CN116672442B - Preparation of medicine for treating osteosarcoma by combining ligustilide and HER2-CAR-T cells - Google Patents
Preparation of medicine for treating osteosarcoma by combining ligustilide and HER2-CAR-T cells Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
-
- 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
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/00113—Growth factors
- A61K39/001131—Epidermal growth factor [EGF]
-
- 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
- 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
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- 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)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- 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
-
- 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
- C12N2510/00—Genetically modified cells
Abstract
The patent discloses a drug for treating osteosarcoma prepared by combining ligustilide and HER2-CAR-T cells, belonging to the field of biological medicine, and comprising ligustilide and HER2-CAR-T cells. The medicine can not only improve the killing effect on malignant tumor cells such as osteosarcoma, reduce the application dosage of HER2-CAR-T cells and reduce toxic and side effects, but also reduce medical cost. Ligustilide can enhance the recognition and killing effect of HER2-CAR-T cells by up-regulating HER2 antigen expression, and edit, modify and remove the skeleton thereof by chemical method, and various derivatives capable of enhancing the killing ability of CAR-T cells are reserved, including pharmaceutically acceptable isomers, salts, metal complexes and the like. In addition, the medicine is simultaneously suitable for other cancers expressing HER2 antigen, such as esophagus cancer, stomach cancer, pancreas cancer, breast cancer, ovarian cancer, liver cancer, leukemia, prostate cancer, melanoma and the like, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of biological medicine, and discloses application of ligustilide and HER2 antigen-targeted CAR-T lymphocyte combination in preparation of osteosarcoma medicines.
Background
Osteosarcoma is one of the common malignant bone tumors worldwide, well developed in children and young people. Osteosarcoma has the characteristics of high malignancy, high recurrence rate, high early metastasis rate and poor prognosis, and seriously affects the life quality and life cycle of patients. At present, surgical excision and adjuvant chemotherapy are common methods for treating osteosarcoma, and although the methods prolong the life span of patients and improve the life quality to a certain extent, the methods still face the problems of limited treatment effect, chemotherapy drug resistance, side effect, tumor recurrence, distant metastasis and the like. Therefore, the treatment of osteosarcoma has been a problem in the medical field that cannot be overcome, and is a research hotspot, and in order to cope with the above-mentioned difficulty, it is important to explore a novel, safe and effective treatment method.
Chimeric antigen receptor T lymphocyte (Chimeric antigen receptor T cell, CAR-T cell) cell therapy is a novel, promising immunotherapy that potentially cures malignant tumors. Briefly, CAR-T cells consist of two parts, CAR and T lymphocytes, the role of CAR is to specifically recognize and bind to tumor cells, while T lymphocytes act to kill tumor cells, and the two, when combined, accomplish the targeting of killing tumor cells. At present, the national administration of supervision has approved the first CAR-T cell preparation (Abiraterone injection) to be marketed, and the U.S. FDA approved the use of anti-CD 19 CAR-T cells for the clinical treatment of recurrent or refractory large B cell lymphoma adult patients, and has obtained curative effects. Based on the great progress of CAR-T cell therapy in hematological tumors, CAR-T cell immunotherapy brings new ideas for the treatment of osteosarcoma. Since osteosarcoma is a solid tumor, there are many differences between solid tumors and hematological tumors. Several studies have found that CAR-T cells still face a number of problems in the treatment of osteosarcoma, such as tumor antigen escape, off-target effects, difficulty in homing and colonization, immunosuppressive tumor microenvironment, and cytokine release syndrome, which directly affect the therapeutic efficacy of CAR-T cells. The immune system of patients with osteosarcoma is often weaker, the number of immune cells in the body is smaller, and the ability to kill tumor cells is not expected to be strong. Therefore, there is a need for further research on how to enhance the anti-tumor effect and reduce the side effects of CAR-T cell therapies.
Ligustilide (LIG), also known as Dongangelide, is an ester compound, and is widely found in Angelica sinensis (oliv) Diels, ligusticum chuanxiong, an umbrella-type plant. Ligustilide has wide application, and has various pharmacological effects of resisting atherosclerosis, inflammation, dementia, and brain blood supply. Ligustilide can inhibit proliferation activity of lung cancer A549 cells, improve Bax, inhibit expression of Bcl-2 protein, and promote apoptosis of lung cancer cells. Studies have also shown that ligustilide can inhibit the viability and migration ability of liver cancer HCC cells, while hardly damaging normal liver cells. Studies have also shown that ligustilide inhibits the activity of human acute leukemia AML cells, has the ability to block proliferation and cell colony formation. Ligustilide can exert the effect of inhibiting tumor cell proliferation or promoting tumor cell apoptosis in various ways, so that ligustilide may also have the effect of inhibiting osteosarcoma. In addition, compared with chemical preparations, the preparation has wide sources, is safe and easy to obtain, and has great excavation potential in the aspect of preventing and treating related cancers. Currently, there is no report in the industry that ligustilide is combined with CAR-T lymphocytes targeting HER2 antigen to prepare anti-osteosarcoma drugs.
Disclosure of Invention
The invention provides a new application of ligustilide and HER2-CAR-T cell combined preparation in treating osteosarcoma, and proves the application result thereof. Although the present invention is exemplified by anti-osteosarcoma, the drug is equally applicable to other cancers expressing HER2 antigen, such as malignant tumors of esophagus, stomach, pancreas, breast, ovary, liver, leukemia, prostate, melanoma, and the like. The advantages of the ligustilide and HER2-CAR-T cell combined preparation are as follows: firstly, the synergistic effect is exerted, and the killing effect on tumor cells such as osteosarcoma and the like is increased; second, lowering the applied dose of HER2-CAR-T cells; third, reducing the toxic side effects of HER2-CAR-T cell therapy; fourth, due to the economical price of ligustilide, the medical cost can be reduced after the ligustilide and the ligustilide are combined.
The patent applicant researches find that ligustilide can enhance the recognition, binding and killing effect of HER2-CAR-T cells on osteosarcoma, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, ovarian cancer, liver cancer, leukemia, prostatic cancer and melanoma by up-regulating the expression of osteosarcoma HER2 antigen. Importantly, ligustilide can be chemically edited, modified and removed by reduction, substitution and other methods to form side chain groups on the skeleton, so that various derivatives capable of enhancing the killing ability of CAR-T cells are reserved, and pharmaceutically acceptable isomers, salts, metal complexes and the like are included.
The CAS number of ligustilide in the invention is 4431-01-0, molecular formula C 12 H 14 O 2 ，
Molecular weight: 190.24,
the structural formula is as follows:
HER2 antigen is highly expressed on the surface of osteosarcoma cells, but is expressed in small amounts in normal tissue cells, which provides a binding target for CAR-T cells. HER2 antigen is also highly expressed in malignant tumors such as esophageal cancer, gastric cancer, pancreatic cancer, breast cancer and ovarian cancer, so ligustilide combined with HER2-CAR-T cell preparation is equally applicable to these malignant tumors, but is not limited thereto. Ligustilide has strong activity against cancers such as breast cancer, bladder cancer, colon cancer and liver cancer, and has wide application prospect in preventing and treating cancers.
The patent constructs a HER2-CAR structure of a target HER2 antigen, and the nucleotide sequence is SEQ ID No.1.HER2-CAR structure includes an extracellular domain, a transmembrane region, and an intracellular domain. Further, the extracellular domain consists of a CD8 a Leader, HER2scFv single chain antibody, and a CD8 a hinge region; the transmembrane region is composed of CD8 alpha; the intracellular domain consists of the CD28 co-stimulatory domain, the CD137 co-stimulatory domain and the cd3ζ intracellular signaling region.
CD8 alpha Leader is chimeric receptor signal peptide in HER2-CAR, and the nucleotide sequence is SEQ ID No.2.
The HER2scFv is a humanized single-chain antibody of HER2 antigen on the surface of a targeted malignant tumor cell in the HER2-CAR, has the advantages of higher safety and avoidance of causing immune response of human bodies, and has a nucleotide sequence of SEQ ID No.3.
CD8 alpha-hinge/CD 8 alpha TM is the hinge and transmembrane regions of HER2-CAR, and the nucleotide sequence is SEQ ID No.4.
CD28 is intracellular co-stimulatory domain in HER2-CAR, has antigen presenting, T lymphocyte activating and anti-tumor cytokine secretion promoting effects, and has nucleotide sequence of SEQ ID No.5.
CD137 in HER2-CAR is another intracellular co-stimulatory domain, similar to CD28 co-stimulatory domain, and also has antigen presenting, T lymphocyte activation promoting and anti-tumor cytokine secretion effects, and has nucleotide sequence of SEQ ID No.6.
CD3 zeta in HER2-CAR is intracellular signal region, has the function of presenting signal to activate T lymphocyte, and has nucleotide sequence as SEQ ID No.7.
The nucleotide sequence of HER2-CAR and its vector is SEQ ID No.8.
SEQ ID No.1 HER2-CAR
1 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg
61 ccgcagatcc aaatgactca gtcaccatct tctgtatctg cttcagtcgg agaTagggtt
121 acaatcactt gcaaagcctc acaagatgtc tcaataggtg tcgCatggta tcaacagaaa
181 cctggtaagg cccctaagtt gttgatctac tcagcctcat acagatacac cggagtacca
241 tcaaggtcct caggttctgg atcaggtact gactttacct tgaccattcc atcattgcag
301 ccaGaggatt ttgcaaccta ttattgccag caatactaca tctaccctta caccttcgga
361 caaggaacaa aagttgagat taagtcatca ggtggaggag gttcaggtgg aggaggttct
421 ggaggtgaag tacaattagt cgaatcagga ggtggtttgg ttcaacctgg aggatcatta
481 aggttgtctt gtgcagcatc tggtttcaca ttcaccgatt acacaatgga ttgggttaga
541 caggcccctg gaaagggttt ggagtgggtc gccgacgtca accctaattc tggtggatca
601 atctataacc agagattcaa gggaaggttc accttgtcag tggataggtc taagaacacc
661 ttgtatttgc agatgaactc attgagggct gaggatacag ccgtttacta ctgtgccaga
721 aatttgggac cttctttcta cttcgactac tggggtcaag gtactttggt taccgtatct
781 tcaacgcgtt ctagaGTGCC GGTCTTCCTG CCAGCGAAGC CCACCACGAC GCCAGCGCCG
841 CGACCACCAA CACCGGCGCC CACCATCGCG TCGCAGCCCC TGTCCCTGCG CCCAGAGGCG
901 TGCCGGCCAG CGGCGGGGGG CGCAGTGCAC ACGAGGGGGC TGGACTTCGC CTGTGATATC
961 TACATCTGGG CGCCCTTGGC CGGGACTTGT GGGGTCCTTC TCCTGTCACT GGTTATCACC
1021 CTTTACTGCA ACCACAGGAA CAGGAGTAAG AGGAGCAGGC TCCTGCACAG TGACTACATG
1081 AACATGACTC CCCGCCGCCC CGGGCCCACC CGCAAGCATT ACCAGCCCTA TGCCCCACCA
1141 CGCGACTTCG CAGCCTATCG CTCCCGTTTC TCTGTTGTTA AACGGGGCAG AAAGAAGCTC
1201 CTGTATATAT TCAAACAACC ATTTATGAGA CCAGTACAAA CTACTCAAGA GGAAGATGGC
1261 TGTAGCTGCC GATTTCCAGA AGAAGAAGAA GGAGGATGTG AACTGAGAGT GAAGTTCAGC
1321 AGGAGCGCAG ACGCCCCCGC GTACCAGCAG GGCCAGAACC AGCTCTATAA CGAGCTCAAT
1381 CTAGGACGAA GAGAGGAGTA CGATGTTTTG GACAAGAGAC GTGGCCGGGA CCCTGAGATG
1441 GGGGGAAAGC CGAGAAGGAA GAACCCTCAG GAAGGCCTGT ACAATGAACT GCAGAAAGAT
1501 AAGATGGCGG AGGCCTACAG TGAGATTGGG ATGAAAGGCG AGCGCCGGAG GGGCAAGGGG
1561 CACGATGGCC TTTACCAGGG TCTCAGTACA GCCACCAAGG ACACCTACGA CGCCCTTCAC
1621 ATGCAGGCCC TGCCCCCTCG C
SEQ ID No.2 CD8 Leader
1 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg
61 ccg
SEQ ID No.3 HER2 scFv
1 cagatccaaa tgactcagtc accatcttct gtatctgctt cagtcggaga Tagggttaca
61 atcacttgca aagcctcaca agatgtctca ataggtgtcg Catggtatca acagaaacct
121 ggtaaggccc ctaagttgtt gatctactca gcctcataca gatacaccgg agtaccatca
181 aggtcctcag gttctggatc aggtactgac tttaccttga ccattccatc attgcagcca
241 Gaggattttg caacctatta ttgccagcaa tactacatct acccttacac cttcggacaa
301 ggaacaaaag ttgagattaa gtcatcaggt ggaggaggtt caggtggagg aggttctgga
361 ggtgaagtac aattagtcga atcaggaggt ggtttggttc aacctggagg atcattaagg
421 ttgtcttgtg cagcatctgg tttcacattc accgattaca caatggattg ggttagacag
481 gcccctggaa agggtttgga gtgggtcgcc gacgtcaacc ctaattctgg tggatcaatc
541 tataaccaga gattcaaggg aaggttcacc ttgtcagtgg ataggtctaa gaacaccttg
601 tatttgcaga tgaactcatt gagggctgag gatacagccg tttactactg tgccagaaat
661 ttgggacctt ctttctactt cgactactgg ggtcaaggta ctttggttac cgtatcttca
721 acgcgt
SEQ ID No.4 CD8-hinge/CD8-TM
1 GTGCCGGTCT TCCTGCCAGC GAAGCCCACC ACGACGCCAG CGCCGCGACC ACCAACACCG
61 GCGCCCACCA TCGCGTCGCA GCCCCTGTCC CTGCGCCCAG AGGCGTGCCG GCCAGCGGCG
121 GGGGGCGCAG TGCACACGAG GGGGCTGGAC TTCGCCTGTG ATATCTACAT CTGGGCGCCC
181 TTGGCCGGGA CTTGTGGGGT CCTTCTCCTG TCACTGGTTA TCACCCTT
SEQ ID No.5 CD28-ICD
1 AGGAGTAAGA GGAGCAGGCT CCTGCACAGT GACTACATGA ACATGACTCC CCGCCGCCCC
61 GGGCCCACCC GCAAGCATTA CCAGCCCTAT GCCCCACCAC GCGACTTCGC AGCCTATCGC
121 TCC
SEQ ID No.6 CD137-ICD
1 CGTTTCTCTG TTGTTAAACG GGGCAGAAAG AAGCTCCTGT ATATATTCAA ACAACCATTT
61 ATGAGACCAG TACAAACTAC TCAAGAGGAA GATGGCTGTA GCTGCCGATT TCCAGAAGAA
121 GAAGAAGGAG GATGTGAACT
SEQ ID No.7 CD3zeta
1 CTGAGAGTGA AGTTCAGCAG GAGCGCAGAC GCCCCCGCGT ACCAGCAGGG CCAGAACCAG
61 CTCTATAACG AGCTCAATCT AGGACGAAGA GAGGAGTACG ATGTTTTGGA CAAGAGACGT
121 GGCCGGGACC CTGAGATGGG GGGAAAGCCG AGAAGGAAGA ACCCTCAGGA AGGCCTGTAC
181 AATGAACTGC AGAAAGATAA GATGGCGGAG GCCTACAGTG AGATTGGGAT GAAAGGCGAG
241 CGCCGGAGGG GCAAGGGGCA CGATGGCCTT TACCAGGGTC TCAGTACAGC CACCAAGGAC
301 ACCTACGACG CCCTTCACAT GCAGGCCCTG CCCCCTCGC
SEQ ID No.8
1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
2161 acagcagaga tccagtttat cgatGGCTCC GGTGCCCGTC AGTGGGCAGA GCGCACATCG
2221 CCCACAGTCC CCGAGAAGTT GGGGGGAGGG GTCGGCAATT GATCCGGTGC CTAGAGAAGG
2281 TGGCGCGGGG TAAACTGGGA AAGTGATGTC GTGTACTGGC TCCGCCTTTT TCCCGAGGGT
2341 GGGGGAGAAC CGTATATAAG TGCAGTAGTC GCCGTGAACG TTCTTTTTCG CAACGGGTTT
2401 GCCGCCAGAA CACAGGggat cgctagcgct accggactca gatctcgagG CCACCatggc
2461 cttaccagtg accgccttgc tcctgccgct ggccttgctg ctccacgccg ccaggccgca
2521 gatccaaatg actcagtcac catcttctgt atctgcttca gtcggagaTa gggttacaat
2581 cacttgcaaa gcctcacaag atgtctcaat aggtgtcgCa tggtatcaac agaaacctgg
2641 taaggcccct aagttgttga tctactcagc ctcatacaga tacaccggag taccatcaag
2701 gtcctcaggt tctggatcag gtactgactt taccttgacc attccatcat tgcagccaGa
2761 ggattttgca acctattatt gccagcaata ctacatctac ccttacacct tcggacaagg
2821 aacaaaagtt gagattaagt catcaggtgg aggaggttca ggtggaggag gttctggagg
2881 tgaagtacaa ttagtcgaat caggaggtgg tttggttcaa cctggaggat cattaaggtt
2941 gtcttgtgca gcatctggtt tcacattcac cgattacaca atggattggg ttagacaggc
3001 ccctggaaag ggtttggagt gggtcgccga cgtcaaccct aattctggtg gatcaatcta
3061 taaccagaga ttcaagggaa ggttcacctt gtcagtggat aggtctaaga acaccttgta
3121 tttgcagatg aactcattga gggctgagga tacagccgtt tactactgtg ccagaaattt
3181 gggaccttct ttctacttcg actactgggg tcaaggtact ttggttaccg tatcttcaac
3241 gcgttctaga GTGCCGGTCT TCCTGCCAGC GAAGCCCACC ACGACGCCAG CGCCGCGACC
3301 ACCAACACCG GCGCCCACCA TCGCGTCGCA GCCCCTGTCC CTGCGCCCAG AGGCGTGCCG
3361 GCCAGCGGCG GGGGGCGCAG TGCACACGAG GGGGCTGGAC TTCGCCTGTG ATATCTACAT
3421 CTGGGCGCCC TTGGCCGGGA CTTGTGGGGT CCTTCTCCTG TCACTGGTTA TCACCCTTTA
3481 CTGCAACCAC AGGAACAGGA GTAAGAGGAG CAGGCTCCTG CACAGTGACT ACATGAACAT
3541 GACTCCCCGC CGCCCCGGGC CCACCCGCAA GCATTACCAG CCCTATGCCC CACCACGCGA
3601 CTTCGCAGCC TATCGCTCCC GTTTCTCTGT TGTTAAACGG GGCAGAAAGA AGCTCCTGTA
3661 TATATTCAAA CAACCATTTA TGAGACCAGT ACAAACTACT CAAGAGGAAG ATGGCTGTAG
3721 CTGCCGATTT CCAGAAGAAG AAGAAGGAGG ATGTGAACTG AGAGTGAAGT TCAGCAGGAG
3781 CGCAGACGCC CCCGCGTACC AGCAGGGCCA GAACCAGCTC TATAACGAGC TCAATCTAGG
3841 ACGAAGAGAG GAGTACGATG TTTTGGACAA GAGACGTGGC CGGGACCCTG AGATGGGGGG
3901 AAAGCCGAGA AGGAAGAACC CTCAGGAAGG CCTGTACAAT GAACTGCAGA AAGATAAGAT
3961 GGCGGAGGCC TACAGTGAGA TTGGGATGAA AGGCGAGCGC CGGAGGGGCA AGGGGCACGA
4021 TGGCCTTTAC CAGGGTCTCA GTACAGCCAC CAAGGACACC TACGACGCCC TTCACATGCA
4081 GGCCCTGCCC CCTCGCACGC GTggaagcgg agccacgaac ttctctctgt taaagcaagc
4141 aggagatgtt gaagaaaacc ccgggcctat ggtgagcaag ggcgaggagc tgttcaccgg
4201 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
4261 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
4321 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
4381 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
4441 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
4501 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
4561 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
4621 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
4681 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
4741 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
4801 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
4861 tctcggcatg gacgagctgt acaagtaaGC GGCCGCccgc gtctggaaca atcaacctct
4921 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
4981 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
5041 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
5101 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
5161 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
5221 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
5281 caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc
5341 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
5401 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
5461 tcagacgagt cggatctccc tttgggccgc ctccccgcct ggaattaatt ctgcagtcga
5521 gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt
5581 gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct
5641 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagagg
5701 ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac
5761 cacacacaag gctacttccc tgattagcag aactacacac cagggccagg ggtcagatat
5821 ccactgacct ttggatggtg ctacaagcta gtaccagttg agccagataa ggtagaagag
5881 gccaataaag gagagaacac cagcttgtta caccctgtga gcctgcatgg gatggatgac
5941 ccggagagag aagtgttaga gtggaggttt gacagccgcc tagcatttca tcacgtggcc
6001 cgagagctgc atccggagta cttcaagaac tgctgatatc gagcttgcta caagggactt
6061 tccgctgggg actttccagg gaggcgtggc ctgggcggga ctggggagtg gcgagccctc
6121 agatcctgca tataagcagc tgctttttgc ctgtactggg tctctctggt tagaccagat
6181 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
6241 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
6301 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat
6361 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc cttgacattg
6421 ctagcgtttt accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
6481 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
6541 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
6601 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
6661 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
6721 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
6781 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
6841 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
6901 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
6961 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
7021 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
7081 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
7141 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
7201 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
7261 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
7321 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
7381 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
7441 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
7501 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
7561 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
7621 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
7681 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
7741 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
7801 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
7861 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
7921 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
7981 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
8041 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
8101 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
8161 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
8221 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
8281 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
8341 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
8401 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
8461 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
8521 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
8581 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
8641 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
8701 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
8761 tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc
8821 ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac ctgtgattcc
8881 tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac aaacttagta
8941 gtttttaaag aaattgtatt tgttaaatat gtactacaaa cttagtagt
The T lymphocytes in HER2-CAR-T cells may be derived from autologous T lymphocytes, allogeneic T lymphocytes or pluripotent stem cell-induced T lymphocytes. Among the various T lymphocytes of the above origin, autologous T lymphocytes from the patient are preferred, which is advantageous in reducing the incidence of immune responses and side effects. Because the HER2 antigen on the surfaces of malignant tumor cells such as osteosarcoma and the like is highly expressed, and normal tissue cells indicate that the HER2 antigen expression level is very low, the CAR-T cells targeting the HER2 antigen can specifically identify and kill the malignant tumor cells, thereby playing an anti-tumor role and avoiding normal tissues from being attacked.
The invention provides a medicine for treating osteosarcoma, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, ovarian cancer and other malignant tumors, wherein the medicine comprises ligustilide and HER2-CAR-T cells, and the use method of the medicine is intravenous injection. Ligustilide and HER2-CAR-T cell can be used independently or in combination. Specifically, common modes of administration of HER2-CAR-T cells are injections, including intravenous, subcutaneous, intradermal, intrathecal or intramuscular, with the usual method being intravenous by dissolving the CAR-T cells in saline or dextrose injection. The common administration mode of ligustilide is intravenous injection, which can be widely distributed and can reach heart, bone, lung and liver. The pharmaceutical composition contains at least one diluent and at least one stabilizer, which can be matched with carrier substances in pharmacy, such as water, physiological saline, glucose aqueous solution, other non-aqueous solvents and the like, but is not limited to the above.
The first administration method is as follows: firstly, infusing ligustilide injection intravenously, and then infusing HER2-CAR-T cells intravenously; and a second administration method is as follows: the ligustilide and HER2-CAR-T cell mixed solution is dissolved in physiological saline and infused intravenously. The advantages of the ligustilide and HER2-CAR-T cell combined preparation are as follows: firstly, ligustilide increases the number of HER2 antigen expression on the surface of osteosarcoma cells, which is beneficial to improving the recognition, combination and killing effect of HER2-CAR-T cells on osteosarcoma cells; second, reducing the applied dose of HER2-CAR-T cells, avoiding adverse reactions caused by HER2-CAR-T cells; thirdly, the therapeutic effect of the combined preparation is better than that of single administration; fourth, patient medical costs are reduced.
In a word, the ligustilide and HER2-CAR-T cell combined preparation can strengthen the treatment effect on malignant tumors such as osteosarcoma and the like which highly express HER2 antigen, exert a synergistic effect, reduce the dosage of HER2-CAR-T cells, reduce toxic and side effects and reduce medical cost on the premise of ensuring the tumor killing efficacy.
Drawings
FIG. 1 is a schematic representation of the structure of a HER2-CAR;
FIG. 2 is a bar graph of LIG, HER2-CAR-T, and LIG+HER2-CAR-T vs. U2OS cell lysis;
FIG. 3 is a bar graph of LIG, HER2-CAR-T, and LIG+HER2-CAR-T for tumor cell killing factors TNF-gamma, IL-2 produced during killing of U2OS cells;
FIG. 4 is a bar graph of LIG, HER2-CAR-T, and LIG+HER2-CAR-T for tumor cell killing factor perforin granzyme B produced during killing of U2OS cells;
wherein LIG represents ligustilide, HER2-CAR-T represents chimeric antigen receptor T lymphocytes targeting HER2 antigen, and U2OS cells are human osteosarcoma cells.
Detailed Description
The invention provides application of ligustilide and HER2-CAR-T cell combined preparation in preparation of medicines for treating malignant tumors. The applicant finds that ligustilide can up-regulate the quantity of HER2 antigen expressed by malignant tumor, and further obviously enhance the recognition, combination and killing effect of HER2-CAR-T cells on tumor cells. The combination of the two shows a synergistic effect, can achieve good tumor treatment effect, and can reduce the dosage of HER2-CAR-T lymphocytes and reduce side effects caused by the treatment of the independent CAR-T lymphocytes. In addition, ligustilide is a small molecular organic compound, has low price, and can reduce the immunotherapy cost of CAR-T cells.
The synergy of ligustilide and HER2-CAR-T cell combination preparations for the treatment of osteosarcoma is demonstrated by specific examples below. As shown in fig. 1, the signal peptide of HER2-CAR sequence selects CD8 a, the extracellular recognition region is a HER2 single chain antibody, then the extracellular region of CD8 a and the transmembrane region of CD8 a are sequentially linked as a linking structure, and then the intracellular regions of CD28, CD137, and CD3 ζ are sequentially linked.
Example 1
Constructing a plasmid: the present application constructs a third generation chimeric antigen receptor plasmid, CD8 a-HER 2scFv-CD8-CD28-CA137-CD3 zeta. Wherein, CD8 alpha is signal peptide, the corresponding nucleotide sequence is 5'-atg gcc tta cca gtg acc gcc ttg ctc ctg ccg ctg gcc ttg ctg ctc cac gcc gcc agg ccg-3' (the nucleotide sequence is SEQ ID No. 2), the extracellular recognition region of HER2scFv is connected (the nucleotide sequence is SEQ ID No. 3), the CD8 alpha hinge region and the CD8 alpha transmembrane region are sequentially connected (the nucleotide sequence is SEQ ID No. 4), and the intracellular region costimulatory signals of CD28 (the nucleotide sequence is SEQ ID No. 5), CD137 (the nucleotide sequence is SEQ ID No. 6) and CD3 zeta (the nucleotide sequence is SEQ ID No. 7). The nucleotide sequence of the third generation chimeric antigen receptor HER2-CAR is SEQ ID No.1. Compared with the first-generation and second-generation CAR-T cells, the third-generation CAR-T cells have the advantages that the co-stimulation structural domain of an intracellular region is increased, the secretion quantity of tumor cell killing factors can be increased, and the tumor killing effect is improved.
Construction of lentiviral vectors and packaging plasmids: inserting a coding gene fragment (with a nucleotide sequence of SEQ ID No. 1) of a chimeric antigen receptor HER2-CAR into a lentivirus eukaryotic expression vector pLV [ Exp ] with cleavage sites AtsI and BstXI, and ensuring the accuracy of the gene fragment through the steps of cleavage, connection, identification and the like, thereby obtaining a HER2-CAR recombinant plasmid pLV [ Exp ] -HER2-CAR; calculating the purity and the content of the nucleic acid, and finally sub-packaging and storing at-20 ℃ for standby.
T lymphocyte lentiviral transfection: collecting and separating peripheral blood mononuclear cells of healthy people, adding CD3/CD28 immunomagnetic beads for incubation, and screening to obtain CD3 positive T lymphocytes; the recombinant lentivirus is added into the cell to culture, HER2-CAR-T lymphocyte is obtained, after transfection is successful, the transfection efficiency is detected by adopting methods such as a flow cytometer, immunofluorescence and the like to be more than or equal to 70%, which indicates that the CAR-T cell targeting HER2 antigen is successfully prepared and is stored in cell freezing solution special for reinfusion for patients.
Example 2
In vitro killing effect of ligustilide and HER2-CAR-T cell combined preparation on tumor cells, target cells select U2OS cells (human osteosarcoma cells) expressing HER2, and subculture in DMEM high sugar medium containing fetal bovine serum. U2OS cells were inoculated into 96-well cell culture plates with a cell number of 1×10 4 Well, culture for 24 hours. According to different treatment methods, 3 experimental groups, namely ligustilide group (ligustilide injection 3 mL), HER2-CAR-T cell group (1×10) 6 Per mL cell suspension) and ligustilide in combination with HER2-CAR-T cells (1X 10) 6 3mL of cell suspension+5% ligustilide injection).
The lactate dehydrogenase release experiment evaluates the killing effect of the drug on tumor cells, the lactate dehydrogenase release method utilizes lactate dehydrogenase release existing in cytoplasm, when cell damage cell membrane is broken, the lactate dehydrogenase release is released into extracellular culture medium, and the amount of lactate dehydrogenase release is measured to evaluate the degree of cell damage; and detecting the activity of releasing lactic dehydrogenase in the cell culture supernatant by using a lactic dehydrogenase releasing cytotoxicity detection kit, and judging the killing effect of the drug on tumor cells. Different effector cells were added to the U2OS cells separately for co-culture. After 24 hours, 20 μl of cell lysate provided by the kit is added to the tumor cell group without lymphocytes, and the mixture is reacted for 1 hour at 37 ℃; centrifugation was performed in a multiwell plate centrifuge at 400g for 5min. 120 μl of supernatant was added to a new 96-well plate; preparing lactic dehydrogenase detection working solution by using 20 μl of lactic acid solution, 20 μl of INT solution (1X) and 20 μl of enzyme solution, and 60 μl of total per well system; 60. Mu.l of the prepared lactate dehydrogenase assay working solution was added to a 96-well plate containing a cell culture supernatant; mixing, incubating at room temperature and about 25 ℃ for 30min under the dark condition; absorbance at 490 nm; setting the supernatant of the pure tumor cell hole as a sample control hole, and setting the supernatant of the lysed tumor cell hole as a maximum enzyme activity control hole; cell killing efficiency calculation: (action sample wells (OD value) -sample blank wells (OD value)/(maximum enzyme activity control wells ((OD value) -sample control wells ((OD value)). Times.100%), lysis of tumor cells by different treatments, as shown in FIG. 2.
Example 3
Pharmacology revealing that ligustilide and HER2-CAR-T cell preparation exert strong tumor killing effect through cytokine detection, and U2OS cells expressing HER2 antigen are expressed at 1×10 per well 5 The drugs of different groups were mixed with target cells in 96-well plates and incubated in CO in a total volume of 200. Mu.l of culture medium (RPMI 1640 medium+10% FBS) 2 The incubator was incubated for 24 hours at 37 ℃. Each group had 3 duplicate wells. Next, 10. Mu.l of the supernatant was centrifuged and the cytokine concentration levels of TNF-. Gamma.and IL-2 were measured using the Alpha LISA assay kit from Perkin Elmer. As shown in fig. 3, the content of TNF-gamma and IL-2 cytokines detected in the ligustilide and HER2-CAR-T cell combined preparation is higher than that of the HER2-CAR-T cell group alone and the ligustilide group alone, which illustrates the reason that the combined application of the two enhances the killing effect on tumor cells.
Example 4
Detecting the granzyme B by an ELISA method, collecting supernatant, and diluting the supernatant by 5 times by using a reagent detection enhancer; add 500. Mu.l of sample dilution to the standard tube; configured to have a concentration of 220ng/ml. The dilution of the double ratio was performed to 10000pg/ml,5000pg/ml,2500pg/ml,1250pg/ml,625pg/ml,313pg/ml,156pg/ml,0 pg/ml. Adding 50 μl of standard substance and sample supernatant into the detection plate, adding 50 μl of antibody mixture, and incubating at room temperature under shaking for 1 hr; the supernatant was removed and washed three times for 2 minutes with 350. Mu.l of wash solution. After the last cleaning, the board is beaten by absorbent paper to wash out the residual liquid; adding 100 μl TMB color development solution, incubating at room temperature for 10min, adding 100 μl stop solution, and mixing; measuring the absorbance value of each hole by using the wavelength of 450nm of the enzyme label instrument; and (3) preparing a standard curve, substituting absorbance values of all samples into a regression equation, and multiplying the obtained result concentration by a dilution factor (5 times) to determine the concentration of the sample to be detected. Figure 4 shows that the content of granzyme B detected in the ligustilide and HER2-CAR-T cell combination formulation is higher than that of the HER2-CAR-T cell group alone and the ligustilide group alone, demonstrating why the combined use of the two enhances the killing effect on tumor cells.
Claims (7)
1. The application of ligustilide and HER2-CAR-T cell in preparing osteosarcoma medicine is characterized in that: ligustilide has CAS number 4431-01-0 and molecular formula C 12 H 14 O 2 Molecular weight: 190.24, the structural formula is as follows:
；
the nucleotide sequence of the HER2-CAR and the carrier thereof is SEQ ID No.8; HER2-CAR structure includes an extracellular domain, a transmembrane region, and an intracellular domain;
the extracellular domain consists of a CD8 a Leader, a HER2scFv single chain antibody, and a CD8 a hinge region; the transmembrane region is composed of CD8 alpha TM; the intracellular domain consists of the CD28 co-stimulatory domain, the CD137 co-stimulatory domain and the cd3ζ intracellular signaling region.
2. The use of ligustilide in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, characterized in that: CD8 alpha Leader is chimeric receptor signal peptide in HER2-CAR, and the nucleotide sequence is SEQ ID No.2.
3. The use of ligustilide in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, characterized in that: the HER2scFv is a humanized single-chain antibody of HER2 antigen on the surface of a malignant tumor cell in the HER2-CAR, and the nucleotide sequence is SEQ ID No.3.
4. The use of ligustilide in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, characterized in that: CD8 alpha-hinge/CD 8 alpha TM is the hinge and transmembrane regions of HER2-CAR, and the nucleotide sequence is SEQ ID No.4.
5. The use of ligustilide in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, characterized in that: CD28 is the intracellular co-stimulatory domain in HER2-CAR and has the nucleotide sequence of SEQ ID No.5.
6. The use of ligustilide in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, characterized in that: CD137 in HER2-CAR is another co-stimulatory domain intracellular and has the nucleotide sequence of SEQ ID No.6.
7. The use of ligustilide in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, characterized in that: CD3 zeta in HER2-CAR is intracellular signal region and the nucleotide sequence is SEQ ID No.7.
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