WO2018219327A1 - 抗cd40抗体、其抗原结合片段及其医药用途 - Google Patents

抗cd40抗体、其抗原结合片段及其医药用途 Download PDF

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WO2018219327A1
WO2018219327A1 PCT/CN2018/089252 CN2018089252W WO2018219327A1 WO 2018219327 A1 WO2018219327 A1 WO 2018219327A1 CN 2018089252 W CN2018089252 W CN 2018089252W WO 2018219327 A1 WO2018219327 A1 WO 2018219327A1
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seq
antibody
variable region
chain variable
antigen
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PCT/CN2018/089252
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English (en)
French (fr)
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闫树德
蒋家骅
胡齐悦
张连山
曹国庆
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to EP18809419.7A priority Critical patent/EP3632932A4/en
Priority to CA3064298A priority patent/CA3064298A1/en
Priority to CN201880010464.7A priority patent/CN110267989B/zh
Priority to RU2019141751A priority patent/RU2779128C2/ru
Priority to MX2019014375A priority patent/MX2019014375A/es
Priority to US16/618,006 priority patent/US11525005B2/en
Priority to KR1020197038295A priority patent/KR20200012920A/ko
Priority to JP2019566666A priority patent/JP7257971B6/ja
Priority to AU2018278051A priority patent/AU2018278051A1/en
Priority to BR112019025048-4A priority patent/BR112019025048A2/pt
Publication of WO2018219327A1 publication Critical patent/WO2018219327A1/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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K16/2875—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 NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
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    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas

Definitions

  • the present invention relates to an anti-CD40 antibody, an antigen-binding fragment against CD40, a chimeric antibody comprising the CDR region of the anti-CD40 antibody, a humanized antibody, and a pharmaceutical composition comprising the human anti-CD40 antibody and antigen-binding fragment thereof, And its use as an anticancer drug.
  • Tumor immunotherapy is a hot spot in the field of cancer therapy, in which T cell tumor immunotherapy is at its core. Tumor immunotherapy is a way to fully utilize and mobilize killer T cells in tumor patients and kill tumors. It may be the most effective and safest way to treat tumors. Tumor immunotherapy currently has good prospects for the treatment of several different types of cancer, including disseminated metastatic tumors.
  • T cells The activation of T cells in humans takes a system of two signaling pathways.
  • APCs antigen presenting cells
  • costimulatory molecules are required to provide a second signal.
  • This dual-signal pathway system plays a crucial role in the balance of the immune system in the body, and it strictly regulates the body's immune response to both self and non-self antigens.
  • a second signal provided by a costimulatory molecule it will result in a non-responsive or sustained specific immune response of the T cell, resulting in tolerance. Therefore, the second signaling pathway plays a very important regulatory role in the whole process of the body's immune response.
  • CD40 is one of the glycoproteins expressed on the cell surface. It is a type I membrane intrinsic glycoprotein with a molecular weight of 48 kDa. It belongs to the tumor necrosis factor receptor (TNFR) superfamily and plays an important role in the immune system. It is expressed in a variety of immune cells, such as B cells, dendritic cells, monocytes and macrophages. When signal transduction occurs through CD40, specialized antigen presenting cells are activated. The natural ligand for CD40 is designated CD154 or CD40L and is known to be predominantly expressed in mature T lymphocytes. CD40L-mediated signaling can trigger a number of cellular biological events, including immune cell activation, proliferation, and production of cytokines and chemokines. CD40 signaling is important for T cell-dependent immune responses, particularly in the context of tumor environment, where CD40-stimulated dendritic cells are capable of activating tumor-specific effector T cells, which have the potential to eradicate tumor cells.
  • TNFR tumor necrosis factor receptor
  • CD40 expression occurs in many normal cells and tumor cells including B lymphocytes.
  • melanoma belongs to a tumor with CD40 expression, while 30% to 70% of solid tumors also have CD40 expression.
  • Activation of CD40 is currently known to be effective in triggering anti-tumor responses (Tong et al, Cancer Gene Therapy, 2003, 10: 1-13), including immune activation of tumor-specific T cell responses, direct apoptosis of CD40 positive tumors.
  • stimulation of the humoral response leading to ADCC, and the observed tumor eradication is strongly associated with the appearance of tumor-specific cytotoxic T lymphocytes.
  • CD40 antibodies are associated with side effects such as shock syndrome and cytokine release syndrome (van Mierlo et al, Proc. Nat 1. Acad. Sci. USA, 2002, 99: 5561-5566). .
  • the present invention is directed to an anti-CD40 antibody having high affinity, high selectivity, high biological activity, and a cancer therapeutic drug/composition and method for inducing immune activation by stimulating CD40 and its pathway.
  • the invention provides a CD40 antibody or antigen-binding fragment thereof, comprising:
  • An antibody light chain variable region comprising at least one LCDR selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NO :14, SEQ ID NO: 15 or SEQ ID NO: 16; SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44; SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60; and/or
  • An antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO :11, SEQ ID NO: 12 or SEQ ID NO: 13; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41; SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57.
  • the anti-CD40 antibody or antigen-binding fragment thereof wherein the antibody light chain variable region comprises SEQ ID NO: 6, SEQ ID NO: 14, SEQ LCD R1 shown as ID NO. 42, SEQ ID NO. 50 or SEQ ID NO.
  • the anti-CD40 antibody or antigen-binding fragment thereof wherein the antibody light chain variable region comprises SEQ ID NO: 7, SEQ ID NO: 15, SEQ LCD R2 shown as ID NO. 43, SEQ ID NO. 51 or SEQ ID NO.
  • the anti-CD40 antibody or antigen-binding fragment thereof wherein the antibody light chain variable region comprises SEQ ID NO: 8, SEQ ID NO: 16, SEQ The LCDR3 region shown by ID NO. 44, SEQ ID NO. 52 or SEQ ID NO.
  • the anti-CD40 antibody or antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises SEQ ID NO: 3, SEQ ID NO: 11, SEQ HCDR1 of ID NO. 39, SEQ ID NO. 47 or SEQ ID NO.
  • the anti-CD40 antibody or antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises SEQ ID NO: 4, SEQ ID NO: 12, SEQ HCDR2 as shown in ID NO. 40, SEQ ID NO. 48 or SEQ ID NO.
  • the anti-CD40 antibody or antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises SEQ ID NO: 5, SEQ ID NO: 13, SEQ HCDR3 as shown in ID NO. 41, SEQ ID NO. 49 or SEQ ID NO.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody light chain variable region comprises SEQ ID NO: 6, SEQ ID NO: 7 and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 8.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody light chain variable region comprises SEQ ID NO: 14, SEQ ID NO: 15 and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 16.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody light chain variable region comprises SEQ ID NO: 42, SEQ ID NO: 43 and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:44.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody light chain variable region comprises SEQ ID NO: 50, SEQ ID NO: 51, and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:52.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody light chain variable region comprises SEQ ID NO: 58, SEQ ID NO: 59, and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:60.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody heavy chain variable region comprises SEQ ID NO: 3, SEQ ID NO: 4, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody heavy chain variable region comprises SEQ ID NO: 11, SEQ ID NO: 12, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:13.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody heavy chain variable region comprises SEQ ID NO: 39, SEQ ID NO: 40, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:41.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody heavy chain variable region comprises SEQ ID NO: 47, SEQ ID NO: 48, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:49.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody heavy chain variable region comprises SEQ ID NO: 55, SEQ ID NO: 56, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:57.
  • an anti-CD40 antibody or antigen-binding fragment thereof, wherein said antibody light chain variable region comprises:
  • LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;
  • LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;
  • LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44;
  • LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52;
  • LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60;
  • antibody heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13;
  • HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57.
  • a particularly preferred anti-CD40 antibody or antigen-binding fragment thereof can be selected from any of the following:
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the antibody heavy chain variable region comprises, for example, SEQ ID NO: 3, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 4 and SEQ ID NO: 5.
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable region comprises, for example, SEQ ID NO: 11, LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 12 and SEQ ID NO: 13.
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, respectively; the antibody heavy chain variable region comprises sequences such as: HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.
  • the antibody light chain variable region comprises the sequences of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, respectively; the antibody heavy chain variable region comprises: HCDR1, HCDR2 and HCDRs set forth in SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49.
  • the antibody light chain variable region comprises the sequences of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, respectively; the antibody heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57.
  • the antibody light chain variable region sequence is selected from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 10; the heavy chain variable region sequence is selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO: 9.
  • the above anti-CD40 antibody or antigen-binding fragment thereof may be a murine antibody or a chimeric antibody.
  • amino acid sequence of the heavy chain variable region of the murine antibody or the chimeric antibody is as shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 2;
  • amino acid sequence of the heavy chain variable region of the murine antibody or the chimeric antibody is set forth in SEQ ID NO. 9, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO.
  • the antibody Or the light chain variable region LCVR of the antigen fragment is the sequence SEQ ID NO: 38, the heavy chain variable region HCVR is the sequence SEQ ID NO: 37; or the light chain variable region LCVR of the antibody or antigen fragment is the sequence SEQ ID NO: 46, the heavy chain variable region HCVR is the sequence of SEQ ID NO: 45; or the light chain variable region LCVR of the antibody or antigen fragment is the sequence SEQ ID NO: 54, and the heavy chain variable region HCVR is the sequence SEQ ID NO:53.
  • the anti-CD40 antibody or antigen-binding fragment thereof wherein the antibody is a murine antibody or a fragment thereof.
  • a murine antibody or fragment thereof as described above, wherein the antibody light chain variable region further comprises a light chain FR region of a murine kappa, lambda chain or variant thereof.
  • a murine antibody or fragment thereof as described above, further comprising a light chain constant region of a murine kappa, lambda chain or variant thereof.
  • a murine antibody or fragment thereof as described above, wherein the antibody heavy chain variable region further comprises a heavy chain FR region of murine IgG1, IgG2, IgG3, IgG4 or variants thereof .
  • a murine antibody or fragment thereof as described above, further comprising a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein the antibody is a chimeric antibody or a fragment thereof.
  • the anti-CD40 chimeric antibody or fragment thereof, wherein the chimeric antibody light chain variable region sequence is: SEQ ID NO: 2 or SEQ ID NO: 10.
  • the anti-CD40 chimeric antibody or fragment thereof, wherein the chimeric antibody heavy chain variable region sequence is: SEQ ID NO: 1 or SEQ ID NO: 9.
  • an anti-CD40 chimeric antibody or fragment thereof as described above, further comprising a light chain constant region of a human kappa, lambda chain or variant thereof.
  • an anti-CD40 chimeric antibody or fragment thereof as described above, further comprising a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein said antibody is a human antibody or a fragment thereof.
  • an anti-CD40 antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody or a fragment thereof.
  • the anti-CD40 human antibody or antigen-binding fragment thereof wherein the human antibody light chain sequence is: SEQ ID NO: 18 or SEQ ID NO: Variant; the variant preferably has a 0-10 amino acid change in the light chain, more preferably a 2 and 3 mutation in the amino acid position, and the amino acids after the mutation in position 2 and 3 are preferably I, V. Or L.
  • the anti-CD40 human antibody or antigen-binding fragment thereof wherein the human antibody heavy chain sequence is: SEQ ID NO: 17 or SEQ ID NO: 19 Variants; the variants preferably have a 0-10 amino acid change in the heavy chain, more preferably a mutation in the 6 and 8 amino acid positions, and the amino acids after both site mutations are preferably I, A or L.
  • an anti-CD40 human antibody or antigen-binding fragment thereof as described above, further comprising a constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising human IgG1 IgG2 or IgG4 constant region.
  • the anti-CD40 humanized antibody or antigen-binding fragment thereof, wherein the humanized antibody light chain variable region further comprises human ⁇ , ⁇ chain or variant thereof Light chain FR zone.
  • the anti-CD40 humanized antibody or fragment thereof, wherein the light chain FR region sequence on the humanized antibody light chain variable region is derived from SEQ.
  • the anti-CD40 humanized antibody or fragment thereof, wherein the humanized antibody light chain variable region sequence is SEQ ID NO: 33 or SEQ ID NO The sequence shown at :34, or a variant thereof.
  • the anti-CD40 humanized antibody or fragment thereof, wherein the humanized antibody light chain sequence is as set forth in SEQ ID NO: 18 or SEQ ID NO: The sequence shown, or a variant thereof.
  • the amino acid change of 10 is more preferably a mutation of amino acid sites 2 and 3, and the amino acids after mutation are preferably I, V or L.
  • an anti-CD40 humanized antibody or fragment thereof as described above, further comprising a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the heavy chain FR region of the body further comprises human IgG1, IgG2, IgG3, IgG4 or a variant thereof
  • the anti-CD40 humanized antibody or fragment thereof, wherein the heavy chain FR region sequence on the heavy chain variable region of the humanized antibody is derived from SEQ.
  • the anti-CD40 humanized antibody or fragment thereof, wherein the humanized antibody heavy chain variable region sequence is SEQ ID NO: 26 or SEQ ID NO The sequence shown in :30, or a variant thereof.
  • the anti-CD40 humanized antibody or fragment thereof wherein the humanized antibody heavy chain sequence is as set forth in SEQ ID NO: 17 or SEQ ID NO:
  • the sequence shown, or a variant thereof; preferably, the variant has an amino acid change of 0-10 in the heavy chain variable region, more preferably a mutation at amino acid positions 6 and 8, and the amino acid after mutation is preferably I, A or L.
  • an anti-CD40 humanized antibody or fragment thereof as described above, further comprising a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising The human IgGl, IgG2 or IgG4 heavy chain FR region, more preferably comprises a human IgGl or IgG2 heavy chain FR region.
  • the anti-CD40 antibody or antigen-binding fragment thereof wherein the antigen-binding fragment is Fab, Fv, sFv, F(ab') 2 , linear antibody, single Chain antibodies, Nanobodies, domain antibodies and multispecific antibodies.
  • the invention further provides a DNA sequence encoding an anti-CD40 antibody or antigen-binding fragment thereof, multispecific antibody or single chain antibody as described above.
  • the invention further provides an expression vector comprising the DNA sequence as described above.
  • the invention further provides a host cell transformed with an expression vector as described above.
  • a host cell as described above characterized in that said host cell is a bacterium, preferably Escherichia coli.
  • a host cell as described above is a yeast, preferably Pichia pastoris.
  • a host cell as described above is a mammalian cell, preferably a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) 293 cell.
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • the present invention further provides a single-chain antibody comprising the anti-CD40 antibody or antigen-binding fragment thereof as described above.
  • the invention further provides a multispecific antibody comprising an anti-CD40 antibody or antigen-binding fragment thereof as described above.
  • the invention further provides an antibody-drug conjugate comprising an anti-CD40 antibody light chain variable region and/or a heavy chain variable region as described above.
  • the antibody-drug conjugates are well known in the art and are formed by the interconnection of antibody-linker-drug (toxin), and known linkers include cleavage linkers, split cleavage linkers, such as linkers including, but not limited to, SMCC, SPDP Etc.; Toxins are also well known in the art, such as DM1, DM4, MMAE, MMAF, and the like.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CD40 antibody or antigen-binding fragment thereof, a multispecific antibody or a single chain antibody as described above, and a pharmaceutically acceptable excipient, dilution or carrier.
  • the invention further provides an anti-CD40 antibody or antigen-binding fragment thereof, a multispecific antibody, a single chain antibody or a pharmaceutical composition comprising the same as described above for the preparation of a disease or condition mediated by CD40 or CD40L.
  • the disease is preferably cancer; and the cancer is most preferably lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder Cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma, and melanoma.
  • the invention further provides a method of treating and preventing a CD40 or CD40L mediated disease or condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-CD40 antibody or antigen-binding fragment thereof, a multispecific antibody, A single-chain antibody or a pharmaceutical composition comprising the same; wherein the disease is preferably cancer; and the cancer is most preferably lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer Colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma and melanoma.
  • the present invention further provides an anti-CD40 antibody or antigen-binding fragment thereof, a multispecific antibody, a single-chain antibody or a pharmaceutical composition comprising the same as described above, for use in the preparation of a medicament for improving symptoms of a patient suffering from an autoimmune disease .
  • the present invention further provides an anti-CD40 antibody or antigen-binding fragment thereof, a multispecific antibody, a single chain antibody or a pharmaceutical composition comprising the same as described above, for use in the preparation of a medicament for ameliorating symptoms of a patient suffering from an inflammatory disease .
  • Figure 1 shows the activation of DC cells by a murine anti-human CD40 antibody based on CD80 activating molecules.
  • Figure 2 is a graph showing the activation of DC cells by a murine anti-human CD40 antibody based on CD86 activating molecules.
  • Figure 3 is a graph showing tumor growth curves of Raji transplanted lymphoma co-transplanted with human PBMC and DC cells.
  • Figure 4 is a graph showing body weight changes of NOG mice transplanted with Raji-transplanted lymphoma co-transplanted with human PBMC and DC cells.
  • antibody refers to an immunoglobulin which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, respectively. , ⁇ chain and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each of the five types of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising human or murine IgGl, 2, 3, 4 or variants thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • Each of the light chain variable region (VL) and the heavy chain variable region (VH) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2. , FR3, CDR3 and FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the invention conform to known IMGT numbering rules in number and position.
  • APC antigen presenting cell
  • T cells recognize this complex using the T cell receptor (TCR).
  • APCs include, but are not limited to, dendritic cells (DC), peripheral blood mononuclear cells (PBMC), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DC).
  • DC dendritic cells
  • PBMC peripheral blood mononuclear cells
  • monocytes B lymphoblasts
  • DC monocyte-derived dendritic cells
  • the term “antigen presentation” refers to the process by which APCs capture antigens and enable them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.
  • CD40 refers to a cell surface receptor that is a member of the TNF receptor superfamily, also known as TNFRSF5.
  • CD40 is ubiquitously expressed on the surface of dendritic cells, B cells and macrophages and is a molecule required for the production and maintenance of T cell immunity.
  • the term “CD40” includes any variant or isoform of CD40 that is naturally expressed by a cell.
  • the antibodies of the invention can be cross-reactive with CD40 from a non-human species. Alternatively, the antibody may be human CD40-specific and may not exhibit cross-reactivity with other species.
  • CD40, or any variant or isoform thereof can be isolated from cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques common in the art and described herein.
  • the anti-CD40 antibody targets human CD40 with a normal glycosylation pattern.
  • recombinant human antibody includes human antibodies which are prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as (1) transgenes from human immunoglobulin genes, transgenic chromosomes.
  • Such recombinant human antibodies comprise variable regions and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.
  • murine antibody is in the present invention a monoclonal antibody to human CD40 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the CD40 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine CD40 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, a lambda chain or a variant thereof, or further comprising a murine IgG1, IgG2 , heavy chain constant region of IgG3 or IgG4 or a variant thereof.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • Human antibodies of the invention can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody does not include an antibody in which a CDR sequence derived from a germline of another mammalian species, such as a mouse, has been grafted onto a human framework sequence (ie, "humanized antibody”).
  • humanized antibody also referred to as a CDR-grafted antibody, refers to an antibody produced by grafting a CDR sequence of a mouse into a human antibody variable region framework. It is possible to overcome the strong immune response induced by chimeric antibodies by carrying a large amount of mouse protein components. To avoid a decrease in activity while reducing immunogenicity, the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the constant region gene of the human antibody is cloned as needed to change the mouse.
  • the region gene and the human constant region gene are ligated into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgGl, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgGl or IgG2 heavy chain constant region.
  • antigen-binding fragment refers to an antigen-binding fragment of an antibody and an antibody analog, which typically includes at least a portion of an antigen binding or variable region (eg, one or more CDRs) of a parental antibody.
  • the antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, Nanobodies, domain antibodies, and multispecific antibodies.
  • Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.
  • a "Fab fragment” consists of a light chain and a heavy chain CH1 and a variable region.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic action of the CH3 domain.
  • a "Fab' fragment” contains a light chain and a portion of a heavy chain comprising a VH domain and a CH1 domain and a region between the CH1 and CH2 domains, thereby being between the two heavy chains of the two Fab' fragments An interchain disulfide bond is formed to form a F(ab')2 molecule.
  • the "F(ab')2 fragment” contains two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains.
  • the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both heavy and light chains, but lacks a constant region.
  • multispecific antibody is used in its broadest sense to encompass antibodies having multi-epitope specificity.
  • These multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; having two or more Antibodies of the VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region Different targets or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked Along with antibody fragments and the like.
  • ADC antibody-drug conjugate
  • single-chain antibody is a single-stranded recombinant protein joined by a heavy chain variable region (VH) and a light chain variable region (VL) of a antibody via a ligation peptide, which is the smallest with complete antigen binding sites.
  • VH heavy chain variable region
  • VL light chain variable region
  • domain antibody fragment is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region chain.
  • two or more VH regions are covalently joined to a peptide linker to form a bivalent domain antibody fragment.
  • the two VH regions of a bivalent domain antibody fragment can target the same or different antigens.
  • binding to CD40 refers to the ability to interact with human CD40.
  • antigen binding site refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • An epitope can be formed by an adjacent amino acid, or a non-adjacent amino acid juxtaposed by a tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, and the like. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.
  • the terms “specifically bind” and “selectively bind” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody has an equilibrium dissociation constant of less than about 10 -7 M or even less when measured by surface plasmon resonance (SPR) techniques in an instrument.
  • SPR surface plasmon resonance
  • K D K D of binding to a predetermined antigen and which bind to the predetermined antigen non-specific affinity of its antigen than the predetermined antigen or a closely-related antigen (e.g., BSA, etc.) binding affinity at least twice.
  • the term "antibody recognizing an antigen” can be used interchangeably herein with the term “specifically bound antibody”.
  • cross-reactive refers to the ability of an antibody of the invention to bind to CD40 from a different species.
  • an antibody of the invention that binds to human CD40 can also bind to CD40 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (eg, SPR and ELISA), or binding or functional interactions with cells that express CD40 physiologically.
  • binding assays eg, SPR and ELISA
  • Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • inhibiting or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • the inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blockade.
  • Inhibition and blockade are also intended to include a decrease in any measurable ligand binding affinity when contacted with an anti-CD40 antibody, compared to a ligand not contacted with an anti-CD40 antibody.
  • inhibiting growth eg, involving cells
  • inhibiting growth is intended to include any measurable reduction in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a particular antigen.
  • inducing for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • the "ADCC” described in the present invention is an antibody-dependent cell-mediated cytotoxicity, which means that a cell expressing an Fc receptor directly kills an antibody by Fc segment of the recognition antibody.
  • Target cells The ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc segment on IgG.
  • the modification refers to mutations in the heavy chain constant region of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2/4 chimera, F235E of IgG4, or L234A/E235A mutation.
  • a mouse can be immunized with human CD40 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region. Human FR germline sequences are available on the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN 014441351.
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • the cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the FC region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified and collected by a conventional technique.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange. The resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • the antibody of the present invention refers to a monoclonal antibody.
  • the monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain.
  • Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases, whether by inducing the degradation of such symptoms or inhibiting the progression of such symptoms to any clinically unmeasurable extent.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount” effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition.
  • Embodiments of the invention e.g., methods of treatment or preparations
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molec ⁇ lar Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Common conservative substitutions for amino acids are as follows:
  • a binding compound consisting essentially of the amino acid sequence recited may also include one or more amino acids that do not significantly affect the properties of the binding compound.
  • naturally occurring refers to the fact that the object can be found in nature.
  • a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified in the laboratory by humans is naturally occurring.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the route and dosage of the method of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living organism, cell, or human body depending on the background.
  • Endogenous refers to a substance produced in a cell, organism or human body depending on the background.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • His-tagged human CD40 (h-CD40-his) recombinant protein, Fc-tagged human CD40 (h-CD40-Fc) recombinant protein, his-tagged mouse CD40 (m-CD40-his) recombinant protein and band His tagged rhesus CD40 (rhesus-CD40-his) recombinant protein (#CD0-C52H7) was purchased from Acrobiosystems, Inc., and the respective sequence sources are shown in Table 1. The protein can be used in the experiments of the following examples.
  • Anti-human CD40 monoclonal antibodies are produced by immunizing mice. Experimental C57BL/6 mice, female, 6-8 weeks old [Zhao Yan (Suzhou) New Drug Research Center Co., Ltd., animal production license number: 201503259]. Feeding environment: SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. The mice that have adapted to the environment are divided into 2 cages, 5 per cage.
  • the immunizing antigen was an Fc-tagged human modified CD40 recombinant protein (h-CD40-Fc, formulated in phosphate buffer solution to 1 ⁇ g/ ⁇ l).
  • Emulsification with Freund's adjuvant (Sigma, Lot No.: F5881/F5506): first use of Freund's complete adjuvant (CFA), remaining booster nucleic acid adjuvant (CpG, Sangon Biotech) and aluminum for injection (Imject Alum) , Thermo, Lot No.: PH203866).
  • the immunization time was 0, 14, 28, 42, 56, and 70 days. On the 21st, 35th, 49th, 63th, and 77th day, blood was collected for blood test, and the serum of the mouse was detected by ELISA to determine the antibody titer in the serum of the mouse.
  • spleen cell fusion was performed in mice with high antibody titer in serum and the titer was platform-oriented.
  • the immunization was boosted 3 days before fusion, and 10 ⁇ g/phosphate buffer solution was administered intraperitoneally (IP).
  • IP intraperitoneally
  • Antigen solution Spleen lymphocytes and myeloma cell Sp2/0 cells were optimized using an optimized PEG-mediated fusion step ( CRL-8287 (TM ) was fused to obtain hybridoma cells, and five monoclonal hybridoma cell lines with good activity in vitro were selected.
  • An ELISA assay was used to detect the binding properties of anti-CD40 antibodies.
  • the CD40 recombinant protein with his-tag was directly coated, and after the antibody was added, the antibody-antigen-binding activity was detected by adding a secondary antibody (HRP-conjugated anti-antibody Fc antibody) and HRP substrate TMB.
  • Human or rhesus CD40-his protein was coated on a 96-well microtiter plate, and incubated at a concentration of 0.5 ⁇ g/ml at 100 ⁇ l per well overnight at 4 °C. Wash the wash three times, 250 ⁇ l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Add 200 ⁇ l/well blocking solution for 2 hours at room temperature. Wash the wash three times, 250 ⁇ l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. 100 ⁇ l of each anti-CD40 antibody to be tested diluted with the dilution was added to each well. Incubate for 1 hour at room temperature. Wash the wash three times, 250 ⁇ l per well.
  • the selected anti-human CD40 antibody blocked the binding of human CD40 and human CD40L by an in vitro blocking assay.
  • the specific method is to coat the Fc-tagged CD40 recombinant protein (h-CD40-Fc) onto a 96-well microtiter plate, add the anti-CD40 antibody to fully bind to the occupied epitope, and then add the his-tagged CD40L to detect his The tag was used to calculate the binding amount of CD40 to CD40L, and the IC50 value of CD40 antibody blocking of CD40 active site was calculated.
  • the human CD40-Fc protein was coated on a 96-well microtiter plate, and incubated at 100 ⁇ l per well at a concentration of 1 ⁇ g/ml overnight at 4 °C. Wash the wash three times, 250 ⁇ l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Add 200 ⁇ l/well blocking solution for 2 hours at room temperature. Wash the wash three times, 250 ⁇ l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. 100 ⁇ l of the anti-CD40 test antibody diluted with the dilution was added to each well, and incubated for 1 hour at room temperature. Wash the wash three times, 250 ⁇ l per well.
  • the human anti-capture antibody was covalently coupled to the Biacore instrument (Biacore X100, GE) biosensor chip CM5 according to the method in the human anti-capture kit (Cat. #BR-1008-39, GE). And capture a certain amount of chimeric or humanized antibody to be tested, and then flow through a series of concentration gradients of CD40 antigen (CD40 antigen purchased from Acrobiosystems) on the surface of the chip, and use Biacore instrument (Biacore X100, GE) to detect the reaction in real time. The signal thus obtains a binding and dissociation curve. After each cycle dissociation is completed, the biochip is washed and regenerated using the regeneration solution disposed in the human anti-capture kit.
  • Biacore instrument Biacore X100, GE
  • the amino coupling kit used in the experiment was purchased from GE (Cat. #BR-1000-50, GE) and the buffer was HBS-EP+10 ⁇ buffer solution (Cat.#BR-1006-69, GE). Dilute to 1 x (pH 7.4) with DIWater.
  • the experimental data were fitted with the (1:1) Binding model using Biacore X100 evaluation software 2.0 GE software to obtain affinity values, as shown in Tables 10 and 11.
  • HEK-Blue CD40L cells were purchased from Invivogen (Cat#hkb-cd40), which stably transfects the human CD40 gene and NF-kB-mediated SEAP genome, and can detect SEAP secreted in the supernatant by the SEAP substrate QUANTI-Blue. To characterize the level of activation of the CD40 signaling pathway. In this experiment, the in vitro cell viability of CD40 antibody was evaluated according to the EC50 size by detecting the activation of cellular HEK-Blue CD40L.
  • the cell HEK-Blue CD40L was cultured in DMEM medium containing 10% FBS, 100 ⁇ g/ml Zeocin and 30 ⁇ g/ml Blasticidin, and passaged 2 to 3 times a week, and the passage ratio was 1:5 or 1:10.
  • the medium was aspirated, the cell layer was washed with 5 ml of 0.25% trypsin, then the trypsin was aspirated, and the cells were digested in an incubator for 3 to 5 minutes, and the cells were resuspended by adding fresh medium.
  • the medium is DMEM containing 10% FBS, 100 ⁇ g/ml Zeocin and 30 ⁇ g/ml Blasticidin. Add 100 ⁇ l of sterile water. The plates were incubated in an incubator for 24 hours (37 ° C, 5% CO 2 ). After the cells were attached, 100 ⁇ l of a gradient dilution of the antibody to be tested was added to each well. Incubate the plates in an incubator for 20-24 hours (37 ° C, 5% CO 2 ). 40 ⁇ l of each cell was supernatanted into a new 96-well flat bottom plate, 160 ⁇ l of QUANTI-Blue substrate solution was added, and the plate was incubated in the incubator for 1-3 hours in the dark. The absorbance at 620 nm was measured with a microplate reader (Thermo M ⁇ lti SkanFc), and the EC50 value was calculated to evaluate the in vitro cell viability of the CD40 antibody.
  • PBMCs peripheral blood and then sorted with CD14 MACS beads for monocytes.
  • Induction culture of MoDC cells was carried out by adding RPMI 1640 medium containing 10 ng/ml of IL4 and 100 ng/ml of GM-CSF for 6 days. After 6 days, the cells were collected, 1 ⁇ 10 5 cells, CD209-PE, CD1a-PerCP/Cy5.5 and CD14-PE/Cy7 stained, and FACS analysis was successful in inducing MoDC (the above operations are all routine operations in the art).
  • the cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and sent to a sequencing company for sequencing. The sequence of 5 murine antibodies was finally obtained.
  • the heavy and light chain variable region sequences of murine mAb 2H6 are as follows:
  • the heavy and light chain variable region sequences of 9E5 are as follows:
  • the heavy and light chain variable region sequences of 1D9 are as follows:
  • the heavy and light chain variable region sequences of 14C10 are as follows:
  • the heavy and light chain variable region sequences of 38B4 are as follows:
  • variable region sequences were respectively ligated to the human antibody IgG1 constant region sequence to obtain a human-mouse chimeric antibody sequence, and the sequence of the chimeric antibody was inserted into the pCP expression vector by molecular cloning technique (purchased from Maibo)
  • molecular biological operation methods such as molecular cloning are carried out according to the conventional operating conditions, and can be referred to the "Molecular Cloning: Laboratory Manual"
  • the HEK293 cell expression system can be used.
  • Human-mouse chimeric antibodies 2H6-C and 9E5-C were obtained.
  • the in vitro activity assays were performed on chimeric antibodies purified by MabSelect SuRe (GE Lifesciences) affinity chromatography. The data are shown in Table 10.
  • the heavy and light chain variable region sequences were compared with the antibody Germline database to obtain a human germline template with high homology.
  • the human germline light chain framework region is derived from a human kappa light chain gene, and the antibody of the invention is preferably a human germline light chain template Vk1-33/JK4 (2H6) or Vk2-28/JK4 (9E5).
  • the human germline heavy chain framework region is derived from a human heavy chain, and the antibody of the present invention is preferably a human germline heavy chain template VH1-69/JH6 (2H6) or VH1-2/JH6 (9E5), as shown below:
  • 2H6 is preferably a human germline heavy chain template IGHV1-69 (SEQ ID NO: 21):
  • 2H6 is preferably a human germline light chain template IGkV1-33 (SEQ ID NO: 22):
  • 9E5 is preferably a human germline heavy chain template IGHV1-2 (SEQ ID NO: 23):
  • 9E5 is preferably a human germline light chain template IGkV2-28 (SEQ ID NO: 24):
  • the CDR regions of the murine antibody are grafted onto the selected humanized template, replacing the humanized variable region, and recombined with the corresponding human IgG constant region (preferably the heavy chain is IgG1 and the light chain is ⁇ ). Then, based on the three-dimensional structure of the murine antibody, the residues which have an important interaction with the CDRs and the residues of the CDRs, and the residues which have an important influence on the conformation of VL and VH, are subjected to back mutation and chemistry of the CDR regions. Unstable amino acid residues are optimized to give the final humanized molecule. Its heavy chain variable region sequences are set forth in SEQ ID NOs: 25-30; the light chain variable region sequences are set forth in SEQ ID NOs: 31-36.
  • the final humanized hu2H6 (using the H1b heavy chain and the L1c light chain) and the hu9E5 antibody molecule (using the H1c heavy chain and the L1a light chain) were selected via the above expression comparison of the light and heavy chain combinations and the number of back mutations, respectively.
  • the complete light and heavy chain sequences are set forth in SEQ ID NOs: 17-20.
  • the binding activity, blocking activity, and the like of the humanized antibodies hu2H6 and hu9E5 of the present invention to human, rhesus CD40 are shown in Table 11.
  • the results showed that the ELISA binding and blocking activity of the humanized anti-human CD40 antibody of the present invention was comparable to that of the positive antibody pfizer/alligator.
  • the Biacore measurement affinity of hu9E5 and human CD40 was more than 10 times that of the positive antibody Alligator reference, and the Pfizer reference was used. 4 times more.
  • the normal human peripheral blood was taken and the healthy human PBMC was isolated by density gradient centrifugation.
  • Mononuclear cells were sorted by CD14+microbeads kit, and CD14+ monocytes were separated according to the protocol provided by the kit, that is, 20 ⁇ l of Anti-CD14 microbeads was added per 10 7 cells, and incubated at 4 ° C for 15 minutes. Then, the cells were added to a magnetic column, and after washing three times, the cells in the magnetic column, that is, CD14+ monocytes were collected.
  • CD14+ monocytes were cultured for 6 days in RPMI 1640 medium containing 10 ng/ml IL4 and 100 ng/ml GM-CSF (culture method is a routine method in the art), induced culture of MoDC cells, and the remaining cells were added with IL-containing RPMI 1640 of 2, the suspended cells were collected after the culture (the culture method and the method of collecting the cells are all conventional methods in the art), and the T cells were sorted using the CD3+microbeads kit.
  • the experiment was divided into human IgG1 antibody control group, hu2H6, hu9E5 and reference antibody G12, and the dose was 3 mg/kg.
  • Five mice per group were administered once a week for six weeks and administered three times in succession.
  • Relative tumor inhibition rate TGI (%): TGI% (1-T/C) ⁇ 100%.
  • T/C% is the relative tumor proliferation rate, that is, the percentage of tumor volume or tumor weight relative to the treatment group and the control group at a certain time point.
  • T and C are the tumor volume (TV) or tumor weight (TW) of the treatment group and the IgG1 control group at a specific time point, respectively.

Abstract

提供了抗CD40抗体、其抗原结合片段及其医药用途。进一步地，提供了包含所述抗CD40抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗CD40抗体及其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。尤其地，提供了一种人源化的抗CD40抗体，在制备用于治疗CD40介导的疾病或病症的药物中的用途。

Description

抗CD40抗体、其抗原结合片段及其医药用途
本申请要求申请日为2017年6月1日的中国专利申请CN201710402559.0的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种抗CD40抗体，抗CD40的抗原结合片段，包含所述抗CD40抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗CD40抗体及其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。
背景技术
癌症是当今人类社会长期面临的最大健康挑战。传统的疗法如手术、化疗和放疗在治疗播散的实体肿瘤中收效甚微。肿瘤免疫治疗是肿瘤治疗领域一个热点，其中T细胞的肿瘤免疫治疗又处于其核心位置。肿瘤免疫治疗是充分利用、调动肿瘤患者体内的杀伤性T细胞，对肿瘤进行杀伤作用，它可能是最有效的也是最安全的***的途径。肿瘤免疫疗法目前对治疗几种不同类型的癌症，包括播散的转移性肿瘤已具有很好的前景。
人体内T细胞的活化采取了两条信号通路的***，除了需要通过抗原呈递细胞(APC)递呈MHC-抗原肽给T细胞提供第一信号外，还需要一系列协同刺激分子提供第二信号，进而才能使T细胞产生正常的免疫应答。这个双信号通路***对体内免疫***的平衡起到着至关重要的作用，它严格调控者机体对自身和非自身抗原产生不同的免疫应答。如果缺少协同刺激分子提供的第二信号，将会导致T细胞的无应答或持续特异性免疫应答，从而产生耐受。因此，第二信号通路在机体免疫应答的整个过程中起着非常关键的调节作用。
CD40是细胞表面表达的糖蛋白之一，为分子量48kDa的I型膜内在糖蛋白，属于肿瘤坏死因子受体(TNFR)超家族，并在免疫***中起重要作用。其表达在多种免疫细胞中，如B细胞、树突细胞、单核细胞和巨噬细胞等。当通过CD40发生信号传导时，专门的抗原呈递细胞会被激活。CD40的天然配体被命名为CD154或CD40L，已知主要表达在成熟T淋巴细胞中。CD40L介导信号的传导会触发一些细胞生物事件，包括免疫细胞激活、增殖，以及细胞因子和趋化因子的产生。CD40信号传导对于T细胞依赖性免疫应答是非常重要的，尤其在肿瘤环境背景中，CD40刺激的树突细胞能够激活肿瘤特异 性效应T细胞，该类T细胞具有根除肿瘤细胞的潜力。
CD40表达发生在包括B淋巴细胞在内的许多正常细胞和肿瘤细胞中。例如黑色素瘤即属于具有CD40表达的肿瘤，而30％至70％的实体肿瘤也具有CD40表达。目前已知CD40的激活可有效触发抗肿瘤反应(Tong等，Cancer Gene Therapy,2003，10:1-13)，包括肿瘤特异性T细胞反应的免疫激活、对CD40阳性肿瘤直接的细胞凋亡作用，以及剌激导致ADCC的体液反应，且观察到的肿瘤根除与肿瘤特异性细胞毒性T淋巴细胞的出现强烈相关。同时也不应被忽视的是，全身施用CD40抗体与副作用相关，如休克综合征和细胞因子释放综合征(van Mierlo等，Proc.Nat1.Acad.Sci.USA,2002,99:5561-5566)。
目前有多家国际制药公司在研发如上述针对CD40的单克隆抗体，它通过特异性刺激免疫激活，最大限度提高患者自身对肿瘤的免疫***反应，从而达到对肿瘤细胞进行杀伤的目的。相关专利如CN1198647、CN1369015、CN1582165、CN100430419、CN101014386、CN101237882、CN101289510、CN101490086、CN103842382、CN104918957、WO2002028904、WO2011123489、WO2012149356、WO2013034904、WO2015091853、WO2016196314、WO2017040932、WO2017004006等。迄今为止，辉瑞(相关产品已许可给罗氏)、Alligator等公司的抗CD40抗体均在临床前动物模型中观测到了良好的肿瘤杀伤效果，并已推进入I期临床试验阶段当中。
本发明旨在提供有着高亲和力，高选择性，高生物活性的抗CD40抗体，用于通过刺激CD40及其通路导致免疫激活的癌症治疗药物/组合物和方法。
发明内容
本发明提供一种CD40抗体或其抗原结合片段，其包含：
抗体轻链可变区，所述的抗体轻链可变区包含至少1个选自如以下序列所示的LCDR：SEQ ID NO:6，SEQ ID NO:7，SEQ ID NO:8；SEQ ID NO:14，SEQ ID NO:15或SEQ ID NO:16；SEQ ID NO:42，SEQ ID NO:43，SEQ ID NO:44；SEQ ID NO:50，SEQ ID NO:51，SEQ ID NO:52；SEQ ID NO:58，SEQ ID NO:59或SEQ ID NO:60；和/或
抗体重链可变区，所述的抗体重链可变区包含至少1个选自如以下序列所述的HCDR：SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:11，SEQ ID NO:12或SEQ ID NO:13；SEQ ID NO:39，SEQ ID NO:40，SEQ ID NO:41；SEQ ID NO:47，SEQ ID NO:48，SEQ ID NO:49；SEQ ID NO:55、SEQ ID NO:56或SEQ ID NO:57。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段， 其中所述的抗体轻链可变区包含如SEQ ID NO:6、SEQ ID NO:14、SEQ ID NO.42、SEQ ID NO.50或者SEQ ID NO.58所示的LCDR1。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体轻链可变区包含如SEQ ID NO:7、SEQ ID NO:15、SEQ ID NO.43、SEQ ID NO.51或者SEQ ID NO.59所示的LCDR2。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体轻链可变区包含如SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO.44、SEQ ID NO.52或者SEQ ID NO.60所示的LCDR3区。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体重链可变区包含如SEQ ID NO:3、SEQ ID NO:11、SEQ ID NO.39、SEQ ID NO.47或者SEQ ID NO.55所示的HCDR1。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体重链可变区包含如SEQ ID NO:4、SEQ ID NO:12、SEQ ID NO.40、SEQ ID NO.48或者SEQ ID NO.56所示的HCDR2。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体重链可变区包含如SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO.41、SEQ ID NO.49或者SEQ ID NO.57所示的HCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体轻链可变区包含分别如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体轻链可变区包含分别如SEQ ID NO:42、SEQ ID NO:43和SEQ ID NO:44所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体轻链可变区包含分别如SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体轻链可变区包含分别如SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:60 所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体重链可变区包含分别如SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体重链可变区包含分别如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体重链可变区包含分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体重链可变区包含分别如SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其所述的抗体重链可变区包含分别如SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体轻链可变区包含分别如：
SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；或
SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；或
SEQ ID NO:42、SEQ ID NO:43和SEQ ID NO:44所示的LCDR1、LCDR2和LCDR3；或
SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的LCDR1、LCDR2和LCDR3；或
SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:60所示的LCDR1、LCDR2和LCDR3；
且其中所述的抗体重链可变区包含分别如：
SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；或
SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3； 或
SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示的HCDR1、HCDR2和HCDR3；或
SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的HCDR1、HCDR2和HCDR；或
SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57所示的HCDR1、HCDR2和HCDR3。
特别优选的抗CD40抗体或其抗原结合片段可选自下述任一种：
(1)抗体轻链可变区包含分别如：SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3。
(2)抗体轻链可变区包含分别如：SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的LCDR1、LCDR2和LCDR3。
(3)抗体轻链可变区包含分别如：SEQ ID NO:42、SEQ ID NO:43和SEQ ID NO:44所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含序列分别如：SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示的HCDR1、HCDR2和HCDR3。
(4)抗体轻链可变区包含序列分别如：SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的HCDR1、HCDR2和HCDR。
(5)抗体轻链可变区包含序列分别如：SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:60所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，所述的抗体轻链可变区序列选自SEQ ID NO:2或SEQ ID NO:10；重链可变区序列选自SEQ ID NO:1或SEQ ID NO:9。
上述的抗CD40抗体或其抗原结合片段可为鼠源抗体或者嵌合抗体。
较佳地：
其中所述鼠源抗体或者所述嵌合抗体的重链可变区的氨基酸序列如SEQ ID NO.1所示，轻链可变区的氨基酸序列如SEQ ID NO.2所示；或者，所述鼠源抗体或者所述嵌合抗体的重链可变区的氨基酸序列如SEQ ID NO.9所示，轻链可变区的氨基酸序列如SEQ ID NO.10所示；或者，所述抗体或抗原片段的轻链可变区LCVR为序列SEQ ID NO:38，重链可变区HCVR为序列SEQ ID NO：37；或者，所述抗体或抗原片段的轻链可变区 LCVR为序列SEQ ID NO:46，重链可变区HCVR为序列SEQ ID NO：45；或者，所述抗体或抗原片段的轻链可变区LCVR为序列SEQ ID NO:54，重链可变区HCVR为序列SEQ ID NO：53。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体为鼠源抗体或其片段。
在本发明一个优选的实施方案中，一种如上所述的鼠源抗体或其片段，其抗体轻链可变区进一步包含鼠源κ、λ链或其变体的轻链FR区。
在本发明一个优选的实施方案中，一种如上所述的鼠源抗体或其片段，其进一步包含鼠源κ、λ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，一种如上所述的鼠源抗体或其片段，其抗体重链可变区进一步包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链FR区。
在本发明一个优选的实施方案中，一种如上所述的鼠源抗体或其片段，其进一步包含鼠源IgG1,IgG2,IgG3,IgG4或其变体的重链恒定区。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体为嵌合抗体或其片段。
在本发明一个优选的实施方案中，一种如上所述的抗CD40嵌合抗体或其片段，其中所述的嵌合抗体轻链可变区序列为：SEQ ID NO:2或SEQ ID NO:10。
在本发明一个优选的实施方案中，一种如上所述的抗CD40嵌合抗体或其片段，其中所述的嵌合抗体重链可变区序列为：SEQ ID NO:1或SEQ ID NO:9。
在本发明一个优选的实施方案中，一种如上所述的抗CD40嵌合抗体或其片段，其进一步包含人源κ、λ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，一种如上所述的抗CD40嵌合抗体或其片段，其进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体为人抗体或其片段。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗体为人源化抗体或其片段。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人抗体或其抗原结合片段，其中所述的人抗体轻链序列为：SEQ ID NO:18或SEQ ID NO:20或其变体；所述的变体优选在轻链有0-10的氨基酸变化，更优选为氨基酸位点在2和3的突变，位点2和位点3突变后的氨基酸均优选为I、V或L。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人抗体或其抗原结合片段，其中所述的人抗体重链序列为：SEQ ID NO:17或SEQ ID NO:19或其变体；所述的变体优选在重链有0-10的氨基酸变化，更优选为氨基酸位点在6和8的突变，两个位点突变后的氨基酸均优选为I、A或L。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人抗体或其抗原结合片段，其进一步包含人IgG1,IgG2,IgG3或IgG4或其变体的恒定区，优选包含人IgG1、IgG2或IgG4恒定区。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其抗原结合片段，其人源化抗体轻链可变区进一步包含人源κ、λ链或其变体的轻链FR区。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其中所述人源化抗体轻链可变区上的轻链FR区序列，来源于如SEQ ID NO:22所示的人种系轻链IGkV1-33序列；或来源于如SEQ ID NO:24所示的人种系轻链IGkV2-28序列。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其中所述人源化抗体轻链可变区序列为如SEQ ID NO:33或SEQ ID NO:34所示的序列，或其变体。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其中所述人源化抗体轻链序列为如SEQ ID NO:18或SEQ ID NO:20所示的序列，或其变体。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其中所述人源化抗体轻链可变区变体优选在轻链可变区有0-10的氨基酸变化，更优选为氨基酸位点在2和3的突变，突变后的氨基酸均优选为I、V或L。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其进一步包含人源κ、λ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其人源化抗体重链可变区进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链FR区。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其中所述人源化抗体重链可变区上的重链FR区序列，来源于如SEQ ID NO:21所示的人种系重链IGHV1-69序列；或来源于如SEQ ID NO:23所示的人种系重链IGHV1-2序列。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其中所述人源化抗体重链可变区序列为如SEQ ID NO:26或SEQ ID NO:30所示的序列，或其变体。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其中所述人源化抗体重链序列为如SEQ ID NO:17或SEQ ID NO:19所示的序列，或其变体；所述变体优选在重链可变区有0-10的氨基酸变化，更优选为氨基酸位点在6和8的突变，突变后的氨基酸均优选为I、A或L。
在本发明一个优选的实施方案中，一种如上所述的抗CD40人源化抗体或其片段，其进一步包含人源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链FR区，更优选包含人源IgG1或IgG2重链FR区。
在本发明一个优选的实施方案中，一种如上所述的抗CD40抗体或其抗原结合片段，其中所述的抗原结合片段为Fab、Fv、sFv、F(ab’) 2、线性抗体、单链抗体、纳米抗体、结构域抗体和多特异性抗体。
本发明进一步提供一种编码如上所述的抗CD40抗体或其抗原结合片段、多特异性抗体或者单链抗体的DNA序列。
本发明进一步提供一种含有如上所述的DNA序列的表达载体。
本发明进一步提供一种用如上所述的表达载体转化的宿主细胞。
在本发明一个优选的实施方案中，一种如上所述的宿主细胞，其特征在于，所述的宿主细胞为细菌，优选为大肠杆菌。
在本发明一个优选的实施方案中，一种如上所述的宿主细胞为酵母菌，优选为毕赤酵母。
在本发明一个优选的实施方案中，一种如上所述的宿主细胞为哺乳动物细胞，优选为中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
本发明进一步提供一种单链抗体，其含有如上所述的抗CD40抗体或其抗原结合片段。
本发明进一步提供一种多特异性抗体，其含有如上所述的抗CD40抗体或其抗原结合片段。
本发明进一步提供一种抗体-药物偶联物，含有如前所述的抗CD40抗体轻链可变区和/或重链可变区。所述的抗体-药物偶联物是本领域公知的，其由抗体-接头-药物(毒素)相互连接形成，已知的接头包括裂解接头、***解接头，例如接头包括但不限于SMCC、SPDP等等；毒素也是本领域公知的，例如DM1、DM4、MMAE、MMAF等。
本发明进一步提供一种药物组合物，其含有如上所述的抗CD40抗体或其抗原结合片段、多特异性抗体或者单链抗体，以及可药用的赋形剂、稀释或载体。
本发明进一步提供一种如上所述的抗CD40抗体或其抗原结合片段、多特异性抗体、单链抗体或包含其的药物组合物，在制备用于治疗CD40或CD40L介导的疾病或病症的药物中的用途；其中所述的疾病优选为癌症；所述的癌症最优选为淋巴癌、乳腺癌、卵巢癌、***癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠直肠癌、膀胱癌、横纹肌肉瘤、食管癌、***、多发性骨髓瘤、白血病、胆囊癌、胶质母细胞瘤和黑色素瘤。
本发明进一步提供一种治疗和预防CD40或CD40L介导的疾病或病症的方法，该方法包括给予所需患者治疗有效量的如上所述的抗CD40抗体或其抗原结合片段、多特异性抗体、单链抗体或包含其的药物组合物；其中所述的疾病优选为癌症；所述的癌症最优选为淋巴癌、乳腺癌、卵巢癌、***癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠直肠癌、膀胱癌、横纹肌肉瘤、食管癌、***、多发性骨髓瘤、白血病、胆囊癌、胶质母细胞瘤和黑色素瘤。
本发明进一步提供一种如上所述的抗CD40抗体或其抗原结合片段、多特异性抗体、单链抗体或包含其的药物组合物，在制备用于改善自身免疫疾病患者的症状的药物的用途。
本发明进一步提供一种如上所述的抗CD40抗体或其抗原结合片段、多特异性抗体、单链抗体或包含其的药物组合物，在制备用于改善炎性疾病患者的症状的药物的用途。
附图说明
图1为鼠源抗人CD40抗体基于CD80活化分子对DC细胞的激活作用。
图2为鼠源抗人CD40抗体基于CD86活化分子对DC细胞的激活作用。
图3为与人PBMC和DC细胞共移植的Raji移植淋巴瘤的肿瘤生长曲线。
图4为与人PBMC和DC细胞共移植的Raji移植淋巴瘤的NOG小鼠的体重变化曲线。
发明详述
一、术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.Biol.Chem,243,p3558(1968)中所述。
本发明所述的术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM，IgD，IgG，IgA和IgE，其相应的重链分别为μ链，δ链，γ链，α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1，IgG2，IgG3，IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
在本发明中，本发明所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、2、3、4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序依次为：FR1、CDR1、FR2、CDR2、FR3、CDR3以及FR4。轻链的3个CDR区指LCDR1、LCDR2和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的IMGT编号规则。
术语“抗原呈递细胞”或“APC”是在其表面上展示与MHC复合的外来抗原的细胞。T细胞利用T细胞受体(TCR)识别这种复合物。APC的实例包括但不限于树突细胞(DC)、外用血单个核细胞(PBMC)、单核细胞、B淋巴母细胞和单核细胞衍生的树突细胞(DC)。术语“抗原呈递”是指APC捕获抗原和使它们能够被T细胞识别的过程，例如作为MHC-I/MHC-II偶联物的组分。
术语“CD40”是指为TNF受体超家族成员的细胞表面受体，又称为TNFRSF5。CD40普遍表达于树突细胞、B细胞和巨噬细胞表面，是产生和维持T细胞免疫所需的分子。术语“CD40”包括由细胞天然表达的CD40的任何变体或同种型。本发明的抗体可与得自非人物种的CD40交叉反应。作为另一种选择，该抗体也可以是人CD40特异性的，可不表现出与其他物种的交叉反应性。CD40或其任何变体或同种型可从天然表达它们的细 胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。优选地，抗CD40抗体靶向具有正常糖基化模式的人源CD40。
术语“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；(3)从重组组合人抗体文库中分离的抗体；以及(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人CD40的单克隆抗体。制备时用CD40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。在本发明一个优选的实施方案中，所述的鼠源CD40抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后***人载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1或IgG2重链恒定区。
术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。抗原结合片段实例包括但不限于：Fab、Fab′、F(ab′)2、Fv片段、线性抗体(linear antibody)、单链抗体、纳米抗体、结构域抗体和多特异性抗体。工程改造的抗体变体综述于Holliger和Hudson(2005)Nat.Biotechnol.23：1126-1136中。
“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。
“Fc”区含有包含抗体的CH1和CH2结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。
“Fab′片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′)2分子。
“F(ab′)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab′)2片段由通过两条重链间的二硫键保持在一起的两个Fab′片段组成。
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双抗体(diabodies)、双特异性双抗体和三抗体(triabodies)、己共价或非共价连接在一起的抗体片段等。
术语“抗体-药物偶联物”(ADC)指与一种或多种异源的化学合成分子，包括但不限于细胞毒剂偶联的抗体或抗体片段。
术语“单链抗体”是由抗体的重链可变区(VH)和轻链可变区(VL)通过一段连接肽连接而成的单链重组蛋白，它是具有完全抗原结合位点的最小抗体片段。
术语“结构域抗体片段”是仅含有重链可变区或轻链可变区链的具有免疫学功能的免疫球蛋白片段。在某些情况下，两个或多个VH区与肽接头共价连接以形成二价结构域 抗体片段。二价结构域抗体片段的两个VH区可靶向相同或不同抗原。
本发明的术语“与CD40结合”，指能与人CD40相互作用。本发明的术语“抗原结合位点”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
本发明所用的术语“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人CD40作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。
术语“交叉反应”是指本发明的抗体与来自不同物种的CD40结合的能力。例如，结合人CD40的本发明的抗体也可以结合另一物种的CD40。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达CD40的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。
术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。配体的抑制/阻断优选地降低或改变无抑制或阻断的情况下发生配体结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗CD40抗体接触时，与未与抗CD40抗体接触的配体相比，任何可测量的配体结合亲和力降低。
术语“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的剌激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指剌激特定的直接细胞杀伤机制。
本发明中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗 体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A，L234A，L235A；IgG2/4chimera，IgG4的F235E,或L234A/E235A突变。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，老鼠可以用人CD40或其片段免疫，所得到的抗体能被复性，纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。相应抗体的cDNA序列可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达***会导致抗体的糖基化，特别是在FC区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本发明的抗体指单克隆抗体。本发明所述的单克隆抗体(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是 通过诱导这类症状退化还是抑制这类症状发展到任何临床不可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本发明的实施方案(例如治疗方法或制品)在缓解每个患都有的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecμlar Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。氨基酸常见的保守取代如下：
Figure PCTCN2018089252-appb-000001
Figure PCTCN2018089252-appb-000002
整个说明书和权利要求书中使用的术语“基本上由……组成”或其变形表示包括所有所述元件或元件组，并且任选包括与所述元件类似或不同性质的其它元件，所述其它元件非显著改变指定给药方案、方法或组合物的基本性质或新性质。作为非限制性例子，基本上由所提及的氨基酸序列组成的结合化合物还可以包括一种或多种氨基酸，其不显著影响结合化合物的性质。
本发明所述的应用于某个对象的术语“天然存在的”是指这样的事实，即该对象可在自然界中发现。例如存在于可从自然界来源分离得到的生物体(包括病毒)、且未经人工在实验室中有意修饰的多肽序列或多核苷酸序列即是天然存在的。
“有效量”包含足以改善或预防医字病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指要据背景在生物、细胞或人体外产生的物质。“内源性”指根据背景在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该 事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
具体实施方式
以下结合实施例用于进一步描述本发明，但这些实施例并非限制着本发明的范围。本发明实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1 免疫抗原、筛选抗原的序列及制备
带his标签的人CD40(h-CD40-his)重组蛋白、带Fc标签的人CD40(h-CD40-Fc)重组蛋白、带his标签的小鼠CD40(m-CD40-his)重组蛋白和带his标签的恒河猴CD40(rhesus-CD40-his)重组蛋白(#CD0-C52H7)均为购买自Acrobiosystems公司的纯化商业蛋白实际，其各自的序列来源见表1。蛋白可用于下述各实施例实验中。
表1.重组蛋白氨基酸序列来源
名称 氨基酸序列起止 Genbank登录号
h-CD40-his Glu21-Arg193 AAH12419.1
h-CD40-Fc Glu21-Arg193 NP_001241.1
m-CD40-his Val24-Arg193 P27512
rhesus-CD40-his Glu21-Arg193 NP_001252791.1
实施例2 抗体杂交瘤的制备
抗人CD40单克隆抗体通过免疫小鼠产生。实验用C57BL/6小鼠，雌性，6～8周龄[昭衍(苏州)新药研究中心有限公司，动物生产许可证号：201503259]。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠分成2笼，每笼5只。
免疫抗原为带Fc标签的人修饰过的CD40重组蛋白(h-CD40-Fc，用磷酸盐缓冲液配制成1μg/μl)。用弗氏佐剂(Sigma，Lot No.：F5881/F5506)乳化：首次用弗氏完全佐剂(CFA)，其余加强免疫用核酸类佐剂(CpG,Sangon Biotech)和注射用铝(Imject Alum，Thermo，Lot No.：PH203866)。免疫注射时间为第0、14、28、42、56、70天。于第21， 35，49，63，77天采血进行血检，用ELISA方法检测小鼠血清，确定小鼠血清中的抗体滴度。
在第4次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合，融合前3天加强免疫，腹膜内(IP)注射10μg/只的磷酸盐缓冲液配制的抗原溶液。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2018089252-appb-000003
CRL-8287 TM)进行融合得到杂交瘤细胞，选出体外活性好的五株单克隆杂交瘤细胞株。
实施例3 ELISA结合实验
ELISA实验被用来检测抗CD40抗体的结合特性。用直接包被带his标签的CD40重组蛋白，抗体加入后，通过加入二抗(HRP偶联的抗一抗Fc的抗体)和HRP底物TMB检测抗体与抗原结合的活性。
人或恒河猴CD40-his蛋白包被96孔酶标板，按0.5μg/ml浓度每孔100μl，4℃孵育过夜。洗液洗三遍，每孔250μl。每次洗涤震荡10秒以保证充分清洗。加入200μl/孔封闭液室温孵育2小时。洗液洗三遍，每孔250μl。每次洗涤震荡10秒以保证充分清洗。每孔加100μl用稀释液稀释好的抗CD40待测抗体。室温孵育1小时。洗液洗三遍，每孔250μl。每孔加入100μl用稀释液按1:20000倍稀释的HRP标记的山羊抗人IgG二抗。室温孵育1小时。洗液洗三遍，每孔250μl。每孔加入100μl TMB，避光反应15分钟。加入50μl每孔的0.16M硫酸。Thermo MμltiSkanFc酶标仪读取450nm OD值，计算CD40抗体对CD40的结合EC50值。
表2.鼠源杂交瘤抗体对不同种系CD40的ELISA结合实验结果
Figure PCTCN2018089252-appb-000004
实施例4 抗CD40抗体阻断CD40和CD40L结合实验
本实验中，通过体外阻断实验，检测所筛选出来的抗人CD40抗体阻断人CD40和人CD40L结合。具体方法是将带Fc标签的CD40重组蛋白(h-CD40-Fc)包被到96孔酶标板上，加入抗CD40抗体充分结合占据表位后，再加入带his标签的CD40L，通过检测his标签，来计算CD40与CD40L的结合量，计算CD40抗体对CD40活性位点阻 断的IC50值。
人CD40-Fc蛋白包被96孔酶标板，按1μg/ml浓度每孔100μl，4℃孵育过夜。洗液洗三遍，每孔250μl。每次洗涤震荡10秒以保证充分清洗。加入200μl/孔封闭液室温孵育2小时。洗液洗三遍，每孔250μl。每次洗涤震荡10秒以保证充分清洗。每孔加100μl用稀释液稀释好的抗CD40待测抗体，室温孵育1小时。洗液洗三遍，每孔250μl。每孔加入100μl稀释好的CD40L-his，室温孵育1小时，洗液洗三遍。每孔加入100μl用稀释液按1:2000倍稀释的HRP标记的抗his标签的二抗。室温孵育1小时。洗液洗三遍，每孔250μl。每孔加入100μl TMB，避光反应15分钟。加入50μl每孔的0.16M硫酸。Thermo MμltiSkanFc酶标仪读取450nm OD值，计算CD40抗体对CD40与CD40L结合阻断的IC50值。
表3.人hCD40/hCD40L ELISA阻断实验结果
Figure PCTCN2018089252-appb-000005
实施例5 Biacore亲和力测定
按照人抗捕获试剂盒(Cat.#BR-1008-39，GE)说明书中的方法将人抗捕获抗体共价偶联于Biacore仪器(Biacore X100，GE)的生物传感芯片CM5上，从而亲和捕获一定量的待测嵌合或人源化抗体，然后于芯片表面流经一系列浓度梯度下的CD40抗原(CD40抗原均购自Acrobiosystems)，利用Biacore仪器(Biacore X100，GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用人抗捕获试剂盒里配置的再生溶液将生物芯片洗净再生。实验中用到的氨基偶联试剂盒购自GE公司(Cat.#BR-1000-50，GE)，缓冲液为HBS-EP+10×缓冲溶液(Cat.#BR-1006-69，GE)用D.I.Water稀释至1×(pH 7.4)。实验得到的数据用BiacoreX100evaluation software2.0GE软件以(1:1)Binding模型进行拟合，得出亲和力数值，见表10和表11。
实施例6 抗CD40抗体报告基因细胞活性实验
HEK-Blue CD40L细胞购自Invivogen(Cat#hkb-cd40)，该细胞稳定转染了人CD40基因和NF-kB介导的SEAP基因组，可以通过SEAP底物QUANTI-Blue检测上清中分泌的SEAP来表征CD40信号通路的活化水平。本实验通过检测细胞HEK-Blue CD40L的 活化，根据EC50大小评价CD40抗体的体外细胞活性。细胞HEK-Blue CD40L培养在含10％FBS，100μg/ml Zeocin和30μg/ml Blasticidin的DMEM培养基中，一周传代2～3次，传代比例1:5或1:10。传代时，吸掉培养基，用5ml 0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞。在96孔细胞培养板中加入100μL的细胞悬液，密度为5×10^5细胞/ml，培养基为含10％FBS，100μg/ml Zeocin和30μg/ml Blasticidin的DMEM，96孔板***只加入100μl无菌水。将培养板在培养箱培养24小时(37℃，5％CO 2)。细胞贴壁后，每孔加入100μl梯度稀释的待测抗体。将培养板在培养箱孵育20-24小时(37℃，5％CO 2)。每孔取40μl细胞上清到一个新的96孔平底板中，加入160μl QUANTI-Blue底物溶液，将培养板在培养箱内避光孵育1-3小时。用酶标仪(Thermo MμltiSkanFc)测定在620nm处的吸光度，计算EC50值评价CD40抗体的体外细胞活性。
表4 抗CD40抗体报告基因细胞活性实验结果
Figure PCTCN2018089252-appb-000006
实施例7 抗CD40抗体激活DC细胞实验
从正常人外周血中分离PBMC，然后用CD14 MACS beads分选单核细胞。加入含10ng/ml IL4和100ng/ml GM-CSF的RPMI 1640培液培养6天，进行MoDC细胞(单一核细胞来源的树突细胞)的诱导培养。6天后收集细胞，取1×10 5个细胞，CD209-PE，CD1a-PerCP/Cy5.5和CD14-PE/Cy7染色，FACS分析是否成功诱导MoDC(上述操作均为本领域的常规操作)。收集成功诱导的DC，加入各待测抗体及参照抗体抗体，分别设置相应梯度浓度稀释(抗体的梯度浓度详见图1)。培养48小时后，收集细胞，进行CD80，CD86和HLA-DR染色，FACS检测收集数据。根据数据通过激活原代DC细胞实验，五个鼠源抗体均显示了明显的活性，激活了DC细胞表面的活化分子CD80和CD86，并呈剂量依赖效应，总体效果与两株参照抗体(分别为Pfizer的CP-870,893和Alligator Bioscience的ADC-1013)相当或略优。见图1和图2。
实施例8 抗CD40抗体的克隆与定序
取以上筛选鉴定出的5株抗体的杂交瘤亚克隆，收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,15596-018)提取RNA(按照试剂盒说明书步骤)，反转录(PrimeScript TM Reverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增后送测序公司测序。最终得到5株鼠源抗体的序列。
鼠单抗2H6的重链和轻链可变区序列如下：
2H6HCVR
Figure PCTCN2018089252-appb-000007
2H6LCVR
Figure PCTCN2018089252-appb-000008
其含有下表5中的CDR序列：
表5
名称 序列 编号
HCDR1 GYAFSDYLIE SEQ ID NO：3
HCDR2 VINPGSGGSNYNEKIKD SEQ ID NO：4
HCDR3 GGGGFTY SEQ ID NO：5
LCDR1 RASQDISNYLN SEQ ID NO：6
LCDR2 FASRLHS SEQ ID NO：7
LCDR3 QQGSTLPWT SEQ ID NO：8
9E5的重链和轻链可变区序列如下：
9E5HCVR
Figure PCTCN2018089252-appb-000009
9E5LCVR
Figure PCTCN2018089252-appb-000010
Figure PCTCN2018089252-appb-000011
其含有下表6中的CDR序列：
表6
名称 序列 编号
HCDR1 GYILTTYWIT SEQ ID NO：11
HCDR2 DIHPGSGSTKYNEKFKS SEQ ID NO：12
HCDR3 RDY SEQ ID NO：13
LCDR1 RSSQNIVNSQGNTYLE SEQ ID NO：14
LCDR2 KVTNRFS SEQ ID NO：15
LCDR3 FQASLVPWT SEQ ID NO：16
1D9的重链和轻链可变区序列如下：
1D9HCVR
Figure PCTCN2018089252-appb-000012
1D9LCVR
Figure PCTCN2018089252-appb-000013
其含有下表7中的CDR序列：
表7
名称 序列 编号
HCDR1 GYAFTNYLIN SEQ ID NO：39
HCDR2 ILNPGSGGTNYNENFKD SEQ ID NO：40
HCDR3 GSPGFAY SEQ ID NO：41
LCDR1 RASQDINIYLN SEQ ID NO：42
LCDR2 STSGLHS SEQ ID NO：43
LCDR3 QQGYTLPYT SEQ ID NO：44
14C10的重链和轻链可变区序列如下：
14C10HCVR
Figure PCTCN2018089252-appb-000014
Figure PCTCN2018089252-appb-000015
14C10LCVR
Figure PCTCN2018089252-appb-000016
其含有下表8中的CDR序列：
表8
名称 序列 编号
HCDR1 GYAFTNYLIE SEQ ID NO：47
HCDR2 VINPEFGGTNYNEKFKG SEQ ID NO：48
HCDR3 GGGGFTY SEQ ID NO：49
LCDR1 RASQDISSHLN SEQ ID NO：50
LCDR2 YTSRLHS SEQ ID NO：51
LCDR3 QQGNTLPWT SEQ ID NO：52
38B4的重链和轻链可变区序列如下：
38B4HCVR
Figure PCTCN2018089252-appb-000017
38B4LCVR
Figure PCTCN2018089252-appb-000018
其含有下表9中的CDR序列：
表9
名称 序列 编号
HCDR1 GYTFTDYYIN SEQ ID NO：55
HCDR2 GIYPGTGNTYYNEKFKG SEQ ID NO：56
HCDR3 RGLPSLCFDY SEQ ID NO：57
LCDR1 SASQGISNYLN SEQ ID NO：58
LCDR2 YTSSLHS SEQ ID NO：59
LCDR3 QQYSKLPPT SEQ ID NO：60
其中最优两株抗体2H6和9E5进入后续开发。将获得的可变区序列分别接上人的抗体IgG1恒定区序列，得到人-鼠嵌合的抗体序列，利用分子克隆技术，把嵌合抗体的序列***到pCP表达载体中(购自迈博斯生物公司)中，经过PCR后测序鉴定(该部分的分子克隆等分子生物学操作方法按常规操作条件进行，具体可以参照《分子克隆：实验室手册》)，利用HEK293细胞表达***，即可获得人-鼠嵌合抗体2H6-C和9E5-C。对经过MabSelect SuRe(GE Lifesciences)亲和力层析纯化后的嵌合抗体进行各项体外活性检测，数据见表10。
表10.嵌合抗体的体外活性
Figure PCTCN2018089252-appb-000019
实施例9 小鼠抗体人源化实验
在所获得的鼠源抗体2H6和9E5的VH/VLCDR典型结构的基础上，将重、轻链可变区序列与抗体Germline数据库比较，获得同源性高的人种系模板。其中人类种系轻链框架区来自人κ轻链基因，本发明抗体优选人种系轻链模版Vk1-33/JK4(2H6)或Vk2-28/JK4(9E5)。人类种系重链框架区来自人重链，本发明抗体优选人种系重链模版VH1-69/JH6(2H6)或VH1-2/JH6(9E5)，如下所示：
2H6优选人种系重链模版IGHV1-69(SEQ ID NO：21):
Figure PCTCN2018089252-appb-000020
2H6优选人种系轻链模板IGkV1-33(SEQ ID NO：22)：
Figure PCTCN2018089252-appb-000021
9E5优选人种系重链模版IGHV1-2(SEQ ID NO：23):
Figure PCTCN2018089252-appb-000022
9E5优选人种系轻链模板IGkV2-28(SEQ ID NO：24)：
Figure PCTCN2018089252-appb-000023
将鼠源抗体的CDR区移植到选择的人源化模板上，替换人源化可变区，再与相应的人源IgG恒定区(优选重链为IgG1，轻链为κ)重组。然后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，并对CDR区化学不稳定氨基酸残基优化，得到最终的人源化分子。其重链可变区序列如SEQ ID NO:25-30所示；轻链可变区序列如SEQ ID NO:31-36所示。
hu2H6-H1a(SEQ ID NO:25)：
Figure PCTCN2018089252-appb-000024
hu2H6-H1b(SEQ ID NO:26)：
Figure PCTCN2018089252-appb-000025
hu2H6-H1c(SEQ ID NO:27)：
Figure PCTCN2018089252-appb-000026
hu9E5-H1a(SEQ ID NO:28)：
Figure PCTCN2018089252-appb-000027
hu9E5-H1b(SEQ ID NO:29)：
Figure PCTCN2018089252-appb-000028
hu9E5-H1c(SEQ ID NO:30)：
Figure PCTCN2018089252-appb-000029
hu2H6-L1a(SEQ ID NO:31)：
Figure PCTCN2018089252-appb-000030
hu2H6-L1b(SEQ ID NO:32)：
Figure PCTCN2018089252-appb-000031
hu2H6-L1c(SEQ ID NO:33)：
Figure PCTCN2018089252-appb-000032
hu9E5-L1a(SEQ ID NO:34)：
Figure PCTCN2018089252-appb-000033
hu9E5-L1b(SEQ ID NO:35)：
Figure PCTCN2018089252-appb-000034
hu9E5-L1c(SEQ ID NO:36)：
Figure PCTCN2018089252-appb-000035
经由以上轻重链组合的表达测试和回复突变数量对比，选择出最终的人源化hu2H6(使用H1b重链和L1c轻链)和hu9E5抗体分子(使用H1c重链和L1a轻链)，其各自的完整轻重链序列如SEQ ID NO:17-20所示。
hu2H6HC
Figure PCTCN2018089252-appb-000036
Figure PCTCN2018089252-appb-000037
hu2H6LC
Figure PCTCN2018089252-appb-000038
hu9E5HC
Figure PCTCN2018089252-appb-000039
hu9E5LC
Figure PCTCN2018089252-appb-000040
实施例10 人源化抗体测试数据
本发明人源化抗体hu2H6和hu9E5与人，恒河猴CD40的结合活性、阻断活性等，见表11。结果表明，本发明人源化抗人CD40抗体的ELISA结合、阻断活性与阳性抗体pfizer/alligator相当，尤其是，hu9E5和人CD40的Biacore测量亲和力是阳性抗体Alligator 参照的10倍以上、Pfizer参照的4倍以上。
表11 人源化hu2H6和hu9E5抗体的体外活性
Figure PCTCN2018089252-appb-000041
实施例11 抗CD40抗体对小鼠肿瘤生长的抑制
取正常人外周血，用密度梯度离心法分离健康人PBMC。用CD14+microbeads试剂盒分选单核细胞，按照试剂盒所提供的protocol进行CD14+单核细胞的分离，即每10 7个细胞加入20μl Anti-CD14microbeads，4℃孵育15分钟。然后，将细胞加入磁柱中，洗三次后，收集磁柱中的细胞，即CD14+单核细胞。CD14+单核细胞中加入含10ng/ml IL4和100ng/ml GM-CSF的RPMI 1640培养液培养6天(培养方法为本领域常规方法)，进行MoDC细胞的诱导培养，剩余的细胞加入含IL-2的RPMI 1640，培养后收集悬浮的细胞(培养方法以及收集细胞的方法均为本领域的常规方法)，使用CD3+microbeads试剂盒分选T细胞。六天后收集并分取MoDC细胞和CD3+T细胞，将他们与Raji细胞(上海生科院细胞库，培养于含10％胎牛血清的RPMI1640培养液中)按1:5:20的比例混合，接种于每只NOG小鼠(南京银河生物，适应性饲养5天)皮下。实验动物均饲养于恒温恒湿的独立通风盒内，饲养室温度18.0-26.0℃，湿度40-70％，10-20次/小时换气，昼夜明暗交替时间12h/12h。
实验分为人IgG1抗体对照组，hu2H6，、hu9E5和参照抗体G12，给药剂量均为3mg/kg。每组5只小鼠，每周一次，六周注射给药，连续给药3次。整个实验过程中，用游标卡尺每周测量2次肿瘤长径和宽径，肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)计算。相对肿瘤抑制率TGI(％)：TGI％＝(1-T/C)×100％。T/C％为相对肿瘤增殖率，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。T和C分别为治疗组和IgG1对照组在某一特定时间点的肿瘤体积(TV)或瘤重(TW)。
结果显示，人源化的抗CD40抗体hu2H6和hu9E5相对IgG1对照均有非常显著的抑瘤作用，给药21天后肿瘤接近完全消除；对比参照抗体G12的抑瘤作用总体相当或稍好，如图3和图4所示。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (26)

  1. 一种抗CD40抗体或其抗原结合片段，其包含：
    抗体轻链可变区，所述的抗体轻链可变区包含至少1个选自如以下序列所示的LCDR：SEQ ID NO:6，SEQ ID NO:7，SEQ ID NO:8；SEQ ID NO:14，SEQ ID NO:15，SEQ ID NO:16；SEQ ID NO:42，SEQ ID NO:43，SEQ ID NO:44；SEQ ID NO:50，SEQ ID NO:51，SEQ ID NO:52；SEQ ID NO:58，SEQ ID NO:59或SEQ ID NO:60；和/或
    抗体重链可变区，所述的抗体重链可变区包含至少1个选自如以下序列所述的HCDR：SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5；SEQ ID NO:11，SEQ ID NO:12，SEQ ID NO:13；SEQ ID NO:39，SEQ ID NO:40，SEQ ID NO:41；SEQ ID NO:47，SEQ ID NO:48，SEQ ID NO:49；SEQ ID NO:55、SEQ ID NO:56或SEQ ID NO:57。
  2. 如权利要求1所述的抗CD40抗体或其抗原结合片段，所述的LCDR包括LCDR1、LCDR2和LCDR3中的一种或多种，所述LCDR1的氨基酸序列如序列表中SEQ ID NO:6、SEQ ID NO:14、SEQ ID NO.42、SEQ ID NO.50或者SEQ ID NO.58所示；所述LCDR2的氨基酸序列如序列表中SEQ ID NO:7、SEQ ID NO:15、SEQ ID NO.43、SEQ ID NO.51或者SEQ ID NO.59；所述LCDR3的氨基酸序列如序列表中SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO.44、SEQ ID NO.52或者SEQ ID NO.60所示；
    和/或，所述HCDR包括HCDR1、HCDR2和HCDR3中的一种或多种，所述HCDR1的氨基酸序列如序列表中SEQ ID NO:3、SEQ ID NO:11、SEQ ID NO.39、SEQ ID NO.47或者SEQ ID NO.55所示；所述HCDR2的氨基酸序列如序列表中SEQ ID NO:4、SEQ ID NO:12、SEQ ID NO.40、SEQ ID NO.48或者SEQ ID NO.56所示；所述HCDR3的氨基酸序列如序列表中SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO.41、SEQ ID NO.49或者SEQ ID NO.57所示。
  3. 如权利要求2所述的抗CD40抗体或其抗原结合片段，其中所述的抗体轻链可变区包含序列分别如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3，或者包含序列分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3，或者包含序列分别如SEQ ID NO:42、SEQ ID NO:43和SEQ ID NO:44所示的LCDR1、LCDR2和LCDR3，或者包含序列分别如SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的LCDR1、LCDR2和LCDR3，或者包含分别如SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:60所示的LCDR1、LCDR2和LCDR3；
    和/或，所述的抗体重链可变区包含序列分别如SEQ ID NO:3、SEQ ID NO:4和SEQ ID  NO:5所示的HCDR1、HCDR2和HCDR3，或者包含序列分别如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3，或者包含序列分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示的HCDR1、HCDR2和HCDR3，或者包含序列分别如SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的HCDR1、HCDR2和HCDR，或者包含序列分别如SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57所示的HCDR1、HCDR2和HCDR3。
  4. 如权利要求3所述的抗CD40抗体或其抗原结合片段，其中所述的抗体轻链可变区包含序列分别如：SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；且抗体重链可变区包含分别如：SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3；
    或者，其中所述的抗体轻链可变区包含分别如：SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的LCDR1、LCDR2和LCDR3；
    或者，其中所述的抗体轻链可变区包含序列分别如：SEQ ID NO:42、SEQ ID NO:43和SEQ ID NO:44所示的LCDR1、LCDR2和LCDR3；且抗体重链可变区包含序列分别如：SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示的HCDR1、HCDR2和HCDR3；
    或者，其中所述的抗体轻链可变区包含序列分别如：SEQ ID NO:50、SEQ ID NO:51和SEQ ID NO:52所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:49所示的HCDR1、HCDR2和HCDR；
    或者，其中所述的抗体轻链可变区包含序列分别如：SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:60所示的LCDR1、LCDR2和LCDR3；抗体重链可变区包含分别如：SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57所示的HCDR1、HCDR2和HCDR3。
  5. 如权利要求1-4任一项所述的抗CD40抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为鼠源抗体或者嵌合抗体。
  6. 如权利要求5所述的抗CD40抗体或其抗原结合片段，其中所述鼠源抗体或者所述嵌合抗体的重链可变区的氨基酸序列如SEQ ID NO.1所示，轻链可变区的氨基酸序列如SEQ ID NO.2所示；
    或者，重链可变区的氨基酸序列如SEQ ID NO.9所示，轻链可变区的氨基酸序列如SEQ ID NO.10所示；
    或者，重链可变区的氨基酸序列如SEQ ID NO.37所示，轻链可变区的氨基酸序列如SEQ ID NO.38所示；
    或者，重链可变区的氨基酸序列如SEQ ID NO.45所示，轻链可变区的氨基酸序列如SEQ ID NO.46所示；
    或者，重链可变区的氨基酸序列如SEQ ID NO.53所示，轻链可变区的氨基酸序列如SEQ ID NO.54所示。
  7. 如权利要求1-4任一项所述的抗CD40抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为人源化抗体或者人抗体。
  8. 如权利要求7所述的抗CD40抗体或其抗原结合片段，其中所述人源化抗体轻链可变区上的轻链FR区序列，来源于如SEQ ID NO:22所示的人种系轻链IGkV1-33序列；或来源于如SEQ ID NO:24所示的人种系轻链IGkV2-28序列。
  9. 如权利要求7所述的抗CD40抗体或其抗原结合片段，其中所述人源化抗体轻链序列为如SEQ ID NO:18或SEQ ID NO:20所示的序列或其变体；所述的变体优选在轻链可变区有0-10的氨基酸变化，更优选为氨基酸位点在2和3的突变，突变后的氨基酸优选为I、V或L。
  10. 如权利要求7～9任一项所述的抗CD40抗体或其抗原结合片段，其中所述人源化抗体重链可变区进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链FR区，优选包含人源IgG1、IgG2或IgG4重链FR区，更优选包含人源IgG1或IgG2重链FR区。
  11. 如权利要求10所述的抗CD40抗体或其抗原结合片段，其中所述人源化抗体重链可变区上的重链FR区序列，来源于如SEQ ID NO:21所示的人种系重链IGHV1-69序列；或来源于如SEQ ID NO:23所示的人种系重链IGHV1-2序列。
  12. 如权利要求7～11任一项所述的抗CD40抗体或其抗原结合片段，其中所述人源化抗体重链序列为如SEQ ID NO:17或SEQ ID NO:19所示的序列或其变体；所述变体优选在重链可变区有0-10的氨基酸变化，更优选为氨基酸位点在6和8的突变，突变后的氨基酸优选为I、A或L。
  13. 如权利要求7～12任一项所述的抗CD40抗体或其抗原结合片段，其中所述人源化抗体为人源化抗体hu9E5或人源化抗体hu2H6，其中所述的人源化抗体可变区序列如下：
    人源化抗体hu2H6：重链可变区序列为SEQ ID NO：26；轻链可变区序列为SEQ ID NO：33；
    人源化抗体hu9E5：重链可变区序列为SEQ ID NO：30，轻链可变区序列为SEQ ID NO：34。
  14. 根据权利要求13所述的抗CD40抗体或其抗原结合片段，其中所述人源化抗体 hu9E5包含重链抗体序列SEQ ID NO:19，和轻链抗体序列SEQ ID NO:20；其中所述人源化抗体hu2H6包含重链抗体序列SEQ ID NO:17，和轻链抗体序列SEQ ID NO:18。
  15. 一种多特异性抗体，含有如权利要求1-14任一项所述的抗CD40抗体或其抗原结合片段的轻链可变区和/或重链可变区。
  16. 一种单链抗体，含有如权利要求1-14任一项所述的抗CD40抗体或其抗原结合片段的轻链可变区和/或重链可变区。
  17. 一种抗体-药物偶联物，其中所述的抗体含有如权利要求1-14任一项所述的抗CD40抗体或其抗原结合片段的轻链可变区和/或重链可变区；较佳地含有如权利要求1-15任一项所述的抗CD40抗体或其抗原结合片段。
  18. 一种编码如权利要求1-14任一项所述的抗CD40抗体或抗原结合片段、权利要求16所述的多特异性抗体或者权利要求17所述的单链抗体的DNA序列。
  19. 一种含有如权利要求18所述的DNA序列的表达载体。
  20. 一种用如权利要求19所述的表达载体转化的宿主细胞。
  21. 如权利要求20所述的宿主细胞，其特征在于，所述的宿主细胞为细菌，所述的细菌优选为大肠杆菌；或者为酵母菌，所述的酵母菌优选为毕赤酵母；或者为哺乳动物细胞，所述的哺乳动物细胞优选为中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
  22. 一种药物组合物，其含有如权利要求1-14任一项所述的抗CD40抗体或其抗原结合片段、权利要求15所述的多特异性抗体、权利要求16所述的单链抗体或者权利要求17所述的抗体-药物偶联物，以及可药用的赋形剂、稀释剂或载体。
  23. 如权利要求1-14任一项所述的抗CD40抗体或其抗原结合片段、权利要求15所述的多特异性抗体、权利要求16所述的单链抗体、权利要求17所述的抗体-药物偶联物或者权利要求22所述的药物组合物，在制备用于治疗或预防CD40或CD40L介导的疾病或病症的药物中的用途；其中所述的疾病优选为癌症；所述的癌症最优选为淋巴癌、乳腺癌、卵巢癌、***癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠直肠癌、膀胱癌、横纹肌肉瘤、食管癌、***、多发性骨髓瘤、白血病、胆囊癌、胶质母细胞瘤和黑色素瘤。
  24. 一种治疗和预防CD40或CD40L介导的疾病或病症的方法，该方法包括给予所需患者治疗有效量的如权利要求1-14任一项所述的抗CD40抗体或其抗原结合片段、权利要求15所述的多特异性抗体、权利要求16所述的单链抗体、权利要求17所述的抗体-药物偶联物或者权利要求23所述的药物组合物，或如权利要求22所述的药物组合物， 其中所述的疾病优选为癌症；所述的癌症最优选为淋巴癌、乳腺癌、卵巢癌、***癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠直肠癌、膀胱癌、横纹肌肉瘤、食管癌、***、多发性骨髓瘤、白血病、胆囊癌、胶质母细胞瘤和黑色素瘤。
  25. 如权利要求1-14任一项所述的抗CD40抗体或其抗原结合片段、权利要求15所述的多特异性抗体、权利要求16所述的单链抗体、权利要求17所述的抗体-药物偶联物或者权利要求22所述的药物组合物，在制备用于改善自身免疫疾病患者的症状的药物的用途。
  26. 如权利要求1-14任一项所述的抗CD40抗体或其抗原结合片段、权利要求15所述的多特异性抗体、权利要求16所述的单链抗体、权利要求17所述的抗体-药物偶联物或者权利要求22所述的药物组合物，在制备用于改善炎性疾病患者的症状的药物的用途。
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BR112019025048A2 (pt) 2020-06-30
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US20200148778A1 (en) 2020-05-14
CN110267989B (zh) 2023-04-04
CN110267989A (zh) 2019-09-20
AU2018278051A1 (en) 2020-01-30
EP3632932A1 (en) 2020-04-08
CA3064298A1 (en) 2018-12-06
JP7257971B6 (ja) 2023-07-24
JP2020521504A (ja) 2020-07-27
TW201902925A (zh) 2019-01-16
JP7257971B2 (ja) 2023-04-14
US11525005B2 (en) 2022-12-13
KR20200012920A (ko) 2020-02-05

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