WO2020143506A1 - 检测多种恶性肿瘤细胞的抗体及其应用 - Google Patents

检测多种恶性肿瘤细胞的抗体及其应用 Download PDF

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WO2020143506A1
WO2020143506A1 PCT/CN2019/130486 CN2019130486W WO2020143506A1 WO 2020143506 A1 WO2020143506 A1 WO 2020143506A1 CN 2019130486 W CN2019130486 W CN 2019130486W WO 2020143506 A1 WO2020143506 A1 WO 2020143506A1
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seq
amino acid
variable region
antibody
chain variable
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PCT/CN2019/130486
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English (en)
French (fr)
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张素平
科佩斯·汤姆
吴柳风
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深圳大学
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Priority claimed from CN201910015053.3A external-priority patent/CN111378040B/zh
Application filed by 深圳大学 filed Critical 深圳大学
Priority to EP19909467.3A priority Critical patent/EP3936523A4/en
Publication of WO2020143506A1 publication Critical patent/WO2020143506A1/zh
Priority to US17/369,988 priority patent/US20210388086A1/en

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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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/49—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells

Definitions

  • the present application relates to the field of biomedicine, in particular to antibodies for detecting multiple tumor cells and their applications, and in particular to an anti-ROR2 antibody and its applications.
  • the tyrosine kinase orphan-like receptor ROR2 is an important development-related regulatory protein. In the early embryonic development, it has a high expression in various tissues and has an important role in tissue value-added and differentiation. After the second trimester, the expression level of ROR2 gradually decreased. Except in some osteoblasts and uterine cells, ROR2 is basically not expressed in adult tissues. The current research shows that ROR2 is highly expressed in many tumor tissues, including osteosarcoma, melanoma, colorectal cancer and gastric cancer, breast cancer, lymphoma, leukemia and other cancers. Therefore, many scholars believe that ROR2 is a disease-related gene and can be used as an ideal drug target.
  • ROR2 is an orphan receptor for tyrosine kinase that is expressed in large amounts during embryonic development and plays an important role. It is difficult to detect in most adult tissues. Studies in recent years have found that although most normal tissues are not ROR2 is expressed, but many malignant tumor cells including breast cancer, lung cancer, pancreatic cancer and other cells express ROR2 highly; and, the higher the ROR2 expression level, the higher the malignancy of the cancer cells, the more prone to metastasis, relapse, and the worse the prognosis. . ROR2 can usually be detected on tumor cells with a low degree of differentiation. Such tumor cells have a strong ability to relapse and metastasize.
  • an object of the present application is to propose an anti-ROR2 antibody and its application.
  • the present application is based on the inventors' research findings as follows: during tumor research, as surface markers of tumor stem cells, such as CD133, CD44, CD24, ALDH1, etc., are expressed in tumor cells or normal adult tissues, so that the anti-tumor stem cell drugs developed for these surface molecules can also cause certain toxicity to normal tissues.
  • ROR2 is also abundantly expressed in cancer stem cells, but is less expressed in normal cells, so the development of therapeutic antibodies against ROR2 will cause less damage to normal tissues. Therefore, ROR2 is an ideal drug target.
  • the drugs developed for ROR2 can be used in the treatment of tumors and cancers, and the damage to normal tissues will be relatively small.
  • the inventors of the present application developed specific antibodies against ROR2 antigen through research, and the antibodies provided in the present application can bind to ROR2 antigen with high specificity, thus being applied to targeted therapy of tumors and specificity of ROR2 antigen Sexual diagnosis and testing.
  • the present application provides an anti-ROR2 antibody or antigen-binding fragment, including at least one of the following: (1) a light chain variable region having the amino acid sequences shown in CSASSSVSYMHWYQ, IYDTSKLAS, and CQQWSSNPPTFGAG, and having The heavy chain variable region of the amino acid sequence shown by YTITSYLMHWV, LEWIGYINPYNDGTKYNEKFKDKAT and CARSDVYYGVRFAYWGQG; (2) The light chain variable region with the amino acid sequence shown by CKASQNVGTNVAWFQ, IYLASYRYS and CQQYNSYPLTFGGGG, and the light chain variable region with YTFTNYWIQN and YTFTNYWIQK Variable region; (3) Light chain variable region with amino acid sequences shown by CSASSSISYMYWYQ, IYDTSILAS and CQQWSSYPFTFGSG, and heavy chain variable region with amino acid sequences
  • the antibody or antigen-binding fragment provided herein has high affinity with ROR2 antigen, and at a concentration of 0.0001 ⁇ g/ml, it shows affinity, and can be used for a variety of detections related to ROR2 antigen. Moreover, it can be applied to the treatment of tumors and exert a targeted therapeutic effect.
  • the antibody or antigen-binding fragment described above may further include the following technical features:
  • the antibody or antigen-binding fragment includes at least one of the following: (a) having SEQ The light chain variable region of the amino acid sequence shown in ID NO: 1 and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 2; (b) The light chain variable region having the amino acid sequence shown in SEQ ID NO: 3 And the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 4; (c) has SEQ The light chain variable region of the amino acid sequence shown in ID NO: 5 and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 6; amino acids with at least one conservative amino acid substitution compared to (a) to (c) sequence.
  • the antibody or antigen-binding fragment includes at least one of the following: a light chain having the amino acid sequence shown in SEQ ID NO: 7 and a heavy chain having the amino acid sequence shown in SEQ ID NO: 8; Light chain with the amino acid sequence shown in SEQ ID NO: 9 and heavy chain with the amino acid sequence shown in SEQ ID NO: 10; light chain with the amino acid sequence shown in SEQ ID NO: 11 and amino acid sequence shown in SEQ ID NO: 12 Heavy chain.
  • the present application provides an isolated polynucleotide encoding the antibody or antigen-binding fragment according to any embodiment of the first aspect of the present application.
  • the polynucleotide includes at least one of the following:
  • a sequence having more than 90% homology optionally a sequence having more than 95% homology, preferably a sequence having more than 98% homology , More preferably a sequence with 99% or more homology;
  • a sequence having more than 90% homology optionally a sequence having more than 95% homology, preferably having more than 98% homology
  • the sequence is more preferably a sequence having 99% or more homology
  • a sequence having 90% or more homology optionally a sequence having 95% or more homology, preferably a sequence having 98% or more homology, more preferably Sequences with more than 99% homology;
  • a sequence having 90% or more homology optionally a sequence having 95% or more homology, preferably a sequence having 98% or more homology, and more preferably a sequence having more than 98% homology Sequences with more than 99% homology.
  • the present application provides an expression vector comprising the polynucleotide according to any embodiment of the second aspect of the present application.
  • the expression vector described above further includes: a control element operably connected to the polynucleotide for controlling the expression of the polynucleotide in a host cell.
  • control element includes at least one of the following: a promoter, an enhancer, and a terminator.
  • the host cell is a mammalian cell.
  • the present application provides a recombinant cell comprising the expression vector according to any embodiment of the third aspect of the present application.
  • the present application provides a method for preparing an anti-ROR2 antibody or antigen-binding fragment thereof, which includes culturing the recombinant cell described in the fourth aspect of the present application.
  • the present application provides the use of an antibody or antigen-binding fragment in the preparation of a medicament for the treatment of cancer, the antibody or antigen-binding fragment is as described in the first aspect of the present application Antibody or antigen binding fragment.
  • the present application provides the use of an antibody or antigen-binding fragment in the preparation of a kit for diagnostic testing of ROR2 antigen, the antibody or antigen-binding fragment is the first The antibody or antigen-binding fragment of one aspect.
  • the kit is used for immunoblotting, immunoprecipitation, and ELISA detection.
  • the present application provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment described in any embodiment of the first aspect of the present application and a pharmaceutically acceptable carrier.
  • the present application provides a chimeric antigen receptor against ROR2, including: an extracellular region, a transmembrane region and an intracellular region, the extracellular region includes antibodies or antigen-binding fragments, so The antibody or antigen-binding fragment is a single chain, and the antibody or antigen-binding fragment is the antibody or antigen-binding fragment according to any embodiment of the first aspect of the present application.
  • the present application provides a Car-T cell that expresses the anti-ROR2 chimeric antigen receptor described in the ninth aspect of the present application.
  • FIG. 1 is a Fab of A12, B22, B30 with different concentrations provided according to an embodiment of the present application Graph of antibody ELISA test results.
  • FIG. 2 is a graph of SDS-PAGE identification results of different antibodies provided according to examples of the present application.
  • FIG. 3 is a graph showing the results of ELISA detection of different concentrations of chimeric antibodies according to the examples of the present application.
  • FIG. 4 is a graph of SDS-PAGE identification results of ROR2 expression in T47D cells and 231 cells according to examples of the present application.
  • FIG. 5 is a flow cytometry result diagram of different chimeric antibodies for detecting ROR2 in T47D cells according to examples of the present application.
  • FIG. 6 is a flow cytometry result diagram of different chimeric antibodies for detecting ROR2 in 231 cells according to examples of the present application.
  • FIG. 7 is a flow cytometry result of detecting the expression of ROR2 in blood cancer patients using A12 chimeric antibody according to an embodiment of the present application.
  • FIG. 8 is a flow cytometry result of detecting the expression of ROR2 in lymphoma patient tissues using A12 chimeric antibody according to an embodiment of the present application.
  • FIG. 9 is a graph showing the results of immunoblotting using different chimeric antibodies according to the examples of the present application.
  • FIG. 10 is a graph showing the results of immunoprecipitation for different chimeric antibodies according to the examples of the present application for the detection of endogenous ROR2 protein.
  • FIG. 11 is a graph showing the results of immunoprecipitation for different chimeric antibodies provided in the examples of the present application for the detection of exogenous ROR2 protein.
  • FIG. 12 is an A12-car structure diagram provided according to an embodiment of the present application.
  • FIG. 13 is a flow cytometry result chart showing the proportion of lymphocytes capable of expressing CAR-T according to an example of the present application.
  • FIG. 14 is a graph showing the killing results of A12-Car-T on T47D cells positive for ROR2 expression and 231 cells negative for ROR2 expression according to an embodiment of the present application.
  • the term "antibody” is an immunoglobulin molecule capable of binding to a specific antigen. It includes two light chains with lighter molecular weight and two heavy chains with heavier molecular weight.
  • the heavy chain (H chain) and light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule.
  • the amino acid sequence of the amino-terminus (N-terminus) of the peptide chain varies greatly, and is called the variable region (V-region), and the carboxyl-terminus (C-terminus) is relatively stable.
  • the V regions of the L chain and the H chain are called VL and VH, respectively.
  • hypervariable region The amino acid composition and arrangement order of certain regions in the variable region have a higher degree of change, called hypervariable region (HVR).
  • the hypervariable region is where the antigen and antibody bind, so it is also called determinant complementarity Area (complementarity-determining region, CDR).
  • CDR complementarity-determining region
  • This application uses the extracellular segment of ROR2 to obtain Fab (antigen-binding fragment) antibody fragments with high specificity and high affinity against ROR2 through immunization.
  • the antibody fragment can specifically bind to ROR2 antigen, so that it can target diseases such as tumors.
  • the present application provides an antibody or antigen-binding fragment having a light chain variable region shown by CSASSSVSYMHWYQ, IYDTSKLAS, and CQQWSSNPPTFGAG, and a light chain variable region shown by YTITSYLMHWV, LEWIGYINPYNDGTKYNEKFKDKAT, and CARSDVYYGVRFAYWGQG Heavy chain variable region.
  • the antibody or antigen-binding fragment has a light chain variable region represented by CKASQNVGTNVAWFQ, IYLASYRYS and CQQYNSYPLTFGGG, and a heavy chain variable region represented by YTFTNYWIQWM, LEWIGEINPSNGRTDYNEKFKNRAT and CANYRPGYWGQG, respectively.
  • the antibody or antigen-binding fragment has light chain variable regions shown by CSASSSISYMYWYQ, IYDTSILAS, and CQQWSSYPFTFGSG, and heavy chain variable regions shown by YTFTSYLIHWV, LEWIGYINPYNDGTKYNEKFKDKAT, and CARSDVYYGVRFAYWGQG, respectively.
  • CSASSSISYMYWYQ As an example.
  • CSASSSISYMYWYQ As a CDR region on the light chain variable region, it can bind to antigen with high specificity.
  • Antibodies or antigen-binding fragments having the above-mentioned CDR regions can specifically bind to ROR2 antigen and exert the function of ROR2 antibody.
  • the antibody or antigen-binding fragment provided herein has more than one conservative amino acid substitution compared to any of the light chain variable region and heavy chain variable region described above.
  • Antigen-binding fragment refers to an antibody fragment that retains the ability to specifically bind antigen (ROR2).
  • Constant amino acid substitution refers to the substitution of another amino acid for a biologically, chemically, or structurally similar residue. Biologically similar means that the substitution does not destroy the biological activity of the ROR2 antibody or ROR2 antigen.
  • Structurally similar means that amino acids have side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size. Chemical similarity means that amino acids have the same charge or are both hydrophilic or hydrophobic.
  • hydrophobic residues isoleucine, valine, leucine or methionine are mutually substituted.
  • polar amino acids such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, etc.
  • the present application provides an antibody or antigen binding fragment having the light chain variable region sequence shown in SEQ ID NO: 1 and the heavy chain shown in SEQ ID NO: 2 Chain variable region sequence.
  • the light chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:1.
  • the heavy chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:2.
  • the present application provides an antibody or antigen-binding fragment having the light chain variable region sequence shown in SEQ ID NO: 3 and the heavy chain shown in SEQ ID NO: 4 Chain variable region sequence.
  • the light chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:3.
  • the heavy chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:4.
  • the present application provides an antibody or antigen-binding fragment having the light chain variable region sequence shown in SEQ ID NO: 5 and the heavy chain shown in SEQ ID NO: 6 Chain variable region sequence.
  • the light chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:5.
  • the heavy chain variable region sequence of the antibody or antigen-binding fragment has more than one conservative amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:6.
  • conservative amino acid substitutions will not change the biological function of antibodies or antigen-binding fragments.
  • these conservative amino acid substitutions can occur in amino acids other than the CDR regions in the heavy chain variable region and the light chain variable region.
  • the antibody having the light chain variable region sequence shown in SEQ ID NO: 1 and the heavy chain variable region sequence shown in SEQ ID NO: 2 is also called A12 antibody, and its light chain variable region sequence VL (SEQ ID NO: 1) is:
  • VH (SEQ ID NO: 2) is:
  • the antibody of heavy chain variable region sequence shown in NO: 4 is also called B22 antibody, and its light chain variable region sequence VL (SEQ ID NO: 3) is:
  • the heavy chain variable region sequence VH (SEQ ID NO: 4) is:
  • the antibody of heavy chain variable region sequence shown in NO: 6 is also called B30 antibody, and its light chain variable region sequence VL (SEQ ID NO: 5) is:
  • the heavy chain variable region sequence VH of B30 antibody (SEQ ID NO: 6)
  • the present application provides an anti-ROR2 antibody, the antibody having SEQ The light chain shown in ID NO: 7 and the heavy chain shown in SEQ ID NO: 8.
  • SEQ ID NO: 7 is:
  • SEQ ID NO: 8 is:
  • the present application provides an anti-ROR2 antibody, the antibody having SEQ The light chain shown in ID NO: 9 and the heavy chain shown in SEQ ID NO: 10.
  • SEQ ID NO: 9 is:
  • SEQ ID NO: 10 is:
  • the present application provides an anti-ROR2 antibody, the antibody having SEQ The light chain shown in ID NO:11 and the heavy chain shown in SEQ ID NO:12.
  • SEQ ID NO: 11 is:
  • SEQ ID NO: 12 is:
  • the polynucleotides expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.
  • the present application also provides an isolated polynucleotide encoding the antibody or antigen-binding fragment described above.
  • the isolated polynucleotide encodes A12 antibody, B22 antibody, and B30 antibody, respectively.
  • the heavy chain variable region SEQ encoding A12 antibody The nucleotide sequence of the amino acid sequence shown in ID NO: 2 is (SEQ ID NO: 14):
  • the heavy chain variable region SEQ encoding B22 antibody The nucleotide sequence of the amino acid sequence shown in ID NO: 4 is (SEQ ID NO: 16):
  • the heavy chain variable region SEQ encoding B30 antibody The nucleotide sequence of the amino acid sequence shown in ID NO: 6 is (SEQ ID NO: 18):
  • the nucleotide sequence of the amino acid sequence shown in ID NO: 8 is (SEQ ID NO: 20)
  • the isolated polynucleotide has at least 90% homology with the above-mentioned amino acid coding sequence, preferably 95% homology, more preferably 98%, 99% homology Homology.
  • the present application also provides an expression vector comprising the above isolated polynucleotide.
  • the polynucleotide may be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide.
  • these control elements can come directly from the carrier itself, or they can be exogenous, that is, not from the carrier itself.
  • These control elements may be promoters, enhancers or terminators, as long as they can regulate gene expression.
  • the polynucleotide and the control element need only be operably connected.
  • operably linked refers to linking a foreign gene to a vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can exert their intended regulation of the transcription and translation of the foreign gene. Function.
  • the polynucleotides used to encode the heavy and light chains of antibodies can be inserted into different vectors independently. It is common to insert them into the same vector.
  • Commonly used vectors can be, for example, plasmids, bacteriophages and the like. For example, pcDNA plasmid.
  • the present application also provides a recombinant cell containing the expression vector.
  • the expression vector can be introduced into mammalian cells (such as 293F cells, CHO cells, etc.) to construct and obtain recombinant cells, and then use these recombinant cells to express the antibodies or antigen-binding fragments provided by the present application. By culturing the recombinant cells, corresponding antibodies can be obtained.
  • compositions, kits and pharmaceutical uses and uses in the preparation of kits are provided.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.
  • the anti-ROR2 antibodies provided herein can be incorporated into a pharmaceutical composition suitable for administration by a subject.
  • these pharmaceutical compositions include the anti-ROR2 antibodies provided herein and a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof.
  • the pharmaceutical composition includes isotonic agents, such as sugars, polyols (such as mannitol, sorbitol) Or sodium chloride.
  • isotonic agents such as sugars, polyols (such as mannitol, sorbitol) Or sodium chloride.
  • the pharmaceutically acceptable carrier can also include minor amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, to extend the shelf life or efficacy of the antibody.
  • the antibodies of the present application can be incorporated into pharmaceutical compositions suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration eg, intravenous, subcutaneous, intraperitoneal, intramuscular
  • These pharmaceutical compositions can be prepared in various forms. Examples include liquid, semi-solid, and solid dosage forms, including, but not limited to, liquid solutions (eg, injection solutions and infusion solutions), dispersing or suspending agents, tablets, pills, powders, liposomes, and suppositories.
  • Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions.
  • the antibody can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
  • the anti-ROR2 antibody herein can also be made into a kit or part of other diagnostic reagents as needed.
  • the present application also provides a kit including the above-mentioned ROR2 antibody.
  • the kit provided by the present application for example, it can be used in immunoblotting, immunoprecipitation, etc., which involve detection using the specific binding performance of ROR2 antigen and antibody.
  • kits can contain any one or more of the following: antagonists, anti-ROR2 antibodies, or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature.
  • Anti-ROR2 antibodies can be used for different types of diagnostic tests.
  • the antibodies provided herein can also be used for radioimmunoassay and radioimmunotherapy of cancer.
  • cancers or tumors can be any unregulated cell growth. Specifically, it may be lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, liver cancer, breast cancer, colorectal cancer, lymphoma, blood cancer, or the like.
  • the anti-ROR2 antibody provided in the present application may be provided to the subject.
  • the present application provides a method for treating cancer, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof provided by the present application.
  • this application developed an antibody-based immunotherapy method based on ROR2 against various tumor cells or even tumor stem cells, including monoclonal antibodies to ROR2, and a new ROR2-Car T cell therapy technology.
  • amino acid sequence of the light chain variable region and the heavy chain variable region of one antibody are SEQ ID NO: 1 and SEQ, respectively ID NO:2, named A12 antibody; the light chain variable region amino acid sequence and heavy chain variable region amino acid sequence of an antibody are SEQ ID NO: 3 and SEQ ID NO: 4, respectively, named B22 antibody; one The light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence of the antibody are SEQ ID NO: 5 and SEQ, respectively ID NO: 6, named B30 antibody.
  • the light chain amino acid sequence of the A12 antibody is SEQ ID NO: 7
  • the heavy chain amino acid sequence is SEQ ID NO: 8
  • the light chain amino acid sequence of the B22 antibody is SEQ ID NO: 9
  • the heavy chain amino acid sequence is SEQ ID NO : 10
  • the light chain amino acid sequence of the B30 antibody is SEQ ID NO: 11
  • the heavy chain amino acid sequence is SEQ ID NO: 12.
  • the DNA sequences of the variable regions of the A12 antibody, B22 antibody and B30 antibody were used to derive the DNA sequence, and the expression sequence of the chimeric antibody was established after species optimization based on the expression vector, and cloned into the pcDNA3.4 vector.
  • the amino acid sequence of the antibody is not changed, only codon optimization is performed, and the optimized nucleotide sequence encoding the amino acid sequence is easier to express in mammalian cells. Therefore, for example, when expressing the A12 chimeric antibody, the amino acid sequence is the same as the above-mentioned A12 antibody amino acid sequence, the difference is that the nucleotide sequence encoding the A12 chimeric antibody is optimized.
  • nucleotide sequence encoding the light chain of the A12 chimeric antibody is SEQ ID NO: 19
  • nucleotide sequence encoding the heavy chain of the A12 chimeric antibody is SEQ ID NO:20;
  • nucleotide sequence encoding the light chain of the B22 chimeric antibody is SEQ ID NO: 21
  • nucleotide sequence encoding the heavy chain of the B22 chimeric antibody is SEQ ID NO:22;
  • the nucleotide sequence encoding the light chain of the B30 chimeric antibody is SEQ ID NO: 23
  • the nucleotide sequence encoding the heavy chain of the B22 chimeric antibody is SEQ ID NO:24.
  • FIG. 1 is the SDS-PAGE identification of purified antibodies of ROR2-B30 (SA120-1), B22 (SA120-2), and A12 (SA120-3). As a result, the results show that our purified antibodies have higher purity.
  • the first, second, and third columns represent the SDS-PAGE gel identification results of non-reducing A12, B22, and B30 antibody samples with a loading volume of 2.0 ⁇ g
  • the fourth column represents the protein marker.
  • the fifth, sixth, and seventh columns represent the SDS-PAGE gel identification results of the reduced A12, B22, and B30 antibody samples with a sample volume of 2.0 ⁇ g
  • the eighth, ninth, and ten columns represent the sample volumes of 0.5, 1.0, and 2 ⁇ g, respectively.
  • BSA fetal bovine serum protein
  • FIG. 3 is the OD450 value of B30 chimeric antibody, B22 chimeric antibody and A12 chimeric antibody at different concentrations. It can be seen that when the chimeric antibody concentration is low (0.001ug/ml or 0.0001ug/ml) At that time, the antibody already has affinity, and it also shows that our chimeric antibody can be used for ELISA and has a high affinity.
  • the purified antibodies were stained by flow cytometry to detect T47D cells (positive ROR2 expression, see Figure 4) and 231 breast cancer cells (negative ROR2 expression, see Figure 4). The result is shown below. Flow cytometry results show that all three chimeric antibodies can specifically bind ROR2 membrane protein.
  • Figure 4 shows the expression level of ROR2 in T47D cells and 231 cells detected by Santa Cruz's ROR2 antibody.
  • Figure 5 shows the results of three chimeric antibodies using flow cytometry to detect the ROR2 expression level of T47D. The concentration of all three chimeric antibodies was 10ug/ml.
  • Fig. 6 shows the results of flow cytometric detection of ROR2 expression level of 231 by three chimeric antibodies. The concentration of all three chimeric antibodies was 10ug/ml.
  • A12 chimeric antibody can detect the ROR2 level of PDX in patients with different blood cancers and lymphomas
  • lymphoma patient tissues from the hospital, and then build a PDX model. Take the tumor tissue grown by PDX mice, use human dissociation kit to separate the tissue into single cells, and then add A12 chimeric antibody (concentration 3 ⁇ g/ml) ), incubated for 1 hour, and then incubated with Alexa-488-conjugated anti-mouse IgG secondary antibody and subjected to flow cytometry to check the ROR2 level of tumor cells.
  • the results are shown in Figure 8.
  • the left panel of Fig. 8 shows the peak of ROR2 fluorescence intensity of tumor cells of lymphoma patients.
  • the two scattergrams on the right show the distribution of cell fluorescence intensity of blank cells and B16 antibody staining tubes of lymphoma patients.
  • ROR2 chimeric antibody can be used in immunoblotting (WB)
  • ROR2 chimeric antibody can be used for immunoprecipitation (IP)
  • ROR2 chimeric antibody To identify whether the ROR2 chimeric antibody can be used for immunoprecipitation, we used 293T cells positive for ROR2 expression for IP. After the cells were lysed, B30 chimeric antibody, B22 chimeric antibody and A12 chimeric antibody were used for IP respectively, and the protein after IP was subjected to western blot to detect ROR2 protein with SantaCruz ROR2 (Cat. No. sc-374174) antibody. Among them, IgG antibody serves as a negative control antibody (cannot precipitate ROR2), and Input is a sample of cell lysate that has not been immunoprecipitated (can express ROR2 protein). The results are shown in Figure 10. The IP results indicate that all three antibodies can be used for IP and can be used to detect endogenous ROR2.
  • Flag tag is a peptide fragment composed of eight hydrophilic amino acids DYKDDDDK, which can be fused with protein and does not occupy protein epitopes or domains, to avoid affecting the protein's function, secretion and transport and other aspects of performance, use Flag tag to mark If the target protein is expressed, the target protein will also be expressed.
  • IP was performed with B30 chimeric antibody, B22 chimeric antibody, A12 chimeric antibody and Flag, respectively, and immunoblot (IB) was performed with Flag and B30 chimeric antibody, B22 chimeric antibody, and A12 chimeric antibody, respectively.
  • IB immunoblot
  • the experiment further verified the ROR2 antibody provided by this application, and developed ROR2-Car T cell therapy technology is used for targeted treatment of diseases.
  • Untransformed T cells (NT), transformed empty CD532A-T, and transformed A12-CAR CAR-A12-T cells were co-cultured with two target cells in 96-well plates.
  • One of them is the T-47D breast cancer cell line that expresses ROR2, and the other is the MDA-MB-231 breast cancer cell line that does not express ROR2.
  • the resulting kill rate was then normalized to the target cell kill rate of the untransformed T cell (NT) group.
  • the results are shown in Figure 14.
  • A12-Car-T can significantly kill T47D cells with positive ROR2 expression, but not 231 cells with negative ROR2 expression.

Abstract

一种抗ROR2抗体及其应用。该ROR2抗体具有CSASSS VSYMHWYQ、IYDTSKLAS和CQQWSSNPPTFGAG所示氨基酸序列的轻链可变区，以及具有YTITSY LMHWV、LEWIGYINPYNDGTKYNEKFKDKAT和CARSDVYYGVRFAYWGQG所示氨基酸序列的重链可变区；或者具有CKASQNVGTNVAWFQ、IYLASYRYS和CQQYNSYPLTFGGG所示氨基酸序列的轻链可变区，以及具有YTFTNYWIQWM、LEWIGEINPSNGRTDYNEKFKNRAT和CANYRPGYWGQG所示氨基酸序列的重链可变区。

Description

检测多种恶性肿瘤细胞的抗体及其应用 技术领域
本申请涉及生物医药领域，尤其涉及检测多种肿瘤细胞的抗体及其应用，具体涉及一种抗ROR2抗体及其应用。
 背景技术
酪氨酸激酶孤儿样受体ROR2是重要的发育相关调节蛋白，在胚胎发育早期，多种组织中均有较高的表达和对组织的增值，分化等具有重要的作用。妊娠中期后，ROR2的表达水平逐渐下降。除了在部分成骨细胞和子宫细胞之外，ROR2在成人组织中基本不表达。而目前研究表明，ROR2在许多肿瘤组织中高表达，包括骨肉瘤，黑色素瘤，结直肠癌和胃癌，乳腺癌，淋巴瘤，白血病等癌症。因此，许多学者认为ROR2是个疾病相关的基因，且可以作为理想的药物靶点。
ROR2是一个在胚胎发育过程中大量表达并起重要作用的一个酪氨酸激酶孤儿受体，而在成体大多数的组织中很难检测到，最近几年的研究发现，虽然大多数正常组织不表达ROR2，但是许多恶性肿瘤细胞包括乳腺癌，肺癌，胰腺癌等细胞却高表达ROR2；并且，ROR2表达水平越高的癌细胞，其恶性程度越高，越容易发生转移，复发，预后越差。ROR2通常在分化程度很低的肿瘤细胞上可以检测到，这类肿瘤细胞具有很强的复发和转移能力。
然而针对ROR2的药物研发还有待改进。
 技术解决方案
本申请旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本申请的一个目的在于提出一种抗ROR2抗体及其应用。
本申请是基于发明人的如下研究所发现的：在肿瘤的研究过程中，作为肿瘤干细胞的表面标记物，例如CD133,CD44, CD24, ALDH1等，无论在肿瘤细胞还是在正常的成体组织中都有表达，致使针对这些表面分子开发的抗肿瘤干细胞的药物对正常组织也会造成一定的毒害。而ROR2在肿瘤干细胞也有丰富的表达，但是在正常细胞中却表达较少，因此开发出针对ROR2的治疗性抗体对正常组织的伤害会比较小。所以，ROR2是理想的药物靶点。而针对ROR2所开发出的药物可以用于肿瘤和癌症的治疗中，且对正常组织的损害会比较小。
为此，本申请的发明人通过研究开发了针对ROR2抗原的特异性抗体，利用本申请所提供的抗体能够高特异性的与ROR2抗原结合，从而应用于肿瘤的靶向治疗以及ROR2抗原的特异性诊断和检测。
具体而言，本申请提供了如下技术方案：
根据本申请的第一方面，本申请提供了一种抗ROR2抗体或抗原结合片段，包括下列至少之一：（1）具有CSASSSVSYMHWYQ、IYDTSKLAS和CQQWSSNPPTFGAG所示氨基酸序列的轻链可变区，以及具有YTITSYLMHWV、LEWIGYINPYNDGTKYNEKFKDKAT和CARSDVYYGVRFAYWGQG所示氨基酸序列的重链可变区；（2）具有CKASQNVGTNVAWFQ、IYLASYRYS和CQQYNSYPLTFGGG所示氨基酸序列的轻链可变区，以及具有YTFTNYWIQWM、LEWIGEINPSNGRTDYNEKFKNRAT和CANYRPGYWGQG所示氨基酸序列的重链可变区；（3）具有CSASSSISYMYWYQ、IYDTSILAS和CQQWSSYPFTFGSG所示氨基酸序列的轻链可变区，以及具有YTFTSYLIHWV、LEWIGYINPYNDGTKYNEKFKDKAT和CARSDVYYGVRFAYWGQG所示氨基酸序列的重链可变区；与（1）~（3）相比，具有至少一个保守氨基酸取代的氨基酸序列。本文提供的抗体或抗原结合片段与ROR2抗原具有高亲和力，在0.0001μg/ml的浓度下，即表现出亲和性，可以用于多种和ROR2抗原相关的检测。而且，能够应用于肿瘤的治疗中，发挥靶向治疗作用。
根据本申请的实施例，以上所述的抗体或抗原结合片段可以进一步包括如下技术特征：
在本申请的一些实施例中，所述抗体或抗原结合片段，包括下列至少之一：（a）具有SEQ ID NO:1所示氨基酸序列的轻链可变区和SEQ ID NO:2所示氨基酸序列的重链可变区；（b）具有SEQ ID NO:3所示氨基酸序列的轻链可变区和SEQ ID NO:4所示氨基酸序列的重链可变区；（c）具有SEQ ID NO:5所示氨基酸序列的轻链可变区和SEQ ID NO:6所示氨基酸序列的重链可变区；与（a）~（c）相比，具有至少一个保守氨基酸取代的氨基酸序列。
在本申请的一些实施例中，所述抗体或抗原结合片段，包括下列至少之一：具有SEQ ID NO:7所示氨基酸序列的轻链和SEQ ID NO:8所示氨基酸序列的重链；具有SEQ ID NO:9所示氨基酸序列的轻链和SEQ ID NO:10所示氨基酸序列的重链；具有SEQ ID NO:11所示氨基酸序列的轻链和SEQ ID NO:12所示氨基酸序列的重链。
根据本申请的第二方面，本申请提供了一种分离的多核苷酸，所述多核苷酸编码本申请第一方面任一实施例所述的抗体或抗原结合片段。
根据本申请的实施例，所述多核苷酸包括下列至少之一：
具有SEQ ID NO:13所示的轻链可变区核苷酸序列和SEQ ID NO:14所示的重链可变区核苷酸序列；
具有SEQ ID NO:15所示的轻链可变区核苷酸序列和SEQ ID NO:16所示的重链可变区核苷酸序列；
具有SEQ ID NO:17所示的轻链可变区核苷酸序列和SEQ ID NO:18所示的重链可变区核苷酸序列；
具有SEQ ID NO:19所示的轻链核苷酸序列和SEQ ID NO:20所示的重链核苷酸序列；
具有SEQ ID NO:21所示的轻链核苷酸序列和SEQ ID NO:22所示的重链核苷酸序列；
具有SEQ ID NO:23所示的轻链核苷酸序列和SEQ ID NO:24所示的重链核苷酸序列；
与所述任一轻链可变区核苷酸序列相比，具有90%以上同源性的序列，任选具有95%以上同源性的序列，优选为具有98%以上同源性的序列，更优选为具有99%以上同源性的序列；
与所述任一的重链可变区核苷酸序列相比，具有90%以上同源性的序列，任选具有95%以上同源性的序列，优选为具有98%以上同源性的序列，更优选为具有99%以上同源性的序列；
与上述任一轻链核苷酸序列相比，具有90%以上同源性的序列，任选具有95%以上同源性的序列，优选为具有98%以上同源性的序列，更优选为具有99%以上同源性的序列；
与上述任一重链核苷酸序列相比，具有90%以上同源性的序列，任选具有95%以上同源性的序列，优选为具有98%以上同源性的序列，更优选为具有99%以上同源性的序列。
根据本申请的第三方面，本申请提供了一种表达载体，包含本申请第二方面任一实施例所述的多核苷酸。
在本申请的一些实施例中，以上所述表达载体进一步包括：控制元件，所述控制元件与所述多核苷酸可操作地连接，用于控制所述多核苷酸在宿主细胞中的表达。
在本申请的一些实施例中，所述控制元件包括下列至少之一：启动子、增强子和终止子。
在本申请的一些实施例中，所述宿主细胞为哺乳动物细胞。
根据本申请的第四方面，本申请提供了一种重组细胞，包含本申请第三方面任一实施例所述的表达载体。
根据本申请的第五方面，本申请提供了一种制备抗ROR2抗体或其抗原结合片段的方法，包括培养本申请第四方面所述的重组细胞。
根据本申请的第六方面，本申请提供了一种抗体或抗原结合片段在制备药物中的用途，所述药物用于治疗癌症，所述抗体或抗原结合片段为本申请第一方面所述的抗体或抗原结合片段。
根据本申请的第七方面，本申请提供了一种抗体或抗原结合片段在制备试剂盒中的用途，所述试剂盒用于ROR2抗原的诊断检测，所述抗体或抗原结合片段为本申请第一方面所述的抗体或抗原结合片段。
在本申请的一些实施例中，所述试剂盒用于免疫印迹、免疫沉淀、ELISA检测。
根据本申请的第八方面，本申请提供了一种药物组合物，包括：本申请第一方面任一实施例所述的抗体或抗原结合片段和药学上可接受的载体。
根据本申请的第九方面，本申请提供了一种抗ROR2的嵌合抗原受体，包括：胞外区、跨膜区和胞内区，所述胞外区包括抗体或抗原结合片段，所述抗体或抗原结合片段为单链，所述抗体或抗原结合片段为本申请第一方面任一实施例所述的抗体或抗原结合片段。
根据本申请的第十方面，本申请提供了一种Car-T细胞，所述Car-T细胞表达本申请第九方面所述的抗ROR2的嵌合抗原受体。
 附图说明
图1是根据本申请的实施例提供的不同浓度的A12，B22，B30的Fab 抗体的ELISA检测结果图。
图2是根据本申请的实施例提供的不同抗体的SDS-PAGE鉴定结果图。
图3是根据本申请的实施例提供的不同浓度的嵌合抗体的ELISA检测结果图。
图4是根据本申请的实施例提供的T47D细胞和231细胞中ROR2表达情况的SDS-PAGE鉴定结果图。
图5是根据本申请的实施例提供的不同嵌合抗体检测T47D细胞中ROR2的流式细胞测定结果图。
图6是根据本申请的实施例提供的不同嵌合抗体检测231细胞中ROR2的流式细胞测定结果图。
图7是根据本申请的实施例提供的利用A12嵌合抗体检测血液癌症病人ROR2表达情况的流式测定结果。
图8是根据本申请的实施例提供的利用A12嵌合抗体检测淋巴瘤病人组织ROR2表达情况的流式测定结果。
图9是根据本申请的实施例提供的利用不同嵌合抗体进行的免疫印迹测定结果图。
图10是根据本申请的实施例提供的不同嵌合抗体用于内源性ROR2蛋白检测的免疫沉淀测定结果图。
图11是根据本申请的实施例提供的不同嵌合抗体用于外源性ROR2蛋白检测的免疫沉淀测定结果图。
图12是根据本申请的实施例提供的A12-car结构图。
图13是根据本申请的实施例提供的能够表达CAR-T的淋巴细胞的比例的流式细胞测定结果图。
图14是根据本申请的实施例提供的A12-Car-T对ROR2表达阳性T47D细胞和ROR2表达阴性231细胞的杀伤结果图。
 本发明的实施方式
下面详细描述本申请的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本申请，而不能理解为对本申请的限制。
在对本申请描述的过程中，对于本文中有关的术语进行了解释和说明，这些解释和说明仅仅是为了方便对于方案的理解，并不能看做是对本申请保护方案的限制。
抗体
本文中，术语“抗体”是能够与特异性抗原结合的免疫球蛋白分子。包括两条分子量较轻的轻链和两条分子量较重的重链，重链（H链）和轻链（L链）由二硫键连接形成一个四肽链分子。其中，肽链的氨基端（N端）氨基酸序列变化很大，称为可变区（V区），羧基端（C端）相对稳定，变化很小，称为恒定区（C区）。L链和H链的V区分别称为VL和VH。
在可变区中某些区域氨基酸组成和排列顺序具有更高的变化程度，称为高变区（Hypervariable region，HVR），高变区为抗原和抗体结合的位置，因此也称为决定簇互补区（complementarity-determining region，CDR）。重链可变区和轻链可变区上均有三个CDR区。
本申请利用ROR2胞外段，通过免疫获得了高特异性的高亲和力的抗ROR2的Fab（antigen-binding fragment）抗体片段。利用该抗体片段能够与ROR2抗原特异性结合，从而可以靶向性***等疾病。
在一些实施方案中，本申请提供了一种抗体或者抗原结合片段，所述抗体或者抗原结合片段具有CSASSSVSYMHWYQ、IYDTSKLAS和CQQWSSNPPTFGAG所示的轻链可变区，以及具有YTITSYLMHWV、LEWIGYINPYNDGTKYNEKFKDKAT和CARSDVYYGVRFAYWGQG所示的重链可变区。在另一些实施方案中，所述抗体或抗原结合片段分别具有CKASQNVGTNVAWFQ、IYLASYRYS和CQQYNSYPLTFGGG所示的轻链可变区，以及具有YTFTNYWIQWM、LEWIGEINPSNGRTDYNEKFKNRAT和CANYRPGYWGQG所示的重链可变区。在又一些实施方案中，所述抗体或抗原结合片段分别具有CSASSSISYMYWYQ、IYDTSILAS和CQQWSSYPFTFGSG所示的轻链可变区，以及YTFTSYLIHWV、LEWIGYINPYNDGTKYNEKFKDKAT和CARSDVYYGVRFAYWGQG所示的重链可变区。以CSASSSVSYMHWYQ为例，其作为轻链可变区上的CDR区，能够高度特异性和抗原结合。具有上述CDR区的抗体或者抗原结合片段，能够特异性结合ROR2抗原，发挥ROR2抗体的功能。
在另一些实施方案中，本申请所提供的抗体或者抗原结合片段与上述任一轻链可变区和重链可变区相比，具有一个以上保守氨基酸取代。“抗原结合片段”是指保持特异性结合抗原（ROR2）能力的抗体片段。“保守氨基酸取代”指的是氨基酸被另一氨基酸发生生物学上、化学上或者结构上相似的残基所取代。生物学上相似的指的是该取代不破坏ROR2抗体或者与ROR2抗原的生物学活性。结构上相似指的是氨基酸具有相似长度的侧链，如丙氨酸、甘氨酸或丝氨酸，或具有相似大小的侧链。化学相似性指的是氨基酸具有相同的荷电或者都是亲水或者疏水的。例如疏水残基异亮氨酸、缬氨酸、亮氨酸或者甲硫氨酸相互取代。或者用极性氨基酸例如用精氨酸取代赖氨酸、谷氨酸取代天冬氨酸、谷氨酰胺取代天冬酰胺，丝氨酸取代苏氨酸等等。
在一些实施方案中，本申请提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段具有SEQ ID NO:1所示的轻链可变区序列和SEQ ID NO:2所示的重链可变区序列。在另一些实施方案中，所述抗体或抗原结合片段的轻链可变区序列与SEQ ID NO:1所示氨基酸序列相比，具有一个以上保守氨基酸取代。在一些实施方案中，所述抗体或抗原结合片段的重链可变区序列与SEQ ID NO:2所示氨基酸序列相比，具有一个以上保守氨基酸取代。
在一些实施方案中，本申请提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段具有SEQ ID NO:3所示的轻链可变区序列和SEQ ID NO:4所示的重链可变区序列。在另一些实施方案中，所述抗体或抗原结合片段的轻链可变区序列与SEQ ID NO:3所示氨基酸序列相比，具有一个以上保守氨基酸取代。在一些实施方案中，所述抗体或抗原结合片段的重链可变区序列与SEQ ID NO:4所示氨基酸序列相比，具有一个以上保守氨基酸取代。
在一些实施方案中，本申请提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段具有SEQ ID NO:5所示的轻链可变区序列和SEQ ID NO:6所示的重链可变区序列。在另一些实施方案中，所述抗体或抗原结合片段的轻链可变区序列与SEQ ID NO:5所示氨基酸序列相比，具有一个以上保守氨基酸取代。在一些实施方案中，所述抗体或抗原结合片段的重链可变区序列与SEQ ID NO:6所示氨基酸序列相比，具有一个以上保守氨基酸取代。
当然，这些保守氨基酸取代不会对抗体或者抗原结合片段的生物学功能带来改变。在一些具体方式中，这些保守氨基酸取代可以发生在重链可变区和轻链可变区中除了CDR区之外的氨基酸上。
其中，具有SEQ ID NO:1所示轻链可变区序列和SEQ ID NO:2所示重链可变区序列的抗体也称为A12抗体，其轻链可变区序列VL（SEQ ID NO：1）为：
DIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQQWSSNPPTFGAGTKLELK；
其重链可变区序列VH（SEQ ID NO:2）为：
QVQLQQSGPELVKPGASVRMSCKAAGYTITSYLMHWVKQRPGQDLEWIGYINPYNDGTKYNEKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARSDVYYGVRFAYWGQGTLVTVS。
其中，具有SEQ ID NO:3所示轻链可变区序列和SEQ ID NO:4所示重链可变区序列的抗体也称为B22抗体，其轻链可变区序列VL（SEQ ID NO:3）为：
DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWFQQKPGQSPKPLIYLASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGGGTNLEIKR；
其中，B22抗体的重链可变区序列VH（SEQ ID NO:4）为：
QVQLQQPGAALVKPGASVKLSCKASGYTFTNYWIQWMKQRPGQGLEWIGEINPSNGRTDYNEKFKNRATLTVDNSSTTAYMQLSSLTSEDSAVYYCANYRPGYWGQGTSVTVSS。
其中，具有SEQ ID NO:5所示轻链可变区序列和SEQ ID NO:6所示重链可变区序列的抗体也称为B30抗体，其轻链可变区序列VL（SEQ ID NO:5）为：
DIVLTQSPVIMSASPGEKVTMTCSASSSISYMYWYQQKPGSSPRLLIYDTSILASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPFTFGSGTKLEIK；
其中，B30抗体的重链可变区序列VH（SEQ ID NO:6）
QVQLQQSGPELVKPGASVRMSCKAAGYTFTSYLIHWVKQRPGQDLEWIGYINPYNDGTKYNEKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARSDVYYGVRFAYWGQGTLVTVSA。
在一些优选方案中，本申请提供了一种抗ROR2抗体，该抗体具有SEQ ID NO:7所示的轻链和SEQ ID NO:8所示的重链。
其中SEQ ID NO:7为：
DIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQQWSSNPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
SEQ ID NO:8为：
QVQLQQSGPELVKPGASVRMSCKAAGYTITSYLMHWVKQRPGQDLEWIGYINPYNDGTKYNEKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARSDVYYGVRFAYWGQGTLVTVSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
在一些优选方案中，本申请提供了一种抗ROR2抗体，该抗体具有SEQ ID NO:9所示的轻链和SEQ ID NO:10所示的重链。
其中SEQ ID NO:9为：
DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWFQQKPGQSPKPLIYLASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGGGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
SEQ ID NO:10为：
QVQLQQPGAALVKPGASVKLSCKASGYTFTNYWIQWMKQRPGQGLEWIGEINPSNGRTDYNEKFKNRATLTVDNSSTTAYMQLSSLTSEDSAVYYCANYRPGYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
在一些优选方案中，本申请提供了一种抗ROR2抗体，该抗体具有SEQ ID NO:11所示的轻链和SEQ ID NO:12所示的重链。
其中SEQ ID NO:11为：
DIVLTQSPVIMSASPGEKVTMTCSASSSISYMYWYQQKPGSSPRLLIYDTSILASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
SEQ ID NO:12为：
QVQLQQSGPELVKPGASVRMSCKAAGYTFTSYLIHWVKQRPGQDLEWIGYINPYNDGTKYNEKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARSDVYYGVRFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
多核苷酸、表达载体、重组细胞
在制备或者获取这些抗体的过程中，可以利用表达这些抗体的多核苷酸，与不同的载体连接，然后在不同细胞中表达，来获得相应抗体。
为此，本申请还提供了一种分离的多核苷酸，所述多核苷酸编码上述所述的抗体或抗原结合片段。
在一些实施方案中，所述分离的多核苷酸分别编码A12抗体、B22抗体和B30抗体。
其中编码A12抗体的轻链可变区SEQ ID NO:1所示氨基酸序列的核苷酸序列为（SEQ ID NO:13）：
Gacatcgtgctgacccagtctccagccatcatgtctgctagccctggcgagaaagtgacaatgacctgctccgcctcctcctccgtgtcctacatgcactggtatcagcagaagtccggcacctctcctaagcggtggatctacgacacctccaagctggctagcggagtgcctgccagattttccggctctggctctggcacctcttactccctgaccatctcctccatggaagccgaggatgccgccatctactactgccagcagtggtctagcaaccctcctacctttggcgctggcaccaagctggaactgaag。
编码A12抗体的重链可变区SEQ ID NO:2所示氨基酸序列的核苷酸序列为（SEQ ID NO:14）：
Caggttcagctgcagcagtctggacctgagctggttaagcctggtgcctccgtccggatgtcttgcaaggctgctggctacaccatcaccagctacctgatgcactgggtcaagcagaggccaggccaggacttggagtggatcggctacatcaacccctacaacgacggcaccaagtacaacgagaagttcaaggacaaggctaccctgacctccgacaagtcctcctccaccgcctacatggaactgtccagcctgacctctgaggactccgccgtgtactactgcgccagatccgatgtgtactatggcgtcagattcgcctactggggccagggcacactggtcacagtttct。
其中编码B22抗体的轻链可变区SEQ ID NO:3所示氨基酸序列的核苷酸序列为（SEQ ID NO:15）：
Gacatcgtgctgacccagtctccagccatcatgtctgctagccctggcgagaaagtgacaatgacctgctccgcctcctcctccgtgtcctacatgcactggtatcagcagaagtccggcacctctcctaagcggtggatctacgacacctccaagctggctagcggagtgcctgccagattttccggctctggctctggcacctcttactccctgaccatctcctccatggaagccgaggatgccgccatctactactgccagcagtggtctagcaaccctcctacctttggcgctggcaccaagctggaactgaag。
编码B22抗体的重链可变区SEQ ID NO:4所示氨基酸序列的核苷酸序列为（SEQ ID NO:16）：
Caggttcagttgcagcaacctggcgctgctctggttaagcctggcgcttctgtgaagctgtcctgcaaggcttccggctacaccttcaccaactattggatccagtggatgaagcagcggccaggccaaggcctggaatggatcggagagatcaacccttctaacggccggaccgactacaacgagaagttcaagaaccgggctaccctgaccgtggacaactcttctaccaccgcctacatgcagctgtccagcctgacctctgaggactccgccgtgtactactgcgccaactacagacctggctattggggccagggcacctctgtgacagtctcttct。
其中编码B30抗体的轻链可变区SEQ ID NO:5所示氨基酸序列的核苷酸序列为（SEQ ID NO:17）：
Gacatcgtgatgacccagagccagaaattcatgtccacctccgtgggcgacagagtgtccgtgacatgcaaggcctctcagaacgtgggcaccaacgtggcctggttccagcagaaacctggccagtctcctaagcctctgatctacctggcctcctaccggtactctggcgtgcccgatagattcaccggctctggatctggcaccgacttcaccctgaccatctccaacgtgcagtctgaggacctggccgagtacttctgccagcagtacaacagctaccctctgacctttggcggaggcaccaacctggaaatcaag。
编码B30抗体的重链可变区SEQ ID NO:6所示氨基酸序列的核苷酸序列为（SEQ ID NO:18）：
Caggttcagctgcagcagtctggacctgagctggttaagcctggtgcctccgtccggatgtcttgcaaggctgccggctacaccttcaccagctacctgatccactgggtcaagcagaggccaggccaggacttggagtggatcggctacatcaacccctacaacgacggcaccaagtacaacgagaagttcaaggacaaggctaccctgacctccgacaagtcctcctccaccgcctacatggaactgtccagcctgacctctgaggactccgccgtgtactactgcgccagatccgatgtgtactatggcgtcagattcgcctactggggccagggcacactggtcacagtttctgct。
其中编码A12抗体的轻链SEQ ID NO:7所示氨基酸序列的核苷酸序列为（SEQ ID NO:19）
Gacatcgtgctgacccagtctccagccatcatgtctgctagccctggcgagaaagtgacaatgacctgctccgcctcctcctccgtgtcctacatgcactggtatcagcagaagtccggcacctctcctaagcggtggatctacgacacctccaagctggctagcggagtgcctgccagattttccggctctggctctggcacctcttactccctgaccatctcctccatggaagccgaggatgccgccatctactactgccagcagtggtctagcaaccctcctacctttggcgctggcaccaagctggaactgaagagaacagtggccgctcctagcgtgttcatcttcccaccttccgacgagcagctgaagtctggcacagcctctgtcgtgtgcctgctgaacaacttctaccccagagaagccaaggtgcagtggaaggtggacaacgccctgcagagcggcaatagccaagagagcgtgaccgagcaggacagcaaggactctacctacagcctgagcagcaccctgacactgagcaaggccgactacgagaagcacaaagtgtacgcctgcgaagtgacccaccagggcctttctagccctgtgaccaagagcttcaaccggggcgaatgt；
其中编码A12抗体的重链SEQ ID NO:8所示氨基酸序列的核苷酸序列为（SEQ ID NO:20）
Caggttcagctgcagcagtctggacctgagctggttaagcctggtgcctccgtccggatgtcttgcaaggctgctggctacaccatcaccagctacctgatgcactgggtcaagcagaggccaggccaggacttggagtggatcggctacatcaacccctacaacgacggcaccaagtacaacgagaagttcaaggacaaggctaccctgacctccgacaagtcctcctccaccgcctacatggaactgtccagcctgacctctgaggactccgccgtgtactactgcgccagatccgatgtgtactatggcgtcagattcgcctactggggccagggcacactggtcacagtttctgcctctacaaagggccctagtgtgttccctctggctcccagcagcaagtctacatctggcggaacagccgctctgggctgcctggtcaaggattactttcccgagcctgtgaccgtgtcctggaatagcggagcactgacaagcggcgtgcacacctttccagctgtgctgcaaagcagcggcctgtactctctgagcagcgtggtcacagtgcctagctctagcctgggcacccagacctacatctgcaatgtgaaccacaagcctagcaacaccaaggtggacaagaaggtggaacccaagagctgcgacaagacccacacctgtcctccatgtcctgctccagaactgctcggcggaccttccgtgttcctgtttcctccaaagcctaaggacaccctgatgatcagcagaacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgctcctatcgagaaaaccatcagcaaggccaagggccagcctagggaaccccaggtttacacactgcctccaagcagggacgagctgaccaagaatcaggtgtccctgacctgcctcgtgaagggcttctacccttccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactacaagacaacccctcctgtgctggacagcgacggctcattcttcctgtacagcaagctgacagtggacaagtccagatggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgtctcctggcaaa。
其中编码B22抗体的轻链SEQ ID NO:9所示氨基酸序列的核苷酸序列为（SEQ ID NO:21）
Gacatcgtgatgacccagagccagaaattcatgtccacctccgtgggcgacagagtgtccgtgacatgcaaggcctctcagaacgtgggcaccaacgtggcctggttccagcagaaacctggccagtctcctaagcctctgatctacctggcctcctaccggtactctggcgtgcccgatagattcaccggctctggatctggcaccgacttcaccctgaccatctccaacgtgcagtctgaggacctggccgagtacttctgccagcagtacaacagctaccctctgacctttggcggaggcaccaacctggaaatcaagagaacagtggccgctcctagcgtgttcatcttcccaccttccgacgagcagctgaagtctggcacagcctctgtcgtgtgcctgctgaacaacttctaccccagagaagccaaggtgcagtggaaggtggacaacgccctgcagagcggcaatagccaagagagcgtgaccgagcaggacagcaaggactctacctacagcctgagcagcaccctgacactgagcaaggccgactacgagaagcacaaagtgtacgcctgcgaagtgacccaccagggcctttctagccctgtgaccaagagcttcaaccggggcgaatgt。
其中编码B22抗体的重链SEQ ID NO:10所示氨基酸序列的核苷酸序列为（SEQ ID NO:22）
Caggttcagttgcagcaacctggcgctgctctggttaagcctggcgcttctgtgaagctgtcctgcaaggcttccggctacaccttcaccaactattggatccagtggatgaagcagcggccaggccaaggcctggaatggatcggagagatcaacccttctaacggccggaccgactacaacgagaagttcaagaaccgggctaccctgaccgtggacaactcttctaccaccgcctacatgcagctgtccagcctgacctctgaggactccgccgtgtactactgcgccaactacagacctggctattggggccagggcacctctgtgacagtctcttctgcctctacaaagggccctagtgtgttccctctggctcccagcagcaagtctacatctggcggaacagccgctctgggctgcctggtcaaggattactttcccgagcctgtgaccgtgtcctggaatagcggagcactgacaagcggcgtgcacacctttccagctgtgctgcaaagcagcggcctgtactctctgagcagcgtggtcacagtgcctagctctagcctgggcacccagacctacatctgcaatgtgaaccacaagcctagcaacaccaaggtggacaagaaggtggaacccaagagctgcgacaagacccacacctgtcctccatgtcctgctccagaactgctcggcggaccttccgtgttcctgtttcctccaaagcctaaggacaccctgatgatcagcagaacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgctcctatcgagaaaaccatcagcaaggccaagggccagcctagggaaccccaggtttacacactgcctccaagcagggacgagctgaccaagaatcaggtgtccctgacctgcctcgtgaagggcttctacccttccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactacaagacaacccctcctgtgctggacagcgacggctcattcttcctgtacagcaagctgacagtggacaagtccagatggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgtctcctggcaaa。
其中编码B30抗体的轻链SEQ ID NO:11所示氨基酸序列的核苷酸序列为（SEQ ID NO:23）
Gacatcgtgctgacccagtctccagtgatcatgtccgcttctcccggcgagaaagtgacaatgacctgctccgcctcctccagcatctcctacatgtactggtatcagcagaagcccggctcctctcctcggctgctgatctacgatacctccatcctggcttccggcgtgccagtgcggttttctggttctggctctggcacctcctacagcctgaccatctccagaatggaagccgaggacgccgccacctactactgtcagcagtggtctagctaccccttcacctttggctccggcaccaagctggaaatcaagagaacagtggccgctcctagcgtgttcatcttcccaccttccgacgagcagctgaagtctggcacagcctctgtcgtgtgcctgctgaacaacttctaccccagagaagccaaggtgcagtggaaggtggacaacgccctgcagagcggcaatagccaagagagcgtgaccgagcaggacagcaaggactctacctacagcctgagcagcaccctgacactgagcaaggccgactacgagaagcacaaagtgtacgcctgcgaagtgacccaccagggcctttctagccctgtgaccaagagcttcaaccggggcgaatgt。
其中编码B30抗体的重链SEQ ID NO:12所示氨基酸序列的核苷酸序列为（SEQ ID NO:24）：
Caggttcagctgcagcagtctggacctgagctggttaagcctggtgcctccgtccggatgtcttgcaaggctgccggctacaccttcaccagctacctgatccactgggtcaagcagaggccaggccaggacttggagtggatcggctacatcaacccctacaacgacggcaccaagtacaacgagaagttcaaggacaaggctaccctgacctccgacaagtcctcctccaccgcctacatggaactgtccagcctgacctctgaggactccgccgtgtactactgcgccagatccgatgtgtactatggcgtcagattcgcctactggggccagggcacactggtcacagtttctgctgcctctacaaagggccctagtgtgttccctctggctcccagcagcaagtctacatctggcggaacagccgctctgggctgcctggtcaaggattactttcccgagcctgtgaccgtgtcctggaatagcggagcactgacaagcggcgtgcacacctttccagctgtgctgcaaagcagcggcctgtactctctgagcagcgtggtcacagtgcctagctctagcctgggcacccagacctacatctgcaatgtgaaccacaagcctagcaacaccaaggtggacaagaaggtggaacccaagagctgcgacaagacccacacctgtcctccatgtcctgctccagaactgctcggcggaccttccgtgttcctgtttcctccaaagcctaaggacaccctgatgatcagcagaacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacagtacaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgctcctatcgagaaaaccatcagcaaggccaagggccagcctagggaaccccaggtttacacactgcctccaagcagggacgagctgaccaagaatcaggtgtccctgacctgcctcgtgaagggcttctacccttccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactacaagacaacccctcctgtgctggacagcgacggctcattcttcctgtacagcaagctgacagtggacaagtccagatggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgtctcctggcaaa。
在又一些实施方案中，所述分离的多核苷酸与上述编码氨基酸的序列至少具有90%以上的同源性，优选具有95%以上的同源性，更优选具有98%、99%以上的同源性。
本申请还提供了一种表达载体，所述表达载体包含上述分离的多核苷酸。在将上述分离的多核苷酸连接到载体上时，可以将多核苷酸与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制多核苷酸的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。这些控制元件可以是启动子、增强子或者终止子等，只要能够调控基因的表达即可。而多核苷酸与控制元件只需可操作地连接即可。本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。当然用来编码抗体重链和轻链的多核苷酸，可以分别独立的***到不同的载体上，常见的是***到同一载体上。常用的载体例如可以为质粒、噬菌体等等。例如pcDNA质粒。
本申请还提供了一种重组细胞，该重组细胞中包含有该表达载体。可以将表达载体导入到哺乳动物细胞（如293F细胞，CHO细胞等）中，构建获得重组细胞，然后利用这些重组细胞表达本申请提供的抗体或者抗原结合片段。通过该重组细胞进行培养，即可以获得相应抗体。
药物组合物、试剂盒及制药用途和在制备试剂盒中的用途。
本申请还提供了一种药物组合物，所述药物组合物包括上述所述的抗体或者抗原结合片段和药学可接受的载体。
本文提供的抗ROR2抗体可以掺入适合受试者施用的药物组合物中。通常，这些药物组合物包括本文提供的抗ROR2抗体以及药学上可接受的载体。“药学上可接受的载体”可以包括生理学上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等等。具体实例可以是水、盐水、磷酸盐缓冲盐水、葡萄糖、甘油、乙醇等以及它们的组合物中的一种或多种。有许多情况下，药物组合物中包括等渗剂，例如糖类、多元醇( 如甘露醇、山梨醇) 或氯化钠等。当然药学上可接受的载体还可包括微量的辅助物质，例如润湿剂或乳化剂、防腐剂或缓冲剂，用来延长抗体的保存限期或效力。
例如，本申请的抗体可掺入适用于胃肠外施用( 例如静脉内、皮下、腹膜内、肌肉内) 的药物组合物中。这些药物组合物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液( 例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。典型的药物组合物为注射溶液或输注溶液形式。所述抗体可通过静脉输注或注射或肌肉内或皮下注射来施用。
当然，本文中的抗ROR2抗体还可以根据需要被制成试剂盒或者其他诊断性试剂的一部分。根据本申请的实施例，本申请还提供了一种试剂盒，所述试剂盒包括上述ROR2抗体。应用本申请提供的试剂盒，例如可以用于免疫印迹、免疫沉淀等涉及到利用ROR2抗原和抗体特异性结合性能，来检测的试剂盒等。这些试剂盒可包含下列中的任意一种或多种：拮抗剂、抗ROR2抗体或者药物参照材料；蛋白纯化柱；免疫球蛋白亲和纯化缓冲剂；细胞的测定稀释剂；说明书或者文献等。抗ROR2抗体可被用于不同类型的诊断测试，例如可以在体外或者体内检测各种各样的疾病或者药物、毒素或者其他蛋白等的存在。例如可以通过对受试者的血清或者血液进行检测，用来测试相关疾病。这种相关疾病可包括ROR2相关疾病，例如各种癌症等等。当然本文提供的抗体也可以用于癌症的放射免疫检测和放射免疫治疗等等。
这些癌症或者肿瘤可以是任何不受调控的细胞生长。具体地，可以是肺癌、胃癌、胰腺癌、卵巢癌、肝癌、乳腺癌、结直肠癌、淋巴癌、血癌等等。
在利用本申请所提供的抗ROR2抗体治疗癌症时，可以将本申请提供的抗ROR2抗体提供给受试者即可。为此，本申请提供了一种用于治疗癌症的方法，包括向有需要的受试者施用本申请所提供的的抗体或其抗原结合片段。
下面将结合实施例对本申请的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本申请，而不应视为限定本申请的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本申请通过研究，开发了基于ROR2的抗多种肿瘤细胞甚至肿瘤干细胞的基于抗体的免疫治疗方法，包括ROR2的单克隆抗体，及新型的ROR2-Car T细胞治疗技术。
实施例1
我们利用ROR2蛋白，通过免疫小鼠，并借助于噬菌体文库技术，筛选得到B30、B22和A12抗体，这些抗体的ELISA检测如图1所示。其中图1中横坐标代表不同抗体的浓度，纵坐标代表所测定的OD450值。
对这些抗体样本进行氨基酸测序，其中一种抗体的轻链可变区氨基酸序列和重链可变区氨基酸序列分别为SEQ ID NO:1和SEQ ID NO:2，命名为A12抗体；一种抗体的轻链可变区氨基酸序列和重链可变区氨基酸序列分别为SEQ ID NO:3和SEQ ID NO:4，命名为B22抗体；一种抗体的轻链可变区氨基酸序列和重链可变区氨基酸序列分别为SEQ ID NO:5和SEQ ID NO:6，命名为B30抗体。
检测得到，A12抗体的轻链氨基酸序列为SEQ ID NO:7，重链氨基酸序列为SEQ ID NO:8；B22抗体的轻链氨基酸序列为SEQ ID NO:9，重链氨基酸序列为SEQ ID NO:10；B30抗体的轻链氨基酸序列为SEQ ID NO:11，重链氨基酸序列为SEQ ID NO:12。
实施例2
（一）ROR2嵌合抗体的表达与纯化
分别利用A12抗体、B22抗体和B30抗体的可变区氨基酸序列推导出其DNA序列，并根据表达载体进行种属优化后建立嵌合抗体的表达序列，并克隆进pcDNA3.4载体内。
其中，经过优化后未改变抗体的氨基酸序列，仅仅进行了密码子优化，优化后的编码氨基酸序列的核苷酸序列更容易在哺乳动物细胞中表达。因此，例如当表述A12嵌合抗体时，其氨基酸序列与上述A12抗体氨基酸序列相同，差别在于编码A12嵌合抗体的核苷酸序列进行了优化。
具体地，编码A12嵌合抗体的轻链的核苷酸序列为SEQ ID NO:19，编码A12嵌合抗体的重链的核苷酸序列为SEQ ID NO:20；
编码B22嵌合抗体的轻链的核苷酸序列为SEQ ID NO:21，编码B22嵌合抗体的重链的核苷酸序列为SEQ ID NO:22；
编码B30嵌合抗体的轻链的核苷酸序列为SEQ ID NO:23，编码B22嵌合抗体的重链的核苷酸序列为SEQ ID NO:24。
再将构建好的质粒用PEI转染到293F细胞中，进行表达48小时。48小时后，回收上清，用ProteinA柱子进行纯化和层析/浓缩。纯化后的蛋白用SDS-PAGE胶进行鉴定，结果如图2所示，图2是ROR2-B30（SA120-1），B22（SA120-2），A12（SA120-3）纯化抗体SDS-PAGE鉴定结果，结果显示我们纯化的抗体具有较高的纯度。
图2中，从左向右，第一、二、三列代表为上样量为2.0μg的非还原性A12、B22、B30抗体样本的SDS-PAGE胶鉴定结果，第四列代表蛋白marker，第五、六、七列代表上样量为2.0μg的还原性A12、B22、B30抗体样本的SDS-PAGE胶鉴定结果，第八、九、十列分别代表上样量为0.5，1.0，2μg的还原性胎牛血清蛋白（BSA）样本的SDS-PAGE胶结果。
从图2可以看出，SDS-PAGE胶中显示还原性和非还原性的A12、B22、B30嵌合抗体条带大小正确，条带灰度值接近2μg的还原性胎牛血清蛋白（BSA）样本，说明我们纯化的抗体具有较高的纯度。
（二）ELASA验证纯化的ROR2嵌合抗体的亲和力
96孔板中加入ROR2胞外段蛋白包被过夜，5%牛奶封闭后，将纯化好的抗体进行稀释成不同浓度加入不同孔中，进行ELASA检测。OD450如图3所示，图3是不同浓度的B30嵌合抗体，B22嵌合抗体和A12嵌合抗体的OD450值，可见当嵌合抗体浓度较低（0.001ug/ml或0.0001ug/ml）时，抗体就已经具有亲和力，同时也说明我们的嵌合抗体能用于ELISA，且具有较高的亲和力。
（三）流式细胞术检测抗体的结合能力
纯化得到的抗体进行流式染色，进行检测T47D细胞（ROR2表达阳性，见图4）和231乳腺癌细胞（ROR2表达阴性，见图4）。结果如下图所示。流式结果显示，三个嵌合抗体都能特异性结合ROR2膜蛋白。
其中图4为SantaCruz公司ROR2抗体检测T47D细胞和231细胞的ROR2的表达水平。图5为三个嵌合抗体流式检测T47D的ROR2的表达水平的结果，其中三个嵌合抗体的浓度均为10ug/ml。图6为三个嵌合抗体流式检测231的ROR2的表达水平的结果，三个嵌合抗体浓度均为10ug/ml。
实施例3 
（一）A12嵌合抗体能检测不同血液癌症病人和淋巴瘤PDX的ROR2水平
获取不同血液癌症的病人的外周血，利用Ficoll分离出血液中的淋巴细胞，然后加入A12嵌合抗体（浓度为3ug/ml），孵育1小时，然后和Alexa-488结合的抗小鼠IgG二抗孵育，并进行流式分析，结果如图7。其中图7中，ALL对应结果代表急性淋巴细胞白血病病人样本的流式检测结果，AML对应结果代表急性髓细胞白血病病人样本的流式检测结果。
从医院取得淋巴瘤的病人组织，然后建立PDX模型。取PDX小鼠长出的肿瘤组织，用human dissociation kit 将组织分离成单细胞，然后加入A12嵌合抗体（浓度为 3μg/ml ），孵育1小时，然后Alexa-488结合的抗小鼠IgG二抗孵育，并进行流式分析，从而对肿瘤细胞的ROR2水平进行检查。结果如图8所示。图8左图显示的是淋巴瘤病人肿瘤细胞的ROR2荧光强度的峰图，右边两个散点图显示淋巴瘤病人肿瘤细胞空白管和B16抗体染色管的细胞荧光强度分布情况。
（二）ROR2嵌合抗体能应用于免疫印迹（WB）
将带有His标签的鼠源的ROR2胞外端（mouseROR2ex）和人源的ROR2胞外端（humanROR2ex）质粒和空载GFP转染到ROR2敲除的293T细胞（293T-ROR2-KO）中，48小时后，裂解细胞，提取总蛋白，变性后，用SDS-PAGE胶分离蛋白并进行免疫印迹反应，用三个ROR2的嵌合抗体（5ug/ml）检测ROR2的表达。结果如图9所示，表明ROR2-B30嵌合抗体，B22嵌合抗体和A12嵌合抗体均能应用于免疫印迹检测，且只能识别人的ROR2蛋白的胞外端。
（三）ROR2嵌合抗体能应用于免疫沉淀（IP）
为了鉴定ROR2嵌合抗体能否用于免疫沉淀，我们用ROR2表达阳性的293T细胞进行IP。将细胞裂解后，分别用B30嵌合抗体，B22嵌合抗体和A12嵌合抗体进行IP，IP后的蛋白用SantaCruz的ROR2（货号：sc-374174）抗体进行免疫印迹检测ROR2蛋白。其中，IgG抗体作为阴性对照抗体（不能将ROR2沉淀下来），Input为未进行免疫沉淀的细胞裂解液样本（能够表达ROR2蛋白）。结果如图10，IP结果表明三个抗体均能用于IP，能用于检测内源性的ROR2。
为了进一步验证ROR2抗体是否能进行IP，我们给293T细胞转染带有Flag标签的pcDNA-ROR2-Flag质粒。Flag标签是由八个亲水氨基酸DYKDDDDK组成的多肽片段，能够与蛋白融合，而且不会占据蛋白表位或者结构域，避免影响蛋白的功能、分泌性以及转运等各方面性能，利用Flag标签标记目的蛋白，若标签有表达，目的蛋白也会有表达。然后分别用B30嵌合抗体，B22嵌合抗体，A12嵌合抗体和Flag进行IP，分别用Flag和B30嵌合抗体，B22嵌合抗体，A12嵌合抗体进行免疫印迹（IB）。结果如图11所示，结果显示不管是ROR2抗体还是Flag标记抗体进行IP，IB结果中都能检测到ROR2（130kD）条带。
从以上实验可以看出，通过利用免疫小鼠的脾脏构建噬菌体文库，筛选高亲和力的特异性识别ROR2的单克隆抗体。然后进行诊断功能的检测，验证了我们筛选获得的特异性识别ROR2的抗体，可以作为临床精确诊断肿瘤细胞做出帮助；为将来进入临床治疗ROR2阳性的肿瘤病人奠定基础。并且可以确定ROR2抗体影响的信号通路，为将来的临床试验中提供用来预测病人对药物的反应的生物标记（biomarker)。
实施例4
实验进一步验证了本申请提供的ROR2抗体，开发ROR2-Car T细胞治疗技术，用于疾病的靶向治疗。
1、ROR2-A12 Car的构建
我们采用二代的Car序列，用筛选出来的具有高亲和力的抗ROR2的抗体A-12构建ROR2-Car。
2、A12 car结构
A12 CAR由CD8 leader，A12 scFv-VL，18氨基酸linker，A12 scFv-VH，CD8铰链区，CD8跨膜区，4-1BB共刺激因子结构，CD3ζ链结构构成，如图12所示。
3、CAR-T能够结合ROR2
用终浓度2μg/ml的生物素化ROR2蛋白与A12-CAR-T细胞冰上共孵育30分钟，然后用带有PE荧光的亲和链霉素标记ROR2蛋白，冰上15分钟，用流式细胞术检测能够与ROR2蛋白结合的CAR-T的比例。流式结果如图13所示。图13中，左图为空白管散点图，右图为ROR2蛋白结合抗体染色散点图，结果显示有59.1%的淋巴细胞表达A12-CarT。
4、CAR-T体外杀伤靶细胞
将未转化的T细胞(NT) ，转化空载的CD532A-T，和转化A12-CAR的CAR-A12-T细胞与两种靶细胞在96孔板中共培养。其中一种是表达ROR2的T-47D乳腺癌细胞系，一种是不表达ROR2的MDA-MB-231乳腺癌细胞系。每孔12000个靶细胞，120000个T细胞，E:T=10:1，共培养12小时，然后用乳酸脱氢酶释放试验检测对靶细胞的杀伤情况。杀伤率根据公式%细胞毒性=（实验组-效应细胞自发-靶细胞自发）/（靶细胞最大-靶细胞自发）x100计算。得出的杀伤率再以未转化的T细胞 (NT) 组对靶细胞的杀伤率为基准进行标准化。结果如图14所示，A12-Car-T能显著杀伤ROR2表达阳性的T47D细胞，但并不能杀伤ROR2表达阴性231细胞。
从以上实验可以看出，开发ROR2-Car T细胞治疗技术，用我们筛选出来的具有高亲和力的ROR2抗体，进一步开发Car-T细胞治疗技术，ROR2-Car T细胞治疗目前国内外没有任何报道。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、 “示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本申请的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本申请的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本申请的限制，本领域的普通技术人员在本申请的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种抗ROR2抗体或抗原结合片段，其中，包括下列至少之一：
    （1）具有CSASSSVSYMHWYQ、IYDTSKLAS和CQQWSSNPPTFGAG所示氨基酸序列的轻链可变区，以及具有YTITSYLMHWV、LEWIGYINPYNDGTKYNEKFKDKAT和CARSDVYYGVRFAYWGQG所示氨基酸序列的重链可变区；
    （2）具有CKASQNVGTNVAWFQ、IYLASYRYS和CQQYNSYPLTFGGG所示氨基酸序列的轻链可变区，以及具有YTFTNYWIQWM、LEWIGEINPSNGRTDYNEKFKNRAT和CANYRPGYWGQG所示氨基酸序列的重链可变区；
    （3）具有CSASSSISYMYWYQ、IYDTSILAS和CQQWSSYPFTFGSG所示氨基酸序列的轻链可变区，以及具有YTFTSYLIHWV、LEWIGYINPYNDGTKYNEKFKDKAT和CARSDVYYGVRFAYWGQG所示氨基酸序列的重链可变区；
    与（1）~（3）相比，具有至少一个保守氨基酸取代的氨基酸序列；
    包括下列至少之一：
    （a）具有SEQ ID NO:1所示氨基酸序列的轻链可变区和SEQ ID NO:2所示氨基酸序列的重链可变区；
    （b）具有SEQ ID NO:3所示氨基酸序列的轻链可变区和SEQ ID NO:4所示氨基酸序列的重链可变区；
    （c）具有SEQ ID NO:5所示氨基酸序列的轻链可变区和SEQ ID NO:6所示氨基酸序列的重链可变区；
    与（a）~（c）相比，具有至少一个保守氨基酸取代的氨基酸序列；
    包括下列至少之一：
    具有SEQ ID NO:7所示氨基酸序列的轻链和SEQ ID NO:8所示氨基酸序列的重链；
    具有SEQ ID NO:9所示氨基酸序列的轻链和SEQ ID NO:10所示氨基酸序列的重链；
    具有SEQ ID NO:11所示氨基酸序列的轻链和SEQ ID NO:12所示氨基酸序列的重链。
  2. 一种分离的多核苷酸，其中，所述多核苷酸编码权利要求1所述的抗体或抗原结合片段；
    所述多核苷酸包括下列至少之一：
    具有SEQ ID NO:13所示的轻链可变区核苷酸序列和SEQ ID NO:14所示的重链可变区核苷酸序列；
    具有SEQ ID NO:15所示的轻链可变区核苷酸序列和SEQ ID NO:16所示的重链可变区核苷酸序列；
    具有SEQ ID NO:17所示的轻链可变区核苷酸序列和SEQ ID NO:18所示的重链可变区核苷酸序列；
    具有SEQ ID NO:19所示的轻链核苷酸序列和SEQ ID NO:20所示的重链核苷酸序列；
    具有SEQ ID NO:21所示的轻链核苷酸序列和SEQ ID NO:22所示的重链核苷酸序列；
    具有SEQ ID NO:23所示的轻链核苷酸序列和SEQ ID NO:24所示的重链核苷酸序列；
    与所述任一轻链可变区核苷酸序列相比，具有90%以上同源性的序列，具有95%以上同源性的序列，具有98%以上同源性的序列，或者，具有99%以上同源性的序列；
    与所述任一的重链可变区核苷酸序列相比，具有90%以上同源性的序列，具有95%以上同源性的序列，具有98%以上同源性的序列，或者，具有99%以上同源性的序列；
    与上述任一轻链核苷酸序列相比，具有90%以上同源性的序列，具有95%以上同源性的序列，具有98%以上同源性的序列，或者，具有99%以上同源性的序列；
    与上述任一重链核苷酸序列相比，具有90%以上同源性的序列，具有95%以上同源性的序列，具有98%以上同源性的序列，或者，具有99%以上同源性的序列。
  3. 一种表达载体，其中，包含权利要求2所述的多核苷酸；
    进一步包括：
    控制元件，所述控制元件与所述多核苷酸可操作地连接，用于控制所述多核苷酸在宿主细胞中的表达；
    所述控制元件包括下列至少之一：启动子、增强子和终止子；
    所述宿主细胞为哺乳动物细胞。
  4. 一种重组细胞，其中，包含权利要求3所述的表达载体。
  5. 一种制备抗ROR2抗体或其抗原结合片段的方法，其中，包括培养权利要求4所述的重组细胞。
  6. 权利要求1所述的抗体或抗原结合片段在制备药物中的用途，所述药物用于治疗癌症。
  7. 权利要求1所述的抗体或抗原结合片段在制备试剂盒中的用途，所述试剂盒用于ROR2抗原的诊断检测；
    所述试剂盒用于免疫印迹、免疫沉淀和/或ELISA检测。
  8. 一种药物组合物，其中，包括：权利要求1所述的抗体或抗原结合片段和药学上可接受的载体。
  9. 一种抗ROR2的嵌合抗原受体，其中，包括：胞外区、跨膜区和胞内区，
    所述胞外区包括抗体或抗原结合片段，所述抗体或抗原结合片段为单链，所述抗体或抗原结合片段为权利要求1所述的抗体或抗原结合片段。
  10. 一种Car-T细胞，其中，所述Car-T细胞表达权利要求9所述的抗ROR2的嵌合抗原受体。
PCT/CN2019/130486 2019-01-08 2019-12-31 检测多种恶性肿瘤细胞的抗体及其应用 WO2020143506A1 (zh)

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