) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 85 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 93 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 87 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 89 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 90 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 91 or a sequence that differs from it by one or two amino acids; (m) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 98 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 103 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 104 or a sequence that differs from it by one or two amino acids; (n) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 98 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 8 or a sequence that differs from it by one or two amino acids; or (o) (i) VH region, which contains: HCDR1 having the amino acid sequence of SEQ ID NO: 2 or a sequence differing from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 98 or a sequence differing from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 4 or a sequence differing from it by one or two amino acids; and (ii) The VL region, which contains: LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a sequence differing from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a sequence differing from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 109 or a sequence that differs from it by one or two amino acids.

37. The antibody or antigen-binding fragment thereof as claimed in claim 36, wherein said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence that differs from the following sequence by no more than two amino acid residues in total: (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) are SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 respectively; (c) are SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 respectively; (d) are SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (e) are SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58, respectively; (f) are SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively; (g) are SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66 respectively; (h) are SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; (i) are SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively; (j) are SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively; (k) are 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; (l) are SEQ ID NO: 85, SEQ ID NO: 93, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, respectively; (m) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 103 and SEQ ID NO: 104 respectively; (n) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or (o) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 109, respectively.

38. The antibody or antigen-binding fragment thereof as claimed in claim 37, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following amino acid 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) are SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 respectively; (c) are SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42 respectively; (d) are SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, respectively; (e) are SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58, respectively; (f) are SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively; (g) are SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66 respectively; (h) are SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; (i) are SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively; (j) are SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively; (k) are 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; (l) are SEQ ID NO: 85, SEQ ID NO: 93, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 90 and SEQ ID NO: 91, respectively; (m) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 103 and SEQ ID NO: 104 respectively; (n) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or (o) are SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 109, respectively.

39. The antibody or antigen-binding fragment thereof as described in any one of claims 36 to 38, comprising: (a) 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: 1, 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: 5; (b) 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: 27, 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: 31; (c) 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: 35, 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: 39; (d) 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: 43, 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: 47; (e) 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: 51, 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: 55; (f) 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: 9 or 11, 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: 20 or 21; (g) 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: 59, 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: 63; (h) 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: 67, 128, 69, or 60, 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: 71; (i) 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: 72, 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: 76; (j) 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: 80, 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: 82 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 with 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 with the amino acid sequence of SEQ ID NO: 88; (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: 92, 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: 94 or 95; (m) 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: 97, 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: 102 or 108; or (n) 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: 100, 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:

106.

40. The antibody or antigen-binding fragment thereof as claimed in claim 39, comprising: (a) The VH region having the amino acid sequence of SEQ ID NO: 1 and the VL region having the amino acid sequence of SEQ ID NO: 27; (b) The VH region having the amino acid sequence of SEQ ID NO: 1 and the VL region having the amino acid sequence of SEQ ID NO: 31; (c) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 35 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 39; (d) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 43 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 47; (e) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 51 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 55; (f) The VH region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 9 or 11 and the VL region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 20 or 21; (g) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 59 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 63; (h) The VH region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 67, 128, 69 or 60 and the VL region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 71; (i) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 72 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 76; (j) The VH region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 80 and the VL region of an amino acid sequence having the amino acid sequence of SEQ ID NO: 82 or 83; (k) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 84 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 88; (l) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 92 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 94 or 95; (m) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 97 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 102 or 108; or (m) The VH region of the amino acid sequence having the amino acid sequence of SEQ ID NO: 100 and the VL region of the amino acid sequence having the amino acid sequence of SEQ ID NO:

106.

41. The antibody or antigen-binding fragment thereof as claimed in any one of claims 36 to 40, wherein the constant region of the IgG1 heavy chain comprises E430G or E345K, wherein the numbering of the constant region is in accordance with the EU index.

42. The antibody or antigen-binding fragment thereof as claimed in any one of claims 36 to 41, wherein the IgG1 heavy chain constant region further comprises one of the following mutations: (a) E233D; (b) G236D or G236A; (c) G237D; (d) P238D; (e) S239D; (f) S267E; (g) H268D or H268F; (h) P271G; (i) S324T; (j) L328Y, L328F or L328E; (k) A330R; (l) I332E; (m) G236D and H268D; (n) S239D and H268D; (o) S239D, H268D, L328Y and I332E; (p) P238D and L328E; (q) G237D, P271G and A330R; (r) G237D, H268D, P271G and A330R; (s) S267E and L328F; (t) S239D and S267E; (u) G236D and S276E; or (v) E233D, G237D, H268D, P271G, and A330R, Or a combination thereof, wherein the numbering of the constant region is in accordance with the EU index.

43. The antibody or antigen-binding fragment thereof as claimed in claim 42, wherein the IgG1 heavy chain constant region comprises the following mutations: S239D and H268D, wherein the numbering of the constant region is in accordance with the EU index.

44. The antibody or antigen-binding fragment thereof as claimed in claim 42, wherein the IgG1 heavy chain constant region comprises the following mutations: E233D, G237D, H268D, P271G, and A330R, wherein the numbering of the constant region is in accordance with the EU index.

45. The antibody or antigen-binding fragment thereof as claimed in claim 42, wherein the IgG1 heavy chain constant region comprises the following mutations: S267E and L328F, wherein the numbering of the constant region is in accordance with the EU index.

46. ​​The antibody or antigen-binding fragment thereof as claimed in any one of claims 36 to 41, wherein the constant region of the IgG1 heavy chain 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:

125.

47. The antibody or antigen-binding fragment thereof as claimed in claim 46, wherein the constant region of the IgG1 heavy chain has the amino acid sequence of SEQ ID NO:

125.

48. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 47, wherein the antibody or antigen-binding fragment thereof is a PD-1 agonist.

49. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 48, wherein the antibody or antigen-binding fragment thereof has enhanced binding to the Fc receptor.

50. The antibody or antigen-binding fragment thereof as claimed in claim 49, wherein the Fc receptor is an inhibitory Fc receptor (e.g., FcγRII).

51. The antibody or antigen-binding fragment thereof as claimed in claim 49, wherein the Fc receptor is an activating Fc receptor (e.g., FcγRI or FcγRIII).

52. The antibody or antigen-binding fragment thereof as claimed in claims 1 to 51, wherein the antibody or antigen-binding fragment thereof has enhanced effector function.

53. The antibody or antigen-binding fragment thereof as claimed in claim 52, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

54. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 53, wherein the antibody or antigen-binding fragment thereof is capable of depleting cells.

55. The antibody or antigen-binding fragment thereof as claimed in claim 54, wherein the cell is a cell expressing PD-1.

56. The antibody or antigen-binding fragment thereof as claimed in claim 54 or 55, wherein the cell is an immune cell.

57. The antibody or antigen-binding fragment thereof as claimed in claim 56, wherein the immune cell is a T cell, B cell, macrophage, natural killer (NK) cell, dendritic cell (DC), monocyte, neutrophil, fibroblast, or epithelial cell.

58. The antibody or antigen-binding fragment thereof as claimed in claim 57, wherein the immune cell is a T cell or a B cell.

59. The antibody or antigen-binding fragment thereof as claimed in any one of claims 56 to 58, wherein the immune cell is activated.

60. The antibody or antigen-binding fragment thereof as claimed in claim 59, wherein the immune cell is an antigen-activated T cell or a B cell.

61. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 61, wherein at least about 60%, about 75%, or about 90% of the antibody or antigen-binding fragment thereof is unfucosylated.

62. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 57, wherein the antibody or antigen-binding fragment thereof is produced in a cell line, said cell line (a) Possessing α-1,6-fucosyltransferase (Fut8) knockout; or (b) Overexpression of β1,4-N-acetylglucosamine transferase III (GnT-III) and optionally overexpression of Golgi μ-mannosidase II (ManII).

63. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 62, wherein the antibody or antigen-binding fragment is capable of binding to human PD-1.

64. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 63, wherein the antibody or antigen-binding fragment is capable of binding to cynomolgus monkey PD-1.

65. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 64, wherein the antibody or antigen-binding fragment is capable of blocking PD-1 / PD-L1 interaction.

66. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 65, wherein the antibody or antigen-binding fragment is capable of inhibiting T cell activation.

67. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 66, wherein the antibody or antigen-binding fragment is capable of inhibiting T cell proliferation.

68. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 67, wherein the antibody or antigen-binding fragment is capable of inhibiting IFN-γ secretion.

69. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 68, wherein the antibody or antigen-binding fragment thereof is conjugated with an agent.

70. The antibody or antigen-binding fragment thereof as claimed in claim 69, wherein the agent is a cytotoxic agent or a label.

71. The antibody or antigen-binding fragment thereof as claimed in claim 70, wherein the agent is a therapeutic agent.

72. A composition comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 71.

73. The composition of claim 72, wherein the antibody or antigen-binding fragment comprises an Fc region and an N-glycoside-linked carbohydrate chain linked to the Fc region, optionally wherein less than 50% of the N-glycoside-linked carbohydrate chain contains fucose residues.

74. The composition of claim 73, wherein the N-glycoside-linked carbohydrate chain contains substantially no fucose residues.

75. A polynucleotide encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 71.

76. An expression vector comprising the polynucleotide as described in claim 75.

77. A host cell comprising the polynucleotide of claim 75 or the expression vector of claim 76.

78. The host cell of claim 77, wherein the host cell is a mammalian or insect cell.

79. The host cell of claim 77 or 78, wherein the host cell (a) comprises Fut8 knockout; and / or (b) overexpresses GnT-III and optionally overexpresses ManII.

80. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 71, and a pharmaceutically acceptable carrier.

81. A method of treating a disease or condition in a subject in need, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 71, a composition as claimed in any one of claims 72 to 75, or a pharmaceutical composition as claimed in claim 80.

82. The method of claim 81, wherein the disease or condition is an autoimmune disease.

83. The method of claim 81, wherein the disease or condition is an allergy.

84. The method of claim 81, wherein the disease or condition is an inflammatory disease.

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