PD-1 binding protein and pharmaceutical use

Abstract

The present disclosure relates to a PD-1 binding protein and the pharmaceutical use. Specifically, the present disclosure relates to an anti-PD-1 antibody, a pharmaceutical composition containing the antibody, a method for treating inflammatory or autoimmune diseases, and the pharmaceutical use.

Description

PD-1 binding protein and medical use Technical Field

[0001] The present disclosure relates to PD-1 binding proteins, and in particular to anti-PD-1 antibodies and methods and pharmaceutical uses thereof for treating inflammatory or autoimmune diseases. Background Art

[0002] Programmed cell death-1 (PD-1) (also known as programmed cell death 1) is a 268-amino acid type I transmembrane protein and a member of the CD28 / CTLA-4 family of T cell regulators. It is a cell surface receptor protein primarily expressed on T cells and other immune cells. PD-1 has two ligands, PD-L1 and PD-L2, which interact with cell surface receptors. Upon binding to its ligands, PD-L1 or PD-L2, PD-1 downregulates T cell function, including reducing T cell activation, differentiation, proliferation, and cytokine secretion.

[0003] The PD-1 pathway is a naturally occurring negative feedback regulatory mechanism in the immune system. Activating the PD-1 pathway for treating immune disorders is novel and has broad therapeutic potential. This disclosure provides highly structured anti-PD-1 antibodies that can better activate the PD-1 signaling pathway and inhibit T cell activity, thereby improving the effectiveness of treating T cell-mediated inflammatory or autoimmune diseases. Summary of the Invention

[0004] The present disclosure provides PD-1 binding proteins and encoding nucleic acids, vectors, host cells, pharmaceutical compositions, and methods for treating inflammatory or autoimmune diseases and related pharmaceutical uses thereof.

[0005] PD-1 binding protein

[0006] In one aspect, the present disclosure provides a PD-1 binding protein comprising at least one immunoglobulin single variable domain comprising a CDR1, CDR2, and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 2 and 24-29, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. The immunoglobulin single variable domain specifically binds to a PD-1 protein or an epitope thereof.

[0007] In some embodiments, a PD-1 binding protein is provided, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 3-5, respectively, which are CDRs defined according to the Kabat numbering system.

[0008] In some embodiments, in the PD-1 binding protein, the immunoglobulin single variable domain comprises any one, two or three of the aforementioned CDR1, CDR2 and CDR3.

[0009] In some embodiments, the immunoglobulin single variable domain is of camel origin; in some specific embodiments, the immunoglobulin single variable domain comprises an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 90% sequence identity thereto.

[0010] In some embodiments, the immunoglobulin single variable domain is humanized and / or affinity matured.

[0011] In some embodiments, the immunoglobulin single variable domain in the aforementioned PD-1 binding protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 24-29, or an amino acid sequence having at least 90% sequence identity thereto.

[0012] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence as shown in SEQ ID NO: 27, or an amino acid sequence having at least 90% sequence identity thereto.

[0013] In some embodiments, the immunoglobulin single variable domain in the aforementioned PD-1 binding protein has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid mutations compared to any one of SEQ ID NOs: 24-29.

[0014] In some specific embodiments, the above-mentioned amino acid mutation is an amino acid replacement, substitution, modification, deletion and / or addition (such as a conservative substitution of an amino acid), and the mutation does not affect or substantially does not affect the function of specifically binding to PD-1.

[0015] In some embodiments, the aforementioned PD-1 binding protein comprises or is an antibody that specifically binds to the PD-1 protein or an epitope thereof. In some specific embodiments, the antibody is a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof.

[0016] In some embodiments, the aforementioned PD-1 binding protein is a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, a multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), or an antigen-binding fragment or conjugate of any of the foregoing.

[0017] In some embodiments, the immunoglobulin single variable domain in the aforementioned PD-1 binding protein is a single domain antibody or VHH.

[0018] In some embodiments, the present disclosure provides a PD-1 binding protein comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, wherein any two of the immunoglobulin single variable domains may be the same or different, and may form a dimer or multimer molecule.

[0019] In some embodiments, the aforementioned PD-1 binding protein further comprises an immunoglobulin Fc region. In some embodiments, the PD-1 binding protein further comprises a human immunoglobulin Fc region. For example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region. The PD-1 binding protein of the present disclosure may have an Fc region of a wild-type IgG or a variant thereof.

[0020] In some embodiments, the Fc has a mutation at position C220, such as C220A.

[0021] In some embodiments, the Fc region is an Fc region that increases effector function, e.g., increases antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) with increased effector function.

[0022] Exemplary IgG1 Fc regions include those with the following substitutions: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 80H / 290S / 298V;243L / 292P / 300L;243L / 292P / 300L / 396L;243L / 292P / 300L / 305I / 396L;236A / 239D / 332E;326A / 333A;326W / 333S;290E / 298G / 299A;290N / 298G / 299A;290E / 298G / 299A / 326E;or 290N / 298G / 299A / 326E;or any combination of any of the above positions. Said mutations are defined according to the EU numbering system.

[0023] Exemplary IgG1 Fc regions include those with the following substitutions: S239D; S239E; S239K, F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; or K290N / S298G / T299A / K326E, or any combination of the foregoing positions.

[0024] In some embodiments, the Fc region is an Fc region with reduced effector function, eg, reduced ADCC, ADCP, and / or CDC. Exemplary Fc regions with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0025] In some embodiments, the Fc region is human IgG1-Fc with a C220A mutation.

[0026] In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO: 6 or 45. In some specific embodiments, the PD-1 binding protein comprises the Fc region shown in SEQ ID NO: 6.

[0027] In some embodiments, a PD-1 binding protein is provided, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 7, 30, and 48, or having at least 90% sequence identity thereto. In some specific embodiments, the PD-1 binding protein comprises the amino acid sequence as set forth in SEQ ID NO: 30.

[0028] In some embodiments, an anti-PD-1 antibody or an antigen-binding fragment thereof is provided, which binds to or competes for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned PD-1 binding protein of the present disclosure.

[0029] In some embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof is provided that does not bind to or compete for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned PD-1 binding protein of the present disclosure.

[0030] In some embodiments, an anti-PD-1 antibody or an antigen-binding fragment thereof is provided, which blocks the binding of the immunoglobulin single variable domain in the aforementioned PD-1 binding protein of the present disclosure to PD-1 (e.g., human PD-1).

[0031] In some embodiments, an anti-PD-1 antibody or an antigen-binding fragment thereof is provided, wherein the binding of the antibody to PD-1 (e.g., human PD-1) is blocked by the immunoglobulin single variable domain in the aforementioned PD-1 binding protein of the present disclosure.

[0032] In some embodiments, the aforementioned PD-1 binding protein has at least one activity selected from the following:

[0033] (a)≤10 -7 K D The value binds to human PD-1 or its epitope;

[0034] (b) does not block or barely blocks the signaling pathway between PD-L1 and PD-1;

[0035] (c) Activation of the PD-1 signaling pathway;

[0036] (d) inhibiting the activity of T cells to release cytokines, such as inhibiting the activity of T cells to secrete IFNγ;

[0037] (e) inhibiting T cell proliferation, such as inhibiting memory CD8+ T cell proliferation;

[0038] (f) reduction of T cells expressing PD-1;

[0039] (g) Maintaining low levels of PD-1-expressing T cells.

[0040] In some embodiments, the aforementioned PD-1 binding protein of the present disclosure binds to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0041] In another aspect, the present disclosure provides a PD-1 binding protein comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0042] The VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 8 and 31-36; and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NOs: 9 and 37-42, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. The VH and VL specifically bind to the PD-1 antigen or an epitope thereof.

[0043] In some embodiments, a PD-1 binding protein is provided, comprising a heavy chain variable region (VH) and a light chain variable region (VL);

[0044] The VH comprises HCDR1, HCDR2 and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NOs: 10-12; and,

[0045] The VL comprises LCDR1, LCDR2 and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NOs: 13 to 15. The CDRs are defined according to the Kabat numbering system.

[0046] In some embodiments, the VH of the PD-1 binding protein comprises any one, two or three of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 10-12, and / or the VL comprises any one, two or three of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 13-15.

[0047] In some embodiments, the PD-1 binding protein is an anti-PD-1 antibody.

[0048] In some embodiments, the heavy chain variable region (VH) and light chain variable region (VL) of the anti-PD-1 antibody are of murine origin; in some specific embodiments, the VH comprises an amino acid sequence as shown in SEQ ID NO: 8 or having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 9 or having at least 90% sequence identity thereto, respectively.

[0049] In some embodiments, the anti-PD-1 antibody is humanized. In some specific embodiments, the humanization process uses the human germline heavy chain template IGHV1-3 and / or the human germline light chain template IGkV1-16.

[0050] In some embodiments, the anti-PD-1 antibody is a murine antibody, a chimeric antibody, or a humanized antibody or an antigen-binding fragment thereof. In some specific embodiments, the humanized antibody is based on the human germline heavy chain template IGHV1-3 and / or the human germline light chain template IGkV1-16.

[0051] In some embodiments, the VH of the aforementioned PD-1 binding protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 8 and 31-36, or a sequence having at least 90% sequence identity thereto; and / or, the VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9 and 37-42, or a sequence having at least 90% sequence identity thereto.

[0052] In some embodiments, the aforementioned PD-1 binding protein comprises VH and VL, wherein:

[0053] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 41, or a sequence having at least 90% sequence identity thereto;

[0054] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 37, or a sequence having at least 90% sequence identity thereto;

[0055] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38, or a sequence having at least 90% sequence identity thereto;

[0056] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 39, or a sequence having at least 90% sequence identity thereto;

[0057] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto;

[0058] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 37, or a sequence having at least 90% sequence identity thereto;

[0059] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38, or a sequence having at least 90% sequence identity thereto;

[0060] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 39, or a sequence having at least 90% sequence identity thereto;

[0061] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto;

[0062] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 37, or a sequence having at least 90% sequence identity thereto;

[0063] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38, or a sequence having at least 90% sequence identity thereto;

[0064] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 39, or a sequence having at least 90% sequence identity thereto;

[0065] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto;

[0066] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto;

[0067] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 42, or a sequence having at least 90% sequence identity thereto;

[0068] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36, or an amino acid sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 39, or an amino acid sequence having at least 90% sequence identity thereto; or

[0069] The VH comprises an amino acid sequence as set forth in SEQ ID NO: 36, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto.

[0070] In some specific embodiments, the aforementioned PD-1 binding protein comprises VH and VL, wherein:

[0071] The VH comprises the amino acid sequence shown in SEQ ID NO: 35, and the VL comprises the amino acid sequence shown in SEQ ID NO: 41;

[0072] The VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 37;

[0073] The VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 38;

[0074] The VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 39;

[0075] The VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40;

[0076] The VH comprises the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises the amino acid sequence shown in SEQ ID NO: 37;

[0077] The VH comprises the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises the amino acid sequence shown in SEQ ID NO: 38;

[0078] The VH comprises the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises the amino acid sequence shown in SEQ ID NO: 39;

[0079] The VH comprises the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40;

[0080] The VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 37;

[0081] The VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 38;

[0082] The VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 39;

[0083] The VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40;

[0084] The VH comprises the amino acid sequence shown in SEQ ID NO: 35, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40;

[0085] The VH comprises the amino acid sequence shown in SEQ ID NO: 35, and the VL comprises the amino acid sequence shown in SEQ ID NO: 42;

[0086] The VH comprises the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises the amino acid sequence shown in SEQ ID NO: 39; or

[0087] The VH comprises the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40.

[0088] In some embodiments, the VH in the aforementioned PD-1 binding protein has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 31-36, and / or the VL has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 37-42.

[0089] In some specific embodiments, the above-mentioned amino acid mutation is an amino acid replacement, substitution, modification, deletion and / or addition (such as a conservative substitution of an amino acid), and the mutation does not affect or substantially does not affect the function of specifically binding to PD-1.

[0090] In some embodiments, the aforementioned PD-1 binding protein comprises or is an antibody that specifically binds to the PD-1 protein or an epitope thereof. In some specific embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof.

[0091] In some embodiments, the aforementioned PD-1 binding protein comprises Fab, Fab', F(ab')2, or single-chain antibody (scFv) that specifically binds to PD-1.

[0092] In some embodiments, the aforementioned PD-1 binding protein further comprises an immunoglobulin Fc region. In some embodiments, the PD-1 binding protein further comprises a human immunoglobulin Fc region. For example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region. The PD-1 binding protein of the present disclosure may have an Fc region of a wild-type IgG or a variant thereof.

[0093] In some embodiments, the Fc region is an Fc region that increases effector function, e.g., increases antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) with increased effector function.

[0094] Exemplary IgG1 Fc regions include those with the following substitutions: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 80H / 290S / 298V;243L / 292P / 300L;243L / 292P / 300L / 396L;243L / 292P / 300L / 305I / 396L;236A / 239D / 332E;326A / 333A;326W / 333S;290E / 298G / 299A;290N / 298G / 299A;290E / 298G / 299A / 326E;or 290N / 298G / 299A / 326E;or any combination of any of the above positions. Said mutations are defined according to the EU numbering system.

[0095] Exemplary IgG1 Fc regions include those with the following substitutions: S239D; S239E; S239K, F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; or K290N / S298G / T299A / K326E, or any combination of the foregoing positions.

[0096] In some embodiments, the Fc region is an Fc region with reduced effector function, eg, reduced ADCC, ADCP, and / or CDC. Exemplary Fc regions with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0097] In some embodiments, the aforementioned PD-1 binding protein further comprises a human immunoglobulin Fc region, wherein the Fc region is as set forth in SEQ ID NO: 6 or 45, or has at least 80% or at least 90% sequence identity to either SEQ ID NO: 6 or 45. In some embodiments, the aforementioned PD-1 binding protein further comprises a human immunoglobulin Fc region, wherein the Fc region is as set forth in SEQ ID NO: 45.

[0098] In some embodiments, the aforementioned PD-1 binding protein further comprises a heavy chain constant region and a light chain constant region.

[0099] In some embodiments, the aforementioned PD-1 binding protein further comprises a human IgG1 heavy chain constant region and a human κ light chain constant region.

[0100] In some embodiments, the human IgG1 heavy chain constant region has the amino acid sequence set forth in SEQ ID NO: 16; and the human kappa light chain constant region has the amino acid sequence set forth in SEQ ID NO: 17.

[0101] In some embodiments, a PD-1 binding protein is provided, comprising a heavy chain and a light chain, wherein the heavy chain comprises a constant region sequence as set forth in SEQ ID NO: 16, or having at least 90% sequence identity thereto, and the light chain comprises a constant region sequence as set forth in SEQ ID NO: 17, or having at least 90% sequence identity thereto.

[0102] In some embodiments, a PD-1 binding protein is provided, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as set forth in either SEQ ID NO: 18 or 43, or having at least 90% sequence identity thereto; and / or the light chain comprises an amino acid sequence as set forth in either SEQ ID NO: 19 or 44, or having at least 90% sequence identity thereto.

[0103] In some embodiments, a PD-1 binding protein is provided, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 43, or a sequence having at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 44, or a sequence having at least 90% sequence identity thereto.

[0104] In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:43, and / or the light chain comprises the amino acid sequence set forth in SEQ ID NO:44.

[0105] In some embodiments, a PD-1 binding protein is provided, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 49, or a sequence having at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 50, or a sequence having at least 90% sequence identity thereto.

[0106] In some embodiments, the Fc region contained in the aforementioned PD-1 binding protein can enable the binding protein to form a dimeric molecule.

[0107] In some embodiments, the Fc region contained in the aforementioned PD-1 binding protein can extend the in vivo half-life of the binding protein.

[0108] In some embodiments, the PD-1 binding protein of the present disclosure is an anti-PD-1 antibody, or a conjugate or fusion protein comprising the antibody.

[0109] In some embodiments, the aforementioned PD-1 binding protein has at least one activity selected from the following:

[0110] (a)≤10 -7 K D The value binds to human PD-1 or its epitope;

[0111] (b) does not block or barely blocks the signaling pathway between PD-L1 and PD-1;

[0112] (c) Activation of the PD-1 signaling pathway;

[0113] (d) inhibiting the activity of T cells to release cytokines, such as inhibiting the activity of T cells to secrete IFNγ;

[0114] (e) inhibiting T cell proliferation, such as inhibiting memory CD8+ T cell proliferation;

[0115] (f) reduction of T cells expressing PD-1;

[0116] (g) Maintaining low levels of PD-1-expressing T cells.

[0117] In some embodiments, the aforementioned PD-1 binding protein of the present disclosure binds to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0118] In some embodiments, an anti-PD-1 antibody or an antigen-binding fragment thereof is provided, which binds to or competes for binding to the same epitope as the aforementioned PD-1 binding protein of the present disclosure.

[0119] In some embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof is provided that does not bind to or compete for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned PD-1 binding protein of the present disclosure.

[0120] In some embodiments, an anti-PD-1 antibody or an antigen-binding fragment thereof is provided, which blocks the binding of the immunoglobulin single variable domain in the aforementioned PD-1 binding protein of the present disclosure to PD-1 (e.g., human PD-1).

[0121] In some embodiments, an anti-PD-1 antibody or an antigen-binding fragment thereof is provided, wherein the binding of the antibody to PD-1 (e.g., human PD-1) is blocked by the immunoglobulin single variable domain in the aforementioned PD-1 binding protein of the present disclosure.

[0122] In the present disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity; "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0123] Polynucleotides and vectors

[0124] The present disclosure provides polynucleotides encoding the PD-1 binding proteins of the present disclosure. The polynucleotides of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the polynucleotides of the present disclosure are substantially isolated polynucleotides.

[0125] The polynucleotides of the present disclosure may also be in the form of a vector, may be present in a vector and / or may be part of a vector, such as a plasmid, cosmid, YAC or viral vector. The vector may in particular be an expression vector, i.e., a vector that provides for expression of the PD-1 binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector generally comprises at least one polynucleotide of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a specific host. Regulatory elements and other elements that are useful or necessary for the expression of the PD-1 binding protein of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0126] The polynucleotides of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0127] host cells

[0128] The present disclosure provides recombinant host cells that express or are capable of expressing one or more PD-1 binding proteins of the present disclosure and / or contain nucleic acids or vectors of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0129] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0130] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0131] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0132] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0133] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0134] Production or preparation method

[0135] The present disclosure provides a method for preparing the PD-1 binding protein of the present disclosure, which generally comprises the following steps:

[0136] - culturing the host cell of the present disclosure under conditions that allow expression of the binding protein of the present disclosure; and

[0137] - recovering the binding protein expressed by the host cell from the culture; and

[0138] - Optionally, further purification and / or modification of the binding protein of the present disclosure is involved.

[0139] The PD-1 binding proteins of the present disclosure can be produced intracellularly in the cells described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0140] Methods and reagents for recombinantly producing polypeptides, such as specific expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, isolation and purification techniques suitable for producing the binding proteins or antibodies disclosed herein are well known to those skilled in the art. Methods for producing and purifying antibodies are well known in the art and can be found, for example, in the Cold Spring Harbor Laboratory Manual (Chapters 5-8 and 15). The engineered antibodies disclosed herein can also be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The expression vectors can be stably transfected into cells. Mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors to produce antibodies. The culture medium containing the secreted antibodies can be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product should be frozen immediately, such as at -70°C, or freeze-dried.

[0141] However, the PD-1 binding proteins of the present disclosure can also be obtained by other methods known in the art for producing proteins, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0142] Composition / pharmaceutical composition

[0143] The present disclosure provides a composition comprising the aforementioned PD-1 binding protein.

[0144] For example, a pharmaceutical composition is provided, which contains a therapeutically, alleviatingly, or preventively effective amount of the PD-1 binding protein described above and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0145] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the PD-1 binding protein in a unit dosage, or the amount of the PD-1 binding protein in a unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some embodiments, 1-1000 mg.

[0146] In some embodiments, a product or kit is provided that comprises at least one container, each independently comprising a PD-1 binding protein as described above. Optionally, the kit comprises a container and a label. Containers such as bottles, syringes, and test tubes contain a composition effective for treating a condition. A label on or associated with the container indicates that the composition is used to treat the selected condition. The composition comprises a PD-1 binding protein as described above.

[0147] Methods for treating diseases and pharmaceutical uses

[0148] The present disclosure provides methods for using the aforementioned PD-1 binding proteins, polynucleotides, and compositions (including pharmaceutical compositions) for treating, alleviating, preventing, or diagnosing diseases or disorders.

[0149] In some embodiments, a method for improving, alleviating, treating or preventing a disease is provided, comprising administering to a subject an effective amount of the aforementioned PD-1 binding protein, polynucleotide or composition (including a pharmaceutical composition) for improving, alleviating, treating or preventing a disease.

[0150] In some embodiments, the present disclosure provides a use of the PD-1 binding protein in the preparation of a drug for improving, alleviating, treating or preventing a disease.

[0151] In some embodiments, the aforementioned disease is an inflammatory or immune disease.

[0152] In some embodiments, the inflammatory or immune disease is selected from the group consisting of graft-versus-host disease (GvHD), transplant rejection, rheumatoid arthritis (RA), and inflammatory bowel disease (CIA), or a combination thereof.

[0153] In some embodiments, a method of inhibiting T cell activity is provided, comprising administering an effective amount of the aforementioned PD-1 binding protein, polynucleotide, or composition (including pharmaceutical composition) to a subject in need thereof.

[0154] In some embodiments, the inhibitory T cell activity is characterized by at least one of the following:

[0155] (a) inhibiting the activity of T cells to release cytokines, such as inhibiting the activity of T cells to secrete IFNγ;

[0156] (b) inhibiting T cell proliferation, such as inhibiting memory CD8+ T cell proliferation;

[0157] (c) reducing PD-1 positive T cells, for example, reducing PD-1 high-expressing T cells.

[0158] Tests and kits

[0159] The present disclosure provides methods, systems, or devices for detecting PD-1 in vivo or in vitro, comprising treating a sample with the aforementioned binding proteins, polynucleotides, and compositions of the present disclosure.

[0160] In some embodiments, an in vitro detection method, system, or device may include, for example:

[0161] (1) contacting a sample with a PD-1 binding protein, polynucleotide, or composition;

[0162] (2) detecting a complex formed between the aforementioned binding protein, polynucleotide, and sample; and / or

[0163] (3) contacting a reference sample (e.g., a control sample) with the binding protein and nucleic acid; and

[0164] (4) Determining the extent of complex formation by comparison with a reference sample. A change (e.g., a statistically significant change) in complex formation in the sample or subject as compared to the control sample or subject indicates the presence of PD-1 in the sample.

[0165] In other embodiments, the in vivo detection method, system or device may include:

[0166] (1) administering the aforementioned binding protein or polynucleotide to a subject; and

[0167] (2) Detecting the formation of a complex between the binding protein, the polynucleotide, and the subject.

[0168] Detection can include determining the location or time of complex formation. The aforementioned binding proteins and polynucleotides are labeled with a detectable substance, and the detection of substances (such as PD-1) that can bind to proteins and polynucleotides is achieved by detecting the label. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. The formation of complexes between binding proteins, polynucleotides and PD-1 can be detected by measuring substances that are bound to or not bound to PD-1 or visualizing them. Conventional detection assays can be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry. For detection purposes, the binding proteins and polynucleotides of the present disclosure can be labeled with fluorophore chromophores. In some embodiments, diagnostic reagents comprising the above-mentioned polynucleotides and binding proteins are also provided, as well as related diagnostic uses.

[0169] In some embodiments, a kit is also provided, comprising the aforementioned binding protein, polynucleotide, and instructions for diagnostic use. The kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the binding protein may be formulated as a pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0170] Figures 1A and 1B show the PD-L1 antagonism experiments of antibodies 2E1-A06 and 113.3, respectively. Camrelizumab served as a positive control, while peresolimab and IgG1 served as negative controls. Figure 1A shows the results for antibody 2E1-A06, and Figure 1B shows the results for antibody 113.3.

[0171] Figures 2A and 2B show Jurkat reporter gene experiments using antibodies 2E1-A06 and 113.3, respectively, to activate the PD-1 signaling pathway. IgG1 served as a negative control. Figure 2A shows the results for antibody 2E1-A06, and Figure 2B shows the results for antibody 113.3.

[0172] Figure 3 shows the antagonistic effect of antibodies 2E1-A06-H4 and 113.3Hbk2L1+4 on PD-L1, with Camrelizumab as the positive control and Peresolimab and IgG1 as the negative controls.

[0173] Figure 4 shows the inhibitory activity of antibodies 2E1-A06-H4 and 113.3Hbk2L1+4 in the PBMC activation experimental system, with Peresolimab as a positive control.

[0174] Figure 5 shows the inhibitory activity of antibodies 2E1-A06-H4 and 113.3Hbk2L1+4 in the CMV recall experimental system, with Peresolimab as the positive control and IgG1 as the negative control.

[0175] FIG6 shows the antibody-dependent cell-mediated cytotoxicity (ADCC) assay of antibody 2E1-A06-H4, with Peresolimab as a positive control and IgG1 as a negative control.

[0176] FIG7 is an experimental flow chart for evaluating the activity of anti-PD-1 antibodies in a graft-versus-host disease (GvHD) mouse model.

[0177] Figures 8A and 8B show the results of administration of the antibody 2E1-A06-H4 in a graft-versus-host disease (GvHD) mouse model, with peresolimab and the model group (Isotype) serving as controls. Figure 8A shows the average weight change of the mice, and Figure 8B shows the average disease rejection score of the mice.

[0178] FIG9 is an experimental flow chart for evaluating the activity of anti-PD-1 antibodies in a collagen-induced arthritis (CIA) mouse model. DETAILED DESCRIPTION

[0179] Definition of terms

[0180] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0181] "Programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PDCD1," and "hPD-1" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one common epitope with PD-1. The complete PD-1 sequence is GenBank Accession #NP_005009.2.

[0182] "Binding to PD-1" means being able to interact with PD-1 or its epitope, which may be of human origin. "Antigen binding site" refers to a discrete three-dimensional site on an antigen that is recognized by the disclosed antibodies or antigen-binding fragments.

[0183] "PD-1 binding protein" encompasses any protein that can specifically bind to PD-1 or any molecule thereof. PD-1 binding proteins may include antibodies or conjugates thereof as defined in the present disclosure directed against PD-1. PD-1 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. The "PD-1 binding protein" of the present disclosure may comprise at least one immunoglobulin single variable domain (such as VHH) that binds to PD-1. In some embodiments, the "PD-1 binding protein" may comprise 2, 3, 4 or more immunoglobulin single variable domains (such as VHH) that bind to PD-1. In addition to comprising an immunoglobulin single variable domain of PD-1, the PD-1 binding protein of the present disclosure may also comprise a linker and / or a portion having an effector function, such as a half-life extending portion (such as an immunoglobulin single variable domain that binds to serum albumin), and / or a fusion partner (such as serum albumin) and / or a conjugated polymer (such as PEG) and / or an Fc region. The “PD-1 binding protein” of the present disclosure may comprise a heavy chain variable region (VH) and a light chain variable region (VL), such as Fab, Fab′, F(ab′)2, single-chain antibody (scFv), or IgG antibody.

[0184] The "PD-1 binding proteins" or "anti-PD-1 antibodies" of the present disclosure may contain one or more effector molecules, for example, in a conjugated manner. Such "effector molecules" include, for example, small molecule compounds, polypeptides or polypeptide fragments, biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides, and the like.

[0185] "Cytokine" is a general term for proteins released by a cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include IFNγ.

[0186] "Antibodies" encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds, such as IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0187] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (V region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs), whose sequences are relatively conserved. These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0188] "Antigen-binding fragment" encompasses single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3)209-217).

[0189] Methods for producing and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies may comprise multispecifics, such as bispecifics, or may be monospecifics (see, for example, WO92 / 22583 and WO05 / 113605), an example of which is Tri-Fab (or TFM) described in WO 92 / 22583.

[0190] For the determination or definition of CDRs, the definitive delineation of CDRs and the identification of residues in the binding site can be accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0191] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the positions of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequences (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, CDR positions can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs. CDR boundaries may be shortened or extended based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art.

[0192] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the well-known Kabat numbering system.

[0193] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly suitable for use in various embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies.

[0194] As used herein, "recombinant" refers broadly to products such as cells, nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0195] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0196] "Immunoglobulin domain" refers to the globular region of an antibody chain. An immunoglobulin domain is characterized in that it maintains the characteristic folding of the antibody molecule.

[0197] An "immunoglobulin variable domain" refers to an immunoglobulin domain consisting essentially of four "framework regions" (Framework Region 1, or FR1), "Framework Region 2, or FR2," "Framework Region 3, or FR3," and "Framework Region 4, or FR4), and three "complementarity determining regions" (Complementarity Determining Region 1, or CDR1), "Complementarity Determining Region 2, or CDR2," and "Complementarity Determining Region 3, or CDR3." Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0198] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0199] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the VH / VL interactions required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs), and antibodies that are VH domains or derived from VH domains. TM ) and (single domain) antibodies (such as dAbs) as the VL domain or derived from the VL domain TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0200] "VHH domain", also known as heavy chain single domain antibody, VHH, V H H domain, VHH antibody fragment, VHH antibody, nanobody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the VHH from the VH and VL present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to the epitope without the need for other antigen-binding domains (however, in conventional tetrapeptide chain structure antibodies, the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH", "V HH domain", "VHH antibody fragment", "VHH antibody", as well as" "VHH domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) can be used interchangeably. "VHH domain" includes but is not limited to natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained by phage display technology. The total number of amino acid residues in a VHH domain will generally be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009 and WO94 / 04678.

[0201] As is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions for VHH include Chothia, IMGT, and AbM.

[0202] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into the variable region framework of a human antibody. This can overcome the strong immune response induced by chimeric antibodies due to the large amount of non-human protein components they carry. To avoid a decrease in activity while also reducing immunogenicity, minimal reverse mutations can be performed on the fully human antibody variable region to maintain activity. Examples of "humanization" include "humanizing" a VHH domain derived from Camelidae by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a VH domain of a conventional human tetrapeptide chain antibody (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved properties of the VHH, such as removal of potential post-translational modification sites). A humanized VHH domain may contain one or more fully human framework region sequences. Another example of "humanization" includes transplanting the CDR sequence of a mouse to the antibody variable region framework of a person, i.e., antibodies produced in different types of human germline antibody framework sequences. The strong antibody variable antibody reaction induced by carrying a large amount of heterologous protein components can be overcome in chimeric antibodies. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework) are about to "transplant" CDRs on other "supports" (including but not limited to human supports or non-immunoglobulin supports). Supports and technologies suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies of the present disclosure also include humanized antibodies further subjected to affinity maturation of CDRs by phage display. In addition, in order to avoid a decrease in activity caused by a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0203] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody, such alterations resulting in an improvement in the affinity of the antibody for the antigen. For example, an "affinity matured" PD-1 binding protein or anti-PD-1 antibody has one or more alterations in one or more CDRs that result in an increase in affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0204] "Fully human antibodies" include antibodies with variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). "Fully human antibodies" do not include "humanized antibodies."

[0205] Generally, "specific binding" or "selective binding" refers to binding of a binding protein to an epitope on an antigen. The PD-1 binding protein of the present disclosure is measured as 10 -7 to 10 -10 Mole / L (M), or 10 -8 to 10 -10 M, or 10 -9 to 10 -10 M, or lower dissociation constant (K D ) binds to the antigen (i.e. PD-1) or its epitope. -4 M's K DValues ​​are generally considered to indicate nonspecific binding.Specific binding of a binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays as described in the present disclosure.

[0206] "Epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining the binding of an epitope to a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0207] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K DThe K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​are compared with those of non-target interactions (e.g., control antibodies known not to bind PD-1). D The value is evaluated.

[0208] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0209] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0210] "Nucleic acid" or "polynucleotide" are used interchangeably herein and refer to any DNA or RNA molecule, whether single-stranded or double-stranded, and its complementary sequence in the case of single-stranded, such as double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0211] "Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporating polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny are not necessarily identical (in morphology or genomic DNA complement) to the parent cell due to natural, accidental, or intentional mutations. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. The term includes mutant progeny that have the same function or biological activity as the cells screened for in the initial transformation.

[0212] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0213] "Inflammatory or autoimmune disease" refers to a disease or condition resulting from inappropriate activation of the immune system, which may include inflammatory diseases or conditions, autoimmune diseases or disorders. In some embodiments, "inflammatory disease" or "inflammatory condition" refers to a disease or condition in which a component of a mammal's immune system causes, mediates, or otherwise contributes to an inflammatory response that results in disease in the mammal. In some embodiments, "autoimmune disease" or "autoimmune disorder" refers to those diseases or disorders resulting from inappropriate activation of immune cells reactive to one's own tissues.

[0214] "Immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0215] "Downregulate" refers to a detectable decrease in the level of an immune response in a subject compared to the level of the response in the subject in the absence of the treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject.

[0216] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0217] "Treatment" means administering to a subject, for internal or external use, a therapeutic agent, such as a binding protein or a pharmaceutical composition thereof, comprising any of the present disclosure, as a therapeutic agent, to a subject who has, is suspected of having, or is predisposed to having one or more immune disorders for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or product) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0218] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0219] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including but not limited to."

[0220] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans.

[0221] Example

[0222] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0223] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0224] Example 1. Screening and preparation of anti-PD-1 nanobody (VHH)

[0225] 1.1 Screening, sequencing, and preparation of antigens

[0226] Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule. An extracellular fragment of the human PD-1 protein (Leu25-Gln167) with a C-terminal his-tag was selected as an immunogen for immunization of animals (camelids). The sequence, source, and use of the recombinant human PD-1 protein used in this disclosure are shown in Table 1. This protein reagent can be used in the experiments described in the following examples.

[0227] Table 1. Sources and uses of recombinant protein amino acid sequences

[0228] The complete PD-1 protein sequence is described as follows: 1-23aa is the signal peptide, 24-170aa is the extracellular region of PD-1 (underlined), 171-191aa is the transmembrane region, and 192-288aa is the intracellular region.

[0229] >Human PD-1 amino acid sequence

[0230] 1.2 Immunization, phage library construction and panning

[0231] After four immunizations, the serum titer of the alpaca was tested. Once the titer was acceptable, PBMCs were isolated, total RNA was extracted, tested for purity, and reverse transcribed into DNA for subsequent antibody gene amplification. The purified vector was then ligated with the target VHH fragment by enzyme digestion. Electroporation was performed, and clones were selected to generate a phage library.

[0232] Specific phage were screened using a solid-phase coating of PD-1 antigen and trypsin elution. After three rounds of panning, 384 clones were randomly selected from the second and third round titer plates. Positive clones were screened using phage ELISA and measured by optical density at 450 nm. Based on the sequencing results, amino acid sequence alignment and phylogenetic tree analysis were performed, ultimately yielding 25 unique sequences.

[0233] The activity of these antibodies was tested, including assays for activation of the PD-1 signaling pathway, inhibitory activity against memory T cells using a CMV recall assay, inhibition of T cell cytokine release using a PBMC activation assay, and FACS analysis of binding to human and monkey PD-1. The antibodies were also tested for drugability (e.g., aggregation, fragmentation, accelerated stability, and post-translational modifications (PTMs)). Anti-PD-1 antibodies with significant activity and drugability were identified, resulting in 2E1-A06-VHH, which was subsequently tested for subsequent testing.

[0234] The 2E1-A06-VHH sequence is as follows:

[0235] >2E1-A06-VHH

[0236] The underlined CDR sequences are determined according to the Kabat numbering system, as shown in Table 2.

[0237] Table 2. CDR sequences of 2E1-A06-VHH (according to the Kabat numbering system)

[0238] 1.3 Preparation of Nanobodies

[0239] The obtained 2E1-A06-VHH is fused with wild-type IgG1-Fc or IgG1-Fc with a mutation to obtain a Nanobody. For example, 2E1-A06-VHH is fused with human wild-type IgG1-Fc (Glu216-Lys447) with a C220A mutation (numbered according to the EU numbering system) to obtain a VHH-Fc chimeric antibody. The sequence of human wild-type IgG1-Fc with a C220A mutation is as follows:

[0240] >Human wild-type IgG1-Fc (Glu216-Lys447) with C220A mutation

[0241] >Human wild-type IgG1-Fc

[0242] The sequence of the 2E1-A06 antibody is as follows:

[0243] >2E1-A06

[0244] The underlined portion is human IgG1 Fc with the C220A mutation.

[0245] The gene sequence encoding the aforementioned antibody was synthesized and subcloned into the pcDNA3.1 expression vector. The expression vector and the transfection reagent PEI were transfected into CHO cells at a ratio of 1:2 and incubated in a CO2 incubator for 4-5 days. The expressed antibody was centrifuged, the supernatant was recovered, and the antibody was purified according to conventional methods. After testing, the target antibody was obtained.

[0246] Example 2. Screening and preparation of anti-PD-1 monoclonal antibodies (IgG)

[0247] 2.1 Screening, sequencing and preparation of antigens

[0248] The His-tagged human PD-1 (h-PD-1-His) recombinant protein and the His-tagged monkey PD-1 (cyno-PD-1-His) recombinant protein were both purified commercial protein reagents purchased from Acrobiosystems. The sources of their respective sequences are shown in Table 3. These protein reagents can be used in the experiments in the following examples.

[0249] Table 3. Sources and uses of recombinant protein amino acid sequences

[0250] 2.2 Preparation and screening of antibody hybridomas

[0251] Anti-human PD-1 monoclonal antibodies were generated by immunizing mice. Balb / C mice, 6 to 8 weeks old, were housed in an SPF-grade environment.

[0252] The immunizing antigen was a His-tagged recombinant human PD-1 protein (h-PD-1-His, prepared at 1 μg / μL in phosphate-buffered saline). It was emulsified with Freund's complete adjuvant (CFA) for the first dose and incomplete adjuvant for the remaining doses. Immunizations were administered on days 0, 14, 28, 42, and 70. Blood samples were collected on days 21, 35, 49, 63, and 77, and mouse serum was assayed by ELISA to determine antibody titers.

[0253] After the third immunization, mice with high antibody titers in the serum and whose titers tended to be stable were selected for spleen cell fusion, and boosted immunization was performed 3 days before fusion. Lymphocytes were fused with myeloma cells Sp2 / 0 cells by electrofusion to obtain hybridoma cells, and the hybridoma supernatant was screened for in vitro activity. The clones with good in vitro activity were sequenced, and the sequences were aligned and analyzed by evolutionary tree analysis based on the sequencing results to finally obtain the mouse monoclonal antibody sequence. The sequences of the heavy chain variable region (HCVR) and light chain variable region (LCVR) of monoclonal antibody m113.3 are as follows:

[0254] >m113.3-HCVR

[0255] >m113.3-LCVR

[0256] The underlined CDR sequences are determined according to the Kabat numbering system, see Table 4

[0257] Table 4. CDR sequences of m113.3 (according to the Kabat numbering system)

[0258] 2.3 Preparation of anti-PD-1 monoclonal antibodies

[0259] The obtained heavy chain variable region and light chain variable region were ligated to the human IgG1 heavy chain constant region and human kappa light chain constant region, respectively, to construct the human-mouse chimeric full-length antibody 113.3. The sequence is as follows:

[0260] >Human IgG1 heavy chain constant region

[0261] >Human kappa light chain constant region

[0262] >113.3-HC

[0263] >113.3-LC

[0264] Peresolimab is a PD-1 agonist developed by Eli Lilly and Company. Its heavy and light chain sequences are as follows, serving as the control antibody in the subsequent examples:

[0265] >Peresolimab HC

[0266] >Peresolimab LC

[0267] Camrelizumab is a PD-1 antagonist developed by Hengrui Medicine that can block the binding of PD-L1 to PD-1 and is used for anti-tumor treatment. Its specific sequence is as follows, used as a control antibody in subsequent examples:

[0268] >Camrelizumab HC

[0269] >Camrelizumab LC

[0270] In the above sequence, the underlined heavy chain is the human IgG1 Fc region, and the underlined light chain is the human Cκ region.

[0271] The gene sequence encoding the aforementioned antibody was synthesized and subcloned into the pcDNA3.1 expression vector. The expression vector and the transfection reagent PEI were transfected into CHO cells at a ratio of 1:2 and incubated in a CO2 incubator for 4-5 days. The expressed antibody was centrifuged, the supernatant was recovered, and the antibody was purified according to conventional methods. After testing, the target antibody was obtained.

[0272] Example 3. Affinity of anti-PD-1 antibodies

[0273] 3.1 SPR affinity determination of anti-PD-1 antibodies for PD-1 protein

[0274] Experimental Methods: Detection was performed using a Biacore 8K instrument (GE Healthcare). A Protein A sensor chip was used, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Each test antibody was prepared in HBS-EP+ buffer as a ligand and captured using Protein A on the chip channel. Human PD-1 antigen protein (ACRO, PD1-H5221) was prepared in HBS-EP+ buffer. A two-fold serial dilution from 100 nM was performed across seven concentration points. The analyte was passed through the experimental and reference channels at a flow rate of 30 μL / min, with an association time of 90 s and a dissociation time of 500 s. Regeneration buffer (10 mM Glycine pH 1.5 (GE Healthcare, 29238268-AA) was used for 30 s at a flow rate of 10 μL / min. Data were processed using Biacore 8K Evaluation software. The signal value of the corresponding reference channel (Fc 1) was subtracted from the signal value of the detection channel (Fc 2) to generate a corrected signal curve. Affinity kinetics curves were fitted to a 1:1 Langmuir binding model, and the association rate, Ka, and dissociation rate, Kd, ​​as well as the dissociation constant (i.e., affinity, KD), were calculated.

[0275] The results showed that antibodies 2E1-A06 and 113.3 bound to human PD-1 protein, and their affinities are shown in Table 5.

[0276] Table 5. Affinity of anti-PD-1 antibodies and PD-1 recombinant protein

[0277] 3.2 Anti-PD-1 Antibody Antagonism Experiment on PD-L1

[0278] The antagonism of chimeric antibodies against PD-L1 was detected using an experimental system in which PD-L1-overexpressing cells were co-cultured with Jurkat reporter gene cell lines.

[0279] Experimental method: 40,000 PD-L1 aAPC / CHO-K1 cells (Promega) were plated in 96-well plates and cultured overnight. The next day, the cells were washed with PBS buffer and 1640 medium, and 50,000 Jurkat PD-1NFAT reporter cells overexpressing human PD-1 and different concentrations of the test antibodies were added. After the cells were cultured in a 37° incubator for 6 hours, Bright-Glo TM (Promega) to detect the expression of reporter genes.

[0280] As shown in Figures 1A and 1B, the PD-1 antagonist control antibody Camrelizumab exhibited antagonistic PD-L1 function and induced significant luciferase expression; while 2E1-A06, 113.3, and Peresolimab did not antagonize the binding of PD-L1 to PD-1.

[0281] 3.3 Reporter gene experiments using anti-PD-1 antibodies to activate the PD-1 signaling pathway

[0282] The activation activity of chimeric antibodies on PD-1 was explored using an experimental system in which THP-1 cells and Jurkat reporter gene cells were co-cultured.

[0283] Experimental method: 100,000 Jurkat PD-1NFAT reporter cells overexpressing human PD-1 were plated in 96-well plates and different concentrations of the test antibodies were added. At the same time, 50,000 THP-1 cells overexpressing CD32a after treatment with mitomycin C were added and incubated at 37° for 30 minutes. Subsequently, 2μg / ml anti-CD3 (Biolegend, OKT3) and 1μg / ml anti-CD28 (Biolegend) were added to activate the Jurkat cells to express the luciferase reporter gene. After the co-incubated cells were incubated at 37° for another 5 hours, the expression of the reporter gene was detected using Bright-GloTM (Promega).

[0284] As shown in Figures 2A and 2B, both 2E1-A06 and 113.3 significantly inhibited the expression of the luciferase reporter gene, indicating that both antibodies can significantly activate the PD-1 signaling pathway.

[0285] Example 4. Humanized modification of anti-PD-1 antibodies

[0286] 4.1 Humanization of anti-PD-1 nanobodies

[0287] Nanobody humanization utilizes a CDR grafting approach. Using the IMGT or NCBI website, the parental PD-1 nanobody sequence is aligned with fully human germline genes in the IMGT database. Human germline genes with high homology to the PD-1 nanobody are selected as humanized framework region templates (FRs). The CDR sequences of the camelid parental antibody are then grafted into the framework regions (FRs) of the corresponding germline genes.

[0288] At the same time, during the humanization process, based on the results of structural analysis of the sequence and antibody homology modeling, key core residues near the CDR region were back-mutated to ultimately obtain humanized antibody mutants. The following is the humanized sequence of 2E1-A06-VHH:

[0289] >2E1-A06-VHH-H1

[0290] >2E1-A06-VHH-H2

[0291] >2E1-A06-VHH-H3

[0292] >2E1-A06-VHH-H4

[0293] >2E1-A06-VHH-H5

[0294] >2E1-A06-VHH-H6

[0295] In the above sequences, the CDRs are underlined (numbered according to the Kabat system).

[0296] The above humanized VHHs were fused to human wild-type IgG1-Fc (Glu216-Lys447) with the C220A mutation (numbered according to the EU numbering system) to obtain the full-length sequence. For example, 2E1-A06-VHH-H4 (SEQ ID NO: 27) was fused to human wild-type IgG1-Fc (Glu216-Lys447) to obtain 2E1-A06-H4 (SEQ ID NO: 30).

[0297] >2E1-A06-H4

[0298] The underlined portion is the human IgG1 Fc region with the C220A mutation.

[0299] The gene sequence encoding the aforementioned antibody was synthesized and subcloned into the pcDNA3.1 expression vector. The expression vector and the transfection reagent PEI were transfected into CHO cells at a ratio of 1:2 and incubated in a CO2 incubator for 4-5 days. The expressed antibody was centrifuged, the supernatant was recovered, and the antibody was purified according to conventional methods. After testing, the target antibody was obtained.

[0300] 4.2 Humanization of anti-PD-1 mouse monoclonal antibody

[0301] Based on the representative structure of mouse mAb 113.3, the heavy and light chain variable region sequences were compared with an antibody germline database to obtain human germline templates with high homology. For antibody 113.3, human germline heavy chain templates IGHV1-3 and human germline light chain templates IGkV1-16 were selected. The CDR regions of the mouse mAb were transplanted onto the selected humanized templates, replacing the existing CDR regions in the humanized templates to construct the variable regions. Based on the three-dimensional structure of the mouse mAb, backmutations were performed on buried residues, residues that directly interact with the CDR regions, and residues that have a significant impact on VH and VL conformation to generate a series of humanized antibodies.

[0302] 113.3 The heavy chain variable regions of each humanized antibody are as follows:

[0303] >113.3 H1CVR

[0304] >113.3 H2CVR

[0305] >113.3 H3CVR

[0306] >113.3 HbkCVR

[0307] >113.3 Hbk2CVR

[0308] >113.3 Hbk3CVR

[0309] In the above sequence, the underlined ones are the heavy chain CDRs.

[0310] 113.3 The light chain variable regions of each humanized antibody are as follows:

[0311] >113.3 L1CVR

[0312] >113.3 L2CVR

[0313] >113.3 L3CVR

[0314] >113.3 L4CVR

[0315] >113.3 L1+4CVR

[0316] >113.3 L2+4CVR

[0317] In the above sequence, the light chain CDR is underlined.

[0318] The above heavy chain variable regions and light chain variable regions were connected to the human IgG1 heavy chain constant region and the human Cκ light chain constant region, respectively, to obtain the full-length sequence.

[0319] The full-length heavy chain and light chain sequences of 113.3Hbk2L1+4 are shown below as an example:

[0320] >113.3 Hbk2C

[0321] >113.3 L1+4C

[0322] In the above sequence, the underlined heavy chain is the human IgG1 Fc region, and the underlined light chain is the human Cκ region.

[0323] The above-mentioned humanized full-length heavy chain sequences and full-length light chain sequences were combined to obtain the following humanized molecules, as shown in Table 6:

[0324] Table 6. Anti-PD-1 humanized antibodies

[0325] The gene sequence encoding the aforementioned antibody was synthesized and subcloned into the pcDNA3.1 expression vector. The expression vector and the transfection reagent PEI were transfected into CHO cells at a ratio of 1:2 and incubated in a CO2 incubator for 4-5 days. The expressed antibody was centrifuged, the supernatant was recovered, and the antibody was purified according to conventional methods. After testing, the target antibody was obtained.

[0326] Example 5. Anti-PD-1 Antibody Affinity Determination

[0327] SPR was used to detect the affinity of anti-PD-1 antibodies to recombinant human PD-1 protein.

[0328] Experimental Methods: Detection was performed using a Biacore 8K instrument (GE Healthcare). A Protein A sensor chip was used, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Each test antibody was prepared in HBS-EP+ buffer as a ligand and captured using Protein A on the chip channel. Human PD-1 antigen protein (ACRO, PD1-H5221) was prepared in HBS-EP+ buffer. A two-fold serial dilution from 100 nM was performed across seven concentration points. The analyte was passed through the experimental and reference channels at a flow rate of 30 μL / min, with an association time of 90 s and a dissociation time of 500 s. Regeneration buffer (10 mM Glycine pH 1.5 (GE Healthcare, 29238268-AA) was used for 30 s at a flow rate of 10 μL / min. Data were processed using Biacore 8K Evaluation software. The signal value of the corresponding reference channel (Fc 1) was subtracted from the signal value of the detection channel (Fc 2) to generate a corrected signal curve. Affinity kinetics curves were fitted to a 1:1 Langmuir binding model, and the association rate, Ka, and dissociation rate, Kd, ​​as well as the dissociation constant (i.e., affinity, KD), were calculated.

[0329] The results showed that both humanized antibodies 2E1-A06-H4 and 113.3Hbk2L1+4 could bind to human PD-1 protein, and the affinities were shown in Table 7.

[0330] Table 7. Affinity test results of anti-PD-1 antibodies to PD-1 recombinant protein

[0331] Example 6. Activity determination of anti-PD-1 antibodies

[0332] 6.1 Anti-PD-1 Antibody Antagonism Experiment on PD-L1

[0333] The antagonism of humanized antibodies against PD-L1 was detected using an experimental system in which PD-L1-overexpressing cells were co-cultured with Jurkat reporter gene cell lines.

[0334] Experimental method: 40,000 PD-L1 aAPC / CHO-K1 cells (Promega) were plated in 96-well plates and cultured overnight. The next day, the cells were washed with PBS buffer and 1640 medium, and 50,000 Jurkat PD-1NFAT reporter cells overexpressing human PD-1 and different concentrations of the test antibodies were added. After the cells were cultured in a 37° incubator for 6 hours, Bright-GloTM (Promega) to detect the expression of reporter genes.

[0335] The results are shown in Figure 3. Only the PD-1 antagonist Camrelizumab induced significant expression of the luciferase reporter gene, while 2E1-A06-H4, 113.3Hbk2L1+4, and Peresolimab did not hinder the binding of PD-L1 to PD-1.

[0336] 6.2 Inhibitory Activity of Anti-PD-1 Antibodies in PBMC Activation Experimental System

[0337] The PBMC activation assay was used to evaluate the activity of humanized antibodies in inhibiting the release of cytokines from T cells.

[0338] Experimental Methods: PBMCs were incubated with 0.05 μg / mL PHA for 72 hours and plated at 200,000 cells / well in 96-well plates pre-coated with 0.01 μg / mL anti-CD3 and various concentrations of humanized antibodies. Anti-CD28 at 0.2 μg / mL was also added and incubated. After overnight incubation at 37°C, IFNγ secretion in the supernatant was measured using the HTRF human IFN gamma kit (Cisbio).

[0339] The results, as shown in Figure 4, show that both 2E1-A06-H4 and 113.3Hbk2L1+4 inhibited IFNγ secretion in a dose-dependent manner, significantly outperforming the agonist molecule peresolimab. The conclusions of this experiment were reproducibly verified in four different PBMC donors (see Table 8).

[0340] Table 8. Anti-PD-1 antibodies inhibit the secretion of IFNγ by T cells from different PBMC donors in the PBMC activation experimental system

[0341] 6.3 Inhibitory activity of humanized antibodies in the CMV recall assay

[0342] The CMV recall assay was used to evaluate the inhibitory activity of humanized antibodies against memory T cells.

[0343] Experimental Methods: 200,000 resuscitated PBMCs were incubated with 3-50 ng / mL of a CMV peptide pool (Mabtech) and various concentrations of humanized antibodies. After 5 days of culture, expression of the proliferation marker Ki67 (BD) in memory CD8+ T cells was detected using various fluorescently labeled antibodies and a fluorescent secondary antibody, anti-human IgG Fc (Invitrogen).

[0344] The results, as shown in Figure 5, show that both 2E1-A06-H4 and 113.3Hbk2L1+4 were able to inhibit the proliferation of memory CD8+ T cells, with inhibitory activity superior to that of the agonist molecule peresolimab. The conclusions of this experiment were reproducibly verified in different PBMC donors, as shown in Table 9.

[0345] Table 9. Humanized antibodies inhibit the activity of memory T cells from different PBMC donors in the CMV recall experimental system

[0346] Example 7. ADCC activity of anti-PD-1 antibodies

[0347] The ADCC assay was used to evaluate the humanized antibody-mediated NK cell killing ability against PD-1 positive cells.

[0348] Experimental method: CD4+ T cells were sorted and enriched from fresh primary human peripheral blood PBMC using a sorting kit (Stem cell). After pre-coating with 2μg / mL anti-CD3 and 1μg / mL anti-CD28 for 48 hours, the cells were detected by CellTrace TM Violet (Invitrogen) was used for light labeling. After 20 minutes of incubation, CD4+ T cells were further incubated with 100 nM of the test antibody. Fresh primary NK cells were added (NK:CD4+ T = 5:1), mixed thoroughly, and incubated at 37° for 4 hours. The proportion of apoptotic cells in CD4+ T cells was determined using PI (Biyuntian) staining.

[0349] The results are shown in FIG6 or Table 10. In two different donors, 2E1-A06-H4 can induce ADCC killing, and the killing activity is better than that of Peresolimab.

[0350] Table 10. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of anti-PD-1 antibodies

[0351] Example 8. Verification of in vivo function and effect of anti-PD-1 antibodies

[0352] 8.1 Anti-PD-1 Antibody Activity Evaluation in Graft-Versus-Host Disease (GvHD) Model

[0353] Experimental method: 6-8 week old female NOG mice (Vitamin B) were adaptively raised in an SPF environment. The mice in average groups were modeled by intravenous injection of primary human peripheral blood PBMC (5,000,000 cells). Each group of mice was treated with equimolar doses starting from the day of modeling, and the body weight and rejection score of each mouse were tested twice a week (evaluated from the skin, coat color, posture and activity), and the mice were euthanized 35 days after induction. The specific experimental process is shown in Figure 7, and the dosing regimen is shown in Table 11. Mice were administered according to the following regimen, and the agonist activity of the humanized antibody was evaluated by two indicators: mouse body weight change and rejection score.

[0354] Table 11. Dosing regimen for graft-versus-host disease (GvHD) model

[0355] The results are shown in Figures 8A and 8B and Table 12. Both 2E1-A06-H4 and the control PD-1 agonist Peresolimab significantly inhibited mouse weight loss and rejection scores (statistical differences were calculated between each group and the model group data, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). 2E1-A06-H4 was significantly more effective than the control Peresolimab in maintaining mouse body weight and inhibiting rejection scores (statistical differences were calculated between the 2E1-A06-H4 group and the Peresolimab group data, #, p < 0.05; ##, p < 0.01; ###, p < 0.001; ####, p < 0.0001), indicating that in this model, 2E1-A06-H4 more significantly inhibited human PBMC activation than Peresolimab and had superior in vivo agonist activity.

[0356] Table 12. Average body weight change and average clinical scores in the graft-versus-host disease (GvHD) model

[0357] 8.2 Anti-PD-1 Antibody Activity Evaluation in Collagen-Induced Arthritis (CIA) Mouse Model

[0358] To better mimic the human mechanism of antibody action in mice, we modified the Fc of 2E1-A06-H4 to mouse IgG2a, renaming it 2E1-A06-H4-mIgG2a. Peresolimab and 113.3Hbk2L1+4 retained only the VH and VL sequences, modified the heavy chain CH1 and Fc to mIgG2a, and the light chain constant region to mouse kappa, renaming them Peresolimab-mIgG2a and 113.3Hbk2L1+4-mIgG2a, respectively. The specific sequences are as follows:

[0359] >Peresolimab-mIgG2a HC

[0360] >Peresolimab-mIgG2a LC

[0361] >2E1-A06-H4-mIgG2a

[0362] >113.3 Hbk2L1+4-mIgG2a HC

[0363] >113.3 Hbk2L1+4-mIgG2a LC

[0364] Experimental method: 8-9 week old male C57BL / 6 strain human PD-1 transgenic mice (Biocytogen) were adaptively raised in an SPF environment. On days 0 and 21, the mice were intradermally injected with 0.2 mg of type II collagen and an adjuvant emulsion at the base of the tail. Starting from the second induction, the mouse limb arthritis scores were performed three times a week. On the 30th day after the first induction, the mice were evenly divided into groups according to the mouse arthritis scores and peripheral C-reactive protein levels, and drug treatment was started at the same time. Thereafter, the scoring frequency was maintained at three times a week, and the mice were euthanized at the end of the experiment on the 57th day to detect pathological changes in the right hind joint. The specific experimental process is shown in Figure 9. Mice were administered according to the following scheme, and the agonist activity of the humanized antibody was evaluated by the mouse arthritis score and the joint pathology score at the end point.

[0365] Table 13. Dosing regimen for collagen-induced arthritis (CIA) mouse model

[0366] The results showed that 2E1-A06-H4-mIgG2a significantly inhibited arthritis scores in mice, while the control peresolimab-mIgG2a had a weaker effect on improving the total disease score. Individual analysis of the changes in scores after dosing compared to grouping on day 30 revealed that 2E1-A06-H4-mIgG2a induced arthritis scores to change below 0 in most animals, indicating arthritis relief. In the control peresolimab-mIgG2a group, some individuals experienced disease remission in the later stages of the disease (Table 14). These data demonstrate that 2E1-A06-H4-mIgG2a has a significant pharmacological advantage over peresolimab-mIgG2a in inhibiting joint damage in a mouse arthritis model.

[0367] Table 14. Arthritis scores and arthritis relief ratios of mice in each group in the arthritis (CIA) mouse model

[0368] Scoring data are Mean ± SEM; the arthritis remission rate is defined as the proportion of individuals whose arthritis score at the time of testing changed < 0 compared with the arthritis score at the time of group administration.

Claims

1. A PD-1 binding protein comprising at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 2 and 24-29, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

2. The PD-1 binding protein according to claim 1, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 3-5.

3. The PD-1 binding protein according to claim 1 or 2, wherein the immunoglobulin single variable domain is of camel origin, humanized, and / or affinity matured.

4. The PD-1 binding protein according to any one of claims 1 to 3, wherein The immunoglobulin single variable domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2 and 24-29, or an amino acid sequence having at least 90% sequence identity thereto.

5. The PD-1 binding protein according to any one of claims 1 to 4, comprising an amino acid sequence as shown in SEQ ID NO: 30, 7 or 48, or an amino acid sequence having at least 90% sequence identity thereto.

6. The PD-1 binding protein according to any one of claims 1 to 5, wherein The PD-1 binding protein is an anti-PD-1 antibody; preferably, the anti-PD-1 antibody is a camel antibody, a chimeric antibody, a humanized antibody or an antigen-binding fragment thereof.

7. A PD-1 binding protein comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 8 and 31-36; and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 9 and 37-42, The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

8. The PD-1 binding protein of claim 7, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3; wherein the HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences set forth in SEQ ID NOs: 10-12; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3; wherein the LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences set forth in SEQ ID NOs: 13-15.

9. The PD-1 binding protein according to claim 7 or 8, wherein the PD-1 binding protein is an anti-PD-1 antibody, and the anti-PD-1 antibody is a murine antibody, a chimeric antibody, or a humanized antibody or an antigen-binding fragment thereof; Preferably, the humanized antibody uses the human germline heavy chain template IGHV1-3, and / or the human germline light chain template IGkV1-16.

10. The PD-1 binding protein according to any one of claims 7 to 9, wherein The VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 8 and 31-36, or a sequence having at least 90% sequence identity thereto; and the VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9 and 37-42, or a sequence having at least 90% sequence identity thereto; Preferably, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 41, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 37, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 39, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 37, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 39, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 37, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 39, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 42, or a sequence having at least 90% sequence identity thereto; the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 39, or a sequence having at least 90% sequence identity thereto; The VH comprises an amino acid sequence as shown in SEQ ID NO: 36, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 40, or a sequence having at least 90% sequence identity thereto; or, The VH comprises an amino acid sequence as set forth in SEQ ID NO: 8, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 9, or a sequence having at least 90% sequence identity thereto. The PD-1 binding protein according to any one of claims 1 to 10, further comprising an immunoglobulin Fc region; preferably, the Fc region is the Fc region of human IgG1, IgG2 or IgG4.

12. The PD-1 binding protein according to claim 11, comprising a heavy chain and a light chain, wherein: The heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 43, or at least 90% identical thereto; and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 44, or at least 90% identical thereto; The heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 49, or a sequence having at least 90% sequence identity thereto; and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 50, or a sequence having at least 90% sequence identity thereto; or The heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence having at least 90% sequence identity thereto; and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having at least 90% sequence identity thereto.

13. A polynucleotide encoding the PD-1 binding protein according to any one of claims 1 to 12. A host cell comprising the polynucleotide of claim 13 .

15. A pharmaceutical composition comprising the PD-1 binding protein according to any one of claims 1 to 12 and at least one pharmaceutically acceptable excipient, diluent or carrier.

16. A method for preparing a PD-1 binding protein, comprising: 1) culturing the host cell according to claim 14, and 2) isolating the expressed PD-1 binding protein from the host cell.

17. Use of the PD-1 binding protein according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 15, in the preparation of a medicament for treating inflammatory or autoimmune diseases; Preferably, the inflammatory or autoimmune disease is selected from graft-versus-host disease (GvHD), transplant rejection, arthritis (CIA), rheumatoid arthritis (RA).

18. A method for treating an inflammatory or autoimmune disease, comprising administering to a subject a therapeutically effective amount of the PD-1 binding protein of any one of claims 1 to 12, the polynucleotide of claim 13, or the pharmaceutical composition of claim 15; Preferably, the inflammatory or autoimmune disease is selected from graft-versus-host disease (GvHD), transplant rejection, arthritis (CIA), or rheumatoid arthritis (RA).

19. A method for downregulating an immune response, comprising administering to a subject a therapeutically effective amount of the PD-1 binding protein of any one of claims 1 to 12, the polynucleotide of claim 13, or the pharmaceutical composition of claim 15.

20. A method for inhibiting T cell activity, comprising administering to a subject in need thereof an effective amount of the PD-1 binding protein of any one of claims 1 to 12, the polynucleotide of claim 13, or the pharmaceutical composition of claim 15; Preferably, the inhibitory T cell activity is selected from at least one of the following features: (a) inhibiting the activity of T cells to release cytokines, such as inhibiting the activity of T cells to secrete IFNγ; (b) inhibiting T cell proliferation, such as inhibiting memory CD8+ T cell proliferation; (c) Reduce PD-1 positive T cells.

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