WO2022017428A1 - 抗ctla-4抗体及其用途 - Google Patents

抗ctla-4抗体及其用途 Download PDF

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WO2022017428A1
WO2022017428A1 PCT/CN2021/107707 CN2021107707W WO2022017428A1 WO 2022017428 A1 WO2022017428 A1 WO 2022017428A1 CN 2021107707 W CN2021107707 W CN 2021107707W WO 2022017428 A1 WO2022017428 A1 WO 2022017428A1
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seq
amino acid
variable region
chain variable
antibody
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PCT/CN2021/107707
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English (en)
French (fr)
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潘志卫
姚剑
张静
周岳华
刘洪川
武海
姚盛
冯辉
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Priority to BR112023000826A priority Critical patent/BR112023000826A2/pt
Priority to CN202180049546.4A priority patent/CN115812081A/zh
Priority to US18/016,643 priority patent/US20230295301A1/en
Priority to EP21846885.8A priority patent/EP4201958A1/en
Priority to CA3186108A priority patent/CA3186108A1/en
Priority to AU2021311701A priority patent/AU2021311701A1/en
Publication of WO2022017428A1 publication Critical patent/WO2022017428A1/zh

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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CTLA-4 and compositions comprising the same. Also provided are nucleic acid molecules encoding the antibodies of the invention or antigen-binding fragments thereof, vectors and host cells for expressing the antibodies of the invention or antigen-binding fragments thereof, and therapeutic and diagnostic methods and uses of the antibodies of the invention or antigen-binding fragments thereof.
  • Cytotoxic T-lymphocyte-associated protein 4 (CTLA4 or CTLA-4, cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a transmembrane protein encoded by the CTLA-4 gene. Located on human chromosome 2, 2q33. CTLA-4 is a member of the immunoglobulin superfamily and consists of an extracellular V domain, a transmembrane domain and a cytoplasmic domain. CTLA-4 has homology with the co-stimulatory molecule receptor CD28 on the surface of T cells, and the two compete for binding to its ligands B7-1 (CD80) and B7-2 (CD86), which are mainly expressed in antigen presentation. cell surface.
  • CTLA-4 Compared with CD28, CTLA-4 has a higher binding affinity to CD80 and CD86, so it can compete and block CD28-mediated activation.
  • CTLA-4 is normally expressed on the surface of regulatory T cells (Treg) and conventional T cells in an activated state. After binding to B7 molecules, it inhibits the activation of T cells, participates in the negative regulation of immune responses, acts as an immune checkpoint and downregulates immune responses, so CTLA-4 plays a very important role in immune regulation.
  • Treg regulatory T cells
  • T cells require the stimulation of two signals.
  • the first signal comes from the T cell receptor (TCR) that specifically binds to the antigen-peptide-MHC complex on the surface of antigen-presenting cells (APC), and the second signal pathway requires co-stimulation.
  • TCR T cell receptor
  • APC antigen-presenting cells
  • CD80/CD86 B7-1/B7-2
  • CTLA-4 down-regulates the function of T cells through the following pathways: First, CTLA-4 can competitively block the co-binding of CD28 with CD80/86 through its high affinity with CD80/CD86.
  • CTLA-4 can reduce CD80/CD86 by reducing the expression level of CD80/CD86 on antigen-presenting cells (APC) or by removing CD80/CD86 molecules from the surface of antigen-presenting cells (APC) by trans-endocytosis. CD28-involved T cell activation.
  • CTLA-4 can inhibit TCR signaling by mediating the binding of dendritic cells to CD80/CD86 and inducing the expression of tryptophan-degrading enzyme IDO.
  • CTLA-4 can also bind to the immune synapse by recruiting inhibitory molecules to induce the production of regulatory cytokines, thereby inhibiting the transmission of APC and TCR signaling.
  • Blockade of CTLA-4 has been shown in many studies to induce tumor regression.
  • Anti-CTLA-4 antibodies can effectively and specifically inhibit cellular and humoral immune responses in vitro and in vivo, have significant therapeutic effects on transplant rejection and various autoimmune diseases, and have low toxic and side effects.
  • CTLA-4 mAbs ipilimumab (Bristol-Myers Squibb) and Tremelimumab (AstraZeneca) are currently used in some cancer treatments and are being tested for other anti-cancer indications, there is still a need to include active Novel anti-CTLA-4 antibodies with improved known antibodies.
  • the present invention provides an anti-CTLA-4 antibody or an antigen-binding fragment thereof, which has the advantages of high affinity and high specificity against human CTLA-4.
  • the anti-CTLA-4 antibodies or antigen-binding fragments thereof provided by the present invention can be used as a stand-alone therapy or in combination with other therapies/or other anti-cancer agents, for example, in the treatment of cancer.
  • the present invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and/or a light chain variable region:
  • the heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively; or with SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in the amino acid sequence shown in: 3; preferably, the HCDR2 having 1, 2 or 3 amino acid differences with the amino acid sequence shown in SEQ ID NO: 2 is SEQ ID NO: 37; preferably, the HCDR3 having 1, 2 or 3 amino acid differences with the amino acid sequence shown in SEQ ID NO: 3 is SEQ ID NO: 38; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 respectively; or with SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences in amino acid sequence shown in: 9; or (III) amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 respectively HCDR1, HCDR2 and HCDR3; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; or with SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences in the amino acid sequence shown in: 21; preferably, the HCDR2 having 1, 2 or 3 amino acid differences with the amino acid sequence shown in SEQ ID NO: 20 is SEQ ID NO: 39; or
  • (V) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or with SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences in amino acid sequences shown in: 24;
  • the light chain variable region comprises:
  • the amino acid sequence shown in: 12 respectively has LCDR1, LCDR2 and LCDR3 with 1, 2 or 3 amino acid differences; or (III) amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 respectively LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 respectively have 1, 2 or 3 amino acid difference LCDR1, LCDR2 and LCDR3; preferably,
  • the LCDR1 having 1, 2 or 3 amino acid differences with the amino acid sequence shown in SEQ ID NO: 16 is SEQ ID NO: 40 or 41; or
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; or with SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO :
  • the amino acid sequences shown in 27 have LCDR1, LCDR2 and LCDR3 with 1, 2 or 3 amino acid differences, respectively.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region:
  • a heavy chain variable region comprising:
  • a light chain variable region comprising:
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:40, SEQ ID NO:17 and SEQ ID NO:18, respectively;
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 is selected from the amino acid sequence shown in SEQ ID NO: 1 or 19; HCDR2 is selected from any one of SEQ ID NO: 2, 20, 37 and 39.
  • the amino acid sequence shown, HCDR3 is selected from the amino acid sequence shown in any one of SEQ ID NOs: 3, 21 and 38; and
  • Described light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the aminoacid sequence of LCDR1 is as shown in SEQ ID NO:72;
  • the aminoacid sequence of LCDR2 is as shown in SEQ ID NO:17;
  • the aminoacid sequence of LCDR3 is as shown in SEQ ID NO:17: 18, wherein said SEQ ID NO:72 has the amino acid sequence shown in the following general formula:
  • LCDR1 XASQNVGTYVA, wherein X is selected from K, R and Q.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and/or a light chain variable region, wherein,
  • the heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively;
  • the light chain variable region comprises:
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; or
  • VI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or
  • VI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 19, SEQ ID NO: 39 and SEQ ID NO: 21, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 40, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or
  • VIII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:37 and SEQ ID NO:38, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 40, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or
  • (IX) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; and a light chain variable region comprising The amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:40, SEQ ID NO:17 and SEQ ID NO:18, respectively.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region:
  • the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 28, 30, 32, 34 or 35, or the same as SEQ ID NO: 28, 30, 32, 34 or 35
  • the amino acid sequence shown in any one of the amino acid sequences has at least 95%, 96%, 97%, 98% or 99% sequence identity
  • the light chain variable region comprises as SEQ ID NO: 29, 31,
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region:
  • the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NO: 28 or 34 or a variant thereof, and
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33 or a variant thereof,
  • the antibody or antigen-binding fragment thereof comprises:
  • (I) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 42, 43, 44, 45, 46, 47 or 48, or the same as SEQ ID NO: 42, 43, 44 An amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in any one of , 45, 46, 47 or 48; and
  • a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61 or 62, or with SEQ ID NO: 54, 55, An amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in any one of 56, 57, 58, 59, 60, 61 or 62; or
  • (II) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 49, 50, 51, 52 or 53, or a combination with SEQ ID NO: 49, 50, 51, 52 or 53 An amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in any one; and
  • a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61 or 62, or with SEQ ID NO: 54, 55, An amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of 56, 57, 58, 59, 60, 61 or 62; or
  • VI a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO:50 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO:55; or
  • VII a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO:50 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO:56; or
  • VIII a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO:46 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO:60; or
  • the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NO:63, 65, 67, or 69, or the same as SEQ ID NO:63
  • the light chain comprises the amino acid sequence shown in any one of SEQ ID NO: 64, 66, 68 or 70, or the amino acid sequence shown in any one of SEQ ID NO: 64, 66, 68 or 70 Amino acid sequences having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the antibody comprises:
  • the antibody or antigen-binding fragment thereof is a murine, chimeric, humanized, or fully human antibody, or antigen-binding fragment thereof.
  • the antigen-binding fragment is a Fab, Fab', F(ab')2, Fv, scFv, or sdAb.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof of the present invention is of any IgG subtype, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1 or IgG4, more preferably IgG1 kappa subtype.
  • the invention provides an isolated anti-CTLA-4 antibody or antigen-binding fragment thereof having one or more of the following properties:
  • amino acid residues 27, 28 and 29 in combination with SEQ ID NO:71, wherein the amino acid residue numbering is amino acid numbering starting from A37 in SEQ ID NO:71;
  • the present invention also provides an isolated antibody or an antigen-binding fragment thereof, which binds to the same or overlapping epitope of human CTLA-4 protein as the above-mentioned anti-CTLA-4 antibody.
  • the present invention also provides an isolated antibody or an antigen-binding fragment thereof, which competes with the above-mentioned anti-CTLA-4 antibody for binding to the epitope of human CTLA-4 protein.
  • the present invention also provides an isolated antibody or antigen-binding fragment thereof, which binds to the same or completely or partially overlapping epitope of human CTLA-4 protein as antibody huJS007-47.
  • the present invention also provides an isolated antibody or antigen-binding fragment thereof, which competes with the antibody huJS007-47 for binding to the epitope of human CTLA-4 protein.
  • the anti-CTLA-4 antibodies or antigen-binding fragments thereof of the invention bind to an epitope consisting of residues 27-29 of SEQ ID NO:71, wherein the amino acid residues are numbered from SEQ ID NO:71 Amino acid numbering starts at A37.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention binds an epitope consisting of residues 63-65 of SEQ ID NO:71, wherein the amino acid residues are numbered from SEQ ID NO:71 Amino acid numbering begins with M1.
  • the anti-CTLA-4 antibodies or antigen-binding fragments thereof of the invention bind to one or more epitopes selected from the group consisting of amino acid residues 27, 28 and 29 of SEQ ID NO:71, wherein the amino acid residues are numbered Amino acid numbering is performed starting from A37 in SEQ ID NO:71.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention binds one or more epitopes selected from the group consisting of amino acid residues 63, 64 and 65 of SEQ ID NO: 71, wherein the amino acid residues are numbered Amino acid numbering is performed starting from M1 in SEQ ID NO:71.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention binds to human CTLA-4 at the epitope at amino acid residue 27 of SEQ ID NO: 71, wherein the amino acid residues are numbered from Amino acid numbering begins at A37 in SEQ ID NO:71.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention binds to human CTLA-4 at the epitope at amino acid residue 28 of SEQ ID NO: 71, wherein the amino acid residues are numbered from Amino acid numbering begins at A37 in SEQ ID NO:71.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention binds to human CTLA-4 at the epitope at amino acid residue 29 of SEQ ID NO: 71, wherein the amino acid residues are numbered from Amino acid numbering begins at A37 in SEQ ID NO:71.
  • the anti-CTLA-4 antibodies or antigen-binding fragments thereof of the invention bind to different epitopes of human CTLA-4 protein with Ipilimumab.
  • the anti-CTLA-4 antibody of the present invention inhibits or blocks the binding of human CTLA-4 protein to human CD80 or to human CD86; preferably, the amino acid residues that compete with CD80 for binding are 135, 137 , one or more of 138 and 140, wherein amino acid residue numbering is amino acid numbering starting from M1 in SEQ ID NO:71.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention binds to residues 36-41 and/or 59-66 and/or 109-110 and/or 133- of SEQ ID NO:71 An epitope consisting of 140, wherein the amino acid residue numbering is amino acid numbering starting from M1 in SEQ ID NO:71.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof of the present invention binds one or more amino acid residues selected from the group consisting of SEQ ID NO: 71 amino acid residues 36, 39, 41, 59, 61, 62, 63, 64, 65, 66, 109, 110, 133, 135, 136, 137, 138 and 140 epitopes, wherein the amino acid residues are numbered starting from M1 in SEQ ID NO:71.
  • the present invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.
  • the present invention also provides a single chain antibody comprising a light chain variable region and a heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.
  • the invention also provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof as described herein conjugated to a therapeutic or diagnostic agent.
  • the present invention provides a polynucleotide encoding an anti-CTLA-4 antibody or antigen-binding fragment thereof as described herein.
  • the present invention provides an expression vector comprising the polynucleotide described herein, preferably, the vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising the polynucleotide described herein or the expression vector described herein, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the present invention provides a method of making an anti-CTLA-4 antibody or antigen-binding fragment thereof described herein, the method comprising expressing herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof The antibody or antigen-binding fragment thereof is expressed in the host cell, and the expressed antibody or antigen-binding fragment thereof is recovered from the host cell.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CTLA-4 antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cells, and pharmaceutically acceptable carriers or excipients.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein
  • a disease or condition is cancer
  • the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, Breast, colon, lung, bone, pancreatic, skin, head and neck, uterine, ovarian and rectal cancers.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein , which is used for the treatment and/or prevention of a CTLA-4 mediated disease or condition, preferably the disease or condition is cancer, more preferably the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer , lung, bone, pancreatic, skin, head and neck, uterine, ovarian and rectal cancers.
  • the present invention provides a method of treating and/or preventing a CTLA-4 mediated disease or disorder, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, herein
  • the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or The pharmaceutical compositions described herein, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or The pharmaceutical composition described herein, preferably it further comprises a delivery device.
  • the present invention provides a method of detecting the presence of CTLA-4 in a sample using the antibody or antigen-binding fragment thereof described herein.
  • Figure 1 FACS detection of hybridoma anti-CTLA-4 antibody blocking the binding of huCTLA-4 to huCD80.
  • Figure 3 Cross-reactivity of hybridoma anti-CTLA-4 antibodies with other antigens (TIGIT, BTLA).
  • Figure 4 Binding of chimeric anti-CTLA-4 antibodies to huCTLA-4 was detected by ELISA.
  • Figure 5 Detection of the binding of chimeric anti-CTLA-4 antibody to huCTLA-4 expressing cells by FACS.
  • Figure 6 The ability of chimeric anti-CTLA-4 antibodies to block the binding of huCTLA-4 to CD80 was detected by ELISA.
  • Figure 7 7a: FACS detection of the ability of chimeric anti-CTLA-4 antibody to block the binding of huCTLA-4 to CD80 expressing cells; 7b: FACS detection of the ability of chimeric anti-CTLA-4 antibody to block the binding of huCTLA-4 to CD86 expressing cells .
  • Figure 8 The biological activity of chimeric anti-CTLA-4 antibody antagonizing huCTLA-4 was detected by luciferase method.
  • Figure 9 Detection of humanized anti-CTLA-4 antibody binding to huCTLA-4 by ELISA.
  • Figure 10 The ability of humanized anti-CTLA-4 antibodies to block the binding of huCTLA-4 to CD80 was detected by ELISA.
  • Figure 11 The biological activity of human anti-CTLA-4ylated antibody was detected by luciferase method.
  • Figure 12 ADCC activity of humanized anti-CTLA-4 antibodies.
  • Figure 13 CDC activity of humanized anti-CTLA-4 antibodies.
  • Figure 14 Inhibition of tumor growth in mice by humanized anti-CTLA-4 antibodies.
  • Figure 15 Fortebio binding assay identifies antigenic epitopes.
  • Figure 16 Identification of antigenic epitopes by hydrogen deuterium exchange mass spectrometry.
  • Figure 17 Binding of monoclonal antibody huJS007-47 to huCTLA-4 mutant was detected by ELISA.
  • Figure 18 Inhibitory effect of huJS007-47 on tumor growth of hCTLA4 humanized mice transplanted with MC38.
  • Figure 19 Inhibitory effect of huJS007-47 on the growth of hCTLA4 humanized mice transplanted with H22 tumors.
  • Figure 20 Overall structure and interaction details of JS007 binding to CTLA-4; the overall structure of the heavy chain variable region (VH) and light chain variable region (VL) of the JS007 antibody in complex with CTLA-4 is shown in Figure 20A Figure, wherein the three heavy chain complementarity determinants (HCDR1-3) and the three light chain complementarity determinants (LCDR1-3) of JS007 are shown in different colors; Figure 20B is the hydrogen formed by the amino acids between JS007 and CTLA-4 In the bond interaction network, the amino acids involved in hydrogen bond interactions are shown as rod-like structures, in which the color of each CDR is consistent with that in Figure 20A, and the color of its amino acids is consistent with the color depth of the corresponding region.
  • VH heavy chain variable region
  • VL light chain variable region
  • Figure 20B is the hydrogen formed by the amino acids between JS007 and CTLA-4
  • the amino acids involved in hydrogen bond interactions are shown as rod-like structures, in which the color of each CDR is consistent with that in Figure 20
  • Figure 21 Molecular basis for the competition between JS007 and B7-1 for binding to CTLA-4; the structure shown in Figure 21A is the complex structure of CTLA-4-JS007 and CTLA-4-B7-1 (PDB: 1I8L) according to CTLA-4
  • CTLA-4 only shows the molecular structure of CTLA-4 in CTLA-4-JS007
  • the FG loop of CTLA-4 mediates the main competitive binding
  • Figure 21B shows the surface structure of CTLA-4 bound to JS007 or B7-1 molecules, in which the amino acids that only bind to the JS007 antibody are shown as the dark part in the upper left of Figure 21B, and only bind to B7
  • the amino acids bound by -1 are shown in the upper right dark part of Figure 21B, while the amino acids involved in the interaction with both JS007 and B7-1 are shown in the middle dark part in Figure 21B.
  • the term "or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” should be construed as inclusive, that is, including at least one of the number or list of elements, but also including more than one, and optionally , additional unlisted items. Only under terms expressly stated to the contrary, such as “only one” or “indeed one” or when “consisting of” is used in a claim, will refer to only one of the listed numbers or one element of the list.
  • cytotoxic T lymphocyte-associated antigen 4" protein CTLA-4
  • CTLA-4 recombinant protein CLA-4
  • CTLA-4 antigen CTL-4 antigen
  • CTLA-4 CTL-4 antigen
  • CTLA-4 refers to the full length of CTLA-4 from human and cynomolgus monkeys or a fragment thereof (such as a mature fragment thereof lacking a signal peptide).
  • human CTLA-4" refers to the human sequence CTLA-4, such as the complete amino acid sequence of human CTLA-4 having NCBI accession number NM_005214.3.
  • Human CTLA-4 sequence may differ from human CTLA-4 with NCBI Accession No. 4 have roughly the same biological function.
  • the biological function of human CTLA-4 is to have epitopes in the extracellular domain of CTLA-4 that are specifically bound by the anti-CTLA-4 constructs of the present disclosure, or the biological function of human CTLA-4 is to regulate T cell activity.
  • huCD80 refers to the human sequence huCD80, such as the complete amino acid sequence of huCD80 with NCBI accession number NM_005191.3.
  • huCD86 refers to the human sequence huCD86, such as the complete amino acid sequence of huCD86 with NCBI accession number NM_006889.3;
  • cynomolgus monkey CD86 refers to the cynomolgus monkey sequence cyno CD86, such as cynomolgus monkey with NCBI accession number NM_102115124 Complete amino acid sequence of monkey CD86.
  • percent (%) amino acid sequence identity or simply “identity” is defined as the maximum percent sequence identity obtained when amino acid sequences are aligned (and where necessary gaps are introduced), and no conservative substitutions are considered to be Following the portion of sequence identity, the percentage of amino acid residues in the candidate amino acid sequence that are identical to those in the reference amino acid sequence.
  • Sequence alignments can be performed to determine percent amino acid sequence identity using various methods in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
  • immune response refers to the action by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines and complement) by these cells or by the liver, which results in selective Damage, destroy or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of growth factors to receptors, that results in the transmission of signals from one part of a cell to another of the cell. part.
  • delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions leading to signal transduction.
  • the penultimate process usually involves nuclear events that lead to changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological signature” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, neutralization or antagonism of CTLA in vivo or in vitro -4 ability activity, IC 50, in vivo stability of the antibody and the immunogenic properties of the antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (ie, generally with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention of The ability of proteins to be expressed at high levels in mammalian cells.
  • antibody refers to any form of antibody that possesses the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric and camelized single domain antibodies.
  • isolated antibody refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media .
  • isolated does not mean the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include large numbers of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.
  • full-length antibody refers to an immunoglobulin molecule comprising at least four peptide chains in nature: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and spaced by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • antibody-binding fragment of an antibody (“parent antibody”) includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding or variable region (eg, one or more CDRs) of the parent antibody that retains the parental At least some binding specificity of an antibody.
  • antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab') 2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
  • a binding fragment or derivative typically retains at least 10% of its antigen-binding activity when the antigen-binding activity is expressed on a molar basis.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of an antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • single-chain Fv or "scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides typically also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • bivalent antibody contains two antigen-binding sites. In some cases, the two binding sites have the same antigenic specificity. However, bivalent antibodies can be bispecific.
  • diabody refers to a small antibody fragment with two antigen-binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH).
  • VL light chain variable domain
  • VH-VL or VL-VH Variable domain
  • murine antibody or "hybridoma antibody” in the present disclosure is a monoclonal antibody against human CTLA-4 prepared according to the knowledge and skill in the art. In preparation, test subjects are injected with CTLA-4 antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domains are obtained from rodent or the like antibodies (“parental antibodies”), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibody induces induction in human subjects as compared to the parental rodent antibody The likelihood of an adverse immune response is low.
  • humanized antibody refers to a form of antibody that contains sequences from human and non-human (eg, mouse, rat) antibodies.
  • humanized antibodies comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • FR hypervariable loops Framework
  • a humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, “mouse antibody” refers to an antibody comprising only mouse immunoglobulin sequences. Alternatively, fully human antibodies may contain rat sugar chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Likewise, “rat antibody” refers to an antibody comprising only rat immunoglobulin sequences.
  • an “isotype” antibody refers to the class of antibody provided by the heavy chain constant region genes (eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4). Isotypes also include modified forms of one of these species, wherein modifications have been made to alter Fc function, eg, to enhance or reduce effector function or binding to Fc receptors.
  • heavy chain constant region genes eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4
  • epitope refers to a protein determinant capable of specific binding by an antibody.
  • Epitopes are usually composed of various chemically active surface molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is the loss of binding to the former but not to the latter in the presence of a denaturing solvent.
  • cross-reactivity refers to the binding of antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat).
  • cross-reactivity should be understood as an inter-species reaction with the same molecule X expressed in different species.
  • the cross-reactivity specificity of monoclonal antibodies recognizing human CTLA-4, monkey, and/or murine CTLA-4 (mouse or rat) can be determined by FACS analysis.
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • affinity molecule X for its partner Y can generally dissociation constant (K D) is represented by equilibrium, the equilibrium dissociation constant and the dissociation rate constant is the rate constant (k dis, respectively and k on) ratio.
  • K D dissociation constant
  • k dis rate constant
  • k on rate constant
  • Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • does not bind to a protein or cell means, does not bind to the protein or cell, or does not bind to it with high affinity, ie binds the protein or cell with a K D of 1.0 x 10 -6 M or higher, more preferably 1.0 x 10 -5 M or higher, more preferably 1.0 ⁇ 10 -4 M or higher, 1.0 ⁇ 10 -3 M or higher, more preferably 1.0 ⁇ 10 -2 M or higher.
  • high affinity for an IgG antibody, the antigen refers to K D of 1.0 ⁇ 10 -6 M or less, preferably 5.0 ⁇ 10 -8 M or less, more preferably 1.0 ⁇ 10 -8 M or lower, 5.0 ⁇ 10 -9 M or lower, more preferably 1.0 ⁇ 10 -9 M or lower.
  • high affinity binding may vary.
  • IgM subtype "high affinity” means a binding K D of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or less.
  • antibody-dependent cytotoxicity refers to cell-mediated immune defense in which immune system effector cells actively associate cell membrane surface antigens with antibodies, such as CTLA- 4 Antibodies that bind to target cells such as cancer cells are lysed.
  • CDC complement-dependent cytotoxicity
  • IgG and IgM antibodies that, when bound to surface antigens, initiate the canonical complement pathway, including formation of membrane attack complexes and lysis of target cells.
  • the antibody of the present invention binds to CTLA-4, it induces CDC to cancer cells.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless expressly limited, the term includes nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, in Kariko et al. Human US Patent No. 8,278,036, which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods of synthesizing such mRNA molecules, and methods for delivering therapeutic proteins in vivo).
  • nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complements thereof, as well as sequences explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construct refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), derived from Any source, capable of integrating with the genome or replicating autonomously, constitutes a polynucleotide molecule in which one or more polynucleotide molecules have been functionally linked (ie, operably linked).
  • the recombinant construct will typically comprise a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Expression of the nucleic acids of the invention can be directed using both heterologous and non-heterologous (ie, endogenous) promoters.
  • Vector refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell).
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors After introduction into the host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell.
  • Expression vectors contain one or more phenotypic selectable markers and origins of replication to ensure maintenance of the vector and to provide for amplification within the host if desired.
  • Activation can have the same meaning, eg, activation, stimulation, or treatment of a cell or receptor with a ligand, unless the context otherwise or clearly dictates.
  • Ligand includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules such as peptidomimetics of cytokines and peptidomimetics of antibodies.
  • Activation can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.
  • a “response/response”, eg, the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (eg, concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment), wherein changes Related to activation, stimulation, or processing, or to internal mechanisms such as genetic programming.
  • the terms “treating” or “treating” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (ie, slowing or arresting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treating” or “treating” refers to alleviating or ameliorating at least one physical parameter, including those physical parameters that may not be discernible by a patient. In another embodiment, “treating” or “treating” refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of physical parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
  • cyno or “cynomolgus monkey” refers to a cynomolgus monkey.
  • Administration "in combination with” one or more other therapeutic agents includes simultaneous (co) administration and sequential administration in any order.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” mean that the CTLA-4 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents, is effective in preventing Or an amount that ameliorates the symptoms of one or more diseases or conditions or the progression of that disease or condition.
  • a therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • the therapeutically effective dose refers to that ingredient only.
  • a therapeutically effective dose refers to the combined amount of active ingredients that elicits a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • An effective amount of the therapeutic agent will result in an improvement in the diagnostic criterion or parameter by at least 10%, usually by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.
  • Cancer and “cancerous” refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Benign and malignant cancers as well as dormant tumors or micrometastases are included in this definition. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, cancer of the stomach or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, Cancer of salivary gland, kidney or kidney, liver, prostate, vulva, thyroid, liver, and various types of head and neck cancer, and B-cell lymphomas (including low-grade/follicular non-HoJ King's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small anucleate NHL, bulky disease (NHL), small lymph
  • the invention provides anti-CTLA-4 antibodies or antigen-binding fragments thereof.
  • anti-CTLA-4 antibody refers to a CTLA-4 protein or fragment thereof with sufficient affinity such that the Antibodies can be used as diagnostic and/or therapeutic agents in targeting CTLA-4.
  • Antibodies of the invention can be produced using any suitable method for producing antibodies.
  • CTLA-4 in any suitable form can be used as an immunogen (antigen) for generating antibodies.
  • any CTLA-4 variant or fragment thereof can be used as an immunogen.
  • hybridoma cells producing murine monoclonal anti-human CTLA-4 antibodies can be produced by methods well known in the art.
  • Antibodies derived from rodents may cause unwanted antibody immunogenicity when used in vivo as therapeutics, and repeated use results in an immune response against the therapeutic antibody that at least results in loss of therapeutic efficacy , and in severe cases lead to a potentially fatal allergic reaction.
  • One approach to reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies in which mouse variable regions are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439- 43). However, the retention of intact rodent variable regions in chimeric antibodies may still cause deleterious immunogenicity in patients.
  • CDR complementarity determining region
  • the chimeric or humanized antibodies of the present invention can be prepared based on the sequences of the prepared murine monoclonal hybridoma antibodies.
  • DNA encoding heavy and light chain immunoglobulins can be obtained from murine hybridomas of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques.
  • the chimeric CTLA-4 antibody of the present invention can use methods known in the art to operably link hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions ( See, eg, US Patent No. 4,816,567 to Cabilly et al.) for obtaining chimeric heavy chains and chimeric light chains for preparation.
  • the chimeric antibodies of the present invention comprise constant regions that may be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG1, IgG4, more preferably IgG1 kappa subtype.
  • the chimeric CTLA-4 antibody of the present invention can be obtained by "mixing and matching" transfected expression cells with chimeric light chain and chimeric heavy chain expression plasmids.
  • CTLA-4 binding can be tested using the binding assays described above and other conventional binding assays (eg, ELISA).
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia based on the three-dimensional structure of the antibody and topology of the CDR loops (Chothia et al.
  • the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. different assignment systems or combinations), resulting in their claimed CDR boundaries being different from the specific CDR boundaries defined by the present invention.
  • Antibodies with different specificities have different CDRs.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
  • the minimal binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • murine CDR regions can be inserted into human germline framework regions using methods known in the art. See US Patent Nos. 5,225,539 to Winter et al. and US Patent Nos. 5,530,101 to Queen et al.; 5,585,089; 5,693,762 and 6,180,370
  • amino acid changes include amino acid deletions, insertions or substitutions.
  • the anti-CTLA-4 antibodies or antigen-binding fragments thereof of the invention include those that have been mutated by amino acid deletions, insertions, or substitutions, but are still related to the antibodies described above (particularly in the CDR regions depicted in the sequences above) Those antibodies that have amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions that have been mutated by amino acid deletions, insertions, or substitutions when compared to the CDR regions depicted in a particular sequence.
  • the polynucleotides encoding the antibodies of the invention include those that have been mutated by nucleotide deletions, insertions, or substitutions, but still have at least about 60, 70, or 60, 70 or more of the coding region corresponding to the CDRs depicted in the sequences described above. , 80, 90, 95 or 100% identical polynucleotides.
  • one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants.
  • An Fc region variant may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are replaced with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl - 1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol,
  • Ipilimumab of the present invention is prepared by Suzhou Junmeng with reference to patent CN1371416B.
  • the present invention provides a polynucleotide encoding an anti-CTLA-4 antibody or antigen-binding fragment thereof as described herein.
  • the polynucleotide may comprise a polynucleotide encoding the amino acid sequence of the light chain variable region and/or heavy chain variable region of an antibody, or a polynucleotide comprising the amino acid sequence encoding the light chain and/or heavy chain of an antibody .
  • the present invention provides an expression vector comprising the polynucleotide as described herein, preferably, the vector is a eukaryotic expression vector.
  • the polynucleotides as described herein are contained in one or more expression vectors.
  • the present invention provides a host cell comprising a polynucleotide as described herein or an expression vector as described herein, preferably the host cell is a eukaryotic cell, more preferably a mammalian cell .
  • the present invention provides a method for preparing an anti-CTLA-4 antibody or antigen-binding fragment thereof as described herein, the method comprising under conditions suitable for expression of the antibody or antigen-binding fragment thereof
  • the antibody or antigen-binding fragment thereof is expressed in a host cell as described herein, and the expressed antibody or antigen-binding fragment thereof is recovered from the host cell.
  • the invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese Hamster Ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, Monkey Kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
  • ATCC American Type Culture Collection
  • the present invention provides a method of making an anti-CTLA-4 antibody, wherein the method comprises, when introducing an expression vector into a mammalian host cell, by culturing the host cell for a period of time sufficient to allow the antibody to develop in the host.
  • the antibody is produced by expression in the cell, or more preferably by secretion of the antibody into the medium in which the host cell is grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • afucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo, and are unlikely to be immunogenic , because their carbohydrate structure is a normal component of native human serum IgG.
  • compositions and pharmaceutical preparations are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CTLA-4 antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, or an antigen-binding fragment thereof as described herein the host cell described above, and a pharmaceutically acceptable carrier or excipient.
  • the anti-CTLA-4 antibodies or pharmaceutical compositions thereof provided by the present invention may incorporate suitable carriers, excipients and other agents in formulations for combined administration, thereby providing improved transfer, delivery, tolerance, and the like.
  • composition refers to a formulation that allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered.
  • compositions of the anti-CTLA-4 antibodies described herein are preferably in the form of aqueous solutions or lyophilized formulations.
  • compositions or formulations of the present invention may also contain one or more other active ingredients required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other .
  • the other active ingredient is a chemotherapeutic agent, an immune checkpoint inhibitor, a growth inhibitory agent, an antibiotic, or various anti-tumor or anti-cancer agents known, in a suitable amount effective for the intended use exist in combination.
  • the pharmaceutical compositions of the present invention further comprise compositions of polynucleotides encoding anti-CTLA-4 antibodies.
  • the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or The pharmaceutical compositions described herein, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or The pharmaceutical composition described herein, preferably it further comprises a delivery device.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein
  • a disease or condition is cancer
  • the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, Breast, colon, lung, bone, pancreatic, skin, head and neck, uterine, ovarian and rectal cancers.
  • the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein , which is used for the treatment and/or prevention of a CTLA-4 mediated disease or condition, preferably the disease or condition is cancer, more preferably the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer , lung, bone, pancreatic, skin, head and neck, uterine, ovarian and rectal cancers.
  • the present invention provides a method of treating and/or preventing a CTLA-4 mediated disease or disorder, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, herein
  • the cancer or tumor described herein can be selected from melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer cancer and rectal cancer.
  • modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (eg, in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration Wait.
  • the present invention also provides for co-administration of a therapeutically effective amount of one or more therapies (eg, therapeutic modalities and/or other therapeutic agents) to a subject.
  • the therapy includes surgery and/or radiation therapy.
  • the methods or uses provided herein further comprise administering to an individual one or more therapies (eg, therapeutic modalities and/or other therapeutic agents).
  • therapies eg, therapeutic modalities and/or other therapeutic agents.
  • Antibodies of the invention can be used alone or in combination with other therapeutic agents in therapy. For example, it can be co-administered with at least one additional therapeutic agent.
  • PD-1 antibody, PD-L1 antibody and LAG-3 antibody for example, PD-1 antibody, PD-L1 antibody and LAG-3 antibody.
  • the present application also provides the above-mentioned anti-CTLA-4 antibody or its antigen-binding fragment, polynucleotide, vector, host cell, immunoconjugate of the antibody or its fragment, or pharmaceutical composition prepared for the prevention and/or treatment of CTLA -4 Use in a medicament for a related disease or disorder, such as a medicament for a tumor.
  • the tumors disclosed herein may be colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, advanced solid tumor or metastases thereof, and the like.
  • the present invention provides a method of detecting the presence of CTLA-4 in a sample using the antibody or antigen-binding fragment thereof described herein.
  • detection includes quantitative or qualitative detection.
  • the sample is a biological sample.
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by their combination with other methods, and equivalent substitutions known to those skilled in the art
  • preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • the present invention adopts the following abbreviations:
  • his-tag stands for histidine tag
  • Fc tag stands for crystallizable fragment tag
  • ECD stands for extracellular domain
  • PEI stands for polyethyleneimine
  • BSA bovine serum albumin
  • PBS stands for phosphate buffered saline
  • CFSE carboxyfluorescein Diacetate succinimidyl ester
  • APC stands for heterotopic phycocyanin
  • NA-PE stands for phycoerythrin-labeled neutravidin
  • PE stands for phycoerythrin
  • TMB stands for 3,3',5,5' - Tetramethylbenzidine
  • HEPES for hydroxyethylpiperazine ethanethiosulfonic acid buffer
  • DTT for dithiothreitol.
  • Example 1 Preparation of recombinant protein for anti-CTLA-4 antibody preparation and testing
  • huCTLA-4 human CTLA-4 (huCTLA-4) gene (Sino Biological), wherein the gene sequence of huCTLA-4 ECD is NCBI accession number NM_005214.3. Corresponding primers were designed, the gene encoding huCTLA-4 ECD was obtained by PCR amplification, and restriction endonuclease sites BSPQI and NotI were added to the upstream and downstream of the gene. The obtained PCR amplified fragment was double digested with BSPQI and NotI, and then cloned into the eukaryotic expression plasmid system (HX1).
  • HX1 eukaryotic expression plasmid system
  • huCTLA-4 ECD his-tag
  • huCTLA-4 ECD FC
  • the cynomolgus monkey CTLA-4 (cynoCTLA-4) gene (Sino Biological) was obtained, wherein the gene sequence of cynoCTLA-4 ECD is NCBI accession number: 102115124. Corresponding primers were designed, and the gene encoding cynoCTLA-4 ECD was obtained by PCR amplification, and restriction endonuclease sites BSPQI and NheI were added to the upstream and downstream of the gene. The obtained PCR-amplified fragment was double digested with BSPQI and NheI, and then cloned into the eukaryotic expression plasmid system (HX1). 293 cells were transfected with this plasmid through PEI, and after 6 days of culture, the culture supernatant was collected and purified to obtain cynoCTLA-4 ECD (FC) recombinant protein.
  • huCD80 Human CD80 (huCD80) gene (Sino Biological), wherein the gene sequence of huCD80 ECD is NCBI accession number NM_005191.3.
  • Corresponding primers were designed, and the gene encoding huCD80 ECD was obtained by PCR amplification, and restriction endonuclease sites EcoRI and NheI were added to the upstream and downstream of the gene.
  • the obtained PCR-amplified fragment was digested with EcoRI and NheI, and then cloned into the eukaryotic expression plasmid system (MX2-FC). 293 cells were transfected with this plasmid through PEI, and after 6 days of culture, the culture supernatant was collected and purified by affinity chromatography to obtain the recombinant protein huCD80 ECD (Fc tag).
  • the human CD86 (huCD86) gene (Sino Biological) was obtained, wherein the gene sequence of huCD86ECD was NCBI accession number NM_006889.3. Corresponding primers were designed, and the gene encoding huCD86ECD was obtained by PCR amplification, and restriction endonuclease sites SapI were added to the upstream and downstream of the gene.
  • the obtained PCR-amplified fragment was digested with SapI and cloned into the eukaryotic expression plasmid system (HX1-FC). 293 cells were transfected with this plasmid through PEI, and after 6 days of culture, the culture supernatant was collected and purified by affinity chromatography to obtain the recombinant protein huCD86ECD (Fc tag).
  • mice Five mice (purchased from Simonsen Laboratories of Gilroy female BALB/c, 8 weeks) were immunized with huCTLA-4 ECD (his tag) as antigen. After the primary immunization (50 ⁇ g/avian), a booster immunization (25 ⁇ g/avian) was performed every 1 week or 2 weeks, and a total of 6 immunizations were performed.
  • huCTLA-4 ECD his tag
  • the inguinal lymph nodes, popliteal lymph nodes and spleen of the mice were taken, and the lymphocyte-rich suspension was obtained after grinding in normal saline. /0 (from ATCC) fusion.
  • the fusion products were cultured in DMEM complete medium containing 1:50 HAT (hypoxanthine, methotrexate, and thymidine) for 5 days to screen for successfully fused cells (ie, hybridomas). It was then changed to DMEM complete medium containing 1:50 HT (hypoxanthine and thymidine) until the end of the selection.
  • the ratio of DMEM complete medium is: 15% FBS (fetal bovine serum)+1:50L-glutamine+100U/mL penicillin-streptomycin+1:100OPI (oxaloacetate, pyruvate and insulin), incubator conditions is 8% CO 2 , 37°C.
  • 461 hybridomas secreting antibodies that can bind to huCTLA-4Fc were screened by ELISA.
  • Antibodies expressed by 24 of the 219 hybridoma cells were able to block the binding of huCTLA-4 to huCD86 by ForteBio-based blocking analysis.
  • 11 polyclonal hybridoma cells expressing antibodies with huCD80 blocking activity were obtained from 24 strains. They were further subcloned.
  • CHO cells expressing huCTLA-4 (derived from ATCC) were incubated with the above hybridoma antibody or control antibody (Ipilimumab) at different dilution concentrations, the initial antibody concentration was 5ug/mL, and the dilution ratio was 1:3, and then 5 ⁇ g/mL was added.
  • hybridoma antibodies (1B2.1, 1A5.1, 3E6.1, 3E6.2, 4B7.1, 4E7.1, 5B9.1) were combined with huCTLA-4 expressing Jurkat effector cells (Promega) and Raji APC target cells (Promega) were mixed (the control antibody was Ipilimumab) to detect the expression of luciferase mediated by the downstream signaling pathway of Jurkat effector cells, so as to determine the biological activity of hybridoma antibodies.
  • 293T-TIGIT, 293T-CTLA4, 293T-BTLA or 293T blast cells were treated with 1 ⁇ g hybridoma antibody 1B2.1, 1A5.1, 3E6.1, 3E6.2, 4B7.1, 4E7.1, 5B9.1 or control Antibodies were incubated at 4°C for 30 minutes, then the above cell mixture was incubated with 5 ⁇ L of APC-labeled murine secondary antibody (Southern Biotech) for 20 minutes at 4°C to detect secondary antibody bound to cells.
  • the control antibody was selected from commercially purchased CTLA4 monoclonal antibody L3D10 (BioLegend) and TIGIT monoclonal antibody MBSA43 (eBioscience).
  • the DNA coding sequence corresponding to the variable region of the anti-CTLA-4 murine antibody was determined by a method based on degenerate primer PCR. Briefly, the hybridoma cell lines were scaled up and cultured, cells were collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol. Using this as a template to synthesize the first-strand cDNA, and then use the first-strand cDNA as a subsequent template to amplify the DNA coding sequence of the variable region by PCR. The PCR primers used were based on Ig-primer sets. The PCR product was recovered and purified. The amplified product was sequenced to obtain the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CTLA-4 murine antibody.
  • the germline and rearranged Ig variable region sequence databases were searched for consensus sequences using NCBI Ig-Blast (http://www.ncbi.nlm.nih.gov/projects/igblast/). Based on Kabat (Wu, TT and Kabat, EA 1970 J. Exp. Med., 132: 211-250) and the IMGT system (Lefranc M.-P.
  • CDR complementarity determining region
  • amino acid sequences of the light and heavy chain variable regions and CDRs of the selected anti-CTLA-4 murine antibodies are shown in Table 2.
  • Table 2 CDR and variable region amino acid sequences of anti-CTLA-4 murine antibodies (KABAT protocol)
  • the light chain and heavy chain variable regions of anti-CTLA-4 murine antibodies 1A5.1, 3E6.1, 4B7.1, and 5B9.1 were selected to construct anti-CTLA-4 chimeric antibodies.
  • the coding sequences of heavy chain constant region Fc and light chain constant region ⁇ were cloned from human B lymphocytes (from Beijing Institute of Blood) and introduced into pCDNA3.1 plasmid.
  • the coding sequences of the heavy chain and light chain variable regions of the aforementioned anti-CTLA-4 murine antibody were synthesized by Genescript Company.
  • the coding sequences of the heavy chain variable regions of various anti-CTLA-4 murine antibodies were digested with BSPQI, and the coding sequences of the light chain variable regions were digested with BSPQI, and various combinations as shown in Table 3 were used.
  • the pCDNA3.1 plasmid of the coding sequence of the region was sequenced to confirm the correct clone.
  • the best chimeric antibodies were screened according to the binding of the chimeric antibodies to huCTLA-4, the ability to block the binding of huCTLA-4 to CD80/CD86 and the biological activity of antagonizing huCTLA-4 in Table 3.
  • the experimental methods and results are shown below.
  • JS007-3, JS007-15, JS007-20 chimeric antibodies have good binding to huCTLA-4, and their EC 50s are 17.23ng/mL, 19.72ng/mL and 12.88ng/mL, respectively, and
  • the control antibody, ipilimumab was comparable or superior to the control antibody.
  • CHO cells expressing huCTLA-4 (self-constructed by Junmeng, CHO derived from ATCC) were mixed with the chimeric antibody and control antibody (Ipilimumab) in Table 3 (initial concentration of 10 ⁇ g/ml, 3-fold concentration gradient dilution) at room temperature. After incubation for 30 minutes, a fluorescent secondary antibody (PE-anti human IgG) was added to detect the binding of the chimeric antibody to huCTLA-4 expressing cells.
  • a fluorescent secondary antibody PE-anti human IgG
  • HX1 huCTLA4 hiss to 1.0 ⁇ g/mL with PBS (Hyclone), add 100 ⁇ l/well to the ELISA plate, incubate at 37°C for 90 min; wash the plate; add 200 ⁇ l/well 2% BSA to the plate, set Incubate in a constant temperature incubator at 37°C for 90 min; wash the plate; take MX2 hCD80 Fc and dilute it with 2% BSA to 5.0 ⁇ g/mL, and dilute the sample with this concentration of MX2 hCD80 Fc.
  • the chimeric antibody and control antibody Ipilimumab were diluted to 100 ⁇ g/ml, and each dilution was not higher than 10 times.
  • the chimeric antibody and the control antibody were sequentially diluted in a 2.5-fold gradient on the sample dilution plate. All chimeric antibody and control antibody solutions were added to the ELISA plate at 100 ⁇ l/well, and incubated in a constant temperature incubator at 37°C for 90 min; washed the plate; HRP-conjugated goat anti-mouse antibody IgG (Fc specific) (Sigma, cat.
  • A2554 Dilute 5000 times with 2% BSA, add 100 ⁇ l/well to the ELISA plate, incubate for 60 min in a constant temperature incubator at 37 °C; wash the plate; add chromogenic solution TMB, 100 ⁇ l/well, avoid air bubbles, and protect from light at 37 °C Color development for 15min; finally add 2M hydrochloric acid solution to stop the reaction, 100 ⁇ l/well, avoid air bubbles, complete the microplate reader reading within 10min (wavelength: 450/620nm)
  • JS007-3, JS007-15, JS007-20 chimeric antibodies have the ability to block huCTLA-4 binding to CD80, an IC 50 were 189.7ng / mL, 189.7ng / mL and 186.6ng /mL, and both were significantly better than the control antibody Ipilimumab.
  • the chimeric antibody and control antibody Ipilimumab in Table 3 (initial concentration of 30 ⁇ g/ml, 3-fold concentration gradient dilution) and a certain concentration of huCTLA4 recombinant protein were added to CHO cells expressing CD80 or CD86 (constructed by Junmeng independently). ), after co-incubating at room temperature for 30 minutes, a fluorescent secondary antibody (PE-anti human IgG4) (Southern biotech) was added to detect the ability of the chimeric antibody to block the binding of huCTLA-4 to CD80/CD86 expressing cells.
  • PE-anti human IgG4 Pacificn biotech
  • JS007-3, JS007-15 and JS007-20 chimeric antibodies all had the ability to block the binding of huCTLA-4 to CD80/CD86 expressing cells.
  • the IC 50s of blocking the binding of huCTLA-4 to CD80-expressing cells were 0.6986ng/mL, 0.398ng/mL and 1.211ng/mL, which were significantly better than the control antibody Ipilimumab; blocking the binding of huCTLA-4 to CD86-expressing cells the IC 50 were 0.196ng / mL, 0.0816ng / mL and 0.2528ng / mL, or control antibody Ipilimumab rather than the control antibody.
  • chimeric antibodies (JS007-3, JS007-15, JS007-20) were mixed with huCTLA-4 expressing Jurkat effector cells and Raji APC target cells to detect fluorescence mediated by downstream signaling pathways of Jurkat effector cells Peptase expression was measured, and the biological activity of chimeric antibodies against huCTLA-4 was determined.
  • JS007-3, JS007-15 and JS007-20 chimeric antibodies have good biological activity of antagonizing huCTLA-4.
  • the humanization transformation process involves the following steps: A. Compare the gene sequence of the antibody secreted by each hybridoma cell with the human embryonic antibody gene sequence to find out the sequence with high homology; B. Analyze and investigate HLA-DR affinity, select the human embryonic framework region sequence with low affinity; C. Using computer simulation technology, apply molecular docking to analyze the amino acid sequence of the variable region and its surrounding framework region, and investigate its spatial three-dimensional binding mode.
  • the gene sequence of the antibody secreted by each hybridoma cell is analyzed for the key amino acid individuals that can interact with hCTLA-4 and maintain the spatial structure, and then graft it into the selected human embryo.
  • the amino acid sites in the framework region that must be retained are marked; D.
  • the embedded residues and residues that have direct interaction with the CDR region As well as backmutation of residues that have important effects on the conformation of VL and VH, and optimization of amino acid residues that lead to chemical instability in the CDR region of the antibody. The optimization results are as follows:
  • HCDR2 YIGYDGSNYYNPSLKN (SEQ ID NO: 2) was optimized YIGYDGSNYYNPSLK S (SEQ ID NO: 37 );
  • HCDR3 NYYSGYFDF (SEQ ID NO: 3) optimization of NYYSGYFD S (SEQ ID NO: 38 );
  • HCDR2 YIGYDGSNNYNPSLKN (SEQ ID NO: 20) was optimized YIGYDGSNNYNPSLK S (SEQ ID NO: 39 );
  • LCDR1 KASQNVGTYVA (SEQ ID NO: 16) optimized to R ASQNVGTYVA (SEQ ID NO: 40);
  • LCDR1 KASQNVGTYVA (SEQ ID NO: 16) was optimized to Q ASQNVGTYVA (SEQ ID NO: 41).
  • variable regions of humanized anti-CTAL-4 antibodies were obtained:
  • 4B7VL-4 SEQ ID NO: 57
  • 4B7VL-5 SEQ ID NO: 58
  • 4B7VL-6 SEQ ID NO: 59
  • 4B7VL-7 SEQ ID NO: 60
  • 4B7VL-8 SEQ ID NO: 61
  • 4B7VL-9 SEQ ID NO: 62
  • variable regions of the humanized anti-CTLA-4 antibodies designed above were used in various combinations as shown in Table 4.
  • the coding sequences of the heavy and light chain variable regions of the humanized anti-CTLA-4 antibodies were synthesized by Genscript Company , the heavy chain variable region coding sequence and light chain variable region coding sequence of various synthetic humanized anti-CTLA-4 antibodies were digested with Bspq I and introduced into the pCDNA3.1 plasmid that already contained the coding sequence of the constant region, and Correct clones were confirmed by sequencing.
  • the humanized anti-CTLA-4 antibody had good binding to huCTLA-4, comparable to or better than the control antibody.
  • HX1 hCTLA4-his Dilute HX1 hCTLA4-his to 1.0 ⁇ g/mL with PBS (Hyclone), add 100 ⁇ l/well to the ELISA plate, incubate at 37°C for 90 min; wash the plate; add 200 ⁇ l/well 2% BSA to the plate, Incubate in a 37°C constant temperature incubator for 90 min; wash the plate; take MX2 hCD80 Fc and dilute it with 2% BSA to 5.0 ⁇ g/mL, and dilute the antibody with this concentration of MX2 hCD80 Fc.
  • the humanized anti-CTLA-4 antibody and control antibody Ipilimumab in Table 4 were diluted to 100 ⁇ g/ml, and each dilution was not higher than 10 times.
  • the humanized anti-CTLA-4 antibody had a good ability to block the binding of huCTLA-4 to CD80, which was comparable to or better than that of the control antibody.
  • Humanized anti-CTLA-4 antibodies huJS007-46, 47, 48, 49, 55, 56, 73, 79, 82, 88, 100 and 106 were selected for biological Activity analysis.
  • Jurkat cells expressing huCTLA-4 were plated at 6' 10 4 cells per well, and 3' 10 4 Raji APC cells were added to each well as well as the above 12 humanized anti-CTLA-4 antibodies or control antibodies (Ipilimumab) at different concentrations. ), and after 6 hours of incubation, the luciferase method was used to measure the T cell activation activity.
  • 293T-CTLA4 cells were labeled with CFSE, and peripheral blood mononuclear cells (PBMC2144896) and different concentrations of the above 12 humanized anti-CTLA-4 antibodies or controls were added at a target cell:effector ratio of 1:25.
  • Antibody Ipilimumab
  • Cells were stained with propidium iodide (PI) and analyzed by flow cytometry. ADCC killing (%) was expressed as the percentage of dead target cells (PI and CFSE positive) to total target cells (CFSE positive).
  • 293T-CTLA4 cells were activated with different concentrations (0.8-100 ⁇ g/mL) of the above 12 humanized anti-CTLA-4 antibodies or control antibody (Ipilimumab) at 37°C for 15 minutes, and different dilution gradients (1:5, 1:1) were added. 10, 1:20) of human serum complement and incubated for 1 hour. After incubation, cells were stained with propidium iodide (PI) and analyzed with a BD FACSCalibur flow cytometer. CDC killing (%) was expressed as a percentage of PI positive target cells to total target cells. The results are shown in Figure 13, indicating that the above 12 humanized anti-CTLA-4 antibodies have no CDC activity or negligible CDC activity.
  • PI propidium iodide
  • the CDR/variable region/light chain/heavy chain amino acid sequences of the four humanized anti-CTLA-4 antibodies are shown in Table 5.
  • the route of administration in all groups was intraperitoneal injection, the dosage was 0.3 mg/kg, and the administration concentration was 0.03 mg/ml.
  • the drug was administered twice a week, 5 times in a row, and the experiment was terminated 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • T0 the mean tumor volume of the treatment group and the positive control group on the 0th day of administration
  • Vi the negative control group on the ith day of administration
  • V0 the mean tumor volume of the negative control group on the 0th day of administration.
  • KLH IgG1 negative control group mean tumor volume was 975 ⁇ 115mm 3
  • the relative tumor inhibition rate was 18.1%; the mean tumor volumes of the huJS007-47 treatment group, huJS007-48 treatment group, huJS007-79 treatment group and huJS007-106 treatment group were 229 ⁇ 85mm 3 , 313 ⁇ 197mm 3 , 550 ⁇ 229mm 3 , respectively and 472 ⁇ 125mm 3 , compared with KLH IgG1, the relative tumor inhibition rates were 89.2%, 79.1%, 50.9% and 60.1%, respectively, indicating that the above humanized anti-CTLA-4 antibodies can inhibit the humanized B-hCTLA4 in vivo.
  • the growth of subcutaneous xenografts of murine MC38-WT cells was significantly better than that of the
  • Embodiment 12 Fortebio binding experiment identifies antigenic epitopes
  • the full-length antibodies 2.7 ⁇ g/mL humanized anti-CTLA-4 antibody huJS007-47 and 2 ⁇ g/mL control antibody (Ipilimumab), were first captured using a Protein A probe (Fortebio). The probe was then immersed in 55nM human CTLA (huCTLA) antigen solution to bind the full-length antibody to the antigen. Finally, the probe was immersed in 600nM Fab solution, including humanized Fab huJS00-47 and control Fab (Ipilimumab), to detect whether the antigen and Fab bind.
  • huCTLA human CTLA
  • 600nM Fab solution including humanized Fab huJS00-47 and control Fab (Ipilimumab)
  • huCTLA-4 could continue to bind to the control Fab (Ipilimumab). 4 different epitopes.
  • Example 13 Identification of antigenic epitopes by hydrogen deuterium exchange mass spectrometry
  • the desalted peptides were separated with a linear gradient of 4-40% acetonitrile and 0.3% formic acid using a 2.1 mm x 5 cm C18 column (1.9 ⁇ m Hypersil Gold, Thermo Fisher) over 6 minutes.
  • Sample processing, protein digestion and peptide isolation were performed at 4°C.
  • Mass spectrometry data for hydrogen deuterium exchange was acquired using an Orbitrap mass spectrometer (Orbitrap Fusion TM Tribrid TM Mass Spectrometer, Thermo Fisher), measured at a resolution of 65,000 (m/z 400). Each sample had three replicates at each time point.
  • Hydrogen-deuterium exchange mass spectrometry data analysis The average m/z centroid value of the mass spectral peak intensity of each digested peptide was calculated by the HDX Workbench software, and then converted into the percentage of deuterium incorporation. The key amino acid sequences involved in the spatial epitope were calculated, and the difference in Delta %D was determined by calculating the difference between the two samples (comparing the change in percent deuterium incorporation on the same peptide). Differences in Delta%D outside -5 to 5% were considered significant. In addition, HDX Workbench detected statistically significant (p ⁇ 0.05) differences between samples at each time point by the student's t test.
  • the upper curve is the hydrogen deuterium exchange rate of the peptide (YASPGKATE, +1) when it is not bound to huJS007-47
  • the lower curve is the hydrogen of the peptide after binding to huJS007-47.
  • Deuterium exchange rate characterization (significant decrease). According to the detection results of hydrogen deuterium exchange mass spectrometry, huJS007-47 and Ipilimumab bind to different epitopes of huCTLA-4.
  • Example 14 Detection of binding of monoclonal antibody huJS007-47 to huCTLA-4 mutant by ELISA
  • the huCTLA-4 mutants used in the ELISA assay included unmutated HXT huCTLA4 his, as well as mutated huCTLA4 N-his P27A, huCTLA4 N-his G28A, huCTLA4 N-his K29A, and huCTLA4 N-his T31A.
  • huCTLA-4 Dilute huCTLA-4 his and its mutants to 1.0 ⁇ g/ml with PBS (Hyclone), and incubate in a constant temperature incubator at 37°C for 90 min; wash the plate and block it with 2% BSA; after washing the plate, add gradient dilution with 2% BSA
  • the monoclonal antibody huJS007-47 (from 1000ng/ml to 0.042ng/ml, 2.5-fold serial dilution) was incubated at 37°C for 60min; washed the plate; HRP-conjugated goat anti-human antibody IgG (Fc specificity) for detection (Sigma, product number A0170), incubate at 37°C for 60 minutes, then incubated with HRP substrate TMB (Sigma, product number T2885) at 37°C for 15 minutes for color development, and finally add 2M hydrochloric acid to stop, avoid air bubbles, and complete within 10 minutes Microplate reader reading (wavelength: 450/
  • Example 15 Inhibitory effect of huJS007-47 on the growth of hCTLA4 humanized mice transplanted with MC38 tumor use
  • mice Take 6-8-week-old female hCTLA4 humanized mice (Biocyto Jiangsu Gene Biotechnology Co., Ltd.), and subcutaneously inoculate 1 x 10 6 MC38 cells (0.1ml/mice (medium containing cells RPMI1640 ( Gibco))).
  • the average tumor volume was about 119 mm 3
  • 40 animals were selected and randomly divided into 5 groups according to the tumor volume, with 8 animals in each group. respectively
  • Anti-KLH hIgG1 negative control group 1mg/kg
  • Ipilimumab positive control group 1mg/kg
  • huJS007-47 treatment group 0.1 mg/kg
  • huJS007-47 treatment group 0.3 mg/kg
  • huJS007-47 treatment group 1mg/kg
  • the groups were administered on the same day, and the route of administration in all groups was intraperitoneal injection, twice a week, 6 times in a row, and the experiment ended 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • TGI% tumor inhibition rate
  • T0 the mean tumor volume of the treatment group on the 0th day of administration
  • Vi the mean tumor volume of the negative control group on the ith day of administration
  • V0 negative The mean tumor volume of the control group on the 0th day of administration
  • anti-KLH hIgG1 mean tumor volume of negative control group at a dose of 1mg / kg for the 1116 ⁇ 106mm 3.
  • the mean tumor volume of the Ipilimumab positive control group at a dose of 1 mg/kg was 255 ⁇ 88 mm 3 and the TGI% was 86.36%.
  • huJS007-47 significantly inhibited the growth of MC38 tumor volume in hCTLA4 humanized mice at doses of 0.1, 0.3 and 1 mg/kg, and showed a good dose effect.
  • -47 has significantly better tumor suppressive effect than ipilimumab.
  • Example 16 Inhibitory effect of huJS007-47 on the growth of hCTLA4 humanized mice transplanted with H22 tumor
  • mice Take 6-8 week old female hCTLA4 humanized mice (Shanghai Southern Model Biotechnology Co., Ltd.), and inoculate 1 x 10 6 H22 cells (0.1 ml/mouse (medium containing cells RPMI1640 (Gibco) subcutaneously on the right back ))).
  • the average tumor volume was about 119 mm 3
  • 35 animals were selected and randomly divided into 5 groups according to the tumor volume, with 7 animals in each group. respectively
  • Anti-KLH hIgG1 negative control group 0.3mg/kg
  • Ipilimumab positive control group 0.1mg/kg
  • huJS007-47 treatment group 0.03mg/kg
  • huJS007-47 treatment group 0.1 mg/kg
  • huJS007-47 treatment group 0.3 mg/kg
  • the groups were administered on the same day, and the route of administration in all groups was intraperitoneal injection, twice a week, 6 times in a row, and the experiment ended 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • the mice were euthanized, and the tumor inhibition rate TGI% was calculated in the same way as in Example 15.
  • the mean tumor volume of the anti-KLH hIgG1 negative control group at the dose of 0.3 mg/kg was 2304 ⁇ 402 mm 3 on the 21st day after the start of administration.
  • the mean tumor volume of the Ipilimumab positive control group at the dose of 0.1 mg/kg was 837 ⁇ 397 mm 3 and the TGI% was 67.14%.
  • huJS007-47 significantly inhibited the growth of H22 tumor volume in hCTLA4 humanized mice at doses of 0.1 and 0.3 mg/kg, and showed a good dose effect.
  • -47 has significantly better tumor suppressive effect than ipilimumab.
  • Embodiment 17 CTLA4-JS007 scFv crystallization method and structure analysis
  • CTLA4-JS007 scFv complex Through protein preparation and crystal screening of CTLA4-JS007 scFv complex, X-ray crystallography was obtained, and the structure of the complex was analyzed by molecular replacement method, and the molecular basis of CTLA4-JS007 scFv interaction was analyzed to evaluate JS007 antibody (i.e. huJS007 -47) Molecular mechanism of binding to CTLA-4 and its mechanism of blocking CTLA-4/B7-1 interaction.
  • JS007 antibody i.e. huJS007 -47
  • JS007 scFv is huJS007-47 scFv
  • CTLA-4 protein is human CTLA-4 protein (SEQ ID NO: 71).
  • CTLA-4 protein C35-P154
  • JS007-scFv The inclusion body form of CTLA-4 protein (C35-P154) and JS007-scFv were prepared by prokaryotic expression system (E.coli BL21 strain), and CTLA4 and JS007-scFv proteins were obtained by in vitro renaturation of inclusion bodies. The two proteins were then purified by gel filtration chromatography (GE, superdex 200 (10/300GL)). The CTLA4 protein and JS007-scFv protein were mixed and incubated on ice at a molar ratio of 1:1 for 30 min, and then the complex was further purified by gel filtration chromatography (GE, superdex 200 (10/300GL)). , the CTLA4-JS07 scFv complex protein was obtained.
  • CTLA4-JS007scFv complex protein with a purity higher than 90% was screened for crystals using a crystal screening kit, and 1 ⁇ l of the protein solution was mixed with 1 ⁇ l of the pool solution at a concentration of 10 mg/ml, and placed at 16° for crystal growth. Finally, CTLA4-JS007 scFv complex crystals were screened under the conditions of MD1-13-20 (0.1M Tris pH: 8.5 30%w/v PEG 4000) of Molecular Dimension Company.
  • the collection of protein crystal diffraction data of CTLA-4-JS007scFv complex was carried out at the Shanghai Light Source (SSRF) BL17U Biomacromolecule Crystallography Beamline Station.
  • the X-ray wavelengths used were Crystal seat position adjusted, exposure, preliminary indexing, and diffraction data collected.
  • the diffraction data were processed using HKL2000 software. It was then modeled and refined with Coot and Phenix. Evaluate the final model with Molprobity.
  • the analyzed three-dimensional structure map and electron density map of the protein were analyzed and displayed using PyMOL software.
  • Numbers without parentheses in the column "Number of interactions" represent the number of van der Waals interactions within a distance of 4.5 ⁇ ; numbers in parentheses represent the number of hydrogen bond interactions within a distance of 3.5 ⁇ .
  • Blocking the interaction between CTLA-4 and its ligands B7-1 (ie CD80) or B7-2 (ie CD86) is one of the molecular mechanisms by which JS007 antibody exerts immune activation.
  • B7-1 ie CD80
  • B7-2 ie CD86
  • JS007 blocking the interaction between CTLA-4 and B7-1 was further investigated. In-depth analysis was carried out.
  • CTLA-4-JS007 and the complex structure of CTLA-4-B7-1 were analyzed according to CTLA-4 as the fixed reference. There is significant steric hindrance between the CTLA-4 molecules, while the FG loop in the CTLA-4 molecule has extensive interactions with both JS007 and B7-1, including hydrogen bond interactions and van der Waals interactions (Figure 21A).

Abstract

一种与CTLA-4特异性结合的抗体或其抗原结合片段和包含其的组合物。还提供了编码抗体或其抗原结合片段的核酸分子，用于表达抗体或其抗原结合片段的载体和宿主细胞，以及抗体或其抗原结合片段的治疗和诊断方法和用途。

Description

抗CTLA-4抗体及其用途 技术领域
本发明提供了与CTLA-4特异性结合的抗体或其抗原结合片段和包含其的组合物。还提供了编码本发明抗体或其抗原结合片段的核酸分子，用于表达本发明抗体或其抗原结合片段的载体和宿主细胞，以及本发明抗体或其抗原结合片段的治疗和诊断方法和用途。
背景技术
细胞毒性T淋巴细胞相关蛋白4(CTLA4或CTLA-4，cytotoxic T-lymphocyte-associated protein 4)，也称为CD152(cluster of differentiation 152)，是由CTLA-4基因编码的跨膜蛋白，该基因位于人类第2号染色体2q33。CTLA-4是免疫球蛋白超家族的一员，由胞外V区、跨膜区和胞浆区组成。CTLA-4与T细胞表面的协同刺激分子受体CD28具有同源性，两者竞争结合其配体B7-1(CD80)和B7-2(CD86)，这类配体主要表达于抗原递呈细胞表面。与CD28相比，CTLA-4与CD80和CD86的结合亲和力更高，因此能够竞争和阻断CD28介导的激活作用。CTLA-4通常表达在调节性T细胞(Treg)和活化状态的常规T细胞表面上。其在与B7类分子结合后，抑制T细胞的激活，参与免疫反应的负调控，充当免疫检查点并下调免疫应答，所以CTLA-4在免疫调节中起到非常重要的作用。
T细胞的激活需要两个信号的刺激，第一个信号来自T细胞受体(TCR)特异性结合抗原递呈细胞(APC)表面的抗原肽-MHC复合物，第二个信号通路需要共刺激分子(如CD28)的参与，当CD28与B7-1/B7-2(CD80/CD86)结合后可以进一步活化T细胞，促进其成熟和增殖。目前的研究表明，在免疫应答过程中，CTLA-4通过下列途径下调T细胞的功能：其一，CTLA-4可以通过其与CD80/CD86的高亲和力，竞争性阻断CD28与CD80/86共刺激信号的传递，进而抑制T细胞的增殖，减少IL-2的分泌。其二，CTLA-4可以通过降低CD80/CD86在抗原递呈细胞(APC)上的表达水平或者通过反式内吞作用将CD80/CD86分子从抗原递呈细胞(APC)表面移除，从而减少了CD28参与的T细胞激活。其三，CTLA-4可以通过介导树突状细胞结合CD80/CD86并诱导色氨酸降解酶IDO的表达，抑制TCR信号。此外，CTLA-4还可以通过招募 抑制性分子结合到免疫突触，诱导产生调节性细胞因子，从而抑制APC和TCR信号的传递。
已在许多研究中证明CTLA-4的阻断可以诱导肿瘤消退。抗CTLA-4抗体能够在体内外有效、特异地抑制细胞和体液免疫应答，对移植排斥反应及各种自身免疫性疾病有显著的治疗作用，毒副作用低。
尽管目前已有CTLA-4的单抗药物Ipilimumab(百时美施贵宝)和Tremelimumab(阿斯利康)用于一些癌症治疗并且正在测试其他抗癌适应症，但是仍需要在各方面包括活性相比于已知抗体改善的新型抗CTLA-4抗体。
发明内容
本发明提供了抗CTLA-4抗体或其抗原结合片段，其具有针对人CTLA-4的高亲和力和高特异性等优势。本发明提供的抗CTLA-4抗体或其抗原结合片段可作为独立的疗法或与其它疗法/或其他抗癌药剂联合，用于诸如癌症的治疗。
在一个方面，本发明提供了一种抗CTLA-4抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区：
所述重链可变区包含：
(Ⅰ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；优选地，所述与SEQ ID NO:2所示氨基酸序列具有1、2或3个氨基酸差异的HCDR2是SEQ ID NO:37；优选地，所述与SEQ ID NO:3所示氨基酸序列具有1、2或3个氨基酸差异的HCDR3是SEQ ID NO:38；或
(Ⅱ)氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或(Ⅲ)氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或
(Ⅳ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的 HCDR1、HCDR2和HCDR3；或与SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；优选地，所述与SEQ ID NO:20所示氨基酸序列具有1、2或3个氨基酸差异的HCDR2是SEQ ID NO:39；或
(Ⅴ)氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；
所述轻链可变区包含：
(Ⅰ)氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或
(Ⅱ)氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或(Ⅲ)氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；优选地，所述与SEQ ID NO:16所示氨基酸序列具有1、2或3个氨基酸差异的LCDR1是SEQ ID NO:40或41；或
(Ⅳ)氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3。
在一些实施方式中，所述抗体或其抗原结合片段包含重链可变区和/或轻链可变区：
重链可变区，所述重链可变区包含：
(Ⅰ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
(Ⅱ)氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；或
(Ⅲ)氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的 HCDR1、HCDR2和HCDR3；或
(Ⅳ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；或
(Ⅴ)氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；或
(Ⅵ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:37和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
(Ⅶ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:37和SEQ ID NO:38所示的HCDR1、HCDR2和HCDR3；或
(Ⅷ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:38所示的HCDR1、HCDR2和HCDR3；或
(Ⅸ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:39和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；
和/或，轻链可变区，所述轻链可变区包含：
(Ⅰ)氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或
(Ⅱ)氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的LCDR1、LCDR2和LCDR3；或
(Ⅲ)氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
(Ⅳ)氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
(Ⅴ)氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
(Ⅵ)氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，
所述重链可变区包含HCDR1、HCDR2和HCDR3，其中HCDR1选自如SEQ ID NO:1或19所示的氨基酸序列；HCDR2选自如SEQ ID NO:2、20、37和39中任一 项所示的氨基酸序列，HCDR3选自如SEQ ID NO:3、21和38中任一项所示的氨基酸序列；和
所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中LCDR1的氨基酸序列如SEQ ID NO:72所示；LCDR2的氨基酸序列如SEQ ID NO:17所示；LCDR3的氨基酸序列如SEQ ID NO:18所示，其中所述的SEQ ID NO:72具有如下通式所示的氨基酸序列：
LCDR1：XASQNVGTYVA，其中，X选自K、R和Q。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含重链可变区和/或轻链可变区，其中，
所述重链可变区包含：
(Ⅰ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
(Ⅱ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:37和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
(Ⅲ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:38所示的HCDR1、HCDR2和HCDR3；或
(Ⅳ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:37和SEQ ID NO:38所示的HCDR1、HCDR2和HCDR3；或
(Ⅴ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；或
(Ⅵ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:39和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；
所述轻链可变区包含：
(Ⅰ)氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
(Ⅱ)氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
(Ⅲ)氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，所述抗体或其抗原结合片段包含：
(Ⅰ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或
(Ⅱ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的LCDR1、LCDR2和LCDR3；或
(Ⅲ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
(Ⅳ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
(Ⅴ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
(Ⅵ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
(Ⅶ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:39和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
(Ⅷ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:37和SEQ ID NO:38所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分 别如SEQ ID NO:40、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
(Ⅸ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，所述抗体或其抗原结合片段包含重链可变区和轻链可变区：
(Ⅰ)所述重链可变区包含如SEQ ID NO:28、30、32、34或35中任一项所示的氨基酸序列，或与SEQ ID NO:28、30、32、34或35中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:29、31、33或36中任一项所示的氨基酸序列，或与SEQ ID NO:29、31、33或36中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；或
(Ⅱ)氨基酸序列如SEQ ID NO:28所示的重链可变区和氨基酸序列如SEQ ID NO:29所示的轻链可变区；或
(Ⅲ)氨基酸序列如SEQ ID NO:30所示的重链可变区和氨基酸序列如SEQ ID NO:31所示的轻链可变区；或
(Ⅳ)氨基酸序列如SEQ ID NO:32所示的重链可变区和氨基酸序列如SEQ ID NO:33所示的轻链可变区；或
(Ⅳ)氨基酸序列如SEQ ID NO:34所示的重链可变区和氨基酸序列如SEQ ID NO:33所示的轻链可变区；或
(Ⅵ)氨基酸序列如SEQ ID NO:35所示的重链可变区和氨基酸序列如SEQ ID NO:36所示的轻链可变区。
在一些实施方式中，所述抗体或其抗原结合片段包含重链可变区和轻链可变区：
所述重链可变区包含如SEQ ID NO:28或34中任一项所示的氨基酸序列或其变体，和
所述轻链可变区包含如SEQ ID NO:33所示的氨基酸序列或其变体，
其中所述变体包含：
在氨基酸序列如SEQ ID NO:28或34中任一项所示的重链可变区中的1、2、3、4、5、6、7、8、9或10个氨基酸差异，和/或
在氨基酸序列如SEQ ID NO:33所示的轻链可变区中的1、2、3、4、5、6、7、8、9或10个氨基酸差异。
在一些实施方式中，所述抗体或其抗原结合片段包含：
(Ⅰ)重链可变区，其包含如SEQ ID NO:42、43、44、45、46、47或48中任一项所示的氨基酸序列，或与SEQ ID NO:42、43、44、45、46、47或48中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；和
轻链可变区，其包含如SEQ ID NO:54、55、56、57、58、59、60、61或62中任一项所示的氨基酸序列，或与SEQ ID NO:54、55、56、57、58、59、60、61或62中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；或
(Ⅱ)重链可变区，其包含如SEQ ID NO:49、50、51、52或53中任一项所示的氨基酸序列，或与SEQ ID NO:49、50、51、52或53中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；和
轻链可变区，其包含如SEQ ID NO:54、55、56、57、58、59、60、61或62中任一项所示的氨基酸序列，或与SEQ ID NO:54、55、56、57、58、59、60、61或62中任一项所述的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；或
(Ⅲ)氨基酸序列如SEQ ID NO:50所示的重链可变区和氨基酸序列如SEQ ID NO:54、55、56或57所示的轻链可变区；或
(Ⅳ)氨基酸序列如SEQ ID NO:51所示的重链可变区和氨基酸序列如SEQ ID NO:54或55所示的轻链可变区；或
(Ⅴ)氨基酸序列如SEQ ID NO:46、47、53所示的重链可变区和氨基酸序列如SEQ ID NO:54或60所示的轻链可变区；或
(Ⅵ)氨基酸序列如SEQ ID NO:50所示的重链可变区和氨基酸序列如SEQ ID NO:55所示的轻链可变区；或
(Ⅶ)氨基酸序列如SEQ ID NO:50所示的重链可变区和氨基酸序列如SEQ ID NO:56所示的轻链可变区；或
(Ⅷ)氨基酸序列如SEQ ID NO:46所示的重链可变区和氨基酸序列如SEQ ID NO:60所示的轻链可变区；或
(Ⅸ)氨基酸序列如SEQ ID NO:53所示的重链可变区和氨基酸序列如SEQ ID  NO:60所示的轻链可变区。
在一些实施方式中，所述抗体包含重链和轻链，所述重链包含如SEQ ID NO:63、65、67或69中任一项所示的氨基酸序列，或与SEQ ID NO:63、65、67或69中任一项所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；
和所述轻链包含如SEQ ID NO:64、66、68或70中任一项所示的氨基酸序列，或与SEQ ID NO:64、66、68或70中任一项所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，所述抗体包含：
(Ⅰ)氨基酸序列如SEQ ID NO:63所示的重链，和氨基酸序列如SEQ ID NO:64所示的轻链；或
(Ⅱ)氨基酸序列如SEQ ID NO:65所示的重链，和氨基酸序列如SEQ ID NO:66所示的轻链；或
(Ⅲ)氨基酸序列如SEQ ID NO:67所示的重链，和氨基酸序列如SEQ ID NO:68所示的轻链；或
(Ⅳ)氨基酸序列如SEQ ID NO:69所示的重链，和氨基酸序列如SEQ ID NO:70所示的轻链。
在一些实施方式中，所述抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体、或全人抗体，或其抗原结合片段。
在一些实施方式中，所述抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段是任何IgG亚型，如IgG1、IgG2、IgG3或IgG4，优选为IgG1或IgG4，更优选为IgG1kappa亚型。
在另一个方面，本发明提供了一种分离的抗CTLA-4抗体或其抗原结合片段，其具有以下特性中的一种或多种：
(1)与本文所述的抗CTLA-4抗体或其抗原结合片段结合相同或者完全或部分重叠的人CTLA-4蛋白的表位；
(2)与本文所述的抗CTLA-4抗体或其抗原结合片段竞争结合人CTLA-4蛋白的表位；
(3)结合由SEQ ID NO:71的残基27-29组成的表位，其中氨基酸残基编号为从 SEQ ID NO:71中A37开始进行氨基酸编号；
(4)结合SEQ ID NO:71的氨基酸残基27，28和29中的一个或多个，其中氨基酸残基编号为从SEQ ID NO:71中A37开始进行氨基酸编号；
(5)抑制或阻断人CTLA-4蛋白与人CD80和/或人CD86或表达人CD80和/或人CD86的细胞的结合；优选的，其与CD80竞争结合的氨基酸残基为135，137，138和140中的一个或多个，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号；
(6)结合由SEQ ID NO:71的残基36-41和/或59-66和/或109-110和/或133-140组成的表位，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号；和
(7)结合SEQ ID NO:71的氨基酸残基36，39，41，59，61，62，63，64，65，66，109，110，133，135，136，137，138和140中的一个或多个，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号。
本发明还提供了一种分离的抗体或其抗原结合片段，其与上述的抗CTLA-4抗体结合相同或重叠的人CTLA-4蛋白的表位。
本发明还提供了一种分离的抗体或其抗原结合片段，其与上述的抗CTLA-4抗体竞争结合人CTLA-4蛋白的表位。
本发明还提供了一种分离的抗体或其抗原结合片段，其与抗体huJS007-47结合相同或者完全或部分重叠的人CTLA-4蛋白的表位。
本发明还提供了一种分离的抗体或其抗原结合片段，其与抗体huJS007-47竞争结合人CTLA-4蛋白的表位。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段结合由SEQ ID NO:71的残基27-29组成的表位，其中氨基酸残基编号为从SEQ ID NO:71中A37开始进行氨基酸编号。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段结合由SEQ ID NO:71的残基63-65组成的表位，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段结合一个或多个选自SEQ ID NO:71的氨基酸残基27，28和29的表位，其中氨基酸残基编号为从SEQ ID NO:71中A37开始进行氨基酸编号。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段结合一个或多个选自SEQ ID NO:71的氨基酸残基63，64和65的表位，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段在SEQ ID NO:71的27的氨基酸残基的表位上与人CTLA-4结合，其中氨基酸残基编号为从SEQ ID NO:71中A37开始进行氨基酸编号。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段在SEQ ID NO:71的28的氨基酸残基的表位上与人CTLA-4结合，其中氨基酸残基编号为从SEQ ID NO:71中A37开始进行氨基酸编号。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段在SEQ ID NO:71的29的氨基酸残基的表位上与人CTLA-4结合，其中氨基酸残基编号为从SEQ ID NO:71中A37开始进行氨基酸编号。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段与Ipilimumab结合人CTLA-4蛋白的不同表位。在一些实施方式中，本发明所述抗CTLA-4抗体抑制或阻断人CTLA-4蛋白与人CD80或与人CD86的结合；优选的，其与CD80竞争结合的氨基酸残基为135，137，138和140中的一个或多个，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段结合由SEQ ID NO:71的残基36-41和/或59-66和/或109-110和/或133-140组成的表位，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号。
在一些实施方式中，本发明所述抗CTLA-4抗体或其抗原结合片段结合一个或多个选自SEQ ID NO:71的氨基酸残基36，39，41，59，61，62，63，64，65，66，109，110，133，135，136，137，138和140的表位，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号。
本发明还提供了一种多特异性抗体，包含本文所述抗体或其抗原结合片段的轻链可变区和重链可变区。
本发明还提供了一种单链抗体，包含本文所述抗体或其抗原结合片段的轻链可变区和重链可变区。
本发明还提供了一种免疫缀合物，其包含与治疗剂或诊断剂缀合的本文所述抗体或其抗原结合片段。
在又一个方面，本发明提供了一种多核苷酸，其编码本文所述的抗CTLA-4抗体或其抗原结合片段。
在又一个方面，本发明提供了一种表达载体，其包含本文所述的多核苷酸，优选地，所述载体为真核表达载体。
在又一个方面，本发明提供了一种宿主细胞，其包含本文所述的多核苷酸或本文所述的表达载体，优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了一种制备本文所述的抗CTLA-4抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下在本文所述的宿主细胞中表达所述抗体或其抗原结合片段，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
在又一个方面，本发明提供了一种药物组合物，其包含本文所述的抗CTLA-4抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物在制备用于治疗和/或预防CTLA-4介导的疾病或病症的药物中的用途，优选所述疾病或病症为癌症，更优选所述癌症选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，其用于治疗和/或预防CTLA-4介导的疾病或病症，优选所述疾病或病症为癌症，更优选所述癌症选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明提供了一种治疗和/或预防CTLA-4介导的疾病或病症的方法，其包括向有需要的受试者施用本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，优选所述疾病或病症为癌症，更优选所述癌症选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明提供了一种药物组合，其包含本文所述的抗体或其抗原 结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，优选其进一步包括给药装置。
在又一个方面，本发明提供了一种使用本文所述的抗体或其抗原结合片段检测CTLA-4在样品中的存在的方法。
附图说明
图1：FACS检测杂交瘤抗CTLA-4抗体阻断huCTLA-4与huCD80的结合。
图2：杂交瘤抗CTLA-4抗体拮抗huCTLA-4的生物学活性。
图3：杂交瘤抗CTLA-4抗体与其他抗原(TIGIT、BTLA)的交叉反应。
图4：ELISA法检测嵌合抗CTLA-4抗体与huCTLA-4的结合。
图5：FACS法检测嵌合抗CTLA-4抗体与huCTLA-4表达细胞的结合。
图6：ELISA法检测嵌合抗CTLA-4抗体阻断huCTLA-4与CD80结合的能力。
图7：7a：FACS检测嵌合抗CTLA-4抗体阻断huCTLA-4与CD80表达细胞结合的能力；7b：FACS检测嵌合抗CTLA-4抗体阻断huCTLA-4与CD86表达细胞结合的能力。
图8：荧光素酶法检测嵌合抗CTLA-4抗体拮抗huCTLA-4的生物学活性。
图9：ELISA法检测人源化抗CTLA-4抗体与huCTLA-4的结合。
图10：ELISA法检测人源化抗CTLA-4抗体阻断huCTLA-4与CD80结合的能力。
图11：荧光素酶法检测人源抗CTLA-4化抗体的生物学活性。
图12：人源化抗CTLA-4抗体的ADCC活性。
图13：人源化抗CTLA-4抗体的CDC活性。
图14：人源化抗CTLA-4抗体对小鼠肿瘤生长的抑制。
图15：Fortebio结合实验鉴定抗原表位。
图16：氢氘交换质谱鉴定抗原表位。
图17：ELISA法检测单克隆抗体huJS007-47与huCTLA-4突变体的结合。
图18：huJS007-47对hCTLA4人源化小鼠移植MC38肿瘤生长的抑制作用。
图19：huJS007-47对hCTLA4人源化小鼠移植H22肿瘤生长的抑制作用。
图20：JS007与CTLA-4结合的整体结构和相互作用细节；图20A中显示JS007抗体的重链可变区(VH)与轻链可变区(VL)与CTLA-4复合物结构的整体图，其中JS007的三个重链互补决定簇(HCDR1-3)和三个轻链互补决定簇(LCDR1-3)显示为不同颜色；图20B为JS007与CTLA-4之间的氨基酸形成的氢键相互作用网络，参与氢键相互作用的氨基酸显示为棒状结构，其中各CDR颜色与20A图中一致，其氨基酸的颜色与相应区域的颜色深浅一致。
图21：JS007与B7-1竞争结合CTLA-4的分子基础；图21A中所示结构为CTLA-4-JS007与CTLA-4-B7-1复合物结构(PDB:1I8L)按照CTLA-4为固定参照物进行叠加后的图，其中CTLA-4仅显示CTLA-4-JS007中的CTLA-4分子结构，CTLA-4的FG loop介导了主要的竞争性结合，显示为图21A中左上方的分子结构图；图21B中所示为CTLA-4中与JS007或B7-1分子结合的表面结构图，其中只与JS007抗体结合的氨基酸显示为图21B的左上方深色部位，只与B7-1结合的氨基酸显示为图21B的右上方深色部位，而同时参与与JS007和B7-1相互作用的氨基酸显示为图21B的中间深色部位。
具体实施方式
定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值，包括但不限于±5％、±2％、±1％和±0.1％，因为这些变化适于进行所公开的方法。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的 组合。
如本文所用，术语“或”应被理解为与如上定义的“和/或”具有相同的含义。例如，当分离列表中的项目时，“或”或“和/或”应被解释为包容性的，即，包括数量或元素列表中的至少一个，但也包括多于一个，以及任选地，额外的未列出的项目。只有明确指出相反的术语下，例如“只有一个”或“的确一个”或者在权利要求中使用“由...组成”时，将指的是仅列出的一个数字或列表的一个元素。
除非明确指出相反的情况，否则如本文所用，词“一”和“一个”应理解为“至少一个”。
术语“细胞毒性T淋巴细胞相关抗原4”、“蛋白CTLA-4”、“CTLA-4重组蛋白”、“CTLA-4”、“CTLA4”、“CTLA-4抗原”可互换使用，并且包括人CTLA-4的变体、亚型、物种同源物或其他物种的CTLA-4和具有CTLA-4的至少一个共同表位的类似物，除非另有说明。该术语涵盖“全长”未加工的CTLA-4以及由细胞内加工产生的任何形式的CTLA-4或其任何片段。在一个实施方式中,CTLA-4是指来自人和食蟹猴CTLA-4全长或其片段(诸如其缺乏信号肽的成熟片段)。
术语“人CTLA-4”是指人序列CTLA-4，诸如具有NCBI登录号NM_005214.3的人CTLA-4的完整氨基酸序列。人CTLA-4序列可由于具有例如保守突变或非保守区域中的突变而不同于具有NCBI登录号NM_005214.3的人CTLA-4，并且CTLA-4与具有NCBI登录号NM_005214.3的人CTLA-4具有大致上相同的生物功能。举例来说，人CTLA-4的生物功能是在CTLA-4的细胞外结构域中具有由本公开的抗CTLA-4构建体特异性结合的表位，或人CTLA-4的生物功能是调节T细胞活性。
术语“huCD80”是指人序列huCD80，诸如具有NCBI登录号NM_005191.3的huCD80的完整氨基酸序列。术语“huCD86”是指人序列huCD86，诸如具有NCBI登录号NM_006889.3的huCD86的完整氨基酸序列；术语“食蟹猴CD86”是指食蟹猴序列cyno CD86，诸如具有NCBI登录号NM_102115124的食蟹猴CD86的完整氨基酸序列。
术语“百分比(％)氨基酸序列同一性”或简称“同一性”定义为在将氨基酸序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守取代视为序列同一性的部分之后，候选氨基酸序列中的氨基酸残基与参比氨基酸序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN 或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
术语“免疫应答”是指由例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝产生可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入的病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。
术语“信号转导途径”或“信号转导活性”是指通常由蛋白质间相互作用诸如生长因子对受体的结合启动的生化因果关系，所述关系导致信号从细胞的一部分传递至细胞的另一部分。一般地，传递包括引起信号转导的系列反应中的一种或多种蛋白质上的一个或多个酪氨酸、丝氨酸或苏氨酸残基的特定磷酸化。倒数第二过程通常包括细胞核事件，从而导致基因表达的变化。
术语“活性”或“生物活性”，或术语“生物性质”或“生物特征”此处可互换使用，包括但不限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗CTLA-4活性的能力、IC 50、抗体的体内稳定性和抗体的免疫原性质。本领域公知的抗体的其它可鉴定的生物学性质或特征包括，例如，交叉反应性(即通常与靶定肽的非人同源物，或与其它蛋白质或组织的交叉反应性)，和保持哺乳动物细胞中蛋白质高表达水平的能力。使用本领域公知的技术观察、测定或评估前面提及的性质或特征，所述技术包括但不局限于ELISA、FACS或BIACORE等离子体共振分析、不受限制的体外或体内中和测定、受体结合、细胞因子或生长因子的产生和/或分泌、信号转导和不同来源(包括人类、灵长类或任何其它来源)的组织切片的免疫组织化学。
术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。
术语“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针 对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“全长抗体”，是指在天然存在时包含至少四条肽链的免疫球蛋白分子：两条重(H)链和两条轻(L)链通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab'，F(ab') 2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。术语“结合化合物”是指抗体及其结合片段两者。
术语“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。Fv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。
术语“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。
术语“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同 的抗原特异性。然而，二价抗体可以是双特异性的。
术语“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。
术语“鼠源抗体”或“杂交瘤抗体”在本公开中为根据本领域知识和技能制备的抗人CTLA-4的单克隆抗体。制备时用CTLA-4抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
术语“全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
术语“表位”指能够与抗体特异性结合的蛋白质决定簇。表位通常由各种化学活性表面分子诸如氨基酸或糖侧链组成，并且通常具有特定三维结构特征以及特定电荷特征。构象性表位和非构象性表位的区别在于在变性溶剂存在下，与前者而非与后者的结合丧失。
本文中所描述的术语“交叉反应”指的是对人类、猴、和/或鼠源(小鼠或大鼠)相 同靶分子的抗原片段的结合。因此，“交叉反应”应被理解为与在不同物种中表达的相同分子X的种属间反应。识别人CTLA-4、猴、和/或鼠CTLA-4(小鼠或大鼠)的单克隆抗体的交叉反应特异性可通过FACS分析确定。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(K D)代表，平衡解离常数是解离速率常数和结合速率常数(分别是k dis和k on)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
术语“不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的K D为1.0×10 -6M或更高，更优选1.0×10 -5M或更高，更优选1.0×10 -4M或更高、1.0×10 -3M或更高，更优选1.0×10 -2M或更高。
术语“高亲和性”对于IgG抗体而言，是指对于抗原的K D为1.0×10 -6M或更低，优选5.0×10 -8M或更低，更优选1.0×10 -8M或更低、5.0×10 -9M或更低，更优选1.0×10 -9M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指K D为10 -6M或更低，优选10 -7M或更低，更优选10 -8M或更低。
术语“抗体依赖的细胞毒性”、“抗体依赖的细胞介导的细胞毒性”或“ADCC”是指细胞介导的免疫防御，其中免疫系统效应细胞主动地将细胞膜表面抗原与抗体，例如CTLA-4抗体，结合的靶细胞例如癌细胞裂解。
术语“补体依赖的细胞毒性”或“CDC”是指IgG和IgM抗体的效应功能，当与表面抗原结合时引发典型的补体途径，包括形成膜攻击复合体以及靶细胞裂解。本发明的抗体，与CTLA-4结合时，引发对癌细胞的CDC。
术语“核酸”或“多核苷酸”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语包括具有与参照核酸相似的结合性质并且以与天然存在的核苷酸相似的方式被代谢的含有已知的天然核苷酸的类似物的核酸(参见，属于Kariko等人的美国专利No.8,278,036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子取代可通过生成其中一个或多个选择的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代的序列来实现(Batzer等人，Nucleic  Acid Res.19:5081(1991)；Ohtsuka等人，J.Biol.Chem.260:2605-2608(1985)；和Rossolini等人，Mol.Cell.Probes 8:91-98(1994))。
“构建体”是指任何重组多核苷酸分子(诸如质粒、粘粒、病毒、自主复制多核苷酸分子、噬菌体或线性或环状单链或双链DNA或RNA多核苷酸分子)，衍生自任何来源，能够与基因组整合或自主复制，构成如下多核苷酸分子，其中已经以功能操作的方式连接(即，可操作地连接)一或多个多核苷酸分子。重组构建体通常会包含可操作地连接至转录起始调节序列的本发明的多核苷酸，这些序列会导引多核苷酸在宿主细胞中的转录。可使用异源及非异源(即，内源)启动子两者导引本发明的核酸的表达。
“载体”是指任何重组多核苷酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。一种类型的载体为“质粒”，是指环状双链DNA环，可将额外DNA区段连接至该环中。另一类型的载体为病毒载体，其中可将额外DNA区段连接至病毒基因组中。某些载体能够在被引入到的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体及游离型哺乳动物载体)。在引入到宿主细胞中后，其他载体(例如，非游离型哺乳动物载体)整合至宿主细胞的基因组中，且因此与宿主基因组一起复制。此外，某些载体能够导引被操作性连接的基因的表达。本文将此类载体称为“表达载体”。
本文所用术语“表达载体”是指能够在转化、转染或转导至宿主细胞中时复制及表达目的基因的核酸分子。表达载体包含一或多个表型选择标记及复制起点，以确保维护载体及以在需要的情况下于宿主内提供扩增。
用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。
如本文中所用，术语任何疾病或病症的“治疗”或“医治”在一个实施方式中是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在 另一个实施方式中，“治疗”或“医治”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些物理参数。在另一个实施方式中，“治疗”或“医治”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。
“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴。
“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的连续施用。
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的CTLA-4抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。
“癌症”和“癌性”指或描述哺乳动物中特征通常为细胞生长不受调控的生理疾患。此定义中包括良性和恶性癌症以及休眠肿瘤或微转移。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤，和白血病。此类癌症的更具体例子包括鳞状细胞癌，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃的癌或胃癌(包括胃肠癌)，胰腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，肝瘤(hepatoma)，乳腺癌，结肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌或肾的癌，肝癌，前列腺癌，外阴癌，甲状腺癌，肝的癌，及各种类型的头和颈癌，以及B细胞淋巴瘤(包括低级/滤泡性非何杰金氏淋巴瘤(NHL)，小淋巴细胞性(SL)NHL，中级/滤泡性NHL，中级弥漫性NHL，高级成免疫细胞性NHL，高级成淋巴细胞性NHL，高级小无核裂细胞性NHL，贮积病(bulky disease)NHL，套细胞淋巴瘤，AIDS相关淋巴瘤，和瓦尔登斯特伦氏(Waldenstrom) 巨球蛋白血症)，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖。
抗CTLA-4抗体
在一个方面，本发明提供了抗CTLA-4抗体或其抗原结合片段。术语“抗CTLA-4抗体”、“抗CTLA-4”、“CTLA-4抗体”或“结合CTLA-4的抗体”是指能够以足够的亲合力结合CTLA-4蛋白或其片段以致所述抗体可以用作靶向CTLA-4中的诊断剂和/或治疗剂。
可采用用于产生抗体的任何合适方法来产生本发明的抗体。任何合适形式的CTLA-4都可用作产生抗体的免疫原(抗原)。通过举例而非限制，任何CTLA-4变体或其片段都可用作免疫原。在一些实施方式中，产生鼠源的单克隆抗人CTLA-4抗体的杂交瘤细胞可通过本领域公知的方法产生。
来源于啮齿动物(如小鼠)的抗体在体内用作治疗药物时可能引起不需要的抗体免疫原性，重复使用导致人体产生针对治疗性抗体的免疫应答，这类免疫应答至少导致丧失治疗功效，而严重的则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA 84:3439-43)。然而，嵌合抗体中的完整啮齿动物可变区的保留仍可能在患者中引起有害的免疫原性。将啮齿动物可变结构域的互补决定区(CDR)环移植到人构架上(即人源化)已被用于进一步将啮齿动物序列减至最低(Jones等(1986)Nature 321:522；Verhoeyen等(1988)Science 239:1534)。
在一些实施方式中，本发明的嵌合或人源化抗体可基于所述制备的鼠单克隆杂交瘤抗体的序列来制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
在一些实施方式中，本发明所述的嵌合CTLA-4抗体，可使用本领域已知的方法将杂交瘤来源的免疫球蛋白重链和轻链可变区与人IgG恒定区有效连接(参见例如属于Cabilly等人的美国专利No.4,816,567)，获得嵌合型重链和嵌合型轻链来制备。在一些实施方式中，本发明的嵌合抗体包含的恒定区可选自任何人IgG亚型，如 IgG1、IgG2、IgG3、IgG4，优选IgG1、IgG4，更优选IgG1kappa亚型。
在一些实施方式中，本发明的嵌合CTLA-4抗体可由嵌合型轻链与嵌合型重链表达质粒“混合和匹配”转染表达细胞获得，此类“混合和匹配”的抗体的CTLA-4结合可使用上述结合测定和其它常规结合测定(例如，ELISA)来进行测试。
本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883；Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))、基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services,National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999Nucleic Acids Research,27,209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia、AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
本发明所述的人源化抗体，可以使用本领域已知的方法将鼠源CDR区插入人种 系框架区。参见Winter等人的美国专利No.5,225,539及Queen等人的美国专利No.5,530,101；5,585,089；5,693,762和6,180,370
在一些实施方式中，氨基酸变化包括氨基酸缺失、插入或置换。在一些实施方式中，本发明的抗CTLA-4抗体或其抗原结合片段包括具有已通过氨基酸缺失、插入或置换突变的，但仍与上述抗体(特别地在上述序列中描绘的CDR区中)有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方式中，本发明的抗体与具体序列中描绘的CDR区相比较时，在CDR区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。
在一些实施方式中，编码本发明抗体的多核苷酸包括已通过核苷酸缺失、插入或置换突变的，但仍然与上文中所述的序列中描绘的CDR对应编码区具有至少约60、70、80、90、95或100％同一性的多核苷酸。
在一些实施方式中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。
在一些实施方式中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。
在一些实施方式中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
本发明Ipilimumab参照专利CN1371416B，由苏州君盟制备。
抗体表达
在又一个方面，本发明提供了一种多核苷酸，其编码本文所述的抗CTLA-4抗体或其抗原结合片段。所述多核苷酸可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列的多核苷酸，或包含编码抗体的轻链和/或重链的氨基酸序列的多核苷酸。
在又一个方面，本发明提供了一种表达载体，其包含如本文所述的多核苷酸，优选地，所述载体为真核表达载体。在一些实施方式中，如本文所述的多核苷酸包含在一个或多个表达载体中。
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸或如本文所述的表达载体，优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了一种用于制备如本文所述的抗CTLA-4抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下在本文所述的宿主细胞中表达所述抗体或其抗原结合片段，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
本发明提供用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别优选的细胞系。
在一个实施方式中，本发明提供制备抗CTLA-4抗体的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中时，通过将宿主细胞培养足够的一段时间，以允许抗体在宿主细胞中表达，或者更优选抗体分泌到宿主细胞生长的培养基中,来产生抗体。可采用标准蛋白质纯化方法从培养基中回收抗体。
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。
药物组合物和药物制剂
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗CTLA-4抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体或本文所述的宿主细胞，和药学上可接受的载体或赋形剂。应理解，本发明提供的抗CTLA-4抗 体或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的抗CTLA-4抗体与一种或多种任选的药用辅料(Remington's Pharmaceutical Sciences，第16版，Osol,A.编辑(1980))混合来制备包含本文所述的抗CTLA-4抗体的药物制剂，优选地以水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。在一些实施方式中，本发明的药物组合物还包含编码抗CTLA-4抗体的多核苷酸的组合物。
在又一个方面，本发明提供了一种药物组合，其包含本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，优选其进一步包括给药装置。
医药用途
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物在制备用于治疗和/或预防CTLA-4介导的疾病或病症的药物中的用途，优选所述疾病或病症为癌症，更优选所述癌症选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明提供了本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，其用于治疗和/或预防CTLA-4介导的疾病或病症，优选所述疾病或病症为癌症，更 优选所述癌症选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在又一个方面，本发明提供了一种治疗和/或预防CTLA-4介导的疾病或病症的方法，其包括向有需要的受试者施用本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，优选所述疾病或病症为癌症，更优选所述癌症选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在一些实施方式中，本文所述的癌症或肿瘤可以选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
在一些实施方式中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
本发明还提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。
在一些实施方式中，本发明提供的方法或用途还包括向个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体。例如，可以与至少一种另外的治疗剂共施用。例如，PD-1抗体、PD-L1抗体和LAG-3抗体。
本申请还提供了上述抗CTLA-4抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、抗体或其片段的免疫缀合物、或药物组合物在制备用于预防和/或治疗CTLA-4相关疾病或病症的药物中的用途，例如肿瘤的药物中的用途。
在一些实施方式中，本文公开的肿瘤可以为结肠癌、黑色素瘤、间皮质瘤、肾细胞癌、淋巴瘤、晚期实体瘤或其转移瘤等。
用于诊断和检测的方法
在又一个方面，本发明提供了一种使用本文所述的抗体或其抗原结合片段检测CTLA-4在样品中的存在的方法。术语“检测”用于本文中时，包括定量或定性检测。在一些实施方式中，所述样品是生物样品。在某些实施方式中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方式中，生物样品包含细胞或组织。
本发明包括所述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明采用下述缩略词：
his-tag代表组氨酸标签；Fc tag代表可结晶片段标签；ECD代表胞外结构域；PEI代表聚乙烯亚胺；BSA代表牛血清白蛋白；PBS代表磷酸缓冲盐溶液；CFSE代表羧基荧光素二醋酸盐琥珀酰亚胺酯；APC代表异位藻蓝蛋白；NA-PE代表藻红蛋白标记的中性亲和素；PE代表藻红蛋白；TMB代表3,3',5,5'-四甲基联苯胺；HEPES代表羟乙基哌嗪乙硫磺酸缓冲液；DTT代表二硫苏糖醇。
实施例
通过以下实施例对本发明进行说明，但并不旨在对本发明作出任何限制。本文已经详细描述了本发明，其中也公开了其具体实施方式。在不脱离本发明精神和范围的情况下，针对本发明具体实施方式进行的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。实施例中所用到的方法和材料，除非另有说明，否则为本领域的常规方法和材料。
实施例1、用于抗CTLA-4抗体制备和测试的重组蛋白的制备
1.1人CTLA-4胞外结构区重组蛋白制备
获得人CTLA-4(huCTLA-4)基因(Sino Biological)，其中huCTLA-4 ECD的基因序列为NCBI登录号NM_005214.3。设计相应引物，通过PCR扩增得到编码huCTLA-4 ECD的基因，在基因的上下游分别添加限制性内切酶位点BSPQI和NotI。获得的PCR扩增片段经BSPQI和NotI双酶切后，克隆到真核表达质粒系统(HX1)中。以此质粒通过PEI转染293细胞，培养6天后，收集培养上清液，通过纯化分 别得到huCTLA-4 ECD(his-tag)和huCTLA-4 ECD(FC)重组蛋白。
1.2食蟹猴CTLA-4胞外结构区重组蛋白制备
获得食蟹猴CTLA-4(cynoCTLA-4)基因(Sino Biological)，其中cynoCTLA-4 ECD的基因序列为NCBI登录号:102115124。设计相应引物，通过PCR扩增得到编码cynoCTLA-4 ECD的基因，在基因的上下游分别添加限制性内切酶位点BSPQI和NheI。获得的PCR扩增片段经BSPQI和NheI双酶切后，克隆到真核表达质粒系统(HX1)中。以此质粒通过PEI转染293细胞，培养6天后，收集培养上清液，通过纯化得到cynoCTLA-4 ECD(FC)重组蛋白。
1.3人CD80胞外结构区重组蛋白制备
获得人CD80(huCD80)基因(Sino Biological)，其中huCD80 ECD的基因序列为NCBI登录号NM_005191.3。设计相应引物，通过PCR扩增得到编码huCD80 ECD的基因，在基因的上下游分别添加限制性内切酶位点EcoRI和NheI。获得的PCR扩增片段经EcoRI和NheI双酶切后，克隆到真核表达质粒系统(MX2-FC)中。以此质粒通过PEI转染293细胞，培养6天后，收集培养上清液，通过亲和层析纯化得到重组蛋白huCD80 ECD(Fc tag)。
1.4人CD86胞外结构区重组蛋白制备
获得人CD86(huCD86)基因(Sino Biological)，其中huCD86ECD的基因序列为NCBI登录号NM_006889.3。设计相应引物，利用PCR扩增得到编码huCD86ECD的基因，在基因的上下游分别添加限制性内切酶位点SapI。获得的PCR扩增片段经SapI酶切后，克隆到真核表达质粒系统(HX1-FC)中。以此质粒通过PEI转染293细胞，培养6天后，收集培养上清液，通过亲和层析纯化得到重组蛋白huCD86ECD(Fc tag)。
实施例2、小鼠杂交瘤细胞的制备
2.1、免疫动物
将huCTLA-4 ECD(his tag)作为抗原免疫5只小鼠(购自Simonsen Laboritories of Gilroy雌性BALB/c，8周)。在初次免疫(50μg/只)后，每隔1周或2周进行一次加强免疫(25μg/只)，共进行6次免疫。
2.2、细胞融合
在最后一针加强免疫4天后，取小鼠腹股沟淋巴结、腘窝淋巴结和脾脏，在生 理盐水中碾磨后取富含淋巴细胞的悬浮液，按常规电转方法将其与小鼠骨髓瘤细胞Sp2/0(来源于ATCC)融合。将融合产物在含1:50HAT(次黄嘌呤、氨甲蝶呤和胸腺嘧啶核苷)的DMEM完全培养基中培养5天以筛选成功融合的细胞(即，杂交瘤细胞)。然后换成含1:50HT(次黄嘌呤和胸腺嘧啶核苷)的DMEM完全培养基直至筛选结束。
DMEM完全培养基配比是：15％FBS(胎牛血清)+1:50L-谷氨酰胺+100U/mL青链霉素+1:100OPI(草酰乙酸、丙酮酸和胰岛素)，培养箱条件是8％CO 2，37℃。
实施例3、小鼠杂交瘤细胞的筛选和所得抗CTLA-4鼠源抗体的性能检测
在11520株多克隆杂交瘤细胞中，通过ELISA筛选出461株分泌可与huCTLA-4Fc结合的抗体的杂交瘤细胞。在这461株杂交瘤细胞中，有219株杂交瘤细胞表达可与cynoCTLA-4Fc结合的抗体。经基于ForteBio的阻断分析，219株中的24株杂交瘤细胞表达的抗体能够阻断huCTLA-4与huCD86结合。再经基于FACS的阻断分析，从24株中获得11株表达具有huCD80阻断活性的抗体的多克隆杂交瘤细胞。进而对它们进行了亚克隆。最终从上述杂交瘤细胞株中筛选出总共7株单克隆杂交瘤细胞，并将它们各自分泌的抗体分别进行了纯化和分析。另外从这7株单克隆杂交瘤细胞分别提取mRNA，对它们的抗体可变区编码序列进行了测序。由此初步筛选出的7种杂交瘤抗体分别编号为1B2.1，1A5.1，3E6.1，3E6.2，4B7.1，4E7.1，5B9.1。另外验证了这7种杂交瘤抗体与其他抗原(TIGIT，BTLA)没有显著的交叉反应。
实验方法和结果如下所示。
3.1、FACS检测杂交瘤抗体阻断huCTLA-4与huCD80的结合
将表达huCTLA-4的CHO细胞(来源于ATCC)与不同稀释浓度的上述杂交瘤抗体或对照抗体(Ipilimumab)一起孵育，初始抗体浓度为5ug/mL，稀释比例为1:3，然后加入5μg/mL生物素标记的huCD80，用NA-PE检测。
如图1，检测结果显示，除了1B2.1外，1A5.1，3E6.1，3E6.2，4B7.1，4E7.1，5B9.1都能够显著阻断huCTLA-4与huCD80的结合。
3.2、杂交瘤抗体拮抗huCTLA-4的生物学活性
将不同稀释浓度的杂交瘤抗体(1B2.1，1A5.1，3E6.1，3E6.2，4B7.1，4E7.1，5B9.1)与表达huCTLA-4的Jurkat效应细胞(Promega)和Raji APC靶细胞(Promega)混合(对照抗体为Ipilimumab)，从而检测Jurkat效应细胞下游信号通路介导的荧光 素酶表达，从而测定杂交瘤抗体的生物学活性。
如图2所示，1B2.1的拮抗huCTLA-4生物学活性较低，4E7.1，5B9.1，3E6.1，3E6.1，4B7.1和1A5.1均有较好的拮抗huCTLA-4生物学活性。
3.3、杂交瘤抗体与其他抗原(TIGIT、BTLA)的交叉反应
将293T-TIGIT，293T-CTLA4，293T-BTLA或293T母细胞与1μg杂交瘤抗体1B2.1，1A5.1，3E6.1，3E6.2，4B7.1，4E7.1，5B9.1或对照抗体在4℃孵育30分钟，然后将上述细胞混合物与5μL的APC-标记的鼠二抗(Southern Biotech)在4℃孵育20分钟，检测结合到细胞上的二抗。其中，对照抗体选自商业采购的CTLA4单抗L3D10(BioLegend)和TIGIT单抗MBSA43(eBioscience)。
如图3所示，上述抗体与其它抗原(TIGIT、BTLA)没有显著的交叉反应。
3.4、杂交瘤抗体与huCTLA-4的亲和力
如表1所示，通过ForteBio仪器测定上述杂交瘤抗体与huCTLA-4之间的亲和力。确认制备的上述杂交瘤抗体能够特异性结合huCTLA-4。
表1：杂交瘤抗体与huCTLA-4的亲和力
样品 1A5.1 1B2.1 3E6.1 3E6.2 4B7.1 4E7.1 5B9.1
KD(M) 1.74E-09 1.02E-9 <1.0E-12 5.24E-10 5.70E-10 1.47E-09 <1.0E-12
实施例4、抗CTLA-4鼠源抗体的可变区序列的测定(根据Kabat或IMGT表 示)
用基于简并引物PCR的方法，测定抗CTLA-4鼠源抗体的可变区对应的DNA编码序列。简言之，将杂交瘤细胞株分别放大培养，1000rpm离心收集细胞，并以Trizol提取总RNA。以此为模板合成第一链cDNA，之后以第一链cDNA作为后续模板，PCR扩增可变区DNA编码序列。所用PCR引物是基于Ig-引物组。回收并纯化PCR产物。对扩增产物测序，得到抗CTLA-4鼠源抗体的重链可变区和轻链可变区氨基酸序列。
用NCBI Ig-Blast(http://www.ncbi.nlm.nih.gov/projects/igblast/)在种系和重排Ig可变区序列数据库中搜索共有序列。基于Kabat(Wu,T.T及Kabat,E.A.1970J.Exp.Med.,132:211-250)及IMGT系统(Lefranc M.-P.等人，1999Nucleic Acids Research,27,209-212)，藉由序列批注及藉由基于因特网的序列分析(http://www.imgt.org/IMGT_vquest/vquest与http://www.ncbi.nlm.nih.gov/igblast/)鉴 定互补决定区(CDR)氨基酸序列。
经选择的抗CTLA-4鼠源抗体的轻链和重链可变区及CDR的氨基酸序列如表2所示。
表2：抗CTLA-4鼠源抗体的CDR和可变区氨基酸序列(KABAT方案)
Figure PCTCN2021107707-appb-000001
注：4B7.1-1和4B7.1-2均来自4B7.1。
实施例5、抗CTLA-4嵌合抗体的构建
根据实施例3的评估结果，选取抗CTLA-4鼠源抗体1A5.1、3E6.1、4B7.1、5B9.1的轻链和重链可变区构建抗CTLA-4嵌合抗体。
从人B淋巴细胞(来自北京血液研究所)中克隆重链恒定区Fc和轻链恒定区κ的编码序列，引入pCDNA3.1质粒中。前述抗CTLA-4鼠源抗体的重链和轻链可变区编码序列由Genescript公司合成。各种抗CTLA-4鼠源抗体的重链可变区编码序列经BSPQI酶切，轻链可变区编码序列经BSPQI酶切后，采用如表3所示的各种组合，引入已经引入恒定区编码序列的pCDNA3.1质粒中，并经测序确定正确克隆。将各种嵌合型重链和轻链表达质粒混合配对转染表达细胞(CHOK1 18,苏州君盟)，获得20种嵌合抗体，其编号和相应可变区氨基酸序列具体见表3。
表3：嵌合抗体的编号及其重链和轻链可变区的来源
Figure PCTCN2021107707-appb-000002
Figure PCTCN2021107707-appb-000003
实施例6、嵌合抗体的筛选
根据表3中的嵌合抗体与huCTLA-4的结合、阻断huCTLA-4与CD80/CD86结合的能力以及拮抗huCTLA-4的生物学活性，筛选最佳嵌合抗体。实验方法和结果如下所示。
6.1ELISA法检测嵌合抗体与huCTLA-4的结合
使用PBS(Hyclone)稀释HX1 hCTLA4 his至1.0μg/mL，以100μl/孔加入酶标板，37℃恒温培养箱中静置包被90min；洗板；加入200μl/孔2％BSA至板内，置37℃恒温培养箱内孵育90min；洗板。将表3中的嵌合抗体及对照抗体(Ipilimumab)用稀释液(2％BSA)稀释至1000ng/ml，每次稀释倍数不高于10倍。然后，在样品稀释板上以2.5倍梯度依次稀释嵌合抗体及对照抗体。所有嵌合抗体及对照抗体溶液以100μl/孔加入酶标板中，置37℃恒温培养箱内孵育60min；洗板；将辣根过氧化物酶(HRP)偶联的羊抗人抗体IgG(Sigma，货号：A0170)(Fc特异性)用2％BSA稀释5000倍，以100μl/孔加入酶标板，置37℃恒温培养箱内孵育60min；洗板；加入显色液TMB，100μl/孔，避免气泡，37℃避光显色15min；最后加2M的盐酸溶液终止反应，100μl/孔，避免气泡，10min内完成酶标仪读数(波长：450/620nm)。
如图4所示，JS007-3、JS007-15、JS007-20嵌合抗体具有与huCTLA-4良好的结合，其EC 50分别为17.23ng/mL、19.72ng/mL和12.88ng/mL，与对照抗体Ipilimumab相当或优于对照抗体。
6.2FACS法检测嵌合抗体与huCTLA-4表达细胞的结合
将表达huCTLA-4的CHO细胞(君盟自主构建，CHO来源于ATCC)分别与表3中的嵌合抗体及对照抗体(Ipilimumab)(起始浓度为10μg/ml，3倍浓度梯度稀释)室温共孵育30分钟，然后加入荧光二抗(PE-anti human IgG)，从而检测嵌合抗体与huCTLA-4表达细胞的结合。
如图5所示，JS007-3、JS007-15和JS007-20嵌合抗体具有与huCTLA-4表达细胞良好的结合，其EC 50分别为249.4ng/mL、189.4ng/mL和486.1ng/mL，均优于对 照抗体Ipilimumab。
6.3ELISA法检测嵌合抗体阻断huCTLA-4与CD80结合的能力
使用PBS(Hyclone)稀释HX1 huCTLA4 his至1.0μg/mL，以100μl/孔加入酶标板，37℃恒温培养箱中静置孵育90min；洗板；加入200μl/孔2％BSA至板内，置37℃恒温培养箱内孵育90min；洗板；取MX2 hCD80 Fc用2％BSA稀释至5.0μg/mL，并以此浓度的MX2 hCD80 Fc来稀释样品。将嵌合抗体及对照抗体Ipilimumab稀释至100μg/ml，每次稀释倍数不高于10倍。然后，在样品稀释板上以2.5倍梯度依次稀释嵌合抗体及对照抗体。所有嵌合抗体及对照抗体溶液以100μl/孔加入酶标板中，置37℃恒温培养箱内孵育90min；洗板；将HRP偶联的羊抗鼠抗体IgG(Fc特异性)(Sigma，货号：A2554)用2％BSA稀释5000倍，以100μl/孔加入酶标板，置37℃恒温培养箱内孵育60min；洗板；加入显色液TMB，100μl/孔，避免气泡，37℃避光显色15min；最后加2M的盐酸溶液终止反应，100μl/孔，避免气泡，10min内完成酶标仪读数(波长：450/620nm)
如图6所示，JS007-3，JS007-15，JS007-20嵌合抗体均具有阻断huCTLA-4与CD80结合的能力，其IC 50分别为189.7ng/mL、189.7ng/mL和186.6ng/mL，且均明显优于对照抗体Ipilimumab。
6.4FACS检测嵌合抗体阻断huCTLA-4与CD80/CD86表达细胞结合的能力
分别将表3中的嵌合抗体和对照抗体Ipilimumab(起始浓度为30μg/ml，3倍浓度梯度稀释)与一定浓度的huCTLA4重组蛋白，加入到表达CD80或CD86的CHO细胞(君盟自主构建)中，室温共孵育30分钟后，加入荧光二抗(PE-anti human IgG4)(Southern biotech)，从而检测嵌合抗体阻断huCTLA-4与CD80/CD86表达细胞结合的能力。
如图7a和7b所示，JS007-3、JS007-15和JS007-20嵌合抗体均具有阻断huCTLA-4与CD80/CD86表达细胞结合的能力。其中，阻断huCTLA-4与CD80表达细胞结合的IC 50分别为0.6986ng/mL、0.398ng/mL和1.211ng/mL，均明显优于对照抗体Ipilimumab；阻断huCTLA-4与CD86表达细胞结合的IC 50分别为0.196ng/mL、0.0816ng/mL和0.2528ng/mL，与对照抗体Ipilimumab相当或明显优于对照抗体。
6.5荧光素酶法检测嵌合抗体拮抗huCTLA-4的生物学活性
将不同稀释浓度的嵌合抗体(JS007-3、JS007-15、JS007-20)与表达huCTLA-4的Jurkat效应细胞及Raji APC靶细胞混合，从而检测Jurkat效应细胞的下游信号通 路介导的荧光素酶表达，测定嵌合抗体拮抗huCTLA-4的生物学活性。
如图8所示，JS007-3、JS007-15和JS007-20嵌合抗体具有较好的拮抗huCTLA-4的生物学活性。
实施例7、抗体可变区的人源化改造
对于抗体可变区的人源化改造，首先在NCBI(http://www.ncbi.nlm.nih.gov/igblast/)网站中的人类免疫球蛋白基因数据库搜寻与鼠源抗体可变区的cDNA序列同源的人类种系IgG基因。再藉由Kabat编号系统或IMGT编号系统定义可变区CDR的氨基酸序列及其精确边界。原则上将与鼠源抗体可变区具有高同源性的人类IGHV及IGKV选为人源化模板，藉由CDR嫁接实施抗体可变区的人源化。
根据前文获得的杂交瘤细胞分泌的抗体的可变区序列，进行人源化改造。简言之，人源化改造过程涉及以下步骤：A、将各杂交瘤细胞分泌的抗体的基因序列与人胚胎系抗体基因序列进行比对，找出同源性高的序列；B、分析考察HLA-DR亲和性，选出亲和力低的人胚胎系框架区序列；C、利用计算机模拟技术，应用分子对接分析可变区及其周边的框架区氨基酸序列，考察其空间立体结合方式。通过计算静电力、范德华力、亲疏水性和熵值，分析各杂交瘤细胞分泌的抗体的基因序列中可与hCTLA-4作用以及维护空间架构的关键氨基酸个体，将其嫁接到所选择的人胚胎系基因框架区中，并在此基础上标示必须保留的框架区氨基酸位点；D、以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并优化导致抗体CDR区化学不稳定的氨基酸残基。优化结果如下：
HCDR2:YIGYDGSNYYNPSLKN(SEQ ID NO:2)优化为YIGYDGSNYYNPSLK S(SEQ ID NO:37)；
HCDR3:NYYSGYFDF(SEQ ID NO:3)优化为NYYSGYFD S(SEQ ID NO:38)；
HCDR2:YIGYDGSNNYNPSLKN(SEQ ID NO:20)优化为YIGYDGSNNYNPSLK S(SEQ ID NO:39)；
LCDR1:KASQNVGTYVA(SEQ ID NO:16)优化为 RASQNVGTYVA(SEQ ID NO:40)；
LCDR1:KASQNVGTYVA(SEQ ID NO:16)优化为 QASQNVGTYVA(SEQ ID NO:41)。
在此基础上得到了以下多种人源化抗CTAL-4抗体可变区：
1A5VH-1：SEQ ID NO:42
Figure PCTCN2021107707-appb-000004
1A5VH-2：SEQ ID NO:43
Figure PCTCN2021107707-appb-000005
1A5VH-3：SEQ ID NO:44
Figure PCTCN2021107707-appb-000006
1A5VH-4：SEQ ID NO:45
Figure PCTCN2021107707-appb-000007
1A5VH-5：SEQ ID NO:46
Figure PCTCN2021107707-appb-000008
1A5VH-6：SEQ ID NO:47
Figure PCTCN2021107707-appb-000009
1A5VH-7：SEQ ID NO:48
Figure PCTCN2021107707-appb-000010
4B7VH2-1：SEQ ID NO:49
Figure PCTCN2021107707-appb-000011
4B7VH2-2：SEQ ID NO:50
Figure PCTCN2021107707-appb-000012
4B7VH2-3：SEQ ID NO:51
Figure PCTCN2021107707-appb-000013
4B7VH2-4：SEQ ID NO:52
Figure PCTCN2021107707-appb-000014
4B7VH2-5：SEQ ID NO:53
Figure PCTCN2021107707-appb-000015
4B7VL-1：SEQ ID NO:54
Figure PCTCN2021107707-appb-000016
4B7VL-2：SEQ ID NO:55
Figure PCTCN2021107707-appb-000017
4B7VL-3：SEQ ID NO:56
Figure PCTCN2021107707-appb-000018
4B7VL-4：SEQ ID NO:57
Figure PCTCN2021107707-appb-000019
4B7VL-5：SEQ ID NO:58
Figure PCTCN2021107707-appb-000020
4B7VL-6：SEQ ID NO:59
Figure PCTCN2021107707-appb-000021
4B7VL-7：SEQ ID NO:60
Figure PCTCN2021107707-appb-000022
4B7VL-8：SEQ ID NO:61
Figure PCTCN2021107707-appb-000023
4B7VL-9：SEQ ID NO:62
Figure PCTCN2021107707-appb-000024
将上文设计的人源化抗CTLA-4抗体可变区采用如表4所示的各种组合，人源化抗CTLA-4抗体的重链和轻链可变区编码序列由Genscript公司合成，各种合成的人源化抗CTLA-4抗体的重链可变区编码序列和轻链可变区编码序列经Bspq I酶切后引入已经含有恒定区编码序列的pCDNA3.1质粒中，并经测序确定正确克隆。将各种人源化型重链和轻链表达质粒混合配对转染表达细胞(CHOK1 18,苏州君盟)，表达的抗体通过离心回收后，按常规方法进行抗体纯化，获得108种人源化抗CTLA-4抗体，其编号和氨基酸序列具体见表4。
Figure PCTCN2021107707-appb-000025
实施例8、人源化抗CTLA-4抗体的筛选
根据上述人源化抗CTLA-4抗体与huCTLA-4的结合、阻断huCTLA-4与CD80/CD86结合的能力以及拮抗huCTLA-4的生物学活性，筛选最佳人源化抗CTLA-4抗体。实验方法和结果如下所示。
8.1 ELISA法检测人源化抗CTLA-4抗体与huCTLA-4的结合
使用PBS(Hyclone)稀释HX1 hCTLA4 his至1.0μg/mL，以100μl/孔加入酶标板，37℃恒温培养箱中静置包被90min；洗板；加入200μl/孔2％BSA至板内，置37℃恒温培养箱内孵育90min；洗板。将表4中的人源化抗CTLA-4抗体及对照抗体Ipilimumab用稀释液(2％BSA)稀释至1000ng/ml，每次稀释倍数不高于10倍。然后，在样品稀释板上以2.5倍梯度依次稀释人源化抗体及对照抗体。所有人源化抗体及对照抗体溶液以100μl/孔加入酶标板中，置37℃恒温培养箱内孵育60min；洗板；将HRP偶联的羊抗人抗体IgG(Fc特异性)用2％BSA稀释5000倍，以100μl/孔加入酶标板，置37℃恒温培养箱内孵育60min；洗板；加入显色液TMB，100μl/孔，避免气泡，37℃避光显色15min；最后加2M的盐酸溶液终止反应，100μl/孔，避免气泡，10min内完成酶标仪读数(波长：450/620nm)。图9为表4中的人源化抗CTLA-4抗体对比对照抗体的相对结合活性。
如图9所示，人源化抗CTLA-4抗体具有与huCTLA-4良好的结合，与对照抗体相当或优于对照抗体。
8.2 ELISA法检测人源化抗CTLA-4抗体阻断huCTLA-4与CD80结合的能力
使用PBS(Hyclone)稀释HX1 hCTLA4-his至1.0μg/mL，以100μl/孔加入酶标板，37℃恒温培养箱中静置孵育90min；洗板；加入200μl/孔2％BSA至板内，置37℃恒温培养箱内孵育90min；洗板；取MX2 hCD80 Fc用2％BSA稀释至5.0μg/mL，并以此浓度的MX2 hCD80 Fc来稀释抗体。将表4中的人源化抗CTLA-4抗体及对照抗体Ipilimumab稀释至100μg/ml，每次稀释倍数不高于10倍。然后，在样品稀释板上以2.5倍梯度依次稀释人源化抗体及对照抗体。所有人源化抗体及对照抗体溶液以100μl/孔加入酶标板中，置37℃恒温培养箱内孵育90min；洗板；将HRP偶联的羊抗鼠抗体IgG(Fc特异性)用2％BSA稀释5000倍，以100μl/孔加入酶标板，置37℃恒温培养箱内孵育60min；洗板；加入显色液TMB，100μl/孔，避免气泡，37℃避光显色15min；最后加2M的盐酸溶液终止反应，100μl/孔，避免气泡，10min内 完成酶标仪读数(波长：450/620nm)。图10为人源化抗CTLA-4抗体对比对照抗体的相对抑制活性。
如图10所示，人源化抗CTLA-4抗体具有良好的阻断huCTLA-4与CD80结合的能力，与对照抗体相当或优于对照抗体。
8.3荧光素酶法检测人源化抗CTLA-4抗体拮抗huCTLA-4的生物学活性
根据上述结合活性、阻断结合活性和人源化程度选择人源化抗CTLA-4抗体huJS007-46、47、48、49、55、56、73、79、82、88、100和106进行生物学活性分析。
将表达huCTLA-4的Jurkat细胞按每孔6′10 4个细胞铺板，每孔加入3′10 4个Raji APC细胞以及不同浓度的上述12种人源化抗CTLA-4抗体或对照抗体(Ipilimumab)，孵育6小时后，采用荧光素酶法测定T细胞激活活性。
如图11所示，12种人源化抗CTLA-4抗体均具有很高的生物学活性，其EC 50均明显优于对照抗体Ipilimumab。
实施例9、人源化抗CTLA-4抗体的ADCC活性
293T-CTLA4细胞用CFSE标记，按1:25的靶细胞:效应细胞(Target:effector)比例添加外周血单核细胞(PBMC2144896)和不同浓度的上述12种人源化抗CTLA-4抗体或对照抗体(Ipilimumab)，孵育过夜。细胞用碘化丙啶(PI)染色，用流式细胞仪进行分析。ADCC杀伤(％)表示为死亡靶细胞(PI和CFSE阳性)占总靶细胞(CFSE阳性)的百分比。
如图12(分成两幅小图仅仅是为了方便显示)所示，12种人源化抗CTLA-4抗体均具有ADCC活性，其中人源化抗CTLA-4抗体46、47、48、73、79及106的ADCC活性与对照抗体相当或优于对照抗体，特别是抗体46和48。
实施例10、人源化抗CTLA-4抗体的CDC活性
293T-CTLA4细胞用不同浓度(0.8-100μg/mL)的上述12种人源化抗CTLA-4抗体或对照抗体(Ipilimumab)在37℃活化15分钟，加入不同稀释梯度(1:5、1:10、1:20)的人血清补体并培养1小时。培养结束后，用碘化丙啶(PI)染色细胞，用BD FACSCalibur流式细胞仪进行分析。CDC杀伤(％)表示为PI阳性靶细胞占总靶 细胞的百分比。结果如图13所述，表明上述12个人源化抗CTLA-4抗体没有CDC活性或者CDC活性可以忽略不计。
综合考虑结合活性、阻断结合能力、生物学活性、ADCC活性和CDC活性，选择huJS007-47、huJS007-48、huJS007-79和huJS007-106人源化抗CTLA-4抗体进行下一步评价。
这4种人源化抗CTLA-4抗体的CDR/可变区/轻链/重链氨基酸序列如表5所示。
表5：4种人源化抗CTLA-4抗体的各部分的氨基酸序列(KABAT方案)
  huJS007-47 huJS007-48 huJS007-79 huJS007-106
HCDR1 SEQ ID NO：19 SEQ ID NO：19 SEQ ID NO：1 SEQ ID NO：19
HCDR2 SEQ ID NO：39 SEQ ID NO：39 SEQ ID NO：37 SEQ ID NO：20
HCDR3 SEQ ID NO：21 SEQ ID NO：21 SEQ ID NO：38 SEQ ID NO：21
LCDR1 SEQ ID NO：40 SEQ ID NO：40 SEQ ID NO：40 SEQ ID NO：40
LCDR2 SEQ ID NO：17 SEQ ID NO：17 SEQ ID NO：17 SEQ ID NO：17
LCDR3 SEQ ID NO：18 SEQ ID NO：18 SEQ ID NO：18 SEQ ID NO：18
VH SEQ ID NO：50 SEQ ID NO：50 SEQ ID NO：46 SEQ ID NO：53
VL SEQ ID NO：55 SEQ ID NO：56 SEQ ID NO：60 SEQ ID NO：60
HC SEQ ID NO：63 SEQ ID NO：65 SEQ ID NO：67 SEQ ID NO：69
LC SEQ ID NO：64 SEQ ID NO：66 SEQ ID NO：68 SEQ ID NO：70
实施例11、人源化抗CTLA-4抗体对小鼠肿瘤生长的抑制
取50只6-8周龄雌性B-hCTLA4人源化小鼠(百奥塞图)，将MC38WT细胞以以1×10 6个/0.1mL浓度接种小鼠的右侧皮下，待肿瘤生长到约138mm 3时将小鼠按肿瘤体积随机分组，每组6只，共6组，分别为：
G1 KLH IgG1(0.3mg/kg)阴性对照组、
G2 Ipilimumab(0.3mg/kg)阳性对照组、
G3 huJS007-47(0.3mg/kg)治疗组、
G4 huJS007-48(0.3mg/kg)治疗组、
G5 huJS007-79(0.3mg/kg)治疗组，和
G6 huJS007-106(0.3mg/kg)治疗组。
所有组的给药途径均为腹腔注射，给药剂量为0.3mg/kg，给药浓度为0.03mg/ml。每周给药2次，连续给药5次，末次给药3天后结束实验。每周测量肿瘤体积及体重 2次，记录小鼠体重和肿瘤体积。实验结束时将小鼠安乐死，计算相对肿瘤抑制率TGI％＝(1-(Ti-T0)/(Vi-V0))×100％。Ti：治疗组和阳性对照组在给药第i天的肿瘤体积均值；T0：治疗组和阳性对照组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值；V0：阴性对照组在给药第0天的肿瘤体积均值。
如图14所示，在小鼠接种肿瘤细胞后第25天，KLH IgG1阴性对照组平均肿瘤体积为975±115mm 3，Ipilimumab阳性对照组平均肿瘤体积为824±267mm 3，与KLH IgG1相比，相对肿瘤抑制率为18.1％；huJS007-47治疗组、huJS007-48治疗组、huJS007-79治疗组和huJS007-106治疗组平均肿瘤体积分别为229±85mm 3、313±197mm 3、550±229mm 3和472±125mm 3，与KLH IgG1相比，相对肿瘤抑制率分别为89.2％、79.1％、50.9％和60.1％，表明上述人源化抗CTLA-4抗体能够体内抑制B-hCTLA4人源化小鼠MC38-WT细胞皮下移植瘤的生长，且明显优于对照抗体Ipilimumab。
实施例12、Fortebio结合实验鉴定抗原表位
使用Protein A探针(Fortebio)先分别捕获全长抗体，即2.7μg/mL的人源化抗CTLA-4抗体huJS007-47与2μg/mL的对照抗体(Ipilimumab)。再将探针浸入55nM的人源CTLA(huCTLA)抗原溶液中，使全长抗体与抗原结合。最后将探针浸入600nM Fab溶液中，包括人源化Fab huJS00-47与对照Fab(Ipilimumab)，检测抗原与Fab是否发生结合。
如图15所示，全长抗体huJS007-47与抗原huCTLA-4结合后，huCTLA-4可以继续与对照Fab(Ipilimumab)结合，但按照Fortebio结合实验检测结果，huJS007-47与Ipilimumab结合于huCTLA-4的不同表位。
实施例13、氢氘交换质谱鉴定抗原表位
氢氘交换质谱方法进行抗原抗体二维多肽谱图的鉴定：通过氢氘交换质谱平台LEAP PAL3.0使用二级质谱(MS/MS)在质谱仪(Orbitrap Fusion TM Tribrid TM Mass Spectrometer，Thermo Fisher)上鉴定蛋白的酶解肽段。将MS/MS数据文件提交给Proteome discover软件进行肽段鉴定。
氢氘交换质谱样品的制备：将5-10微摩尔抗原或抗体或抗原抗体复合物(1:1摩 尔比)(50mM HEPES，pH 7.4，150mM NaCl，2mM DTT)分别在4℃下放置1小时，使之形成复合物的稳定状态。在4℃条件下，将5微升上述复合物在交换缓冲液(50mM HEPES，pH 7.4,150mM NaCl，2mM DTT)上稀释到20微升D 2O(氘)中并放置各种不同的HDX时间点(例如，0、60、300、900秒)。氢氘交换一段时间后，通过与25μL冰的4M盐酸胍和1％三氟乙酸混合来停止。在停止反应后立即将样品管置于干冰上直至样品注入HDX LEAP PAL3.0平台。注射到全自动氢氘交换平台后，样品以200μL/min通过固定的胃蛋白酶柱，并将酶解的肽段捕获在C 18捕获柱上并脱盐。在6分钟内，用2.1mm×5cm C 18柱(1.9μm Hypersil Gold，Thermo Fisher)以4-40％乙腈和0.3％甲酸的线性梯度分离脱盐的肽段。样品处理，蛋白质酶解和肽段分离都在4℃下进行。使用Orbitrap质谱仪(Orbitrap Fusion TM Tribrid TM Mass Spectrometer，Thermo Fisher)获得氢氘交换的质谱数据，测量的分辨率为65,000(m/z400)。每一个样品在每一个时间点都有三个重复的样本。
氢氘交换质谱数据分析：通过HDX Workbench软件来计算每个酶解的肽段的质谱峰强度的平均m/z质心值，随后转换成氘掺入的百分比。计算参与空间表位的关键氨基酸序列，通过计算两个样本之间的差异来确定Delta％D的差值(比较同一肽段上氘掺入百分比的变化)。Delta％D在-5至5％之外的差异被认为是有显著性差异的。此外，HDX Workbench通过student’s t test检测每个时间点的样品之间的统计学显著性(p<0.05)差异。
如图16所示，位于上方的曲线是肽段(YASPGKATE，+1)在未结合huJS007-47时的氢氘交换速率表征，位于下方的曲线是该肽段在与huJS007-47结合后的氢氘交换速率表征(显著性降低)。按照氢氘交换质谱实验检测结果，huJS007-47与Ipilimumab结合于huCTLA-4的不同表位。
实施例14、ELISA法检测单克隆抗体huJS007-47与huCTLA-4突变体的结合
根据氢氘交换质谱结果，针对huCTLA-4中的“YASPGKATE”突变为P27A“YASAGKATE”、G28A“YASPAKATE”、K29A“YASPGAATE”、T31A“YASPGKAAE”而获得的PCR扩增片段经EcoRI和NotI双酶切后，克隆到真核表达质粒(HXT)中。以此质粒通过PEI转染293细胞，培养6天后，收集培养上清液，通过纯化得到huCTLA-4 ECD(his-tag)重组蛋白。其氨基酸序列为SEQ ID NO: 71中第37个氨基酸(A)开始到第162个氨基酸(F)的序列。
ELISA检测使用的huCTLA-4突变体包括未突变的HXT huCTLA4 his，以及突变的huCTLA4 N-his P27A、huCTLA4 N-his G28A、huCTLA4 N-his K29A和huCTLA4 N-his T31A。用PBS(Hyclone)将huCTLA-4 his及其突变体稀释至1.0μg/ml包板，37℃恒温培养箱中孵育90min；洗板并用2％BSA封闭；洗板后加入用2％BSA梯度稀释的单克隆抗体huJS007-47(从1000ng/ml至0.042ng/ml，2.5倍梯度稀释)，置37℃孵育60min；洗板；用1:5000稀释的HRP偶联的羊抗人抗体IgG(Fc特异性)进行检测(Sigma公司，货号A0170)，37℃孵育60min，然后与HRP底物TMB(Sigma，货号T2885)37℃，孵育15min显色，最后加2M盐酸终止，避免气泡，10min内完成酶标仪读数(波长：450/620nm)。
如图17所示，根据ELISA结果，当huJS007-47结合抗原时，推测huCTLA-4表面与huJS007-47抗体结合相关表位在抗原的27位氨基酸P、28位氨基酸G、29位氨基酸K。单独突变27位氨基酸P或29位氨基酸K皆可大幅降低结合活性。单独突变28位氨基酸G可基本完全抑制结合活性。本次ELISA结果与氢氘交换质谱结果相符合，huJS007-47与Ipilimumab结合于huCTLA-4的不同表位。
实施例15、huJS007-47对hCTLA4人源化小鼠移植MC38肿瘤生长的抑制作 用
取6-8周龄雌性hCTLA4人源化小鼠(百奥赛图江苏基因生物技术有限公司)，于右侧背部皮下接种1 x 10 6MC38细胞(0.1ml/只(含细胞的培养基RPMI1640(Gibco)))。待平均肿瘤体积约为119mm 3时，挑选40只动物，根据肿瘤体积随机分为5组，每组8只动物。分别为
Anti-KLH hIgG1阴性对照组，1mg/kg；
Ipilimumab阳性对照组，1mg/kg；
huJS007-47治疗组，0.1mg/kg；
huJS007-47治疗组，0.3mg/kg；
huJS007-47治疗组，1mg/kg；
分组当天给药，所有组给药途径均为腹腔注射，每周给药2次，连续给药6次，末次给药3天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI％(TGI％＝ [1-(Ti-T0)/(Vi-V0)]×100％)。(Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值，V0：阴性对照组在给药第0天的肿瘤体积均值)。
如图18所示，在开始给药后第21天，anti-KLH hIgG1阴性对照组在1mg/kg的剂量下的平均肿瘤体积为1116±106mm 3。Ipilimumab阳性对照组在1mg/kg的剂量下的平均肿瘤体积为255±88mm 3，TGI％为86.36％。huJS007-47在0.1，0.3和1mg/kg的剂量下，平均肿瘤体积分别为736±203mm 3，47±12mm 3，33±15mm 3，TGI％分别为38.11％、107.22％和108.63％。表明huJS007-47在0.1，0.3和1mg/kg的剂量下，显著抑制hCTLA4人源化小鼠移植MC38肿瘤体积增长，并呈现良好的剂量效应，且在等剂量条件下(1mg/kg)，huJS007-47抑瘤作用显著优于Ipilimumab。
实施例16、huJS007-47对hCTLA4人源化小鼠移植H22肿瘤生长的抑制作用
取6-8周龄雌性hCTLA4人源化小鼠(上海南方模式生物科技股份有限公司)，于右侧背部皮下接种1 x 10 6H22细胞(0.1ml/只(含细胞的培养基RPMI1640(Gibco)))。待平均肿瘤体积约为119mm 3时，挑选35只动物，根据肿瘤体积随机分为5组，每组7只动物。分别为
Anti-KLH hIgG1阴性对照组，0.3mg/kg；
Ipilimumab阳性对照组，0.1mg/kg；
huJS007-47治疗组，0.03mg/kg；
huJS007-47治疗组，0.1mg/kg；
huJS007-47治疗组，0.3mg/kg；
分组当天给药，所有组给药途径均为腹腔注射，每周给药2次，连续给药6次，末次给药3天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI％，计算方式与实施例15相同。
如图19所示，在开始给药后第21天，anti-KLH hIgG1阴性对照组在0.3mg/kg的剂量下的平均肿瘤体积为2304±402mm 3。Ipilimumab阳性对照组在0.1mg/kg的剂量下的平均肿瘤体积为837±397mm 3，TGI％为67.14％。huJS007-47在0.03，0.1和0.3mg/kg的剂量下，平均肿瘤体积分别为1674±508mm 3，466±171mm 3，271±155mm 3，TGI％分别为28.83％、84.12％和93.04％。表明huJS007-47在0.1和0.3mg/kg的剂量下，显著抑制hCTLA4人源化小鼠移植H22肿瘤体积增长，并呈现良好 的剂量效应，且在等剂量条件下(0.1mg/kg)，huJS007-47抑瘤作用显著优于Ipilimumab。
实施例17、CTLA4-JS007 scFv结晶方法和结构解析
通过CTLA4-JS007 scFv复合物蛋白制备和晶体筛选，获得X射线晶体衍射，并通过分子置换法解析复合物结构，并对CTLA4-JS007 scFv相互作用的分子基础进行分析，以评价JS007抗体(即huJS007-47)与CTLA-4结合的分子机制及其阻断CTLA-4/B7-1相互作用的机制。
实施例中JS007 scFv为huJS007-47 scFv，CTLA-4蛋白为人源CTLA-4蛋白(SEQ ID NO:71)。
17.1实验方法
17.1.1 CTLA4-JS007 scFv复合物蛋白制备及晶体筛选
利用原核表达体系(E.coli BL21菌株)制备CTLA-4蛋白(C35-P154)和JS007-scFv的包涵体形式蛋白，通过体外包涵体复性方式分别获得CTLA4和JS007-scFv蛋白。然后通过凝胶过滤层析方法(GE，superdex 200(10/300GL))将两种蛋白进行纯化。将CTLA4蛋白和JS007-scFv蛋白以摩尔比1：1的比例置于冰上进行混合孵育30min，之后通过凝胶过滤层析方法(GE，superdex 200(10/300GL))对复合物进行进一步纯化，获得CTLA4-JS07 scFv复合物蛋白。将纯度高于90％的CTLA4-JS007scFv复合物蛋白利用晶体筛选试剂盒进行晶体筛选，以10mg/ml的浓度将蛋白溶液1μl与池液1μl混匀，置于16°进行晶体生长。最终在Molecular Dimension公司MD1-13-20(0.1M Tris pH：8.5 30％w/v PEG 4000)的条件下筛选到了CTLA4-JS007 scFv复合物晶体。
17.1.2 CTLA4-JS007 scFv复合物结构解析
在上海光源(SSRF)BL17U生物大分子晶体学光束线站进行CTLA-4-JS007scFv复合物蛋白晶体衍射数据的收集工作，所使用的X射线波长为
Figure PCTCN2021107707-appb-000026
调整晶体座位置，曝光，初步指标化(index)，收集衍射数据。利用HKL2000软件对衍射数据进行处理。然后用Coot和Phenix进行建模和精修。用Molprobity评估最终模型。解析后的蛋白质三维结构图及电子密度图使用PyMOL软件分析并展示。
17.2结构分析
17.2.1 JS007与CTLA-4结合的复合物的整体结构和相互作用的分子基础
通过对JS007与CTLA-4复合物的结构分析，发现JS007抗体的所有6个CDR均参与了与CTLA-4的相互作用(图20A)，其中重链与轻链相比贡献了更多的与CTLA-4的相互作用(表6)，重链CDR1中的Y33、重链CDR2中的Y54以及重链CDR3中的Y100、Y101、S102和Y104与CTLA-4的BC loop(K65、P63)和FG loop(Y135、P136、Y140)发生广泛的氢键相互作用。而轻链CDR1中的T31、轻链CDR2中的Y53以及轻链CDR3中的D92与CTLA-4的BC loop(E59、K65)和N110形成了广泛的氢键相互作用(图20B)。
表6.JS007抗体轻链/重链与CTLA-4相互作用
Figure PCTCN2021107707-appb-000027
轻链 CTLA-4 相互作用数量
T31 N110 13(1)
Y32 K65,G64,G109,N110 3,1,7,7
Y49 H39,A61,S62 1,8,3
S50 E59,A61,S62,N110 1,2,1,4
S52 E59 1
Y53 H39,A41,E59,A61 1,1,13(1),10
Y91 S62,G64,K65 1,2,1
D92 K65 8(1)
注：“相互作用数量”一栏中的未加括号的数字代表4.5埃距离内的范德华力相互作用数量；在括号内的数字代表3.5埃距离内的氢键相互作用数量。
17.2.2 JS007阻断CTLA-4与B7-1之间的相互作用的结构基础
阻断CTLA-4与其配体B7-1(即CD80)或B7-2(即CD86)之间的相互作用是JS007抗体发挥免疫活化作用的分子机制之一。通过对CTLA-4-JS007的复合物结构与CTLA-4-B7-1的复合物结构进行比较(PDB:1I8L)，进一步对JS007阻断CTLA-4与B7-1之间的相互作用的机制进行了深入分析。
将CTLA-4-JS007的复合物结构与CTLA-4-B7-1的复合物结构(PDB:1I8L)按照CTLA-4为固定参照进行重叠分析，结果表明，JS007的重链与B7-1之间存在显著的空间位阻，而CTLA-4分子中的FG loop与JS007和B7-1均具有广泛的相互作用，包括氢键相互作用和范德华力相互作用(图21A)。通过对CTLA-4分子中与JS007和B7-1的相互作用的氨基酸分析，发现CTLA-4分子FG loop上的Y135，P137，P138和Y140同时参与了与JS007或B7-1的结合，介导了JS007与B7-1对CTLA-4的竞争性结合(图21B)。
序列表
Figure PCTCN2021107707-appb-000028
Figure PCTCN2021107707-appb-000029
Figure PCTCN2021107707-appb-000030
Figure PCTCN2021107707-appb-000031
Figure PCTCN2021107707-appb-000032
Figure PCTCN2021107707-appb-000033
Figure PCTCN2021107707-appb-000034
Figure PCTCN2021107707-appb-000035

Claims (20)

  1. 一种抗CTLA-4抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区：
    所述重链可变区包含：
    (Ⅰ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；优选地，所述与SEQ ID NO:2所示氨基酸序列具有1、2或3个氨基酸差异的HCDR2是SEQ ID NO:37；优选地，所述与SEQ ID NO:3所示氨基酸序列具有1、2或3个氨基酸差异的HCDR3是SEQ ID NO:38；或
    (Ⅱ)氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或
    (Ⅲ)氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；或
    (Ⅳ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；优选地，所述与SEQ ID NO:20所示氨基酸序列具有1、2或3个氨基酸差异的HCDR2是SEQ ID NO:39；或
    (Ⅴ)氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；或与SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示氨基酸序列分别具有1、2或3个氨基酸差异的HCDR1、HCDR2和HCDR3；
    所述轻链可变区包含：
    (Ⅰ)氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或
    (Ⅱ)氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的 LCDR1、LCDR2和LCDR3；或与SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；或
    (Ⅲ)氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3；优选地，所述与SEQ ID NO:16所示氨基酸序列具有1、2或3个氨基酸差异的LCDR1是SEQ ID NO:40或41；或
    (Ⅳ)氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或与SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示氨基酸序列分别具有1、2或3个氨基酸差异的LCDR1、LCDR2和LCDR3。
  2. 如权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含重链可变区和/或轻链可变区：
    重链可变区，所述重链可变区包含：
    (Ⅰ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
    (Ⅱ)氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；或
    (Ⅲ)氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3；或
    (Ⅳ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；或
    (Ⅴ)氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；或
    (Ⅵ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:37和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
    (Ⅶ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:37和SEQ ID NO:38所示的HCDR1、HCDR2和HCDR3；或
    (Ⅷ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:38所示的HCDR1、HCDR2和HCDR3；或
    (Ⅸ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:39和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；
    和/或，轻链可变区，所述轻链可变区包含：
    (Ⅰ)氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或
    (Ⅱ)氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的LCDR1、LCDR2和LCDR3；或
    (Ⅲ)氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
    (Ⅳ)氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
    (Ⅴ)氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
    (Ⅵ)氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3。
  3. 如权利要求2所述的抗体或其抗原结合片段，其包含：
    (Ⅰ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或
    (Ⅱ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的LCDR1、LCDR2和LCDR3；或
    (Ⅲ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
    (Ⅳ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分 别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
    (Ⅴ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
    (Ⅵ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
    (Ⅶ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:39和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
    (Ⅷ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:37和SEQ ID NO:38所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3；或
    (Ⅸ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:40、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3。
  4. 如权利要求1-3中任一项所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区：
    (Ⅰ)所述重链可变区包含如SEQ ID NO:28、30、32、34或35中任一项所示的氨基酸序列，或与SEQ ID NO:28、30、32、34或35中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:29、31、33或36中任一项所示的氨基酸序列，或与SEQ ID NO:29、31、33或36中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序 列同一性的氨基酸序列；或
    (Ⅱ)氨基酸序列如SEQ ID NO:28所示的重链可变区和氨基酸序列如SEQ ID NO:29所示的轻链可变区；或
    (Ⅲ)氨基酸序列如SEQ ID NO:30所示的重链可变区和氨基酸序列如SEQ ID NO:31所示的轻链可变区；或
    (Ⅳ)氨基酸序列如SEQ ID NO:32所示的重链可变区和氨基酸序列如SEQ ID NO:33所示的轻链可变区；或
    (Ⅳ)氨基酸序列如SEQ ID NO:34所示的重链可变区和氨基酸序列如SEQ ID NO:33所示的轻链可变区；或
    (Ⅵ)氨基酸序列如SEQ ID NO:35所示的重链可变区和氨基酸序列如SEQ ID NO:36所示的轻链可变区。
  5. 如权利要求1-3中任一项所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区：
    所述重链可变区包含如SEQ ID NO:28或34中任一项所示的氨基酸序列或其变体，和
    所述轻链可变区包含如SEQ ID NO:33所示的氨基酸序列或其变体，
    其中所述变体包含：
    在氨基酸序列如SEQ ID NO:28或34中任一项所示的重链可变区中的1、2、3、4、5、6、7、8、9或10个氨基酸差异，和/或
    在氨基酸序列如SEQ ID NO:33所示的轻链可变区中的1、2、3、4、5、6、7、8、9或10个氨基酸差异。
  6. 如权利要求5所述的抗体或其抗原结合片段，其包含：
    (Ⅰ)重链可变区，其包含如SEQ ID NO:42、43、44、45、46、47或48中任一项所示的氨基酸序列，或与SEQ ID NO:42、43、44、45、46、47或48中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO:54、55、56、57、58、59、60、61或62中任一项所示的氨基酸序列，或与SEQ ID NO:54、55、56、57、58、59、60、61或62中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；或
    (Ⅱ)重链可变区，其包含如SEQ ID NO:49、50、51、52或53中任一项所示的氨基酸序列，或与SEQ ID NO:49、50、51、52或53中任一项所示的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO:54、55、56、57、58、59、60、61或62中任一项所示的氨基酸序列，或与SEQ ID NO:54、55、56、57、58、59、60、61或62中任一项所述的氨基酸序列具有至少95％，96％，97％，98％或99％序列同一性的氨基酸序列；或
    (Ⅲ)氨基酸序列如SEQ ID NO:50所示的重链可变区和氨基酸序列如SEQ ID NO:54、55、56或57所示的轻链可变区；或
    (Ⅳ)氨基酸序列如SEQ ID NO:51所示的重链可变区和氨基酸序列如SEQ ID NO:54或55所示的轻链可变区；或
    (Ⅴ)氨基酸序列如SEQ ID NO:46、47、53所示的重链可变区和氨基酸序列如SEQ ID NO:54或60所示的轻链可变区；或
    (Ⅵ)氨基酸序列如SEQ ID NO:50所示的重链可变区和氨基酸序列如SEQ ID NO:55所示的轻链可变区；或
    (Ⅶ)氨基酸序列如SEQ ID NO:50所示的重链可变区和氨基酸序列如SEQ ID NO:56所示的轻链可变区；或
    (Ⅷ)氨基酸序列如SEQ ID NO:46所示的重链可变区和氨基酸序列如SEQ ID NO:60所示的轻链可变区；或
    (Ⅸ)氨基酸序列如SEQ ID NO:53所示的重链可变区和氨基酸序列如SEQ ID NO:60所示的轻链可变区。
  7. 如权利要求6所述的抗体或其抗原结合片段，其中所述抗体包含重链和轻链，所述重链包含如SEQ ID NO:63、65、67或69中任一项所示的氨基酸序列，或与SEQ ID NO:63、65、67或69中任一项所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；
    和所述轻链包含如SEQ ID NO:64、66、68或70中任一项所示的氨基酸序列，或与SEQ ID NO:64、66、68或70中任一项所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  8. 如权利要求7所述的抗体或其抗原结合片段，其中所述抗体包含：
    (Ⅰ)氨基酸序列如SEQ ID NO:63所示的重链，和氨基酸序列如SEQ ID NO:64所示的轻链；或
    (Ⅱ)氨基酸序列如SEQ ID NO:65所示的重链，和氨基酸序列如SEQ ID NO:66所示的轻链；或
    (Ⅲ)氨基酸序列如SEQ ID NO:67所示的重链，和氨基酸序列如SEQ ID NO:68所示的轻链；或
    (Ⅳ)氨基酸序列如SEQ ID NO:69所示的重链，和氨基酸序列如SEQ ID NO:70所示的轻链。
  9. 如权利要求1-8中任一项所述的抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体、人源化抗体、或全人抗体，或其抗原结合片段。
  10. 如权利要求1-9中任一项所述的抗体或其抗原结合片段，其中所述抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb；优选地，所述抗CTLA-4抗体或其抗原结合片段是任何IgG亚型，如IgG1、IgG2、IgG3或IgG4，优选为IgG1。
  11. 一种分离的抗CTLA-4抗体或其抗原结合片段，其具有以下特性中的一种或多种：
    (1)与权利要求1-10中任一项所述的抗CTLA-4抗体或其抗原结合片段结合相同或者完全或部分重叠的人CTLA-4蛋白的表位；
    (2)与权利要求1-10中任一项所述的抗CTLA-4抗体或其抗原结合片段竞争结合人CTLA-4蛋白的表位；
    (3)结合由SEQ ID NO:71的残基27-29组成的表位，其中氨基酸残基编号为从SEQ ID NO:71中A37开始进行氨基酸编号；
    (4)结合SEQ ID NO:71的氨基酸残基27，28和29中的一个或多个，其中氨基酸残基编号为从SEQ ID NO:71中A37开始进行氨基酸编号；
    (5)抑制或阻断人CTLA-4蛋白与人CD80和/或人CD86或表达人CD80和/或人CD86的细胞的结合；优选的，其与CD80竞争结合的氨基酸残基为135，137，138和140中的一个或多个，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号；
    (6)结合由SEQ ID NO:71的氨基酸残基36-41和/或59-66和/或109-110和/或133-140组成的表位，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基 酸编号；和
    (7)结合SEQ ID NO:71的氨基酸残基36，39，41，59，61，62，63，64，65，66，109，110，133，135，136，137，138和140中的一个或多个，其中氨基酸残基编号为从SEQ ID NO:71中M1开始进行氨基酸编号。
  12. 一种多核苷酸，其编码如权利要求1-11中任一项所述的抗CTLA-4抗体或其抗原结合片段。
  13. 一种表达载体，其包含如权利要求12所述的多核苷酸，优选地，所述载体为真核表达载体。
  14. 一种宿主细胞，其包含如权利要求12所述的多核苷酸或如权利要求13所述的表达载体，优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
  15. 一种制备如权利要求1-11中任一项所述的抗CTLA-4抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下在权利要求14所述的宿主细胞中表达所述抗体或其抗原结合片段，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
  16. 一种药物组合物，其包含如权利要求1-11中任一项所述的抗CTLA-4抗体或其抗原结合片段、权利要求12所述的多核苷酸、权利要求13所述的表达载体、权利要求14所述的宿主细胞，和药学上可接受的载体或赋形剂。
  17. 如权利要求1-11中任一项所述的抗体或其抗原结合片段、权利要求12所述的多核苷酸、权利要求13所述的表达载体、权利要求14所述的宿主细胞、或权利要求16所述的药物组合物在制备用于治疗和/或预防CTLA-4介导的疾病或病症的药物中的用途，优选所述疾病或病症为癌症，更优选所述癌症选自黑素瘤、肾癌、前列腺癌、乳腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌和直肠癌。
  18. 一种药物组合，其包含如权利要求1-11中任一项所述的抗体或其抗原结合片段、权利要求12所述的多核苷酸、权利要求13所述的表达载体、权利要求14所述的宿主细胞、或权利要求16所述的药物组合物，以及一种或多种另外的治疗剂。
  19. 一种试剂盒，其包括如权利要求1-11中任一项所述的抗体或其抗原结合片段、权利要求12所述的多核苷酸、权利要求13所述的表达载体、权利要求14所述的宿主细胞、或权利要求16所述的药物组合物，优选其进一步包括给药装置。
  20. 一种使用如权利要求1-11中任一项所述的抗体或其抗原结合片段检测CTLA-4在样品中的存在的方法。
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