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WO2024187743A1 - 抗cd27单克隆抗体及其应用 - Google Patents

抗cd27单克隆抗体及其应用 Download PDF

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Publication number
WO2024187743A1
WO2024187743A1 PCT/CN2023/124588 CN2023124588W WO2024187743A1 WO 2024187743 A1 WO2024187743 A1 WO 2024187743A1 CN 2023124588 W CN2023124588 W CN 2023124588W WO 2024187743 A1 WO2024187743 A1 WO 2024187743A1
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Prior art keywords
antibody
variable region
chain variable
heavy chain
seq
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PCT/CN2023/124588
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English (en)
French (fr)
Inventor
蔡则玲
陈羿
何巧巧
王雅玲
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上海赛金生物医药有限公司
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Publication of WO2024187743A1 publication Critical patent/WO2024187743A1/zh

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    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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

Definitions

  • the present invention relates to the field of medicine, and in particular to anti-CD27 humanized monoclonal antibodies and preparations thereof.
  • CD27 is a member of the TNFR superfamily. It is a type I transmembrane glycoprotein with a molecular weight of about 55kD, usually a homodimer linked by disulfide bridges. CD27 is expressed as a surface antigen on most T cells, natural killer cells, antibody-secreting plasma cells, and memory B cells. CD70, as a ligand of CD27, interacts with CD27 and recruits intracellular TRAF proteins to the intracellular domain of CD27, thereby activating downstream signals. Generally, after binding to CD70, CD27 inside the cell activates NF- ⁇ B and JNK signaling pathways by linking TNF receptor-related molecules TRAF2 and TRAF5 to promote T cell proliferation and the secretion of corresponding cytokines. Studies have shown that CD27-CD70-mediated co-stimulation plays an important role in the growth, differentiation, and survival of T cells. It can also promote B cell proliferation, differentiation into plasma cells, production of immunoglobulins, and induce NK cell activation.
  • CD27 is considered a promising target for cancer immunotherapy.
  • CD27 antibodies are in clinical trials, including Varlilumab, a CD27 monoclonal antibody from Celldex Therapeutics, which has entered Phase II clinical trials.
  • Varlilumab a CD27 monoclonal antibody from Celldex Therapeutics, which has entered Phase II clinical trials.
  • this anti-human agonist CD27 antibody is disclosed, which activates CD27 when cross-linked.
  • In vivo experiments can enhance the proliferation of mouse immune cells and cytokine release, enhance the immune response of mice to vaccines, and enhance the anti-tumor activity of mice in different tumor models.
  • CD27 is a new immunotherapy target with great potential.
  • CD27 monoclonal antibody drug on the market at home and abroad, so it is necessary to develop CD27 monoclonal antibodies with better clinical efficacy.
  • the purpose of the present invention is to provide a CD27 antibody with high affinity and high biological activity and its application.
  • a heavy chain variable region of an antibody wherein the heavy chain variable region has three complementary determining regions (CDRs) selected from the following group:
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • a complementarity determining region CDR1 wherein the amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID No: 2;
  • Complementarity determining region CDR3 the amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID No:4.
  • the heavy chain variable region includes four framework regions FR, and the four framework regions FR are separated by the above-mentioned CDR1, CDR2 and CDR3.
  • the heavy chain variable region has an amino acid sequence shown in SEQ ID No: 1, 17, 18, 19, or 20.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID No:9.
  • an antibody heavy chain is provided, wherein the antibody heavy chain has the heavy chain variable region of the antibody according to the first aspect of the present invention.
  • the constant region of the heavy chain is of human origin.
  • the heavy chain constant region is the heavy chain constant region of IgG1, IgG2 or IgG4.
  • the heavy chain constant region is a wild-type Fc or a mutant Fc.
  • the mutant Fc causes the ADCC effect of the antibody to be lost or substantially lost.
  • the mutant Fc has L234A and L235A mutations.
  • the heavy chain sequence is as shown in SEQ ID NO: 27, and the constant region of the heavy chain is wild-type Fc.
  • the heavy chain sequence is as shown in SEQ ID NO: 28, and the constant region of the heavy chain is a mutant Fc having L234A and L235A mutations on the Fc of IgG1.
  • a light chain variable region of an antibody wherein the light chain variable region has three complementary determining regions L-CDRs as shown in SEQ ID No: 6, 7, and 8;
  • the light chain variable region has three complementary determining regions L-CDRs with sequences as shown in SEQ ID No: 14, 15, and 16.
  • the light chain variable region includes the following three complementarity determining regions L-CDR:
  • a complementarity determining region L-CDR1 wherein the amino acid sequence of the complementarity determining region L-CDR1 is shown in SEQ ID No: 6;
  • Complementarity determining region L-CDR3 the amino acid sequence of the complementarity determining region L-CDR3 is shown in SEQ ID No:8.
  • the light chain variable region includes four framework regions FR, and the four framework regions FR are separated by the above-mentioned CDR1, CDR2 and CDR3.
  • the light chain variable region has an amino acid sequence shown in SEQ ID No: 5, 21, 22, 23 or 24.
  • the light chain variable region has the amino acid sequence shown in SEQ ID No:13.
  • an antibody light chain is provided, wherein the antibody light chain has the light chain variable region of the antibody according to the third aspect of the present invention.
  • the constant region of the light chain is of human origin.
  • the constant region of the light chain is a Kappa or Lambda light chain constant region.
  • an antibody wherein the antibody has:
  • the antibody has: the heavy chain as described in the second aspect of the present invention; and/or the light chain as described in the fourth aspect of the present invention.
  • the antibody is an anti-CD27 antibody.
  • the antibody further comprises a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region of the antibody is a wild-type Fc or a mutant Fc.
  • the mutant Fc causes the ADCC effect of the antibody to be lost or substantially lost.
  • the mutant Fc has L234A and L235A mutations.
  • the heavy chain sequence of the antibody is as shown in SEQ ID NO: 27, and the heavy chain The constant region was wild-type Fc.
  • the heavy chain sequence of the antibody is as shown in SEQ ID NO: 28, and the constant region of the heavy chain is a mutant Fc having L234A and L235A mutations on the Fc of IgG1.
  • the antibody has a heavy chain variable region as shown in SEQ ID No: 1, 17, 18, 19 or 20; and/or a light chain variable region as shown in SEQ ID No: 5, 21, 22, 23 or 24.
  • the antibody has a heavy chain variable region as shown in SEQ ID No:9; and/or a light chain variable region as shown in SEQ ID No:13.
  • the antibody is selected from the following group:
  • (Z2) an antibody having a heavy chain variable region shown in SEQ ID No: 18 and a light chain variable region shown in SEQ ID No: 22;
  • the antibody is a humanized antibody, a chimeric antibody, or a murine antibody.
  • the antibody specifically binds to CD27.
  • the antibody has the function of blocking CD70 from binding to CD27.
  • the antibody has the function of activating T cells and promoting T cell proliferation.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is in the form of a drug conjugate.
  • a recombinant protein is provided, wherein the recombinant protein has:
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a polymer.
  • an antibody preparation comprising:
  • the preparation is a pharmaceutical composition.
  • the excipient or carrier is a pharmaceutically acceptable carrier or excipient.
  • the carrier includes: a buffer, sterile water, and optionally a surfactant.
  • the concentration of the antibody is 5-100 mg/mL; preferably 10-70 mg/mL, more preferably 20-60 mg/mL.
  • the buffer is selected from the following group: PBS buffer system, citric acid buffer system, histidine buffer system, or a combination thereof.
  • the pH range of the preparation is 5.0-7.5, preferably 5.5-7.
  • the preparation is an injection preparation.
  • kits comprising the antibody according to the fifth aspect of the present invention and a container for containing the antibody.
  • a CAR construct wherein the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to CD27, and the scFv has the heavy chain variable region as described in the first aspect of the present invention and the light chain variable region as described in the third aspect of the present invention.
  • a recombinant immune cell is provided, wherein the immune cell expresses an exogenous CAR construct as described in the ninth aspect of the present invention.
  • the immune cells are selected from the following group: NK cells and T cells.
  • the immune cells are from humans or non-human mammals (such as mice).
  • an antibody-drug conjugate comprising:
  • an antibody portion the antibody portion being selected from the group consisting of a heavy chain variable region as described in the first aspect of the invention, a heavy chain as described in the second aspect of the invention, a light chain variable region as described in the third aspect of the invention, a light chain as described in the fourth aspect of the invention, or an antibody as described in the fifth aspect of the invention, or a combination thereof; and
  • conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody portion and the coupling portion are coupled via a chemical bond or a linker.
  • an active ingredient wherein the active ingredient is selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the immune cell as described in the tenth aspect of the present invention, the antibody-drug conjugate as described in the eleventh aspect of the present invention, or a combination thereof, wherein the active ingredient is used for
  • the tumor is selected from the following group: a blood tumor, a solid tumor, or a combination thereof.
  • the blood tumor is selected from the following group: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • Hodgkin's lymphoma or a combination thereof.
  • the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.
  • gastric cancer gastric cancer peritoneal metastasis
  • liver cancer leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.
  • NSCLC non-small cell lung cancer
  • the medicine or preparation is used for preparing a medicine or preparation for preventing and/or treating a disease associated with CD27 (positive expression).
  • the antibody is in the form of an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the detection reagent or kit is used to diagnose CD27-related diseases.
  • the detection reagent or kit is used to detect CD27 protein in a sample.
  • the detection reagent is a detection sheet.
  • a pharmaceutical composition wherein the pharmaceutical composition comprises:
  • an active ingredient wherein the active ingredient is selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the immune cell as described in the tenth aspect of the present invention, the antibody as described in the eleventh aspect of the present invention. a poly(vinylidene phosphodiesterase inhibitor) or a combination thereof; and
  • the pharmaceutical composition further comprises a second anti-tumor active ingredient.
  • the second active ingredient is selected from the following group: cytotoxic drugs, toxins, cytokines, enzymes, antibodies, or a combination thereof.
  • the second active ingredient includes: an antibody targeting EGFR and an antibody targeting HER2.
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used to treat tumors.
  • a polynucleotide encodes a polypeptide selected from the group consisting of:
  • a vector is provided, wherein the vector contains the polynucleotide as described in the fourteenth aspect of the present invention.
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a genetically engineered host cell wherein the host cell contains the vector as described in the fifteenth aspect of the present invention or the polynucleotide as described in the fourteenth aspect of the present invention is integrated into its genome.
  • a method for in vitro detection (including diagnostic or non-diagnostic) of CD27 protein in a sample comprising the steps of:
  • a detection plate comprising: a substrate (support plate) and a test strip, wherein the test strip contains the antibody as described in the fifth aspect of the present invention or the antibody-drug conjugate as described in the eleventh aspect of the present invention.
  • a kit comprising:
  • the kit contains the detection plate as described in the eighteenth aspect of the present invention.
  • a method for preparing a recombinant polypeptide comprising:
  • a method for treating a CD27-related disease comprising: administering to a subject in need thereof the antibody as described in the fifth aspect of the present invention, the antibody-drug conjugate of the antibody, or the CAR-T cell expressing the antibody, or a combination thereof.
  • the subject includes humans and non-human mammals.
  • the subject is a human.
  • FIG1 shows SDS-PAGE analysis of the antibodies of the present invention.
  • FIG2 shows the results of ELISA test of the antibody of the present invention.
  • FIG3 shows the binding effect of the antibody of the present invention with the CD27 protein on the CHO cell membrane.
  • FIG4 shows the binding effect of human-mouse chimeric CD27 antibody on CD27 protein on the surface of human T and B cells detected by FACS.
  • FIG5 shows the inhibitory effect of human-mouse chimeric CD27 antibody on the binding of CD27 and CD70.
  • FIG6 shows the human-mouse chimeric CD27 monoclonal antibody binding epitope experiment.
  • Figure 7 shows an epitope experiment of 13B3 and 1F5 binding.
  • FIG8 shows the effect of anti-CD27 antibodies in promoting the proliferation of CD3+ T cells.
  • FIG. 9 shows that anti-CD27 antibodies inhibit the NF ⁇ B signaling pathway downstream of CD27 by blocking the binding of CD70 on the surface of Raji cells and CD27 on the surface of Jurkat cells.
  • FIG. 10 shows the ELISA results of the in vitro binding activity of humanized monoclonal CD27 antibodies to antigen CD27.
  • FIG11 shows the flow cytometry results of the binding of humanized CD27 monoclonal antibodies to CD27 protein on the cell membrane.
  • FIG. 12 shows the inhibitory effect of humanized CD27 antibodies on the binding of CD27 to CD70.
  • FIG. 13 shows the promoting effect of humanized antibodies on T cell proliferation.
  • Figure 14 shows the changes in the percentage of T and B lymphocytes in the peripheral blood of mice 3 days after the last administration of the antibody of the present invention.
  • Figure 14A CD3+T lymphocytes
  • Figure 14B CD3+CD4+T lymphocytes
  • Figure 14C CD3+CD4-T lymphocytes
  • Figure 14D CD19+B lymphocytes.
  • Figure 15 shows the changes in the percentage of T lymphocytes in the peripheral blood of mice 10 days after the last administration of the antibody of the present invention.
  • Figure 15A CD3+T lymphocytes
  • Figure 15B CD3+CD4+T lymphocytes
  • Figure 15C CD3+CD8+T lymphocytes.
  • Figure 16 shows the changes in the percentage of T lymphocytes in the spleen of mice 10 days after the last administration of the antibody of the present invention.
  • Figure 16A CD3+T lymphocytes
  • Figure 16B CD3+CD4+T lymphocytes
  • Figure 16C CD3+CD8+T lymphocytes.
  • Figure 17 shows the effects of humanized antibodies alone or in combination with other immune checkpoint inhibitor antibodies on IFN ⁇ production by T cells in the exhaustion stage.
  • FIG. 18 shows the in vitro binding activity of wild-type and variant CD27 antibodies to CD27.
  • FIG. 19 shows the inhibitory effects of wild-type and variant CD27 antibodies on the binding of CD27 to CD70 in vitro.
  • Figure 20 shows the activity of wild-type and variant CD27 antibodies in binding to cell surface Fc receptors CD32a and CD64.
  • Figure 20A Binding to cell surface CD32a;
  • Figure 20B Binding to cell surface CD64.
  • Figure 21 shows the changes in the percentage of T lymphocytes in the peripheral blood of mice 3 days after the last administration of the wild-type and variant CD27 antibodies of the present invention.
  • Figure 21A CD3+T lymphocytes
  • Figure 21B CD3+CD4+T lymphocytes
  • Figure 21C CD3+CD8+T lymphocytes.
  • Figure 22 shows the changes in the percentage of T lymphocytes in the peripheral blood of mice 10 days after the last administration of the wild-type and variant CD27 antibodies of the present invention.
  • Figure 22A CD3+T lymphocytes
  • Figure 22B CD3+CD4+T lymphocytes
  • Figure 22C CD3+CD8+T lymphocytes.
  • the inventors have conducted extensive and in-depth research and obtained a number of mouse antibodies with excellent properties such as high affinity for human CD27 after a large number of screenings, and have also obtained a mouse antibody gene with high affinity and high anti-tumor activity. On this basis, chimeric antibodies and humanized transformations were further prepared.
  • the antibodies of the present invention can effectively bind to human CD27, have excellent activity, and can be used as monoclonal antibody drugs for targeted therapy. On this basis, the present invention was completed.
  • the term “about” can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
  • the term “comprising” or “including (comprising)” may be open, semi-closed and closed. In other words, the term also includes “consisting essentially of” or “consisting of”.
  • Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage.
  • a predetermined comparison window which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein
  • VL light chain variable region
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • antibodies of the present invention are used interchangeably, and all refer to antibodies that specifically bind to CD27, such as proteins or polypeptides having a heavy chain variable region (such as the amino acid sequence of SEQ ID No: 27 or 35) and/or a light chain variable region (such as the amino acid sequence of SEQ ID No: 31 or 39). They may or may not contain an initial methionine.
  • CD27 As a co-stimulatory T cell receptor, CD27 also promotes the survival of activated T cells through co-stimulation with OX40 (CD134) and 4-1BB, and is the key to T cell activation and memory differentiation; CD70 is mainly expressed in activated lymphocytes under normal circumstances, but under pathological conditions, CD70 is highly expressed in a variety of tumor cells. Tumor cells bind to the T cell receptor CD27 by expressing CD70. Chronic co-stimulation causes T cells to express immune checkpoints such as PD-1 and TIM-3, leading to immune exhaustion. The high expression of CD70 in tumors may suggest that tumors use CD70 to control tumor-infiltrating lymphocytes (TIL) expressing CD27, thereby causing immune escape.
  • TIL tumor-infiltrating lymphocytes
  • CD27 target antibodies have the effect of strengthening tumor immunity and blocking immune escape from a mechanistic perspective.
  • antibody refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the Kabat scheme (Kabat et al., 1991) is based on the location of regions of high sequence variation between sequences of the same domain type, with antibody heavy (VH) and light (V ⁇ and V ⁇ ) variable domains numbered differently.
  • Chothia's program (Al-Lazikani, 1997) is the same as Kabat's program, but corrects the position of the insertion annotation around the first VH complementarity determining region (CDR) to correspond to the structural loop.
  • CDR VH complementarity determining region
  • the enhanced Chothia program (Abhinandan and Martin, 2008) makes further structural corrections to the insertion position.
  • IMGT Lefranc, 2003
  • AHo Hegger and Plückthun, 2001
  • IMGT and AHo differ in the number of positions they annotate (128 and 149, respectively) and in the positions where they consider indels to occur.
  • immunoglobulins can be divided into five categories, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain.
  • immunoglobulin isotypes namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the disulfide bonds of the heavy chain, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into ⁇ chain or ⁇ chain according to the difference in the constant region.
  • Each of the five classes of Ig can have a ⁇ chain or a ⁇ chain.
  • the subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known to those in the art.
  • the antibody light chain of the present invention may further comprise a light chain constant region, wherein the light chain constant region comprises a human or mouse ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises IgG1, IgG2, IgG3, IgG4 or variants thereof of human or mouse origin.
  • the sequences of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region.
  • the variable region includes three hypervariable regions (HVRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of three CDR regions and four FR regions, and the order arranged from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.
  • the six CDRs of the CD27 antibody are divided in combination with the Kabat method.
  • mouse antibody in the present invention refers to a monoclonal antibody against CD27 prepared according to the knowledge and skills in the art.
  • the test subject is injected with CD27 antigen, and then the mouse antibody expressing the desired sequence or functional characteristics is isolated.
  • the murine CD27 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or a variant thereof.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody.
  • humanized antibody also known as CDR-grafted antibody, refers to antibodies produced by transplanting mouse CDR sequences into human antibody variable region frameworks, that is, different types of human germline antibody framework sequences. Humanized antibodies can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components they carry. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. In order to avoid a decrease in activity caused by a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • antigen-binding fragment of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CLDN18.2). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term “antigen-binding fragment of an antibody” include
  • Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • F(ab') 2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;
  • Fv antibody contains the variable region of antibody heavy chain and variable region of light chain, but has no constant region, and has the smallest antibody fragment of all antigen binding sites. Generally, Fv antibody also contains a polypeptide linker between VH and VL domains, and can form the structure required for antigen binding.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E.A et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • epitope refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a CD27 molecule).
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation.
  • binding refers to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody binds with an affinity (KD) of less than about 10-7 M, such as less than about 10-8 M, 10-9 M or 10-10 M or less.
  • competitive binding refers to an antibody that recognizes the same epitope (also called antigenic determinant) or a portion of the same epitope on the extracellular region of CD27 as the monoclonal antibody of the present invention and binds to the antigen.
  • the antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of CD27 recognized by the monoclonal antibody of the present invention.
  • KD or "Kd” refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
  • antibodies of the invention bind to CD27 with a dissociation equilibrium constant (KD) of less than about 10-7 M, such as less than about 10-8 M, 10-9 M, or 10-10 M or less.
  • antigenic determinant refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be prepared using DNA recombinant techniques well known in the art.
  • the term "monoclonal antibody” refers to an antibody secreted from a clone of a single cell source. Monoclonal antibodies are highly specific, directed against a single antigenic epitope.
  • the cell may be a eukaryotic, prokaryotic, or phage clone.
  • the antibodies may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention.
  • conservative variant polypeptides are preferably produced by amino acid substitution according to the following table.
  • the heavy chain constant region and/or light chain constant region of the antibody of the present invention may be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region of human IgG1, IgG2, etc., or a human kappa, Lambda light chain constant region.
  • sequence formed by adding, deleting, modifying and/or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may preferably be a fully humanized antibody.
  • the antibody derivatives of the present invention can be single-chain antibodies and/or antibody fragments, such as Fab, Fab', (Fab')2, or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG, IgM antibodies or other subtypes of antibodies.
  • the antibody of the present invention may be a humanized antibody, a CDR-grafted and/or modified antibody targeting CD27.
  • the number of added, deleted, modified and/or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • any method suitable for producing monoclonal antibodies can be used to produce the CD27 antibodies of the present invention.
  • animals can be immunized with linked or naturally occurring CD27 proteins or fragments thereof.
  • Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • CD27 can be used as an immunogen (antigen) to generate non-human antibodies specific for CD27 and screen the biological activity of the antibodies.
  • the immunogen can be used alone or in combination with any of the known One or more immunogenicity enhancers are used in combination.
  • the immunogen can be purified from a natural source or produced in a genetically modified cell.
  • the DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin.
  • the DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to adenoviral vectors, baculovirus vectors, plasmids, and non-viral vectors.
  • sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.
  • the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • artificial synthesis methods can also be used to synthesize the relevant sequence, especially when the fragment length is short.
  • a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the term "host cell” refers to a cell into which an expression vector has been introduced.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant or animal cell (such as a mammalian cell).
  • the step of transforming host cells with recombinant DNA described in the present invention can be carried out by techniques well known in the art.
  • the obtained transformants can be cultured by conventional methods, and the transformants express the polypeptide encoded by the gene of the present invention.
  • conventional culture medium is used to cultivate under suitable conditions.
  • the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). To determine.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies have different stabilities in different formulation buffers, which are manifested as changes in charge heterogeneity, degradation of antibody molecules, and aggregation. These changes in quality properties are related to the physical and chemical properties of the antibodies themselves. Therefore, in the process of antibody drug development, it is necessary to screen formulation buffers suitable for different antibodies based on their physical and chemical properties.
  • commonly used antibody formulation buffer systems include phosphate buffer, citrate buffer, histidine buffer, etc.
  • salt ions or excipients such as sorbitol, trehalose, sucrose, etc.
  • surfactants such as Tween
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the nature of the formulated substance and the condition to be treated.
  • the prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.
  • the antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used to bind to CD27 protein molecules, and thus can be used to prevent and treat CD27-related diseases.
  • other therapeutic agents can also be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the polypeptide of the present invention can
  • a safe and effective amount of the pharmaceutical composition is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 milligrams/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 20 milligrams/kg body weight.
  • the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.
  • the antibodies of the invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.
  • the sample (specimen) used includes cells, tissue samples and biopsy specimens.
  • the term "biopsy” used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or fragment thereof) of the present invention.
  • the kit further comprises a container, instructions for use, a buffer, etc.
  • the antibody of the present invention can also be fixed to a detection plate.
  • the present inventors obtained highly specific and high affinity anti-CD27 mouse monoclonal antibodies 13B3, 16B1 and 17H3 through hybridoma fusion technology, and prepared human-mouse chimeric monoclonal antibodies and humanized antibodies based on them.
  • the antibodies of the present invention have high affinity and high specificity for human CD27, and can bind not only to solid-phase human CD27 but also to membrane-formed human CD27 with high efficiency.
  • the antibodies of the present invention have significant in vitro activity. They not only efficiently bind to human CD27, efficiently activate T cells and promote T cell proliferation, but also efficiently block the binding of CD27 to CD70, thereby inhibiting immunity and being used for the treatment of autoimmune diseases.
  • the humanized antibodies of the present invention have low or no immunogenicity when applied to humans.
  • the antibodies of the present invention have some binding to the solid phase-bound cynomolgus monkey CD27 recombinant protein, but do not bind to mouse CD27, thus facilitating in vivo experiments in non-human primates.
  • the antibody of the present invention has ADCC effect, and therefore has a significant specific killing effect on leukemia (myeloid leukemia, lymphocytic leukemia, T cell leukemia, B cell leukemia, etc.) and lymphoma that highly express CD27.
  • leukemia myeloid leukemia, lymphocytic leukemia, T cell leukemia, B cell leukemia, etc.
  • lymphoma that highly express CD27.
  • the ACDD loss-of-function mutant antibody of the present invention has essentially no direct killing effect on T lymphocytes expressing CD27, and is suitable for use in applications requiring activation of the immune function of T cells and maintenance of the number of T cells, such as treatment of solid tumors and treatment of non-T lymphocyte blood tumors.
  • Mouse spleen cells were used to fuse with mouse myeloma cells.
  • the medium was completely replaced, and the culture medium was IMDM culture medium containing 10% FBS, 200ul/well, incubated at 37°C, 5% CO 2.
  • ELISA test was performed according to the cell growth density. The cells in the positive wells tested were subcloned for the first and second time. The detection steps are as follows:
  • Post-fusion binding antibody screening CD27-mFc coating: Dilute CD27 with PBS to a concentration of 1ug/ml, mix well and aspirate into a 96-well ELISA plate, 50ul/well, seal with a sealing film and incubate at 4°C overnight.
  • ELISA plate blocking Wash twice with 250ul/well PBS and pat dry. Add 150ul/well of blocking solution, seal with a sealing film and incubate at 37°C for 1.5 hours.
  • Primary antibody incubation Take 100ul/well of hybridoma supernatant and add it to the ELISA plate, seal with a sealing film and incubate at 37°C for 1 hour.
  • Secondary antibody incubation Discard the blocking solution, wash 3 times with 250ul PBST and pat dry. Dilute the goat anti-mouse Fab HRP secondary antibody with a binding solution at 1:3000, add 100ul/well to the ELISA plate, seal with a sealing film and incubate at 37°C for 1 hour.
  • TMB incubation Discard the blocking solution, wash 3 times with 250ul PBST and pat dry. Add TMB 50ul/well to the ELISA plate, seal the plate with a sealing film and place at 37°C for 15-25 minutes.
  • HCl termination terminate with 2M HCl 50ul/well.
  • ELISA reader detection dual wavelength 450nm/655nm detection, the results are calculated as OD450-OD655.
  • CD27-mFc coating Dilute CD27-mFc with PBS to a concentration of 1ug/ml, mix well, and then use a multi-well pipette to aspirate into a 96-well ELISA plate, 50ul/well, seal with a sealing film, and place in a 4°C refrigerator overnight.
  • ELISA plate blocking Wash twice with 250ul/well PBS and pat dry. Then add 150ul/well of blocking solution, seal with a sealing film, and place at 37°C for 1.5 hours.
  • Primary antibody incubation Wash twice with 250ul/well PBS and pat dry. Dilute 0.6ug/ml CD70-hFc in the binding solution, and add 50ul/well to the 96-well dilution plate.
  • the expression cassette for expressing human CD27 was introduced into CHO cells to obtain a CHO cell line expressing human CD27 (transmembrane protein), which was designated as CHO-CD27.
  • the three clones are 13B3, 16B1 and 17H8, among which 13B3 and 16B1 have obvious function of blocking CD70 binding to CD27, while 17H8 has weaker blocking ability.
  • Total RNA Miniprep Kit catalog number RTN70. Take about 8 ⁇ L of total RNA for subsequent reverse transcription PCR, first at 65°C for 5 min to eliminate the secondary structure of RNA, then add 2 ⁇ L of 5 ⁇ gDNA wiper, 42°C for 2 min to remove genomic DNA contamination. Finally, add 2 ⁇ L of 10 ⁇ RT Mix, 2 ⁇ L of HiscriptIII Enzyme Mix and 1 ⁇ L of random hexamer (Random Hexamers, Norveg, R312). Tap the tube wall to mix, centrifuge briefly to throw the liquid to the bottom of the tube, and finally perform reverse transcription reaction under the following conditions: 25°C, 5 min; 42°C, 45 min; 85°C, 5 s.
  • the above cDNA was used as a template and PCR was used to amplify the hybridoma antibody with different primer combinations.
  • the primers used in this experiment mainly refer to von Boehmer, L. et al.,
  • Reaction conditions denaturation at 94°C for 3 minutes, followed by 94°C for 30 seconds, 55°C for 30 seconds, and 72°C. 40 cycles of 35 sec each were performed with a 5 min incubation at the end of the cycle at 72 °C.
  • the present invention screened 3 mouse hybridomas that bind to solid phase and membrane CD27, namely 13B3, 16B1 and 17H8. Each clone was further cloned and the V region sequence of the heavy chain and light chain was analyzed.
  • the sequences of three strains of mouse hybridoma antibodies against human CD27 were sequenced and analyzed in the early stage. The sequences of two clones were consistent. In this experiment, two clones with different sequences were recombinantly expressed and analyzed for activity.
  • specific PCR primers were designed according to different sequences. The PCR reaction conditions were as follows: after denaturation at 94°C for 3 minutes, 40 cycles were performed at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 35 seconds, and incubated at 72°C for 5 minutes at the end of the cycle.
  • the heavy chain expression vector (plasmid P378) and the light chain expression vector (P379) were digested with Xho I.
  • the digested vector was identified by agarose gel electrophoresis, and after confirming that the bands were correct, it was recovered and purified.
  • the digested vector and fragments were quantified and homologously recombined, and the ligated products were transformed, and the plasmids were extracted for transient cell transfection. All 293F cells, culture media and transfection reagents in this experiment were purchased from Sino Biological.
  • the cell culture fluid harvested by transient transfection was centrifuged at 4000rp for 10min, and the cell pellet was discarded.
  • the binding of two CD27 monoclonal antibodies to the CD27 antigen (Kangdai homemade) was tested by ELISA using standard methods and steps. The specific steps are as follows: dilute the antigen CD27-mFc (Kangdai homemade) to 1ug/mL with NaHCO 3 solution at pH 9.6, add 50uL to each well in a 96-well ELISA plate, and store in a refrigerator at 4°C overnight. The next day, after washing twice with PBS, block with 3% BSA, 100uL per well, and incubate at 37°C for 1.5h. After washing 4 times with PBST, add the antibody diluent and continue incubating at 37°C for 2h.
  • FACS buffer i.e., 1 ⁇ PBS+0.5% BSA solution.
  • FACS buffer i.e., 1 ⁇ PBS+0.5% BSA solution.
  • the amount of cells required for one sample is about 1-5 ⁇ 10 5 .
  • Wash the cells once with FACS buffer dilute the secondary antibody FITC-anti-human IgG (Abcam: 6854) with FACS buffer, resuspend the cells and incubate in a 4°C refrigerator for 30min.
  • wash the cells with FACS buffer resuspend the cells with 200uL FACS buffer, and detect them on the machine.
  • the results are shown in Figure 3 and Table 3.
  • the 13B3-hFc chimeric monoclonal antibody binds well to CHO-CD27, with an EC 50 of 2.486 ug/mL for binding to CHO-CD27, and the positive control 1F5 has an EC 50 of 2.869 ug/mL, which is equivalent to the binding ability.
  • the data show that the 13B3-hFc antibody of the present invention not only has a strong binding ability to solid-phase antigens (ELISA method), but also has a strong binding ability to cells.
  • the binding activity of 17H8-hFc chimeric monoclonal antibody to CHO-CD27 was also high.
  • the cells were resuspended again with 100uL of FACS working solution, and the secondary antibody Cy5-AffiniPure F(ab') 2 Fragment Goat Anti-Human IgG, Fc ⁇ fragment specific (JacksonImmuno Research, 109-176-098) and FITC-anti-human CD3 antibody (BD Bioscience, 556611) PE-anti-human CD19 (biolegend,) were added and gently mixed, and incubated at 4°C for 30min. Finally, wash the cells with FACS working solution, resuspend them in 300uL buffer and detect them on the machine.
  • the results are shown in Figure 4.
  • the first row shows the expression of CD27 on CD3+ T cells, and the second row shows the expression of CD27 on CD19+ B cells.
  • the dissociation constant (Kd) of human-mouse chimeric CD27 monoclonal antibody was detected using GATOR (ProbeLife) detection instrument and AHC (Pall, 185064), Human antibody Capture probe.
  • the human-mouse chimeric CD27 monoclonal antibody was diluted to 50nM in binding buffer (Q buffer [PBS (10mM PH7.4) + 0.02% Tween 20 + 0.2% BSA].
  • the chimeric CD27 antibody was diluted 2-fold in Q buffer starting from the highest concentration of 100nM.
  • the kinetic association assay was started by placing the antibody capture sensor in the above serially diluted antigen solution. Open Octet Data Acquisition software and select New Kinetics Experiment-Basic Kinetics mode;
  • CD70 (R&D, 9328-CL-100) was diluted to 1ug/mL with PBS, and 50uL was added to each well of the 96-well ELISA plate and stored in a refrigerator at 4°C overnight. The next day, after washing twice with PBS, the plate was blocked with 3% BSA, 100uL was added to each well, and the plate was incubated at 37°C for 1.5h. After washing 4 times with PBST, a fixed concentration of CD27-mFc (final concentration of 4.3ug/mL) and antibody diluent were added, and the plate was incubated at 37°C for 2h.
  • the 13B3-hFc chimeric CD27 monoclonal antibody can specifically block the binding of CD27 and CD70, with an IC 50 of 1.474 ug/mL.
  • 17H8-hFc did not block the binding of CD27 to CD70, suggesting that the binding sites of 17H8-hFc and CD27 are different. Same as 13B3-hFc.
  • 13B3-hFc was diluted to 2ug/mL with NaHCO3 solution at pH 9.6, and 50uL was added to each well of a 96-well ELISA plate and stored in a refrigerator at 4°C overnight. The next day, after washing twice with PBS, the plate was blocked with 3% BSA, 100uL was added to each well, and incubated at 37°C for 1.5h. After washing 4 times with PBST, a fixed concentration of CD27-mFc (final concentration of 0.8ug/mL) and 1F5 antibody diluent were added, and the plate was incubated at 37°C for 2h.
  • Example 5 Human-mouse chimeric CD27 monoclonal antibody promotes IFN ⁇ secretion and T cell proliferation in vitro 5.1 Promotes IFN ⁇ secretion
  • lymphocytes secrete cytokine IFN ⁇ and promote cell proliferation.
  • human-mouse chimeric CD27 monoclonal antibody disclosed in the present invention as a positive regulator of T cell activation, we conducted the following experiments to demonstrate the enhancing effect of the disclosed antibody on IFN ⁇ production of PBMC cells and its effect on T cell proliferation.
  • PBMC cells Resuscitate PBMC cells ( termells), remove the cells from the liquid nitrogen tank, quickly place them in a 37°C water bath to melt, transfer them to a 15mL centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, and adjust the cell density to 5 ⁇ 10 ⁇ 6/mL. Pipette 100uL of PHA dilution into a 96-well plate, then pipette 100uL of cell fluid into the corresponding wells, shake gently, and place in a 37°C, 5% CO2 incubator for 72h. After 72h, collect the cell supernatant and use the IFN ⁇ detection kit (Biolegend, 430115) to detect the IFN ⁇ content.
  • IFN ⁇ detection kit Biolegend, 430115
  • anti-CD3 antibody Biolegend, 317303
  • the antibody to be tested were diluted to a fixed concentration with PBS, and 50uL was added to each well of the cell culture plate. After gently mixing, it was placed in a 4°C environment overnight. The next day, the PBS antibody dilution was discarded, and the plate was placed open on a clean bench for a few minutes to dry the remaining liquid. Resuscitated purified CD3 + T cells (Hycells, donor ID: PBZ1017), and the cells were collected by centrifugation and labeled with CFSE (Biyuntian, C0051). The relevant operations were carried out according to the instructions. Then the cell density was adjusted to 0.5 ⁇ 10 ⁇ 6/mL with complete culture medium, and 200uL was added to the above 96-well plate per well. Placed in a 37°C, 5% CO 2 incubator for 72h.
  • anti-CD27 antibodies can significantly promote the proliferation of CD3+T cells.
  • the proliferation ratios of cells in the 1F5 and 13B3-hFc treatment groups were 73.69% and 78.54%, respectively, among which 13B3-hFc was more effective than 1F5.
  • Two cell lines were used in this experiment: 1.
  • the specific cell construction method is as follows: Jurkat was electroporated with two plasmids at the same time, one for expressing CD27 (i.e., CD27 cDNA was constructed into a conventional expression plasmid driven by CMV promoter), and the other was pNL3.2.NF- ⁇ B-RE[NlucP/NF- ⁇ B-RE/Hygro]Vector (Promega, N111A). After hygromycin and G418 pressure screening for 2 weeks, CD27-positive single cells were sorted by flow cytometry. After the single cells grew into a cell population, the cells were spread on anti-CD3 mAb gradient-coated cell culture plates.
  • Raji cells This cell is a lymphoma cell line that highly expresses molecules such as CD80, CD86, and CD70.
  • the experimental steps are as follows: prepare anti-CD27mAbs into 2 ⁇ working solution, dilute the antibody to 60ug/mL with complete culture medium (RPMI1640+10% FBS), then perform 3-fold gradient dilution, take 50uL and add it to a dedicated 96-well plate (Cornning, 3903), then add 50uL of the mixture of Jurkat-CD27-NF ⁇ B-Luc and Raji cells (number ratio of 2:1, density 4E6/mL) to the culture plate, and detect after 16h (Promega, N1120).
  • anti-CD27 antibodies inhibit the CD27 downstream NF ⁇ B signaling pathway by blocking the binding of CD70 on the surface of Raji cells and CD27 on the surface of Jurkat cells. This effect becomes more obvious as the antibody concentration increases. Although 1F5 and 13B3-hFc bind to different epitopes of CD27, both have this function and the effect is comparable.
  • the humanized antibody sequence was designed. The specific steps are as follows: First, use Discovery Studio and Antibody Modeling, using homology modeling method to construct a three-dimensional molecular model of the variable region. Next, by comparing the existing antibody structures in the database, the variable region and CDR of the parent antibody are structurally simulated. At the same time, cDNA-derived human germline sequences with high homology to the murine parent antibody VH and VL are selected for comparison.
  • the heavy chain VH of 13B3 selects IGHV1 with the highest homology as the humanized design template and designs the sequence.
  • the light chain VL selects IGKV6 and IGKV3 as the humanized design template and designs the sequence.
  • the original heavy chain mVH sequence of the murine parent antibody numbered 13B3 was designed into four humanized sequences: 13B3-huVH1 (SEQ ID No: 17), 13B3-huVH2 (SEQ ID No: 18), 13B3-huVH3 (SEQ ID No: 19), and 13B3-huVH4 (SEQ ID No: 20).
  • the original light chain mVL of the antibody was designed into four humanized sequences. Vl sequences: 13B3-huVL1 (SEQ ID No: 21), 13B3-huVL2 (SEQ ID No: 22) and 13B3-huVL3 (SEQ ID No: 23), 13B3-huVL4 (SEQ ID No: 24).
  • CD27 monoclonal antibody Varlilumab (1F5) was used as a positive control.
  • test method is the same as in Example 3.1, and the results are shown in Figure 10 and Table 10.
  • test method is the same as in Example 3.2, and the results are shown in Figure 11 and Table 11.
  • test method is the same as in Example 3.5, and the results are shown in Figure 12 and Table 12.
  • Fix the ligand to the probe After preparing the ligand, set the flow rate to 10uL/min and fix the ligand to the probe.
  • the affinity of some humanized antibodies to CD27 is better than that of the chimeric antibody 13B3-hFc.
  • the supernatants were collected for detection of IFN ⁇ , and the cells were collected for detection of cell proliferation.
  • T cell proliferation Compared with the isotype control IgG1, all anti-CD27 antibodies can significantly promote CD3+ T cell proliferation.
  • the isotype control IgG1 proliferation ratio was 2.03%, and the five humanized antibodies (13B3-huH4L1 (P33425), 13B3-huH2L2 (P33426), 13B3-huH2L3 (P33430), 13B3-huH2L4 (P33433), 13B3-huH3L4 (P33434)) were 7.74%, 8.03%, 10.64%, 12.83%, 8.9%, respectively.
  • the proliferation ratio of the isotype control IgG1 was 3.35%, and the ratios of the five humanized antibodies (13B3-huH4L1 (P33425), 13B3-huH2L2 (P33426), 13B3-huH2L3 (P33430), 13B3-huH2L4 (P33433), 13B3-huH3L4 (P33434)) were 12.59%, 12.24%, 14.83%, 15.83%, and 13.04%, respectively.
  • all anti-CD27 antibodies showed a significant effect in promoting T cell proliferation.
  • IFN ⁇ secretion When T cells receive the first signal from anti-CD3mAb and the costimulatory signal from anti-CD27mAb, in addition to massive proliferation, they also secrete cytokines, including IFN ⁇ . We also detected the content of IFN ⁇ in the supernatant, and the five humanized antibodies (13B3-huH4L1 (P33425), 13B3-huH2L2 (P33426), 13B3-huH2L3 (P33430), 13B3-huH2L4 (P33433), 13B3-huH3L4 (P33434)) increased by 1.62, 1.86, 1.52, 1.86, and 1.51 times, respectively. In summary, all anti-CD27 antibodies showed a significant promotion of T cell secretion of IFN ⁇ .
  • Example 13 Effect of humanized antibodies on T and B lymphocyte levels in vivo
  • Human CD27 knock-in (KI) mice were used to study the effects of humanized antibody 13B3-huH2L4 on T and B lymphocyte content in vivo.
  • mice 24 KI mice were divided into 4 groups, 6 mice in each group (3 females and 3 males).
  • the mice in the 4 groups were intraperitoneally injected with PBS, 13B3-huH2L4 (P33433) 1 ⁇ g/mL and 10 ⁇ g/mL, and positive reference antibody 1F5 10 ⁇ g/mL, twice a week, for a total of 3 times. Blood was collected 3 days and 10 days after the last administration to detect lymphocytes.
  • the anti-CD27 antibody of the present invention efficiently kills T lymphocytes that highly express CD27.
  • Example 14 Activation effect of humanized antibodies combined with anti-PD-1 or CTLA-4 antibodies on T lymphocytes activated by PHA
  • Human PBMC (TPCS, A19K214031) was added to a 24-well culture plate and cultured in RPMI1640 containing 10% FBS.
  • PHA (Sigma NO 9019) was added to a final concentration of 1 ⁇ g/mL to activate T lymphocytes.
  • the supernatant was discarded, the cells were washed once, and then the cells were added to a 96-well culture plate, 5 x 10 5 cells per well, cultured in RPMI1640 containing 10% FBS, PHA was added to a final concentration of 1 ⁇ g/mL, and 13B3-huH2L4 0.3 ⁇ g/mL or 3 ⁇ g/mL, and anti-PD-1 or CTLA-4 or TIGIT antibodies were added to a final concentration of 3 ⁇ g/mL (as shown in Figure 17).
  • the anti-PD-1 antibody was Tepli, and the anti-CTLA-4 and TIGIT antibodies were both commercially available antibodies (Sakin). After 3 days of culture, the supernatant was collected to detect the IFN ⁇ content.
  • the two amino acids L234/L235 (EU numbering) in the heavy chain CH1 region of the humanized antibodies 13B3-huH2L3 and 13B3-huH2L4 were replaced with amino acid Ala (LALA variant) to eliminate the antibody's ADCC/ADCP/CDC function.
  • the heavy chain sequences of 13B3-huH2L3 and 13B3-huH2L4 are shown in SEQ ID NO:27, and the heavy chain sequence of the LALA variant is shown in SEQ ID NO:28.
  • the variant gene was obtained by artificial gene synthesis.
  • the two variant antibodies 13B3-huH2L3 (LALA) and 13B3-huH2L4 (LALA) share the LALA variant heavy chain (SEQ ID No:28).
  • CD27-mFc coating Dilute CD27 to a concentration of 1 ⁇ g/mL with 1XPBS, mix well, and then use a multi-well pipette to aspirate into a 96-well ELISA plate at 50 ⁇ L/well. Seal with a sealing film and place in a 4°C refrigerator overnight. Primary antibody incubation: 13B3-huH2L3 (LALA), 13B3-huH2L4 (LALA) antibodies and various control antibodies (as shown in Figure 18) were graded diluted, 100 ⁇ L/well were added to the ELISA plate, the plate was sealed with a sealing film, and then placed at 37°C for 1 hour.
  • LALA 13B3-huH2L3
  • LALA 13B3-huH2L4
  • Secondary antibody incubation Discard the blocking solution, wash 3 times with 250 ⁇ L PBST, and pat dry. Dilute the AP-labeled anti-human Fc secondary antibody with the binding solution at 1:3000, add 100 ⁇ L/well to the ELISA plate, seal with a sealing film, and place at 37°C for 1 hour.
  • PNPP incubation Discard the blocking solution, wash 3 times with 250 ⁇ L PBST, and pat dry. Take 1 PNPP and dissolve it in 1mL 5X diethanolamine substrate, then add 4mL deionized water to dissolve it, add 50 ⁇ L/well to the ELISA plate, seal the plate with a sealing film and place it at 37°C for 15-25 minutes.
  • ELISA detection dual wavelength 405nm/490nm detection, the result is calculated as OD405-OD490.
  • the variant antibody 13B3-huH2L4 (LALA) and its parent antibody (wild type) 13B3-huH2L4 have the same in vitro binding activity to human CD27 (EC 50 is 29 ng/mL), and there is no significant difference in the in vitro binding activity to CD27 between the variant antibody 13B3-huH2L3 (LALA) and other control antibodies (1F5 and MK5890) ( Figure 18 ).
  • CD70 coating Dilute CD70 to a concentration of 1 ⁇ g/mL with 1XPBS, mix well, and aspirate into a 96-well ELISA plate with a multi-well pipette, 50 ⁇ L/well, seal with a sealing film, and place in a 4°C refrigerator overnight.
  • ELISA plate blocking Wash twice with 250 ⁇ L/well PBS, pat dry. Add 150 ⁇ L/well of blocking solution, seal with a sealing film, and place at 37°C for 1.5 hours.
  • TMB incubation discard the blocking solution, wash 3 times with 250 ⁇ L PBST, and pat dry. Add TMB 50 ⁇ L/well to the ELISA plate, seal with sealing film and incubate at 37°C for 15-25 minutes.
  • HCL termination The reaction was terminated with 2M HCL 50 ⁇ L/well.
  • Detection by microplate reader dual wavelength 450nm/655nm detection, the result was calculated as OD450-OD655.
  • variant antibody 13B3-huH2L4 LALA
  • parent antibody 13B3-huH2L4 1.31 ⁇ g/mL
  • the variant antibody 13B3-huH2L3 also has excellent in vitro inhibitory activity on the binding of CD27 and CD70, with an IC 50 of 1.09 ⁇ g/mL, which is better than the control antibody 1F5 (IC 50 of 1.31 ⁇ g/mL) and significantly better than the control antibody MK5890 (IC 50 of 1.89 ⁇ g/mL) ( FIG. 19 ).
  • the binding activity of the variant antibodies to membrane CD32a and membrane CD64 was studied using Jurkat cells stably transfected with the human CD32A gene (Jurkat-CD32a) and CHO-K cells stably transfected with the human CD64 gene (CHO-CD64).
  • Jurkat-CD32a and CHO-CD64 cells were suspended in 0.5% BSA PBS and added to 96-well round-bottom plates, with 50 ⁇ L per well containing 5 x 10 5 cells.
  • 13B3-huH2L4 and 13B3-huH2L4(LALA) were diluted 3-fold and incubated at 4°C for 1 hour.
  • 300-fold diluted FITC-labeled anti-human Fc antibody Jackson ImmunoResearch Lab., #109-116-170 was added at 100 ⁇ L/well and incubated at 4°C for 0.5 hour.
  • the cells were washed twice with 0.5% BSA PBS, suspended in 200 ⁇ L PBS buffer, and analyzed by flow cytometry.
  • the variant antibody 13B3-huH2L4 did not bind to CD32a expressed on the cell surface, while its parent wild-type antibody 13B3-huH2L4 could normally bind to CD32a on the cell surface ( FIG. 20A ).
  • the variant antibody 13B3-huH2L4 did not bind to CD64 expressed on the cell surface, while its parent wild-type antibody 13B3-huH2L4 could normally bind to CD64 on the cell surface ( FIG. 20B ).
  • Example 17 Effects of variant antibodies on T lymphocytes in vivo
  • mice Fourteen KI mice were divided into three groups, two in the PBS control group and four in each of the other two groups, and 13B3-huH2L4 (LALA) 10 mg/kg and positive reference antibody 1F5 10 mg/kg were administered intraperitoneally twice a week for a total of three times. Blood samples were collected for testing at 3 and 10 days after the last administration.
  • LALA 13B3-huH2L4
  • the positive reference antibody 1F5 significantly reduced the proportion of CD3+ (Figure 21A), CD3+CD4+ ( Figure 21B) and CD3+CD8+ ( Figure 21C) T lymphocytes in the peripheral blood compared with the PBS control group, but 13B3-huH2L4 (LALA) had no effect on the content of these T lymphocytes and did not change their proportion in the peripheral blood.
  • test results of the number of each T lymphocyte in the peripheral blood showed that the trend of the number change was consistent with the trend of the respective percentage change.
  • the variant antibody 13B3-huH2L4 substantially eliminated the ADCC function, and thus had substantially no direct killing effect on T lymphocytes expressing CD27.
  • Such variant antibodies of the present invention with significantly reduced or eliminated ADCC are particularly suitable for use in applications requiring activation of the immune function of T cells and maintenance of the number of T cells, such as treatment of solid tumors, and treatment of non-T lymphocyte blood tumors (such as B cell tumors).

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Abstract

本发明提供了抗CD27单克隆抗体及其制剂。具体地，本发明提供了一种新的抗CD27抗体。本发明的抗体能够高特异性地结合抗原，具有高亲和力和高生物活性，可特异性结合人CD27抗原分子，有望用于生产在体外和体内均可高效进行免疫治疗的药物。

Description

抗CD27单克隆抗体及其应用 技术领域
本发明涉及医药领域，具体地涉及抗CD27人源化单克隆抗体及其制剂。
背景技术
CD27是TNFR超家族成员之一，是一种分子量约为55kD的I型跨膜糖蛋白,通常是由二硫桥连结的同源二聚体。CD27在大多数T细胞、自然杀伤细胞以及抗体分泌浆细胞和记忆B细胞上作为表面抗原表达。CD70作为CD27的配体，与CD27相互作用，并将细胞内TRAF蛋白募集到CD27的胞内结构域，从而激活下游信号。一般情况下,与CD70结合后，细胞内部的CD27通过链接TNF受体相关分子TRAF2和TRAF5，激活NF-κB以及JNK信号通路，来促进T细胞的增殖及相应细胞因子的分泌。研究表明，CD27-CD70介导的共刺激对T细胞的生长、分化和存活中起重要作用，同时还可以促进B细胞增殖、分化为浆细胞及产生免疫球蛋白以及诱导NK细胞活化。
CD27被认为是一种很有前途的癌症免疫治疗靶点，目前，有几个CD27抗体处在临床试验中,包括已进入临床II期的Celldex Therapeutics(塞德斯医疗公司)的CD27单抗Varlilumab。在WO2011/130434中，公开了这种抗人激动型CD27抗体，其在交联时激活CD27，体内实验中可以增强小鼠免疫细胞增值和细胞因子释放、增强小鼠对疫苗的免疫应答以及在不同肿瘤模型中增强小鼠的抗肿瘤活性。Varlilumab与抗PD-1单抗以及PD-L1单抗联合治疗的临床试验正在进行中，目前已显示出具有良好的耐受性，为通常对PD-1抑制剂单一疗法有抗药性的肿瘤患者提供了新的治疗前景。Aduro BioTech基于其单B细胞筛选单克隆抗体技术平台，开发了抗CD27激动型单抗MK-5890,目前也处于II期临床试验中，在披露的WO2012/004367专利中，描述了第一抗人激动性抗体(命名为hCD27.15)，可以激活CD27介导的NF-κB免疫反应的共刺激。另外，百时美施贵宝公司(BMS)公开的专利WO2019195452A1中披露了一种非配体阻断型的CD27抗体，可以激活T细胞，并在在小鼠肿瘤模型中具有抗肿瘤活性。
综上所述，CD27是极具潜力的免疫治疗新靶点。然而目前国内外还没有CD27的单克隆抗体药物上市，因此需要开发出更优的临床药效的CD27单克隆抗体。
发明内容
本发明目的是提供了一种高亲和力和高生物活性的CD27抗体及其应用。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区具有选自下组的三个互补决定区CDR：
(a)SEQ ID No:2、3和4所示的CDR1、CDR2和CDR3；
(b)SEQ ID No:2、25和4所示的CDR1、CDR2和CDR3；
(c)SEQ ID No:2、26和4所示的CDR1、CDR2和CDR3；
(d)SEQ ID No:10、11和12所示的CDR1、CDR2和CDR3。
在另一优选例中，所述的重链可变区包括以下三个互补决定区CDR：
(1)互补决定区CDR1，所述互补决定区CDR1的氨基酸序列如SEQ ID No:2所示；
(2)互补决定区CDR2，所述互补决定区CDR2的氨基酸序列如SEQ ID No:3、25或26所示；及
(3)互补决定区CDR3，所述互补决定区CDR3的氨基酸序列如SEQ ID No:4所示。
在另一优选例中，所述重链可变区包括四个骨架区FR，所述4个骨架区FR被上述CDR1、CDR2和CDR3间隔开。
在另一优选例中，所述的重链可变区具有SEQ ID No:1、17、18、19、或20所示的氨基酸序列。
在另一优选例中，所述的重链可变区具有SEQ ID No:9所示的氨基酸序列。
在本发明的第二方面，提供了一种抗体重链，所述抗体重链具有如本发明的第一方面所述的抗体的重链可变区。
在另一优选例中，所述重链的恒定区是人源的。
在另一优选例中，所述重链的恒定区为IgG1、IgG2或IgG4的重链恒定区。
在另一优选例中，所述重链恒定区为野生型Fc或突变型Fc。
在另一优选例中，所述突变型Fc导致抗体的ADCC作用丧失或基本丧失。
在另一优选例中，所述突变型Fc具有L234A和L235A突变。
在另一优选例中，所述重链序列如SEQ ID NO:27所示，所述重链的恒定区为野生型Fc。
在另一优选例中，所述重链序列如SEQ ID NO:28所示，所述重链的恒定区为突变型Fc，其具有在IgG1的Fc上的L234A和L235A突变。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区具有序列如SEQ ID No:6、7、和8所示的三个互补决定区L-CDR；
或所述的轻链可变区具有序列如SEQ ID No:14、15、和16所示的三个互补决定区L-CDR。
在另一优选例中，所述的轻链可变区包括以下三个互补决定区L-CDR：
(1)互补决定区L-CDR1，所述互补决定区L-CDR1的氨基酸序列如SEQ ID No:6所示；
(2)互补决定区L-CDR2，所述互补决定区L-CDR2的氨基酸序列如SEQ ID No:7所示；及
(3)互补决定区L-CDR3，所述互补决定区L-CDR3的氨基酸序列如SEQ ID No:8所示。
在另一优选例中，所述轻链可变区包括四个骨架区FR，所述4个骨架区FR被上述CDR1、CDR2和CDR3间隔开。
在另一优选例中，所述的轻链可变区具有SEQ ID No:5、21、22、23或24所示的氨基酸序列。
在另一优选例中，所述的轻链可变区具有SEQ ID No:13所示的氨基酸序列。
在本发明的第四方面，提供了一种抗体轻链，所述抗体轻链具有如本发明的第三方面所述的抗体的轻链可变区。
在另一优选例中，所述轻链的恒定区是人源的。
在另一优选例中，所述轻链的恒定区为Kappa、Lambda轻链恒定区。
在本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明的第一方面所述的重链可变区；和/或
(2)如本发明的第三方面所述的轻链可变区。
或者，所述抗体具有：如本发明的第二方面所述的重链；和/或如本发明的第四方面所述的轻链。
在另一优选例中，所述的抗体是抗CD27的抗体。
在另一优选例中，所述抗体还具有重链恒定区和轻链恒定区。
在另一优选例中，所述抗体的重链恒定区为野生型Fc或突变型Fc。
在另一优选例中，所述突变型Fc导致抗体的ADCC作用丧失或基本丧失。
在另一优选例中，所述突变型Fc具有L234A和L235A突变。
在另一优选例中，所述抗体的重链序列如SEQ ID NO:27所示，所述重链的 恒定区为野生型Fc。
在另一优选例中，所述抗体的重链序列如SEQ ID NO:28所示，所述重链的恒定区为突变型Fc，其具有在IgG1的Fc上的L234A和L235A突变。
在另一优选例中，所述抗体具有如SEQ ID No:1、17、18、19或20所示的重链可变区；和/或如SEQ ID No:5、21、22、23或24所示的轻链可变区。
在另一优选例中，所述抗体具有如SEQ ID No:9所示的重链可变区；和/或如SEQ ID No:13所示的轻链可变区。
在另一优选例中，所述抗体选自下组：
(Z1)具有SEQ ID No:1所示的重链可变区和SEQ ID No:5所示的轻链可变区的抗体：
(Z2)具有SEQ ID No:18所示的重链可变区和SEQ ID No:22所示的轻链可变区的抗体；
(Z3)具有SEQ ID No:18所示的重链可变区和SEQ ID No:23所示的轻链可变区的抗体；
(Z4)具有SEQ ID No:18所示的重链可变区和SEQ ID No:24所示的轻链可变区的抗体；
(Z5)具有SEQ ID No:19所示的重链可变区和SEQ ID No:24所示的轻链可变区的抗体；
(Z6)具有SEQ ID No:20所示的重链可变区和SEQ ID No:21所示的轻链可变区的抗体。
在另一优选例中，所述抗体为人源化抗体、嵌合抗体、或鼠源抗体。
在另一优选例中，所述抗体为特异性结合CD27。
在另一优选例中，所述的抗体具有阻断CD70结合CD27功能。
在另一优选例中，所述的抗体具有激活T细胞、促进T细胞增殖的功能。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体为双特异性抗体或多特异性抗体。
在另一优选例中，所述的抗体为药物偶联物形式。
在本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在本发明的第七方面，提供了一种抗体制剂，所述的抗体制剂包括：
(a)如本发明的第五方面所述的抗体；以及
(b)载体或赋形剂。
在另一优选例中，所述的制剂为药物组合物。
在另一优选例中，所述的赋形剂或载体为药学上可接受的载体或赋形剂。
在另一优选例中，所述的载体包括：缓冲剂、无菌水，任选的表面活性剂
在另一优选例中，在所述的制剂中，所述抗体的浓度为5-100mg/mL；较佳地10-70mg/mL，更佳地20-60mg/mL。
在另一优选例中，所述缓冲剂选自下组：PBS缓冲体系、柠檬酸缓冲体系、组氨酸缓冲体系、或其组合。
在另一优选例中，所述的制剂pH范围为5.0-7.5，较佳地为5.5-7。
在另一优选例中，所述的制剂为注射制剂。
在本发明的第八方面，提供了一种试剂盒，所述的试剂盒含有本发明的第五方面所述的抗体，以及盛装所述抗体的容器。
在本发明的第九方面，提供了一种CAR构建物，所述的CAR构建物的抗原结合区域的scFv段为特异性结合于CD27的结合区，并且所述scFv具有如本发明的第一方面所述的重链可变区和如本发明的第三方面所述的轻链可变区。
在本发明的第十方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明的第九方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第十一方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在本发明的第十二方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的免疫细胞、如本发明第十一方面所述的抗体药物偶联物、或其组合，所述活性成分用于
(a)制备检测试剂或试剂盒；
(b)制备预防和/或治疗CD27相关疾病的药物或制剂；和/或
(c)制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、霍奇金淋巴瘤、或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、或其组合。
在另一优选例中，所述药物或制剂用于制备预防和/或治疗与CD27(表达阳性的)相关的疾病的药物或制剂。
在另一优选例中，所述的抗体为药物偶联物(ADC)形式。
在另一优选例中，所述的检测试剂或试剂盒用于诊断CD27相关疾病。
在另一优选例中，所述检测试剂或试剂盒用于检测样品中CD27蛋白。
在另一优选例中，所述的检测试剂为检测片。
在本发明的第十三方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的免疫细胞、如本发明第十一方面所述的抗 体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物还包含抗肿瘤的第二活性成分。
在另一优选例中，所述的第二活性成分选自下组：细胞毒性药物、毒素、细胞因子、酶、抗体、或其组合。
在另一优选例中，所述的第二活性成分包括：靶向EGFR的抗体、靶向HER2的抗体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物用于治疗肿瘤。
在本发明的第十四方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体；或
(2)如本发明第六方面所述的重组蛋白；和/或
(3)如本发明第九方面所述的CAR构建物。
在本发明的第十五方面，提供了一种载体，所述的载体含有如本发明第十四方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第十六方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本发明第十五方面所述的载体或基因组中整合有如本发明第十四方面所述的多核苷酸。
在本发明的第十七方面，提供了一种体外检测(包括诊断性或非诊断性)样品中CD27蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD27蛋白。
在本发明的第十八方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明第五方面所述的抗体或如本发明第十一方面所述的抗体药物偶联物。
在本发明的第十九方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗如本发明第五方面所述的抗体的二抗；
或者，所述试剂盒含有如本发明第十八方面所述的检测板。
在本发明的第二十方面，提供了一种重组多肽的制备方法，所述方法包括：
(a)在适合表达的条件下，培养如本发明第十四方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明第五方面所述的抗体或如本发明第六方面所述的重组蛋白。
在本发明的第二十一方面，提供了一种治疗CD27相关疾病，例如癌症，的方法，所述方法包括：给需要的对象施用如本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
在另一优选例中，所述对象包括人和非人哺乳动物。
在另一优选例中，所述对象为人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明抗体的SDS-PAGE分析。
图2显示了本发明抗体的ELISA试验结果。
图3显示了本发明抗体与CHO细胞膜上CD27蛋白的结合效果。
图4显示了FACS检测人鼠嵌合CD27抗体与人T、B细胞表面CD27蛋白的结合效果。
图5显示了人鼠嵌合CD27抗体对CD27与CD70的结合的抑制效果。
图6显示了人鼠嵌合CD27单克隆抗体结合表位实验。
图7显示了13B3和1F5结合的表位实验。
图8显示了抗CD27的抗体促进CD3+T细胞的增殖的效果。
图9显示了抗CD27抗体通过阻断Raji细胞表面CD70和Jurkat细胞表面的CD27的结合，而抑制CD27下游NFκB信号通路。
图10显示了人源化单克隆CD27抗体与抗原CD27的体外结合活性的ELISA结果。
图11显示了人源化CD27单克隆抗体与细胞膜上CD27蛋白的结合的流式细胞术结果。
图12显示了人源化CD27抗体对CD27与CD70的结合的抑制效果。
图13显示了人源化抗体对T细胞增殖的促进效果。
图14显示了本发明抗体最后1次给药后3天小鼠外周血中T和B淋巴细胞百分比的变化。图14A：CD3+T淋巴细胞；图14B：CD3+CD4+T淋巴细胞；图14C：CD3+CD4-T淋巴细胞；图14D：CD19+B淋巴细胞。
图15显示了本发明抗体最后1次给药后10天小鼠外周血中T淋巴细胞百分比的变化。图15A：CD3+T淋巴细胞；图15B：CD3+CD4+T淋巴细胞；图15C：CD3+CD8+T淋巴细胞。
图16显示了本发明抗体最后1次给药后10天小鼠脾脏中T淋巴细胞百分比的变化。图16A：CD3+T淋巴细胞；图16B：CD3+CD4+T淋巴细胞；图16C：CD3+CD8+T淋巴细胞。
图17显示了人源化抗体单独作用或联合其它免疫检查点抑制剂抗体对处于衰竭阶段的T细胞IFNγ产生的影响。
图18显示了野生型和变异的CD27抗体体外结合CD27的活性。
图19显示了野生型和变异的CD27抗体体外对CD27与CD70的结合的抑制效果。
图20显示了野生型和变异的CD27抗体结合细胞表面Fc受体CD32a和CD64的活性。图20A：与细胞表面CD32a的结合；图20B：与细胞表面CD64的结合。
图21显示了本发明野生型和变异的CD27抗体最后1次给药后3天小鼠外周血中T淋巴细胞百分比的变化。图21A：CD3+T淋巴细胞；图21B：CD3+CD4+T淋巴细胞；图21C：CD3+CD8+T淋巴细胞。
图22显示了本发明野生型和变异的CD27抗体最后1次给药后10天小鼠外周血中T淋巴细胞百分比的变化。图22A：CD3+T淋巴细胞；图22B：CD3+CD4+T淋巴细胞；图22C：CD3+CD8+T淋巴细胞。
具体实施方式
本发明人经过广泛而深入的研究，经过大量筛选，获得了多株对人CD27具有高亲和力等优良性能的鼠源抗体，并在高亲和力和高抗肿瘤活性的鼠源抗体基 础上，进一步制备了嵌合抗体和人源化改造。本发明的抗体能有效结合人CD27，具有优良的活性，可作为靶向治疗的单抗药物。在此基础上完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
序列同一性通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“轻链可变区”与“VL”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合CD27的抗体，例如具有重链可变区(如SEQ ID No:27或35的氨基酸序列)和/或轻链可变区(如SEQ ID No:31或39的氨基酸序列)的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
CD27
CD27作为一种协同刺激的T细胞受体，与OX40(CD134)、4-1BB的共刺激还促进活化T细胞的存活，是T细胞启动和记忆分化的关键；CD70在正常情况下，主要表达于活化的淋巴细胞，但在病理情况下，CD70高表达于多种肿瘤细胞中，肿瘤细胞通过表达CD70结合T细胞受体CD27，慢性的共刺激导致T细胞表达PD-1，TIM-3等免疫检查点，从而导致免疫功能耗竭。CD70在肿瘤中的高表达可能暗示了肿瘤利用CD70来控制表达CD27的肿瘤浸润淋巴细胞(TIL)，从而发生免疫逃逸。CD27靶点抗体从机制上具有强化肿瘤免疫、阻断免疫逃逸的功效。
抗体
如本文所用，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。
已有的抗体编号方案包括：
1.Kabat方案(Kabat等，1991)是基于相同结构域类型的序列之间的高序列变异区域的位置，抗体重(VH)和轻(Vλ和Vκ)可变结构域的编号不同。
2.Chothia的方案(Al-Lazikani，1997)与Kabat的方案相同，但校正了在第一个VH互补决定区(CDR)周围插入注释的位置，使其对应于结构环。同样，增强型Chothia计划(Abhinandan和Martin，2008)对插入位置进行了进一步的结构修正。
3.与这些类似Kabat的方案相反，IMGT(Lefranc，2003)和AHo(Honegger和Plückthun，2001)都定义了抗体和T细胞受体(TCR)(Vα和Vβ)可变结构域的独特方案。因此，可以容易地在域类型之间比较等效残基位置。IMGT和AHo在他们注释的位置数量(分别为128和149)以及他们认为indel发生的位置上有所不同。
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根据其较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。轻链根据恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
本发明所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4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(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。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(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成，从氨基端到竣基端依次排列的顺序序为:FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。轻链的3个CDR区指LCDR1、LCDR2和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。本发明的实施例中，结合kabat方法，对CD27抗体的6个CDR进行划分。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的抗CD27的单克隆抗体。制备时用CD27抗原注射试验对象，然后分离表达具有所需序列或功能特 性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源CD27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或其变体的重链恒定区。
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。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
术语“抗体的抗原结合片段”(或简称“抗体片段”)是指抗体的保持特异性结合抗原(例如，CLDN18.2)的能力的一个或多个片段。己显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语“抗体的抗原结合片段”中包含的结合片段的实例包括
(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；
(ii)F(ab')2片段，包含通过较链区上的二硫桥连接的两个Fab片段的二价片段；
(iii)由VH和CH1结构域组成的Fd片段；
(iv)由抗体的单臂的VH和VL结构域组成的Fv片段。
Fv抗体含有抗体重链可变区、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。一般的，Fv抗体还包含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。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，CD27分子上的特定部位)。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10-7M，例如大约小于10-8M、10-9M或l0-10M或更小的亲和力(KD)结合。
术语“竞争结合”是指与本发明的单克隆抗体识别CD27的胞外区上的相同表位(也称为抗原决定簇)或相同表位的一部分并与所述抗原结合的抗体。与本发明 的单克隆抗体结合相同表位的抗体是指识别并结合于本发明的单克隆抗体所识别的CD27的氨基酸序列的抗体。
术语“KD”或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本发明的抗体以小于大约10-7M，例如小于大约10-8M、10-9M或l0-10M或更小的解离平衡常数(KD)结合CD27。
如本文所用，术语“抗原决定簇”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。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。
如本文所用，术语“单克隆抗体”指得自单个细胞来源的克隆分泌的抗体。单克隆抗体是高度特异性的，针对单个抗原表位。所述的细胞可能是真核的、原核的或噬菌体的克隆细胞株。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据下表进行氨基酸替换而产生。

抗CD27人源化抗体
在另一优选例中，本发明抗体的重链恒定区和/或轻链恒定区可以是人源化的重链恒定区或轻链恒定区。更优选地，所述的人源化的重链恒定区或轻链恒定区为人IgG1、IgG2等的重链恒定区或人kappa、Lambda轻链恒定区。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本发明的抗体可以是双链或单链抗体，并且可以优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2、或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
本发明抗体可以是靶向CD27的人源化抗体、CDR嫁接和/或修饰的抗体。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
抗体的制备
任何适于产生单克隆抗体的方法都可用于产生本发明的CD27抗体。例如，可以用连接或天然存在的CD27蛋白或其片段免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的CD27都可以作为免疫原(抗原)，用于产生对CD27特异的非人抗体，筛选所述抗体的生物学活性。免疫原可以单独使用，或与本领域已知的 一种或多种免疫原性增强剂组合使用。免疫原可以由天然来源纯化，或者在遗传修饰的细胞中产生。编码免疫原的DNA在来源上可以是基因组或非基因组的(例如cDNA)。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于腺病毒载体、杆状病毒载体、质粒和非病毒载体。
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输己与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物或动物细胞(如哺乳动物细胞)。
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本发明的基因所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA)) 来测定。
抗体制剂
抗体在不同的制剂缓冲液中具有不同的稳定性，表现为电荷异质性的变化、抗体分子的降解、聚合等，这些质量性质的变化与抗体本身的理化性质相关，因此，在抗体药物开发过程，需根据不同抗体的理化性质筛选适合其自身的制剂缓冲液。目前常用的抗体制剂缓冲体系有磷酸盐缓冲液、柠檬酸缓冲液、组氨酸缓冲液等，同时根据抗体性质会添加不同浓度的盐离子或山梨醇、海藻糖、蔗糖等赋形剂，以及适量的诸如吐温等表面活性剂，以维持抗体的稳定性。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的CAR-T细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明的药物组合物可直接用于结合CD27蛋白分子，因而可用于预防和治疗CD27相关的疾病。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的药物组合物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内。
检测用途和试剂盒
本发明的抗体可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒。在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。本发明的抗体也可以固定于检测板。
本发明的主要优点包括：
(a)本发明人通过杂交瘤融合技术获得高特异性和高亲和力的抗CD27的鼠单抗13B3、16B1和17H3，并在此基础上制备了人鼠嵌合单克隆抗体和人源化抗体。
(b)本发明抗体对人CD27具有高亲和力和高特异性，不仅可结合固相人CD27还高效结合膜形式的人CD27。
(c)本发明抗体具有显著的体外活性，不仅高效结合人CD27，高效地激活T细胞和促进T细胞增殖，还可高效阻断CD27与CD70结合的结合，从而抑制免疫并用于自身免疫疾病的治疗。
(d)本发明抗体的结构稳定。
(e)本发明的人源化抗体应用于人体时，具有低免疫原性或无免疫原性。
(f)本发明抗体与结合固相的食蟹猴CD27重组蛋白有一定结合，但不结合小鼠CD27，因此有助于开展非人灵长动物的体内实验。
(g)本发明抗体具有ADCC作用，因此，对高表达CD27的白血病(髓系白血病、淋巴细胞白血病、T细胞白血病、B细胞白血病等)和淋巴瘤等有显著特异性杀伤作用。
(h)本发明的ACDD作用丧失型突变抗体对表达CD27的T淋巴细胞基本无直接的杀伤作用，适合用于需要激活T细胞的免疫功能且维持T细胞数量的应用场合，如针对实体瘤的治疗，以及针对非T淋巴细胞的血液肿瘤的治疗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold  Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非特别说明，否则本发明实施例中所用材料和试剂均为市售产品。
实施例1抗人CD27鼠单克隆抗体的产生
1.1免疫
将5只C57小鼠以重组制备的人CD27重组蛋白在完全弗氏佐剂(complete Freund's adjuvant CFA)存在下经由腹腔和皮下来进行多点免疫。在不完全弗氏佐剂(incomplete Freund's)存在下以相同的免疫原CD27分别在第14天、第21天进行免疫追加。
1.2融合
使用小鼠的脾细胞与小鼠骨髓瘤细胞融合。
1.3杂交瘤筛选
融合后第5天～10天，根据细胞生长密度，全换液，培养基为含10％FBS的IMDM培养基，200ul/孔，37℃，5％CO2孵育。融合后第10～14天，根据细胞生长密度，进行ELISA检测。检测的阳性孔细胞进行第一次和第二次亚克隆。检测步骤如下：
融合后结合抗体筛选：CD27-mFc包被：用PBS将CD27稀释至浓度为1ug/ml，混匀后用吸入96孔酶标板中，50ul/孔，用封板膜密封后4℃过夜。酶标板封闭：250ul/孔PBS洗涤2次，拍干。再加入150ul/孔的封闭液，封板膜密封后置37℃，1.5小时。一抗孵育：取杂交瘤上清100ul/孔加入酶标板，封板膜密封后置37℃，1小时。二抗孵育：弃封闭液，250ul PBST洗3遍，拍干。以结合液1:3000稀释山羊抗鼠Fab HRP二抗，100ul/孔加入酶标板，封板膜密封后置37℃，1小时。TMB孵育：弃封闭液，250ul PBST洗3遍，拍干。TMB 50ul/孔加入酶标板，封板膜密封后置37℃，15-25分钟。HCl终止：用2M HCl 50ul/孔终止。酶标仪检测：双波长450nm/655nm检测，结果以OD450-OD655计算。
竞争抗体检测：
CD27-mFc包被：用PBS将CD27-mFc稀释至浓度为1ug/ml，混匀后用多孔移液器吸入96孔酶标板中，50ul/孔，用封板膜密封后置于4℃冰箱过夜。酶标板封闭：250ul/孔PBS洗涤2次，拍干。再加入150ul/孔的封闭液，封板膜密封后置37℃，1.5小时。一抗孵育：250ul/孔PBS洗涤2次，拍干。分别稀释0.6ug/ml的CD70-hFc在结合液中，50ul/孔加入96孔稀释板中。50ul/孔杂交瘤上清加入上面的相应稀释板中，混匀后，再将100ul/孔加入包被好的96孔酶标板中。封板膜密封后置37℃，1.5小时。二抗孵育：弃封闭液，250ulPBST洗3遍，拍干。以 结合液1:3000稀释AP anti-human Fc，100ul/孔加入酶标板，封板膜密封后置37℃，1小时。PNPP孵育：弃封闭液，250ul PBST洗3遍，拍干。取1颗PNPP于1ml5X二乙醇胺底物，再加入4ml去离子水溶解，50ul/孔加入酶标板，封板膜密封后置37℃，15-25分钟。酶标仪检测：双波长405nm/490nm检测，结果以OD405-OD490计算。
FACS检测：
将表达人CD27的表达盒导入CHO细胞，获得表达人CD27(跨膜蛋白)的CHO细胞系，记为CHO-CD27。
将CHO-CD27细胞计数并1000rpm 5min离心。用0.5％BSA PBS重悬，取5E5个/孔加入96孔尖底板中。400g 5min离心，并弃去上清。每株阳性克隆选两个亚克隆，100ul/孔上清加入96孔尖底板，4℃，30min。0.5％BSA PBS 250ul/孔洗两次，1:100稀释Anti-mouse-IgG-FITC，100ul/孔加入96孔尖底板，4℃，30min。0.5％BSA PBS 250ul/孔洗两次，200ul/孔加入PBS。样品用Beckman CytoFLEX.进行检测。
实验结果与分析：
从众多克隆中，筛选出3株性能优异的克隆。这3株单克隆抗体都结合固相人CD27以及膜形式的人CD27，与结合固相的食蟹猴CD27重组蛋白有一定结合，但不结合小鼠CD27。3株克隆分别为13B3、16B1和17H8，其中13B3和16B1具有明显阻断CD70结合CD27的功能，17H8的阻断能力较弱。
1.4重链和轻链可变区测序
1.4.1 RNA提取及cDNA获得
取约1～2×105个杂交瘤细胞进行RNA提取(Sigma，GenEluteTMMammalian
Total RNA Miniprep Kit，货号RTN70)。取约8μL的总RNA进行后续的反转录PCR，首先65℃，5min，以消除RNA的二级结构，再加2μL的5×gDNA wiper，42℃，2min，以去除基因组DNA的污染。最后再加2μL的10×RT Mix，2μL的HiscriptIII Enzyme Mix和1μL的随机六聚体(Random Hexamers，诺唯赞，R312)。轻弹管壁混匀，瞬时离心将液体甩到管底，最后用如下条件进行逆转录反应：25℃，5min；42℃，45min；85℃，5s。
1.4.2抗体可变区基因的扩增
将上述cDNA做为模板，用PCR方法以不同的引物组合扩增出杂交瘤抗
体的VH和VL基因。本实验所用的引物主要参考文献von Boehmer,L.等,
Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.Nat Protoc,2016.11(10):p.1908-1923.，
反应条件：在94℃变性3分钟后，在94℃，30秒、55℃，30秒、72℃， 35秒下各40个循环，在72℃下的循环末端孵育5分钟。
本发明前期筛选到3株结合固相和膜形式CD27的小鼠杂交瘤，分别为13B3、16B1和17H8。对每株克隆进行进一步克隆，并进行重链和轻链的V区序列分析。
结果：获得的重轻链PCR的条带明亮，大小正确，测序反应峰型正常单一。NCBI Blast结果，符合正常的抗体V区序列特征，即有完整的Framework区，CDR区，无终止密码子，无移码突变，有VDJ/VJ基因的重排。成功获得所有抗体的V区，其中13B3和16B1序列相同，具体序列如下表1所示：
表1

实施例2.人鼠嵌合单克隆抗体的制备
前期测序分析了3株抗人CD27的鼠杂交瘤抗体的序列，其中两个克隆的序列一致，本次实验将2个序列不一样的克隆进行重组表达和活性分析。首先根据不同的序列设计特异性的PCR引物。PCR反应条件如下：在94℃变性3分钟后，在94℃，30秒、55℃，30秒、72℃，35秒下各40个循环，在72℃下的循环末端孵育5分钟。
将重链的表达载体(质粒P378)和轻链表达载体(P379)，进行Xho I单酶切。酶切后的载体进行琼脂糖凝胶电泳鉴定，确认条带无误后，进行回收纯化。将酶切后的载体和片段进行定量并同源重组连接,取连接产物进行转化,抽取质粒,进行细胞瞬时转染。本实验所有的293F细胞、培养基和转染试剂均购买于义翘神州，将瞬转收获的细胞培养液4000rp，离心10min，弃去细胞沉淀。取适量proteinA beads加入到上清中，室温孵育4h。离心弃去上清后，用PBS洗涤proteinA beads两次，最后用pH＝3的枸橼酸洗脱，最后用1M Tris调整pH为中性。纯化后的蛋白进行了SDS-PAGE分析。
结果如图1所示，各抗体还原与非还原的条带大小正常。
实施例3.人鼠嵌合CD27单克隆抗体与抗原CD27的结合
3.1用ELISA方法研究人鼠嵌合CD27单克隆抗体与抗原CD27的体外结合活性
为了确定本公开人鼠嵌合单克隆抗体是否可以结合CD27抗原，进行体外测试。将CD27单抗Varlilumab(1F5)用作阳性对照。
通过ELISA，使用标准方法和步骤测试了2株CD27单克隆抗体与CD27抗原(康岱自制)的结合，具体步骤如下：用pH 9.6的NaHCO3溶液稀释抗原CD27-mFc(康岱自制)至1ug/mL，每孔50uL加入到96孔酶标板中，4℃冰箱过夜。次日，PBS洗涤两遍之后，用3％的BSA封闭，每孔100uL，37℃孵育1.5h。PBST洗涤4次之后，加入抗体稀释液后继续37℃孵育2h，继续用PBST洗涤4次后，加入一定比例稀释的二抗Ap-anti-human IgG Fc(JacksonImmuno Research，109-055-098)，37℃孵 育1.5h，最后PBST洗涤4次后，加入PNPP显色液，37℃孵育10-15min后，酶标仪读数。1F5为对照抗体。
实验结果与分析：
ELISA试验结果显示(图2、表2)：2株CD27人鼠嵌合单克隆抗体(编号13B3-hFc和17B8-hFc)能特异性结合CD27。结合的亲和力EC50分别为：0.0344ug/mL和0.0754ug/mL，阳性对照1F5的EC50为0.0381ug/mL，该结果表明，重组表达的7104嵌合抗体结合CD27活性很好，与阳性对照1F5相当。
表2
3.2用流式细胞术方法(flow cytometry)研究人鼠嵌合CD27单克隆抗体与细胞膜上CD27蛋白的结合
为了确定本公开人鼠嵌合单克隆抗体是否可以结合CHO细胞膜上的CD27蛋白，进行体外测试。将1F5用作阳性对照。
首先配制FACS buffer，即1×PBS+0.5％BSA溶液。取稳定高表达人CD27的CHO细胞系，一个样品所需的细胞量约为1-5×105，用FACS buffer将抗体以一定的比例稀释后重悬细胞，体积约为100uL，4℃冰箱孵育30min，FACS buffer将细胞洗涤一次后，用FACS buffer稀释二抗FITC-anti-human IgG(Abcam：6854)，将细胞重悬后置于4℃冰箱孵育30min，最后，FACS buffer洗涤细胞后，用200uL的FACS buffer重悬细胞，上机检测。
表3
实验结果与分析：
结果如图3和表3所示，13B3-hFc嵌合单克隆抗体与CHO-CD27结合较好，结合CHO-CD27的EC50为2.486ug/mL，阳性对照1F5为2.869ug/mL，结合能力相当。该数据表明本发明的13B3-hFc抗体不仅与固相抗原(ELISA法)结合能力很强，与细 胞膜上的CD27结合活性也很高。而17H8-hFc嵌合单克隆抗体则与CHO-CD27结合较差。
3.3 FACS检测人鼠嵌合CD27抗体与人T、B细胞表面CD27蛋白的结合
为了确定本公开人鼠嵌合7104单克隆抗体是否可以结合人T、B细胞膜上的CD27蛋白，进行体外测试。将1F5用作阳性对照。
取新鲜采集的人血，经密度梯度离心后获得外周血单核细胞，每个样品约5×10^5个细胞，体积100uL(PBS+0.5％BSA)分别加入本试验待测抗体1F5、13B3-hFc、17H8-hFc，终浓度5ug/mL，4℃孵育30min。用FACS(PBS+0.5％BSA)工作液洗涤细胞一次。再次用100uL的FACS工作液重悬细胞，加入二抗Cy5-AffiniPure F(ab')2Fragment Goat Anti-Human IgG,Fcγfragment specific(JacksonImmuno Research，109-176-098)和FITC-anti-human CD3抗体(BD Bioscience，556611)PE-anti-human CD19(biolegend，)温和混匀后，4℃孵育30min。最后FACS工作液洗涤细胞后，300uLBuffer重悬，上机检测。
实验结果：
结果如图4所示，第一排显示的是CD27在CD3+的T细胞上面表达，第二排显示CD27在CD19+的B细胞上面的表达。
结果显示：T细胞上的CD27表达较高，约有75％，B细胞的表达则较低约为20％。13B3可以很好的结合T、B细胞，染色效果与对照1F5相当，而17H8结合T、B细胞的能力则较差。
3.4用生物膜层光学干涉技术(BLI)研究人鼠嵌合CD27单克隆抗体与CD27的结合活性
用GATOR(ProbeLife)检测仪器以及AHC(Pall，185064),Human antibody Capture探针来检测人鼠嵌合CD27单克隆抗体的解离常数(Kd)。将人鼠嵌合CD27单克隆抗体在结合缓冲液(Q buffer[PBS(10mM PH7.4)+0.02％Tween 20+0.2％BSA]中稀释至50nM。将嵌合CD27抗体在Q buffer中从最高浓度100nM开始2倍梯度稀释。通过将抗体捕获传感器置于上述连续稀释的抗原溶液中来开始进行动力学关联测定。打开Octet Data Acquisition软件，选择New Kinetics Experiment-Basic Kinetics模式；
按照下面表格设置流程：
表4用生物膜层光学干涉技术(BLI)研究CD27抗体结合活性的流程

表5结合于抗原CD27的人鼠嵌合CD27单克隆抗体的动力学分析结果
实验结果如上表所示，所有抗体在Global拟合模式下的相关系数R2都大于0.95，符合系统适应性要求，结果可靠。
3.5人鼠嵌合CD27抗体对CD27与CD70的结合的抑制
为了确定本公开人鼠嵌合CD27单克隆抗体是否可以抑制CD27与配体CD70的结合，进行体外ELISA测试。
用PBS稀释CD70(R&D，9328-CL-100)至1ug/mL，每孔50uL加入到96孔酶标板中，4℃冰箱过夜。次日，PBS洗涤两遍之后，用3％的BSA封闭，每孔100uL，37℃孵育1.5h。PBST洗涤4次之后，加入固定浓度的CD27-mFc(终浓度为4.3ug/mL)和抗体稀释液后继续37℃孵育2h，继续用PBST洗涤4次后，加入1:300稀释的二抗HRP-anti-mouse Fc(JacksonImmuno Research，115-035-164)，37℃孵育1h，最后PBST洗涤4次后，加入TMB显色液(碧云天，P0209)，37℃孵育10-15min后，用2M HCl终止，酶标仪读数。
表6
实验结果：
实验结果如表6和图5所示,13B3-hFc嵌合CD27单克隆抗体能特异性阻断CD27与CD70的结合,IC50为1.474ug/mL。
17H8-hFc不阻断CD27与CD70的结合，这提示17H8-hFc与CD27的结合位点不 同于13B3-hFc。
实施例4.人鼠嵌合CD27单克隆抗体结合表位分析
4.1动力学结合方法研究
方法同3.4实施例中亲和力测定一致，不同的是在抗原CD27加载之后，继续加载不同的抗体，如抗体可以继续结合，则说明两个抗体表位没有重叠，若不能继续结合，则说明表位重叠或者是相同的表位。
实验结果与分析：
结果如图6显示，在13B3结合到探针后，继续加载抗原CD27，抗原可以结合，再继续加载不同的抗体，13B3则不能继续结合，1F5与17H8则可以继续结合，说明13B3和17H8/1F5结合CD27的表位不同。同理，用17H8或1F5结合探针后，得到相同的实验结果，该结果表明，13B3、17H8以及1F5与CD27的结合表位各不相同。
4.2 ELISA方法研究
pH 9.6的NaHCO3溶液稀释13B3-hFc至2ug/mL，每孔50uL加入到96孔酶标板中，4℃冰箱过夜。次日，PBS洗涤两遍之后，用3％的BSA封闭，每孔100uL，37℃孵育1.5h。PBST洗涤4次之后，加入固定浓度的CD27-mFc(终浓度为0.8ug/mL)和1F5抗体稀释液后继续37℃孵育2h，继续用PBST洗涤4次后，加入1:3000稀释的二抗HRP-anti-mouse Fc(JacksonImmuno Research，115-035-164)，37℃孵育1h，最后PBST洗涤4次后，加入TMB显色液(碧云天，P0209)，37℃孵育10-15min后，用2M HCl终止，酶标仪读数。
实验结果与分析：
如图7所示，与IgG1相比，即使在1F5较高的浓度下，CD27和13B3的结合也没有明显的减弱，这说明13B3和1F5结合的表位是不同的。
实施例5.人鼠嵌合CD27单克隆抗体体外促进IFNγ分泌和T细胞的增殖5.1促进IFNγ分泌
T细胞激活后，淋巴细胞分泌细胞因子IFNγ，同时促进细胞增生。为了测试本公开人鼠嵌合CD27单克隆抗体作为T细胞活化的阳性调节剂功能，我们进行了以下实验，证明了本公开抗体对PBMC细胞IFNγ产生的增强作用以及对T细胞的增值的作用。
试验前一天，用PBS将待测抗体分别稀释至30ug/mL，然后每孔50uL加至96孔板对应的孔中，放至4℃过夜。次日，取出96孔板，将孔中的PBS吸弃， 放置在超净工作台中。用完全培养基(RPMI1640+10％FBS)将PHA(Sigma，货号：L9019)稀释至2ug/mL，放置在一旁备用。复苏PBMC细胞(Oricells)，从液氮罐中取出细胞，迅速置于37℃水浴锅中融化，吸至15mL离心管中，1000rpm离心5min，吸弃上清，将细胞密度调整至5×10^6/mL。吸100uLPHA稀释液至96孔板中，然后再吸100uL的细胞液至相应的孔中，轻轻摇晃后，置于37℃，5％CO2培养箱中培养72h。72h后，收集细胞上清，IFNγ检测试剂盒(Biolegend，430115)检测IFNγ的含量。
实验结论与分析：
上清IFNγ的结果表明：与空白对照相比，13B3抗体可以明显的促进细胞IFNγ的产生，变化倍数达到52倍，与阳性对照1F5，17H8的IFNγ释放的变化倍数为38，略低于阳性对照1F5。
表7IFNγ检测结果
5.2促进T细胞的增生
试验前一天，用PBS将anti-CD3抗体(Biolegend，317303)和待测抗体稀释至固定浓度后，每孔50uL加至细胞培养板中，轻轻混匀后，放置于4℃环境中过夜。次日，吸弃PBS抗体稀释液，敞口放置于超净工作台数分钟，吹干残余的液体。复苏纯化的CD3+T细胞(Hycells，donor ID：PBZ1017)，离心收集细胞后用CFSE(碧云天，C0051)标记细胞，相关操作按其说明书进行。然后用完全培养基将细胞密度调整至0.5×10^6/mL，每孔200uL加至上述96孔板中。放置于37℃，5％CO2培养箱中培养72h。
72h后，收集细胞检测细胞增殖情况。
实验结论与分析：
如图8所示，抗CD27的抗体可以非常明显的促进CD3+T细胞的增殖,与对照IgG1相比(细胞增殖比例为19.03％),1F5和13B3-hFc处理组细胞的增殖比例分别为73.69％、78.54％，其中13B3-hFc效果优于1F5。
实施例6.人鼠嵌合CD27单克隆抗体对CD70-CD27下游信号通路的阻断
本试验用到两个细胞株：1、Jurkat-CD27-NFκB-Luc细胞，具体的细胞构建方法如下：Jurkat同时电转两个质粒，一个为表达CD27的质粒(即将CD27的cDNA构建到CMV启动子驱动的常规表达质粒上)，另一个pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro]Vector(Promega，N111A)，经过潮霉素和G418压力筛选2个星期，流式细胞仪分选CD27阳性的单细胞。待到单细胞生长到一个细胞群体后，将细胞铺到anti-CD3 mAb梯度包被的细胞培养板中，16h后，检测(Promega，N1120)，选择有剂量依赖的细胞株，同时检测CD27的表达。选取CD27高表达的细胞株进行下游试验。2、Raji细胞，此细胞为淋巴瘤细胞系，高表达CD80、CD86、CD70等分子。
本试验步骤如下：将anti-CD27mAbs制备成2×工作液，用完全培养基(RPMI1640+10％FBS)将抗体稀释支60ug/mL，然后进行3倍梯度稀释，取50uL加至专用的96孔板中(Cornning，3903)，然后将Jurkat-CD27-NFκB-Luc和Raji细胞的混合液(数目比为2：1，密度4E6/mL)50uL加入培养板中，16h后检测(Promega，N1120)。
实验结果与分析：
如图9所示，抗CD27抗体通过阻断Raji细胞表面CD70和Jurkat细胞表面的CD27的结合，而抑制CD27下游NFκB信号通路，随着抗体浓度的增加，这种效果更加明显。1F5和13B3-hFc虽然结合CD27不同的表位，但二者都具备这种功能，且效果相当。
实施例8.人源化CD27单克隆抗体设计和制备
8.1人源化CD27单克隆抗体的设计
根据13B3鼠源母本抗体序列，设计了人源化抗体序列，具体步骤如下：首先使用Discovery Studio和Antibody Modeling，采用同源建模方法构建可变区的三维分子模型。接下来，通过比对数据库已有抗体结构，分别对母本抗体可变区及CDR进行结构模拟。同时，选择分别与鼠源母本抗体VH和VL具有高度同源性的cDNA衍生的人种系(Germline)序列进行比对。13B3的重链VH选择同源度最高的IGHV1为人源化设计模板，设计序列。轻链VL选择IGKV6和IGKV3为人源化设计模板，设计序列。
结果
将鼠源母本抗体编号13B3原始重链mVH序列设计为4个人源化序列：13B3-huVH1(SEQ ID No：17)、13B3-huVH2(SEQ ID No：18)、13B3-huVH3(SEQ ID No：19)，13B3-huVH4(SEQ ID No：20)。将抗体原始轻链mVL设计为4个人源 化序列：13B3-huVL1(SEQ ID No：21)，13B3-huVL2(SEQ ID No：22)和13B3-huVL3(SEQ ID No：23)，13B3-huVL4(SEQ ID No：24)。
表8基于13B2的人源化抗体的VH和VL序列

分别对上述的人源化VH和VL进行组合，得到相应的人源化抗体：
表9示例性的人源化抗体轻重链不同组合方式以及对应编号：

实施例9人源化抗体与抗原CD27的结合
9.1用ELISA方法研究人源化单克隆CD27抗体与抗原CD27的体外结合活性
为了确定本公开人源化CD27单克隆抗体是否可以结合CD27抗原，进行体外测试。将CD27单抗Varlilumab(1F5)用作阳性对照。
试验方法同实施例3.1，结果如图10与表10所示。
表10
实验结果与分析：
5株人源化单克隆抗体(13B3-huH4L1(P33425)、13B3-huH2L2(P33426)、13B3-huH2L3(P33430)、13B3-huH2L4(P33433)、13B3-huH3L4(P33434))均能较好的结合固相的CD27，其EC50值分别为0.249nM、0.325nM、0.280nM、0.274nM、0.242nM。嵌合抗体13B3-hFc和阳性对照1F5的EC50分别为0.337nM、0.404nM。人源化抗体的结合能力优于1F5。
9.2用流式细胞术方法(flow cytometry)研究人源化CD27单克隆抗体与细胞膜上CD27蛋白的结合
为了确定本发明人源化CD27单克隆抗体是否可以结合CHO细胞膜上的CD27蛋白，进行体外测试。将1F5用作阳性对照。
试验方法同实施例3.2，结果如图11与表11所示。
表11

实验结果与分析：
5株人源化单克隆抗体(13B3-huH4L1(P33425)、13B3-huH2L2(P33426)、13B3-huH2L3(P33430)、13B3-huH2L4(P33433)、13B3-huH3L4(P33434))均能较好的结合膜形式的CD27，其EC50值分别为1.270nM、1.478nM、1.411nM、1.501nM、1.620nM。阳性对照1F5的EC50为、2.584nM。由此可见，人源化抗体的结合能力优于阳性对照1F5。
实施例10.人源化CD27抗体对CD27与CD70的结合的抑制
为了确定本公开人源化CD27单克隆抗体是否可以抑制CD27与配体CD70的结合，进行体外ELISA测试。将1F5用作阳性对照。
试验方法同实施例3.5，结果如图12与表12所示。
表12
实验结果与分析：
5株人源化单克隆抗体(13B3-huH2L2(P33426)、13B3-huH2L3(P33430)、13B3-huH4L3(P33432)、13B3-huH2L4(P33433)、13B3-huH3L4(P33434))均能较好阻断CD70和CD27的结合，其IC50值分别为12.837nM、13.092nM、12.804nM、12.804nM、11.698nM。嵌合抗体13B3-hFc的IC50为13.795nM。
实施例11人源化抗体与抗原CD27的结合的亲和力研究(SPR法)
仪器：Biacore T200(Cytiva)，探针：CM5(批号；10313498)。
固定配体到探针上：配制配体后，设置流速为10uL/min,将配体固定到探针上。
进行动力学参数设置：将1×HBS-P+缓冲液将供试品调整至饱和浓度后，再分别倍数稀释至少5个浓度后，设置流速为30uL/min，并设置相应的结合及解离时间，反应温度默认25℃，进行检测。
表13
实验结果与分析：
一些人源化后的抗体与CD27的亲和力优于嵌合抗体13B3-hFc。
实施例12人源化抗体对T细胞增殖的检测
试验前一天，用PBS将anti-CD3抗体(Biolegend，317303)和待测抗体稀释至固定浓度后，每孔50uL加至细胞培养板中，轻轻混匀后，放置于4℃环境中过夜。次日，吸弃PBS抗体稀释液，敞口放置于超净工作台数分钟，吹干残余的液体。复苏纯化的CD3+T细胞(Hycells，donor ID：PAZ022)，离心收集细胞后用CFSE(碧云天，C0051)标记细胞，相关操作按其说明书进行。然后用完全培养基将细胞密度调整至0.5×10^6/mL，每孔200uL加至上述96孔板中。放置于37℃，5％CO2培养箱中培养72h。
72h后，分别上集上清用于检测IFNγ，收集细胞检测细胞增殖。
细胞增殖如图13所示；
表14 IFNγ检测结果：

实验结果与分析：
T细胞增殖：与同型对照IgG1相比，所有抗CD27的抗体均能明显促进CD3+的T细胞增殖。就CD3+CD4+的细胞增殖比例来看，同型对照IgG1的增殖比例为2.03％，五株人源化抗体(13B3-huH4L1(P33425)、13B3-huH2L2(P33426)、13B3-huH2L3(P33430)、13B3-huH2L4(P33433)、13B3-huH3L4(P33434))的比例分别为7.74％、8.03％、10.64％、12.83％、8.9％，。就CD3+CD4-的细胞增殖比例来看，同型对照IgG1的增殖比例为3.35％，，五株人源化抗体(13B3-huH4L1(P33425)、13B3-huH2L2(P33426)、13B3-huH2L3(P33430)、13B3-huH2L4(P33433)、13B3-huH3L4(P33434))的比例分别为12.59％、12.24％、14.83％、15.83％、13.04％。综上，所有抗CD27的抗体都表现出明显的促进T细胞增殖的效果。
IFNγ分泌：T细胞受到来自于anti-CD3mAb的第一信号和来自于anti-CD27mAb的共刺激信号时，除了大量增殖外，还会分泌细胞因子，其中就有IFNγ。我们同时检测了上清中IFNγ的含量，五株人源化抗体(13B3-huH4L1(P33425)、13B3-huH2L2(P33426)、13B3-huH2L3(P33430)、13B3-huH2L4(P33433)、13B3-huH3L4(P33434))分别有1.62、1.86、1.52、1.86、1.51倍的增加。综上，所有抗CD27的抗体都表现出明显的促进T细胞分泌IFNγ。
实施例13.人源化抗体体内对T和B淋巴细胞含量的影响
用人CD27基因敲入(KI)小鼠研究人源化抗体13B3-huH2L4体内对T和B淋巴细胞含量的影响。
将24只KI小鼠分为4组，每组6只(3雌3雄)，4组小鼠分别腹腔注射PBS、13B3-huH2L4(P33433)1μg/mL和10μg/mL以及阳性参比抗体1F5 10μg/mL，每周2次，共给药3次。分别在最后1次给药后3天和10天取血检测淋巴细胞。
在每只小鼠100μL的血样中，各加入1μL的anti-mouse CD8 APC(BD 553035)、anti-mouse CD4 PE(BD 553653)、anti-mouse CD3 FITC(BD 555274)和anti-mouse CD19 APC(BioLegend 115512)，混匀后4℃孵育30min。然后各加入1mL用ddH2O  10倍稀释的lysing buffer(BD 349202)，涡旋震荡15s，冰上静置40min，裂解红细胞。500g转速离心5min，弃上清，用1mL 0.5％BSA的PBS清洗沉淀。再500g转速离心5min，弃上清。每个样品加入200μL lysing buffer悬浮，用流式分析仪进行检测。
实验结果与分析：
末次给药后3天，与对照组相比，13B3-huH2L4的2个剂量组和阳性对照组的外周血中CD3+(图14A)、CD3+CD4+(图14B)和CD3+CD4-(图14C)T淋巴细胞占比均显著下降，尤其是CD3+CD4+T淋巴细胞。这提示13B3-huH2L4抗体对于细胞表面存在高表达的CD27的CD3+T淋巴细胞有杀伤。此外，B淋巴细胞的表面无CD27，故未观察到对CD19+B淋巴细胞的杀伤作用，故CD19+B淋巴细胞占比有一定上升(图14D)。
末次给药后10天，外周血中CD3+(图15A)、CD3+CD4+(图15B)和CD3+CD8+(图15C)T淋巴细胞占比仍显著低于PBS对照组，和末次给药后3天相比还在继续下降。
末次给药后10天，与外周血中一样，脾脏中CD3+(图16A)、CD3+CD4+(图16B)和CD3+CD8+(图16C)T淋巴细胞占比显著低于PBS对照组。
同时检测了外周血中各T淋巴细胞的数量，其变化与它们的百分比变化一致。
结果显示，本发明的抗CD27抗体高效地杀伤高表达CD27的T淋巴细胞。
实施例14.人源化抗体联合抗PD-1或CTLA-4抗体对PHA激活后的T淋巴细胞的激活作用
把人PBMC(TPCS，A19K214031)加入到24-孔培养板，培养在含10％FBS的RPMI1640培养里，加入PHA(Sigma NO 9019)至终浓度1μg/mL，激活T淋巴细胞。培养3天后，弃上清，清洗细胞一次，然后把细胞加入到96-孔培养板中，每孔5 x 105个细胞，培养在含10％FBS的RPMI1640培养里，加入PHA至终浓度1μg/mL，并分别入13B3-huH2L4 0.3μg/mL或3μg/mL，以及抗PD-1或CTLA-4或TIGIT抗体，至终浓度3μg/mL(如图17所示)。抗PD-1抗体是特瑞普利，抗CTLA-4和TIGIT抗体均是为市售的抗体(赛金公司)。培养3天后取上清检测IFNγ含量。
实验结果与分析：
实验结果显示(图17)，13B3-huH2L4单独作用不会增强衰竭的T细胞IFNγ的产生，但抗PD-1或CTLA-4抗体单独作用能促进衰竭的T细胞IFNγ的产生，而且在与13B3-huH2L4连用时能进一步促进IFNγ的产生。
实施例15.人源化CD27抗体的修饰
15.1降低人源化CD27抗体与Fc受体结合能力的变异
为了降低抗体介导的ADCC/ADCP/CDC活性，把人源化抗体13B3-huH2L3和13B3-huH2L4的重链CH1区域的2个氨基酸L234/L235(EU编号方式)用氨基酸Ala取代(LALA变异体)，消除抗体的ADCC/ADCP/CDC功能。13B3-huH2L3和13B3-huH2L4的重链序列如SEQ ID NO:27所示，LALA变异的重链序列如SEQ ID NO:28所示。变异的基因通过人工基因合成获得。2个变异抗体13B3-huH2L3(LALA)和13B3-huH2L4(LALA)共用LALA变异的重链(SEQ ID No:28)。
15.2变异抗体与CD27体外结合活性
CD27-mFc包被：用1XPBS将CD27稀释至浓度为1μg/mL，混匀后用多孔移液器吸入96well酶标板中，50μL/well，用封板膜密封后置于4℃冰箱过夜。一抗孵育：梯度稀释13B3-huH2L3(LALA)、13B3-huH2L4(LALA)抗体以及各种对照抗体(如图18所示)，100μL/well加入酶标板，封板膜密封后置37℃，1小时。二抗孵育：弃封闭液，250μL PBST洗3遍，拍干。以结合液1:3000稀释AP标记的抗人Fc二抗，100μL/well加入酶标板，封板膜密封后置37℃，1小时。PNPP孵育：弃封闭液，250μL PBST洗3遍，拍干。取1颗PNPP于1mL 5X二乙醇胺底物，再加入4mL去离子水溶解，50μL/well加入酶标板，封板膜密封后置37℃，15-25分钟。酶标仪检测：双波长405nm/490nm检测，结果以OD405-OD490计算。
实验结果与分析：
变异抗体13B3-huH2L4(LALA)和其母本抗体(野生型)13B3-huH2L4与人CD27体外结合活性相同(EC50均为29ng/mL)，与变异抗体13B3-huH2L3(LALA)以及其它对照抗体(1F5和MK5890)的CD27体外结合活性也没有显著差别(图18)。
15.3.变异抗体阻断CD70与CD27结合的活性
CD70包被：用1XPBS将CD70稀释至浓度为1μg/mL，混匀后用多孔移液器吸入96well酶标板中，50μL/well，用封板膜密封后置于4℃冰箱过夜。酶标板封闭：250μL/well PBS洗涤2次，拍干。再加入150μL/well的封闭液，封板膜密封后置37℃，1.5小时。一抗孵育：将变异13B3-huH2L3(LALA)、13B3-huH2L4(LALA)抗体以及各种对照抗体(如图？所示)梯度稀释在CD27-mFc 4μg/mL中，100μL/well加入酶标板，封板膜密封后置37℃，1小时。弃封闭液，250μL PBST洗3遍，拍干。以结合液1:3000稀释HRP标记的抗小鼠Fc二抗，100μL/well加入酶标板，封板膜密封后置37℃，1小时。TMB孵育：弃封闭液，250μL PBST洗3遍，拍干。TMB 50μL/well加入酶标板，封板膜密封后置37℃，15-25分钟。HCL终止： 用2MHCL 50μL/well终止反应。酶标仪检测：双波长450nm/655nm检测，结果以OD450-OD655计算。
实验结果与分析：
变异抗体13B3-huH2L4(LALA)和其母本抗体13B3-huH2L4体外抑制CD27和CD70结合的活性没有本质上的差别。此外，出乎意料地，变异抗体13B3-huH2L3(LALA)的抑制活性有一定改善，IC50为1.12μg/mL，而母本抗体13B3-huH2L4的IC50＝1.31μg/mL)。
此外，变异抗体13B3-huH2L3(LALA)也具有优异的体外抑制CD27和CD70结合的活性，IC50为1.09μg/mL，优于对照抗体1F5(IC50为1.31μg/mL)，明显优于对照抗体MK5890(IC50为1.89μg/mL)(图19)。
实施例16.变异抗体与CD32和CD64结合活性
用人CD32A基因稳定转染的Jurkat细胞(Jurkat-CD32a)和人CD64基因稳定转染的CHO-K细胞(CHO-CD64)研究变异抗体与膜CD32a和膜CD64的结合活性。
把Jurkat-CD32a和CHO-CD64细胞悬浮在0.5％BSA PBS中，加入96-孔圆底板中，每孔50μL含5 x 105个细胞。3倍梯度稀释13B3-huH2L4和13B3-huH2L4(LALA)，4℃孵育1小时，用0.5％BSA PBS洗两次后，加入300倍稀释的FITC标记的anti-human Fc抗体(Jackson ImmunoResearch Lab.，#109-116-170)，100μL/孔，4℃孵育0.5小时，用0.5％BSA PBS洗两次，悬浮在200μL PBS缓冲液里，用流式细胞仪分析。
实验结果与分析：
结果显示，变异抗体13B3-huH2L4(LALA)不结合表达在细胞表面上的CD32a，而其母本野生型抗体13B3-huH2L4能正常的与细胞表面上的CD32a(图20A)。同样的，变异抗体13B3-huH2L4(LALA)不结合表达在细胞表面上的CD64，而其母本野生型抗体13B3-huH2L4能正常的与细胞表面上的CD64结合(图20B)。
实施例17.变异抗体体内对T淋巴细胞影响
用人CD27基因敲入(KI)小鼠研究变异抗体13B3-huH2L4(LALA)体内对T淋巴细胞含量的影响。
将14只KI小鼠分为3组，PBS对照组2只，其余2组各4只，分别给予13B3-huH2L4(LALA)10mg/kg和阳性参照抗体1F5 10mg/kg。腹腔给药，一周两次，共给药3次。在最后一次给药3天和10天分别取血检测。
在每只小鼠100μL的血样中，各加入1μL的anti-mouse CD8 APC(BD 553035)、anti-mouse CD4 PE(BD 553653)和anti-mouse CD3 FITC(BD 555274)， 混匀后4℃孵育30min。然后各加入1mL用ddH2O 10倍稀释的lysing buffer(BD 349202)，涡旋震荡15s，冰上静置40min，裂解红细胞。500g转速离心5min，弃上清，用1mL 0.5％BSA的PBS清洗沉淀。再500g转速离心5min，弃上清。每个样品加入200μL lysing buffer悬浮，用流式分析仪进行检测。
实验结果与分析：
末次给药后3天，与PBS对照组相比，阳性参照抗体1F5显著降低外周血中CD3+(图21A)、CD3+CD4+(图21B)和CD3+CD8+(图21C)T淋巴细胞占比，但13B3-huH2L4(LALA)对这些T淋巴细胞的含量没有影响，没有改变它们在外周血中的占比。
末次给药后10天，阳性参照抗体1F5组里小鼠外周血中CD3+(图22A)、CD3+CD4+(图22B)和CD3+CD8+(图22C)T淋巴细胞占比仍显著低于PBS对照组，和末次给药后3天相比没有变化。13B3-huH2L4(LALA)组与PBS对照组相比没有差别。
此外，对于外周血中各T淋巴细胞数量的检测结果显示，数量变化趋势与各自的百分比变化趋势是一致的。
结果显示，与阳性参照抗体相比，变异抗体13B3-huH2L4(LALA)基本消除了ADCC功能，因而对表达CD27的T淋巴细胞基本无直接的杀伤作用。本发明的这类ADCC显著下降或消除的变异抗体，特别适合用于需要激活T细胞的免疫功能且维持T细胞数量的应用场合，如针对实体瘤的治疗，以及针对非T淋巴细胞的血液肿瘤(如B细胞瘤)的治疗。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (20)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区具有选自下组的三个互补决定区CDR：
    (a)SEQ ID No:2、3和4所示的CDR1、CDR2和CDR3；
    (b)SEQ ID No:2、25和4所示的CDR1、CDR2和CDR3；
    (c)SEQ ID No:2、26和4所示的CDR1、CDR2和CDR3；
    (d)SEQ ID No:10、11和12所示的CDR1、CDR2和CDR3。
  2. 如权利要求1所述的重链可变区，其特征在于，所述重链可变区包括以下三个互补决定区CDR：
    (1)互补决定区CDR1，所述互补决定区CDR1的氨基酸序列如SEQ ID No:2所示；
    (2)互补决定区CDR2，所述互补决定区CDR2的氨基酸序列如SEQ ID No:3、25或26所示；及
    (3)互补决定区CDR3，所述互补决定区CDR3的氨基酸序列如SEQ ID No:4所示。
  3. 一种抗体重链，其特征在于，所述抗体重链具有如权利要求1所述的抗体的重链可变区。
  4. 如权利要求3所述的抗体重链，其特征在于，所述抗体重链的Fc具有导致抗体ADCC作用丧失或基本丧失的突变。
  5. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区具有序列如SEQ ID No:6、7、和8所示的三个互补决定区L-CDR；
    或所述的轻链可变区具有序列如SEQ ID No:14、15、和16所示的三个互补决定区L-CDR。
  6. 如权利要求5所述的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区L-CDR：
    (1)互补决定区L-CDR1，所述互补决定区L-CDR1的氨基酸序列如SEQ ID No:6所示；
    (2)互补决定区L-CDR2，所述互补决定区L-CDR2的氨基酸序列如SEQ ID No:7所示；及
    (3)互补决定区L-CDR3，所述互补决定区L-CDR3的氨基酸序列如SEQ ID No:8所示。
  7. 一种抗体轻链，其特征在于，所述抗体轻链具有如权利要求5所述的抗体 的轻链可变区。
  8. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求5所述的轻链可变区；
    或者，所述抗体具有：如权利要求3所述的重链；和/或如权利要求7所述的轻链。
  9. 如权利要求8所述的抗体，其特征在于，所述抗体的Fc具有导致抗体ADCC作用丧失或基本丧失的突变。
  10. 如权利要求8所述的抗体，其特征在于，所述抗体选自下组：
    (Z1)具有SEQ ID No:1所示的重链可变区和SEQ ID No:5所示的轻链可变区的抗体：
    (Z2)具有SEQ ID No:18所示的重链可变区和SEQ ID No:22所示的轻链可变区的抗体；
    (Z3)具有SEQ ID No:18所示的重链可变区和SEQ ID No:23所示的轻链可变区的抗体；
    (Z4)具有SEQ ID No:18所示的重链可变区和SEQ ID No:24所示的轻链可变区的抗体；
    (Z5)具有SEQ ID No:19所示的重链可变区和SEQ ID No:24所示的轻链可变区的抗体；
    (Z6)具有SEQ ID No:20所示的重链可变区和SEQ ID No:21所示的轻链可变区的抗体。
  11. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的重链可变区、如权利要求3所述的重链、如权利要求5所述的轻链可变区、如权利要求7所述的轻链、或如权利要求8所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  12. 一种抗体制剂，其特征在于，所述的抗体制剂包括：
    (a)如权利要求8所述的抗体；以及
    (b)载体或赋形剂。
  13. 一种试剂盒，其特征在于，所述的试剂盒含有权利要求8所述的抗体，以及盛装所述抗体的容器。
  14. 一种CAR构建物，其特征在于，所述的CAR构建物的抗原结合区域的scFv段为特异性结合于CD27的结合区，并且所述scFv具有如权利要求1所述的重链可变区和如权利要求5所述的轻链可变区。
  15. 一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如权利要求14所述的CAR构建物。
  16. 一种抗体药物偶联物，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求3所述的重链、如权利要求5所述的轻链可变区、如权利要求7所述的轻链、或如权利要求8所述的抗体、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  17. 一种活性成分的用途，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求3所述的重链、如权利要求5所述的轻链可变区、如权利要求7所述的轻链、或如权利要求8所述的抗体、或其组合；所述活性成分用于
    (a)制备检测试剂或试剂盒；
    (b)制备预防和/或治疗CD27相关疾病的药物或制剂；和/或
    (c)制备预防和/或治疗癌症或肿瘤的药物或制剂。
  18. 一种药物组合物，所述的药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求3所述的重链、如权利要求5所述的轻链可变区、如权利要求7所述的轻链、或如权利要求8所述的抗体、或如权利要求15所述的免疫细胞、或其组合；以及
    (ii)药学上可接受的载体。
  19. 一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
    (1)如权利要求1所述的重链可变区、如权利要求3所述的重链、如权利要求5所述的轻链可变区、如权利要求7所述的轻链、或如权利要求8所述的抗体；或
    (2)如权利要求11所述的重组蛋白；和/或
    (3)如权利要求14所述的CAR构建物。
  20. 一种治疗CD27相关疾病的方法，所述方法包括：给需要的对象施用如权利要求8所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
PCT/CN2023/124588 2023-03-16 2023-10-13 抗cd27单克隆抗体及其应用 WO2024187743A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102007147A (zh) * 2008-06-30 2011-04-06 协和发酵麒麟株式会社 抗cd27抗体
CN104284678A (zh) * 2012-03-15 2015-01-14 詹森生物科技公司 人抗cd27抗体、方法和用途
CN109890846A (zh) * 2016-09-26 2019-06-14 默沙东公司 抗-cd27抗体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102007147A (zh) * 2008-06-30 2011-04-06 协和发酵麒麟株式会社 抗cd27抗体
CN104284678A (zh) * 2012-03-15 2015-01-14 詹森生物科技公司 人抗cd27抗体、方法和用途
CN109890846A (zh) * 2016-09-26 2019-06-14 默沙东公司 抗-cd27抗体

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