WO2020108611A1 - 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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WO2020108611A1
WO2020108611A1 PCT/CN2019/121941 CN2019121941W WO2020108611A1 WO 2020108611 A1 WO2020108611 A1 WO 2020108611A1 CN 2019121941 W CN2019121941 W CN 2019121941W WO 2020108611 A1 WO2020108611 A1 WO 2020108611A1
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seq
antibody
variable region
heavy chain
light chain
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PCT/CN2019/121941
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English (en)
French (fr)
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廖成
蒋家骅
徐祖朋
张连山
林�源
林侃
钱雪明
滕菲
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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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Priority to CA3120793A priority Critical patent/CA3120793A1/en
Priority to BR112021009835-6A priority patent/BR112021009835A2/pt
Priority to US17/296,519 priority patent/US20220025060A1/en
Priority to JP2021528960A priority patent/JP2022509156A/ja
Priority to KR1020217019296A priority patent/KR20210099027A/ko
Priority to EP19891086.1A priority patent/EP3892634A4/en
Priority to AU2019389354A priority patent/AU2019389354A1/en
Priority to CN201980072271.9A priority patent/CN112969714B/zh
Priority to MX2021005823A priority patent/MX2021005823A/es
Publication of WO2020108611A1 publication Critical patent/WO2020108611A1/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
    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
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    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/75—Agonist effect on antigen
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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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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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

Definitions

  • the present disclosure relates to an anti-CD40 antibody comprising a mutation in a heavy chain constant region, an antigen-binding fragment thereof, a chimeric antibody comprising the CDR region of the anti-CD40 antibody, a humanized antibody, and a human anti-CD40 antibody or antigen binding thereof Fragmented pharmaceutical composition and its use as an anti-cancer drug.
  • Tumor immunotherapy is a hot spot in the field of tumor therapy, in which T cell tumor immunotherapy is at its core position. Tumor immunotherapy makes full use of and mobilizes killer T cells in tumor patients to kill tumors. Tumor immunotherapy may be one of the most effective and safest ways to treat tumors. Tumor immunotherapy currently has good prospects for treating several different types of cancer, including disseminated metastatic tumors.
  • T cells adopts a dual signal pathway system: MHC-antigen peptides are presented to T cells through antigen presenting cells (APC), thereby providing a first signal; a series of co-stimulatory molecules are needed to provide a second signal, and T cells produce a normal immune response.
  • APC antigen presenting cells
  • This dual-signal pathway system plays a vital role in the balance of the body's immune system. It strictly regulates the body's immune response to self and non-self antigens. If the second signal provided by the costimulatory molecule is lacking, it will lead to a non-response or sustained specific immune response of the T cells, resulting in tolerance. Therefore, the second signaling pathway plays a very critical 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 about 48 kDa. It belongs to the tumor necrosis factor receptor (TNFR) superfamily and plays an important role in the immune system.
  • CD40 is expressed in a variety of immune cells, such as B cells, dendritic cells, monocytes, and macrophages. When signaling occurs through CD40, specialized antigen-presenting cells are activated.
  • the natural ligand for CD40 is named CD154 or CD40L and is known to be mainly expressed in mature T lymphocytes.
  • CD40L-mediated signaling triggers some cellular biological events, including immune cell activation, proliferation, and the production of cytokines and chemokines.
  • CD40 signaling is very important for T cell-dependent immune responses, especially in the context of tumor environments. CD40-stimulated dendritic cells can activate tumor-specific effector T cells, which have the potential to eradicate tumor cells.
  • CD40 expression occurs in many normal cells and tumor cells including B lymphocytes.
  • melanoma is a tumor with CD40 expression
  • 30% to 70% of solid tumors also have CD40 expression.
  • CD40 can effectively trigger an anti-tumor response (Tong et al. Cancer Gene Therapy, 2003, 10:1-13), including immune activation of tumor-specific T cell responses and direct apoptosis of CD40 positive tumors
  • the effects, as well as the stimulation led to the humoral response of ADCC, and a strong correlation between tumor eradication and the appearance of tumor-specific cytotoxic T lymphocytes was observed.
  • CD40 antibodies are associated with side effects, such as shock syndrome and cytokine release syndrome (van Mierlo et al., Proc. Nat1. Acad. Sci. USA, 2002, 99:5561-5566) .
  • WO2006019447, WO2014145806, US8734791, US9657106, US8084582, WO2008150494, WO2004099249 disclose mutations of S267E, L328F, N325S of antibody heavy chains, which caused the antibody to remove the binding to Fc ⁇ RIII, but enhanced the binding to Fc ⁇ RIIB Binding, thereby removing antibody-dependent cytotoxicity (ADCC), but enhancing Fc ⁇ RIIB-mediated cross-linking, thereby enhancing CD40 activation and enhancing antigen presentation by dendritic cells.
  • ADCC antibody-dependent cytotoxicity
  • the present disclosure aims to provide an anti-CD40 antibody with high affinity, high selectivity, and high biological activity that lacks antibody-dependent cytotoxicity (ADCC), but has enhanced Fc ⁇ RIIB-mediated crosslinking, thereby inhibiting tumor growth in vivo .
  • the antibodies of the present disclosure can be used as drugs or compositions for the treatment of cancer mediated by stimulation of CD40 and its pathway.
  • the present disclosure provides a CD40 antibody or antigen-binding fragment thereof, which comprises:
  • Antibody light chain variable region comprises at least one LCDR selected from the following sequence: 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
  • Antibody heavy chain variable region comprises at least one HCDR selected from the following sequence: 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.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO. 42, LCDR1 shown in SEQ ID NO.50 or SEQ ID NO.58.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO. 43, LCDR2 shown in SEQ ID NO.51 or SEQ ID NO.59.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO. 44, LCDR3 area shown in SEQ ID NO.52 or SEQ ID NO.60.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the antibody heavy chain variable region comprises SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO. 39, HCDR1 shown in SEQ ID NO. 47 or SEQ ID NO. 55.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above wherein the antibody heavy chain variable region comprises, for example, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO. 40, HCDR2 shown in SEQ ID NO. 48 or SEQ ID NO. 56.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above the antibody light chain variable region comprises SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively The LCDR1, LCDR2, and LCDR3 shown.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above the antibody light chain variable region comprising SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively The LCDR1, LCDR2, and LCDR3 shown.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above the antibody light chain variable region comprising SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44 respectively The LCDR1, LCDR2, and LCDR3 shown.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above the light chain variable region of the antibody comprises SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively The LCDR1, LCDR2, and LCDR3 shown.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the antibody heavy chain variable region comprises SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively HCDR1, HCDR2 and HCDR3 shown.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the antibody heavy chain variable region comprises SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 respectively HCDR1, HCDR2 and HCDR3 shown.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the antibody heavy chain variable region comprises SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, respectively HCDR1, HCDR2 and HCDR3 shown.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the antibody heavy chain variable region comprises SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, respectively HCDR1, HCDR2 and HCDR3 shown.
  • the anti-CD40 antibody or antigen-binding fragment thereof, wherein the light chain variable region of the antibody comprises, for example:
  • LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; or
  • LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; or
  • LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44; or
  • LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52; or
  • LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60;
  • variable region of the heavy chain of the antibody comprises as follows:
  • HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; or
  • HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; or
  • HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41; or
  • HCDR1, HCDR2 and HCDR as shown in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49; or
  • HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57.
  • the anti-CD40 antibody or antigen-binding fragment thereof may be selected from any of the following:
  • Antibody light chain variable region contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; antibody heavy chain variable region contains SEQ HCDR1, HCDR2 and HCDR3 shown in ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
  • the antibody light chain variable region contains 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 contains SEQ LCDR1, LCDR2 and LCDR3 shown in ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
  • the antibody light chain variable region contains 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 contains sequences such as: HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.
  • Antibody light chain variable regions contain sequences such as: SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 52; antibody heavy chain variable regions contain respectively: HCDR1, HCDR2 and HCDR shown in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49.
  • the antibody light chain variable region contains sequences such as: SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60 as shown in LCDR1, LCDR2 and LCDR3; the antibody heavy chain variable region contains as follows: HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57.
  • the antibody light chain variable region sequence is selected from SEQ ID NO: 2 or SEQ ID NO: 10; the heavy chain variable region sequence is selected from SEQ ID NO: 1 or SEQ ID NO: 9.
  • the aforementioned 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 shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2. Show.
  • amino acid sequence of the heavy chain variable region of the murine antibody or the chimeric antibody is shown in SEQ ID NO. 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10. As shown.
  • the light chain variable region LCVR of the antibody or antigen fragment has the sequence SEQ ID NO: 38
  • the heavy chain variable region HCVR has the sequence SEQ ID NO: 37.
  • the light chain variable region LCVR of the antibody or antigen fragment has the sequence SEQ ID NO: 46
  • the heavy chain variable region HCVR has the sequence SEQ ID NO: 45.
  • the light chain variable region LCVR of the antibody or antigen fragment has the sequence SEQ ID NO: 54
  • the heavy chain variable region HCVR has the sequence SEQ ID NO: 53.
  • the anti-CD40 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, or a fragment thereof.
  • the light chain variable region of the antibody when the anti-CD40 antibody or antigen-binding fragment thereof is a murine antibody or a fragment thereof, the light chain variable region of the antibody further comprises a light chain FR region of a murine kappa, lambda chain or variant thereof Or the light chain constant region, and/or the antibody heavy chain variable region thereof further comprises a heavy chain FR region or a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the anti-CD40 antibody or antigen-binding fragment thereof when it is a chimeric antibody or fragment thereof, it comprises a light chain constant region of human ⁇ , ⁇ chain or a variant thereof, and/or comprises human origin The heavy chain constant region of IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
  • the light chain variable region sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 10
  • the heavy chain variable region sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 9 .
  • the antibody light chain sequence is: SEQ ID NO: 18 or SEQ ID NO: 20 or a variant thereof;
  • the specific variant has 0-10 amino acid changes in the light chain, more specifically the mutations at the amino acid positions 2 and 3, and the amino acids after the position 2 and position 3 mutations are independently selected from I, V or L;
  • the variant specifically has 0-10 amino acid changes in the heavy chain, more specifically the 6th and 8th positions Mutation of amino acid positions.
  • the amino acids after mutation at two positions are independently selected from I, A or L.
  • an anti-CD40 humanized antibody or fragment thereof as described above the humanized antibody heavy chain variable region further comprises the weight of human IgG1, IgG2, IgG3, IgG4, or a variant thereof Chain constant region or FR region, specifically comprising human IgG1, IgG2 or IgG4 heavy chain constant region or FR region, more specifically human IgG1 or IgG2 heavy chain constant region or FR region, and/or human ⁇ , ⁇ chain Or a light chain FR region thereof.
  • an anti-CD40 humanized antibody or fragment thereof as described above, wherein the light chain FR region sequence on the light chain variable region of the humanized antibody is derived from SEQ ID NO: 22 is the human germline light chain IGkV1-33 sequence; or derived from the human germline light chain IGkV2-28 sequence shown in SEQ ID NO:24.
  • an anti-CD40 humanized antibody or fragment thereof as described above, wherein the humanized antibody light chain variable region variant specifically has 0-10 amino acids in the light chain variable region The changes are more specifically mutations at amino acid positions 2 and 3.
  • the amino acids after mutation are specifically I, V or L.
  • an anti-CD40 humanized antibody or fragment thereof as described above further comprises a light chain constant region of a human kappa, lambda chain, or variant thereof.
  • an anti-CD40 humanized antibody or fragment thereof as described above wherein the sequence of the heavy chain FR region on the heavy chain variable region of the humanized antibody is derived from SEQ ID NO:
  • the anti-CD40 humanized antibody or fragment thereof, the heavy chain variable region is selected from the sequence shown in one of SEQ ID NO: 25-30 or a variant thereof, and the light chain variable region is selected from SEQ ID NO: The sequence shown in one of 31-36 or a variant thereof.
  • the heavy chain variable region is the sequence shown in SEQ ID NO: 26 or a variant thereof, and the light chain variable region is selected from SEQ ID NO: 33. Shown sequence or a variant thereof.
  • the heavy chain variable region is the sequence shown in SEQ ID NO: 30 or a variant thereof, and the light chain variable region is selected from the sequence shown in SEQ ID NO: 34 or a variant thereof.
  • the heavy chain sequence of the humanized anti-CD40 antibody is shown in SEQ ID NO: 17, and the light chain sequence is shown in SEQ ID NO: 18.
  • the heavy chain sequence is shown in SEQ ID NO: 19, and the light chain sequence is shown in SEQ ID NO: 20.
  • the anti-CD40 humanized antibody or fragment thereof wherein the humanized antibody heavy chain sequence is the sequence shown in SEQ ID NO: 61, 62, 63, 64, or 67 or a variant thereof
  • the light chain variable region is the sequence shown in SEQ ID NO: 18, 20 or a variant thereof.
  • the heavy chain sequence of the anti-CD40 humanized antibody or fragment thereof is shown in SEQ ID NO: 61 or 62 or its variant, and the light chain sequence is shown in SEQ ID NO: 18 or its variant
  • the heavy chain sequence is shown in SEQ ID NO: 63, 64, or 67 or a variant thereof, and the light chain sequence is shown in SEQ ID NO: 20 or a variant thereof.
  • the variant has 0-10 amino acid changes in the variable region of the heavy chain, more specifically mutations at amino acid positions 6 and 8, and the amino acids after mutation are specifically I, A or L.
  • SEQ ID NO: 61 contains the amino acid residue at position 266 relative to SEQ ID NO: 17 mutated to glutamic acid (E) (eg S266E);
  • the SEQ ID NO: 62 contains a mutation of amino acid residue 266 relative to SEQ ID NO: 17 to glutamic acid (E) (eg S266E), and a mutation of amino acid residue 324 to serine (S) (eg N324S ), the amino acid residue at position 327 is mutated to phenylalanine (F) (eg L327F);
  • the SEQ ID NO: 63 contains the amino acid residue at position 262 relative to SEQ ID NO: 19 which is mutated to glutamic acid (E) (eg S262E);
  • the SEQ ID NO: 64 contains a mutation of amino acid residue 262 relative to SEQ ID NO: 19 to glutamic acid (E) (eg S262E), and a mutation of amino acid residue 323 to phenylalanine (F) (Eg L323F);
  • the SEQ ID NO: 67 contains a mutation to glutamic acid (E) at position 262 relative to SEQ ID NO: 19 (e.g. S262E), and a mutation at position 320 to serine (S) (e.g. N320S ), the amino acid residue at position 323 is mutated to phenylalanine (F) (for example, L323F). Among them, the numbering of amino acid sites is marked in natural order.
  • the aforementioned anti-CD40 antibody or antigen-binding fragment thereof is mutated from the last amino acid (eg, lysine) of the heavy chain amino acid sequence to alanine (A).
  • the last amino acid of the heavy chain sequence shown in SEQ ID NO: 61, 62, 63, 64, 67 is mutated to A.
  • an antibody which comprises a heavy chain shown in SEQ ID NO: 69 and a light chain shown in SEQ ID NO: 66.
  • an antibody which comprises a heavy chain shown in SEQ ID NO: 68 and a light chain shown in SEQ ID NO: 66.
  • an anti-CD40 antibody or antigen-binding fragment thereof as described above wherein the antigen-binding fragment is Fab, Fv, sFv, F(ab') 2 , linear antibody, single-chain antibody, Nanobodies, domain antibodies and multispecific antibodies.
  • the present disclosure further provides a single-chain antibody containing the heavy chain variable region and the light chain variable region of the anti-CD40 antibody or antigen-binding fragment thereof as described above.
  • the present disclosure further provides a multispecific antibody containing the heavy chain variable region and the light chain variable region of the anti-CD40 antibody or antigen-binding fragment thereof as described above.
  • the present disclosure further provides a nucleic acid molecule (DNA or RNA) that encodes an anti-CD40 antibody or antigen-binding fragment thereof, a multispecific antibody, or a single chain antibody as described above.
  • DNA or RNA nucleic acid molecule
  • the present disclosure further provides an expression vector containing the nucleic acid molecule as described above.
  • the present disclosure further provides a host cell comprising or transformed with the expression vector as described above.
  • the host cells are bacteria, yeast, mammalian cells, specifically E. coli, Pichia pastoris, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells.
  • the present disclosure further provides an antibody-drug conjugate containing the light chain variable region and/or heavy chain variable region of the anti-CD40 antibody as described above.
  • the antibody-drug conjugate is well known in the art, and it is formed by the antibody-linker-drug interconnection.
  • the known linkers include a cleavage linker and a split cleavage linker.
  • Drugs are also known in the art, such as DM1, DM4, MMAE, MMAF, etc.
  • the present disclosure further provides a pharmaceutical composition containing 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 unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of anti-CD40 antibody or fragment thereof, or the amount of CD40 antibody or fragment thereof in the unit dose of the pharmaceutical composition is 0.1-2000 mg, In some embodiments, it is 1-1000 mg.
  • the present disclosure further provides an anti-CD40 antibody or antigen-binding fragment thereof, a multispecific antibody, a single-chain antibody, or a pharmaceutical composition containing the same as described above, in the preparation for the treatment of CD40 or CD40L-mediated diseases or disorders Use in medicine; wherein the disease is specifically cancer; the cancer is most specifically 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 present disclosure further provides a method of treating and preventing a CD40 or CD40L-mediated disease or disorder, the method comprising contacting a subject with a prophylactically effective or therapeutically effective amount of an anti-CD40 antibody or antigen-binding fragment thereof as described above, Multispecific antibodies, single chain antibodies or pharmaceutical compositions thereof; wherein the disease or disorder is specifically cancer; the cancer is most specifically lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, renal 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 present disclosure further provides the use of the above-mentioned anti-CD40 antibody or antigen-binding fragment thereof, multispecific antibody, single chain antibody or pharmaceutical composition thereof in the preparation of a medicament for improving the symptoms of patients with autoimmune diseases.
  • the present disclosure further provides the use of the above-mentioned anti-CD40 antibody or antigen-binding fragment thereof, multispecific antibody, single chain antibody or pharmaceutical composition thereof for the preparation of a medicament for improving the symptoms of patients with inflammatory diseases.
  • Figure 1 shows the activation of DC cells by murine anti-human CD40 antibodies based on CD80 activation molecules.
  • Figure 2 shows the activation of DC cells by murine anti-human CD40 antibodies based on CD86 activating molecules.
  • Figure 3 shows the growth curve of Raji transplanted lymphoma after co-transplantation of Raji transplanted lymphoma with human PBMC and DC cells.
  • Figure 4 shows that the body weight change curve of NOG mice after co-transplantation of Raji transplanted lymphoma with human PBMC and DC cells into NOG mice.
  • Figure 5 shows the tumor growth curve after intraperitoneal injection of a single dose of anti-CD40 antibody into the MC38 colon cancer mouse model.
  • Figure 6 shows the activation of DC cells by anti-CD40 antibodies with heavy chain constant region mutations.
  • 7A and 7B show the activation of DC cells and promotion of cytokine production by anti-CD40 antibodies with heavy chain constant region mutations.
  • antibody in the present disclosure refers to immunoglobulin, which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain and ⁇ chain, respectively. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3 and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by different constant regions.
  • Each of the five types of Ig can have a ⁇ chain or a ⁇ chain.
  • the antibody light chain described in the present disclosure may further comprise a light chain constant region, the light chain constant region comprising a human- or murine-derived kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region, which comprises human or murine IgG1, 2, 3, 4 or variants thereof.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is a variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences.
  • the three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxy terminal 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.
  • APC antigen presenting cell
  • T cells use T cell receptors (TCRs) to recognize this complex.
  • APC include, but are not limited to, dendritic cells (DC), topical blood mononuclear cells (PBMC), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DC).
  • DC dendritic cells
  • PBMC topical 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 includes any variant or isotype of CD40 that is naturally expressed by the cell.
  • the antibodies of the present disclosure can cross-react with CD40 of non-human species. Alternatively, the antibody may be specific for human CD40 and may not exhibit cross-reactivity with other species.
  • CD40, or any variant or isotype thereof can be isolated from cells or tissues in which they are naturally expressed, or produced by recombinant techniques using those techniques commonly used in the art and those described herein.
  • the anti-CD40 antibody targets human CD40 with a normal glycosylation pattern.
  • mouse antibody in the present disclosure is a monoclonal antibody against human CD40 prepared according to the knowledge and skills in the art. During preparation, the test subjects are injected with CD40 antigen, and then hybridomas expressing antibodies with desired sequence or functional properties are isolated.
  • the mouse-derived CD40 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of the mouse-derived ⁇ , ⁇ chain or variant thereof, or further comprise mouse-derived IgG1, IgG2 , IgG3 or IgG4 or its heavy chain constant region.
  • human antibody includes antibodies with variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human backbone sequences (ie, "humanized antibodies”) .
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by grafting a non-human CDR sequence into the framework of a human antibody variable region. It can overcome the strong immune response induced by the chimeric antibody due to carrying a large number of heterogeneous protein components. In order to avoid a decrease in activity at the same time as a decrease in immunogenicity, the variable region of the antibody may be subjected to minimal reverse mutation to maintain activity.
  • chimeric antibody is an antibody that is a fusion of the variable region of an antibody of the first species (such as a mouse) and the constant region of an antibody of another species (such as a human), which can reduce the induction of heterologous antibodies Immune response.
  • the region gene and the human constant region gene are connected 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 the human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and preferably comprises a human heavy chain constant region of IgG1 or IgG2.
  • antigen-binding fragment refers to antigen-binding fragments and antibody analogs of antibodies, which generally include at least a portion of the antigen-binding region or variable region (eg, one or more CDRs) of the parental antibody.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when activity is expressed on a molar basis, antibody fragments retain at least 10% of the maternal binding activity. Preferably, the antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95% 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.
  • Fab fragment is composed of a light chain and a heavy chain CH1 and variable regions.
  • 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 that contain the CH2 and CH3 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.
  • the "F(ab')2 fragment” contains two light chains and two heavy chains including a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
  • the "Fv region” contains variable regions from both heavy and light chains, but lacks constant regions.
  • multispecific antibody is used in its broadest sense and encompasses antibodies with multi-epitope specificity.
  • These multispecific antibodies include, but are not limited to: antibodies containing heavy chain variable regions (VH) and light chain variable regions (VL), where the VH-VL unit has multi-epitope specificity; has two or more Antibodies of VL and VH regions, each VH-VL unit binds to a different target or different epitope of the same target; antibodies with 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 together Antibody fragments etc.
  • the term "corresponding to SEQ ID NO: the nth position of m" refers to: in different antibody coding systems, a mutation site is in position
  • the nth bit of SEQ ID NO: m is comparable or equivalent.
  • the current antibody coding systems include but are not limited to EU, Kabat, Chothia, IMGT (Lefranc, 2003) and AHo (Honegger and Plückthun, 2001), etc.
  • EU Kabat
  • Chothia Chothia
  • IMGT Lefranc, 2003
  • AHo Hegger and Plückthun, 2001
  • ADC antibody-drug conjugate
  • single-chain antibody is a single-chain recombinant protein composed of an antibody's heavy chain variable region (VH) and light chain variable region (VL) connected by a linking peptide, which is the smallest with a complete antigen binding site Antibody fragments.
  • domain antibody fragment is an immunoglobulin fragment having immunological functions containing only heavy chain variable region or light chain variable region chain.
  • two or more VH regions are covalently linked 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.
  • association with CD40 refers to the ability to interact with human CD40.
  • antigen binding site refers to a discontinuous three-dimensional site on the antigen recognized by the antibody or antigen-binding fragment of the present disclosure.
  • epitope refers to a site on an antigen that specifically binds to immunoglobulins or antibodies.
  • the epitope may be formed by adjacent amino acids or non-adjacent amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost after denaturing solvent treatment. Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of epitopes include techniques in the art and techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • telomere binding refers to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody is measured at an equilibrium dissociation constant of less than about 10 -7 M or even smaller when measured in the instrument by surface plasmon resonance (SPR) technology ( K D ) binds to a predetermined antigen, and its affinity to bind to the predetermined antigen is at least twice that of its binding affinity to non-specific antigens other than the predetermined antigen or closely related antigens (such as BSA, etc.).
  • SPR surface plasmon resonance
  • K D surface plasmon resonance
  • the term "antigen-recognizing antibody” may be used interchangeably herein with the term “specifically bound antibody”.
  • cross-reactivity refers to the ability of the antibodies of the present disclosure to bind to CD40 from different species.
  • an antibody of the present disclosure that binds human CD40 can also bind CD40 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (eg, SPR and ELISA), or binding or functional interaction with physiologically expressing CD40 cells.
  • binding assays eg, SPR and ELISA
  • Methods to determine cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • inhibitor or “block” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of ligands preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduced ligand binding affinity when in contact with anti-CD40 antibodies compared to ligands not in contact with anti-CD40 antibodies.
  • inhibit growth e.g. referring to cells
  • inhibit growth is intended to include any measurable reduction in cell growth.
  • inducing an immune response and “enhancing the immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a specific antigen.
  • induction for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the ADCC effect function of the antibody can be reduced or eliminated by modifying the Fc segment on IgG.
  • the modification refers to mutation in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2/4 chimera, F235E of IgG4, or L234A/E235A mutation.
  • mice can be immunized with human CD40 or fragments thereof, the obtained antibodies can be renatured and purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the disclosed antibodies or antigen-binding fragments are genetically engineered to add one or more human FR regions to CDR regions of non-human origin.
  • Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequence of the corresponding antibody can be cloned and recombined into the GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of FC regions.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in the bioreactor to produce antibodies.
  • the antibody-secreted culture fluid can be purified and collected by conventional techniques.
  • Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the resulting product should be immediately frozen, such as -70 °C, or lyophilized.
  • the antibodies of the present disclosure refer to monoclonal antibodies.
  • the monoclonal antibody (mAb) described in the present disclosure refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions and animals , Humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes the contact of reagents with cells and the contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering “administration,” and “treatment” also mean in vitro and ex vivo treatment of, for example, cells by an agent, diagnosis, composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • Treatment means administration of a therapeutic agent for internal or external use to a subject, such as a composition comprising any one of the antibodies or antigen-binding fragments thereof of the present disclosure, the subject has one or more symptoms of the disease. It is known that the therapeutic agent has a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in an amount or amount effective to relieve one or more disease symptoms in the subject or population being treated, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically unmeasurable extent.
  • the amount of therapeutic agent effective to relieve the symptoms of any specific disease may vary according to various factors, such as the disease state, age and weight of the subject, and the drug in the subject in need ability. It is possible to assess whether the symptoms of the disease have been alleviated by any clinical testing methods commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms.
  • embodiments of the present disclosure may be ineffective in relieving the target disease symptoms of each subject, according to any statistical test methods known in the art (such as Student's test, chi-square test, According to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test), it should alleviate the target disease symptoms in a statistically significant number of subjects.
  • statistical test methods such as Student's test, chi-square test, According to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test
  • Constant modification or “conservative substitution or substitution” means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) replace amino acids in the protein so that frequent Make changes without changing the biological activity of the protein.
  • Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change biological activity (see, for example, Watson et al. (1987) Molec ⁇ lar Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Common conservative substitutions of amino acids are as follows:
  • Effective amount includes an amount sufficient to ameliorate or prevent medical symptoms or conditions.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular such or veterinary subject may vary depending on factors such as the condition to be treated, the general health condition described, the route and dosage of the method of administration, and the severity of side effects.
  • the effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to substances that are produced outside the body of organisms, cells, or humans according to context.
  • Endogenous refers to substances produced in a living body, cell, or human body according to context.
  • “Homology” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid residue (for example, if each position of two DNA molecules is occupied by adenine), then the molecules are homologous at that position.
  • the percentage of homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions ⁇ 100%. For example, when the sequences are optimally aligned, if there are 6 matches or homology at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, the comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.
  • cell includes primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to intentional or unintentional mutations, all progeny may not be exactly the same in terms of DNA composition and/or content. The term includes mutant offspring that have the same function or biological activity as the original cell.
  • Recombinant protein with his-tagged human CD40 (h-CD40-his), recombinant protein with Fc-tagged human CD40 (h-CD40-Fc), recombinant protein with his-tagged mouse CD40 (m-CD40-his) and Rhesus monkey CD40 (rhesus-CD40-his) recombinant protein (#CD0-C52H7) of his tag are all purified commercial protein reagents purchased from Acrobiosystems. The protein reagent 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 to 8 weeks old (Zhaoyan (Suzhou) New Drug Research Center Co., Ltd., animal production license number: 201503259). Feeding environment: SPF level.
  • mice After the mice were purchased, they were kept in the laboratory environment for 1 week, adjusted for 12/12 hour light/dark cycle, with a temperature of 20-25°C and a humidity of 40-60%. Divide the acclimated mice into 2 cages, 5 mice per cage.
  • the immune antigen is an Fc-tagged human modified CD40 recombinant protein (h-CD40-Fc, formulated into 1 ⁇ g/ ⁇ l with phosphate buffer).
  • Emulsified with Freund's adjuvant (Sigma, Lot No.: F5881/F5506): Freund's complete adjuvant (CFA) for the first time, nucleic acid adjuvant (CpG, Sangon Biotech) and aluminum for injection (Imject Alum) , Thermo, Lot No.: PH203866).
  • the time of immunization injection is day 0, 14, 28, 42, 56, 70. Blood was collected on the 21st, 35th, 49th, 63th, and 77th days for blood test, and the mouse serum was tested by ELISA to determine the antibody titer in the mouse serum.
  • mice with high antibody titers and stable titers were selected for fusion of spleen cells.
  • the immunization was boosted 3 days before fusion, and intraperitoneal (IP) was injected with 10 ⁇ g/phosphate phosphate buffer Antigen solution.
  • IP intraperitoneal
  • the optimized PEG-mediated fusion step was used to divide splenic lymphocytes and myeloma cells Sp2/0 cells ( CRL-8287 TM ) to obtain hybridoma cells, and select five monoclonal hybridoma cell lines with good activity in vitro.
  • ELISA experiments were used to detect the binding properties of anti-CD40 antibodies.
  • the His-tagged CD40 recombinant protein was directly coated. After the antibody was added, the antibody-antigen-binding activity was detected by adding a secondary antibody (HRP-conjugated anti-anti-Fc antibody) and HRP substrate TMB.
  • the selected anti-human CD40 antibody was tested to block the binding between human CD40 and human CD40L.
  • the specific method is to coat the Fc-tagged CD40 recombinant protein (h-CD40-Fc) on a 96-well microtiter plate, add anti-CD40 antibody to fully bind to occupy the epitope, and then add his-tagged CD40L to detect his Label to calculate the amount of binding of CD40 and CD40L, and calculate the IC50 value of the CD40 antibody blocking the CD40 active site.
  • h-CD40-Fc Fc-tagged CD40 recombinant protein
  • Human CD40-Fc protein was coated on a 96-well microtiter plate, 100 ⁇ l per well at a concentration of 1 ⁇ g/mL, and incubated overnight at 4°C. Wash the lotion three times, 250 ⁇ l per well. Each wash was shaken for 10 seconds to ensure full cleaning. Add 200 ⁇ l/well blocking solution and incubate for 2 hours at room temperature. Wash the lotion three times, 250 ⁇ l per well. Each wash was shaken for 10 seconds to ensure full cleaning. Add 100 ⁇ l of anti-CD40 test antibody diluted with diluent to each well and incubate for 1 hour at room temperature. Wash the lotion three times, 250 ⁇ l per well.
  • the human anti-capture antibody was covalently coupled to the biosensor chip CM5 of the Biacore instrument (Biacore X100, GE) according to the method in the instructions of the human anti-capture kit (Cat.#BR-1008-39, GE), thereby pro And capture a certain amount of chimeric or humanized antibody to be tested, and then flow through a series of concentration gradient CD40 antigens on the chip surface (CD40 antigens are all purchased from Acrobiosystems), using Biacore instrument (Biacore X100, GE) to detect the reaction in real time Signal to obtain binding and dissociation curves.
  • Biacore instrument Biacore X100, GE
  • the biochip is washed and regenerated with the regeneration solution provided in the human anti-capture kit.
  • the amino coupling kit used in the experiment was purchased from GE Corporation (Cat.#BR-1000-50, GE), and HBS-EP+10 ⁇ buffer solution (Cat.#BR-1006-69, GE) was double distilled Dilute with water to 1 ⁇ (pH 7.4).
  • HEK-Blue CD40L cells were purchased from Invivogen (Cat#hkb-cd40). The cells were stably transfected with human CD40 gene and NF-kB-mediated SEAP genome. SEAP secreted in supernatant can be detected by SEAP substrate QUANTI-Blue To characterize the activation level of the CD40 signaling pathway. In this experiment, by detecting the activation of the cell HEK-Blue CD40L, the in vitro cell activity of the CD40 antibody was evaluated based on the EC50 size.
  • the cells HEK-Blue CD40L were cultured in DMEM medium containing 10% FBS, 100 ⁇ g/mL Zeocin and 30 ⁇ g/mL Blasticidin, and passaged 2 to 3 times a week, with a passage ratio of 1:5 or 1:10.
  • aspirate the culture medium rinse the cell layer with 5 mL of 0.25% pancreatin, then aspirate the pancreatin, place the cells in an incubator and digest for 3 to 5 minutes, add fresh medium to resuspend the cells.
  • the culture medium contains 10% FBS, 100 ⁇ g/mL bleomycin Zeocin, and 30 ⁇ g/mL blasticidin Blasticidin.
  • DMEM only add 100 ⁇ l of sterile water to the periphery of the 96-well plate. The culture plate was incubated in an incubator for 24 hours (37°C, 5% CO 2 ). After the cells adhere to the cells, add 100 ⁇ l of the diluted test antibody to each well. The culture plate was incubated in an incubator for 20-24 hours (37°C, 5% CO 2 ).
  • PBMC peripheral blood cells were isolated from normal human peripheral blood, and then monocytes were sorted with CD14 MACS beads.
  • RPMI 1640 culture medium containing 10 ng/mL IL4 and 100 ng/mL GM-CSF was added and cultured for 6 days to induce culture of MoDC cells (monocyte-derived dendritic cells). After 6 days, the cells were collected, 1 ⁇ 10 5 cells were taken, stained with CD209-PE, CD1a-PerCP/Cy5.5 and CD14-PE/Cy7, and the successful induction of MoDC was analyzed by FACS (the above operations are routine operations in the art ).
  • 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
  • variable region sequences were respectively connected to human antibody IgG1 constant region sequences to obtain human-mouse chimeric antibody sequences.
  • pCP expression vector purchased from Maibosi Biotechnology Co., Ltd.
  • sequence identification after PCR For details, please refer to "Molecular Cloning: Laboratory Manual”).
  • human-mouse chimeric antibodies 2H6-C and 9E5-C can be obtained.
  • the heavy and light chain variable region sequences were compared with the antibody germline database to obtain human germline templates with high homology.
  • the antibodies of the present disclosure are preferably human germline light chain templates Vk1-33/JK4 (for 2H6) or Vk2-28/JK4 (for 9E5).
  • the human germline heavy chain framework region is derived from the human heavy chain.
  • the antibodies of the present disclosure are preferably human germline heavy chain templates VH1-69/JH6 (for 2H6) or VH1-2/JH6 (for 9E5), as shown below:
  • 2H6 is the preferred human germline heavy chain template IGHV1-69
  • the CDR region of the murine antibody is grafted onto the selected humanized template, replacing the humanized variable region, and then 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, back-mutate the embedded residues, the residues that have direct interaction with the CDR region, and the residues that have an important influence on the conformation of VL and VH, and CDR region chemistry Unstable amino acid residues are optimized to obtain the final humanized molecule.
  • the heavy chain variable region sequence is shown in SEQ ID NO: 25-30;
  • the sequence of the light chain variable region is shown in SEQ ID NO: 31-36.
  • the results show that the ELISA binding and blocking activity of the humanized anti-human CD40 antibody of the present disclosure is comparable to the positive antibody pfizer/alligator.
  • the Biacore measurement affinity of hu9E5 and human CD40 is more than 10 times that of the positive antibody Alligator control and more than 4 times that of the Pfizer control.
  • the peripheral blood of normal persons was taken and PBMC of healthy persons were separated by density gradient centrifugation.
  • the monocytes were sorted with the CD14+microbeads kit, and the CD14+monocytes were separated according to the protocol provided by the kit, that is, 20 ⁇ l of anti-CD14 microbeads were added every 10 7 cells and incubated at 4°C for 15 minutes. Then, the cells were added to the magnetic column, and after washing three times, the cells in the magnetic column, that is, CD14+ monocytes were collected.
  • RPMI 1640 medium containing 10 ng/mL IL-4 and 100 ng/mL GM-CSF was added to CD14+ monocytes and cultured for 6 days (cultivation method is a conventional method in the art). Induction culture of MoDC cells was carried out. RPMI 1640 of IL-2, after culturing, collect the suspended cells (the culturing method and the method of collecting cells are conventional methods in the art), and sort the T cells using the CD3+ microbead kit.
  • mice in each group were administered by injection once a week, six weeks, and three consecutive times.
  • TGI% (1-T/C) ⁇ 100%.
  • T/C% is the relative tumor proliferation rate, that is, the percentage of the tumor volume or tumor weight of 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.
  • the heavy chain sequences of hu2H6-M and hu2H6-SELFNS are shown in SEQ ID NO: 61 and 62, and the light chain sequences are shown in SEQ ID NO: 18.
  • the heavy chain sequence of hu9E5-M, hu9E5-SELF, hu9E5-SELFNS is shown in SEQ ID NO: 63, 64, 67, and the light chain sequence is shown in SEQ ID NO: 20.
  • the last amino acid K of SEQ ID NO: 61, 62, 63, 64, 67 can be replaced with A. This mutation does not affect the activity of the antibody, but can improve the drug-forming ability of the antibody to a certain extent.
  • antibody 005M heavy chain amino acid sequence 121-450 and hu9E5- The amino acid sequences of positions 113-442 of the M heavy chain are the same.
  • the specific sequence of 005M is as follows:
  • the amino acid at position 331 of the 005M heavy chain SEQ ID NO: 65 was mutated from leucine (L) to phenylalanine (F) to obtain the mutant APX005-SELF; the 005M heavy chain SEQ ID NO: 65 Amino acid at position 328 was mutated from asparagine (N) to serine (S), and amino acid at position 331 was mutated from leucine (L) to phenylalanine (F) to obtain mutant APX005-SELFNS.
  • PBMC peripheral blood cells were isolated from normal human peripheral blood, and then monocytes were sorted with CD14 MACS beads.
  • APX005M-SELFNS and 2H6-SELFNS both showed extremely strong agonist activity, activated the activation molecule CD86 on the surface of DC cells, and showed a dose-dependent effect, which was stronger than Alligator's control antibody G12. See Figure 6.
  • Example 14 Experiment of anti-CD40 antibody mutated in constant region of heavy chain to activate DC cells to produce cytokines
  • PBMC peripheral blood cells were isolated from normal human peripheral blood, and then CD14MACS beads were used to sort monocytes.
  • APX005M-SELFNS, APX005M-SELF, 2H6-SELFNS, 9E5-SELFNS all show extremely strong agonist activity, promote DC cells to secrete cytokine IL-12 p40, and show a dose-dependent effect, the effect is stronger than Alligator's control antibody G12.
  • the results are shown in Figures 7A and 7B and Tables 12 and 13.
  • Example 15 Anti-CD40 antibodies with mutations in the constant region of the heavy chain can inhibit the growth of mouse tumors
  • the antitumor effect and safety of the CD40 antibody drug were evaluated by the size of the tumor and the weight of the mouse.
  • MC38 mouse colon cancer cell lines were cultured using DMEM (10% FBS, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 10 mM HEPES), and the cells proliferated to 80% of the culture dish. At a density of 90%, trypsin-EDTA (0.25%) was added, placed at 37°C for 3 to 5 minutes for digestion, then terminated with a medium containing 10% FBS, then centrifuged, washed twice with PBS, and finally with PBS Resuspend to prepare a single cell suspension and adjust the cell density to 107 cells/mL for later use.
  • DMEM % FBS, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 10 mM HEPES
  • Method of establishing MC38 tumor model Using the MC38 single cell suspension prepared above, on the 7th day, 2 ⁇ 10 6 MC38 cells (200 ⁇ L) were inoculated into 32 humanized hFc ⁇ R/hCD40 C57BL/6 mice (by Shanghai Jiaotong University) Li Fubin's research group in the medical school breeds, SPF-level feeding) subcutaneously on the right rib. When the average tumor volume of the mice reaches about 55mm 3 , they are randomly divided into 4 groups, 8 in each group.
  • a single dose of anti-CD40 antibody was intraperitoneally injected according to the schedule as shown in Table 12, the tumor volume was measured twice a week, the body weight was weighed, and the data was recorded.
  • the control IgG, hu9E5, hu9E5-M were all provided by Shanghai Hengrui Medicine Co., Ltd., diluted with PBS to make the final concentration 0.3mg/mL.
  • the tumor volume of the mice is continuously measured, and the tumor volume is used as an index to evaluate the anti-tumor activity of the test antibody.
  • L short is the shortest diameter of the tumor
  • L long is the longest diameter of the tumor
  • T/C% is the relative tumor proliferation rate, that is, the percentage of the tumor volume of the treatment group and the control group at a certain time point. Calculated as follows:
  • T/C% (TT 0 )/(CC 0 ) ⁇ 100
  • T and C are the tumor volume at the end of the experiment; T 0 and C 0 are the tumor volume at the beginning of the experiment.
  • the in vivo activity results of each group of antibodies in the MC38 tumor model of hFc ⁇ R/hCD40Tg mice can be judged by the change in tumor volume.
  • the tumor volume growth of the hu9E5 group, hu9E5-M group, and 005M group was inhibited, and the relative tumor inhibition rates were 42.0%, 68.9%, and 53.8%, respectively.
  • hu9E5 has certain anti-tumor activity (p>0.05), hu9E5-M and 005M have strong anti-tumor activity (p ⁇ 0.05), and hu9E5-M has a more beneficial effect than 005M, as shown in Table 15 and Figure 5 .

Abstract

本公开涉及抗CD40抗体、其抗原结合片段及其医药用途。所述抗CD40抗体、其抗原结合片段的重链恒定区含有突变，所述突变使得抗CD40抗体丧失了其与FcγRIII结合的活性，同时增强了抗CD40抗体与FcγRIIB的结合，从而丧失了抗体依赖的细胞毒性(ADCC)，但是增强了FcγRIIB介导的抗体交联。重链恒定区中的突变导致增强了对CD40的激活，以及增强了树突细胞对抗原的递呈。本公开的抗CD40抗体及其抗原结合片段作为抗癌药物具有治疗CD40介导的疾病或病症的用途。

Description

抗CD40抗体、其抗原结合片段及其医药用途
本申请要求2018年11月30日提出的中国专利申请《抗CD40抗体、其抗原结合片段及其医药用途》(申请号CN201811448228.1)的优先权。
技术领域
本公开涉及一种在重链恒定区包含突变的抗CD40抗体、其抗原结合片段、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抗体或其抗原结合片段的药物组合物、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性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。肿瘤免疫治疗可能是最有效的也是最安全的***的途径之一。肿瘤免疫疗法目前对治疗几种不同类型的癌症，包括播散性转移性肿瘤已具有很好的前景。
人体内T细胞的激活采取了双信号通路的***：通过抗原递呈细胞(APC)递呈MHC-抗原肽给T细胞，从而提供第一信号；需要一系列协同刺激分子提供第二信号，进而使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，为分子量约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。CD40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。当通过CD40发生信号传导时，专门的抗原递呈细胞会被激活。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介导的信号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的表达发生在包括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，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，并且观察到肿瘤的根除与肿瘤特异性细胞毒性T淋巴细胞的出现存在强烈相关性。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PCT/CN2018/089252、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。
对于抗体恒定区突变，WO2006019447、WO2014145806、US8734791、US9657106、US8084582、WO2008150494、WO2004099249公开了抗体重链的S267E、L328F、N325S的突变，所述突变使得抗体去掉了与FcγRIII的结合，但是增强了与FcγRIIB的结合，从而去除了抗体依赖的细胞毒性(ADCC)，但是增强了FcγRIIB介导的交联，从而增强对CD40的激活，增强树突细胞的抗原递呈。
本公开旨在提供一种高亲和力、高选择性、高生物活性的抗CD40抗体，其缺失抗体依赖的细胞毒性(ADCC)，但是具有增强的FcγRIIB介导的交联，从而抑制体内的肿瘤生长。本公开的抗体能够用作药物或组合物用于治疗刺激CD40及其通路介导的癌症。
发明内容
本公开提供一种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示的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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些实施方案中，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。
一些具体实施方案中，抗CD40抗体或其抗原结合片段，其中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。
一些具体实施方案种，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。
一些具体实施方案中，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。
一些具体实施方案中，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。
另一些具体实施方案中，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.9所示，轻链可变区的氨基酸序列如SEQ ID NO.10所示。
另一些具体实施方案中，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。
另一些具体实施方案中，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。
另一些具体实施方案中，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。
一些具体实施方案中，抗CD40抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体、人抗体或其片段。
一些具体实施方案中，当所述抗CD40抗体或其抗原结合片段为鼠源抗体或其片段时，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区或轻链恒定区，和/或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区或重链恒定区。
一些具体实施方案中，当所述抗CD40抗体或其抗原结合片段为嵌合抗体或其片段时，其包含人源κ、λ链或其变体的轻链恒定区，和/或包含人源IgG1、IgG2、 IgG3或IgG4或其变体的重链恒定区。一些具体实施方案中，轻链可变区序列为SEQ ID NO:2或SEQ ID NO:10所示，和/或重链可变区序列为SEQ ID NO:1或SEQ ID NO:9所示。
一些具体实施方案中，当所述抗CD40抗体或其抗原结合片段为人源化抗体或其片段时，抗体轻链序列为：SEQ ID NO:18或SEQ ID NO:20或其变体；所述的变体具体在轻链有0-10的氨基酸变化，更具体为第2和3位的氨基酸位点的突变，位点2和位点3突变后的氨基酸各自独立地选自I、V或L；抗体重链序列为：SEQ ID NO:17或SEQ ID NO:19或其变体；所述的变体具体在重链有0-10的氨基酸变化，更具体为第6和8位的氨基酸位点的突变，两个位点突变后的氨基酸各自独立地选自I、A或L。
一些具体的实施方案中，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或其变体的重链恒定区或FR区，具体包含人源IgG1、IgG2或IgG4重链恒定区或FR区，更具体包含人源IgG1或IgG2重链恒定区或FR区，和/或包含人源κ、λ链或其变体的轻链FR区。
一些具体的实施方案中，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。
一些具体的实施方案中，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具体在轻链可变区有0-10的氨基酸变化，更具体为氨基酸位点在2和3位的突变，突变后的氨基酸均具体为I、V或L。
一些具体的实施方案中，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。
一些具体的实施方案中，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，和/或来源于如SEQ ID NO:22所示的人种系重链IGkV1-33序列；来源于如SEQ ID NO:23所示的人种系重链IGHV1-2序列，和/或来源于如SEQ ID NO:24所示的人种系重链IGkV2-28序列。
一些具体的实施方案中，抗CD40人源化抗体或其片段，重链可变区选自SEQ ID NO：25-30之一所示的序列或其变体，轻链可变区选自SEQ ID NO：31-36之一所示的序列或其变体。
一些具体的实施方案中，抗CD40人源化抗体或其片段，重链可变区为SEQ ID NO：26所示的序列或其变体，轻链可变区选自SEQ ID NO：33所示的序列或其变体。
一些具体的实施方案中，重链可变区为SEQ ID NO：30所示的序列或其变体， 轻链可变区选自SEQ ID NO：34所示的序列或其变体。
一些具体实施方案中，人源化抗CD40抗体的重链序列如SEQ ID NO：17所示，轻链序列如SEQ ID NO：18所示。
一些具体的实施方案中，重链序列如SEQ ID NO：19所示，轻链序列如SEQ ID NO：20所示。
一些具体的实施方案中，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:61、62、63、64或67所示的序列或其变体，和/或轻链可变区为SEQ ID NO:18、20所示序列或其变体。
一些具体实施方案中，抗CD40人源化抗体或其片段的重链序列如SEQ ID NO:61或62所示序列或其变体，轻链序列如SEQ ID NO:18所示序列或其变体；重链序列如SEQ ID NO:63、64或67所示序列或其变体，轻链序列如SEQ ID NO：20所示序列或其变体。
所述变体在重链可变区有0-10的氨基酸变化，更具体为氨基酸位点在6和8的突变，突变后的氨基酸均具体为I、A或L。
其中，所述SEQ ID NO:61包含相对于SEQ ID NO:17的第266位氨基酸残基突变为谷氨酸(E)(例如S266E)；
所述SEQ ID NO：62包含相对于SEQ ID NO:17的第266位氨基酸残基突变为谷氨酸(E)(例如S266E)，第324位氨基酸残基突变为丝氨酸(S)(例如N324S)，第327位氨基酸残基突变为苯丙氨酸(F)(例如L327F)；
所述SEQ ID NO:63包含在相对于SEQ ID NO:19的第262位氨基酸残基突变为谷氨酸(E)(例如S262E)；
所述SEQ ID NO：64包含相对于SEQ ID NO:19的第262位氨基酸残基突变为谷氨酸(E)(例如S262E)，第323位氨基酸残基突变为苯丙氨酸(F)(例如L323F)；
所述SEQ ID NO：67包含相对于SEQ ID NO:19的第262位氨基酸残基突变为谷氨酸(E)(例如S262E)，第320位氨基酸残基突变为丝氨酸(S)(例如N320S)，第323位氨基酸残基突变为苯丙氨酸(F)(例如L323F)。其中，氨基酸位点的编号是按照自然顺序标号。一些实施方案中，上述抗CD40抗体或其抗原结合片段在重链氨基酸序列的最后一位氨基酸(例如赖氨酸)到丙氨酸(A)的突变。
一些具体实施方案中，在SEQ ID NO:61、62、63、64、67所示重链序列的最后一位氨基酸突变为A。
另一些具体的实施方案中，提供了一种抗体，其包含SEQ ID NO:69所示重链和SEQ ID NO:66所示轻链。
另一些具体的实施方案中，提供了一种抗体，其包含SEQ ID NO:68所示重链和SEQ ID NO:66所示轻链。
一些具体的实施方案中，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。
本公开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的重链可变区和轻链可变区。
本公开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的重链可变区和轻链可变区。
本公开进一步提供一种核酸分子(DNA或RNA)，其编码如上所述的抗CD40抗体或其抗原结合片段、多特异性抗体或者单链抗体。
本公开进一步提供一种表达载体，其含有如上所述的核酸分子。
本公开进一步提供一种宿主细胞，其包含或转化有如上所述的表达载体。一些具体实施方案中，宿主细胞为细菌、酵母菌、哺乳动物细胞，具体为大肠杆菌、毕赤酵母、中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
本公开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。药物也是本领域公知的，例如DM1、DM4、MMAE、MMAF等。
本公开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。
在一些具体实施方式中，所述药物组合物单位剂量中可含有0.01至99重量％的抗CD40抗体或其片段，或药物组合物单位剂量中含CD40抗体或其片段的量为0.1-2000mg，在一些具体实施方式中为1-1000mg。
本公开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；其中所述的疾病具体为癌症；所述的癌症最具体为淋巴癌、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。
本公开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，该方法包括使受试者接触预防有效量或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、单链抗体或其药物组合物；其中所述的疾病或病症具体为癌症；所述的癌症最具体为淋巴癌、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。
本公开进一步提供上述抗CD40抗体或其抗原结合片段、多特异性抗体、单链 抗体或其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。
本公开进一步提供一种上述抗CD40抗体或其抗原结合片段、多特异性抗体、单链抗体或其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显示鼠源抗人CD40抗体基于CD80激活分子对DC细胞的激活作用。
图2显示鼠源抗人CD40抗体基于CD86激活分子对DC细胞的激活作用。
图3显示：Raji移植淋巴瘤与人PBMC和DC细胞共移植之后，Raji移植淋巴瘤的肿瘤生长曲线。
图4显示：将Raji移植淋巴瘤与人PBMC和DC细胞共移植至NOG小鼠中，NOG小鼠的体重变化曲线。
图5显示：向MC38结肠癌小鼠模型腹腔注射单剂量的抗CD40抗体之后的肿瘤生长曲线。
图6显示具有重链恒定区突变的抗CD40抗体对DC细胞的激活作用。
图7A和图7B显示具有重链恒定区突变的抗CD40抗体对DC细胞的激活并促进细胞因子的产生。
具体实施方式
术语
为了更容易理解本公开，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公开所属领域的一般技术人员通常理解的含义。
本公开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。
本公开所述的术语“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。
在本公开中，本公开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。
在本公开中，本公开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。
术语“抗原递呈细胞”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“抗原递呈”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”包括由细胞天然表达的CD40的任何变体或同种型。本公开的抗体可与非人物种的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“鼠源抗体”在本公开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。在本公开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。本公开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)，是指将非人的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异种蛋白成分，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，可对所述抗体的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)”，是将第一物种(如鼠源)抗体的可变区与另一物种(如人)抗体的恒定区融合而成的抗体，可以减轻异源抗体诱发的免疫应答反应。建立鼠-人嵌合抗体，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CH2和CH3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。
“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)、共价或非共价连接在一起的抗体片段等。
在本申请上下文中，当提及重链恒定区的突变位点时，术语“对应于SEQ ID NO：m的第n位”是指：在不同的抗体编码体系中，一个突变位点在地位上与SEQ ID NO：m的第n位是可比较的或等价的。技术人员知晓，现行的抗体编码体系包括但不限于EU、Kabat、Chothia、IMGT(Lefranc，2003)和AHo(Honegger和Plückthun，2001)等。在一种编码体系下，当某个位点被确定为第n位时，它在另一个编码体系下可能被确定为第n’位。技术人员根据常识，能够容易地确定特定位点在不同编码体系下的对应关系(例如，EU编码)。
术语“抗体-药物偶联物”(ADC)指与一种或多种化学合成分子(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。
本公开的术语“与CD40结合”，指能与人CD40相互作用。本公开的术语“抗原结合位点”指抗原上不连续的，由本公开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。
本公开所用的术语“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”是指本公开的抗体与来自不同物种的CD40结合的能力。例如，结合人CD40的本公开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)旨在包括任何可测量的细胞生长的降低。
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。
本公开中所述的“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；IgG2/4嵌合体、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，可以用人CD40或其片段免疫小鼠，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。公开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。
本公开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。
本公开的抗体指单克隆抗体。本公开所述的单克隆抗体(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-嫁接)，或其它现有技术进行重组得到。
“施用”、“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。“施用”、“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。“施用”、“给予”和“处理”还意指通过试剂、诊断、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”意指给予受试者内用或外用的治疗剂，诸如包含本公开的任一种抗体或其抗原结合片段的组合物，所述受试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，在受治疗受试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，例如受试者的疾病状态、年龄和体重，以及药物在受试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。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个受试者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受试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 PCTCN2019121941-appb-000001
Figure PCTCN2019121941-appb-000002
“有效量”包含足以改善或预防医学症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定所述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、所述的总体健康情况、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”指根据背景在生物、细胞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残基占据时(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，术语“转化的细胞”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组成和或含量方面不可能精确相同。该术语包括与最初的细胞具有相同的功能或生物学活性的突变后代。
“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。
实施例
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试剂，其各自的序列来源见表1。所述蛋白试剂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至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稳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 PCTCN2019121941-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 PCTCN2019121941-appb-000004
实施例4 抗CD40抗体阻断CD40和CD40L结合的实验
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之间的结合。
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.阻断人hCD40/hCD40L结合的ELISA实验结果
抗体株 IC50(μg/mL)
1D9 0.2634
2H6 0.2682
9E5 0.2787
14C10 0.3001
38B4 0.2934
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)，HBS-EP+10×缓冲溶液(Cat.#BR-1006-69，GE)用双蒸水稀释至1×(pH 7.4)。
实验得到的数据用BiacoreX100 evaluation software2.0 GE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抗体的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信号通路的激活水平。本实验通过检测细胞HEK-Blue CD40L的激活，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至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至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博莱霉素Zeocin和30μg/mL杀稻瘟素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抗体的报告基因细胞活性实验结果
Figure PCTCN2019121941-appb-000005
实施例7 抗CD40抗体激活DC细胞的实验
从正常人外周血中分离PBMC，然后用CD14 MACS珠分选单核细胞。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，用CD209-PE、CD1a-PerCP/Cy5.5和CD14-PE/Cy7染色，通过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，加入各待测抗体及对照抗体，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细胞表面的激活分子CD80和CD86，并呈剂量依赖效应，总体效果与两株对照抗体(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)，进行反转录(PrimeScript TM Reverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用小鼠Ig-Primer Set(Novagen,TB326 Rev.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。
(1)鼠单抗2H6的重链和轻链可变区序列如下：
2H6 HCVR
Figure PCTCN2019121941-appb-000006
2H6 LCVR
Figure PCTCN2019121941-appb-000007
其含有下表5中的CDR序列：
表5. 2H6的CDR序列
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
(2)9E5的重链和轻链可变区序列如下：
9E5 HCVR
Figure PCTCN2019121941-appb-000008
Figure PCTCN2019121941-appb-000009
9E5 LCVR
Figure PCTCN2019121941-appb-000010
其含有下表6中的CDR序列：
表6. 9E5的CDR序列
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
(3)1D9的重链和轻链可变区序列如下：
1D9 HCVR
Figure PCTCN2019121941-appb-000011
1D9 LCVR
Figure PCTCN2019121941-appb-000012
其含有下表7中的CDR序列：
表7. 1D9的CDR序列
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
(4)14C10的重链和轻链可变区序列如下：
14C10 HCVR
Figure PCTCN2019121941-appb-000013
14C10 LCVR
Figure PCTCN2019121941-appb-000014
其含有下表8中的CDR序列：
表8. 14C10的CDR序列
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
(5)38B4的重链和轻链可变区序列如下：
38B4 HCVR
Figure PCTCN2019121941-appb-000015
38B4 LCVR
Figure PCTCN2019121941-appb-000016
其含有下表9中的CDR序列：
表9. 38B4的CDR序列
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 PCTCN2019121941-appb-000017
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种系数据库比较，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，本公开抗体优选人种系轻链模版Vk1-33/JK4(用于2H6)或Vk2-28/JK4(用于9E5)。
人类种系重链框架区来自人重链，本公开抗体优选人种系重链模版VH1-69/JH6(用于2H6)或VH1-2/JH6(用于9E5)，如下所示：
2H6优选人种系重链模版IGHV1-69
Figure PCTCN2019121941-appb-000018
2H6优选人种系轻链模板IGkV1-33
Figure PCTCN2019121941-appb-000019
9E5优选人种系重链模版IGHV1-2
Figure PCTCN2019121941-appb-000020
9E5优选人种系轻链模板IGkV2-28
Figure PCTCN2019121941-appb-000021
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。
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：
Figure PCTCN2019121941-appb-000022
hu2H6-H1b：
Figure PCTCN2019121941-appb-000023
hu2H6-H1c：
Figure PCTCN2019121941-appb-000024
hu9E5-H1a：
Figure PCTCN2019121941-appb-000025
hu9E5-H1b：
Figure PCTCN2019121941-appb-000026
hu9E5-H1c：
Figure PCTCN2019121941-appb-000027
hu2H6-L1a：
Figure PCTCN2019121941-appb-000028
hu2H6-L1b：
Figure PCTCN2019121941-appb-000029
hu2H6-L1c：
Figure PCTCN2019121941-appb-000030
Figure PCTCN2019121941-appb-000031
hu9E5-L1a：
Figure PCTCN2019121941-appb-000032
hu9E5-L1b：
Figure PCTCN2019121941-appb-000033
hu9E5-L1c：
Figure PCTCN2019121941-appb-000034
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)，其各自的完整轻链和重链序列如SEQ ID NO:17-20所示。
hu2H6 HC：
Figure PCTCN2019121941-appb-000035
hu2H6 LC：
Figure PCTCN2019121941-appb-000036
hu9E5 HC：
Figure PCTCN2019121941-appb-000037
Figure PCTCN2019121941-appb-000038
hu9E5 LC：
Figure PCTCN2019121941-appb-000039
实施例10 人源化抗体的测试数据
本公开人源化抗体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，本公开人源化抗人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对照的10倍以上、Pfizer对照的4倍以上。
表11 人源化hu2H6和hu9E5抗体的体外活性
Figure PCTCN2019121941-appb-000040
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+微珠(microbeads)试剂盒分选单核细胞，按照试剂盒所提供的规程进行CD14+单核细胞的分离，即每10 7个细胞加入20μl抗-CD14微珠，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 IL-4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+微珠试剂盒分选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和对照抗体G12(即Alligator Bioscience的ADC-1013)，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：
(1)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。
(2)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；对比对照抗体G12的抑瘤作用总体相当或稍好，如图3和图4所示。
实施例12 重链恒定区突变的抗CD40抗体的制备
本实施例中制备了前述抗CD40抗体在重链恒定区突变的变体。
具体而言：
-将hu2H6的重链SEQ ID NO:17的第266位氨基酸从丝氨酸(S)突变为谷氨酸(E)，以获得突变体hu2H6-M；
-将hu2H6的重链SEQ ID NO:17的第266位氨基酸从丝氨酸(S)突变为谷氨酸(E)，且第324位氨基酸从天门冬酰胺(N)突变为丝氨酸(S)，且第327位氨基酸从亮氨酸(L)突变为苯丙氨酸(F)，以获得突变体hu2H6-SELFNS；
-将hu9E5的重链SEQ ID NO:19的第262位氨基酸从丝氨酸(S)突变为谷氨酸(E)，以获得突变体hu9E5-M；
-将hu9E5的重链SEQ ID NO:19的第262位氨基酸从丝氨酸(S)突变为谷氨酸(E)，且第323位氨基酸从亮氨酸(L)突变为苯丙氨酸(F)，以获得突变体hu9E5-SELF；
-将hu9E5的重链SEQ ID NO:19的第262位氨基酸从丝氨酸(S)突变为谷氨酸(E)，且第320位氨基酸从天门冬酰胺(N)突变为丝氨酸(S)，且第323位氨基酸从亮氨酸(L)突变为苯丙氨酸(F)，以获得突变体hu9E5-SELFNS。
hu2H6-M、hu2H6-SELFNS的重链序列如下SEQ ID NO：61、62所示，轻链 序列都如SEQ ID NO：18所示。
hu9E5-M、hu9E5-SELF、hu9E5-SELFNS的重链序列如下SEQ ID NO：63、64、67所示，其轻链序列都如SEQ ID NO：20所示。
hu2H6-M HC：
Figure PCTCN2019121941-appb-000041
hu2H6-SELFNS HC：
Figure PCTCN2019121941-appb-000042
hu9E5-M HC：
Figure PCTCN2019121941-appb-000043
hu9E5-SELF HC：
Figure PCTCN2019121941-appb-000044
Figure PCTCN2019121941-appb-000045
hu9E5-SELFNS HC：
Figure PCTCN2019121941-appb-000046
此外，SEQ ID NO：61、62、63、64、67的最后一位氨基酸K可以替换为A，该突变不影响抗体的活性，但可以一定程度地提高抗体的成药性。
以及，根据CN104918957A中另一种抗CD40抗体APX005S267E的可变区(即其第1-120位氨基酸)制备获得抗体005M用作阳性对照，抗体005M重链的第121-450位氨基酸序列和hu9E5-M重链的113-442位氨基酸序列相同。005M具体序列如下：
005M-HC：
Figure PCTCN2019121941-appb-000047
005M-LC：
Figure PCTCN2019121941-appb-000048
将005M的重链SEQ ID NO:65的第331位氨基酸从亮氨酸(L)突变为苯丙氨酸(F)，以获得突变体APX005-SELF；将005M的重链SEQ ID NO:65的第328位氨基酸从天门冬酰胺(N)突变为丝氨酸(S)，第331位氨基酸从亮氨酸(L)突变为苯丙氨酸(F)，以获得突变体APX005-SELFNS。
APX005-SELF HC(L331F)：
Figure PCTCN2019121941-appb-000049
APX005-SELFNS HC(L331F、N328S)：
Figure PCTCN2019121941-appb-000050
经测序确认，制备获得抗体hu2H6-M、hu2H6-SELFNS、hu9E5-M、hu9E5-SELF、hu9E5-SELFNS和005M、APX005-SELF、APX005-SELFNS。
实施例13 含有重链恒定区突变的抗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珠分选单核细胞。加入含25ng/mL IL-4和50ng/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，用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，加入各待测抗体及对照抗体，分别设置相应梯度浓度稀释，获得0.01nM、0.16nM、0.8nM、4nM、20nM、100nM的抗体梯度。培养48小时后，收集细胞，进行CD86和HLA-DR染色，FACS检测收集数据。APX005M-SELFNS与2H6-SELFNS均表现出极强的激动剂活性，激活了DC细胞表面的激活分子CD86，并呈剂量依赖效应，效果强于Alligator的对照抗体G12。参见图6。
实施例14 重链恒定区突变的抗CD40抗体激活DC细胞产生细胞因子实验
从正常人外周血中分离PBMC，然后用CD14MACS beads分选单核细胞。加 入含25ng/mL IL4和50ng/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，用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，加入各待测抗体及对照抗体，分别设置相应梯度浓度稀释，获得0.01nM、0.16nM、0.8nM、4nM、20nM、100nM的抗体梯度。培养48小时后，收集上清，用ELISA检测IL-12 p40的含量。
APX005M-SELFNS、APX005M-SELF、2H6-SELFNS、9E5-SELFNS均表现出极强的激动剂活性，促进DC细胞分泌细胞因子IL-12 p40，并呈剂量依赖效应，效果均强于Alligator的对照抗体G12。结果参见图7A、7B和表12、13。
表12.抗CD40抗体促进DC细胞分泌细胞因子IL-12 p40的结果
Figure PCTCN2019121941-appb-000051
(注：“/”表示未测试该浓度。)
表13.抗CD40抗体促进DC细胞分泌细胞因子IL-12 p40的结果
Figure PCTCN2019121941-appb-000052
实施例15 重链恒定区突变的抗CD40抗体能够抑制小鼠肿瘤的生长
本实施例在人源化hFcγR/hCD40 C57BL/6小鼠的MC38肿瘤模上，通过肿瘤 的大小及小鼠体重来评价CD40抗体用药对抗肿瘤作用及其安全性。
MC38细胞的培养与准备方法：MC38小鼠结肠癌细胞系，使用DMEM培养(10％FBS、1％青霉素-链霉素、1mM丙酮酸钠、10mM HEPES),细胞增殖到培养皿的80％-90％的密度时，加入胰蛋白酶-EDTA(0.25％)，于37℃放置3到5分钟消化，之后用含有10％FBS的培养基终止，然后离心，并用PBS洗净2次，最后用PBS重悬制备单细胞悬液，并调整细胞密度到10 7细胞/mL备用。
MC38肿瘤模型的建立方法：使用上述制备的MC38单细胞悬液，在第7天时将2x10 6个MC38细胞(200μL)接种于32只人源化hFcγR/hCD40 C57BL/6小鼠(由上海交通大学医学院李福彬课题组繁殖，SPF级饲养)右肋部皮下，待小鼠平均肿瘤体积达到55mm 3左右时，随机分成4组，每组8只。
分组后，根据如表12的方案给予单剂腹腔注射抗CD40抗体，每周两次测量肿瘤体积，称体重，记录数据。其中，对照IgG、hu9E5、hu9E5-M均由上海恒瑞医药股份有限公司提供，用PBS稀释，使最终浓度为0.3mg/mL。
抗体抗肿瘤活性的评价指标：
1)从受试小鼠分组开始，连续测量小鼠的肿瘤体积，使用肿瘤体积大小作为评价受试抗体的抗肿瘤活性指标，肿瘤体积(TV)计算公式：
TV＝0.5×L 短×L 短×L 长,
其中L 短是肿瘤的最短直径,L 长是肿瘤的最长直径。
2)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的百分比值。计算公式如下：
T/C％＝(T-T 0)/(C-C 0)×100
其中，T、C为实验结束时的肿瘤体积；T 0、C 0为实验开始时的肿瘤体积。
3)相对肿瘤抑制率TGI(％)＝(1-T/C)×100％。
数据表达和统计学处理：所有数据采用GraphPad Prism 5.0软件分析。数据用平均值±标准差表示，各组间采用单因素方差分析。P<0.05为差异有统计学意义。
表14.实验分组及给药方案
Figure PCTCN2019121941-appb-000053
(注：三天一次，给药3次；ip：腹腔注射)
各组抗体在hFcγR/hCD40Tg小鼠的MC38肿瘤模型中的体内活性结果，通过肿瘤体积大小的变化可判断，在第0天、第3天和第6天给予对照抗体和受试抗 体结束以后，与对照组(对照IgG)相比，hu9E5组、hu9E5-M组和005M组小鼠肿瘤体积的增长受到抑制，相对肿瘤抑制率分别为42.0％、68.9％、53.8％。hu9E5有一定的抗肿瘤活性(p>0.05),hu9E5-M和005M具有强抗肿瘤活性(p<0.05)，并且，hu9E5-M具有比005M更有益的效果，如表15和图5所示。
表15.肿瘤体积(cm 3)
Figure PCTCN2019121941-appb-000054
(注：*p<0.05及以下表示具有统计学意义。)
结果显示，在对应于SEQ ID NO:17的第266位氨基酸或SEQ ID NO:19的第262位氨基酸将S突变为E，能显著提高本申请中抗CD40抗体在体内的抗肿瘤效果。
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 (23)

  1. 一种抗CD40抗体或其抗原结合片段，其在重链恒定区存在突变，其中：
    1)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，
    2)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；且
    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；
    或者，
    3)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，
    4)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；且
    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，
    5)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；且
    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。
  2. 如权利要求1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、嵌合抗体、人源化抗体、人抗体或其抗原结合片段。
  3. 如权利要求2所述的抗CD40抗体或其抗原结合片段，其中所述鼠源抗体或嵌合抗体可变区的氨基酸序列选自：
    1)重链可变区如SEQ ID NO.1所示，轻链可变区如SEQ ID NO.2所示；
    2)重链可变区如SEQ ID NO.9所示，轻链可变区如SEQ ID NO.10所示；
    3)重链可变区如SEQ ID NO.37所示，轻链可变区如SEQ ID NO.38所示；
    4)重链可变区如SEQ ID NO.45所示，轻链可变区如SEQ ID NO.46所示；
    5)重链可变区如SEQ ID NO.53所示，轻链可变区如SEQ ID NO.54所示。
  4. 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，
    优选地，人源IgG1、IgG2或IgG4重链FR区，
    更优选，人源IgG1或IgG2重链FR区。
  5. 如权利要求2所述的抗CD40抗体或其抗原结合片段，其中，
    所述人源化抗体的轻链可变区上的轻链FR区来源于：
    如SEQ ID NO:22所示的人种系轻链IGkV1-33序列、或
    如SEQ ID NO:24所示的人种系轻链IGkV2-28序列；
    所述人源化抗体的重链可变区上的重链FR区来源于：
    如SEQ ID NO:21所示的人种系重链IGHV1-69序列、或
    如SEQ ID NO:23所示的人种系重链IGHV1-2序列；
    优选地，人源化抗体的轻链可变区上的轻链FR区来源于如SEQ ID NO:22所示的人种系轻链IGkV1-33，且人源化抗体的重链可变区上的重链FR区来源于如SEQ ID NO:21所示的人种系重链IGHV1-69；或，
    人源化抗体的轻链可变区上的轻链FR区来源于如SEQ ID NO:24所示的人种系轻链IGkV2-28，且重链可变区上的重链FR区来源于如SEQ ID NO:23所示的人种系重链IGHV1-2。
  6. 如权利要求5所述的抗CD40抗体或其抗原结合片段，其中：
    所述人源化抗体轻链为如SEQ ID NO:18或SEQ ID NO:20所示或其变体，所述的变体在轻链可变区有0至10个氨基酸突变，优选在第2位和/或第3位氨基酸位点的突变，更优选突变后的氨基酸残基为I、V或L；和/或，
    重链恒定区突变前，所述人源化抗体重链为如SEQ ID NO:17或SEQ ID NO:19所示或其变体；所述变体在重链可变区有0至10个氨基酸突变，优选在第6位和/或第8位氨基酸位点的突变，更优选突变后的氨基酸为I、A或L。
  7. 如权利要求6所述的抗CD40抗体或其抗原结合片段，其中：
    重链的可变区如SEQ ID NO：25-30之一或其变体所示，
    轻链的可变区如SEQ ID NO：31-36之一或其变体所示；
    优选地，选自以下：
    重链的可变区如SEQ ID NO：26或其变体所示，轻链的可变区如SEQ ID NO：33或其变体所示；或
    重链的可变区如SEQ ID NO：30或其变体所示，轻链的可变区如SEQ ID NO：34或其变体所示；或
    重链的可变区如SEQ ID NO：17或其变体所示，轻链的可变区如SEQ ID NO：18或其变体所示；或
    重链的可变区如SEQ ID NO：19或其变体所示，轻链的可变区如SEQ ID NO：20或其变体所示。
  8. 如权利要求1-7任一项所述的抗CD40抗体或其抗原结合片段，其中所述重链在选自以下的位点处包含氨基酸残基的突变：
    1)对应于SEQ ID NO：19的第262位或对应于SEQ ID NO：17的第266位；和/或
    2)对应于SEQ ID NO：19的第320位或对应于SEQ ID NO：17的第324位；和/或
    3)对应于SEQ ID NO：19的第323位或对应于SEQ ID NO：17的第327位。
  9. 如权利要求8所述的抗CD40抗体或其抗原结合片段，所述重链包含选自以下的突变：
    1)在对应于SEQ ID NO：19的第262位或SEQ ID NO：17的第266位突变为谷氨酸；和/或
    2)在对应于SEQ ID NO：19的第320位或SEQ ID NO：17的第324位突变为丝氨酸；和/或
    3)在对应于SEQ ID NO：19的第323位或SEQ ID NO：17的第327位突变为苯丙氨酸。
  10. 如权利要求9所述的抗CD40抗体或其抗原结合片段，所述重链包含选自以下的突变或其组合：
    1)在对应于SEQ ID NO：19的262E；
    2)在对应于SEQ ID NO：19的262E和323F；
    3)在对应于SEQ ID NO：19的262E、320S和323F；
    4)在对应于SEQ ID NO：17的266E；
    5)在对应于SEQ ID NO：17的266E、324S和327F。
  11. 根据权利要求1-2和4-10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包含：
    如SEQ ID NO:61或62所示的重链，和如SEQ ID NO:18所示的轻链；
    或，所述人源化抗体包含：
    如SEQ ID NO:63、64、67所示的重链，和如SEQ ID NO:20所示的轻链。
  12. 根据权利要求1-11任一项所述的抗CD40抗体或其抗原结合片段，其中所述重链羧基末端的氨基酸残基突变为丙氨酸残基；
    优选地，SEQ ID NO:61、62、63、64或67所示重链羧基末端的氨基酸残基突变为丙氨酸残基。
  13. 一种单链抗体，其含有：
    如权利要求1-12任一项所限定的轻链可变区和重链可变区。
  14. 一种抗体-药物偶联物，其中所述的抗体含有如权利要求1-12任一项所限定的轻链可变区和重链可变区；优选地含有如权利要求1-12任一项所述的抗CD40抗体或其抗原结合片段。
  15. 一种核酸分子，其编码权利要求1-12任一项所述的抗CD40抗体或抗原结合片段、权利要求13所述的单链抗体。
  16. 一种载体，其含有如权利要求15所述的核酸分子。
  17. 一种宿主细胞，其含有或表达权利要求16所述的载体。
  18. 如权利要求17所述的宿主细胞，所述的宿主细胞选自：细菌、酵母菌、哺乳动物细胞；
    优选地，所述的细菌为大肠杆菌；
    优选地，所述的酵母菌为毕赤酵母；
    优选地，所述的哺乳动物细胞为中国仓鼠卵巢细胞或人胚肾293细胞。
  19. 一种药物组合物，其含有：
    如权利要求1-12任一项所述的抗CD40抗体或其抗原结合片段、权利要求13所述的单链抗体、或者权利要求14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。
  20. 选自以下的任一项在制备药物中的用途：
    如权利要求1-12任一项所述的抗CD40抗体或其抗原结合片段、权利要求13所述的单链抗体、或者权利要求14所述的抗体-药物偶联物、权利要求19所述的药物组合物；
    所述药物用于治疗或预防CD40介导的或CD40L介导的疾病或病症；
    优选地，所述的疾病或病症为癌症；
    最优选，所述的癌症选自：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。
  21. 选自以下的任一项在制备药物中的用途：
    如权利要求1-12任一项所述的抗CD40抗体或其抗原结合片段、权利要求13所述的单链抗体、或者权利要求14所述的抗体-药物偶联物、权利要求19所述的药物组合物；
    所述药物用于改善自身免疫性疾病患者或炎性疾病患者的症状。
  22. 一种预防或治疗CD40介导的或CD40L介导的疾病的方法，所述方法包括使受试者接触预防有效量或治疗有效量的选自以下的任一项：
    权利要求1-12任一项所述的抗CD40抗体或其抗原结合片段、权利要求13所述的单链抗体、权利要求14所述的抗体-药物偶联物、权利要求19所述的药物组合物；
    优选地，所述的疾病为癌症；
    最优选，所述的癌症选自：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。
  23. 一种改善自身免疫性疾病或炎性疾病症状的方法，所述方法包括使受试者接触预防有效量或治疗有效量的选自以下的任一项：
    权利要求1-12任一项所述的抗CD40抗体或其抗原结合片段、权利要求13所述的单链抗体、权利要求14所述的抗体-药物偶联物、权利要求19所述的药物组合物。
PCT/CN2019/121941 2018-11-30 2019-11-29 抗cd40抗体、其抗原结合片段及其医药用途 WO2020108611A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023025194A1 (zh) 2021-08-24 2023-03-02 江苏恒瑞医药股份有限公司 Fap/cd40结合分子及其医药用途

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114573698B (zh) * 2022-03-16 2023-01-06 沈阳三生制药有限责任公司 一种FcRn抗原结合蛋白及其制备方法和应用
WO2023232036A1 (zh) * 2022-05-31 2023-12-07 明济生物制药（北京）有限公司 抗cd40抗体和抗pd-l1×cd40双特异抗体及其应用
CN117229396A (zh) * 2022-06-06 2023-12-15 普米斯生物技术(珠海)有限公司 抗cd40抗体及其用途
WO2024076969A1 (en) * 2022-10-04 2024-04-11 Memorial Sloan-Kettering Cancer Center Anti-cd40 antibodies and uses thereof

Citations (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1198647A (zh) 1997-05-07 1998-11-11 三菱电机株式会社 扬声器安装结构
WO2002028904A2 (en) 2000-10-02 2002-04-11 Chiron Corporation Human anti-cd40 antibodies
CN1369015A (zh) 1999-06-08 2002-09-11 西雅图基因公司 重组抗－cd40抗体及其应用
WO2004099249A2 (en) 2003-05-02 2004-11-18 Xencor, Inc. Optimized fc variants and methods for their generation
CN1582165A (zh) 2001-11-09 2005-02-16 辉瑞产品公司 Cd40的抗体
WO2006019447A1 (en) 2004-07-15 2006-02-23 Xencor, Inc. Optimized fc variants
CN101014386A (zh) 2004-04-27 2007-08-08 诺华疫苗和诊断公司 拮抗性抗－cd40单克隆抗体及其使用方法
CN101237882A (zh) 2005-05-26 2008-08-06 西雅图基因公司 人源化的抗-cd40抗体及其使用方法
CN101289510A (zh) 2001-04-27 2008-10-22 麒麟医药株式会社 抗cd40单克隆抗体
WO2008150494A1 (en) 2007-05-30 2008-12-11 Xencor, Inc. Methods and compositions for inhibiting cd32b expressing cells
CN101490086A (zh) 2006-05-09 2009-07-22 潘詹奈蒂克斯有限公司 来自ch5D12抗体的去免疫拮抗性抗人CD40单克隆抗体
CN101508734A (zh) * 2001-04-27 2009-08-19 协和发酵麒麟株式会社 抗cd40单克隆抗体
WO2011123489A2 (en) 2010-03-31 2011-10-06 Boehringer Ingelheim International Gmbh Anti-cd40 antibodies
US8084582B2 (en) 2003-03-03 2011-12-27 Xencor, Inc. Optimized anti-CD20 monoclonal antibodies having Fc variants
WO2012149356A2 (en) 2011-04-29 2012-11-01 Apexigen, Inc. Anti-cd40 antibodies and methods of use
WO2013034904A1 (en) 2011-09-05 2013-03-14 Alligator Bioscience Ab Anti-cd40 antibodies, uses and methods
US8734791B2 (en) 2002-03-01 2014-05-27 Xencor, Inc. Optimized fc variants and methods for their generation
CN103842382A (zh) 2011-04-21 2014-06-04 百时美施贵宝公司 拮抗cd40之抗体多肽
WO2014145806A2 (en) 2013-03-15 2014-09-18 Xencor, Inc. Heterodimeric proteins
WO2015009853A1 (en) 2013-07-16 2015-01-22 University Of Louisville Research Foundation, Inc. Compositions for mucusal delivery, useful for treating papillomavirus infections
WO2015091853A2 (en) * 2013-12-19 2015-06-25 Alligator Bioscience Ab Antibodies
CN104918957A (zh) 2012-10-30 2015-09-16 埃派斯进有限公司 抗-cd40抗体及其使用方法
WO2016196314A1 (en) 2015-05-29 2016-12-08 Abbvie Inc. Anti-cd40 antibodies and uses thereof
WO2017004006A1 (en) 2015-06-29 2017-01-05 Bristol-Myers Squibb Company Antibodies to cd40
WO2017040932A1 (en) 2015-09-04 2017-03-09 Primatope Therapeutics Inc. Humanized anti-cd40 antibodies and uses thereof
US9657106B2 (en) 2003-03-03 2017-05-23 Xencor, Inc. Optimized Fc variants
WO2017205742A1 (en) * 2016-05-27 2017-11-30 Abbvie Biotherapeutics Inc. Anti-cd40 antibodies and their uses
WO2018219327A1 (zh) * 2017-06-01 2018-12-06 江苏恒瑞医药股份有限公司 抗cd40抗体、其抗原结合片段及其医药用途

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2004309275B2 (en) * 2003-12-25 2010-12-23 Kyowa Kirin Co., Ltd. Mutants of anti-CD40 antibody
US20070292439A1 (en) * 2004-04-27 2007-12-20 Novartis Vaccines And Diagnostics, Inc. Antagonist Anti-Cd40 Monoclonal Antibodies and Methods for Their Use
WO2011139974A2 (en) * 2010-05-03 2011-11-10 Abbott Laboratories Anti-pai-1 antibodies and methods of use thereof
JP6654187B2 (ja) * 2014-08-12 2020-02-26 アリゲーター・バイオサイエンス・アーベー 抗ｃｄ４０抗体を用いた併用療法
BR112017020952A2 (pt) * 2015-04-13 2018-07-10 Five Prime Therapeutics Inc método de tratamento de câncer, composição e uso da composição
EA201890162A1 (ru) * 2015-06-29 2018-07-31 Бристол-Маерс Сквибб Компани Антитела к cd40 с повышенной агонистической активностью
CN106957366B (zh) * 2016-01-12 2022-02-01 上海昀怡健康科技发展有限公司 一种C5aR抗体及其制备方法和应用
RU2019106663A (ru) * 2016-08-12 2020-09-14 Мерк Патент Гмбх Комбинированная терапия рака
AU2018241654A1 (en) * 2017-03-30 2019-11-07 Jiangsu Hengrui Medicine Co., Ltd. Method for preparing antibody-drug conjugate

Patent Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1198647A (zh) 1997-05-07 1998-11-11 三菱电机株式会社 扬声器安装结构
CN1369015A (zh) 1999-06-08 2002-09-11 西雅图基因公司 重组抗－cd40抗体及其应用
WO2002028904A2 (en) 2000-10-02 2002-04-11 Chiron Corporation Human anti-cd40 antibodies
CN101508734A (zh) * 2001-04-27 2009-08-19 协和发酵麒麟株式会社 抗cd40单克隆抗体
CN101289510A (zh) 2001-04-27 2008-10-22 麒麟医药株式会社 抗cd40单克隆抗体
CN100430419C (zh) 2001-04-27 2008-11-05 麒麟医药株式会社 抗cd40单克隆抗体
CN1582165A (zh) 2001-11-09 2005-02-16 辉瑞产品公司 Cd40的抗体
US8734791B2 (en) 2002-03-01 2014-05-27 Xencor, Inc. Optimized fc variants and methods for their generation
US8084582B2 (en) 2003-03-03 2011-12-27 Xencor, Inc. Optimized anti-CD20 monoclonal antibodies having Fc variants
US9657106B2 (en) 2003-03-03 2017-05-23 Xencor, Inc. Optimized Fc variants
WO2004099249A2 (en) 2003-05-02 2004-11-18 Xencor, Inc. Optimized fc variants and methods for their generation
CN104788565A (zh) * 2003-05-02 2015-07-22 赞科股份有限公司 优化的Fc变体及其产生方法
CN101014386A (zh) 2004-04-27 2007-08-08 诺华疫苗和诊断公司 拮抗性抗－cd40单克隆抗体及其使用方法
WO2006019447A1 (en) 2004-07-15 2006-02-23 Xencor, Inc. Optimized fc variants
CN101237882A (zh) 2005-05-26 2008-08-06 西雅图基因公司 人源化的抗-cd40抗体及其使用方法
CN101490086A (zh) 2006-05-09 2009-07-22 潘詹奈蒂克斯有限公司 来自ch5D12抗体的去免疫拮抗性抗人CD40单克隆抗体
WO2008150494A1 (en) 2007-05-30 2008-12-11 Xencor, Inc. Methods and compositions for inhibiting cd32b expressing cells
CN102918063A (zh) * 2010-03-31 2013-02-06 贝林格尔.英格海姆国际有限公司 抗-cd40抗体
WO2011123489A2 (en) 2010-03-31 2011-10-06 Boehringer Ingelheim International Gmbh Anti-cd40 antibodies
CN103842382A (zh) 2011-04-21 2014-06-04 百时美施贵宝公司 拮抗cd40之抗体多肽
WO2012149356A2 (en) 2011-04-29 2012-11-01 Apexigen, Inc. Anti-cd40 antibodies and methods of use
WO2013034904A1 (en) 2011-09-05 2013-03-14 Alligator Bioscience Ab Anti-cd40 antibodies, uses and methods
US20180273630A1 (en) * 2012-10-30 2018-09-27 Apexigen, Inc. Anti-cd40 antibodies and methods of use
CN104918957A (zh) 2012-10-30 2015-09-16 埃派斯进有限公司 抗-cd40抗体及其使用方法
WO2014145806A2 (en) 2013-03-15 2014-09-18 Xencor, Inc. Heterodimeric proteins
WO2015009853A1 (en) 2013-07-16 2015-01-22 University Of Louisville Research Foundation, Inc. Compositions for mucusal delivery, useful for treating papillomavirus infections
WO2015091853A2 (en) * 2013-12-19 2015-06-25 Alligator Bioscience Ab Antibodies
WO2016196314A1 (en) 2015-05-29 2016-12-08 Abbvie Inc. Anti-cd40 antibodies and uses thereof
WO2017004006A1 (en) 2015-06-29 2017-01-05 Bristol-Myers Squibb Company Antibodies to cd40
WO2017040932A1 (en) 2015-09-04 2017-03-09 Primatope Therapeutics Inc. Humanized anti-cd40 antibodies and uses thereof
WO2017205742A1 (en) * 2016-05-27 2017-11-30 Abbvie Biotherapeutics Inc. Anti-cd40 antibodies and their uses
WO2018219327A1 (zh) * 2017-06-01 2018-12-06 江苏恒瑞医药股份有限公司 抗cd40抗体、其抗原结合片段及其医药用途

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Antibodies: A Laboratory Manual"
"The Immunoglobulin FactsBook", 2001
HOLLIGERHUDSON, NAT. BIOTECHNOL., vol. 23, 2005, pages 1126 - 1136
J. BIOL. CHEM, vol. 243, 1968, pages 3558
See also references of EP3892634A4
TONG ET AL., CANCER GENE THERAPY, vol. 10, 2003, pages 1 - 13
VAN MIERLO ET AL., PROC. NATL. ACAD. SCI. USA, vol. 99, 2002, pages 5561 - 5566
WATSON ET AL.: "Molecular Biology of the Gene", 1987, THE BENJAMIN/CUMMINGS PUB. CO., pages: 224

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023025194A1 (zh) 2021-08-24 2023-03-02 江苏恒瑞医药股份有限公司 Fap/cd40结合分子及其医药用途

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