WO2020056790A1 - 特异结合人及猴cd38抗原的单克隆抗体及其制备方法与应用 - Google Patents

特异结合人及猴cd38抗原的单克隆抗体及其制备方法与应用 Download PDF

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WO2020056790A1
WO2020056790A1 PCT/CN2018/107812 CN2018107812W WO2020056790A1 WO 2020056790 A1 WO2020056790 A1 WO 2020056790A1 CN 2018107812 W CN2018107812 W CN 2018107812W WO 2020056790 A1 WO2020056790 A1 WO 2020056790A1
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antibody
human
seq
variable region
monoclonal antibody
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French (fr)
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胡红群
宋晓琦
陈蕞
马晓晓
袁燕萍
周群敏
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苏州思坦维生物技术股份有限公司
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Priority to US17/274,439 priority Critical patent/US11976129B2/en
Priority to EP18934071.4A priority patent/EP3851455B1/en
Priority to KR1020217011519A priority patent/KR102648583B1/ko
Publication of WO2020056790A1 publication Critical patent/WO2020056790A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • the invention belongs to the field of biotechnology-monoclonal antibodies.
  • the invention relates to a monoclonal antibody that specifically binds human and monkey CD38 antigens, a coding sequence thereof, and a preparation method and use thereof.
  • CD38 (also known as T10, an antigen recognized by OKT10 monoclonal antibody) is a type II single-transmembrane glycoprotein with a molecular weight of about 45 kDa.
  • CDNAs encoding human and mouse CD38 genes have been cloned and reported as early as the early 1990s (Jackson DG and Bell JI. J. Immunol. 1990, 144: 2811-2815; Harada et al. J Immunol. 1993, 151: 3111-8); cDNA encoding the cynomolgus macaque CD38 gene was also cloned and reported in 2004 (Ferrero et al, BMC Immunol. 2004, 5:21).
  • the human CD38 protein has a total length of 300 amino acids, of which 21 amino acids in the N-terminal region are located in the cell, 22 amino acids in the cell membrane, and 257 amino acids in the C-terminal region outside the cell membrane.
  • the cynomolgus CD38 protein has a total length of 301 amino acids, which share 92% amino acid sequence homology with the human CD38 protein, while the mouse CD38 protein has 304 amino acids, which share 70% amino acid sequence homology with the human CD38 protein. about.
  • CD38 is also known as ADP-ribosyl cyclease / cyclic ADP-ribose hydrolase 1 (ADPR1 ).
  • CD38 protein molecule also has dehydrogenase activity, which can further catalyze cADPR to generate ADPR (Howard et al. Science. 1993; 262: 105; Summerhill RJ, Jackson DG, Galione A .FEBS Lett. 1993, 335: 231-3; Prasad GS et al, Nature Structural Biology 1996, 3: 957–964). Therefore, CD38 has the activity of a bifunctional enzyme, and its active sites are located in the extracellular membrane structural region.
  • the CD38 (T10) antigen was first reported and discovered by Feinnerz E and colleagues in 1980: they observed that human-derived normal thymocytes and T-lymphoma cell line MOLT-4 express a type that can be coded as OKT10
  • the antigen specifically bound by the monoclonal antibody was called T10 at that time (Feinnerz et al. PNAS 1980, 77: 1588-1592).
  • T10 antigen see review articles: Mehta K, Shahid U and Malavasi F: Human CD38, a cell-surface protein with multiple function. FASEB J 10, 1408-1417).
  • CD38 antigen is relatively highest expressed in multiple myeloma (MM) and B-lymphoma. Therefore, CD38 antigen was considered to treat multiple myeloma as early as the early 1990s. It is an ideal target for B-lymphoma, and has multiple monoclonal antibodies targeting CD38 antigen as therapeutic drugs. It has been tested and reported in vivo and in vitro for the treatment of multiple myeloma and B-lymphoma. :
  • Multiple myeloma is a malignant plasma cell disease, which is characterized by the potential accumulation of plasma cells secreted in the bone marrow, accompanied by low proliferation and prolonged cell growth cycle, which can lead to hypercalcemia, kidney damage, and adjacent bone marrow tissue Destruction and anemia.
  • chemotherapy drugs such as Vincristine, Cyclophosphamide, Melphalan, and Adriamycin.
  • IMDs Immunomodulators
  • PIs Proteasome Inhibitors
  • Bisphosphonates bisphosphonates
  • hormones such as prednisone, Dexamethasone and autologous stem cell transplantation.
  • the representative drugs of immunomodulators are Thalomid (commonly known as Thalidomide, which was approved for marketing by the US FDA in July 1998), and lenalidomide Revlimid (commonly known as lenalidomide, 2005) It was approved by the US FDA in December of this year and Pomalyst (common name Pomalidomide, which was approved by the US FDA in February 2013).
  • Representative drugs for proteasome inhibitors include Velcade Wanke (common name Bortezomib bortezomib, which was approved by the US FDA in May 2003) developed by Takeda / Millennium, and Kyprolis Carfilzoil developed by Onyx Corporation in the United States. Rice (common name Carfilzomib, approved by the US FDA in July 2012).
  • Representative bisphosphonate drugs are Aredia (generic name Pamidronate, which was approved by the US FDA in October 1991) and Zometa (generic name Zoledronic acid, which was approved by the US FDA in August 2001).
  • CRR Complete Response Rate
  • MST median survival time
  • Daratumumab's trade name is Darzalex, which was jointly developed by Johnson & Johnson's Johnson & Johnson subsidiary Janssen Biotech and Danish Genmab.
  • Daratumumab is a fully human IgG1 / kappa monoclonal antibody that specifically recognizes and binds human CD38 antigen. It was originally derived from Genmab's anti-human CD38 monoclonal antibody of the generation number 005 (de Weers et al. J Immunol 2001; 186 : 1840-8; International PCT Application No .: PCT / DK2006 / 000166, US Patent No .: US7829673B2).
  • Daratumumab / 005 monoclonal antibody can be used in complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cells Phagocytosis (antibody-dependent cell-mediated phagocytosis (ADCP)) and direct induction of tumor cell apoptosis (apoptotosis) and other mechanisms quickly kill CD38 antigen-positive tumors such as myeloma.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Phagocytosis antibody-dependent cell-mediated phagocytosis
  • apoptosis direct induction of tumor cell apoptosis
  • other mechanisms quickly kill CD38 antigen-positive tumors such as myeloma.
  • Daratumumab / 005 monoclonal antibody can also inhibit CD38-mediated enzyme activity (ADP-ribosyl cyclase) to play a role.
  • POLLUX Phase III clinical studies
  • the POLLUX study evaluated Daratumumab and lenalidomide lenalidomide and Dexamethasone dexamethasone.
  • Clinical efficacy of combined therapy for third-line myeloma patients 569 patients with myeloma were included in the study, of which 286 patients were treated with Daratumumab and lenalidomide lenalidomide and Dexamethasone dexamethasone (treatment group), lenalidomide lena 283 patients were treated with doxamine and Dexamethasone dexamethasone (control group).
  • the CASTOR study evaluated the clinical efficacy of Daratumumab and Bortezomib bortezomib / Vanke and Dexamethasone dexamethasone in third-line treatment of myeloma patients.
  • the study included 498 patients with myeloma, of which Daratumumab and Bortezomib bortezomib and Dexamethasone There were 251 patients in the combination group of dexamethasone (treatment group) and 247 patients in the control group of combination of Bortezomib bortezomib and Dexamethasone dexamethasone.
  • EQUULEUS MMY1001
  • Daratumumab was first approved by the US FDA in November 2015, it has been gradually promoted from the original single-line four-line treatment of myeloma to first-line treatment in just 3 years, and soon became the market.
  • the best-selling blockbuster variety Daratumumab is currently the only anti-CD38 monoclonal antibody approved in the world; other anti-CD38 monoclonal antibodies currently in clinical trials are only human-mouse chimeric monoclonal antibodies SAR650984 developed by Sanofi / Immungen. (Isatuximab) and MorphoSys, a German pharmaceutical company, screened fully human monoclonal antibody MOR202 from the phage-display antibody library. The main clinical indications for the development of these antibody drugs are also myeloma.
  • These new monoclonal antibodies can be used as a single drug, or combined with other drugs currently on the market for treating myeloma, such as Bortezomib bortezomib, Lenalidomide lenalidomide and other drugs, for the treatment of CD38 overexpressing tumors such as bone marrow Tumors and lymphomas.
  • One of the technical problems to be solved by the present invention is to provide a tumor that has a different antigen binding region / binding site (epitope) from the existing Daratumumab, which binds human and monkey CD38 antigens, and specifically kills CD38 antigens that are highly expressed inside and outside.
  • Other biologically active monoclonal antibodies or derivatives thereof such as antibody Fab fragments, single chain antibodies and the like.
  • the second technical problem to be solved by the present invention is to provide DNA molecules or genes encoding the above antibodies.
  • the third technical problem to be solved by the present invention is to provide a medicine or a pharmaceutical composition containing the above antibody.
  • the fourth technical problem to be solved by the present invention is to provide the application of the medicine or the pharmaceutical composition containing the above antibody in the treatment of CD38 antigen high expression positive tumors.
  • the fifth technical problem to be solved by the present invention is to provide a method for preparing the above antibody.
  • the present invention adopts the following technical solutions:
  • a novel anti-CD38 monoclonal antibody or a derivative thereof that is different from an existing Daratumumab in an antigen binding region / epitope which comprises a first variable region and The second variable region, wherein the first variable region is an antibody light chain variable region, and its antigenic complementarity determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:
  • the amino acid sequence shown in 5; wherein the second variable region is an antibody heavy chain variable region, and its antigenic complementarity determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO : Amino acid sequence shown by 10.
  • the antibodies include human-mouse chimeric, semi-chimeric / semi-human, humanized monoclonal antibodies, and the derivatives include antibody Fab fragments, single-chain Fab-fragments, Fv-fragments, single-chain antibodies, and bispecific Antibodies (bi-specific), antibody-drug-conjugated (ADC), chimeric antigen receptor (T-Cell, CAR-T) and the like.
  • the first variable region is an antibody light chain variable region, which is an amino acid sequence shown in SEQ ID NO: 2;
  • the second variable region is an antibody heavy chain variable region, It is the amino acid sequence shown in SEQ ID NO: 7.
  • the first variable region is an antibody light chain variable region, which is an amino acid sequence shown in SEQ ID NO: 11;
  • the second variable region is an antibody heavy chain variable region, It is the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 7.
  • the present invention comprises the antibody light chain variable region and the human antibody light chain constant region, and the hinge region, the CH1 region, comprising the antibody heavy chain variable region and the human antibody heavy chain constant region, CH 2 and CH3.
  • the human antibody light chain constant region is derived from a human antibody kappa chain or an antibody lamda chain
  • the human antibody heavy chain constant region is derived from a human subtype such as IgG1, IgG2, IgG3, or IgG4. Is IgG1.
  • variable region of the antibody light chain is the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 13
  • the variable region of the antibody heavy chain is the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 14.
  • a third aspect of the present invention is to provide an expression vector containing a DNA molecule / gene nucleotide sequence encoding the above antibody or a derivative thereof and an expression control sequence operatively linked to the sequence.
  • a fourth aspect of the present invention provides a recombinant host cell, which is transformed from the above-mentioned expression vector.
  • the recombinant host cell or its progeny cells express the above-mentioned antibody or its derivative.
  • the antibodies include humanized monoclonal antibodies, and derivatives include antibody Fab fragments, single chain antibodies, bi-specific antibodies, and the like.
  • a fifth aspect of the present invention is to provide a medicament or a pharmaceutical composition comprising a pharmaceutically effective amount of the above-mentioned antibody or a derivative thereof, and a pharmaceutically acceptable carrier.
  • a sixth aspect of the present invention is the application of the above-mentioned antibody drug or pharmaceutical composition in the preparation of a medicament for treating tumors, especially the application of a medicament for treating tumors with positive CD38 expression.
  • the CD38-positive tumor is preferably human myeloma, lymphoma (B-lymphoma), and the like.
  • the present invention describes the use of the antibody to inhibit the growth of human B-lymphoma Raji with high expression of CD38 in vivo.
  • the anti-CD38 antibody of the present invention when used to treat tumors with positive CD38 expression, more consideration may be given to using the constant regions of wild-type or genetically modified human IgG1 and IgM subtype antibodies in order to Maintain or increase the ADCC or CDC of the antibody to achieve a stronger killing effect on tumor tissues and cells.
  • the constant regions of the wild-type or genetically modified human IgG1 and IgM subtype antibodies can be cloned by genetic engineering techniques known to those skilled in the art, or synthesized in vitro.
  • the anti-CD38 antibody or its derivative in the present invention can also be used as a targeting carrier to graft or wrap with other anti-tumor drugs or toxins to form antibody-drug-conjugated (ADC) and target together.
  • ADC antibody-drug-conjugated
  • the method of grafting or encapsulating an antibody with a drug or a toxin can use a general technique known to those skilled in the art.
  • the anti-CD38 antibody or a derivative thereof in the present invention may also be used sequentially or in combination with an antitumor angiogenesis drug or a drug that targets immune checkpoint molecules.
  • an antitumor angiogenesis drug or a drug that targets immune checkpoint molecules for the treatment of tumors with positive CD38 expression.
  • the antitumor angiogenesis drug used sequentially or in combination with the anti-CD38 antibody or the derivative thereof in the present invention may preferably be a macromolecular biological drug or a small molecule chemical drug that targets VEGF or VEGF receptor (VEGF-R).
  • VEGF-R VEGF receptor
  • preferred macromolecule biological drugs targeting VEGF / or VEGF-R include the anti-VEGF monoclonal antibody drug Bevacizumab (trade name Avastin), and the anti-VEGF monoclonal antibody Fab fragment Ranibizumab (trade name) Lucentis); anti-VEGFR2 monoclonal antibody Ramucirumab (trade name Gyramza) and anti-VEGF monoclonal antibody codenamed hPV19 (Stanway is currently researching drugs, see Chinese patent literature: authorized patent number: 201210540692X Patent name: Monoclonal antibody antagonizing the inhibition of vascular endothelial cell growth factor binding to its receptor, and its coding sequence and use; and US patent literature with authorized patent number US9580498B2); or VEGF receptor-Fc fusion protein drugs such as Arp Albercept (trade name: Eylea), Compercept (combercept) and the like.
  • Preferred small molecule chemistries that target the VEGF receptor include Sunitini
  • the preferred drugs for targeted immunosuppression checkpoints used sequentially or in combination with the anti-CD38 antibody or its derivative in the present invention include anti-CTLA4 (Cytotoxic T-lymphocyte Antigen-4) Ipilimumab Ipilimumab (trade name Yervoy) ; Anti-PD-1 (programmed death protein-1) monoclonal antibody Nivolumab (trade name Opdivo), Pembrolizumab (trade name Keytruda) and anti-PD-1 monoclonal antibody hAB21 under development by Stanway (see PCT patent application document: PCT / CN2017 / 089282, Monoclonal antibodies that antagonize the binding of human PD-1 antigen and its ligand, and their preparation methods and applications); Anti-PD-L1 monoclonal antibody drugs include Atezolizumab (trade name Tecentriq), Avelumab (trade name Bavencio), Durvalumab (trade name Imfinzi) and the like.
  • CTLA4
  • the anti-CD38 antibody in the present invention can also be first prepared into a chimeric antigen T cell receptor (CAR-T) and introduced into the peripheral blood from a tumor patient in vitro. Immune cells such as T-lymphocytes isolated from the kidneys, and then cultured and expanded in vitro, these lymphocytes that recognize the CD38 antigen are injected back into the body to play a role in targeting tumors that highly express the CD38 antigen in the body, thereby achieving treatment of tumors Effect.
  • the preparation of the anti-CD38 antibody in the present invention into a chimeric antigen T cell receptor (CAR-T) can use conventional techniques known to those skilled in the art
  • a seventh aspect of the present invention provides a method for preparing the above-mentioned antibody or a derivative thereof, the method comprising:
  • step b transforming the host cell with the expression vector described in step a);
  • step b) culturing the host cell obtained in step b) under conditions suitable for expression of said antibody
  • the antibody is isolated and purified from the host cell culture solution.
  • monoclonal antibody refers to an immunoglobulin obtained from a pure line of cells, has the same structural and chemical characteristics, and is specific for a single epitope.
  • Monoclonal antibodies differ from conventional polyclonal antibody preparations (usually having different antibodies directed against different determinants), and each monoclonal antibody is directed against a single determinant on the antigen.
  • the benefits of monoclonal antibodies are that they are obtained through hybridoma or recombinantly engineered cell culture, and are not mixed with other immunoglobulins.
  • the modifier "monoclonal” indicates the characteristics of the antibody and is obtained from a homogeneous antibody population, which should not be interpreted as requiring any special method to produce the antibody.
  • humanized monoclonal antibody refers to the amino acid sequence of a mouse monoclonal antibody, in addition to retaining complementaryarity-determining regions (CDRs), other sequences (including the framework region sequence in the variable region) ) All or most of the amino acid sequences of adult immunoglobulins are replaced in order to minimize the immunogenicity of mouse-derived monoclonal antibodies by genetic engineering means.
  • CDRs complementaryarity-determining regions
  • antibody and immunoglobulin are isotetrasaccharide proteins of about 150,000 daltons having the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains ( H) composition. Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds differs between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (V H ) at one end. This is followed by multiple constant regions.
  • V H variable region
  • Each light chain has a variable region (V L ) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain and the variable region of the heavy chain relatively. Special amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain parts of the variable regions of an antibody differ in sequence, which forms the binding and specificity of various specific antibodies for their specific antigen. However, the variability is not evenly distributed throughout the antibody variable region. It focuses on three fragments in the variable regions of the light and heavy chains that become complementarity determining regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called framework regions (FR).
  • the variable regions of the antibody heavy and light chains each contain four FR regions, which are generally in a ⁇ -sheet configuration, connected by three CDRs forming a linking loop, and in some cases may form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely held together by the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, 647-669 (1991)).
  • Antibody constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity , CDC).
  • Antibodies of the invention can generally be prepared by the following methods:
  • a gene containing an antibody encoding the present invention is inserted into an expression vector containing an appropriate expression control sequence.
  • expression control sequence generally refers to a sequence involved in controlling the expression of a gene.
  • Expression control sequences include a promoter and a termination signal operatively linked to the target gene.
  • the gene (DNA) sequence encoding the antibody of the present invention can be obtained by conventional means well known to those skilled in the art, such as artificially synthesizing the protein sequence disclosed by the present invention or amplifying by PCR method. Thereafter, the synthetic or PCR amplified DNA fragment can be inserted into a suitable expression vector by a variety of methods well known in the art.
  • the expression vector used in the present invention may be a commercially available expression vector known to those skilled in the art, such as the pCDNA3.1 expression vector of Invitrogen Company.
  • Suitable host cells for transformation with the expression vector generally include prokaryotic and eukaryotic cells.
  • prokaryotic host cells include E. coli, Bacillus subtilis, and the like.
  • eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.
  • preferred host cells are mammalian cells, especially Chinese hamster ovary (CHO) cells.
  • the host cell transformed with the expression vector is cultured under appropriate conditions (such as adherent or suspension culture in a cell culture flask or bioreactor in serum-free medium), the culture supernatant is harvested, and then the protein-A affinity is included. Chromatography, ion exchange chromatography, filtration and sterilization, and other conventional separation steps or means well known to those skilled in the art can be purified to obtain the antibodies of the present invention.
  • the purified antibody of the present invention can be dissolved in a suitable solvent such as a sterile physiological saline solution, and the solubility can be prepared between 0.01 and 100 mg / ml, and the ideal final solubility can be prepared between 1 and 20 mg / ml.
  • a suitable solvent such as a sterile physiological saline solution
  • the present invention selects mammalian-expressed recombinant human CD38 extracellular membrane protein as the immune antigen, and repeats the small dose repeatedly
  • the mouse was subcutaneously immunized to obtain a polyclonal antibody secreting anti-CD38 protein.
  • the mouse containing the high titer antibody was selected from the mouse, and the spleen cells were taken out and fused with mouse myeloma cells in vitro, and then screened by drugs and subcloned.
  • the mouse hybridoma cell line number m29 was identified by ELISA, flow cytometry and other methods, and it was confirmed that the monoclonal antibodies it secreted could not only specifically bind to human CD38 protein and a source of positive CD38 expression with high affinity. Many human tumor cell lines. In addition, antibodies secreted by the m29 mouse hybridoma cell line can kill CD38-positive tumor cells via CDC in vitro.
  • gene fragments encoding the heavy chain variable region and light chain variable region of the mouse-derived antibody are obtained by means of genetic engineering and the like.
  • the antibody is genetically engineered to obtain a human-mouse chimeric expression.
  • Antibodies, or vectors of humanized antibodies are transfected into Chinese hamster ovary (CHO) cells to obtain recombinant engineered cells that stably and efficiently secrete human-mouse chimeric antibodies or humanized antibodies, and are isolated and purified from the recombinant engineered cell culture solution to obtain Biologically active human-mouse chimeric antibody ch29 protein or humanized antibody HH29 protein.
  • mice monoclonal antibody m29 and its human-mouse chimeric antibody (ch29) had different binding sites (epitope) from the CD38 antigen than Daratumumab.
  • the results of direct in vitro ELISA and flow cytometry analysis also showed that the mouse monoclonal antibody m29 and its human-mouse chimeric antibody ch29 can also be highly specific for monkey CD38 recombinant protein and CHO cell lines expressing monkey CD38 recombinant gene. Binding; while Daratumumab monoclonal antibody only binds human CD38 antigen, but does not significantly bind to monkey CD38 antigen.
  • mice The anti-tumor efficacy of mouse-derived m29 monoclonal antibodies and human-mouse chimeric m29 monoclonal antibodies (ch29) was tested in immunodeficiency mice (node mice). The results showed that mouse-derived monoclonal antibody m29 and its human-mouse chimeric antibody ch29 were in vivo. After administration, it has a significant inhibitory effect on tumor growth, and its efficacy is no less than that of Rituximab (trade name Rituxan, human-mouse chimeric anti-human CD20 monoclonal antibody) or Daratumumab, which have been marketed as a positive control drug.
  • Rituximab trade name Rituxan, human-mouse chimeric anti-human CD20 monoclonal antibody
  • Daratumumab which have been marketed as a positive control drug.
  • FIG. 1 is a schematic diagram of an amino acid sequence alignment analysis of human CD38 and mouse CD38 protein in Example 1 of the present invention.
  • FIG. 2A is a schematic diagram of the code and detection OD value of each hybridoma cell line secreting anti-CD38 antibody obtained by ELISA screening in Example 1 of the present invention.
  • FIG. 2B Schematic diagram of the results of using the CDC method to verify the activity of killing Daudi target cells by the supernatant of an anti-CD38 antibody-secreting hybridoma cell line obtained by ELISA screening in Example 2 of the present invention, where the target cells used are Daudi and RFU is Relative Abbreviation for Fluorescence Unit.
  • FIG. 3A is a schematic diagram of the CDC activity of the mouse hybridoma cell line m29 supernatant obtained by the CDC method in Example 3 of the present invention.
  • the target cell used is Daudi, the positive control sample is Daratumumab, and the negative control sample is small.
  • 3B is a schematic diagram of a CDC test result of Daratumumab, a positive control sample in Example 3 of the present invention
  • FIG. 3C is a schematic diagram of CDC detection results of a mouse hybridoma cell line m29 supernatant sample in Example 3 of the present invention.
  • FIG. 4 is a diagram showing the representative results of the analysis of the mouse hybridoma cell m29 supernatant sample and the CD38 antigen-positive human tumor cell strain by flow cytometry in Example 4 of the present invention.
  • the negative control sample is non-correlated Murine monoclonal antibody mAB21 (mouse anti-human PD-1 monoclonal antibody); of which:
  • FIG. 4A is a flow cytometry test result chart of human B-lymphoma cell line Daudi;
  • FIG. 4B is a flow cytometry test result diagram of human myeloma cell line RPMI-8226; FIG.
  • FIG. 4C is a flow cytometric detection result chart of human T-lymphoma cell line MOLT-4 cells.
  • FIG. 5 is a representative result diagram of binding analysis of a mouse monoclonal antibody m29 sample and a positive control sample Daratumumab to CHO cells (CHO-hCD38) transfected with human CD38 gene by flow cytometry in Example 5 of the present invention; negative
  • the control samples were unrelated mouse mAb mAB21 (mouse anti-human PD-1 mAb) or humanized hAB21 mAb; of which:
  • FIG. 5A is a flow cytometry detection result diagram of a mouse monoclonal antibody m29 sample
  • FIG. 5B is a flow cytometry test result of Daratumumab as a positive control sample.
  • FIG. 6 Schematic diagram of competitive ELISA detection of purified mouse monoclonal antibody m29 and Daratumumab binding to CD38 in Example 6 of the present invention; unrelated humanized monoclonal antibody sample hPV19 (anti-VEGF monoclonal antibody) is used as a negative control Samples; of which:
  • FIG. 6A is a graph showing the results of competition between a mouse monoclonal antibody m29 and Daratumumab and a Biotin-labeled Daratumumab sample for binding CD38;
  • FIG. 6A is a graph showing the results of competition between a mouse monoclonal antibody m29 and Daratumumab and a Biotin-labeled Daratumumab sample for binding CD38;
  • FIG. 6B is a graph showing the results of competition between mouse monoclonal antibody m29 and Daratumumab and Biotin-labeled m29 monoclonal antibody for CD38.
  • FIG. 7 is an alignment analysis diagram of the amino acid sequence of the variable region of the mouse monoclonal antibody m29 and the variable region amino acid sequence of Daratumumab in Example 7 of the present invention, wherein:
  • FIG. 7A is an analysis analysis of the amino acid sequence of the variable region of the light chain; the amino acids of m29 monoclonal antibodies that are different from Daratumumbab are represented by “X” symbols, and the amino acid sequence region identified by the box is the CDR1, CDR2, and CDR3;
  • Figure 7B is an analysis of the amino acid sequence alignment of the variable region of the heavy chain; the amino acid sequence of the m29 monoclonal antibody that is different from the Daratumumab monoclonal antibody is indicated by the "X" symbol, and the amino acid sequence region identified by the box is the heavy chain variable region.
  • Example 8 is a flow cytometric detection and analysis of a mouse monoclonal antibody m29 sample, a human-mouse chimeric antibody ch29G and a positive control sample Daratumumab or Rituximab (Rituxan, human-mouse chimeric anti-human CD20 monoclonal antibody) in Example 9 of the present invention.
  • FIG. 8A is a flow cytometric detection result chart of human B-lymphoma cell line Raji;
  • FIG. 8B is a flow cytometric detection result diagram of a human myeloma tumor cell line RPMI-8226; FIG.
  • FIG. 8C is a flow cytometric detection result chart of human T-lymphoma cell line MOLT-4; FIG.
  • FIG. 8D is a flow cytometric detection result chart of human T-lymphoma cell line Jurkat.
  • FIG. 9 is a graph showing the results of in vitro CDC detection and analysis of murine monoclonal antibody m29 sample, human-mouse chimeric antibody ch29G sample, and control sample Daratumumab activity in Example 10 of the present invention, wherein the target cell used is the human B-lymphoma cell line Daudi
  • the complement source was rabbit serum, and the negative control sample was unrelated humanized hPV19 mAb (anti-VEGF mAb).
  • FIG. 10 is a flow cytometric detection of a mouse monoclonal antibody m29 sample, a human-mouse chimeric antibody ch29G sample and a control sample Daratumumab in Example 11 of the present invention and a CHO cell stably expressing wild-type human CD38 (CHO- human CD38 / wild type), or stable transfection of CHO cells expressing S 274F point mutation CD38 (CHO-human CD38 / S 274F mutation).
  • the negative control sample is an unrelated humanized hAB21 monoclonal antibody (antibody). Human PD-1 mAb).
  • FIG. 10A is a detection result diagram of CHO cells expressing wild-type human CD38
  • FIG. 10B is a detection result diagram of CHO cells (CHO / hCD38-S 274F ) expressing point-mutated human CD38.
  • FIG. 11A is a graph of amino acid sequence alignment analysis results of human CD38, orangutan CD38, and cynomolgus CD38 protein in Example 12 of the present invention.
  • FIG. 11B is a flow cytometric detection of monoclonal antibody m29 sample, human-mouse chimeric antibody ch29G sample and control sample Daratumumab and CHO cells (CHO-cynomolgus CD38) stably transfected with cynomolgus CD38 gene in flow cytometry in Example 12 ) Binding result graph, where the negative control sample is an unrelated humanized hAB21 mAb (anti-human PD-1 mAb).
  • FIG. 12 is a diagram comparing the binding results of the human-mouse chimeric antibody ch29G sample and Daratumumab monoclonal antibody with recombinant human CD38 protein (FIG. 12A) and recombinant cynomolgus CD38 protein (FIG. 12B) in Example 13 of the present invention by ELISA detection and comparison.
  • the negative control sample is Rituximab (Rituxan, human-mouse chimeric anti-human CD20 monoclonal antibody).
  • FIG. 13 is a graph showing the results of CDC detection and analysis of human-mouse chimeric antibody ch29G sample, Daratumumab, and positive control sample Rituximab (human-mouse chimeric anti-human CD20 monoclonal antibody) activity in Example 14 of the present invention.
  • the complement source is human serum and negative
  • the control sample was an unrelated humanized hPV19 mAb (anti-VEGF mAb), of which:
  • FIG. 13A is a graph of CDC detection results using a human B-lymphoma cell line Daudi as a target cell;
  • FIG. 13B is a graph of CDC detection results using a human B-lymphoma cell line Raji as a target cell;
  • FIG. 13C is a graph of CDC detection results using human T-lymphoma cell line MOLT-4 as a target cell
  • FIG. 13D is a graph of CDC detection results using a human T-lymphoma cell line Jurkat as a target cell.
  • FIG. 14 is a graph showing in vivo antitumor efficacy of a mouse-derived m29 monoclonal antibody in a nude mouse subcutaneously implanted human B-lymphoma Raji tumor model in Example 17.
  • FIG. 14A is a schematic diagram of the average growth volume in the 10 days before tumor implantation
  • FIG. 14B is a schematic diagram of the average growth volume in the later stage of tumor implantation.
  • Example 15 is a graph showing the in vivo antitumor efficacy of a human-mouse chimeric antibody ch29 sample in a nude mouse subcutaneously implanted human B-lymphoma Raji tumor model in Example 18 of the present invention.
  • the tumor cell lines and DNA primers used in the present invention are shown in Tables 1 and 2, respectively:
  • Table 1 Names and characteristics of tumor cell lines used in the present invention
  • Example 1 Establishment and screening of mouse hybridoma cell lines that secrete anti-CD38 antibodies
  • CD38 monoclonal antibodies are different from existing CD38 monoclonal antibodies such as Daratumumab due to their antigen-binding regions / epitope, and their biological activity and efficacy in vivo and in vitro are expected to be different or even better than these monoclonal antibodies.
  • This new type of CD38 monoclonal antibody that recognizes new sites can be used in combination or sequentially with myeloma treatment drugs currently on the market, such as Bortezomib, lenalidomide, and lenalidomide, to enhance bone marrow.
  • these monoclonal antibody drugs are also expected to be developed to treat other tumors such as B-lymphoma and T-lymphoma, which are positive for CD38 expression.
  • the present invention has carried out the development and preparation of such brand-new CD38 monoclonal antibodies, and the specific preparation steps are as follows:
  • Step 1 Source of recombinant human CD38 protein (immune antigen) and animal immunity
  • the antigen used for immunization is recombinant human CD38 extracellular membrane protein (Human CD38 Protein-His Tag, product of Beijing Yiqiao Shenzhou Company, Catalog: 10818-H08H) expressed by mammals.
  • the recombinant human CD38 protein was mixed with Freund's complete adjuvant (product of Sigma, USA), and Balb / c mice (100 ⁇ l / head, 10 ⁇ g CD38 protein each time) were injected subcutaneously at multiple points.
  • the CD38 protein was mixed with an incomplete adjuvant, and multiple points were subcutaneously immunized at a dose of 10 ⁇ g / head.
  • ELISA enzyme-linked immunosorbent assay
  • the ELISA detection procedure is as follows: a 96-well microtiter plate is coated with recombinant human CD38 protein (2 ⁇ g / ml, pH 9.6, 0.1M NaHCO3 solution), and coated at 37 ° C for 2 hours, and then 2% bovine serum albumin ( (BSA) Block at 4 ° C overnight.
  • BSA bovine serum albumin
  • the coated plate was washed with PBS-0.1% Tween20 solution, and the diluted mouse serum samples (with non-immunized mouse serum as a negative control) were added at a dilution ratio and incubated at 37 ° C for 2 hours; PBS-0.1% Tween20 After washing with liquid, add horseradish peroxidase HRP-labeled goat anti-mouse IgG (product of American Sigma), and incubate for 1 hour at 37 ° C; and then fully wash with PBS-0.1% Tween20 solution, and then add o-phenylenediamine (OPD) -0.1% H 2 O 2 substrate solution developed color for 10-15 min, then 0.1M HCl was added to stop the reaction. Thereafter, the OD value at 492 nm was read in a multifunctional microplate reader (PerkinElmer Victor X3). Spleen cells from mice with relatively high antibody titers were taken for the next cell fusion.
  • mice spleen cell suspension was prepared aseptically with mouse SP2 / 0 myeloma cells (purchased from the Cell Preservation Center of the Shanghai Academy of Life Sciences, Chinese Academy of Sciences), and 5 : 1 or 10: 1 ratio was fused under the action of 50% PEG-1000 (product of American Sigma).
  • the fusion was performed according to the conventional method (Kohler G. and Milstein C: Nature 1975, 256: 495-497), the amount of PEG was 1 ml, and the addition was slowly completed within 60 seconds.
  • reaction was terminated in serum-free RPMI-1640 medium, centrifuged at 1000 rpm for 10 minutes, the supernatant was removed, and the cells precipitated by centrifugation were treated with 10% HAT (H is hypoxanthine, A aminoplatin, T-thymidine, a product of American Sigma Company) in RPMI 1640-10% FCS medium to adjust the cell concentration to 1x10 6 / ml, and added to a 96-well flat-bottom cell culture plate (200 ⁇ l per well) at 37 ° C, 5% Cultivate in a CO2 incubator (product of Thermo Corporation, USA) for 2-3 weeks.
  • HAT hypoxanthine, A aminoplatin, T-thymidine, a product of American Sigma Company
  • ELISA enzyme-linked immunosorbent assay
  • the ELISA detection procedure is as follows: a 96-well microtiter plate is coated with recombinant human CD38 protein (2 ⁇ g / ml, pH 9.6, 0.1M NaHCO3 solution), and coated at 37 ° C for 2 hours, and then 2% bovine serum albumin ( (BSA) Block at 4 ° C overnight. The next day, the coated plate was washed with PBS-0.1% Tween 20 solution, and then a sample of the hybridoma cell culture supernatant was added (the unfused SP2 / 0 myeloma fine culture supernatant was used as a negative control. The CD38 antigen was small after immunization.
  • Mouse serum samples (diluted at 1: 200) as a positive control) were incubated at 37 ° C for 2 hours; after washing with PBS-0.1% Tween 20 solution, horseradish peroxidase HRP-labeled goat anti-mouse IgG (Sigma Company, USA) was added. Product), incubate at 37 ° C for 1 hour; and then fully washed with PBS-0.1% Tween20 solution, then add o-phenylenediamine (OPD) -0.1% H 2 O 2 substrate solution to develop color for 10-15min, and then add 0.1M HCl Stop the reaction. Thereafter, the OD value at 492 nm was read in a multifunctional microplate reader (PerkinElmer Victor X3).
  • OPD o-phenylenediamine
  • a total of more than 600 mouse hybridoma cell clones were screened. As a result, more than 10 positive hybridoma clones secreting anti-CD38 antibodies were obtained (the judgment criterion of positive clones: the OD value was more than 5 times higher than the OD value of the negative control sample).
  • FIG. 2A is the code and detection OD value of each hybridoma cell line that secretes and binds to the CD38 antigen positively obtained by the ELISA screening. These positive hybridoma cells were further expanded and cultured and frozen at -70 ° C.
  • Example 2 Complement-dependent cytotoxicity (CDC) method Screening and detection of in vitro anti-CD38 antibody-positive hybridoma cell line supernatants
  • CDC complement-dependent cytotoxicity
  • Fetal bovine serum American GIBCO company products
  • Target cells human B-lymphoma cell line Daudi (human CD38 + , CD20 + B-lymphoma cell line or other tumor cell lines, purchased from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences)
  • Source of complement human serum / young rabbit serum / rabbit complement (healthy) (home-made)
  • Test article supernatant of anti-CD38 monoclonal antibody positive hybridoma cell line (home-made)
  • Positive control samples Daratumumab (anti-CD38 humanized monoclonal antibody), or Rituximab (anti-CD20 human-mouse chimeric monoclonal antibody)
  • Negative control sample hPV19 mAb (anti-VEGF humanized mAb)
  • the CDC activity can be directly identified by the fluorescence intensity RFU or the CDC cell killing rate can be calculated according to the following formula:
  • FIG. 2B shows the results of using the CDC method to analyze and analyze the activity of killing Daudi target cells from the supernatant of hybridoma cell lines secreting anti-CD38 antibodies (RFU direct value); the supernatant of hybridoma cells of the generation number m29 showed obvious performance CDC activity (compared with other samples, RFU values decreased by more than 95%), while other hybridoma cell culture supernatant samples had no significant CDC activity (RFU values did not significantly decrease).
  • Example 3 Subtype identification of mouse m29 monoclonal antibody and revalidation of complement dependent cytotoxicity (CDC)
  • the mouse-derived m29 monoclonal antibody was identified using a commercially available mouse monoclonal antibody IgG subclass detection card (product of Beijing Boaolong Immunology Technology Co., Ltd., article number BF06038), and the results showed that it was an IgG2b antibody. Thereafter, the same method as in Example 2 was used to verify again the activity of killing Daudi target cells by CDC in vitro from the supernatant of the m29 hybridoma cell line. The results are shown in FIG. 3.
  • Figure 3A is the RFU reading result of the m29 hybridoma cell line supernatant sample directly added to the CDC test plate without dilution; the results again show that the m29 hybridoma cell line supernatant has a significant CDC activity (compared with the negative control sample). In comparison, the RFU value has dropped by more than 95%).
  • FIG. 3B shows the results of the in vitro CDC killing of Daudi target cells by positive control samples Daratumumab with different solubility, and the results show a clear dose-response curve.
  • FIG. 3C shows the results of in vitro CDC killing Daudi target cells containing m29 monoclonal antibody supernatants with different solubilities. The results also show a clear dose-response curve.
  • Example 4 Detection and analysis of the binding of murine monoclonal antibody m29 to the source and human CD38 antigen-expressing tumor cell lines by flow cytometry
  • a sample of the supernatant of the m29 hybridoma cell line, or a sample of an unrelated mouse monoclonal antibody MAB21 (mouse anti-human PD-1 monoclonal antibody) is used as the primary antibody, and FITC fluorescently labeled goat anti-mouse IgG is a secondary antibody, and the binding of the sample to human tumor cell lines positive for CD38 antigen expression was detected by flow cytometry.
  • MAB21 mouse anti-human PD-1 monoclonal antibody
  • human tumor cell lines (B-lymphoma cell line Daudi, myeloma cell line RPMI-8226, and T-lymphoma cell line MOLT-4) known to be positive for CD38 antigen were purchased from the Chinese Academy of Sciences in Shanghai Cell Preservation Center, College of Life Sciences) and m29 hybridoma cell line supernatant samples, or non-related mAb mouse-derived mAB21 (mouse anti-human PD-1 mAb) samples were incubated at 4 ° C for 1 hour, and were subjected to PBS-0.1% FCS After washing with liquid, add FITC-labeled goat anti-mouse IgG (1: 200 dilution, product of Sigma); incubate at 4 ° C for 1 hour, and then wash with PBS-0.1% FCS solution, and then load the sample to Accuri C6Plus Flow Cytometer flow cytometry detection (Becton Dickinson Company, Mountain View, CA, USA).
  • Figure 4 shows the representative results of the flow cytometry.
  • the supernatant of the m29 hybridoma cell line can be clearly compared with the human B-lymphoma cell line positive for CD38 antigen expression.
  • Daudi Figure 4A
  • myeloma cell line RPMI-8226 Figure 4B
  • T-lymphoma cell line MOLT-4 Figure 4C
  • Example 5 Flow cytometry detection and analysis of m29 hybridoma cell line supernatant samples and Daratumumab binding to CHO cells (CHO-hCD38) stably transfected with human CD38 gene expression
  • a mouse-derived m29 hybridoma supernatant or positive sample Daratumumab was used as the primary antibody, and FITC fluorescently labeled goat anti-mouse IgG or FITC fluorescently labeled goat anti-human IgG was used as the secondary antibody.
  • the analysis sample was analyzed by cytometry to bind to CHO cells (CHO-hCD38) stably expressing human CD38 gene.
  • CHO-hCD38 cells were separately sampled with mouse-derived m29 hybridoma supernatants, positive monoclonal antibody samples Daratumumab, or unrelated mouse-derived mAb mAB21 (mouse anti-human PD-1 monoclonal antibody) or humanized them.
  • hAB21 monoclonal antibody samples were incubated at 4 ° C for 1 hour, washed with PBS-0.1% FCS solution, and then added with FITC-labeled goat anti-mouse IgG (1: 200 dilution, Sigma), or for Daratumumab samples, FITC- Labeled goat anti-human IgG-Fc, 1: 200 dilution, Sigma product); Incubate at 4 ° C for 1 hour, and then wash with PBS-0.1% FCS solution, then load the sample to Accuri C6Plus Flow Cytometer for flow cytometry detection (Becton Dickinson Company, Mountain View, CA).
  • Figure 5 shows the representative results of CHO-hCD38 detected by the flow cytometer.
  • Figure 5 Compared with the negative control samples mAB21 or hAB21, the mouse m29 hybridoma supernatant sample ( Figure 5A) and Daratumumab sample ( Figure 5B) can obviously bind to CHO-hCD38 cells, of which m29 monoclonal antibody The binding strength of the sample to CHO-hCD38 cells was no less than that of Daratumumab monoclonal antibody.
  • Example 6 Isolation and purification of mouse m29 monoclonal antibody and in vitro competition ELISA to detect m29 monoclonal antibody competing with Daratumumab for CD38
  • the basic principle and process of the competitive ELISA method are as follows: first, different concentrations of m29 monoclonal antibody samples or Daratumumab and a fixed concentration of biotin-labeled m29 monoclonal antibody (biotin-m29) or biotin-labeled Daratumumab (biotin-Daratumumab) After mixing, transfer the mixture to a 96-well plate pre-coated with CD38-His recombinant protein. After incubation and elution, add enzyme-labeled Avidin (such as horseradish peroxidase-labeled Avidin). After incubation and elution, the substrate was added to display and determine the OD value.
  • biotin-m29 monoclonal antibody biotin-m29
  • biotin-Daratumumab biotin-labeled Daratumumab
  • enzyme-labeled Avidin such as horseradish peroxidase-labeled Avidin
  • FIG. 6 is a schematic diagram of a representative test result of the competitive ELISA method
  • Figure 6A shows the results of in vitro competition between m29 mAb and Daratumumab and biotin-labeled Daratumumab (biotin-Daratumumab) for binding to CD38 protein; as shown in the figure: adding different concentrations of m29 mAb or Daratumumab and a fixed concentration of biotin-Daratumumab samples
  • the OD value of the color reaction of each well is inversely proportional to the amount of labeled monoclonal antibody sample: that is, the higher the amount of m29 monoclonal antibody or Daratumumab monoclonal antibody added, the lower the color development OD value; this result shows that the same as Daratumumab , M29 monoclonal antibody can compete with biotin-Daratumumab sample to compete with CD38; and m29 monoclonal antibody can almost achieve the same effect as Daratumumab to compete with CD38
  • Figure 6B shows the results of in vitro competition between m29 mAb and Daratumumab and biotin-labeled m29 mAb (biotin-m29 mAb) for binding to CD38 protein; as shown in the figure, m29 mAb with different solubility and fixed concentration of biotin
  • the OD value of the color reaction of each well is inversely proportional to the amount of unlabeled m29 monoclonal antibody sample added: That is, the higher the amount of m29 monoclonal antibody added, the lower the color development OD value; and Daratumumab or uncorrelated
  • the amount of hPV19 added to the monoclonal antibody sample had little effect on the OD value of each well. This result indicates that Daratumumab does not compete with m29 mAb for binding to CD38 in vitro.
  • RNA was extracted from mouse hybridoma m29 cells, and then the RNA was used as a template, degenerate primers were used, and the reverse transcription-polymerase chain reaction (RT-PCR) method was used (Wang et al.) : Degenerated Primer Design Amplify the chain variable region from immunoglobulin cDNA. BMC Bioinformatics. 2006; 7Suppl (4): S9) were cloned and amplified to obtain the cDNA gene fragment of the variable region of the heavy chain and the variable region of the light chain of m29 antibody.
  • RT-PCR reverse transcription-polymerase chain reaction
  • the cDNA gene cloning steps are as follows:
  • Step 1 Use a kit (product of Jiangsu Haimen Biyuntian Co., Ltd.) to extract total RNA from mouse m29 hybridoma cells;
  • Step 2 A reverse transcription PCR (RT-PCR) method was used to obtain a cDNA template in an eppendorf tube.
  • the mKaRT sequence of reverse transcription PCR primers for the light chain variable region of m29 antibody is: TGT, CGT, TCA, CTG, CCA, TCA, AT;
  • the mGaRT sequence of the reverse transcription PCR primer mGaRT for the heavy chain variable region of the m29 antibody is: GCA, AGG, CTT, ACA, ACC, ACA, ATC;
  • the RT-PCR reaction system is as follows:
  • the reaction was performed at a temperature of 42 ° C for 1 hour, and then the temperature was raised to 75 ° C. After inactivation for 15 minutes, the obtained cDNA was placed at -20 ° C and stored for future use.
  • Step 3 PCR cloning and amplification of genes encoding the light chain variable region and heavy chain variable region of the m29 antibody
  • a pair of primers used for degenerate primers PCR method to clone and amplify the light chain variable region gene of m29 antibody are:
  • Reverse primer mIgLCR440 CTGAGGCACCTCCAGATGTT.
  • the pair of primers used for degenerate primers PCR cloning and amplification of the variable region gene of m29 antibody heavy chain is:
  • Reverse primer mIgHCR135 GTGCTGGAGGGGACAGTCACT.
  • the DNA products obtained by PCR amplification were analyzed by electrophoresis in 1.5% agarose gel. After the electrophoresis is completed, the separated DNA bands are cut and sequenced to obtain the nucleotide sequences of the antibody light chain and heavy chain variable region DNA.
  • the measured nucleotide sequence of the light chain variable region DNA of the antibody is shown in SEQ ID NO.:1, and the amino acid sequence of the antibody light chain variable region inferred from the DNA nucleotide sequence is shown in SEQ ID NO.:2.
  • CDR1, CDR2, and CDR3 of the complementarity-determining regions (CDRs) of the light chain antigen are shown in SEQ ID NO .: 3, SEQ ID NO .: 4, and SEQ ID NO .: 5 respectively.
  • the measured nucleotide sequence of the heavy chain variable region DNA of the antibody can be found in SEQ ID NO.:6, and the amino acid sequence of the antibody heavy chain variable region inferred from the nucleotide sequence of the DNA can be found in SEQ ID NO.:7 .
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain antigenic complementarity determining region are shown in SEQ ID NO .: 8, SEQ ID NO.:9, and SEQ ID NO.:10, respectively.
  • FIG. 7A is an amino acid sequence comparison analysis result of the light chain variable region encoding the m29 antibody and the variable region of the Daratumumab light chain: wherein the amino acid sequence of the light chain variable region of the m29 monoclonal antibody differs from Daratumumab with an “X” symbol Indicated that the amino acid sequence identified by the box is the CDR1, CDR2 and CDR3 of the variable region of each monoclonal antibody light chain; the comparative analysis results show that the variable region of the light chain of m29 monoclonal antibody and its CDR sequence are significantly different from Daratumumab.
  • Figure 7B shows the amino acid sequence comparison analysis of the heavy chain variable region encoding the m29 antibody and the heavy chain variable region of Daratumumab: where the amino acid sequence of the heavy chain variable region of m29 monoclonal antibody differs from Daratumumab by "X"
  • the symbol indicates that the amino acid sequence identified by the box is the CDR1, CDR2, and CDR3 of the heavy chain variable region of each monoclonal antibody; the comparative analysis results show that the variable region of the light chain of m29 monoclonal antibody and its CDR sequence are significantly different from Daratumumab.
  • Example 8 Construction of human-mouse chimeric antibody (ch29G) of murine m29 antibody
  • the m29 antibody light chain variable region gene and the heavy chain variable region gene obtained by cloning and amplification in Example 7 above were respectively linked with human-kappa light chain constant region (C-domain) and human IgG1-heavy chain constant region gene fragments. Fusion to obtain a human-mouse chimeric light chain gene (ch29L) and a human-mouse chimeric heavy chain gene (ch29H). Thereafter, the light chain chimeric gene and the heavy chain chimeric gene were cloned into the pQY-DHFR-Hex expression plasmid successively, transferred into E. coli for amplification, and a large number of expression plasmids containing human-mouse chimeric antibody genes were isolated.
  • transfected cells are passaged to a petri dish, and a conditioned medium is added to screen for stable expression strains.
  • a conditioned medium is added to screen for stable expression strains.
  • select well-grown cell clones to 96-well plate culture and take the supernatant after 2-3 days to detect the supernatant protein expression by ELISA method, and finally select CHO cells that express higher ch29 antibody protein Strain.
  • This cell line was acclimated with a serum-free medium (CHOM-B01, Shanghai Bai'an Medical Investment Co., Ltd.). After the acclimation was successful, it was expanded and cultured and the supernatant was collected.
  • a Protein-A affinity chromatography column proteinA-Sepharose Fast Flow, product of GE, USA
  • Example 9 Comparison of murine monoclonal antibody m29, human-mouse chimeric antibody ch29G and Daratumumab binding to human tumor cells expressing CD38 antigen by flow cytometry
  • a purified mouse-derived m29 monoclonal antibody, human-mouse chimeric antibody ch29G, and Daratumumab or unrelated hAB21 monoclonal antibody were used as primary antibodies, and FITC fluorescence was used.
  • the labeled goat anti-mouse IgG or goat anti-human IgG was used as the secondary antibody, and the flow cytometry method as in Example 4 was used to detect the binding of the analysis sample to the tumor cell line with positive CD38 antigen expression.
  • human tumor cell lines human Burkitt B-lymphoma cell line Raji, human myeloma cell line RPMI-8226, human T-lymphoma cell line MOLT-4, and human T-lymphoma
  • human tumor cell lines purchased from the Cell Preservation Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
  • mouse m29 monoclonal antibody, human-mouse chimeric antibody ch29G, and Daratumumab or unrelated hAB21 monoclonal antibody samples at 4 ° C for 1 hour.
  • FITC-labeled goat anti-mouse IgG (1: 200 dilution, Sigma product) or FITC-labeled goat anti-human IgG (1: 200 dilution, Sigma product) were added and incubated at 4 ° C. After 1 hour, the sample was washed with PBS-0.1% FCS solution, and then the sample was loaded onto an Accuri C6Plus Flow Cytometer for flow cytometry detection (Becton Dickinson Company, Mountain View, CA, USA).
  • Figure 8 shows the representative results of the flow cytometry test
  • Figure 8A shows the detection results of human Burkitt B-lymphoma cell line Raji: compared with non-related monoclonal antibody hAB21, mouse m29 monoclonal antibody, human-mouse chimeric antibody ch29G, and Daratumumab can obviously interact with human Burkitt B-lymphoma cells.
  • the positive ratio and signal intensity of murine m29 monoclonal antibody and its human-mouse chimeric antibody ch29G binding to Raji cells were almost the same as Daratumumab samples.
  • Figure 8B shows the detection results of human myeloma cell line RPMI-8226.
  • mouse m29 monoclonal antibody, human-mouse chimeric antibody ch29G, and Daratumumab can obviously interact with human Burkitt B-lymphoma cell line.
  • the binding ratio and signal intensity of murine m29 monoclonal antibody and its human-mouse chimeric antibody ch29G to RPMI-8226 cells were almost the same as those of Daratumumab samples.
  • FIG. 8C is the detection result of human T-lymphoma cell line MOLT-4: the positive ratio and signal intensity of murine m29 monoclonal antibody and its human-mouse chimeric antibody ch29G binding to MOLT-4 cells are slightly lower than Daratumumab.
  • FIG. 8D is the detection result of human T-lymphoma cell line Jurkat: the positive ratio and signal intensity of murine m29 monoclonal antibody and its human-mouse chimeric antibody ch29G binding to Jurkat cells are lower than Daratumumab.
  • Example 10 In vitro analysis and comparison of CDC activity of mouse monoclonal antibody m29, human-mouse chimeric monoclonal antibody ch29G and Daratumumab
  • the same CDC detection method as in Example 2 was used to analyze and compare the CDC activity of mouse-derived m29 monoclonal antibody, human-mouse chimeric monoclonal antibody ch29G and Daratumumab.
  • the target cells used are classic Daudi cells, and the complement used is 10% rabbit serum (home-made).
  • Figure 9 shows the test results of the CDC; the results show that both the mouse m29 monoclonal antibody and its human-mouse chimeric monoclonal antibody ch29G have stronger CDC activity than Daratumumab.
  • the highest CDC activity of mouse m29 monoclonal antibody and its human-mouse chimeric monoclonal antibody ch29G was above 95%, and the EC50 value was about 30ng / ml.
  • the highest CDC activity of Daratumumab was only about 55%, and the EC50 value was 100ng / ml.
  • Example 11 Detection of the binding of murine monoclonal antibody m29, human-mouse chimeric antibody ch29G and Daratumumab to point mutation CD38 by flow cytometry
  • the obtained DNA product was electrophoretically separated in a 1.5% agarose gel, and the recovered DNA fragment was used as a template to perform the second round of PCR using the following primers to obtain the complete hCD38-S274F gene:
  • the hCD38-S 274F gene fragment was cloned into the cell expression vector pQY-DHFR (self-built), transformed into E. coli, and the positive recombinant expression plasmid pQY-DHFR-hCD38-S 274F was identified by enzyme digestion verification method.
  • the recombinant plasmid pQY-DHFR-hCD38S274F was mixed with Fugen-6 liposomes (Roche) and co-transfected into CHO-dhfr- cells. Then, the human CD38 gene was stably expressed using IMDM medium containing fetal bovine serum. Mutated CHO cell line (CHO / hCD38-S 274F ).
  • CHO cells stably expressing wild-type or point-mutated human CD38 and samples containing mouse-derived monoclonal antibody m29, human-mouse chimeric monoclonal antibody ch29G or Daratumumab were respectively 4 ° C.
  • the sample was applied to a flow cytometer (Accuri, C6Plus, Flow Cytometer, BD, USA) for detection.
  • FIG. 10 is a schematic diagram of representative results of the flow cytometry detection, wherein:
  • FIG. 10A is the detection result of CHO cells expressing wild-type human CD38, and the results show that, like mouse monoclonal antibody m29, human-mouse chimeric monoclonal antibody ch29 retains CHO cells expressing wild-type CD38 (CHO / hCD38-wild-typ), the binding strength of the two is similar to that of Daratumumab.
  • FIG. 10B shows the detection results of CHO cells (CHO / hCD38-S 274F ) expressing point mutation human CD38.
  • the results show that, as with the mouse monoclonal antibody m29, the human-mouse chimeric monoclonal antibody ch29G also expresses the point mutation CD38.
  • CHO cells (CHO / hCD38-wild-typ), while Daratumumab did not significantly bind to CHO cells expressing this point mutation.
  • Example 12 Establishment of CHO cell line (CHO / mkCD38) stably expressing cynomolgus CD38 and detection of murine monoclonal antibody m29, human-mouse chimeric antibody (ch29G) and Daratumumab by flow cytometry
  • the amino acid sequence of the human CD38 protein is compared with that of the CD38 protein of Macaca fascicularis (Cynomolgus monkey) and CD38 protein of chimpanzee (Phimtrozee, Pantroglodytes) as shown in FIG. 11A; as shown in FIG. 11A: the chimpanzee CD38 (ChiCD38) protein
  • the amino acid sequence of human CD38 protein (huCD38) is almost the same, while the amino acid sequence of cynomolgus CD38 (mkCD38) protein is 91% overall homology with the human CD38 protein amino acid sequence. There are as many as 16.
  • the recombinant plasmid pQY-DHFR-mkCD38 was mixed with Fugen-6 liposomes (Roche) and co-transfected into CHO-dhfr - cells, and then the stable expression of cynomolgus CD38 was selected using IMDM medium containing fetal bovine serum. Protein CHO cell line (CHO-cynomolgus CD38).
  • Flow cytometry was used to detect and analyze murine monoclonal antibody m29, human-mouse chimeric antibody (ch29G), Daratumumab, and a CHO cell line stably expressing cynomolgus CD38 protein (CHO -cynomolgus CD38).
  • FIG. 11B is a schematic diagram showing the representative results of the flow cytometry detection.
  • the results show that the human-mouse chimeric monoclonal antibody ch29G retains almost the same high affinity as the mouse monoclonal antibody m29 and binds to a CHO cell line expressing cynomolgus CD38.
  • the intensity of the binding activity of Daratumumab and CHO cell line expressing cynomolgus CD38 decreased by more than 90%.
  • Example 13 Detection of the binding of human-mouse chimeric antibodies ch29G and Daratumumab to human CD38 protein or cynomolgus CD38 protein by ELISA
  • a direct ELISA method is used to detect the binding of human-mouse chimeric antibodies ch29 and Daratumumab to recombinant human CD38 protein or recombinant cynomolgus CD38 protein.
  • 96-well plates (1ug / ml, 50ul / well, 4 degrees) were coated with recombinant human CD38-his protein or recombinant cynomolgus CD38-his protein (both products of Beijing Yiqiao Shenzhou Company).
  • FIG. 12 shows the detection results of the ELISA.
  • both the human-mouse chimeric antibody ch29G and Daratumumab can bind to human CD38 protein with high affinity ( Figure 12A).
  • the human-mouse chimeric antibody ch29G can also bind to cynomolgus CD38 protein with high affinity; while Daratumumab does not significantly bind to cynomolgus CD38 protein ( Figure 12B).
  • Example 14 Comparative analysis of complement-dependent cytotoxicity (CDC) detection in vitro of human-mouse chimeric monoclonal antibodies ch29G and Daratumumab
  • the in vitro complement-dependent cytotoxicity (CDC) method as in Example 2 was used to detect human-mouse chimeric monoclonal antibody ch29G against a variety of target cells (including Daudi, Raji, MOLT-4). And Jurket et al.) And compared the results with those of Daratumumab.
  • the complement used was derived from healthy human serum (10%, homemade), the CDC positive control sample was Rituximab (anti-CD20 human-mouse chimeric monoclonal antibody), and the negative control sample was hPV19 monoclonal antibody (anti-VEGF humanized monoclonal antibody). .
  • Figure 13 shows the test results of the CDC, where:
  • FIG. 13A shows the results of a CDC test using Daudi as a target cell.
  • the results show that the human-mouse chimeric antibody ch29G has a CDC activity similar to that of the positive control sample Rituximab, and both have stronger CDC activities than Daratumumab.
  • FIG. 13B is the result of the CDC test using Raji as a target cell.
  • the results show that the human-mouse chimeric antibody ch29G also has a CDC activity similar to that of the positive control sample Rituximab, and the CDC activity of Daratumumab is similar to that of the human-mouse chimeric antibody ch29G .
  • FIG. 13C shows the results of a CDC test using MOLT-4 as a target cell. The results show that human-mouse chimeric antibodies ch29G, Daratumumab, and Rituximab have no significant CDC activity.
  • FIG. 13D shows the results of the CDC test with Jurket as the target cell. The results show that the human-mouse chimeric antibody ch29G, Daratumumab and Rituximab also have no significant CDC activity.
  • Example 15 Humanized genetic engineering of murine m29 antibody
  • the human-mouse chimeric antibody ch29G has been shown to retain high affinity binding to human CD38 protein and CDC activity.
  • the chimeric antibody light chain was cloned using a series of genetic engineering cloning methods such as PCR.
  • the CDR gene fragment was transferred to the corresponding human kappa-light chain variable region framework (FR) to obtain a humanized antibody light chain, and this light chain was combined with a chimeric heavy chain. That is, a light chain humanized HH29G antibody was obtained.
  • FR human kappa-light chain variable region framework
  • Example 16 Establishment of a CHO cell engineering strain that stably and efficiently secretes humanized or semi-humanized HH29 mAb (HH29) and isolation and purification of antibody protein
  • the human-mouse chimeric heavy chain gene (ch29H) and humanized light chain gene (HH29L) were cloned into the pQY-Dhfr-Hex expression vector step by step, transferred into E. coli and amplified and isolated to obtain humanized or semi-human Humanized HH29 mAb (HH29) mAb expression plasmid. Thereafter, a recombinant plasmid expressing the light chain humanized antibody HH29 was transiently transfected into CHO cells.
  • the cell culture supernatant in the wells was aspirated, CD38-his protein was used as the coating antigen, and HRP enzyme-labeled Goat-anti-human-IgG was used as the detection secondary antibody (purchased from Shanghai Xitang Biological Company).
  • the OPD was The substrate was developed, and the activity of antibody binding to human CD38 protein antigen in the supernatant of transfected cells was detected by a direct ELISA method.
  • Table 4 below shows the representative test results of the ELISA.
  • the semi-humanized HH29 monoclonal antibody maintained the activity of binding to human CD38 protein.
  • transfected cells were screened by cloning and acclimatized by suspension culture in serum-free medium, several CHO cell engineering strains stably and efficiently secreting and expressing semi-humanized monoclonal antibodies (humanized light chain) HH29 were successfully obtained.
  • a cell engineering strain was selected therefrom and expanded and cultured in a serum-free medium, and the culture supernatant was collected.
  • the supernatant was centrifuged and filtered through a 0.45 ⁇ m filter, and then loaded to include a protein-containing affinity layer.
  • separation and purification steps including proteinA-Sepharose, Fast Flow (GE, USA), ion-exchange analytical column, virus removal / inactivation, and filtration and sterilization (0.22 ⁇ m filter membrane filtration), finally obtained High purity (more than 99% protein purity) HH29 antibody.
  • Example 17 Test of antitumor efficacy of mouse-derived m29 monoclonal antibody in mice
  • a nude mouse subcutaneously implanted human B-lymphoma Raji tumor model was used to test the antitumor efficacy of mouse-derived m29 mAb in animals, and a human-mouse chimeric anti-CD20 monoclonal antibody Rituximab was used as a positive control drug.
  • a human B-lymphoma cell line Raji was first implanted subcutaneously in nude mice; after the tumors of the test animals became tumors, they were administered in groups and observed to record tumor growth.
  • nude mice purchased from the Animal Center of Nanjing University
  • a human-derived B-lymphoma cell line Raji cells Cell Bank of the Chinese Academy of Sciences
  • the tumor volume to be inoculated When they grow to about the size of soybeans (about 100 mm 3 , about 6-7 days after tumor cell inoculation), the animals are randomly divided into the following 3 groups:
  • Figure 14 shows the average growth volume trend of animal tumors in each experimental group, where:
  • FIG. 14A is a schematic diagram of the average growth volume 10 days before tumor implantation, and the results show that there is no significant difference between the groups;
  • Figure 14B is a schematic diagram of the average growth volume in the late stage of tumor implantation. The results show that compared with the normal saline negative control group, the tumors of the m29 monoclonal antibody drug treatment group and the positive control drug Rituximab monoclonal antibody treatment group significantly shrink or even disappear completely.
  • Example 18 Antitumor efficacy of human-mouse chimeric ch29G antibodies tested in mice
  • Example 17 the same nude mouse subcutaneously implanted human B-lymphoma Raji tumor model as in Example 17 was used to test the antitumor efficacy of human-mouse chimeric ch29G monoclonal antibody, and Daratumumab monoclonal antibody was used as a positive control drug.
  • human B-lymphoma Raji cells were first implanted subcutaneously in nude mice; after the tumors of the test animals became tumors, they were administered in groups and observed and recorded tumor growth.
  • human-derived B-lymphoma Raji cells were inoculated in nude mice (purchased from the Animal Center of Nanjing University) in a quantity of 1 ⁇ 10 7 and the tumor volume to be inoculated was about the size of soybeans (approximately 100 mm 3 , tumor cells were inoculated).
  • the animals were randomly divided into the following three groups at the next 6-7 days:
  • Figure 15 shows the trend of the average growth volume of tumors in the experimental groups of each group. The results show that compared with the saline-negative control group, the treatment growth of the ch29G monoclonal antibody drug treatment group and the positive control drug Daratumumab treatment group is significantly inhibited.
  • the efficacy of ch29G monoclonal antibody at a dose of 2.5 mg / kg body weight is similar to that of Daratumumab at a dose of 5 mg / kg body weight.

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Abstract

本发明公开了一种特异结合人及猴CD38抗原的单克隆抗体或其衍生体，其包含抗体轻链可变区的抗原互补决定区CDR1，CDR2和CDR3分别为SEQ ID NO：3，SEQ ID NO：4及SEQ ID NO：5的氨基酸序列；抗体重链可变区的抗原互补决定区CDR1，CDR2和CDR3分别为SEQ ID NO：8，SEQ ID NO：9及SEQ ID NO：10的氨基酸序列。该抗体或其衍生体可作为药物组合物成分或制备成合适药物制剂，单独给药或与其他治疗手段如化疗药物等合并使用，用于治疗CD38表达阳性的肿瘤如人骨髓瘤，人淋巴细胞瘤等。

Description

特异结合人及猴CD38抗原的单克隆抗体及其制备方法与应用 技术领域
本发明属于生物技术-单克隆抗体领域。本发明涉及一种特异结合人及猴CD38抗原的单克隆抗体及其编码序列与其制备方法和用途。
背景技术
CD38(也称为T10，一种被OKT10单抗识别的抗原)是一个分子量在45kDa左右的Ⅱ型单次跨膜糖蛋白。编码人类和小鼠CD38基因的cDNA早在1990年代初就已被克隆与报道(Jackson DG and Bell JI.J.Immunol.1990，144:2811-2815；Harada et al.J Immunol.1993，151:3111-8)；编码食蟹猴(cynomolgus macaque)CD38基因的cDNA也在2004年被克隆与报道(Ferrero E et al，BMC Immunol.2004，5:21)。人CD38蛋白全长共有300个氨基酸，其中N-端区21个氨基酸位于细胞内，位于细胞膜上的有22个氨基酸，位于细胞膜外的C-端区有257个氨基酸。食蟹猴CD38蛋白全长共有301个氨基酸，与人CD38蛋白氨基酸序列同源性为92％，而小鼠CD38蛋白全长则有304个氨基酸，与人CD38蛋白氨基酸序列同源性为70％左右。
最早的一项有关CD38生物学功能的报道则来源于States DJ等于1992年发表的文章(States DJ,Walseth TF and Lee HC.Trends Biochem.Sci.1992，17：495)。在该研究报道中，States DJ等首次注意到了CD38分子与来源于海兔(Aplysia)的ADP核糖环化酶(Aplysia ADP-ribosyl cyclase)具有序列和结构上的同源性，并推测CD38应具有ADP核糖环化酶活性，可催化NAD+生成cADPR，而cADPR在细胞内作为第二信使，参与钙离子(Ca2+)的动员转运；CD38故又称为ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1(ADPR1)。之后的其他研究也证实了这种推测并发现CD38蛋白分子还具有脱氧氢酶活性，可以进一步催化cADPR生成ADPR(Howard M et al.Science.1993；262:105；Summerhill RJ,Jackson DG,Galione A.FEBS Lett.1993，335:231-3；Prasad GS et al,Nature Structural Biology 1996，3:957–964)。因此CD38具有双功能酶的活性，且其发挥这双功能酶活性位点均位于胞膜外结构区域。有关CD38分子结构及其酶活性与功能的研究可参见综述文章(Mehta K,Shahid U and Malavasi F:Human CD38,a cell-surface protein with multiple function.FASEB J.1996，10；1408-1417；George Shubinsky,Michael Schlesinger：The CD38 lymphocyte differentiation marker:new insight into its ectoenzymatic activity and its role as a signal transducer.Immunity 1997,7:315–324)。
CD38(T10)抗原则最早是由Feinnerz E及其同事于1980年报道与发现的：他们观察到来源于人的正常胸腺细胞和T-淋巴瘤细胞株MOLT-4表达一种可以被代号为OKT10的单克隆抗体特异结合的抗原，该抗原当时称之为T10(Feinnerz E et al.PNAS 1980,77:1588-1592)。此后的其他研究证明CD38(T10)抗原除了在T-淋巴细胞表达之外，还广泛表达在人体中的其他细胞如B-淋巴细胞、巨噬细胞、树突状细胞、血小板、骨髓浆细胞等血液、淋巴组织细胞中；此外，其他组织细胞如中枢神经系统中的神经细胞和胶质细胞、周围神经细胞、胰腺中的胰岛细胞、骨组织中的破骨细胞、骨骼肌细胞、心肌细胞和支气管上皮细胞等也表达不同水平的CD38(T10)抗原(参见综述文章：Mehta K,Shahid U and Malavasi F:Human CD38, a cell-surface protein with multiple function.FASEB J 10,1408-1417)。
但与其他组织细胞相比，CD38抗原在多发性骨髓瘤(multiple myeloma,MM)及B-淋巴细胞瘤中表达水平相对最高，因此早在1990年代初CD38抗原就被认为是治疗多发性骨髓瘤及B-淋巴细胞瘤的理想靶点，并且先后有多个以靶向CD38抗原的单克隆抗体作为治疗药物，在动物体内外测试其治疗多发性骨髓瘤、B-淋巴细胞瘤的研究与报道：
如Stevenson FK等在1991年报道了OKT10单抗经基因工程改造后，获得的人-鼠嵌合抗体体外可以通过抗体介导的依赖细胞毒性(antibody-dependent cellular cytotoxicity，ADCC)杀伤CD38表达阳性淋巴肿瘤细胞(Stevenson FK et al,Blood.1991，7:1071-1079)；
Goldmacher VS等在1994年报道了通过嫁接蓖麻毒素(ricin)到鼠抗人CD38单抗HB7，获得具免疫毒性(immunotoxin)的抗体-药物复合物(antibody-drug-conjugated，ADC)，该抗体-药物复合物在体外表现出更强的杀伤CD38表达阳性肿瘤细胞的活性(Goldmacher VS et al.Blood.1994，84:3017-25)；
Ellis JH等则在1995年报道了另一个鼠源抗CD38单抗AT13/5经基因工程改造后的人源化抗体比其前身鼠源抗体具有更强的ADCC活性(Ellis JH et al,J Immunol.1995，155:925-37)。
然而，这些早期的研究结果其后并没有成功转化成有用的临床应用或进入临床研究。
多发性骨髓瘤是一种恶性浆细胞病，其特征是骨髓中分泌的浆细胞潜在的积累，伴随低度增殖和延长细胞生长周期，可导致高钙血症、肾功能损伤、相邻骨髓组织摧毁及贫血等症状。在2015年之前，国内外临床上用于治疗多发性骨髓瘤的主要方法包括化疗药物如长春新碱(Vincristine)、环磷酰胺(Cyclophosphamide)、美法仑(Melphalan)、阿霉素(Adriamycin)，免疫调节剂(Immunomodulators,IMiDs)，蛋白酶体抑制剂(Proteasome Inhibitors,PIs)、双磷酸盐(Bisphosphonates)及激素如强的松(Prednisone)、地塞米松(Dexamethasone)及自体干细胞移植等。
其中，免疫调节剂的代表性药物有美国新基制药(Celgene)公司先后开发上市的Thalomid(通用名Thalidomide，1998年07月获美国FDA批准上市)、来那度胺Revlimid(通用名lenalidomide，2005年12月获美国FDA批准上市)和泊马度胺Pomalyst(通用名Pomalidomide，2013年02月获美国FDA批准上市)。
蛋白酶体抑制剂的代表性药物有日本武田制药(Takeda/Millennium)开发的Velcade万珂(通用名Bortezomib硼替佐米，2003年05月获美国FDA批准上市)、美国Onyx公司开发的Kyprolis卡非佐米(通用名Carfilzomib，2012年07月获美国FDA批准上市)。双磷酸盐的代表性药物有诺华(Novartis)先后开发的Aredia(通用名Pamidronate，1991年10月获美国FDA批准上市)和Zometa(通用名Zoledronic acid，2001年08月获美国FDA批准上市)。
但这些药物的联合用药后的完全反应率(Complete Response Rate,CRR)一般也只有5％，患者中位生存期(Median Survival Time,MST)在首次被诊断后通常为36-48个月。因此，多发性骨髓瘤的治疗领域迫切需要开发出新的更有效的药物或治疗方法。
多发性骨髓瘤的治疗领域在2015年11月因美国FDA批准了全球首个抗CD38单抗药物Daratumumab(达雷木单抗)用于治疗既往至少接受过三次包括Bortezomib硼替佐米和Lenalidomide来那度胺或同时对 硼替佐米与来那度胺治疗后耐药的多发性骨髓瘤患者，而步入了具有革命性意义的、单抗药物靶向治疗的新时代。
Daratumumab商品名为Darzalex，是由美国强生Johnson&Johnson子公司Janssen Biotech与丹麦Genmab公司联合开发的。Daratumumab是一种特异识别与结合人CD38抗原的全人源IgG1/kappa单抗，其最初来源于Genmab公司开发的一代号为005的抗人CD38单抗(de Weers et al.J Immunol 2001；186:1840-8；国际PCT申请号：PCT/DK2006/000166，美国专利号：US7829673B2)。前期非临床研究结果显示Daratumumab/005单抗可以通过补体依赖性细胞毒性(complement-dependent cytotoxicity，CDC)，抗体依赖的细胞介导作用(antibody-dependent cell-mediated cytotoxicity，ADCC),抗体依赖性细胞吞噬作用(antibody-dependent cell-mediated phagocytosis，ADCP)，及直接诱导肿瘤细胞发生调亡(apoptotosis)等多种机制快速杀死CD38抗原表达阳性的肿瘤如骨髓瘤。此外，Daratumumab/005单抗还能抑制CD38介导的酶活性(ADP-ribosyl cyclase)而发挥作用。
2011年美国强生Johnson&Johnson与Genmab公司达成临床合作开发Daratumumab的协议。2013年Daratumumab因其在治疗骨髓瘤临床试验中显示出良好的单药活性，获得美国FDA快速通道指定(Fast Track Designation)及突破性疗法(Breakthrough Therapy Designation)药物产品认定。2015年11月美国FDA主要基于两项分别名为GEN501(Lokhorst HM et al，N Engl J Med.2015，373:1207–19)和SIRIUS(Lonial S et al，Lancet.2016，387:1551-60)的I/II期临床试验结果，快速批准了Daratumumab药物上市，用于四线治疗既往至少接受过三次包括bortezomib硼替佐米和lenalidomide来那度胺或同时对bortezomib硼替佐米和lenalidomide来那度胺治疗后耐药的多发性骨髓瘤患者。在GEN501和SIRIUS临床试验中，结果均显示注射剂量为16mg/kg体重的Daratumumab单抗治疗组中，患者的客观缓解率(Objective response rate,ORR)及中位无进展生存期(Progression-free survival,PFS)都有非常显著的改善。
Johnson & Johnson强生/Genmab公司在Daratumumab获批上市之后，其后又开展了代号分别为POLLUX和CASTOR的两项III期临床研究，其中POLLUX研究是评估Daratumumab与lenalidomide来那度胺及Dexamethasone地塞米松联合用药用于三线治疗骨髓瘤患者的临床疗效；该研究共入组骨髓瘤患者569例，其中Daratumumab与lenalidomide来那度胺及Dexamethasone地塞米松联合用药组286例(治疗组)，lenalidomide来那度胺及Dexamethasone地塞米松联合用药治疗组283例(对照组)。研究结果显示：联用Daratumumab治疗组在12个月时无进展生存期(PFS)患者的比例为83.2％，客观缓解率(ORR)为92.9％，均显著高于对照组(PFS比例为60.1％，ORR为76.4％)(Dimopoulos et al.POLLUX Investigators,N Engl J Med.2016，375:1319–31)。
CASTOR研究则是评估Daratumumab与Bortezomib硼替佐米/万珂及Dexamethasone地塞米松用于三线治疗骨髓瘤患者的临床疗效；该研究共入组骨髓瘤患者498例，其中Daratumumab与Bortezomib硼替佐米及Dexamethasone地塞米松联合用药组251例(治疗组)Bortezomib硼替佐米及Dexamethasone地塞米松联合用药对照组247例。研究结果显示联用Daratumumab治疗组在12个月时无进展生存期PFS患者的比例为60.7％，客观缓解率(ORR)为82.9％，均显著高于对照组(PFS比例为26.9％，ORR为63.2％)(Palumbo A et al,CASTOR Investigators.N Engl J Med.2016，375:754-66)。
基于上述POLLUX和CASTOR的两项临床III期研究，美国FDA于2016年11月批准了Daratumumab与lenalidomide来那度胺及Dexamethasone地塞米松联合用药或Daratumumab与Bortezomib硼替佐米及Dexamethasone地塞米松联合用药，用于三线治疗骨髓瘤。
基于代号为EQUULEUS(MMY1001)的临床研究结果，美国FDA于2017年06月又批准了Daratumumab联合Pomalidomide泊马度胺及Dexamethasone地塞米松用于二线治疗骨髓瘤。在EQUULEUS(MMY1001)临床研究中显示Daratumumab联合Pomalidomide泊马度胺及Dexamethasone地塞米松治疗骨髓瘤患者的客观缓解率(ORR)为60％，中位无进展生存期PFS为8.8个月，中位总生存期17.5个月(Ajai Chari,et al，EQUULEUS；MMY1001Investigators：Blood.2017，130:974–981)。
基于代号为ALCYONE的临床研究结果，美国FDA于2018年05月又最新批准了Daratumumab与Bortezomib硼替佐米、Melphalan美法仑和Prednisone强的松一起联合用药，用于一线治疗未接受高剂量化疗和自体干细胞移植(ASCT)的新诊断骨髓瘤患者。ALCYONE研究共入组了706例骨髓瘤患者，结果显示Daratumumab联合用药组在18个月时无进展生存期(PFS)患者的比例为71.6％，客观缓解率(ORR)为90.9％，均显著高于对照组(PFS比例为50.2％，ORR为73.9％(Mateos M-V et al，ALCYONE Investigators.N Engl J Med.2018,378:518-528)。
因此，Daratumumab自2015年11月首次获美国FDA批准上市之后，在短短的3年时间里便从当初的单药四线治疗骨髓瘤药物一步步晋升为一线治疗药物，并很快成为市场上畅销的重磅级药物品种。Daratumumab是目前全球唯一已获批上市的抗CD38单抗药物；目前其他已进入临床试验的抗CD38单抗药物仅有赛诺菲(Sanofi)/Immungen公司合作开发的人-鼠嵌合单抗SAR650984(Isatuximab)及德国制药公司MorphoSys从phage-display抗体库中筛选获得的全人源单抗MOR202单抗，这几个抗体药物目前所开发的临床主要适应症也都是骨髓瘤。
在中国，目前骨髓瘤的治疗主要药物为进口的Bortezomib硼替佐米/万珂、lenalidomide来那度胺等。随着Bortezomib(硼替佐米/万珂)等药物的专利到期，国内已有几十家企业研制其仿制药，其中齐鲁制药、正大天晴药业和江苏豪森药业研制的Bortezomib硼替佐米仿制药物近期均已获中国药监局(CFDA)批准上市销售，用于治疗骨髓瘤。而以CD38为靶点的单克隆抗体药物，仅有上述强生公司的Daratumumab，赛诺菲公司的SAR650984(Isatuximab)单抗等个别进口品种在国内已完成提交临床试验申请或刚进入临床试验，目前还未见有国内企业自主研发的抗CD38单抗药物进入临床试验申请或临床试验阶段。
鉴于国内外骨髓瘤患者发病人数众多，用于治疗骨髓瘤药物尤其是抗体药物的严重匮乏，因此，研究与开发出新的治疗骨髓瘤的药物、尤其是靶向CD38抗原的单抗药物则很有意义与必要。
Daratumumab尽管自海外上市以来获得了临床医生、患者及市场的广泛好评，但其也还存在不少缺陷与不足，其缺陷与不足至少包括如下几点：
1)患者接受静脉滴注Daratumumab药物时，临床上发生输注不良反应的比例很高，且给药时间较长，如首次静脉注射需要历经8小时，此后再次注射也需5-6小时，且为期8周的第一疗程中患者需要每周接受一次治疗；
2)部分骨髓瘤患者临床上对Daratumumab治疗无应答疗效；另还有一些患者在接受Daratumumab治疗后疾病还是继续进展或发生耐药现象；目前还不清楚患者对Daratumumab治疗产生耐药或失败的原因与机理；
3)Daratumumab不识别猴CD38抗原，限制了其在猴等非人类灵长类动物中开展单药或与其他药物组合使用的临床前药效、药理毒理等研究与开发应用。
鉴于小鼠CD38与人CD38蛋白氨基酸序列同源性为70％左右，因此,理论上推测采用传统的抗原蛋白免疫小鼠及杂交瘤技术，应可以制造或开发出全新的、或针对CD38抗原不同表位(epitope)的单克隆抗体。而从这些全新的、或识别CD38抗原特异表位(epitope)的单克隆抗体中，有望开发出比现已上市的Daratumumab或正在开展临床研究的其他CD38单抗具更强的生物活性或更安全的药物。这些新的单抗可作为单药，或与目前已上市的治疗骨髓瘤的其他药物如Bortezomib硼替佐米、Lenalidomide来那度胺等药物联合或序贯使用，用于治疗CD38高表达肿瘤如骨髓瘤及淋巴瘤等疾病。
发明内容
本发明要解决的技术问题之一是提供一种抗原结合区域/结合位点(epitope)不同与现有的Daratumumab的、全新的结合人及猴CD38抗原的、并具体内外杀伤CD38抗原高表达肿瘤等生物活性的单克隆抗体或其衍生体如抗体Fab片段、单链抗体等。
本发明要解决的技术问题之二是提供编码上述抗体的DNA分子或基因。
本发明要解决的技术问题之三是提供含有上述抗体的药物或药物组合物。
本发明要解决的技术问题之四是提供含有上述抗体的药物或药物组合物在治疗CD38抗原高表达阳性肿瘤的应用。
本发明要解决的技术问题之五是提供制备上述抗体的方法。
为解决上述技术问题，本发明采用如下技术方案：
在本发明第一方面，提供了一种抗原结合区域/结合位点(epitope)不同于现有的Daratumumab的、全新的抗CD38的单克隆抗体或其衍生体，其包含第一可变区和第二可变区，其中所述第一可变区是抗体轻链可变区，其抗原互补决定区CDR1，CDR2和CDR3分别为SEQ ID NO：3，SEQ ID NO：4及SEQ ID NO：5所示的氨基酸序列；其中所述第二可变区是抗体重链可变区，其抗原互补决定区CDR1，CDR2和CDR3分别为SEQ ID NO：8，SEQ ID NO：9及SEQ ID NO：10所示的氨基酸序列。
所述抗体包括人-鼠嵌合、半嵌合/半人源、人源化单克隆抗体，所述衍生体包括抗体Fab片段、单链Fab-片段、Fv-片段、单链抗体、双特异抗体(bi-specific)、抗体-药物嫁接物(antibody-drug-conjugated，ADC)、嵌合抗原T细胞受体(chimeric antigen receptor T-Cell,CAR-T)等。
作为本发明优选的技术方案，所述第一可变区是抗体轻链可变区，为SEQ ID NO：2所示的氨基酸序列；所述第二可变区是抗体重链可变区，为SEQ ID NO：7所示的氨基酸序列。
作为本发明优选的技术方案，所述第一可变区是抗体轻链可变区，为SEQ ID NO：11所示的氨基酸序列；所述第二可变区是抗体重链可变区，为SEQ ID NO：12所示的氨基酸序列或为SEQ ID NO：7所示的氨基酸序列。
作为本发明优选的技术方案，其包含所述抗体轻链可变区和人抗体轻链恒定区，及包含所述抗体重链可变区和人抗体重链恒定区的铰链区，CH1区，CH 2区和CH3区。
作为本发明优选的技术方案，所述人抗体轻链恒定区来自人抗体kappa链或抗体lamda链，所述人抗体重链恒定区来自人IgG1，IgG2，IgG3或IgG4等亚型，其中优选的为IgG1。
在本发明第二方面，提供了一种编码上述抗体或其衍生体的DNA分子或基因，其抗体轻链可变区为SEQ ID NO：1或SEQ ID NO：13所示的核苷酸序列，抗体重链可变区为SEQ ID NO：6或SEQ ID NO：14的核苷酸序列。
本发明的第三方面是提供了一种表达载体，它含有编码上述抗体或其衍生体的DNA分子/基因核苷酸序列以及与该序列操作性相连的表达调控序列。
本发明的第四方面提供了一种重组宿主细胞，它由上述表达载体转化而成。该重组宿主细胞或其子代细胞表达上述抗体或其衍生体。该抗体包括人源化单克隆抗体，衍生体包括抗体Fab片段、单链抗体、双特异抗体(bi-specific)等。
本发明的第五方面是提供一种药物或药物组合物，它含有药学上有效量的上述抗体或其衍生体，以及药学上可接受的载体。
本发明的第六方面是提供上述抗体的药物或药物组合物在制备治疗肿瘤的药物中的应用，尤其在治疗CD38表达阳性的肿瘤的药物中的应用。所述CD38表达阳性的肿瘤优选为人骨髓瘤，淋巴细胞瘤(B-淋巴细胞瘤)等。在本发明的具体实施实例中，本发明描述了该抗体在体内抑制高表达CD38的人B-淋巴细胞瘤Raji生长的应用。
作为本发明优选的另一技术方案，本发明中的抗CD38抗体在治疗CD38表达阳性的肿瘤时，可以更多考虑采用野生型或基因改造过的人IgG1、IgM亚型抗体的恒定区，以保持或提高抗体的ADCC或CDC，达到更强的杀伤肿瘤组织与细胞的效果。野生型或基因改造过的人IgG1、IgM亚型抗体的恒定区可以采用本领域人员已知的基因工程技术克隆获得，或体外合成制备。
本发明中的抗CD38抗体或其衍生体，还可作为靶向载体与其他抗肿瘤药物或毒素嫁接或包裹在一起，形成抗体-药物偶联物(antibody-drug-conjugated，ADC)，一同靶向肿瘤组织，达到更好的杀伤肿瘤的效果。抗体与药物或毒素的嫁接或包裹方法可以采用本领域人员已知的通用技术。
作为本发明优选的另一技术方案，本发明中的抗CD38抗体或其衍生体，还可与抗肿瘤血管增生药物或靶向免疫抑制检查点(inhibitory immune checkpoint molecules)的药物序贯或合并使用，用于治疗CD38表达阳性的的肿瘤。
其中与本发明中的抗CD38抗体或其衍生体序贯或合并使用的抗肿瘤血管增生药物可以优选靶向VEGF或VEGF受体(VEGF-R)的大分子生物药物或小分子化学药物。其中优选的靶向VEGF/或VEGF-R的大分子生物药物包括抗-VEGF单抗药物贝伐单抗(Bevacizumab，商品名Avastin)、抗VEGF单抗Fab 片段雷珠单抗(Ranibizumab，商品名Lucentis)；抗-VEGFR2单抗雷莫卢单抗(Ramucirumab，商品名Gyramza)及代号为hPV19的抗-VEGF单抗(思坦维公司目前在研药物，见中国专利文献：授权专利号：201210540692X，专利名称：拮抗抑制血管内皮细胞生长因子与其受体结合的单克隆抗体及其编码序列与用途；及授权专利号为US9580498B2的美国专利文献)；或VEGF受体-Fc融合蛋白药物如阿普西普(Albercept，商品名:Eylea)、康帕西普(conbercept)等。优选的靶向VEGF受体的小分子化学药物包括舒尼替尼(Sunitinib)、索拉非尼(sorafenib)、阿帕替尼(apatinib)、帕唑帕尼(Pazopanib)等。
而与本发明中的抗CD38抗体或其衍生体序贯或合并使用的靶向免疫抑制检查点优选药物包括抗-CTLA4(Cytotoxic T-lymphocyte Antigen-4)Ipilimumab伊匹单抗(商品名Yervoy)；抗PD-1(programmed death protein-1)单抗Nivolumab(商品名Opdivo)、Pembrolizumab(商品名Keytruda)及思坦维公司在研的抗PD-1单抗hAB21(见PCT专利申请文件：PCT/CN2017/089282，拮抗抑制人PD-1抗原与其配体结合的单克隆抗体及其制备方法与应用)；抗PD-L1单抗药物包括Atezolizumab(商品名Tecentriq)、Avelumab(商品名Bavencio)、Durvalumab(商品名Imfinzi)等。
作为本发明优选的另一技术方案，本发明中的抗CD38抗体还可先制备成嵌合抗原T细胞受体(chimeric antigen receptor T-Cell,CAR-T)，体外导入到从肿瘤患者外周血液中分离获得的免疫细胞如T-淋巴细胞中，再经体外培养扩增后，这些具识别CD38抗原的淋巴细胞再回注入体内，在体内发挥靶向高表达CD38抗原的肿瘤，从而达到治疗肿瘤的效果。将本发明中的抗CD38抗体制备成嵌合抗原T细胞受体(CAR-T)可以采用本领域技术人员已知的常规技术
本发明第七方面是提供制备上述抗体或其衍生体的方法，该方法包括：
a)提供一表达载体，该表达载体含有上述DNA序列以及与该序列操作性相连的表达调控序列；
b)用步骤a)所述的表达载体转化宿主细胞；
c)在适合所述抗体表达的条件下培养步骤b)所得的宿主细胞，和
d)从该宿主细胞培养液中分离纯化获得所述抗体。
本文所采用的术语“单克隆抗体(单抗)”指从一纯系细胞得到的免疫球蛋白，具有相同的结构和化学特性，对单一抗原决定簇有特异性。单克隆抗体与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤或重组工程细胞培养获得，不会混杂有其它免疫球蛋白。修饰语“单克隆”表示了抗体的特性，是从均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本文所采用的术语“人源化单克隆抗体”系将鼠源单克隆抗体的氨基酸序列除保留互补决定区(complementarity-determining regions，CDR)外，其它序列(包括可变区中的框架区序列)全部或大部分替换成人免疫球蛋白的氨基酸序列，以达到通过基因工程手段最大限度地降低鼠源性单克隆抗体的免疫原性。
本文所用的术语“抗体”和“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端 有可变区(V H)。其后是多个恒定区。每条轻链的一端有可变区(V L)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
本文所用的术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中成为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(Framework regions，FR)。抗体重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷1，647-669页(1991))。抗体恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性(Antibody-dependent cellular cytotoxicity，ADCC)或补体介导毒性(Complemnt-dependent cytotoxicity，CDC)。
本发明的抗体通常可以通过以下方法来制备：
首先，将含有编码本发明的抗体的基因插入到含有合适的表达调控序列的表达载体中。
本文所用的术语“表达调控序列”通常指参与控制基因表达的序列。表达调控序列包括与目标基因操作性相连的启动子和终止信号。编码本发明抗体的基因(DNA)序列可用本领域技术人员熟知的常规手段，如根据本发明公开的蛋白质序列人工合成或用PCR法扩增得到。其后可用本领域熟知的各种方法将合成或PCR扩增得到的DNA片段插入到合适的表达载体中。本发明中所用的表达载体可以是本领域技术人员已知的市售表达载体，如Invitrogen公司的pCDNA3.1表达载体。
用于接纳表达载体转化的合适宿主细胞一般包括原核细胞和真核细胞。常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。常用的真核宿主细胞包括酵母细胞、昆虫细胞、哺乳动物细胞等。在本发明中，较佳的宿主细胞是哺乳动物细胞，尤其是中华仓鼠卵巢(CHO)细胞。
表达载体转化的宿主细胞在合适的条件下(如以无血清培养基在细胞培养瓶或生物反应器中贴壁或悬浮培养)培养后，收获培养上清液，然后用包括protein-A亲和层析、离子交换层析、过滤除菌等本领域技术人员熟知的常规分离步骤或手段纯化得到本发明的抗体。
纯化得到的本发明抗体可以溶于适当的溶剂如无菌生理盐水液体中，溶度可以制备成0.01至100mg/ml之间，理想的最终溶度可以制备成1至20mg/ml之间。
为获得一种全新的特异结合人CD38抗原的鼠源单克隆抗体及分泌它的杂交瘤细胞系，本发明选取哺乳动物表达的重组人CD38胞膜外蛋白为免疫抗原，通过反复多次小剂量的小鼠皮下免疫，获得分泌抗CD38蛋白的多克隆抗体；再从中挑取含高效价抗体的小鼠，取其脾脏细胞，通过体外与小鼠骨髓瘤细胞融合、再经药物筛选及亚克隆等步骤而建立了多株稳定分泌抗人CD38蛋白的抗体的杂交瘤单克隆细胞。其中一代号为m29的小鼠杂交瘤细胞株，经ELISA、流式细胞仪等多种方法鉴定，证实其所分泌的单克隆抗体不但能够高亲合力特异结合人CD38蛋白及CD38表达阳性的来源与人的多种肿瘤细胞株。此外，m29的小鼠杂交瘤细胞株分泌的抗体在体外可通过CDC杀死CD38抗原表达阳性的肿瘤细胞。
本发明通过基因工程等手段获得了编码该鼠源抗体重链可变区及轻链可变区的基因片断，在此基础 上对该抗体进行了基因工程改造，获得表达其人-鼠嵌合抗体，或人源化抗体的载体。该表达载体经转染进入中华仓鼠卵巢(CHO)细胞，获得稳定高效分泌表达人-鼠嵌合抗体，或人源化抗体的重组工程细胞，并从重组工程细胞培养液中分离纯化获得到具有生物活性的人-鼠嵌合抗体ch29蛋白或人源化抗体HH29蛋白。
体外采用包括竞争ELISA，流式细胞仪等多种方法鉴定分析表明：该鼠源单抗m29及其人-鼠嵌合抗体(ch29)与CD38抗原的结合位点(epitope)不同与Daratumumab。体外直接ELISA及流式细胞仪等分析结果还表明：该鼠源单抗m29及其人-鼠嵌合抗体ch29也可以与猴CD38重组蛋白及表达猴CD38重组基因的CHO细胞株高亲合力特异结合；而Daratumumab单抗仅结合人CD38抗原，与猴CD38抗原无明显结合。
在免疫缺陷小鼠(node mice)体内测试鼠源m29单抗及人-鼠嵌合m29单抗(ch29)的抗肿瘤疗效，结果表明鼠源单抗m29及其人-鼠嵌合抗体ch29体内给药后对肿瘤生长有明显的抑制作用,且疗效不亚于已上市的阳性对照药物Rituximab(商品名Rituxan,人-鼠嵌合抗人CD20单抗)或Daratumumab。
附图说明
图1为本发明实施例1中人CD38与小鼠CD38蛋白氨基酸序列比对分析示意图。
图2A为本发明实施例1中以ELISA法筛选获得的分泌抗CD38抗体的各杂交瘤细胞株的代号及检测OD值的示意图。
图2B本发明实施例2中以CDC方法验证经ELISA筛选获得的分泌抗CD38抗体的杂交瘤细胞株上清液体外杀伤Daudi靶细胞的活性结果示意图，其中使用的靶细胞为Daudi，RFU为Relative Fluorescence Unit(相对荧光单位)的缩写。
图3A为本发明实施例3中以CDC方法进一步检测分析小鼠杂交瘤细胞株m29上清液的CDC活性结果示意图，其中使用的靶细胞为Daudi，阳性对照样品为Daratumumab，阴性对照样品为小鼠SP2/0骨髓瘤细胞细胞株；
图3B为本发明实施例3中阳性对照样品Daratumumab的CDC检测结果示意图；
图3C为本发明实施例3中小鼠杂交瘤细胞株m29上清液样品的CDC检测结果示意图。
图4为本发明实施例4中以流式细胞仪检测分析小鼠杂交瘤细胞株m29上清样品与CD38抗原表达阳性的人肿瘤细胞株结的合代表性结果图，阴性对照样品为非相关鼠源单抗mAB21(小鼠抗人PD-1单抗)；其中：
图4A为人B-淋巴瘤细胞株Daudi的流式细胞仪检测结果图；
图4B为人骨髓瘤细胞株RPMI-8226的流式细胞仪检测结果图；
图4C为人T-淋巴瘤细胞株MOLT-4细胞的流式细胞仪检测结果图。
图5为本发明实施例5中以流式细胞仪检测分析鼠源单抗m29样品及阳性对照样品Daratumumab与转染表达人CD38基因的CHO细胞(CHO-hCD38)结合的代表性结果图；阴性对照样品为非相关鼠源单抗mAB21(小鼠抗人PD-1单抗)或人源化hAB21单抗；其中：
图5A为鼠源单抗m29样品的流式细胞仪检测结果图；
图5B为阳性对照样品Daratumumab的流式细胞仪检测结果图。
图6本发明实施例6中以竞争性ELISA检测纯化的鼠源单抗m29样品与Daratumumab相互之间竞争结合CD38的示意图；非相关人源化单抗样品hPV19(抗VEGF单抗)为阴性对照样品；其中：
图6A为鼠源单抗m29及Daratumumab与Biotin标记的Daratumumab样品竞争结合CD38的结果图；
图6B为鼠源单抗m29及Daratumumab与Biotin标记的m29单抗竞争结合CD38的结果图。
图7为本发明实施例7中的鼠源单抗m29的可变区氨基酸序列与Daratumumab可变区氨基酸序列比对分析图，其中：
图7A为轻链可变区氨基酸序列比对分析图；m29单抗不同于Daratumumbab之处氨基酸均以“X”符号表示，方框标识的氨基酸序列区域为轻链可变区的CDR1，CDR2及CDR3；
图7B则为重链可变区氨基酸序列比对分析图；m29单抗不同于Daratumumab单抗之处氨基酸均均以“X”符号表示，方框标识的氨基酸序列区域为重链可变区的CDR1，CDR2及CDR3。
图8为本发明实施例9中以流式细胞仪检测分析鼠源单抗m29样品、人-鼠嵌合抗体ch29G及阳性对照样品Daratumumab或Rituximab(Rituxan，人-鼠嵌合抗人CD20单抗)与来源于人的肿瘤细胞株结合的代表性结果图；其中：
图8A为人B-淋巴瘤细胞株Raji的流式细胞仪检测结果图；
图8B为人骨髓瘤肿瘤细胞株RPMI-8226的流式细胞仪检测结果图；
图8C为人T-淋巴瘤细胞株MOLT-4的流式细胞仪检测结果图；
图8D为人T-淋巴瘤细胞株Jurkat的流式细胞仪检测结果图。
图9为本发明实施例10中体外CDC检测分析鼠源单抗m29样品、人-鼠嵌合抗体ch29G样品及对照样品Daratumumab活性的结果图，其中使用的靶细胞为人B-淋巴瘤细胞株Daudi，补体来源为兔血清，阴性对照样品为非相关人源化hPV19单抗(抗VEGF单抗)。
图10为本发明实施例11中以流式细胞仪检测鼠源单抗m29样品、人-鼠嵌合抗体ch29G样品及对照样品Daratumumab分别与稳定转染表达野生型人CD38的CHO细胞(CHO-human CD38/wild type)，或稳定转染表达表达S 274F点突变CD38的CHO细胞(CHO-human CD38/S 274F mutation)的结合结果图，阴性对照样品为非相关人源化hAB21单抗(抗人PD-1单抗)。其中，图10A为表达野生型(wild-type)人CD38的CHO细胞的检测结果图；图10B则为表达点突变人CD38的CHO细胞(CHO/hCD38-S 274F)的检测结果图。
图11A为本发明实施例12中人CD38、猩猩CD38及食蟹猴CD38蛋白氨基酸序列比对分析结果图。
图11B为本发明实施例12中以流式细胞仪检测单抗m29样品、人-鼠嵌合抗体ch29G样品及对照样品Daratumumab与稳定转染表达食蟹猴CD38基因的CHO细胞(CHO-cynomolgus CD38)结合结果图，其中阴性对照样品为非相关人源化hAB21单抗(抗人PD-1单抗)。
图12为本发明实施例13中以ELISA检测比较人-鼠嵌合抗体ch29G样品及Daratumumab单抗分别与重组人CD38蛋白(图12A)及重组食蟹猴CD38蛋白(图12B)的结合结果图，其中阴性对照样品为Rituximab(Rituxan,人-鼠嵌合抗人CD20单抗)。
图13为本发明实施例14中CDC检测分析人-鼠嵌合抗体ch29G样品、Daratumumab及阳性对照样品Rituximab(人-鼠嵌合抗人CD20单抗)活性的结果图，补体来源为人血清，阴性对照样品为非相关人源 化hPV19单抗(抗VEGF单抗)，其中：
图13A为以人B-淋巴瘤细胞株Daudi为靶细胞的CDC检测结果图；
图13B为以人B-淋巴瘤细胞株Raji为靶细胞的CDC检测结果图；
图13C为以人T-淋巴瘤细胞株MOLT-4为靶细胞的CDC检测结果图；
图13D为以人T-淋巴瘤细胞株Jurkat为靶细胞的CDC检测结果图。
图14为本发明实施例17中在裸鼠皮下种植人B-淋巴瘤Raji肿瘤模型中测试鼠源m29单抗的体内抗肿瘤疗效结果图。其中：图14A为种植肿瘤前10天的平均增长体积示意图；图14B为种植肿瘤后期的平均增长体积示意图。
图15为本发明实施例18中在裸鼠皮下种植人B-淋巴瘤Raji肿瘤模型中测试人-鼠嵌合抗体ch29样品的体内抗肿瘤疗效结果图。
具体实施方式
下面将结合实施实例来进一步描述本发明，这些实施例只是为了起说明作用，而不是用来限制本发明。
本发明中使用的肿瘤细胞株及DNA引物(primer)分别见表1及表2：
表1：本发明中使用的肿瘤细胞株名称及其特征
Figure PCTCN2018107812-appb-000001
Figure PCTCN2018107812-appb-000002
表2：本发明中使用的引物名称(primer)及其序列
Figure PCTCN2018107812-appb-000003
实施例1:分泌抗CD38抗体的小鼠杂交瘤细胞系的建立与筛选鉴定
1.1人CD38蛋白的氨基酸序列与小鼠CD38蛋白的氨基酸序列比对分析
人CD38蛋白的氨基酸序列与小鼠CD38蛋白的氨基酸序列比对分析如图1(其中的以方框斜体标注的氨基酸序列为跨细胞模区域)。如图1所示：人CD38与小鼠CD38蛋白氨基酸序列整体相同性仅有59％，因此推测，如采用传统的抗原蛋白免疫小鼠及杂交瘤制备技术，应可以制备出针对各种不同结合区域或氨基酸结合位点的鼠抗人CD38单克隆抗体。这些抗人CD38单克隆抗体，或因其抗原结合区域/结合位点(epitope)不同于已有CD38单抗如Daratumumab，其体内外生物活性及疗效有望不同于或甚至更优于这些单抗。这类识别新位点的、全新的CD38单抗作为药物成分，一方面可与目前已上市的骨髓瘤治疗药物如Bortezomib硼替佐米、lenalidomide来那度胺等联合或序贯使用，达到增强骨髓瘤治疗疗效的作用；另一方面，这些单抗药物也有望开发成为用于治疗CD38表达阳性的其他肿瘤如B-淋巴细胞瘤，T-淋巴细胞瘤等。
为此，本发明开展了这类全新的CD38单抗研发与制备，其具体制备步骤如下：
1.2分泌抗CD38抗体的小鼠杂交瘤细胞系的建立与筛选鉴定
步骤1.重组人CD38蛋白(免疫抗原)的来源与动物免疫
在本发明实施例中，用于免疫的抗原为由哺乳动物表达的重组人CD38胞膜外蛋白(Human CD38 Protein-His Tag,北京义翘神州公司产品,Catalog：10818-H08H)。该重组人CD38蛋白与弗氏完全佐剂(美国Sigma公司产品)混合后，于皮下多点注射Balb/c小鼠(100μl/只，每次10μg CD38蛋白)。首次免疫3周后用不完全佐剂混合CD38蛋白，按10μg/只的剂量皮下多点免疫；此后每隔2周用同样方法加强免疫2-3次,第3次加强免疫一周后取小鼠尾静脉血收集血清，用包被重组人CD38蛋白的96-板以酶联免疫吸附试验(ELISA)方法检测小鼠血清中抗CD38抗体的滴度。
该ELISA检测步骤如下：以重组人CD38蛋白(2μg/ml，pH 9.6，0.1M NaHCO3液)包被96-孔酶标板，37℃包被2小时后，再加入2％牛血清白蛋白(BSA)4℃封闭过夜。次日，包被板经PBS-0.1％Tween20液洗涤后，加入倍比稀释的免疫小鼠血清样品(以未免疫的小鼠血清为阴性对照)37℃孵育2小时；经PBS-0.1％Tween20液洗涤后，加入辣根过氧化物酶HRP-标记的羊抗小鼠IgG(美国Sigma公司产品)，37℃孵育1小时；再经PBS-0.1％Tween20液充分洗涤后，加入邻苯二胺(OPD)-0.1％H 2O 2底物液显色10-15min后再加入0.1M HCl终止反应。其后在多功能酶标仪(PerkinElmer Victor X3)中读取492nm处OD值。取抗体滴度相对较高的小鼠的脾细胞用于下一步的细胞融合。
步骤2、细胞融合
在末次免疫(第4次加强免疫)后3-4天，无菌制备小鼠脾细胞悬液，与小鼠SP2/0骨髓瘤细胞(购自中国科学院上海生命科学院细胞保藏中心)，以5：1或10：1的比例在50％PEG-1000(美国Sigma公司产品)作用下融合。融合按常规法(Kohler G.and Milstein C:Nature 1975，256:495-497),PEG用量1ml，60秒内缓慢加完。反应90秒后，以无血清的RPMI-1640培养基终止反应，1000rpm离心10min,去除上清液，再将离心沉淀下的细胞以含10％HAT(H为次黄嘌呤、A氨基碟呤、T胸腺嘧啶核苷，为美国Sigma公司产品)的RPMI 1640-10％FCS培养基将细胞浓度调节至1x10 6/ml，加入96孔平底细胞培养板(每孔200μl)，于37℃，5％CO2培养箱中(美国Thermo公司产品)培养2-3周。
步骤3、酶联免疫吸附试验(ELISA)筛选抗CD38抗体分泌阳性的小鼠杂交瘤细胞
采用上述筛选检测小鼠血清中抗CD38抗体的滴度的相同的酶联免疫吸附试验(ELISA)方法筛选小鼠杂交瘤细胞培养上清液中是否含有抗CD38抗体。
该ELISA检测步骤如下：以重组人CD38蛋白(2μg/ml，pH 9.6，0.1M NaHCO3液)包被96-孔酶标板，37℃包被2小时后，再加入2％牛血清白蛋白(BSA)4℃封闭过夜。次日，包被板经PBS-0.1％Tween20液洗涤后，加入待检杂交瘤细胞培养上清液样品(以未融合的SP2/0骨髓瘤细培养上清为阴性对照，CD38抗原免疫后小鼠血清样品(以1：200稀释)为阳性对照)37℃孵育2小时；经PBS-0.1％Tween20液洗涤后，加入辣根过氧化物酶HRP-标记的羊抗小鼠IgG(美国Sigma公司产品)，37℃孵育1小时；再经PBS-0.1％Tween20液充分洗涤后，加入邻苯二胺(OPD)-0.1％H 2O 2底物液显色10-15min后再加入0.1M HCl终止反应。其后在多功能酶标仪(PerkinElmer Victor X3)中读取492nm处OD值。
一共筛选了600多个小鼠杂交瘤细胞克隆，结果获得10多个分泌抗CD38抗体的阳性杂交瘤克隆(阳性克隆的判断标准：OD值比阴性对照样品的OD值高5倍以上)。
图2A为经该ELISA筛选获得的分泌结合CD38抗原的阳性的各杂交瘤细胞株的代号及检测OD值。这些阳性杂交瘤细胞再经扩增培养后于-70℃下冻存。
实施例2：补体依赖细胞毒性(complement-dependent cytotoxicity，CDC)方法体外筛选与检测分析分泌抗CD38抗体阳性的杂交瘤细胞株上清液
在本实施例中，用于体外筛选与检测分析抗CD38抗体阳性的杂交瘤细胞株上清液的补体依赖细胞毒性(complement-dependent cytotoxicity，CDC)的方法概述如下：
2.1实验材料
细胞培养基：RPMI-1640Hyclone
胎牛血清：美国GIBCO公司产品
靶细胞：人B-淋巴瘤细胞株Daudi(人CD38 +，CD20 +B-lymphoma cell line或其他肿瘤细胞株，购自中国科学院典型培养物保藏委员会细胞库)
补体来源：人血清/幼兔血清/兔补体(健康)(自制)
供试品：抗CD38单克隆抗体阳性杂交瘤细胞株上清液(自制)
阳性对照样品：Daratumumab(抗CD38人源化单抗)，或Rituximab(抗CD20人-鼠嵌合单抗)
阴性对照样品：hPV19单抗(抗VEGF人源化单抗)
细胞活力检测盒：
Figure PCTCN2018107812-appb-000004
Luminescent Cell Viability Assay Kit(Promega产品)
96-孔细胞培养板：Corning-3610
2.2实验步骤
1)取10μl稀释后的测试样品(抗体样品起始最高浓度为20-200μg/ml，5-倍或3-倍梯度稀释；杂交瘤上清则直接取10μl上样)加入96孔板相应孔中；
2)收集对数生长期靶细胞(Daudi细胞或其他CD38抗原表达阳性靶细胞)，用CDC稀释液(1％FBS RPMI1640培养基)洗涤一遍，计数，细胞活率应大于90％，将重悬的细胞浓度调整至2.5×10 5个/ml；
3)加入靶细胞至96孔板中(Corning-3610)，80μl/孔，每孔约2×10 ^4个细胞，放置37℃-5％CO2培养箱孵育30分钟；
4)加入稀释后的幼兔血清或人血清(均先用CDC稀释液1:10或1:20稀释)，10μl/孔，放置37度-5％CO2培养箱孵育1-2小时；
5)在加入细胞活力检测试剂前，将细胞培养板和细胞活力检测(
Figure PCTCN2018107812-appb-000005
Luminescent Cell Viability kit)和96孔板避光放置30分钟平衡至室温，再每孔加入100uL配置好的试剂，室温避光显色10-15分钟后，应用多功能酶标仪(PerkinElmer Victor X3)检测荧光强度RFU(Relative Fluorescence Unit，或 Relative Luminescent Unit，RLU相对荧光单位)。
2.3CDC活性结果计算
CDC活性可以以荧光强度RFU直接数值标识或按下列公式计算CDC细胞杀伤率：
细胞杀伤率(％)＝100×(1-(RFU样品孔)/((RFU细胞+血清孔))；
细胞活率则以下列公式进行计算：
细胞活率(％)＝100×((RFU样品孔)/(RFU细胞+血清孔))
2.4CDC活性检测结果
图2B为以该CDC法检测分析分泌抗CD38抗体的杂交瘤细胞株上清液体外杀伤Daudi靶细胞的活性结果(RFU直接数值)；其中一代号为m29的杂交瘤细胞上清液表现有明显的CDC活性(与其他样品相比，RFU数值下降达95％以上)，而其他杂交瘤细胞培养上清液样品均无明显的CDC活性(RFU数值无明显下降)。
实施例3：鼠源m29单抗的亚型鉴定及其补体依赖细胞毒性(CDC)的再次验证
鼠源m29单抗的经采用市售的小鼠单克隆抗体IgG亚类检测卡(北京博奥龙免疫技术有限公司产品，货号BF06038)进行鉴定，结果显示为IgG2b型抗体。其后，使用与实施例2中相同的方法再次验证了m29杂交瘤细胞株上清液样品的体外经CDC杀伤Daudi靶细胞的活性，结果如图3。
其中图3A为m29杂交瘤细胞株上清液样品未经稀释后直接加入CDC测试板中的RFU读数结果；结果再次显示m29杂交瘤细胞株上清液表现有明显的CDC活性(与阴性对照样品相比，RFU数值下降达95％以上)。
图3B为不同溶度的阳性对照样品Daratumumab的体外CDC杀伤Daudi靶细胞的活性测试结果，结果显示有明显的剂量-反应曲线。
图3C为含不同溶度的m29单抗上清的体外CDC杀伤Daudi靶细胞的活性CDC活性测试结果，结果也显示有明显的剂量-反应曲线。
实施例4:流式细胞仪检测分析鼠源单抗m29与来源与人的CD38抗原表达肿瘤细胞株的结合
在本实施例中，以m29杂交瘤细胞株上清液样品，或非相关鼠源单抗mAB21(小鼠抗人PD-1单抗)样品为一抗，以FITC荧光标记的羊抗小鼠IgG为二抗，采用流式细胞仪检测分析样品与CD38抗原表达阳性的人肿瘤细胞株的结合。
为此,将已知CD38抗原表达阳性的人肿瘤细胞株(B-淋巴瘤细胞株Daudi、骨髓瘤细胞株RPMI-8226及T-淋巴瘤细胞株MOLT-4，均购自购自中国科学院上海生命科学院细胞保藏中心)分别与m29杂交瘤细胞株上清液样品，或非相关单抗鼠源mAB21(小鼠抗人PD-1单抗)样品4℃孵育1小时，经PBS-0.1％FCS液洗涤后，加入FITC-标记的羊抗小鼠IgG(1：200稀释，Sigma公司产品)；4℃孵育1小时，再经PBS-0.1％FCS液洗涤后，将样品上样至Accuri C6Plus Flow Cytometer流式细胞仪检测(美国Becton  Dickinson公司,Mountain view,CA)。
图4为该流式细胞仪检测的代表性结果。如图4所示：与非相关单抗mAB21(鼠抗人PD-1单抗)样品相比，m29杂交瘤细胞株上清液样品可明显与CD38抗原表达阳性的人B-淋巴瘤细胞株Daudi(图4A)、骨髓瘤细胞株RPMI-8226(图4B)及T-淋巴瘤细胞株MOLT-4(图4C)特异结合。
实施例5：流式细胞仪检测分析m29杂交瘤细胞株上清液样品及Daratumumab与稳定转染表达人CD38基因的CHO细胞(CHO-hCD38)结合
5.1稳定表达人CD38基因的CHO细胞株(CHO-hCD38)的建立
根据Genebank数据库公布的人CD38基因序列(Gene ID:952)，委托苏州金唯智生物科技有限公司合成编码CD38的cDNA片段，并将其克隆到细胞表达载体pQY-DHFR(自建)中，转化大肠杆菌，通过酶切验证方法鉴定阳性重组表达质粒pQY-DHFR-hCD38。其后将重组质粒pQY-DHFR-hCD38与Fugen-6脂质体(Roche公司产品)混合后共转染入CHO-dhfr-细胞后，利用含胎牛血清的IMDM培养基筛选出稳定表达人CD38基因的CHO细胞株(CHO-hCD38)
5.2流式细胞仪检测分析鼠源m29杂交瘤上清液样品及Daratumumab与稳定表达人CD38基因的CHO细胞(CHO-hCD38)结合
在本实施例中，以鼠源m29杂交瘤上清液样品或阳性样品Daratumumab为一抗，分别以FITC荧光标记的羊抗小鼠IgG或FITC荧光标记的羊抗人IgG为二抗，采用流式细胞仪检测分析样品与稳定表达人CD38基因的CHO细胞(CHO-hCD38)结合。
为此,将CHO-hCD38细胞分别与鼠源m29杂交瘤上清液样品，阳性单抗样品Daratumumab，或非相关鼠源单抗mAB21(小鼠抗人PD-1单抗)或其人源化hAB21单抗样品4℃孵育1小时，经PBS-0.1％FCS液洗涤后，分别加入FITC-标记的羊抗小鼠IgG(1：200稀释，Sigma公司产品)或对于Daratumumab样品，则加入FITC-标记的羊抗人IgG-Fc，1：200稀释，Sigma公司产品)；4℃孵育1小时，再经PBS-0.1％FCS液洗涤后，将样品上样至Accuri C6Plus Flow Cytometer流式细胞仪检测(美国Becton Dickinson公司,Mountain view,CA)。
图5为该流式细胞仪检测CHO-hCD38代表性结果。如图5所示：与阴性对照样品mAB21或hAB21相比，鼠源m29杂交瘤上清液样品(图5A)及Daratumumab样品(图5B)均可明显与CHO-hCD38细胞结合，其中m29单抗样品与CHO-hCD38细胞的结合强度不亚于Daratumumab单抗。
实施例6鼠源m29单抗的分离纯化及体外竞争ELISA法检测m29单抗与Daratumumab竞争结合CD38
6.1鼠源m29单抗的分离纯化
将m29杂交瘤细胞扩增，并驯化为无血清培养基(KD-Hybri,珠海恺瑞公司产品)培养。收集一定量的无血清细胞培养上清，经离心及0.45μm滤膜过滤后，上样至Protein-G亲合层析柱(Protein G-Sepharose Fast Flow，美国通用电气GE公司产品)，经1xPBS漂洗后，用缓冲液(0.05M醋酸钠，pH＝3.2)洗脱并获得m29抗体，然后使用超滤管(Millipore UFC903096，30kD)离心更换缓冲液为甘氨酸-Tris(pH＝6.0)后，经测试定量标记后，将纯化获得的m29抗体置于4℃保存。
6.2体外竞争性ELISA法检测分析鼠源m29单抗与Daratumumab竞争结合CD38
该竞争性ELISA法的基本原理与过程是：先将不同浓度的m29单抗样品或Daratumumab与固定浓度的生物素标记的m29单抗(biotin-m29)或生物素标记的Daratumumab(biotin-Daratumumab)混合，之后再将混合物转入预先包被有CD38-His重组蛋白的96-孔板，经孵育及洗脱后，加入酶标记的Avidin(如辣根过氧化物酶标记的Avidin)；再经孵育及洗脱后，加入底物显示并测定OD值。
其中，在实施例中，该竞争性ELISA法检测的具体步骤如下：
1)用重组人CD38胞膜外蛋白(北京义翘神州公司产品)包被96-孔板(包被浓度：2μg/ml,50μl/孔)，4℃过夜；
2)经PBS液漂洗及5％牛奶(稀释在PBS-0.1％tween20液中)室温封闭后,分别加入不同浓度的抗CD38单抗(m29,Daratumumab)或非相关抗体(抗VEGF单抗hPV19)与含固定浓度的生物素标记的生物素标记Daratumumab(biotin-Daratumumab，1:1000稀释)或m29单抗(biotin-m29，1:1000稀释)，37℃孵育1.5h；
3)经PBS-T洗脱后，加入辣根过氧化物酶标记的Avidin(1：5000稀释)，37℃孵育1h；
4)经PBS-T洗脱后，加入显色液(邻苯二胺)-3％双氧水，室温10min至显色；
5)加入1MHCL终止反应，以酶联免疫仪测定492nm波长处各孔的吸光值。
图6为该竞争性ELISA法的代表性检测结果示意图；其中：
图6A为m29单抗及Daratumumab与生物素标记Daratumumab(biotin-Daratumumab)的体外竞争结合CD38蛋白的结果；如图所示：加入不同浓度的m29单抗或Daratumumab与固定浓度的biotin-Daratumumab样品中，各孔显色反应OD值与加入的标记的单抗样品量成反比关系：即加入的m29单抗或Daratumumab单抗量越高，其显色OD值越低；此结果表明，与Daratumumab一样，m29单抗体外可与biotin-Daratumumab样品拮抗竞争结合CD38；且m29单抗几乎也达到与Daratumumab一样的竞争结合CD38的效果
图6B则为m29单抗及Daratumumab与生物素标记m29单抗(biotin-m29单抗)的体外竞争结合CD38蛋白的结果；如图所示，加入不同溶度的m29单抗与固定浓度的biotin-m29样品中，各孔显色反应OD值与加入的未标记的m29单抗样品量成反比关系：即加入的m29单抗量越高，其显色OD值越低；而Daratumumab或非相关单抗样品hPV19的加入量多少对各孔OD值影响不大。此结果表明Daratumumab体外不与m29单抗竞争结合CD38。
综合分析该竞争性ELISA结果可知m29单抗结合人CD38蛋白的位点(epitope)不同与Daratumumab，m29单抗可竞争阻断Daratumumab与人CD38蛋白的结合，而Daratumuma无法竞争阻断m29单抗与人CD38蛋白的结合
实施例7：鼠源m29单抗可变区编码基因的克隆
在此，先从小鼠杂交瘤m29细胞中提取出总RNA中，再以该RNA为模板，采用简并引物(degenerate  primers)，以Reverse transcription-polymerase chain reaction(RT-PCR)法(Wang Y等：Degenerated primer design to amplify the heavy chain variable region from immunoglobulin cDNA.BMC Bioinformatics.2006；7Suppl(4):S9)分别克隆扩增获得m29抗体重链可变区及轻链可变区的cDNA基因片段。
其中该cDNA基因克隆步骤如下：
步骤1、采用试剂盒(江苏海门碧云天公司产品)从小鼠m29杂交瘤细胞中提取出总RNA；
步骤2、采用逆转录PCR(RT-PCR)方法在eppendorf管获得cDNA模板。
其中用于m29抗体轻链可变区逆转录PCR引物mKaRT序列为：TGT CGT TCA CTG CCA TCA AT；
用于m29抗体重链可变区逆转录PCR引物mGaRT序列为：GCA AGG CTT ACA ACC ACA ATC；
RT-PCR反应体系如下：
引物                                     2μl
RNA模板                                  30μl
72℃孵育10分钟，然后冰上放置2分钟；
随后加入：
Figure PCTCN2018107812-appb-000006
于42℃温度下反应1小时，随后温度升至75℃，15分钟灭活后将获得的cDNA置于-20℃，保存备用。
步骤3、编码m29抗体轻链可变区及重链可变区基因的PCR克隆扩增
用于简并引物(degenerate primers)PCR法克隆扩增该m29抗体轻链可变区基因的一对引物为：
正向引物mIgLF1：GACATTGTGATGWCM CA；
反向引物mIgLCR440：CTGAGGCACCTCCAGATGTT.
其中W＝A或T，M＝A或C。
而用于简并引物(degenerate primers)PCR法克隆扩增m29抗体重链可变区基因的一对引物为：
正向引物mIgHset1：CARCTGCARCARYCTG；
其中R＝A或G，Y＝C或T。
反向引物mIgHCR135：GTGCTGGAGGGGACAGTCACT.
PCR扩增得到的DNA产物在1.5％agarose胶中电泳分析。电泳结束后，将分离的DNA条带切下并分别进行测序获得抗体轻链及重链可变区DNA的核苷酸序列。测得的该抗体轻链可变区DNA的核苷 酸序列见SEQ ID NO.:1，由该DNA核苷酸序列推测得到的抗体轻链可变区氨基酸序列见SEQ ID NO.:2。该轻链抗原互补决定区(complementarity-determining regions，CDR)的CDR1、CDR2及CDR3的氨基酸序列分别见SEQ ID NO.:3、SEQ ID NO.:4和SEQ ID NO.:5。
测得的该抗体重链可变区DNA的核苷酸序列见SEQ ID NO.:6，由该DNA的核苷酸序列推测得到的抗体重链可变区氨基酸序列见SEQ ID NO.:7。该重链抗原互补决定区的CDR1、CDR2及CDR3的氨基酸序列分别见SEQ ID NO.:8、SEQ ID NO.:9和SEQ ID NO.:10。
图7A为编码m29抗体的轻链可变区与Daratumumab轻链可变区的氨基酸序列比对分析结果:其中m29单抗轻链可变区氨基酸序列中不同于Daratumumab之处均以“X”符号表示，方框标识的氨基酸序列为各单抗轻链可变区的CDR1，CDR2及CDR3；对比分析结果显示m29单抗轻链可变区及其CDR序列显著不同于Daratumumab。
图7B则为编码m29抗体的重链可变区与Daratumumab重链可变区的氨基酸序列比对分析结果:其中m29单抗重链可变区氨基酸序列中不同于Daratumumab之处均以“X”符号表示，方框标识的氨基酸序列为各单抗重链可变区的CDR1，CDR2及CDR3；对比分析结果显示m29单抗轻链可变区及其CDR序列显著不同于Daratumumab。
实施例8：鼠源m29抗体的人-鼠嵌合抗体(ch29G)的构建
将上述实施例7中克隆扩增获得的m29抗体轻链可变区基因和重链可变区基因分别与人-kappa轻链恒定区(C-domain)和人IgG1-重链恒定区基因片段融合，获得人-鼠嵌合轻链基因(ch29L)及人-鼠嵌合重链基因(ch29H)。其后将轻链嵌合基因与重链嵌合基因先后克隆至pQY-DHFR-Hex表达质粒中，转入大肠杆菌扩增，分离获得大量含人-鼠嵌合抗体基因的表达质粒。
含人-鼠嵌合抗体基因的表达质粒再与X-tremeGENE HP DNA Transfection Reagent脂质体(Roche产品)混合后共转染入CHO-dhfr -细胞。细胞转染后2-3天，取培养上清液，用包被人CD38-his蛋白的96-孔板，用HRP酶标记的羊抗人IgG(Goat-anti-human-IgG，购自上海西塘生物公司)为检测二抗，以ELISA法检测上清中的嵌合抗体(ch29G)与人CD38-his蛋白结合。
下表3为该ELISA代表性检测结果：
表3 ELISA法检测瞬时转染嵌合抗体ch29G基因的CHO细胞培养上清液与人CD38-his蛋白的结合
Figure PCTCN2018107812-appb-000007
表3结果表明，转染人-鼠嵌合抗体基因ch29表达质粒转染的CHO细胞培养上清可与人CD38的蛋白特异结合。
之后，将上述转染细胞传代至培养皿，并加入条件培养基进行稳定表达株的筛选。经过2-3周的培养，将生长良好的细胞克隆挑选至96孔板培养，2-3天后取上清用ELISA方法检测上清蛋白表达量，最后筛选出表达较高ch29抗体蛋白的CHO细胞株。将此细胞株用无血清培养基(CHOM-B01,上海百安医疗投资有限公司)进行驯化，驯化成功后，扩增培养并收集上清。上清经离心及0.45μm滤膜过滤后，上样至Protein-A亲合层析柱(proteinA-Sepharose Fast Flow，美国通用电气GE公司产品)，经分离纯化，及置换成(甘氨酸-Tris,pH＝7.0)，获得纯度达99％以上的人-鼠嵌合抗体(ch29G，或简称ch29)抗体。
实施例9：流式细胞仪检测比较鼠源单抗m29、人-鼠嵌合抗体ch29G及Daratumumab与表达CD38抗原的人肿瘤细胞的结合
在本实施例中，以纯化的鼠源m29单抗、人-鼠嵌合抗体ch29G及Daratumumab或非相关hAB21单抗(人源化抗人PD-1单抗)样品为一抗，以FITC荧光标记的羊抗小鼠IgG或羊抗人IgG为二抗，采用如实施例4中的流式细胞仪法检测分析样品与CD38抗原表达阳性的肿瘤细胞株的结合。
为此,将已知表达CD38抗原的人肿瘤细胞株(人Burkitt B-淋巴瘤细胞株Raji、人骨髓瘤细胞株RPMI-8226、人T-淋巴瘤细胞株MOLT-4及人T-淋巴瘤细胞株Jurkat细胞，均购自中国科学院上海生命科学院细胞保藏中心)分别与鼠源m29单抗、人-鼠嵌合抗体ch29G及Daratumumab或非相关hAB21单抗样品4℃孵育1小时，经PBS-0.1％FCS液洗涤后，分别加入FITC-标记的羊抗小鼠IgG(1：200稀释，Sigma公司产品)或FITC-标记的羊抗人IgG(1：200稀释，Sigma公司产品)4℃孵育1小时，再经PBS-0.1％FCS液洗涤后，将样品上样至Accuri C6Plus Flow Cytometer流式细胞仪检测(美国Becton Dickinson公司,Mountain view,CA)。
图8为该流式细胞仪检测的代表性结果；其中：
图8A为人Burkitt B-淋巴瘤细胞株Raji的检测结果：与非相关单抗hAB21相比，小鼠m29单抗、人-鼠嵌合抗体ch29G及Daratumumab均可明显与人Burkitt B-淋巴瘤细胞株结合；其中鼠源m29单抗及其人-鼠嵌合抗体ch29G与Raji细胞结合的阳性比例与信号强度与Daratumumab样品几乎相同。
图8B为人骨髓瘤细胞株RPMI-8226的检测结果：与非相关单抗hAB21相比，小鼠m29单抗、人-鼠嵌合抗体ch29G及Daratumumab均可明显与人Burkitt B-淋巴瘤细胞株结合；其中鼠源m29单抗及其人-鼠嵌合抗体ch29G与RPMI-8226细胞结合的阳性比例与信号强度与Daratumumab样品也几乎相同。
图8C为人T-淋巴瘤细胞株MOLT-4的检测结果：其中鼠源m29单抗及其人-鼠嵌合抗体ch29G与MOLT-4细胞结合的阳性比例与信号强度稍低于Daratumumab。
图8D为人T-淋巴瘤细胞株Jurkat的检测结果：其中鼠源m29单抗及其人-鼠嵌合抗体ch29G与Jurkat细胞结合的阳性比例与信号强度低于Daratumumab。
实施例10：体外分析对比鼠源单抗m29、人-鼠嵌合单抗ch29G及Daratumumab的CDC活性
采用与实施例2中相同的CDC检测方法体外分析对比了鼠源m29单抗、人-鼠嵌合单抗ch29G及Daratumumab的CDC活性。其中在本实施例中，使用的靶细胞为经典的Daudi细胞，使用的补体为10％的兔血清(自制)。
图9为该CDC的测试结果；结果显示鼠源m29单抗及其人-鼠嵌合单抗ch29G均具有比Daratumumab更强的CDC活性。鼠源m29单抗及其人-鼠嵌合单抗ch29G的CDC活性最高值达95％以上，EC50值在30ng/ml左右；而Daratumumab的CDC活性最高值仅为55％左右，EC50值为100ng/ml。
实施例11：流式细胞仪方法检测鼠源单抗m29、人-鼠嵌合抗体ch29G及Daratumumab与点突变CD38的结合
11.1稳定表达人CD38基因点突变的CHO细胞(CHO/hCD38-S 274F)株的建立
为将人CD38中C-端第274位丝氨酸(serine，S)突变为苯丙氨酸(F)，以实施例5.1中合成的人CD38cDNA为模板，设计引物，进行PCR扩增：
上游引物huCD38F-HindIII-2：
Figure PCTCN2018107812-appb-000008
下游引物huCD38-S274F-R1：
Figure PCTCN2018107812-appb-000009
得到的DNA产物在1.5％agarose胶中电泳分离目的DNA条带，并以回收的DNA片段为模板，利用如下引物进行第二轮PCR，得到完整的hCD38-S274F基因：
上游引物huCD38F-HindIII-2：
Figure PCTCN2018107812-appb-000010
下游引物huCD38-S274F-R2-XhoI：
Figure PCTCN2018107812-appb-000011
将hCD38-S 274F基因片段克隆到细胞表达载体pQY-DHFR(自建)中，转化大肠杆菌，通过酶切验证方法鉴定阳性重组表达质粒pQY-DHFR-hCD38-S 274F。
其后将重组质粒pQY-DHFR-hCD38S274F与Fugen-6脂质体(Roche)混合后共转染入CHO-dhfr-细胞后，利用含胎牛血清的IMDM培养基筛选出稳定表达人CD38基因点突变的CHO细胞细胞株(CHO/hCD38-S 274F)。
11.2流式细胞仪方法检测鼠源单抗m29、人-鼠嵌合单抗ch29G及Daratumumab与表达野生型(wild-type)或点突变人CD38的CHO细胞(CHO/hCD38-S 274F)结合
在本实施例中，采用流式细胞仪检测分析了鼠源单抗m29、人-鼠嵌合单抗ch29G单抗及Daratumumab与表达野生型(wild-type)及点突变人CD38的CHO细胞结合。
其中检测主要步骤如下：为此,分别将稳定表达野生型(wild-type)或点突变人CD38的CHO细胞与含鼠源单抗m29、人-鼠嵌合单抗ch29G或Daratumumab的样品4℃孵育1小时，经PBS-0.1％FCS液洗涤后，加入FITC荧光标记的羊抗小鼠IgG或FITC荧光标记的羊抗人IgG 4℃孵育1小时，再经PBS-0.1％FCS液洗涤后，将样品上样至流式细胞仪(Accuri C6Plus Flow Cytometer，美国BD公司)检测。
图10为该流式细胞仪检测的代表性结果示意图，其中：
图10A为表达野生型(wild-type)人CD38的CHO细胞的检测结果，结果显示与鼠源单抗m29一样，人-鼠嵌合单抗ch29保持有与表达野生型CD38的CHO细胞(CHO/hCD38-wild-typ)的结合，其两者的结合强度与Daratumumab的结合强度相近。
图10B则为表达点突变人CD38的CHO细胞(CHO/hCD38-S 274F)的检测结果，结果显示与鼠源单抗m29一样，人-鼠嵌合单抗ch29G也保持有表达该点突变CD38的CHO细胞(CHO/hCD38-wild-typ)的结合，而Daratumumab与表达该点突变CD38的CHO细胞无明显结合。
实施例12：稳定表达食蟹猴CD38的CHO细胞株(CHO/mkCD38)的建立及流式细胞仪法检测鼠源单抗m29，人-鼠嵌合抗体(ch29G)及Daratumumab与其结合
12.1稳定表达食蟹猴CD38(CHO/mkCD38)株的建立
人CD38蛋白的氨基酸序列与食蟹猴(Macaca fascicularis，Cynomolgus monkey)CD38及黑猩猩(Chimpanzee，Pan troglodytes)CD38蛋白的氨基酸序列比对分析见图11A；如图11A所示：黑猩猩CD38(ChiCD38)蛋白的氨基酸序列与人CD38蛋白(huCD38)的氨基酸序列几乎相同，而食蟹猴CD38(mkCD38)蛋白的氨基酸序列与人CD38蛋白氨基酸序列整体同源性为91％，两者氨基酸序列不同之处则有16个之多。
根据Genebank数据库公布的食蟹猴CD38基因序列(Gene ID:102126394)，委托苏州金唯智生物科技有限公司合成编码食蟹猴(cynomolgus)CD38的cDNA片段，并将其克隆到细胞表达载体pQY-DHFR(自建)中，转化大肠杆菌，通过酶切验证方法鉴定阳性重组表达质粒pQY-DHFR-mkCD38。
其后将重组质粒pQY-DHFR-mkCD38与Fugen-6脂质体(Roche)混合后共转染入CHO-dhfr -细胞后，利用含胎牛血清的IMDM培养基筛选出稳定表达食蟹猴CD38蛋白的CHO细胞株(CHO-cynomolgus CD38)。
12.2流式细胞仪方法检测鼠源单抗m29、人-鼠嵌合抗体(ch29G)及Daratumumab与稳定表达食蟹猴CD38(CHO-cynomolgus CD38)细胞株的的结合
采用与实施例11中11.2节描述的方法以流式细胞仪检测分析了鼠源单抗m29、人-鼠嵌合抗体(ch29G)及Daratumumab与稳定表达食蟹猴CD38蛋白的CHO细胞株(CHO-cynomolgus CD38)。
图11B为该流式细胞仪检测的代表性结果示意图，结果显示：人-鼠嵌合单抗ch29G保持有与鼠源单抗m29几乎一样的高亲和力结合表达食蟹猴CD38的CHO细胞株，而Daratumumab与表达食蟹猴CD38的CHO细胞株的结合活性强度下降达90％以上。
实施例13：ELISA检测人-鼠嵌合抗体ch29G及Daratumumab与人CD38蛋白或食蟹猴CD38蛋白的结合
本实施例中，采用直接ELISA方法来检测人-鼠嵌合抗体ch29及Daratumumab与重组人CD38蛋白或重组食蟹猴CD38蛋白的结合。为此，分别以重组人CD38-his蛋白或重组食蟹猴(cynomolgus)CD38-his蛋白(均为北京义翘神州公司产品)包被96-孔板(1ug/ml,50ul/孔，4度过夜)，5％牛奶封闭后，将人-鼠嵌合抗体ch29样品，Daratumumab及阴性对照抗体Rituximab(抗人CD20单抗)用稀释液(含0.5％牛 奶的PBST)稀释至1ug/ml，上样至包被了CD38蛋白的96-孔板中，2倍梯度稀释12孔，室温孵育1小时，PBS-0.1％Tween20液充分洗涤后，再加入以HRP酶标记的Goat-anti-human-IgG为检测二抗(1:1000稀释，购自上海西塘生物公司)室温孵育1小时，再经PBS-0.1％Tween20液充分洗涤后，加入邻苯二胺(OPD)-0.1％H 2O 2底物液显色10-15min后再加入0.1M HCl终止反应。其后在多功能酶标仪(PerkinElmer Victor X3)中读取492nm处OD值。
图12为该ELISA的检测结果。如图所示：人-鼠嵌合抗体ch29G及Daratumumab均可高亲和力与人CD38蛋白结合(图12A)。人-鼠嵌合抗体ch29G也可与食蟹猴CD38蛋白高亲和力结合；而Daratumumab与食蟹猴CD38蛋白无明显结合(图12B)。
实施例14：体外补体依赖细胞毒性(complement-dependent cytotoxicity，CDC)检测对比分析人-鼠嵌合单抗ch29G及Daratumumab的活性
14.1实验材料及方法
本实施例中，采用如本实施例中2的体外补体依赖细胞毒性(complement-dependent cytotoxicity，CDC)方法检测人-鼠嵌合单抗ch29G对多种靶细胞(包括Daudi，Raji,MOLT-4及Jurket等)的CDC活性，并将结果与Daratumumab的CDC活性结果作对比。其中采用的补体来源于健康人血清(10％，自制)，CDC阳性对照样品为Rituximab(抗CD20人-鼠嵌合单抗)，阴性对照样品为hPV19单抗(抗VEGF人源化单抗)。
图13为该CDC的检测结果，其中：
图13A为以Daudi为靶细胞的CDC测试结果，结果显示人-鼠嵌合抗体ch29G具有与阳性对照样品Rituximab相近的CDC活性，且两者的CDC活性均强于Daratumumab。
图13B为以Raji为靶细胞的CDC测试结果，结果显示人-鼠嵌合抗体ch29G也具有与阳性对照样品Rituximab相近的CDC活性，Daratumumab的CDC活性与人-鼠嵌合抗体ch29G的活性也相近。
图13C为以MOLT-4为靶细胞的CDC测试结果，结果显示人-鼠嵌合抗体ch29G，Daratumumab及Rituximab均无明显的CDC活性。
图13D为以Jurket为靶细胞的CDC测试结果，结果显示人-鼠嵌合抗体ch29G，Daratumumab及Rituximab也均无明显的CDC活性。
实施例15：鼠源m29抗体的人源化基因工程改造
在ELISA法、CDC等检测初步证明人-鼠嵌合抗体ch29G保持有与人CD38蛋白高亲合力结合，及CDC活性的基础上，采用PCR等系列基因工程克隆手段将该嵌合抗体轻链中的抗原互补决定区(CDR)基因片段移植到对应人kappa-轻链可变区骨架(framework regions，FR)上，获得人源化抗体轻链，再将此轻链与嵌合重链组合，即获得轻链人源化HH29G抗体。
15.1鼠源m29抗体轻链的人源化基因工程改造
通过氨基酸序列分析，确定人免疫球蛋白Kappa轻链第一V区胚系基因的表达产物(IgKV2D-29， NCBI Gene ID:28882)与鼠源m29抗体轻链可变区具有最高同源性。据此，将m29抗体轻链骨架区(FR)用人IgKV2D-29的同源序列替换，然后将替换后的可变区基因与人免疫球蛋白IgG-Kappa轻链的恒定区编码序列拼接，最后成功获得人源化的轻链编码基因(h29-L)。其中人源化m29抗体轻链可变区的氨基酸序列见SEQ ID NO.:11，其核苷酸序列为SEQ ID NO：13所示。
15.2鼠源m29抗体重链的人源化基因工程改造
通过氨基酸序列分析，确定人免疫球蛋白Kappa重链第一V区胚系基因的表达产物(IGHV1-69,NCBI Gene ID:28461)与m29重链可变区具有最高同源性。据此，将m29重链骨架区(FR)用人IGHV1-69的同源序列替换，然后将替换后的可变区基因与人免疫球蛋白IgG-的重链恒定区编码序列拼接，最后成功获得人源化的轻链编码基因(h29-h)。其中人源化m29抗体重链可变区的氨基酸序列见SEQ ID NO.:12，其核苷酸序列为SEQ ID NO：14所示。
实施例16：稳定高效分泌表达人源化或半人源化HH29单抗(HH29)的CHO细胞工程株的建立及抗体蛋白的分离纯化
将含人-鼠嵌合重链基因(ch29H)、人源化轻链基因(HH29L)分步克隆到pQY-Dhfr-Hex表达载体，转入大肠杆菌后扩增分离获得含人源化或半人源化HH29单抗(HH29)单抗表达质粒。其后将表达轻链人源化抗体HH29的重组质粒瞬时转染CHO细胞。转染24小时后，吸取孔内细胞培养上清，以CD38-his蛋白为包被抗原，HRP酶标记的Goat-anti-human-IgG为检测二抗(购自上海西塘生物公司)，OPD为显色底物，以直接ELISA法检测转染细胞上清中抗体与人CD38蛋白抗原结合的活性。
下表4为该ELISA代表性检测结果。
表4 ELISA分析瞬时转染细胞培养上清结合人CD38蛋白活性
Figure PCTCN2018107812-appb-000012
如表4中结果所示：与人-鼠嵌合型ch29G单抗抗体一样，半人源化HH29单抗(轻链人源化)保持与人CD38蛋白结合的活性。
上述转染细胞经克隆筛选及无血清培养基悬浮培养驯化后，成功获得多个稳定高效分泌表达半人源化 单抗(轻链人源化)HH29的CHO细胞工程株。
其后，从中选取一细胞工程株再经无血清培养基放大扩增培养后，收集培养上清液，上清液经离心及0.45μm滤膜过滤后，上样至包括含Protein A亲合层析柱(proteinA-Sepharose Fast Flow，美国通用电气GE公司)、离子交换析柱、病毒去除/灭活、及过滤除菌(0.22μm滤膜过滤)在内的多个分离纯化步骤后，最终获得高纯度(蛋白纯度达99％以上)的HH29抗体。纯化的HH29单抗抗体溶于甘氨酸-Tris缓冲液(pH＝7.0)中(1-10mg/ml)，低温(4℃左右)保存。
实施例17：小鼠体内测试鼠源m29单抗抗肿瘤疗效
在本实施例中，采用裸鼠皮下种植人B-淋巴瘤Raji肿瘤模型在动物体内测试鼠源m29单抗的抗肿瘤疗效，并采用人-鼠嵌合抗CD20单抗Rituximab为阳性对照药物。该试验首先在裸鼠皮下种植人B-淋巴瘤细胞株Raji；试验动物成瘤后，再分组给药及观察记录肿瘤生长。
为此，将数量为1x10 7的来源于人的B-淋巴瘤细胞株Raji细胞(中国科学院典型培养物保藏委员会细胞库)接种于裸鼠(购自南京大学动物中心)，待接种的肿瘤体积长至约黄豆大小(约100mm 3，肿瘤细胞接种后第6-7天左右)时将动物随机分为以下3组：
A组：生理盐水阴性对照组(n＝2，等体积生理盐水)
B组：阳性对照药物Rituximab单抗治疗组(n＝4，给药剂量为10mg/kg体重)
C组：m29单抗药物治疗组(n＝4，给药剂量为10mg/kg体重)
动物自分组当天起(即接种肿瘤后第6-7天)，每周腹腔注射(i.p.)给药2次(每隔3-4天)，连续给药6次(给药共3周)。期间每天观察动物一般临床症状，每隔3-4天测量肿瘤长径(mm)和短径(mm)及动物体重。肿瘤体积计算公式为：体积(mm 3)＝长径(mm)x短径(mm)x短径(mm)x0.5。如测量时肿瘤体积超过3000mm 3则对测试动物实行安乐死(euthanized)。
试验结果：
图14为各组试验组动物肿瘤的平均增长体积趋势，其中：
图14A为种植肿瘤前10天的平均增长体积示意图，结果显示各组之间无明显差别；
图14B为种植肿瘤后期的平均增长体积示意图，结果显示，与生理盐水阴性对照组相比，m29单抗药物治疗组及阳性对照药Rituximab单抗治疗组的肿瘤明显缩小甚至彻底消失。
实施例18：小鼠体内测试人-鼠嵌合ch29G抗体的抗肿瘤疗效
在本实施例中，采用与上述实施例17中相同的裸鼠皮下种植人B-淋巴瘤Raji肿瘤模型测试人-鼠嵌合ch29G单抗的抗肿瘤疗效，并采用Daratumumab单抗为阳性对照药物。该试验首先在裸鼠皮下种植人B-淋巴瘤Raji细胞；在试验动物在成瘤后，再分组给药分组及观察记录肿瘤生长。
为此，将数量为1x10 7的来源于人的B-淋巴瘤Raji细胞接种于裸鼠(购自南京大学动物中心)，待接种的肿瘤体积长至约黄豆大小(约100mm 3，肿瘤细胞接种后第6-7天左右)时将动物随机分为以下3组：
A组：生理盐水阴性对照组(n＝10，等体积生理盐水)
B组：阳性对照药物Daratumumab组(n＝10，给药剂量为5mg/kg体重)
C组：ch29G单抗药物治疗组(n＝10，给药剂量为2.5mg/kg体重)
动物自分组当天起(即接种肿瘤后第6-7天)，每周腹腔注射(i.p.)给药2次(每隔3-4天)，连续给药6次(给药共3周)。期间每天观察动物一般临床症状，每隔3-4天测量肿瘤长径(mm)和短径(mm)及动 物体重。肿瘤体积计算公式为：体积(mm 3)＝长径(mm)x短径(mm)x短径(mm)x0.5。如测量时肿瘤体积超过3000mm 3则对测试动物实行安乐死(euthanized)。
试验结果：
图15为各组试验组动物肿瘤的平均增长体积趋势：结果显示，与生理盐水阴性对照组相比，ch29G单抗药物治疗组及阳性对照药Daratumumab治疗组的治疗增长受到明显抑制，其中给药剂量为2.5mg/kg体重的ch29G单抗的疗效与给药剂量为5mg/kg体重的Daratumumab疗效相近似。
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Claims (14)

  1. 一种特异结合人及猴CD38抗原的单克隆抗体或其衍生体，其特征在于，其包含第一可变区和第二可变区，其中所述第一可变区是抗体轻链可变区，其抗原互补决定区CDR1，CDR2和CDR3分别为SEQ ID NO：3，SEQ ID NO：4及SEQ ID NO：5所示的氨基酸序列；其中所述第二可变区是抗体重链可变区，其抗原互补决定区CDR1，CDR2和CDR3分别为SEQ ID NO：8，SEQ ID NO：9及SEQ ID NO：10所示的氨基酸序列。
  2. 根据权利要求1所述的抗体或其衍生体，其特征在于，所述第一可变区是抗体轻链可变区，为SEQ ID NO：2所示的氨基酸序列；所述第二可变区是抗体重链可变区，为SEQ ID NO：7所示的氨基酸序列。
  3. 根据权利要求1所述的抗体或其衍生体，其特征在于，所述第一可变区是抗体轻链可变区，为SEQ ID NO：11所示的氨基酸序列；所述第二可变区是抗体重链可变区，为SEQ ID NO：12所示的氨基酸序列或为SEQ ID NO：7所示的氨基酸序列。
  4. 根据权利要求1、2或3所述的抗体或其衍生体，其特征在于，其包含所述抗体轻链可变区和人抗体轻链恒定区，及包含所述抗体重链可变区和人抗体重链恒定区的铰链区，CH1区，CH2区和CH3区。
  5. 根据权利要求4所述的抗体或其衍生体，其特征在于，所述人抗体轻链恒定区来自人抗体kappa链或抗体lamda链，所述人抗体重链恒定区来自人IgG1，IgG2，IgG3或IgG4亚型。
  6. 一种编码权利要求3所述抗体或其衍生体的DNA分子或基因，其特征在于，其抗体轻链可变区为SEQ ID NO：1或SEQ ID NO：13所示的核苷酸序列，抗体重链可变区为SEQ ID NO：6或SEQ ID NO：14的核苷酸序列。
  7. 一种表达载体，其特征在于，它含有权利要求6所述的DNA分子序列以及与该序列操作性相连的表达调控序列。
  8. 一种重组宿主细胞，其特征在于，它由权利要求7所述的表达载体转化而成。
  9. 根据权利要求8所述的重组宿主细胞或其子代细胞，其中所述重组宿主细胞或其子代细胞表达权利要求1或5所述的抗体或其衍生体。
  10. 一种药物或药物组合物，其特征在于，它含有药学上有效量的如权利要求1或5所述的抗体或其衍生体，以及药学上可接受的载体。
  11. 根据权利要求10所述的药物或药物组合物在制备治疗肿瘤的药物中的应用。
  12. 根据权利要求11所述的应用，其特征在于，所述肿瘤为CD38表达阳性的肿瘤。
  13. 根据权利要求12所述的应用，其特征在于，所述CD38表达阳性的肿瘤为人骨髓瘤， 或人淋巴细胞瘤。
  14. 一种制备权利要求1或5所述的抗体或其衍生体的方法，其特征在于，该方法包括如下步骤：
    a)提供一表达载体，该表达载体含有权利要求1或5所述的DNA分子序列以及与该序列操作性相连的表达调控序列；
    b)用步骤a)所述的表达载体转化宿主细胞；
    c)在适合所述抗体表达的条件下培养步骤b)所得的宿主细胞：和
    d)采用亲合层析从宿主细胞培养液中分离纯化获得所述抗体。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022033535A1 (zh) * 2020-08-12 2022-02-17 三生国健药业（上海）股份有限公司 结合人cd38的抗体、其制备方法和用途
TWI793714B (zh) * 2020-08-12 2023-02-21 大陸商三生國健藥業(上海)股份有限公司 結合人cd38的抗體、其製備方法和用途

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US20210324102A1 (en) 2021-10-21
EP3851455A1 (en) 2021-07-21
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