WO2018059465A1 - 抗cd27抗体、其抗原结合片段及其医药用途 - Google Patents

抗cd27抗体、其抗原结合片段及其医药用途 Download PDF

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WO2018059465A1
WO2018059465A1 PCT/CN2017/103842 CN2017103842W WO2018059465A1 WO 2018059465 A1 WO2018059465 A1 WO 2018059465A1 CN 2017103842 W CN2017103842 W CN 2017103842W WO 2018059465 A1 WO2018059465 A1 WO 2018059465A1
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antibody
seq
antigen
variable region
chain variable
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PCT/CN2017/103842
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French (fr)
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闫树德
蒋家骅
张连山
曹国庆
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江苏恒瑞医药股份有限公司
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Priority to EP17854915.0A priority Critical patent/EP3524624A4/en
Priority to CN201780005087.3A priority patent/CN108473586B/zh
Priority to US16/335,961 priority patent/US20190225698A1/en
Priority to JP2019517054A priority patent/JP7014783B2/ja
Publication of WO2018059465A1 publication Critical patent/WO2018059465A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—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
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an anti-CD27 antibody, an antigen-binding fragment of CD27, a chimeric antibody comprising the CDR region of the antibody, a humanized antibody, and a pharmaceutical composition comprising the human anti-CD27 antibody and antigen-binding fragment thereof, and as The use of anticancer drugs.
  • Tumor immunotherapy is a long-term hot spot in the field of anticancer drugs. It makes full use of and mobilizes killer T cells in tumor patients, killing and destroying tumors, and it may be one of the most effective and safe tumor treatment methods.
  • Activation of T cells in humans is a system with two signaling pathways, in addition to providing the first signal to T cells via antigen presenting cells to present MHC antigen peptides, and a series of costimulatory molecules to provide a second signal to enable T cells Produces a normal immune response.
  • T cells and antigen presenting cells includes a variety of accessory molecules that facilitate the production of immune responses, and CD27 is one of them. It belongs to the tumor necrosis factor receptor (TNF-R) superfamily and binds to T cells. Surface of the CD70.
  • CD27 is a glycosylated type I transmembrane protein, usually a homodimer linked by a disulfide bridge. The disulfide bridge is in the near cell membrane region of the extracellular domain (Camerini et al, J Immunol. 147: 3165-69, 1991).
  • TNF-R tumor necrosis factor receptor
  • CD27 is a glycosylated type I transmembrane protein, usually a homodimer linked by a disulfide bridge. The disulfide bridge is in the near cell membrane region of the extracellular domain (Camerini et al, J Immunol. 147: 3165-69, 1991).
  • the cross-linked CD27 antigen provides a synergistic
  • CD27 is expressed on mature thymocytes, most CD4+ and CD8+ topical blood T cells, natural killer cells, and B cells, and is also highly expressed in B-cell non-Hodgkin's lymphoma and B-cell chronic lymphocytic leukemia; In cytomegalovirus (CMV) infection, multiple sclerosis, sarcoidosis, and B-cell chronic lymphocytic leukemia, elevated levels of soluble CD27 protein are also observed in serum or disease-active sites (Kobata T et al., Proc. Natl .Acad. Sci. USA. 1995 (24): 11249-53; Ranheim EA et al, Blood. 1995 85(12): 3556-65; Loenen WA et al, Eur. J. Immunol. 1992 22:447).
  • CMV cytomegalovirus
  • the present patent application aims to provide anti-CD27 antibodies with high affinity, high selectivity and high biological activity, with specific functional properties that can be associated with the desired beneficial therapeutic effects.
  • the present invention provides an anti-CD27 antibody or antigen-binding fragment thereof, comprising:
  • An antibody light chain variable region comprising at least one LCDR selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NO : 14, SEQ ID NO: 15 or SEQ ID NO: 16;
  • An antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO :11, SEQ ID NO: 12 or SEQ ID NO: 13.
  • the anti-CD27 antibody or antigen-binding fragment thereof, wherein the antibody light chain variable region comprises as set forth in SEQ ID NO: 6 or SEQ ID NO: 14. LCDR1.
  • the anti-CD27 antibody or antigen-binding fragment thereof, wherein the antibody light chain variable region comprises as set forth in SEQ ID NO:7 or SEQ ID NO:15 LCDR2.
  • the anti-CD27 antibody or antigen-binding fragment thereof, wherein the antibody light chain variable region comprises as set forth in SEQ ID NO: 8 or SEQ ID NO: LCDR3 area.
  • the anti-CD27 antibody or antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises as set forth in SEQ ID NO: 3 or SEQ ID NO: HCDR1.
  • the anti-CD27 antibody or antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises as set forth in SEQ ID NO: 4 or SEQ ID NO: HCDR2.
  • the anti-CD27 antibody or antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises as set forth in SEQ ID NO: 5 or SEQ ID NO: HCDR3.
  • an anti-CD27 antibody or antigen-binding fragment thereof as described above wherein said antibody light chain variable region package is SEQ ID NO: 6, SEQ ID NO: 7 and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 8.
  • an anti-CD27 antibody or antigen-binding fragment thereof as described above wherein said antibody light chain variable region package is SEQ ID NO: 14, SEQ ID NO: 15 and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 16.
  • an anti-CD27 antibody or antigen-binding fragment thereof wherein said antibody heavy chain variable region comprises SEQ ID NO: 3, SEQ ID NO: 4, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5.
  • an anti-CD27 antibody or antigen-binding fragment thereof, wherein said antibody light chain variable region comprises:
  • LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;
  • LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;
  • antibody heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
  • a particularly preferred anti-CD27 antibody or antigen-binding fragment thereof can be selected from any of the following:
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the antibody heavy chain variable region comprises, for example, SEQ ID NO: 3, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 4 and SEQ ID NO: 5.
  • the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable region comprises, for example, SEQ ID NO: 11, LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 12 and SEQ ID NO: 13.
  • the antibody light chain variable region sequence is selected from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 10; the heavy chain variable region sequence is selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO: 9.
  • a particularly preferred anti-CD27 antibody or antigen-binding fragment thereof can be selected from any of the following:
  • the antibody light chain variable region sequence is SEQ ID NO: 2
  • the heavy chain variable region sequence is SEQ ID NO: 1.
  • the antibody light chain variable region sequence is SEQ ID NO: 10
  • the heavy chain variable region sequence is SEQ ID NO: 9.
  • an anti-CD27 antibody or antigen-binding fragment thereof wherein said antibody is a murine antibody or a fragment thereof.
  • an anti-CD27 antibody or antigen-binding fragment thereof wherein the antibody is a chimeric antibody or a fragment thereof.
  • the anti-CD27 antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody or a fragment thereof.
  • an anti-CD27 antibody or antigen-binding fragment thereof wherein said antibody is a human antibody or a fragment thereof.
  • an anti-CD27 humanized antibody or fragment thereof as described above, further comprising a constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human source IgG1, IgG2 or IgG4 constant region. Because IgG2 or IgG4 is not ADCC toxic, or IgG1 is toxic without ADCC (antibody-dependent cell-mediated cytotoxicity).
  • the anti-CD27 antibody or antigen-binding fragment thereof wherein the antigen-binding fragment is Fab, Fv, sFv, F(ab') 2 .
  • the invention further provides a DNA sequence encoding an anti-CD27 antibody or antigen-binding fragment thereof as described above.
  • the invention further provides an expression vector comprising the DNA sequence as described above.
  • the invention further provides a host cell transformed with an expression vector as described above.
  • a host cell as described above characterized in that said host cell is a bacterium, preferably Escherichia coli.
  • a host cell as described above is a yeast, preferably Pichia pastoris.
  • a host cell as described above is a mammalian cell, preferably a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) 293 cell.
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CD27 antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient, dilution or carrier.
  • the invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region as previously described.
  • the invention also provides a single chain antibody comprising a light chain variable region and a heavy chain variable region as previously described.
  • the invention also provides an antibody-drug conjugate comprising a light chain variable region and a heavy chain variable region as previously described.
  • the antibody-drug conjugates are well known in the art and are formed by the interconnection of antibody-linker-drug (toxin), known linkers include cleavage linkers, non-cleavage linkers, such as linkers including, but not limited to, SMCC, SPDP Etc.; Toxins are also well known in the art, such as DM1, DM4, MMAE, MMAF, and the like.
  • the invention further provides an anti-CD27 antibody or antigen-binding fragment thereof as described above, or a pharmaceutical composition comprising the same, including but not limited to a multispecific antibody, a single chain antibody, an antibody-drug conjugate, and the like, Use of a medicament for the treatment of a CD27 mediated disease or condition; wherein the disease is preferably a cancer and an autoimmune disease; more preferably a CD27 expressing cancer and an autoimmune disease; said cancer being most preferably rectal Cancer, brain cancer, kidney cancer, lung cancer, liver cancer, multiple myeloma, and melanoma; the autoimmune diseases are most preferably autoimmune encephalomyelitis, systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis.
  • the disease is preferably a cancer and an autoimmune disease; more preferably a CD27 expressing cancer and an autoimmune disease; said cancer being most preferably rectal Cancer, brain cancer, kidney cancer, lung cancer, liver cancer, multiple mye
  • the invention further provides a method of treating and preventing a CD27 mediated disease or condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-CD27 antibody or antigen-binding fragment thereof as described above, or A pharmaceutical composition comprising the same, including but not limited to a multispecific antibody, a single chain antibody, an antibody-drug conjugate, etc.; wherein the disease is preferably a cancer and an autoimmune disease; more preferably a CD27 expressing cancer And autoimmune diseases; the cancer is most preferably rectal cancer, brain cancer, kidney cancer, lung cancer, liver cancer, multiple myeloma and melanoma; the autoimmune disease is most preferably autoimmune encephalomyelitis, system Lupus erythematosus, multiple sclerosis and rheumatoid arthritis.
  • Figure 1 Affinity and kinetic parameters of anti-CD27 antibodies mAb076 and mAb077 with antigen, which were determined by Data Acquisition 7.1 software as described in the respective examples.
  • Figure 2 Data for binding of human anti-CD27 antibody mAb076 and mAb077 to human or mouse CD27 antigen expressed by HEK293 cells using flow cytometry, but not to the remaining TNFR superfamily receptors (cells with binding signals) The proportion of total cells).
  • Figure 3 Gradient dilution titration curve of anti-CD27 antibody mAb076 binding to human or mouse CD27 antigen expressed by HEK293 cells as determined by flow cytometry.
  • Figure 4 Data for binding of human anti-CD27 antibodies mAb076 and mAb077 to mouse T cells as determined by flow cytometry (the ratio of cells with binding signals to the total number of cells).
  • Figure 6 Gradient dilution titration curves for activation of the NF-kB signaling pathway by antibodies mAb076 and mAb077, detected using the luciferase reporter gene method.
  • Figure 7 Figure of activation of human peripheral blood mononuclear T cells by antibody mAb076 and mAb077 as determined by flow cytometry: (A).1F5, mAb076 and mAb077 promote T cell IFN- ⁇ expression. (B). Activation of T cell proliferation by the mAb076 antibody.
  • Figure 8 Affinity and kinetic parameters of binding of the anti-CD27 antibody mAb076 and mAb077 to the FcyRI receptor, as determined by Data Acquisition 7.1 software, as described in the corresponding examples.
  • FIG. 9 E.G7-OVA transplanted T lymphoma in mice treated with vehicle treated with control human IgGl antibody or with anti-CD27 mAb076-IgG antibody in mice treated with tumors Tumor size in days (in mm 3 ).
  • A is the tumor growth curve;
  • B is the relative change in body weight of the mouse. The error is expressed in standard error.
  • Figure 10 Tumors of human Raji-transplanted lymphoma versus days after tumor inoculation in PBS-treated mice, mice treated with control human IgG1 antibody or mice treated with anti-CD27 mAb077-IgG antibody Size (in mm 3 ).
  • A is the tumor growth curve;
  • B is the relative change in body weight of the mouse. The error is expressed in standard error.
  • antibody refers to an immunoglobulin which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, respectively. , ⁇ chain and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each of the five types of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, 2, 3, 4 or a variant thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • Each of the light chain variable region (VL) and the heavy chain variable region (VH) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or conform to the Kabat and Chothia numbering rules (HCDR1). ).
  • APC antigen presenting cell
  • T cells recognize this complex using the T cell receptor (TCR).
  • APCs include, but are not limited to, dendritic cells (DC), peripheral blood mononuclear cells (PBMC), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DC).
  • DC dendritic cells
  • PBMC peripheral blood mononuclear cells
  • monocytes B lymphoblasts
  • DC monocyte-derived dendritic cells
  • the term “antigen presentation” refers to the process by which APCs capture antigens and enable them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.
  • CD27 refers to a cell surface receptor that is a member of the TNF receptor superfamily.
  • CD27 is a molecule essential for the production and long-term maintenance of T cell immunity and plays a key role in regulating B cell activation and immunoglobulin synthesis.
  • CD27 includes any variant or isoform of CD27 (e.g., human CD27 registered with GENBANK under accession number AAH12160.1) naturally expressed by the cell.
  • the antibodies of the invention can be cross-reactive with CD27 from a non-human species. Alternatively, the antibody may also be specific for human CD27 and may not exhibit cross-reactivity with other species.
  • CD27 or any variant or isoform thereof can be expressed from nature Their cells or tissues are isolated or produced by recombinant techniques using techniques common in the art and described herein.
  • the anti-CD27 antibody targets human CD27 with a normal glycosylation pattern.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • Human antibodies of the invention can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody does not include an antibody in which a CDR sequence derived from a germline of another mammalian species, such as a mouse, has been grafted onto a human framework sequence (ie, "humanized antibody”).
  • humanized antibody also referred to as a CDR-grafted antibody, refers to an antibody produced by grafting a CDR sequence of a mouse into a human antibody variable region framework. It is possible to overcome the strong immune response induced by chimeric antibodies by carrying a large amount of mouse protein components. To avoid a decrease in activity while reducing immunogenicity, the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the constant region gene of the human antibody is cloned as needed to change the mouse.
  • the region gene and the human constant region gene are ligated into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC (antibody-dependent) after amino acid mutation Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.
  • the "antigen-binding fragment” as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, an F(ab')2 fragment, and an Fv fragment sFv fragment which binds to human CD27; and the antibody of the present invention One or more CDR regions selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 8.
  • the Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region and has the smallest antibody fragment of the entire antigen binding site.
  • Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • the two antibody variable regions can also be joined by a different linker into a single polypeptide chain, referred to as a single chain antibody or a single chain Fv (sFv).
  • binding to CD27 refers to the ability to interact with human CD27.
  • antigen binding site refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.
  • multispecific antibody is used in its broadest sense to encompass antibodies having multi-epitope specificity.
  • These multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; having two or more Antibodies of the VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region Different targets or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, Antibody fragments, etc., covalently or non-covalently linked together.
  • ADC antibody-drug conjugate
  • single-chain antibody is a single-stranded recombinant protein joined by a heavy chain variable region (VH) and a light chain variable region (VL) of a antibody via a ligation peptide, which is the smallest with complete antigen binding sites.
  • VH heavy chain variable region
  • VL light chain variable region
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • An epitope can be formed by an adjacent amino acid, or a non-adjacent amino acid juxtaposed by a tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, and the like. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.
  • the terms “specifically bind” and “selectively bind” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody has an equilibrium dissociation constant of less than about 10 -7 M or even less when measured by surface plasmon resonance (SPR) techniques in an instrument.
  • SPR surface plasmon resonance
  • K D binds to a predetermined antigen, and its affinity for binding to a predetermined antigen is at least twice its affinity for binding to a non-specific antigen other than a predetermined antigen or a closely related antigen such as BSA or the like.
  • the term “antibody recognizing an antigen” can be used interchangeably herein with the term “specifically bound antibody”.
  • cross-reactive refers to the ability of an antibody of the invention to bind to CD27 from a different species.
  • an antibody of the invention that binds to human CD27 can also bind to CD27 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (eg, SPR and ELISA), or binding or functional interactions with cells that express CD27 physiologically. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • Inhibiting or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of CD70 preferably reduces or alters the normal level or type of activity that occurs when CD70 binding occurs without inhibition or blockade. Inhibition and blockade are also intended to include a decrease in any measurable CD70 binding affinity compared to CD70 that is not contacted with an anti-CD27 antibody when contacted with an anti-CD27 antibody.
  • inhibiting growth eg, involving cells
  • inhibiting growth is intended to include any measurable reduction in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a particular antigen.
  • inducing for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • the "ADCC” described in the present invention is an antibody-dependent cell-mediated cytotoxicity, which means that a cell expressing an Fc receptor directly kills an antibody by Fc segment of the recognition antibody.
  • Target cells The ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc segment on IgG.
  • the modification refers to mutations in the heavy chain constant region of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2/4 chimera, F235E of IgG4, or L234A/E235A mutation.
  • a mouse can be immunized with human CD27 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region. Human FR germline sequences are available on the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN 014441351.
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • the cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the FC region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified and collected by a conventional technique.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange. The resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • the antibody of the present invention refers to a monoclonal antibody.
  • the monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain.
  • Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases, whether by inducing such symptoms to degenerate or inhibiting the progression of such symptoms to any degree of clinical right measurement.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount” effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition.
  • Embodiments of the invention eg, methods of treatment or preparations
  • a binding compound consisting essentially of the amino acid sequence recited may also include one or more amino acids that do not significantly affect the properties of the binding compound.
  • naturally occurring refers to the fact that the object can be found in nature.
  • polypeptide sequences or polynuclear acid sequences that are present in organisms (including viruses) that can be isolated from natural sources and that have not been intentionally modified in the laboratory by humans are naturally occurring.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the route and dosage of the method of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living organism, cell, or human body depending on the background.
  • Endogenous refers to a substance produced in a cell, organism or human body depending on the background.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • An in vitro phage display system was used for screening human antibody fragments directed against human CD27 antigen.
  • the antigens used included CD27-HisFc (Sino Biological #10039-H03H), CD27-Fc (Sino Biological #10039-H31H) and CD27-His (Sino Biological #10039-H08B1).
  • the antigen was directly adsorbed and immobilized on a Maxisorp 96-well screening plate (Thermo Scientific 446469), or biotinylated and adsorbed onto Dynabeads microparticles (M280, Streptavidin, Invitrogen #60210) and automatically screened using KingFisher (Thermo Scientific).
  • the standard phage display screening process was screened.
  • Fab uses the ForteBio Octet RED96 system to determine its affinity for human CD27 by first capturing the human CD27-HisFc fusion protein using the AHC (human IgG antibody Fc fragment specific) sensor of the BLI system and then immersing it in 5-10 ⁇ g/ Ml of each purified Fab protein in assay buffer. The collected data was processed by Data Acquisition 7.1 software and fitted to a 1:1 combined Langmuir model curve. The antibodies mAb076 and mAb077 are the two antibodies with optimal binding and were selected for further analysis and development experiments. Its binding to CD27 and its kinetic constant are shown in Figure 1.
  • the heavy and light chain variable region sequences of human mAb mAb076 are as follows:
  • the heavy and light chain variable region sequences of human mAb mAb077 are as follows:
  • Each antibody fragment was separately cloned into a mammalian cell expression vector expressing the light chain and heavy chain of the IgG1 subtype antibody.
  • the paired vectors were transfected into HEK293 cells in a transient manner according to standard procedures. After the expression, the supernatant was collected by centrifugation, and then IgG was purified by protein A affinity chromatography. The eluted proteins were neutralized and ion exchanged into PB buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). The protein concentration is determined by an ultraviolet spectrophotometer and its purity and properties are determined under denaturing, reducing or non-reducing conditions.
  • Example 4 Surface plasmon resonance (SPR) assay for binding of antibodies to different species of CD27
  • Affinity and kinetic parameters antibody binds to human CD27 was measured using the T200 Biacore TM.
  • the anti-human IgG (Fc) antibody was first immobilized on the CM5 chip of the Biacore system, and the IgG1 portion of the mAb076, mAb077 and the reference antibody 1F5 was captured thereby (flow rate 10 ⁇ L/min, 12 seconds).
  • different concentrations of human CD27 antigen (3.13, 6.25, 12.5, 25, 50, 100 nM) were analyzed at 30 ⁇ L/min, 300 seconds, then 300 seconds dissociation time.
  • the system buffer was 1 ⁇ HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% v/v P20 surfactant, pH 7.4, 25 ° C, the same experiment below), and each experiment was equipped with a chip that did not immobilize any protein. Blank control. At the end of each time, the chip was regenerated with 10 mM glycine (pH 1.5) at a flow rate of 30 ⁇ L/min for 30 seconds. Experimental data was processed by BIAevaluation 3.2 software and fitted to a 1:1 combined Langmuir model curve. The result is shown in Figure 1.
  • the affinity of the antibody for binding to human CD27 was also determined using SPR.
  • the murine CD27-FcHis fusion protein was directly immobilized on a CM5 chip (600 RU) by amino coupling.
  • Antibodies at different concentrations (0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 nM) were loaded at a flow rate of 30 ⁇ L/min for 30 seconds with a dissociation time of 1800 seconds.
  • the results showed that both mAb076 and 077 had pM-level affinity for mouse CD27, whereas the reference antibody 1F5 did not.
  • the data is shown in Figure 1.
  • Affinity and kinetic parameters of antibody binding to monkey CD27 were determined using a ForteBio Octet RED96. After biotinylation of each IgG antibody, it was immobilized on a Streptavidin-containing biosensor by the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Fisher Scientific) method, and the sensor was immersed in the Ganges River. The monkey CD27-Fc protein buffer was incubated for 300 seconds and the blank buffer was dissociated for 300 seconds. The collected data was processed by Data Acquisition 7.1 software and fitted to a 1:1 combined Langmuir model curve. As shown in Figure 1, the affinity of mAb076 and mAb077 was higher than that of 1F5.
  • Example 5 Determination of specific binding of antibodies to cells expressing human/mouse CD27
  • Antibody binding to human or murine CD27 expressed on the cell surface is determined by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the corresponding antigen was transiently transfected into HEK293 cells using the vector plasmid and expression was performed. After 48 hours, the cells were collected and washed once with cold FACS buffer (PBS, 1% BSA), and then incubated with 100 nM of the corresponding antibody for 1 hour on ice. After washing the buffer twice, the mouse anti-human Fc antibody conjugated with Alexa Fluor 647 was bound to ice for 30 minutes, washed once and loaded with Beckman. CytoFlex flow cytometry detects readings and calculates the percentage of cells with binding signals.
  • the antibodies mAb076 and mAb077 bind significantly to human or murine CD27 on the cell surface, and to the remaining TNF receptor superfamily member antigen proteins including human or murine CD40, OX40, CD137, FAS, TNFRSF1A. There is no combination.
  • the EC50 of mAb076 antibody binding to human or murine CD27 expressed on the cell surface was determined by a 1:3 stepwise dilution curve (100 nM to 0.05 nM). As shown in Figure 3, mAb076 has strong binding to both reference antibody 1F5 and cell surface human CD27, while 1F5 binds to murine CD27 with weak signal.
  • This antibody binding assay was performed using activated mouse spleen T cells.
  • BALB/c spleen T cells were purified using Stemcell Technologies' mouse T cell isolation kit and activated with 50 ng/ml PMA and 1 ⁇ g/ml ionomycin (Ionomycin) for 2 days, then 10 5 cells were collected per sample. Incubate for 1 hour on ice using 50 ⁇ l of 100 nM corresponding antibody. After washing once in FACS buffer, the cells were stained with 100 ⁇ l of 1:500 diluted mouse anti-human Fc-APC (Biolegend) for 30 minutes on ice.
  • the buffer was washed once more and resuspended, and then loaded to a CytoFlex flow cytometer to detect readings, and the percentage of cells with binding signals was calculated. As shown in Figure 4, both mAb076 and mAb077 have strong binding to activated mouse T cells.
  • the test by which antibodies block the binding of CD27 to its ligand CD70 was detected by immunofluorescence staining and FACS.
  • the vector plasmid was transiently transfected into HEK293 cells for expression. After 48 hours, the cells were harvested and washed once with cold FACS buffer, and then incubated with 100 nM of the corresponding antibody on ice for 1 hour. Additional 16 nM biotinylated labeled CD70 was added. After 30 minutes of incubation, the buffer was washed twice and incubated with Alexa Fluor647-conjugated streptavidin for 30 minutes.
  • the buffer was resuspended after washing once more, and then loaded to a CytoFlex flow cytometer to detect readings, and the percentage of cells having a CD70-specific binding signal was calculated.
  • both mAb076, mAb077 and the reference antibody were effective in blocking the binding of CD27 to CD70.
  • Example 8 Activation of NF-kB cell signaling pathway by anti-CD27 antibody
  • Activation of the cell surface CD27 molecule triggers activation of a series of downstream signaling pathways, including NK-kB, which can be detected by the luciferase reporter gene method.
  • NK-kB a series of downstream signaling pathways
  • 5x10 5 HEK293T cells were transfected into a plasmid expressing the human CD27 gene, as well as the NFkB-luc and renilla luciferase reporter gene (Promega). After 4 hours, fractionate into 96-well plates, 2 x 10 4 cells per well.
  • HEK293T cells were added and incubated for 18 hours in a 37 ° C incubator at 5% CO 2 .
  • Cells were lysed and luciferase intensities were measured on a SpectraMax i3x plate reader using Promega's Dual-luciferase kit. Analysis and mapping relative to Luciferase Unit (RLU) GraphPad Prism software. As shown in Figure 6, both mAb076 and mAb077 significantly activated the NF-kB reporter gene.
  • 96-well cell culture plates were pre-treated with 100 ul of 0.5 ug/ml anti-CD3 antibody plus different concentrations of the test antibody (30, 10, 3, 1, 0 ug/ml) overnight at 4 °C.
  • Human peripheral blood mononuclear cells (PBMC) using a kit from Sigma Histopaque1077 isolated and purified using purification kit Stemcell Technologies, and resuspended in RPMI1640 medium containing 10% FBS in, 2.5x10 5 / ml. 200 ul (5 x 10 4 ) of the above cells were added to each well of a 96-well plate, and 5 ug/ml of anti-CD28 stimulating antibody was simultaneously used as a positive control.
  • PBMC peripheral blood mononuclear cells
  • Example 10 SPR assay for binding of an antibody to an Fc ⁇ R receptor
  • Fc ⁇ R receptor binding of the antibody on the detection by SPR Biacore TM T200 method Protein L was immobilized on a CM5 sensor chip for antibody capture (10 ⁇ l/min, 12 sec loading). Then, different concentrations of Fc ⁇ RI receptor (0.39, 0.78, 1.56, 3.13, 6.25 nM, in system buffer) were applied at 10 ⁇ l/min for 300 seconds and dissociated for 600 seconds. For both FcyRIIa (CD32a) or Fc ⁇ RIIb (CD32b) receptors, the same method was used, but at a concentration of 25, 50, 100, 200, 400 nM. The collected data was processed by Data Acquisition 7.1 software and fitted to a 1:1 combined Langmuir model curve. mAb076 and mAb077 bind only to the Fc ⁇ RI receptor but not to Fc ⁇ RIIa and Fc ⁇ RIIb. The data results are shown in Figure 8.
  • Example 11 Efficacy of mAb076-IgG in a murine tumor model
  • E.G7-OVA cells were cultured in vitro, and transplanted under subcutaneous conditions in C57BL/6 mice under sterile conditions, and each mouse was inoculated with 1 ⁇ 10 4 cells. The day of tumor inoculation was day 0. The first dose was started on the third day of inoculation, and then proceeded as follows:
  • G1 vehicle control group
  • Example 12 Pharmacodynamics of mAb077-IgG in a xenograft tumor model
  • CB17-SCID mice female, 5-6 weeks, 18-20 g, purchased from Beijing Vital Lihua, divided into 5 groups, 9 in each group.
  • Raji was cultured in vitro in RPMI-1640 medium containing 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • Raji cells in exponential growth phase were collected, under sterile conditions, transplanted subcutaneously CB17-SCID mice were inoculated with 2x10 6 per mouse cells. The day of tumor inoculation was day 0. The first dose was started on the third day of inoculation, and then proceeded as follows:
  • SEM standard error
  • mAb077-IgG showed significant anti-tumor effect and dose-dependent, and the tumor inhibition rate (TGI) was 41%, 49%, and 70% in the low, medium, and high dose groups, respectively. . There was no significant decrease in mouse body weight in each of the drug-administered groups, indicating that the mice were well tolerated at this dose of mAb077-IgG.

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Abstract

一种抗CD27抗体、其抗原结合片段及其医药用途。进一步地，包含所述抗CD27抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗CD27抗体及其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。尤其涉及一种人抗CD27抗体及其在制备用于治疗CD27介导的疾病或病症的药物中的用途。

Description

抗CD27抗体、其抗原结合片段及其医药用途 技术领域
本发明涉及一种抗CD27抗体，CD27的抗原结合片段，包含所述抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗CD27抗体及其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。
背景技术
肿瘤免疫疗法是抗癌药物领域的一个长期热点。它充分利用和调动肿瘤患者体内的杀伤性T细胞，对肿瘤进行杀伤摧毁，可能是最有效且最安全的肿瘤治疗途径之一。人体内T细胞的活化是具备两条信号通路的系统，除了需通过抗原呈递细胞递呈MHC抗原肽给T细胞提供第一信号，同时需要一系列协同刺激分子提供第二信号，才能使T细胞产生正常的免疫应答。
T细胞和抗原呈递细胞之间的相互作用包括有利于产生免疫应答的多种辅助分子，而CD27正是其中重要一种，它属于肿瘤坏死因子受体(TNF-R)超家族并结合T细胞表面的CD70。CD27是糖基化的I型跨膜蛋白，通常是由二硫桥连结的同源二聚体。二硫桥处在胞外域的近细胞膜区域(Camerini等，J Immunol.147:3165-69,1991)。在T细胞上，交联的CD27抗原可提供协同剌激信号，而与T细胞受体一起交联时可诱导T细胞增殖和细胞免疫活化。
CD27在成熟胸腺细胞、大多数CD4+和CD8+外用血T细胞、自然杀伤细胞和B细胞上表达，也在B细胞非霍奇金淋巴瘤和B细胞慢性淋巴细胞白血病中高表达；而在寄生虫、巨细胞病毒(CMV)等感染、多发性硬化、结节病和B细胞慢性淋巴细胞白血病中，血清或疾病活动性部位也观测到可溶CD27蛋白水平升高(Kobata T等，Proc.Nat l.Acad.Sci.USA.1995(24):11249-53；Ranheim EA等，Blood.1995 85(12):3556-65；Loenen WA等，Eur.J.Immunol.1992 22:447)。
近年的研究表明，抗CD27单克隆抗体激动剂可促进T细胞应答，并可作为潜在的抗癌治疗剂(参见专利WO2008051424，WO2011130434)。虽然迄今获得的研究结果已将CD27鉴定为免疫疗法的有用靶标，但抗CD27单克隆抗体哪些具体特征对治疗尤为有利则仍然有待探明。本领域仍然需要深入了解抗CD27抗体的哪些具体功能特性在治疗学上有效，以至进一步获得潜在的对防治疾病疾病更有效的改进型抗CD27抗体。
目前已有多家国际公司研发针对CD27靶点的肿瘤抗体药物。美国Celldex的抗CD27抗体已处在II期临床试验阶段，而默克、Aduro和Apogenix等公司的相关产品尚在临床前开发之中。
本专利发明旨在提供具备高亲和力、高选择性和高生物活性的抗CD27抗体，具有可与期望的有利治疗效果相关联的特定功能特性。
发明内容
本发明提供一种抗CD27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；和
抗体重链可变区，所述的抗体重链可变区包含至少1个选自如以下序列所述的HCDR：SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:11，SEQ ID NO:12或SEQ ID NO:13。
在本发明一个优选的实施方案中，一种如上所述的抗CD27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或SEQ ID NO:14所示的LCDR1。
在本发明一个优选的实施方案中，一种如上所述的抗CD27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或SEQ ID NO:15所示的LCDR2。
在本发明一个优选的实施方案中，一种如上所述的抗CD27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或SEQ ID NO:16所示的LCDR3区。
在本发明一个优选的实施方案中，一种如上所述的抗CD27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或SEQ ID NO:11所示的HCDR1。
在本发明一个优选的实施方案中，一种如上所述的抗CD27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或SEQ ID NO:12所示的HCDR2。
在本发明一个优选的实施方案中，一种如上所述的抗CD27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或SEQ ID NO:13所示的HCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD27抗体或其抗原结合片段，其所述的抗体轻链可变区包分别如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD27抗体或其抗原结合片段，其所述的抗体轻链可变区包分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD27抗体或其抗原结合片段，其所述的抗体重链可变区包含分别如SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD27抗体或其抗原结合片段，其所述的抗体重链可变区包含分别如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，一种如上所述的抗CD27抗体或其抗原结合片段，其中所述的抗体轻链可变区包含分别如：
SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；或
SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；
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。
特别优选的抗CD27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。
在本发明一个优选的实施方案中，所述的抗体轻链可变区序列选自SEQ ID NO:2或SEQ ID NO:10；重链可变区序列选自SEQ ID NO:1或SEQ ID NO:9。
特别优选的抗CD27抗体或其抗原结合片段可选自下述任一种：
(1)抗体轻链可变区序列为SEQ ID NO:2，重链可变区序列为SEQ ID NO:1。
(2)抗体轻链可变区序列为SEQ ID NO:10，重链可变区序列为SEQ ID NO:9。
在本发明一个优选的实施方案中，一种如上所述的抗CD27抗体或其抗原结合片段，其中所述的抗体为鼠源抗体或其片段。
在本发明一个优选的实施方案中，一种如上所述的抗CD27抗体或其抗原结合片段，其中所述的抗体为嵌合抗体或其片段。
在本发明一个优选的实施方案中，一种如上所述的抗CD27抗体或其抗原结合片段，其中所述的抗体为人源化抗体或其片段。
在本发明一个优选的实施方案中，一种如上所述的抗CD27抗体或其抗原结合片段，其中所述的抗体为人抗体或其片段。
在本发明一个优选的实施方案中，一种如上所述的抗CD27人源化抗体或其片段，其进一步包含人源IgG1,IgG2,IgG3或IgG4或其变体的恒定区，优选包含人源IgG1，IgG2或IgG4恒定区。因为IgG2或IgG4没有ADCC毒性，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
在本发明一个优选的实施方案中，一种如上所述的抗CD27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。
本发明进一步提供一种编码如上所述的抗CD27抗体或其抗原结合片段的DNA序列。
本发明进一步提供一种含有如上所述的DNA序列的表达载体。
本发明进一步提供一种用如上所述的表达载体转化的宿主细胞。
在本发明一个优选的实施方案中，一种如上所述的宿主细胞，其特征在于，所述的宿主细胞为细菌，优选为大肠杆菌。
在本发明一个优选的实施方案中，一种如上所述的宿主细胞为酵母菌，优选为毕赤酵母。
在本发明一个优选的实施方案中，一种如上所述的宿主细胞为哺乳动物细胞，优选为中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
本发明进一步提供一种药物组合物，其含有如上所述的抗CD27抗体或其抗原结合片段和可药用的赋形剂、稀释或载体。
本发明还提供了一种多特异性抗体，含有如前所述的轻链可变区和重链可变区。
本发明还提供了一种单链抗体，含有如前所述的轻链可变区和重链可变区。
本发明还提供了一种抗体-药物偶联物，含有如前所述的轻链可变区和重链可变区。所述的抗体-药物偶联物是本领域公知的，其由抗体-接头-药物(毒素)相互连接形成，已知的接头包括裂解接头、非裂解接头，例如接头包括但不限于SMCC、SPDP等等；毒素也是本领域公知的，例如DM1、DM4、MMAE、MMAF等。
本发明进一步提供一种如上所述的抗CD27抗体或其抗原结合片段、或包含其的药物组合物，包括但不限于多特异性抗体、单链抗体、抗体-药物偶联物等形式，在制备用于治疗CD27介导的疾病或病症的药物中的用途；其中所述的疾病优选为癌症和自身免疫疾病；更优选为表达CD27的癌症和自身免疫疾病；所述的癌症最优选为直肠癌、脑癌、肾癌、肺癌、肝癌、多发性骨髓瘤和黑色素瘤；所述的自身免疫疾病最优选为自身免疫性脑脊髓炎、系统性红斑狼疮、多发性硬化和类风湿关节炎。
本发明进一步提供一种治疗和预防CD27介导的疾病或病症的方法，该方法包括给予所需患者治疗有效量的如上所述的抗CD27抗体或其抗原结合片段、或 包含其的的药物组合物，包括但不限于多特异性抗体、单链抗体、抗体-药物偶联物等形式；其中所述的疾病优选为癌症和自身免疫疾病；更优选为表达CD27的癌症和自身免疫疾病；所述的癌症最优选为直肠癌、脑癌、肾癌、肺癌、肝癌、多发性骨髓瘤和黑色素瘤；所述的自身免疫疾病最优选为自身免疫性脑脊髓炎、系统性红斑狼疮、多发性硬化和类风湿关节炎。
附图说明：
图1：抗CD27抗体mAb076和mAb077与抗原的亲和力和动力学参数，其通过Data Acquisition 7.1软件，按相应实施例中所描述的方法进行测定。
图2：使用流式细胞分析测定的人抗CD27抗体mAb076和mAb077与HEK293细胞表达的人或小鼠CD27抗原结合、而与其余TNFR超家族受体均无结合的数据(有结合信号的细胞占细胞总数比例)。
图3：使用流式细胞分析测定的，抗CD27抗体mAb076与HEK293细胞表达的人或小鼠CD27抗原结合的梯度稀释滴定曲线。
图4：使用流式细胞分析测定的人抗CD27抗体mAb076和mAb077与小鼠T细胞结合的数据(有结合信号的细胞占细胞总数比例)。
图5：使用流式细胞分析测定的抗体mAb076和mAb077有效阻断细胞表达CD27与其配体CD70结合的数据(有CD70特异结合信号的细胞占细胞总数比例)
图6：使用荧光素酶报告基因方法检测的，抗体mAb076和mAb077激活NF-kB信号通路的梯度稀释滴定曲线。
图7：使用流式细胞分析测定的，抗体mAb076和mAb077激活人外周血单核T细胞的图：(A).1F5、mAb076和mAb077对T细胞IFN-γ表达的促进作用。(B).mAb076抗体对T细胞增殖的活化。
图8：抗CD27抗体mAb076和mAb077与FcγRI受体结合的亲和力和动力学参数，其通过Data Acquisition 7.1软件，按相应实施例中所描述的方法进行测定。
图9：在溶媒处理的小鼠中、用对照人IgG1抗体处理的小鼠中或用抗CD27 mAb076-IgG抗体处理的小鼠中，E.G7-OVA移植T淋巴瘤相对于肿瘤接种后的天数的肿瘤大小(以mm3表示)。(A)为肿瘤生长曲线；(B)为小鼠体重相对变化。误差用标准误差表示。
图10：在PBS处理的小鼠中、用对照人IgG1抗体处理的小鼠中或用抗CD27 mAb077-IgG抗体处理的小鼠中，人源Raji移植淋巴瘤相对于肿瘤接种后的天数的肿瘤大小(以mm3表示)。(A)为肿瘤生长曲线；(B)为小鼠体重相对变化。误差用标准误差表示。
具体实施方式
一、术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.Biol.Chem,243,p3558(1968)中所述。
本发明所述的术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM，IgD，IgG，IgA和IgE，其相应的重链分别为μ链，δ链，γ链，α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1，IgG2，IgG3，IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
在本发明中，本发明所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可变区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为：FR1，CDR1，FR2，CDR2，FR3，CDR3，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。发明所述的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合Kabat和Chothia编号规则(HCDR1)。
术语“抗原呈递细胞”或“APC”是在其表面上展示与MHC复合的外来抗原的细胞。T细胞利用T细胞受体(TCR)识别这种复合物。APC的实例包括但不限于树突细胞(DC)、外用血单个核细胞(PBMC)、单核细胞、B淋巴母细胞和单核细胞衍生的树突细胞(DC)。术语“抗原呈递”是指APC捕获抗原和使它们能够被T细胞识别的过程，例如作为MHC-I/MHC-II偶联物的组分。
术语“CD27”是指为TNF受体超家族成员的细胞表面受体。CD27是产生和长期维持T细胞免疫所必需的分子，并在调节B细胞活化和免疫球蛋白合成中发挥关键作用。术语“CD27”包括由细胞天然表达的CD27(例如以登录号AAH12160.1登记于GENBANK的人CD27)的任何变体或同种型。本发明的抗体可与得自非人物种的CD27交叉反应。作为另一种选择，该抗体也可以是人CD27特异性的，可不表现出与其他物种的交叉反应性。CD27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CD27抗体靶向具有正常糖基化模式的人源CD27。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统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IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
本发明中所述的“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，以及与人CD27结合的Fv片段sFv片段；包含本发明所述抗体的选自SEQ ID NO:3至SEQ ID NO:8中的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本发明的术语“与CD27结合”，指能与人CD27相互作用。本发明的术语“抗原结合位点”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双抗体(diabodies)、双特异性双抗体和三抗体(triabodies)、己 共价或非共价连接在一起的抗体片段等。
术语“抗体-药物偶联物”(ADC)指与一种或多种异源的化学合成分子，包括但不限于细胞毒剂偶联的抗体或抗体片段。
术语“单链抗体”是由抗体的重链可变区(VH)和轻链可变区(VL)通过一段连接肽连接而成的单链重组蛋白，它是具有完全抗原结合位点的最小抗体片段。
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
本发明所用的术语“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人CD27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(KD)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。
术语“交叉反应”是指本发明的抗体与来自不同物种的CD27结合的能力。例如，结合人CD27的本发明的抗体也可以结合另一物种的CD27。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CD27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。CD70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CD70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CD27抗体接触时，与未与抗CD27抗体接触的CD70相比，任何可测量的CD70结合亲和力降低。
术语“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的剌激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指剌激特定的直接细胞杀伤机制。
本发明中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A，L234A，L235A；IgG2/4chimera，IgG4的F235E,或L234A/E235A突变。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，老鼠可以用人CD27或其片段免疫，所得到的抗体能被复性，纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。相应抗体的cDNA序列可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在FC区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本发明的抗体指单克隆抗体。本发明所述的单克隆抗体(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本发明的实施方案(例如治疗方法或制品)在缓解每个患都有的目标疾病症状方面可 能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
整个说明书和权利要求书中使用的术语“基本上由……组成”或其变形表示包括所有所述元件或元件组，并且任选包括与所述元件类似或不同性质的其它元件，所述其它元件非显著改变指定给药方案、方法或组合物的基本性质或新性质。作为非限制性例子，基本上由所提及的氨基酸序列组成的结合化合物还可以包括一种或多种氨基酸，其不显著影响结合化合物的性质。
本发明所述的应用于某个对象的术语“天然存在的”是指这样的事实，即该对象可在自然界中发现。例如存在于可从自然界来源分离得到的生物体(包括病毒)、且未经人工在实验室中有意修饰的多肽序列或多核昔酸序列即是天然存在的。
“有效量”包含足以改善或预防医字病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指要据背景在生物、细胞或人体外产生的物质。“内源性”指根据背景在细胞、生物或人体内产生的物质。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
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：人CD27抗原结合抗体片段(Fab)的筛选发现
针对人CD27抗原的人源抗体片段筛选使用了体外噬菌粒展示系统。所用抗原包括CD27-HisFc(Sino Biological#10039-H03H),CD27-Fc(Sino Biological #10039-H31H)和CD27-His(Sino Biological#10039-H08B1)。抗原被直接吸附固定在Maxisorp 96孔筛选板(Thermo Scientific 446469),或经生物素化后吸附在Dynabeads微颗粒(M280,Streptavidin,Invitrogen#60210)上，并使用KingFisher(Thermo Scientific)自动筛选工作站经标准噬菌体展示筛选流程进行筛选。针对每种抗原进行了3-4轮以上筛选，在每轮中使用人源抗体Fc片段以消除背景。在最后一轮筛选之后，挑取单菌落克隆孵育上清，通过ELISA方法初步鉴定结合人CD27的抗体片段，初选标准为ELISA读数至少是背景信号的2倍以上。
实施例2：生物膜层干涉(BLI)测定抗体片段与人CD27亲和力
每个独特序列的抗体Fab片段在其C端增加组氨酸标记后使用大肠杆菌表达系统过表达，然后由Ni-NTA亲和树脂进行纯化。Fab使用ForteBio Octet RED96系统由以下方法测定其与人CD27的亲和力：首先使用BLI系统的AHC(人源IgG抗体Fc片段特异)传感器捕获人CD27-HisFc融合蛋白，然后将其浸入含有5-10μg/ml每种纯化Fab蛋白的测量缓冲液中。收集的数据由Data Acquisition 7.1软件进行处理，并拟合为1:1结合的Langmuir模型曲线。抗体mAb076和mAb077为结合力最优的两株抗体，被选取用于进一步的分析和开发试验。其与CD27结合与动力学常数如图1所示。
人单抗mAb076的重链和轻链可变区序列如下：
Figure PCTCN2017103842-appb-000001
Figure PCTCN2017103842-appb-000002
其含有下列CDR序列：
名称 序列 编号
HCDR1 DYGIH SEQID NO：3
HCDR2 WINPNRGSTNYAQKFQG SEQID NO：4
HCDR3 DYGYTWYFDV SEQID NO：5
LCDR1 RASQDISSVLA SEQID NO：6
LCDR2 AASSLQS SEQID NO：7
LCDR3 QQRDAWPLT SEQID NO：8
人单抗mAb077的重链和轻链可变区序列如下：
Figure PCTCN2017103842-appb-000003
Figure PCTCN2017103842-appb-000004
其含有下列CDR序列：
名称 序列 编号
HCDR1 GYGIH SEQID NO：11
HCDR2 WINPNRGSTKYAQKFQG SEQID NO：12
HCDR3 DPGYTWYFDV SEQID NO：13
LCDR1 RASQDISSDLA SEQID NO：14
LCDR2 AASTLQS SEQID NO：15
LCDR3 QQRDAWPPT SEQID NO：16
实施例3：抗体Fab片段转为IgG及其表达纯化
将每个抗体片段分别克隆进表达IgG1亚型抗体轻链和重链的哺乳动物细胞表达载体。配对的载体以瞬转方式按标准流程转染入HEK293细胞中。表达后收集上清离心过滤，然后通过protein A亲和力层析色谱纯化IgG。洗脱的蛋白经中和后离子交换转入PB缓冲液(20mM磷酸钠,150mM NaCl,pH 7.0)。蛋白浓度通过紫外分光光度计测定，并在变性、还原或非还原条件下测定其纯度和性质。
实施例4：表面等离子共振(SPR)测定抗体与不同物种CD27的结合
抗体与人CD27结合的亲和力和动力学参数使用BiacoreTMT200测定。首先在Biacore系统的CM5芯片上固定抗人IgG(Fc)抗体，并以此捕获mAb076、mAb077和参照抗体1F5的IgG1部分(流速10μL/min，12秒)。其后的动力学实验中，不同浓度的人CD27抗原(3.13,6.25,12.5,25,50,100nM)以30μL/min，300秒,然后300秒解离时间进行分析。系统缓冲液为1×HBS-EP(10mM HEPES,150mM NaCl,3mM EDTA,0.005％v/v P20表面活性剂,pH 7.4，25℃，以下实验同)，每个实验配备芯片未固定任何蛋白的空白对照。每次结束后以10mM甘氨酸(pH 1.5)，流速30μL/min，30秒再生芯片。实验数据由BIAevaluation 3.2软件进行处理，并拟合为 1:1结合的Langmuir模型曲线。结果如图1所示。
抗体与人CD27结合的亲和力同样使用SPR测定。在本实验中，鼠CD27-FcHis融合蛋白通过氨基偶联直接固定于CM5芯片上(600RU)。不同浓度(0.39,0.78,1.56,3.13,6.25,12.5,25,50,100nM)的抗体于30μL/min流速，30秒上样，解离时间1800秒。结果显示mAb076和077均对小鼠CD27有pM级别亲和力，而参照抗体1F5则没有。数据如图1所示。
抗体与猴CD27结合的亲和力和动力学参数使用ForteBio Octet RED96测定。各IgG抗体经生物素化标记后，通过EZ-Link Sulfo-NHS-Biotinylation试剂盒(Thermo Fisher Scientific)方法固定于含链亲和素(Streptavidin)的生物传感器上，再将传感器先后浸入含恒河猴CD27-Fc蛋白的缓冲液中300秒结合、空白缓冲液300秒解离。收集的数据由Data Acquisition 7.1软件进行处理，并拟合为1:1结合的Langmuir模型曲线。如图1所示，mAb076和mAb077的亲和力较1F5相比均更高。
实施例5：抗体与细胞表达人/鼠CD27特异结合的测定
抗体与细胞表面表达的人或鼠CD27结合通过荧光激活细胞分选(FACS)测定。相应抗原使用载体质粒瞬转入HEK293细胞后进行表达，48小时后收集细胞并使用冷的FACS缓冲液(PBS，1％BSA)洗一次，然后分别与100nM相应抗体在冰上孵育结合1小时。缓冲液洗2次后，再与Alexa Fluor 647偶联的鼠抗人Fc抗体在冰上结合30分钟，洗1次后上样Beckman
Figure PCTCN2017103842-appb-000005
CytoFlex流式细胞仪检测读数，计算有结合信号的细胞百分比例。如图2所示，抗体mAb076和mAb077与细胞表面的人或鼠CD27均有明显结合，而与包括人或鼠CD40,OX40,CD137,FAS,TNFRSF1A在内的其余TNF受体超家族成员抗原蛋白均无结合。
mAb076抗体与细胞表面表达的人或鼠CD27结合的EC50由1:3逐级梯度稀释曲线(100nM至0.05nM)测定。如图3所示，mAb076与参照抗体1F5和细胞表面人CD27均有较强结合，而1F5与鼠CD27结合信号很弱。
实施例6：抗体与小鼠T细胞结合的测定
该项抗体结合实验使用活化的小鼠脾脏T细胞进行。BALB/c脾脏T细胞使用Stemcell Technologies的小鼠T细胞分离试剂盒进行纯化，并使用50ng/ml PMA和1μg/ml离子霉素(Ionomycin)激活2天，然后每个样品收集105数量的细胞，使用50μl 100nM相应抗体在冰上孵育1小时。FACS缓冲液洗一次后，用100μl 1:500稀释的鼠抗人Fc-APC(Biolegend)冰上孵育染色30分钟。缓冲液再洗一次后重悬浮，然后上样至CytoFlex流式细胞仪检测读数，计算有结合信号的细胞百分比例。如图4所示，mAb076与mAb077均与活化的小鼠T细胞有很强结合力。
实施例7：抗体的CD27配体阻断实验
抗体阻断CD27与其配体CD70结合的测试通过免疫荧光染色和FACS检测。使用载体质粒瞬转CD27进入HEK293细胞后进行表达，48小时后收集细胞并使用冷的FACS缓冲液洗一次，然后分别与100nM相应抗体在冰上孵育结合1小时， 再加入16nM生物素化标记的CD70。孵育30分钟后，缓冲液洗2次并与Alexa Fluor647偶联的链亲和素孵育结合30分钟。缓冲液再洗一次后重悬浮，然后上样至CytoFlex流式细胞仪检测读数，计算有CD70特异结合信号的细胞百分比例。如图5所示，mAb076、mAb077与参照抗体均可有效阻断CD27与CD70的结合。
实施例8：抗CD27抗体对NF-kB细胞信号通路的激活
细胞表面CD27分子的激活会引发下游一系列信号通路的活化，包括NK-kB，可通过荧光素酶报告基因方法检测。在本例中，5x105HEK293T细胞转染入表达人CD27基因的质粒，以及NFkB-luc和renilla荧光素酶报告基因(Promega)。4小时后，分装入96孔板，2x104细胞每孔。将梯度稀释的相应抗体与F(ab')2羊抗人IgG Fcγ二抗预混合后，加入HEK293T细胞并在5％CO2的37℃培养箱中孵育18小时。裂解细胞，并使用Promega的Dual-luciferase试剂盒，在SpectraMax i3x读板仪上测量荧光素酶强度。相对荧光素酶单位(RLU)GraphPad Prism软件分析并作图。如图6所示，mAb076和mAb077对NF-kB报告基因均有显著的激活。
实施例9：抗CD27抗体的T细胞激活实验
在T细胞激活功能实验中，先使用100ul 0.5ug/ml抗CD3抗体加不同浓度的受测抗体(30,10,3,1,0ug/ml)预处理96孔细胞培养板，4℃过夜。人外周血单核细胞(PBMC)使用Sigma的Histopaque1077试剂盒分离，然后用Stemcell Technologies的纯化试剂盒纯化，并重悬浮在含10％FBS的RPMI1640培养基中，2.5x105/ml。在96孔板每孔中加入200ul(5x104)上述细胞，并同时使用5ug/ml抗CD28刺激抗体作为阳性对照。在5％CO2的37℃培养箱中孵育4天后，每孔取100ul转移到新的样品板(剩余部分用于IFN-γ检测)。加入80ul CellTiter-Glo
Figure PCTCN2017103842-appb-000006
试剂孵育10分钟稳定信号，然后用SpectraMax i3x读板仪检测冷发光强度。如图7所示，IgG1形式mAb076和mAb077对T细胞IFN-γ表达的促进作用均比参照抗体1F5更高；而在另一实验中，mAb076抗体对T细胞的活化也比相应形式的1F5更高。
实施例10：SPR测定抗体与FcγR受体的结合
抗体与FcγR受体的结合通过SPR方法在BiacoreTMT200上检测。Protein L固定在CM5传感器芯片上，用于捕获抗体(10μl/min，12秒上样)。然后使用不同浓度的FcγRI受体(0.39,0.78,1.56,3.13,6.25nM，系统缓冲液中)10μl/min上样300秒并解离600秒。对于FcγRIIa(CD32a)or FcγRIIb(CD32b)两种受体，使用相同的方法，但浓度为25,50,100,200,400nM。收集的数据由Data Acquisition 7.1软件进行处理，并拟合为1:1结合的Langmuir模型曲线。mAb076和mAb077仅结合FcγRI受体，而与FcγRIIa和FcγRIIb没有结合。数据结果如图8所示。
实施例11：mAb076-IgG在鼠源肿瘤模型中的药效
40只C57BL/6小鼠，雌性，5-6周，18-20g，分5组，每组8只。体外培养E.G7-OVA细胞，无菌条件下，移植于C57BL/6小鼠皮下，每只小鼠接种1X104 个细胞。肿瘤接种当天为第0天。在接种第3天开始第一次给药，之后按如下方案进行：
G1：溶媒对照组；
G2：IgG1 0.2mg/只,i.p.,N＝8,Q2D,3,5,7,9,11,13天；
G3：mAb076-IgG 0.07mg/只,i.p.,N＝8,Q2D,3,5,7,9,11,13天；
G4：mAb076-IgG 0.2mg/只,i.p.,N＝8,Q2D,3,5,7,9,11,13天；
G5：mAb076-IgG 0.6mg/只,i.p.,N＝8,Q2D,3,5,7,9,11,13天；
给药后每天监测动物日常行为表现，共进行27天。用游标卡尺测量肿瘤的长径及短径后，用长×宽2/2计算肿瘤体积。肿瘤抑制率采用公式TGI％＝(1-T/C)×100％。T和C分别为给药组和对照组在某一特定时间点的肿瘤体积(TV)。体重变化用公式RCBW％＝(BWi–BW0)/BW0×100％，BWi是小鼠当前体重，BW0是分组当日的小鼠体重。所有实验结果以平均瘤体积±标准误差(SEM)表示。用T-Test检验方法比较治疗组肿瘤体积和瘤重与对照组相比有无显著性差异。所有的数据均用Graphpad进行分析，p<0.05为具有显著性差异。如图9所示，在E.G7-OVA肿瘤模型中，mAb076-IgG表现出抗肿瘤作用，各组终末平均肿瘤体积分别为：1355.3mm3、1365.87mm3、0mm3、404.88mm3、0mm3；低、中、高剂量组抑瘤率(TGI)分别为100％、70.36％和100％。各给药组均没有出现明显的C57BL/6小鼠体重下降情况，表明C57BL/6小鼠对该剂量下的mAb076-IgG耐受性良好。
实施例12：mAb077-IgG在异种移植瘤肿瘤模型中的药效
45只CB17-SCID小鼠，雌性，5-6周，18-20g，购自北京维通利华，分5组，每组9只。体外培养Raji在含10％胎牛血清(FBS)的RPMI-1640培养液中。收集指数生长期的Raji细胞，无菌条件下，移植于CB17-SCID小鼠皮下，每只小鼠接种2x106个细胞。肿瘤接种当天为第0天。在接种第3天开始第一次给药，之后按如下方案进行：
第1组：PBS溶媒,i.p.,N＝9,Q2D,3,5,7,9,11,13,15天；
第2组：IgG1 30mg/kg,i.p.,N＝9,Q2D,3,5,7,9,11,13,15天；
第3组：mAb077-IgG 3mg/kg,i.p.,N＝9,Q2D,3,5,7,9,11,13,15天；
第4组：mAb077-IgG 10mg/kg,i.p.,N＝9,Q2D,3,5,7,9,11,13,15天；
第5组：mAb077-IgG 30mg/kg,i.p.,N＝9,Q2D,3,5,7,9,11,13,15天；
给药后每天监测动物日常行为表现，共进行18天。用游标卡尺测量肿瘤的长径及短径后，用长×宽2/2计算肿瘤体积。肿瘤抑制率采用公式TGI％＝(1-T/C)×100％。T和C分别为给药组和对照组在某一特定时间点的肿瘤体积(TV)。体重变化用公式RCBW％＝(BWi–BW0)/BW0×100％，BWi是小鼠当前体重，BW0是分组当日的小鼠体重。所有实验结果以平均瘤体积±标准误差(SEM)表 示。用T-Test检验方法比较治疗组肿瘤体积和瘤重与对照组相比有无显著性差异。所有的数据均用Graphpad进行分析，p<0.05为具有显著性差异。
如图10所示，在Raji移植肿瘤模型中，mAb077-IgG表现出明显的抗肿瘤作用及剂量依赖，低、中、高剂量组抑瘤率(TGI)分别为41％、49％和70％。各给药组均没有出现明显的小鼠体重下降情况，表明小鼠对该剂量下的mAb077-IgG耐受性良好。

Claims (35)

  1. 一种抗CD27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；和
    抗体重链可变区，所述的抗体重链可变区包含至少1个选自如以下序列所述的HCDR：SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:11，SEQ ID NO:12或SEQ ID NO:13。
  2. 如权利要求1所述的抗CD27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或SEQ ID NO:14所示的LCDR1。
  3. 如权利要求1所述的抗CD27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或SEQ ID NO:15所示的LCDR2。
  4. 如权利要求1所述的抗CD27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或SEQ ID NO:16所示的LCDR3。
  5. 如权利要求1所述的抗CD27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或SEQ ID NO:11所示的HCDR1。
  6. 如权利要求1所述的抗CD27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或SEQ ID NO:12所示的HCDR2。
  7. 如权利要求1所述的抗CD27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或SEQ ID NO:13所示的HCDR3。
  8. 如权利要求1所述的抗CD27抗体或其抗原结合片段，其中所述的抗体轻链可变区包含分别如SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3。
  9. 如权利要求1所述的抗CD27抗体或其抗原结合片段，其中所述的抗体轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3。
  10. 如权利要求1所述的抗CD27抗体或其抗原结合片段，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。
  11. 如权利要求1所述的抗CD27抗体或其抗原结合片段，其中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。
  12. 如权利要求1所述的抗CD27抗体或其抗原结合片段，其中所述的抗体轻链可变区包含分别如：
    SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；或
    SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3；
    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LCDR1、LCDR2和LCDR3。
  13. 如权利要求12所述的抗CD27抗体或其抗原结合片段，其中所述的抗体轻链可变区包含分别如：SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的LCDR1、LCDR2和LCDR3；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。
  14. 如权利要求12所述的抗CD27抗体或其抗原结合片段，其中所述的抗体轻链可变区包含分别如：SEQ ID NO: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。
  15. 如权利要求12所述的抗CD27抗体或其抗原结合片段，其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。
  16. 如权利要求12所述的抗CD27抗体或其抗原结合片段，其中所述的抗体轻链可变区序列为SEQ ID NO:2，重链可变区序列为SEQ ID NO:1。
  17. 如权利要求12所述的抗CD27抗体或其抗原结合片段，其中所述的抗体 轻链可变区序列为SEQ ID NO:10，重链可变区序列为SEQ ID NO:9。
  18. 如权利要求1-17任一项所述的抗CD27抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为鼠源抗体或其片段。
  19. 如权利要求1-17任一项所述的抗CD27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嵌合抗体或其片段。
  20. 如权利要求1-17任一项所述的抗CD27抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为人源化抗体或其片段。
  21. 如权利要求1-17任一项所述的抗CD27抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为人抗体或其片段。
  22. 如权利要求21所述的抗CD27抗体或其抗原结合片段，其中所述的抗体轻链可变区序列为：SEQ ID NO:2或SEQ ID NO:10或其变体；所述的变体优选在轻链可变区有0-10的氨基酸变化。
  23. 如权利要求21所述的抗CD27抗体或其抗原结合片段，其中所述的抗体重链可变区序列为：SEQ ID NO:1或SEQ ID NO:9或其变体；所述的变体优选在重链可变区有0-10的氨基酸变化。
  24. 一种编码如权利要求1-23任一项所述的抗体的DNA序列。
  25. 一种含有如权利要求24所述的DNA序列的表达载体。
  26. 一种用如权利要求25所述的表达载体转化的宿主细胞。
  27. 如权利要求25所述的宿主细胞，其特征在于，所述的宿主细胞为细菌，优选为大肠杆菌。
  28. 如权利要求25所述的宿主细胞，其特征在于，所述的宿主细胞为酵母菌，优选为毕赤酵母。
  29. 如权利要求25所述的宿主细胞，其特征在于，所述的宿主细胞为哺乳动物细胞，优选为中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
  30. 一种药物组合物，其含有如权利要求1-17任一项所述的抗CD27抗体或 其抗原结合片段和可药用的赋形剂、稀释或载体。
  31. 一种多特异性抗体，含有如权利要求1-17任一项所述的轻链可变区和重链可变区。
  32. 一种单链抗体，含有如权利要求1-17任一项所述的轻链可变区和重链可变区。
  33. 一种抗体-药物偶联物，其中所述的抗体含有如权利要求1-17任一项所述的轻链可变区和重链可变区。
  34. 如权利要求1-17任一项所述的抗CD27抗体或其抗原结合片段，或如权利要求30所述的药物组合物，或如权利要求31所述的多特异性抗体，或如权利要求32所述的单链抗体，或如权利要求33所述的抗体-药物偶联物在制备用于治疗或预防CD27介导的疾病或病症的药物中的用途；其中所述的疾病优选为癌症和自身免疫疾病；更优选为表达CD27的癌症和自身免疫疾病；所述的癌症最优选为直肠癌、脑癌、肾癌、肺癌、肝癌、多发性骨髓瘤和黑色素瘤；所述的自身免疫疾病最优选为自身免疫性脑脊髓炎、系统性红斑狼疮、多发性硬化和类风湿关节炎。
  35. 一种治疗和预防CD27介导的疾病或病症的方法，该方法包括给予所需患者治疗有效量的如权利要求1-17任一项所述的抗CD27抗体或其抗原结合片段，或如权利要求30所述的药物组合物，或如权利要求31所述的多特异性抗体，或如权利要求32所述的单链抗体，或如权利要求33所述的抗体-药物偶联物；其中所述的疾病优选为癌症和自身免疫疾病；更优选为表达CD27的癌症和自身免疫疾病；所述的癌症最优选为直肠癌、脑癌、肾癌、肺癌、肝癌、多发性骨髓瘤和黑色素瘤；所述的自身免疫疾病最优选为自身免疫性脑脊髓炎、系统性红斑狼疮、多发性硬化和类风湿关节炎。
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