CN116763909B - Preparation of medicine for treating osteosarcoma by combining berberine and HER2-CAR-T cells - Google Patents
Preparation of medicine for treating osteosarcoma by combining berberine and HER2-CAR-T cells Download PDFInfo
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- CN116763909B CN116763909B CN202311007660.8A CN202311007660A CN116763909B CN 116763909 B CN116763909 B CN 116763909B CN 202311007660 A CN202311007660 A CN 202311007660A CN 116763909 B CN116763909 B CN 116763909B
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- Cell Biology (AREA)
Abstract
The invention discloses a drug for treating osteosarcoma prepared by combining berberine and HER2-CAR-T cells, belonging to the field of biological medicine. Comprises berberine and HER2-CAR-T cells, wherein the CAS number of berberine is 633-65-8, and the molecular formula is as follows: C20H18ClNO4, molecular weight: 372.822. the invention takes osteosarcoma as an example, and the medicine is also applicable to other cancers expressing HER2 antigen, such as malignant tumors of esophagus cancer, stomach cancer, pancreas cancer, breast cancer, ovarian cancer and the like. The berberine and HER2-CAR-T cell combined preparation has the following advantages: 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, the berberine is economical in price, and the medical cost can be reduced after the berberine and the berberine are combined.
Description
Technical Field
The invention relates to the technical field of biological medicine, and discloses application of berberine 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.
Berberine (BBR), also known as Berberine, is an isoquinoline alkaloid with anti-tumor, antioxidant and antiinflammatory effects. Berberine can play an anti-tumor role by inhibiting proliferation of tumor cells, blocking tumor cell cycle, inducing apoptosis of tumor cells, regulating autophagy of tumor cells, inhibiting invasion and metastasis of tumor cells, and regulating tumor microenvironment. 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, no report exists in the industry that berberine is combined with CAR-T lymphocytes targeting HER2 antigen to prepare anti-osteosarcoma drugs.
Disclosure of Invention
The invention provides a new application of berberine and HER2-CAR-T cell combined preparation in treating osteosarcoma, and proves the application result. 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, and ovary. The berberine and HER2-CAR-T cell combined preparation has the following advantages: 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, the berberine is economical in price, and the medical cost can be reduced after the berberine and the berberine are combined.
Berberine can inhibit tumor cell proliferation by decreasing the activity of citrate synthase, inhibiting the activation of sterol regulatory element binding protein-1, wnt/β -catenin signaling pathway, down-regulating glucose regulatory protein 78, inducing senescence-associated β -galactosidase activity and a wild-type p53 activation fragment of the cell cycle inhibitor p21, decreasing the expression of cyclin dependent kinase 1; berberine activates mitochondrial related pathways to induce apoptosis mainly by affecting active oxygen related signaling pathway JNK/p38, mitogen activated protein kinase, protein kinase C, extracellular regulatory protein kinase, etc.; berberine can also block tumor cell cycle by inhibiting expression of Cyclin D1 and Cyclin E1, inhibiting expression of CDK4 and regulating Cyclin D1, activating PI3K/Akt and p38 signaling pathway, and inhibiting phosphorylation of Rb protein; berberine can also inhibit tumor cell migration and invasion by inhibiting PI3K/Akt signaling pathway, EGFR/MEK/ERK signaling pathway, down-regulating vascular endothelial growth factor, and TGF-beta/Smad signaling pathway; in addition, berberine activates autophagy by targeting AMPK/mTOR/ULK1 signaling pathway, regulating JNK phosphorylation and promoting Bcl-2/beclin-1 complex separation, increasing binding capacity of cancer cells to glucose regulatory protein 78 and type III phosphatidylinositol kinase VPS 34. Importantly, the berberine can be chemically edited, modified and removed by reduction, substitution and other methods on the side chain groups on the skeleton, and various derivatives, isomers, salts, metal complexes and the like which can enhance the killing capacity of the CAR-T cells are reserved. The CAS number of berberine in the patent of the invention is 633-65-8, and the molecular formula is: c (C) 20 H 18 ClNO 4 Molecular weight: 372.822, 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 berberine in combination with HER2-CAR-T cell preparations is equally applicable to these malignant tumors, but is not limited thereto. The berberine has strong activity of resisting cancers such as breast cancer, bladder cancer, colon cancer, liver cancer and the like, and has wide application prospect in the prevention and treatment of cancers. The patent applicant researches find that berberine can enhance the recognition, binding and killing effect of HER2-CAR-T cells on osteosarcoma cells by up-regulating the expression of osteosarcoma HER2 antigen.
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 berberine and HER2-CAR-T cells, and the use method of the medicine is intravenous injection. The berberine 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 modes of berberine are intravenous injection and oral administration, the berberine emulsion can be used for intravenous injection, the medicine distribution is wide through intravenous injection, and the berberine emulsion can reach heart, bones, lung and liver, only trace amount of berberine is left after 24 hours, and most of the medicine is metabolized and removed in the body; poor oral absorption and short maintenance time of blood concentration. 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, intravenous infusion of berberine injection is carried out, and then HER2-CAR-T cells are intravenous input; and a second administration method is as follows: the berberine emulsion and HER2-CAR-T cell mixed solution are dissolved in normal saline and infused intravenously. The benefits of the berberine and HER2-CAR-T cell combined preparation are as follows: firstly, berberine 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 berberine 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, play a synergistic role, reduce the dosage of HER2-CAR-T cells, reduce toxic and side effects and 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 BBR, HER2-CAR-T, and BBR+HER2-CAR-T versus U2OS cell lysis;
FIG. 3 is a bar graph of BBR, HER2-CAR-T, and BBR+HER2-CAR-T for the production of tumor cell killing factors TNF-gamma, IL-2, and IL-10 during killing of U2OS cells;
wherein BBR represents berberine, 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 berberine and HER2-CAR-T cell combined preparation in preparation of medicines for treating malignant tumors. The applicant finds that the berberine 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, the berberine is a small molecular organic compound, has low price, and can reduce the immunotherapy cost of the CAR-T cells.
The synergy of berberine 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 berberine 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 berberine group (berberine injection 3 mL), HER2-CAR-T cell group (1×10) 6 Per mL cell suspension) and berberine and HER2-CAR-T cell combination preparation group (1×10) 6 3mL of cell suspension+5% berberine 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
Pharmacological tests revealed that berberine and HER2-CAR-T cell preparation exert potent tumor killing effect by cytokine detection, inoculating U2OS cells expressing HER2 antigen into 96-well plate at 100000/well, mixing different groups of drugs with target cells, and adding 200 μl of culture solution (RPMI 1640 culture medium+10% FBS) into CO 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 TNF-gamma, IL-2 cytokine content detected in the berberine and HER2-CAR-T cell combination preparation was higher than that of the HER2-CAR-T cell group alone and the berberine group alone, and in addition, the IL-10 cytokine content was lower than that of the HER2-CAR-T cell group alone and the berberine group alone. TNF-gamma and IL-2 have killing effect on tumor cells, and IL-10 has negative regulation effect on cytokines for killing tumor cells, which clarifies the reason that the combined application of the two has enhanced killing effect on tumor cells.
Sequence table information:
DTD version v1_3
File name, sequence listing dna1.Xml
Software name WIPO Sequence
Software version 2.3.0
Date of production 2023-08-02
Basic information:
the current application/applicant archive name: jilin University
Applicant name or name Jilin university
Applicant name or name/language zh
Applicant name or name/Latin name Jilin University
The name of the inventor is healthy navigation
Inventor name/language zh
Name of inventor/Latin name of GUANJIE ZHAO
The name of the invention is berberine and HER2-CAR-T cell combined preparation of medicament (zh) for treating osteosarcoma
Total sequence 1
Sequence:
serial number (ID) 1
Length 12270
Molecular type DNA
Feature location/qualifier:
- source, 1..12270
>mol_type, unassigned DNA
>organism, DNA
residues:
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgcagatcc aaatgactca gtcaccatct tctgtatctg cttcagtcgg agatagggtt 120
acaatcactt gcaaagcctc acaagatgtc tcaataggtg tcgcatggta tcaacagaaa 180
cctggtaagg cccctaagtt gttgatctac tcagcctcat acagatacac cggagtacca 240
tcaaggtcct caggttctgg atcaggtact gactttacct tgaccattcc atcattgcag 300
ccacaggatt ttgcaaccta ttattgccag caatactaca tctaccctta caccttcgga 360
caaggaacaa aagttgagat taagtcatca ggtggaggag gttcaggtgg aggaggttct 420
ggaggtgaag tacaattagt cgaatcagga ggtggtttgg ttcaacctgg aggatcatta 480
aggttgtctt gtgcagcatc tggtttcaca ttcaccgatt acacaatgga ttgggttaga 540
caggcccctg gaaagggttt ggagtgggtc gccgacgtca accctaattc tggtggatca 600
atctataacc agagattcaa gggaaggttc accttgtcag tggataggtc taagaacacc 660
ttgtatttgc agatgaactc attgagggct gaggatacag ccgtttacta ctgtgccaga 720
aatttgggac cttctttcta cttcgactac tggggtcaag gtactttggt taccgtatct 780
tcaacgcgtt ctagagtgcc ggtcttcctg ccagcgaagc ccaccacgac gccagcgccg 840
cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 900
tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 960
tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 1020
ctttactgca accacaggaa caggagtaag aggagcaggc tcctgcacag tgactacatg 1080
aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 1140
cgcgacttcg cagcctatcg ctcccgtttc tctgttgtta aacggggcag aaagaagctc 1200
ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1260
tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1320
aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1380
ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1440
gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1500
aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1560
cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1620
atgcaggccc tgccccctcg cdnaatggcc ttaccagtga ccgccttgct cctgccgctg 1680
gccttgctgc tccacgccgc caggccgdna cagatccaaa tgactcagtc accatcttct 1740
gtatctgctt cagtcggaga tagggttaca atcacttgca aagcctcaca agatgtctca 1800
ataggtgtcg catggtatca acagaaacct ggtaaggccc ctaagttgtt gatctactca 1860
gcctcataca gatacaccgg agtaccatca aggtcctcag gttctggatc aggtactgac 1920
tttaccttga ccattccatc attgcagcca gaggattttg caacctatta ttgccagcaa 1980
tactacatct acccttacac cttcggacaa ggaacaaaag ttgagattaa gtcatcaggt 2040
ggaggaggtt caggtggagg aggttctgga ggtgaagtac aattagtcga atcaggaggt 2100
ggtttggttc aacctggagg atcattaagg ttgtcttgtg cagcatctgg tttcacattc 2160
accgattaca caatggattg ggttagacag gcccctggaa agggtttgga gtgggtcgcc 2220
gacgtcaacc ctaattctgg tggatcaatc tataaccaga gattcaaggg aaggttcacc 2280
ttgtcagtgg ataggtctaa gaacaccttg tatttgcaga tgaactcatt gagggctgag 2340
gatacagccg tttactactg tgccagaaat ttgggacctt ctttctactt cgactactgg 2400
ggtcaaggta ctttggttac cgtatcttca acgcgtdnag tgccggtctt cctgccagcg 2460
aagcccacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 2520
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 2580
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 2640
cttctcctgt cactggttat cacccttdna aggagtaaga ggagcaggct cctgcacagt 2700
gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 2760
gccccaccac gcgacttcgc agcctatcgc tccdnacgtt tctctgttgt taaacggggc 2820
agaaagaagc tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 2880
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactdnac 2940
tgagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc cagaaccagc 3000
tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac aagagacgtg 3060
gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca 3120
atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg aaaggcgagc 3180
gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc accaaggaca 3240
cctacgacgc ccttcacatg caggccctgc cccctcgcdn atggaagggc taattcactc 3300
ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga 3360
ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta 3420
caagctagta ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag 3480
cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg 3540
gaggtttgac agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt 3600
caagaactgc tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag 3660
gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc 3720
tttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct 3780
aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt 3840
gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt 3900
ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga 3960
gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg 4020
actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga 4080
gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc 4140
agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg 4200
attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca 4260
gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc 4320
aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa 4380
gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg gccgctgatc 4440
ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa 4500
gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag 4560
agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga 4620
agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt 4680
atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa 4740
ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta 4800
aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct 4860
gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc 4920
tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa 4980
gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca 5040
agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg 5100
atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga 5160
gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc 5220
gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga 5280
ttagtgaacg gatctcgacg gtatcgcctt taaaagaaaa ggggggattg gggggtacag 5340
tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa 5400
acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccagttta 5460
tcgatggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 5520
tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg 5580
aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 5640
gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagggga 5700
tcgctagcgc taccggactc agatctcgag gccaccatgg ccttaccagt gaccgccttg 5760
ctcctgccgc tggccttgct gctccacgcc gccaggccgc agatccaaat gactcagtca 5820
ccatcttctg tatctgcttc agtcggagat agggttacaa tcacttgcaa agcctcacaa 5880
gatgtctcaa taggtgtcgc atggtatcaa cagaaacctg gtaaggcccc taagttgttg 5940
atctactcag cctcatacag atacaccgga gtaccatcaa ggtcctcagg ttctggatca 6000
ggtactgact ttaccttgac cattccatca ttgcagccag aggattttgc aacctattat 6060
tgccagcaat actacatcta cccttacacc ttcggacaag gaacaaaagt tgagattaag 6120
tcatcaggtg gaggaggttc aggtggagga ggttctggag gtgaagtaca attagtcgaa 6180
tcaggaggtg gtttggttca acctggagga tcattaaggt tgtcttgtgc agcatctggt 6240
ttcacattca ccgattacac aatggattgg gttagacagg cccctggaaa gggtttggag 6300
tgggtcgccg acgtcaaccc taattctggt ggatcaatct ataaccagag attcaaggga 6360
aggttcacct tgtcagtgga taggtctaag aacaccttgt atttgcagat gaactcattg 6420
agggctgagg atacagccgt ttactactgt gccagaaatt tgggaccttc tttctacttc 6480
gactactggg gtcaaggtac tttggttacc gtatcttcaa cgcgttctag agtgccggtc 6540
ttcctgccag cgaagcccac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 6600
atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 6660
gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 6720
acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaacca caggaacagg 6780
agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 6840
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 6900
cgtttctctg ttgttaaacg gggcagaaag aagctcctgt atatattcaa acaaccattt 6960
atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 7020
gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 7080
cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 7140
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 7200
cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 7260
attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 7320
agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcacg 7380
cgtggaagcg gagccacgaa cttctctctg ttaaagcaag caggagatgt tgaagaaaac 7440
cccgggccta tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 7500
gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 7560
gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 7620
tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 7680
cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 7740
accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 7800
gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 7860
ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 7920
cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 7980
cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 8040
gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 8100
cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 8160
tacaagtaag cggccgcccg cgtctggaac aatcaacctc tggattacaa aatttgtgaa 8220
agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta 8280
atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa 8340
tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg 8400
tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc 8460
ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc 8520
cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg 8580
gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg 8640
acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg 8700
ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc 8760
ctttgggccg cctccccgcc tggaattaat tctgcagtcg agacctagaa aaacatggag 8820
caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag 8880
aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca 8940
aggcagctgt agatcttagc cactttttaa aagaaaagag gggactggaa gggctaattc 9000
actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc 9060
ctgattagca gaactacaca ccagggccag gggtcagata tccactgacc tttggatggt 9120
gctacaagct agtaccagtt gagccagata aggtagaaga ggccaataaa ggagagaaca 9180
ccagcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtgttag 9240
agtggaggtt tgacagccgc ctagcatttc atcacgtggc ccgagagctg catccggagt 9300
acttcaagaa ctgctgatat cgagcttgct acaagggact ttccgctggg gactttccag 9360
ggaggcgtgg cctgggcggg actggggagt ggcgagccct cagatcctgc atataagcag 9420
ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 9480
ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta 9540
gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 9600
gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt 9660
gcaaagaaat gaatatcaga gagtgagagg ccttgacatt gctagcgttt taccgtcgac 9720
ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 9780
gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 9840
atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 9900
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 9960
tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 10020
agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 10080
aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 10140
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 10200
tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 10260
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 10320
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 10380
cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 10440
atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 10500
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 10560
gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 10620
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 10680
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 10740
agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 10800
gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 10860
aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 10920
aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 10980
ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 11040
gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 11100
aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 11160
ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 11220
tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 11280
ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 11340
cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 11400
agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 11460
gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 11520
gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 11580
acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 11640
acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 11700
agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 11760
aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 11820
gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 11880
tccccgaaaa gtgccacctg acgtcgacgg atcgggagat caacttgttt attgcagctt 11940
ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 12000
tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaact 12060
ggataactca agctaaccaa aatcatccca aacttcccac cccataccct attaccactg 12120
ccaattacct gtggtttcat ttactctaaa cctgtgattc ctctgaatta ttttcatttt 12180
aaagaaattg tatttgttaa atatgtacta caaacttagt agtttttaaa gaaattgtat 12240
ttgttaaata tgtactacaa acttagtagt 12270
END
Claims (6)
1. the application of berberine and HER2-CAR-T cell combination in preparing osteosarcoma medicine is characterized in that: berberine 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 a CD28 co-stimulatory domain, a CD137 co-stimulatory domain and a cd3ζ intracellular signaling region;
CD8 alpha Leader is chimeric receptor signal peptide in HER2-CAR, and the nucleotide sequence is SEQ ID No.2.
2. The use of berberine in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, wherein: 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.
3. The use of berberine in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, wherein: 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.
4. The use of berberine in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, wherein: CD28 is the intracellular co-stimulatory domain in HER2-CAR and has the nucleotide sequence of SEQ ID No.5.
5. The use of berberine in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, wherein: CD137 in HER2-CAR is another intracellular co-stimulatory domain, similar to the CD28 co-stimulatory domain, with the nucleotide sequence of SEQ ID No.6.
6. The use of berberine in combination with HER2-CAR-T cells according to claim 1 for the manufacture of a medicament for osteosarcoma, wherein: CD3 zeta in HER2-CAR is intracellular signal region and the nucleotide sequence is SEQ ID No.7.
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| CN105384824A (en) * | 2014-08-26 | 2016-03-09 | 中国人民解放军总医院 | Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered HER2 targeting NKT cell and application thereof |
| CN109824781A (en) * | 2019-01-21 | 2019-05-31 | 卢英 | Specific chimeric antigen receptor, encoding gene and the expression vector of anti-human 2 antigen of HER and application |
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| CN105384824A (en) * | 2014-08-26 | 2016-03-09 | 中国人民解放军总医院 | Chimeric antigen receptor and gene and recombinant expression vector thereof, engineered HER2 targeting NKT cell and application thereof |
| CN109824781A (en) * | 2019-01-21 | 2019-05-31 | 卢英 | Specific chimeric antigen receptor, encoding gene and the expression vector of anti-human 2 antigen of HER and application |
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| Title |
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| 盐酸小檗碱治疗骨肉瘤的作用及其机制;徐西强;中国博士学位论文全文数据库 医药卫生科技辑(第9期);摘要 * |
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