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WO2025195358A1 - Protéine de liaison à pd-1 et utilisation pharmaceutique - Google Patents

Protéine de liaison à pd-1 et utilisation pharmaceutique

Info

Publication number
WO2025195358A1
WO2025195358A1 PCT/CN2025/083096 CN2025083096W WO2025195358A1 WO 2025195358 A1 WO2025195358 A1 WO 2025195358A1 CN 2025083096 W CN2025083096 W CN 2025083096W WO 2025195358 A1 WO2025195358 A1 WO 2025195358A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
acid sequence
seq
sequence identity
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/083096
Other languages
English (en)
Chinese (zh)
Inventor
粟璐
温泽
徐娜
高焓
林�源
廖成
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
Original Assignee
Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Hengrui Pharmaceutical Co Ltd, Shanghai Shengdi Pharmaceutical Co Ltd filed Critical Jiangsu Hengrui Pharmaceutical Co Ltd
Publication of WO2025195358A1 publication Critical patent/WO2025195358A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • 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.
  • Programmed cell death-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.
  • 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.
  • 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.
  • 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.
  • a PD-1 binding protein wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 3-5, respectively.
  • CDRs are defined according to the Kabat numbering system.
  • the immunoglobulin single variable domain comprises any one, two or three of the aforementioned CDR1, CDR2 and CDR3.
  • 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 having at least 90% sequence identity thereto.
  • the immunoglobulin single variable domain is humanized and/or affinity matured.
  • 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 having at least 90% sequence identity therewith.
  • the immunoglobulin single variable domain comprises an amino acid sequence as shown in SEQ ID NO:27 or having at least 90% sequence identity thereto.
  • 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.
  • 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.
  • the aforementioned PD-1 binding protein comprises or is an antibody that specifically binds to the PD-1 protein or an epitope thereof.
  • the antibody is a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof.
  • 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.
  • the immunoglobulin single variable domain in the aforementioned PD-1 binding protein is a single domain antibody or VHH.
  • 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.
  • the aforementioned PD-1 binding protein further comprises an immunoglobulin Fc region.
  • the PD-1 binding protein further comprises a human immunoglobulin Fc region.
  • 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.
  • the Fc has a mutation at position C220, such as C220A.
  • 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.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • 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/29 8D;280H/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
  • 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/S29 8D;D280H/K290S/S298V;F243L/R292P/Y300L;F243L/R292P/Y300L/P396L;F243L/R292P/Y300L/V305I/P396L;G236
  • the Fc region is an Fc region with reduced effector function, eg, reduced ADCC, ADCP, and/or CDC.
  • 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).
  • the Fc region is human IgG1-Fc with a C220A mutation.
  • the Fc region comprises the amino acid sequence shown in SEQ ID NO: 6 or 45.
  • the PD-1 binding protein comprises the Fc region shown in SEQ ID NO: 6.
  • a PD-1 binding protein 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.
  • the PD-1 binding protein comprises the amino acid sequence as set forth in SEQ ID NO: 30.
  • an anti-PD-1 antibody or an antigen-binding fragment thereof 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.
  • 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.
  • an anti-PD-1 antibody or an antigen-binding fragment thereof 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).
  • 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.
  • PD-1 e.g., human PD-1
  • the aforementioned PD-1 binding protein has at least one activity selected from the following:
  • T cell proliferation such as inhibiting memory CD8+ T cell proliferation
  • the aforementioned PD-1 binding proteins of the present disclosure may bind to PD-1 with a KD value of ⁇ 1 ⁇ 10 ⁇ 7 M, e.g., ⁇ 1 ⁇ 10 ⁇ 8 M, or ⁇ 1 ⁇ 10 ⁇ 9 M, or ⁇ 1 ⁇ 10 ⁇ 10 M.
  • the present disclosure provides 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 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.
  • a PD-1 binding protein comprising a heavy chain variable region (VH) and a light chain variable region (VL);
  • the VH comprises HCDR1, HCDR2 and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NOs: 10-12; and,
  • the VL comprises LCDR1, LCDR2, and LCDR3, each comprising an amino acid sequence as shown in SEQ ID NOs: 13-15.
  • the CDRs are defined according to the Kabat numbering system.
  • the VH of the PD-1 binding protein comprises any one, two or three of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 10-12, and/or the VL comprises any one, two or three of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 13-15.
  • the PD-1 binding protein is an anti-PD-1 antibody.
  • 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.
  • the anti-PD-1 antibody is humanized.
  • the humanization process uses the human germline heavy chain template IGHV1-3 and/or the human germline light chain template IGkV1-16.
  • the anti-PD-1 antibody is a murine antibody, a chimeric antibody, or a humanized antibody or an antigen-binding fragment thereof.
  • the humanized antibody is based on the human germline heavy chain template IGHV1-3 and/or the human germline light chain template IGkV1-16.
  • 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 therewith; 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 therewith.
  • the aforementioned PD-1 binding protein comprises VH and VL, wherein:
  • the VH comprises an amino acid sequence as shown in SEQ ID NO: 35, or an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO: 41, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31 or having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as set forth in SEQ ID NO: 37 or having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31 or having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38 or having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as set forth in SEQ ID NO: 31 or having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as set forth in SEQ ID NO: 39 or having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as shown in SEQ ID NO: 31 or a sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as shown 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 an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as set forth in SEQ ID NO: 37, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, or an amino acid sequence having at least 90% sequence identity thereto
  • 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;
  • the VH comprises an amino acid sequence as shown in SEQ ID NO: 32, or an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO: 40, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as set forth in SEQ ID NO: 37, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as set forth in SEQ ID NO: 33, or an amino acid sequence having at least 90% sequence identity thereto
  • 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;
  • the VH comprises an amino acid sequence as shown in SEQ ID NO: 33, or an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO: 40, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as shown in SEQ ID NO: 35, or an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO: 40, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as shown in SEQ ID NO: 35, or an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO: 42, or an amino acid sequence having at least 90% sequence identity thereto;
  • the VH comprises an amino acid sequence as shown in SEQ ID NO: 36, or an amino acid sequence having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO: 39, or an amino acid sequence having at least 90% sequence identity thereto; or
  • the VH comprises an amino acid sequence as shown in SEQ ID NO: 36 or having at least 90% sequence identity thereto
  • the VL comprises an amino acid sequence as shown in SEQ ID NO: 40 or having at least 90% sequence identity thereto.
  • the aforementioned PD-1 binding protein comprises VH and VL, wherein:
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • 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;
  • the VH comprises the amino acid sequence shown in SEQ ID NO: 36
  • the VL comprises the amino acid sequence shown in SEQ ID NO: 39; or
  • the VH comprises the amino acid sequence shown as SEQ ID NO: 36
  • the VL comprises the amino acid sequence shown as SEQ ID NO: 40.
  • 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.
  • 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.
  • the aforementioned PD-1 binding protein comprises or is an antibody that specifically binds to the PD-1 protein or an epitope thereof.
  • the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof.
  • the aforementioned PD-1 binding protein comprises Fab, Fab', F(ab') 2 , or single-chain antibody (scFv) that specifically binds to PD-1.
  • the aforementioned PD-1 binding protein further comprises an immunoglobulin Fc region.
  • the PD-1 binding protein further comprises a human immunoglobulin Fc region.
  • 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.
  • 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.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • 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/29 8D;280H/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
  • 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/S29 8D;D280H/K290S/S298V;F243L/R292P/Y300L;F243L/R292P/Y300L/P396L;F243L/R292P/Y300L/V305I/P396L;G236
  • the Fc region is an Fc region with reduced effector function, eg, reduced ADCC, ADCP, and/or CDC.
  • 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).
  • 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.
  • 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.
  • the aforementioned PD-1 binding protein further comprises a heavy chain constant region and a light chain constant region.
  • the aforementioned PD-1 binding protein further comprises a human IgG1 heavy chain constant region and a human ⁇ light chain constant region.
  • the human IgG1 heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 16; and the human ⁇ light chain constant region has the amino acid sequence shown in SEQ ID NO: 17.
  • a PD-1 binding protein 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.
  • a PD-1 binding protein 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.
  • a PD-1 binding protein which includes a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 43 or having at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 44 or having at least 90% sequence identity thereto.
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 43, and/or the light chain comprises the amino acid sequence shown in SEQ ID NO: 44.
  • a PD-1 binding protein which includes a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 49 or having at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 50 or having at least 90% sequence identity thereto.
  • the Fc region contained in the aforementioned PD-1 binding protein can enable the binding protein to form a dimeric molecule.
  • the Fc region contained in the aforementioned PD-1 binding protein can extend the in vivo half-life of the binding protein.
  • the PD-1 binding protein of the present disclosure is an anti-PD-1 antibody, or a conjugate or fusion protein comprising the antibody.
  • the aforementioned PD-1 binding protein has at least one activity selected from the following:
  • T cell proliferation such as inhibiting memory CD8+ T cell proliferation
  • the aforementioned PD-1 binding proteins of the present disclosure may bind to PD-1 with a KD value of ⁇ 1 ⁇ 10 ⁇ 7 M, e.g., ⁇ 1 ⁇ 10 ⁇ 8 M, or ⁇ 1 ⁇ 10 ⁇ 9 M, or ⁇ 1 ⁇ 10 ⁇ 10 M.
  • an anti-PD-1 antibody or an antigen-binding fragment thereof which binds to or competes for binding to the same epitope as the aforementioned PD-1 binding protein of the present disclosure.
  • 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.
  • an anti-PD-1 antibody or an antigen-binding fragment thereof 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).
  • 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.
  • PD-1 e.g., human PD-1
  • 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.
  • 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.
  • 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.).
  • 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.
  • 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.
  • 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.
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • Bacterial cells include, for example, cells of Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains).
  • Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains.
  • Fungal cells include, for example, 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.
  • Saccharomyces e.g., Saccharomyces cerevisiae
  • Schizosaccharomyces e.g., Schizosaccharomyces pombe
  • Pichia e.g., Pichia pastoris and Pichia methanolica
  • Hansenula Hansenula
  • mammalian cells examples include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • 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.
  • the cells of the present disclosure are incapable of developing into complete plants or animals.
  • the present disclosure provides a method for preparing the PD-1 binding protein of the present disclosure, which generally comprises the following steps:
  • 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.
  • 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.
  • 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.
  • composition/pharmaceutical composition comprising
  • the present disclosure provides a composition comprising the aforementioned PD-1 binding protein.
  • a pharmaceutical composition 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.
  • 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.
  • a product or kit comprising at least one container, each independently comprising a PD-1 binding protein as described above.
  • 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.
  • 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.
  • a method for improving, alleviating, treating or preventing a disease 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.
  • 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.
  • the aforementioned disease is an inflammatory or immune disease.
  • 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.
  • GvHD graft-versus-host disease
  • RA rheumatoid arthritis
  • CIA inflammatory bowel disease
  • a method of inhibiting T cell activity comprising administering an effective amount of the aforementioned PD-1 binding protein, polynucleotide, or composition (including pharmaceutical composition) to a subject in need thereof.
  • the inhibitory T cell activity is characterized by at least one of the following:
  • T cell proliferation such as inhibiting memory CD8+ T cell proliferation
  • reducing PD-1 positive T cells for example, reducing PD-1 high-expressing T cells.
  • 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.
  • an in vitro detection method, system, or device may include, for example:
  • a change e.g., a statistically significant change
  • a 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.
  • the in vivo detection method, system or device may include:
  • 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.
  • binding proteins and polynucleotides of the present disclosure can be labeled with fluorophore chromophores.
  • diagnostic reagents comprising the above-mentioned polynucleotides and binding proteins are also provided, as well as related diagnostic uses.
  • a kit 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.
  • the binding protein may be formulated as a pharmaceutical composition.
  • 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
  • Figure 1B shows the results for antibody 113.3.
  • 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
  • Figure 2B shows the results for antibody 113.3.
  • 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.
  • 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.
  • Figure 5 shows the inhibitory activity of antibodies 2E1-A06-H4 and 113.3Hbk2L1+4 in the CMV recall experimental system, Peresolimab is the positive control, and IgG1 is the negative control.
  • 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.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FIG7 is an experimental flow chart for evaluating the activity of anti-PD-1 antibodies in a graft-versus-host disease (GvHD) mouse model.
  • 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
  • Figure 8B shows the average disease rejection score of the mice.
  • FIG9 is an experimental flow chart for evaluating the activity of anti-PD-1 antibodies in a collagen-induced arthritis (CIA) mouse model.
  • Programmed death 1 "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.
  • 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.
  • 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.
  • the "PD-1 binding protein” may comprise 2, 3, 4 or more immunoglobulin single variable domains (such as VHH) that bind to 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.
  • 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.
  • 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.
  • 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 IFN ⁇ .
  • 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.
  • 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 ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , 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 ⁇ or ⁇ chains based on differences in their constant regions.
  • Each of the five Ig classes can have either kappa or lambda chains.
  • variable region 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.
  • 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).
  • Fab-Fv format was first disclosed in WO2009/040562, and its disulfide-stabilized form Fab-dsFv was first disclosed in WO2010/035012.
  • the 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 multiple specificities, such as bispecifics, or may be monospecific (see, for example, WO92/22583 and WO05/113605), an example of the latter being Tri-Fab (or TFM) described in WO 92/22583.
  • 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.
  • 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 that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; “AbMTM, 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 the primary sequence (see those described in Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198).
  • CDR may refer to a CDR defined by any method known in the art, including a combination of methods. The correspondence between the various numbering systems is well known to those skilled in the art.
  • 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.
  • 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.
  • the antibodies of the present disclosure are recombinant antibodies.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.”
  • 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.
  • 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.
  • Immunoglobulin single variable domain is generally used to refer to an immunoglobulin variable domain (which may be a heavy chain 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 interaction required between the VH and VL domains of conventional four-chain monoclonal antibodies).
  • immunoglobulin single variable domains include nanobodies (including VHH, humanized VHH and/or camelized VH, e.g., camelized human VH), IgNARs, domains, (single domain) antibodies (such as dAbs TM ) that are VH domains or derived from VH domains, and (single domain) antibodies (such as dAbs TM ) that are VL domains or derived from VL domains.
  • Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains are generally preferred.
  • a specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
  • VHH domain also known as heavy chain single domain antibody, VHH, VHH 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)).
  • 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 an epitope without the need for additional 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”, “ VHH 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.
  • 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).
  • 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.
  • Humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the human antibody variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the large amount of non-human protein components they carry. In order to avoid a decrease in activity while also reducing immunogenicity, the fully human antibody variable region can be subjected to minimal reverse mutations to maintain activity.
  • humanization examples include the “humanization” of 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 the corresponding positions in the VH domain of a human conventional tetrapeptide chain structure antibody (also referred to as “sequence optimization” in this disclosure; in addition to humanization, “sequence optimization” may also cover other modifications to the sequence by one or more mutations that provide improved properties of the VHH, such as removing potential post-translational modification sites).
  • a humanized VHH domain may contain one or more fully human framework region sequences.
  • Humanization includes transplanting mouse CDR sequences into human antibody variable region frameworks, i.e., antibodies produced in different types of human germline antibody framework sequences. This can overcome the strong antibody-variable antibody reaction induced by chimeric antibodies due to the large amount of heterologous protein components they carry.
  • Humanization methods include protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), which involves “grafting” CDRs onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for such CDR transplantation are known in the art.
  • humanized antibodies disclosed herein also include humanized antibodies after further affinity maturation of CDRs by phage display.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.
  • 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.
  • HVRs hypervariable regions
  • an “affinity matured” PD-1 binding protein or anti-PD-1 antibody has one or more changes 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, for example, by methods known in the art as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad.
  • 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.”
  • PD-1 binding proteins of the present disclosure bind to the antigen (i.e., PD-1) or an epitope thereof with a dissociation constant ( KD ) of 10-7 to 10-10 moles/liter (M), or 10-8 to 10-10 M, or 10-9 to 10-10 M, or less, as measured in a Biacore, KinExA, or Fortibio assay. Any KD value greater than 10-4 M is generally considered to indicate nonspecific binding.
  • KD dissociation constant
  • M dissociation constant
  • Any KD value greater than 10-4 M is generally considered to indicate nonspecific binding.
  • Specific binding of a binding protein to an antigen or epitope can be determined by any suitable known method, including, for example, surface plasmon resonance (SPR) assays as described herein.
  • SPR surface plasmon resonance
  • 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.
  • Binding affinity or “affinity” is used in the present 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 quantified by determining the dissociation constant ( KD ).
  • KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore).
  • SPR surface plasmon resonance
  • the rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • 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).
  • KD 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).
  • 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 cytometric analysis, as well as other assays exemplified in the present disclosure.
  • binding kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using a Biacore TM system or KinExA.
  • Binding affinities associated with different molecular interactions can be compared by comparing the KD values of individual antibody/antigen complexes, for example, a comparison of the binding affinities of different antibodies for a given antigen.
  • the specificity of an interaction can be evaluated by determining and comparing the KD value of an interaction of interest (e.g., a specific interaction between an antibody and an antigen) with the KD value of a non-target interaction (e.g., a control antibody known not to bind PD -1).
  • a “conservative substitution” refers to a substitution with another amino acid residue having properties similar to the original amino acid residue.
  • lysine, arginine, and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains.
  • 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.
  • “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.
  • 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.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • 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.
  • 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.
  • “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.
  • "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.
  • "autoimmune disease” or “autoimmune disorder” refers to those diseases or disorders resulting from inappropriate activation of immune cells reactive to one's own tissues.
  • Immuno 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.
  • 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).
  • 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.
  • administering refers 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.
  • administering 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.
  • 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.
  • 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 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.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of the target disease in a subject, they 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.
  • 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.
  • 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.
  • the “subject” and “patient” of the present disclosure refer to mammals, especially primates, and especially humans.
  • Programmed death receptor 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.
  • 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.
  • 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.
  • 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.
  • the activity of the above antibodies was tested, including: measuring the antibody's activation activity on the PD-1 signaling pathway, measuring the antibody's inhibitory activity on memory T cells using a CMV recall assay, measuring the antibody's activity in inhibiting T cell cytokine release using a PBMC activation assay, and measuring the antibody's binding to human and monkey PD-1 using FACS.
  • the antibody's drugability (aggregation, fragmentation, accelerated stability, post-translational modifications (PTMs), etc.) was also tested.
  • Anti-PD-1 antibodies with significant advantages in activity and drugability were selected, and 2E1-A06-VHH was obtained for further testing.
  • the 2E1-A06-VHH sequence is as follows:
  • the underlined CDR sequences are determined according to the Kabat numbering system, as shown in Table 2.
  • the obtained 2E1-A06-VHH was fused with wild-type IgG1-Fc or IgG1-Fc with a mutation to obtain a nanobody.
  • 2E1-A06-VHH was 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:
  • the sequence of the 2E1-A06 antibody is as follows:
  • the underlined part is human IgG1 Fc with C220A mutation.
  • the gene sequence encoding the above-mentioned 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.
  • 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.
  • 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.
  • the immunizing antigen was a His-tagged recombinant human PD-1 protein (h-PD-1-His, prepared at 1 ⁇ g/ ⁇ L 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.
  • CFA complete adjuvant
  • 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:
  • 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:
  • 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:
  • 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:
  • the underlined heavy chain is the human IgG1 Fc region
  • the underlined light chain is the human C ⁇ region.
  • the gene sequence encoding the above-mentioned 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.
  • Detection was performed using a Biacore 8K instrument (GE Healthcare).
  • a Protein A sensor chip was used, and the mobile phase was HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20).
  • 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
  • the analyte was diluted two-fold from 100 nM to a total of seven concentration points.
  • Regeneration buffer 10 mM Glycine pH 1.5 (GE Healthcare, 29238268-AA) was run at a flow rate of 10 ⁇ l/min for 30 s.
  • the signal value of the corresponding reference channel (Fc 1) was subtracted from the signal value of the detection channel (Fc 2) to obtain the corrected signal curve.
  • Affinity kinetics curves were fitted according to the 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.
  • 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.
  • 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 were added, along with various concentrations of the test antibody. After incubation at 37°C for 6 hours, reporter gene expression was detected using Bright-Glo TM (Promega).
  • 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.
  • 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.
  • 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.
  • Nanobody humanization utilizes a CDR grafting approach.
  • 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.
  • the CDRs are underlined (numbered according to the Kabat system).
  • VHHs were fused to wild-type human IgG1-Fc (Glu216-Lys447) with the C220A mutation (according to the EU numbering system) to obtain full-length sequences.
  • 2E1-A06-VHH-H4 SEQ ID NO: 27
  • wild-type human IgG1-Fc Glu216-Lys447
  • 2E1-A06-H4 SEQ ID NO: 30
  • the underlined area is the human IgG1 Fc region with the C220A mutation.
  • the gene sequence encoding the above-mentioned 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.
  • mouse mAb 113.3 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.
  • the underlined ones are the heavy chain CDRs.
  • the above heavy chain variable regions and light chain variable regions were connected to the human IgG1 heavy chain constant region and the human C ⁇ light chain constant region, respectively, to obtain the full-length sequence.
  • the underlined heavy chain is the human IgG1 Fc region
  • the underlined light chain is the human C ⁇ region.
  • the gene sequence encoding the above-mentioned 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.
  • SPR was used to detect the affinity of anti-PD-1 antibodies to recombinant human PD-1 protein.
  • Detection was performed using a Biacore 8K instrument (GE Healthcare).
  • a Protein A sensor chip was used, and the mobile phase was HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20).
  • 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
  • the analyte was diluted two-fold from 100 nM to a total of seven concentration points.
  • Regeneration buffer 10 mM Glycine pH 1.5 (GE Healthcare, 29238268-AA) was run at a flow rate of 10 ⁇ l/min for 30 s.
  • the signal value of the corresponding reference channel (Fc 1) was subtracted from the signal value of the detection channel (Fc 2) to obtain the corrected signal curve.
  • Affinity kinetics curves were fitted according to the 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.
  • 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.
  • 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 were added, along with various concentrations of the test antibody. After incubation at 37°C for 6 hours, reporter gene expression was detected using Bright-Glo TM (Promega).
  • the PBMC activation assay was used to evaluate the activity of humanized antibodies in inhibiting the release of cytokines from T cells.
  • PBMCs were incubated with 0.05 ⁇ g/mL PHA for 72 hours and plated at 200,000 cells/well in 96-well plates pre-coated with 0.01 ⁇ g/mL anti-CD3 and various concentrations of humanized antibodies.
  • Anti-CD28 0.2 ⁇ g/mL was also added for incubation. After overnight incubation at 37°C, IFN ⁇ secretion in the supernatant was measured using the HTRF human IFN gamma kit (Cisbio).
  • Anti-PD-1 antibodies inhibit the secretion of IFN ⁇ by T cells from different PBMC donors in the PBMC activation experimental system
  • the CMV recall experimental system was used to evaluate the inhibitory activity of humanized antibodies on memory T cells.
  • PBMCs 200,000 resuscitated PBMCs were incubated with 3-50 ng/mL 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 assessed using various fluorescently labeled antibodies and a fluorescent secondary antibody, anti-human IgG Fc (Invitrogen).
  • Ki67 proliferation marker Ki67
  • the ADCC assay was used to evaluate the ability of humanized antibodies to cytotoxic NK cells against PD-1 positive cells.
  • PI Biyuntian
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • mice 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).
  • PBMC primary human peripheral blood PBMC
  • 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.
  • 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.
  • mice 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 adjuvant emulsion at the base of the tail. Starting from the second induction, the mice were scored for arthritis in their limbs 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 day 57 to detect pathological changes in the right hind joint. The specific experimental process is shown in Figure 9. Mice were dosed according to the following scheme, and the agonist activity of the humanized antibody was evaluated by the mouse arthritis score and the end-point joint pathology score.
  • 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.

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Abstract

La présente divulgation concerne une protéine de liaison à PD-1 et l'utilisation pharmaceutique. Plus particulièrement, la présente invention concerne un anticorps anti-PD-1, une composition pharmaceutique contenant l'anticorps, une méthode de traitement de maladies inflammatoires ou auto-immunes, et l'utilisation pharmaceutique.
PCT/CN2025/083096 2024-03-19 2025-03-18 Protéine de liaison à pd-1 et utilisation pharmaceutique Pending WO2025195358A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108697791A (zh) * 2015-11-03 2018-10-23 詹森生物科技公司 特异性结合pd-1的抗体及其用途
CN110691795A (zh) * 2017-05-30 2020-01-14 百时美施贵宝公司 包含抗-lag3抗体、pd-1途径抑制剂和免疫治疗剂组合的组合物
WO2023040945A1 (fr) * 2021-09-15 2023-03-23 江苏恒瑞医药股份有限公司 Protéine se liant de manière spécifique à pd-1 et son utilisation pharmaceutique
CN117120470A (zh) * 2021-03-10 2023-11-24 北京拓界生物医药科技有限公司 Pd-1结合蛋白及其医药用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108697791A (zh) * 2015-11-03 2018-10-23 詹森生物科技公司 特异性结合pd-1的抗体及其用途
CN110691795A (zh) * 2017-05-30 2020-01-14 百时美施贵宝公司 包含抗-lag3抗体、pd-1途径抑制剂和免疫治疗剂组合的组合物
CN117120470A (zh) * 2021-03-10 2023-11-24 北京拓界生物医药科技有限公司 Pd-1结合蛋白及其医药用途
WO2023040945A1 (fr) * 2021-09-15 2023-03-23 江苏恒瑞医药股份有限公司 Protéine se liant de manière spécifique à pd-1 et son utilisation pharmaceutique

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