CN116785417A - 芦荟苷与her2-car-t细胞联合制备治疗骨肉瘤的药物 - Google Patents
芦荟苷与her2-car-t细胞联合制备治疗骨肉瘤的药物 Download PDFInfo
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Abstract
本发明公开了一种芦荟苷与HER2‑CAR‑T细胞联合制备治疗骨肉瘤的药物，属于生物医药领域。包括芦荟苷和HER2‑CAR‑T细胞；芦荟苷的CAS号为1415‑73‑2，分子式C21H22O9，分子量：418.394，芦荟苷与HER2‑CAR‑T细胞联合制剂的优点如下：第一，发挥协同作用，增加对骨肉瘤等肿瘤细胞的杀伤作用；第二，降低HER2‑CAR‑T细胞的应用剂量；第三，减少HER2‑CAR‑T细胞治疗产生的毒副作用；第四，由于芦荟苷价格经济，二者联合应用后能够降低医疗费用。
Description
技术领域
本发明涉及生物医药技术领域，包括芦荟苷与靶向HER2抗原的CAR-T淋巴细胞联合在制备骨肉瘤药物中的用途。
背景技术
骨肉瘤是全球范围内常见恶性骨肿瘤之一，好发于儿童和年轻人。骨肉瘤具有恶性程度高、复发率高、早期转移率高和预后差的特点，严重影响患者的生活质量和生存期。目前，手术切除和辅助化疗是治疗骨肉瘤的常用方法，尽管这些方法在一定程度上延长了患者生存期、改善了生活质量，但仍面临治疗效果有限、化疗耐药性、副作用、肿瘤复发和远处转移等问题。因此，骨肉瘤的治疗一直是医学领域的未能攻克的难题也是研究热点，为了应对上述困难，探索新型安全有效的治疗方法至关重要。
嵌合抗原受体T淋巴细胞（Chimeric antigen receptor T cell, CAR-T cell）细胞疗法是一种新型、有前景、可能治愈恶性肿瘤的免疫疗法。简单地说，CAR-T细胞由CAR和T淋巴细胞两部分构成，CAR的作用是特异性识别和结合肿瘤细胞，而T淋巴细胞的作用是杀伤肿瘤细胞，二者组合后完成靶向杀伤肿瘤细胞。目前，我国国家药品监督管理局已批准了首个CAR-T细胞制剂（阿基仑赛注射液）上市，美国FDA的批准了将抗CD19的CAR-T细胞用于临床治疗复发或难治性大B细胞淋巴瘤成人患者，并已获得了治愈的效果。基于CAR-T细胞疗法在血液肿瘤中取得的巨大进展，CAR-T细胞免疫疗法为骨肉瘤的治疗带来了新思路。由于骨肉瘤属于实体瘤，实体瘤与血液肿瘤有许多不同之处。多项研究发现CAR-T细胞治疗骨肉瘤仍面临许多问题，例如，肿瘤抗原逃逸、脱靶效应、难以归巢和定殖、免疫抑制肿瘤微环境以及细胞因子释放综合征，这些问题直接影响CAR-T细胞的治疗效果。骨肉瘤患者的免疫系统往往比较弱，体内免疫细胞的数量较少、杀伤肿瘤细胞的能力并没有预想的强。因此，有必要进一步研究如何增强CAR-T细胞疗法的抗肿瘤作用和降低其副作用。
芦荟是一种多年生绿色草本植物，有明亮的黄色管状花，广泛分布于北非，亚洲中东，地中海南部和加那利群岛的炎热干燥地区。芦荟源自“Allaeh”（阿拉伯语，意思是“发光的苦味物质”）和“Vera”（拉丁语，意思是“真”）。芦荟中的多种提取物近年来已广泛应用于抗癌，抗氧化剂，抗糖尿病药、降血脂药以及创面处理敷料等。芦荟大约包含超过 75 种不同化合物，包括维生素（维生素 A，C，E 和 B12 为主），酶（淀粉酶，过氧化氢酶和过氧化物酶），矿物质（主要为锌，铜，硒和钙），糖（6 磷酸甘露糖和葡甘露聚糖等多糖），蒽醌（芦荟苷和大黄素），脂肪酸（羽扇豆酚和菜油甾醇），激素（生长素和赤霉素）和其他生物因子（即水杨酸，木质素和皂苷）。芦荟苷（Aloin，Alo）是从芦荟叶分泌物中提取的主要蒽醌类成分之一，即芦荟蒽酮的天 然 C-糖苷：（10-glucopyranosyl-1,8-dihydroxy-3-hydroxymethyl-9(10H)-anthracenone），据报道其具有抗炎、抗菌、抗氧化、抗病毒和抗癌等药理作用。研究表明，芦荟苷能够诱导多种种系肿瘤细胞的凋亡，包括乳腺癌、卵巢癌、B16-F10 小鼠黑色素瘤和人类 Jurkat T 淋巴细胞。另外，与化学制剂相比，其来源广泛，安全易得，在预防和治疗相关癌症方面具有较大的挖掘潜力。目前，业界内尚无芦荟苷联合靶向HER2抗原的CAR-T淋巴细胞来制备抗骨肉瘤药物的报告。
发明内容
本发明提供了芦荟苷与HER2-CAR-T细胞联合制剂治疗骨肉瘤的新用途，并证明了其应用结果。尽管本发明专利以抗骨肉瘤为例，该药物同样适用于表达HER2抗原的其他癌症，如乳腺癌、卵巢癌、黑色素瘤等等恶性肿瘤。芦荟苷与HER2-CAR-T细胞联合制剂的优点如下：第一，发挥协同作用，增加对骨肉瘤等肿瘤细胞的杀伤作用；第二，降低HER2-CAR-T细胞的应用剂量；第三，减少HER2-CAR-T细胞治疗产生的毒副作用；第四，由于芦荟苷价格经济，二者联合应用后能够降低医疗费用。
专利申请人研究发现芦荟苷可通过上调骨肉瘤HER2抗原表达来增强HER2-CAR-T细胞对骨肉瘤、乳腺癌、卵巢癌、黑色素瘤的识别、结合以及杀伤作用。重要的是，芦荟苷可以通过还原、取代等方法对其骨架上的侧链基团进行编辑、修饰以及去除，保留了能够增强CAR-T细胞杀伤能力的多种衍生物，并且包括药学上可以接受的异构体、盐、金属络等合物等。本发明专利中芦荟苷的CAS 号为1415-73-2，分子式C21H22O9，分子量：418.394，结构式如下：
HER2抗原在骨肉瘤细胞表面呈高表达，但是在正常组织细胞中表达量却很少，这为CAR-T细胞提供了结合靶点。HER2抗原同样高表达于食管癌、胃癌、胰腺癌、乳腺癌和卵巢癌等恶性肿瘤，所以芦荟苷联合HER2-CAR-T细胞制剂同样适用于这些恶性肿瘤，但不限于此。芦荟苷具有较强的抗乳腺癌、膀胱癌、结肠癌和肝癌等癌症的活性，在癌症的预防和治疗中有广泛的应用前景。
本专利构建了靶向HER2抗原的HER2-CAR结构，核苷酸序列为SEQ ID No.1。HER2-CAR结构包括胞外结构域、跨膜区和胞内结构域。进一步，胞外结构域由CD8α Leader、HER2scFv单链抗体以及CD8α铰链区组成；跨膜区由CD8α构成；胞内结构域由CD28共刺激结构域、CD137共刺激结构域以及CD3ζ胞内信号区共同组成。
CD8α Leader为HER2-CAR中的嵌合受体信号肽，核苷酸序列为SEQ ID No.2。
HER2 scFv为HER2-CAR中的靶向恶性肿瘤细胞表面HER2抗原的人源化单链抗体，具有较高的安全性，避免引起人机体的免疫反应的优点，核苷酸序列为SEQ ID No.3。
CD8α-hinge/CD8α TM为HER2-CAR中的铰链区和跨膜区，核苷酸序列为SEQ IDNo.4。
CD28为HER2-CAR中的胞内的共刺激结构域，具有抗原提呈、促进T淋巴细胞活化和分泌抗肿瘤的细胞因子的作用，核苷酸序列为SEQ ID No.5。
HER2-CAR中的CD137为胞内的另一个共刺激结构域，与CD28共刺激结构域相似，也具有抗原提呈、促进T淋巴细胞活化和分泌抗肿瘤的细胞因子的作用，核苷酸序列为SEQ IDNo.6。
HER2-CAR中的CD3ζ为胞内信号区，具有提呈信号活化T淋巴细胞的作用，核苷酸序列为SEQ ID No.7。
HER2-CAR及其载体的核苷酸序列为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 CD8α 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 CD8α-hinge/CD8α-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 CD3ζ
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
HER2-CAR-T细胞中的T淋巴细胞可以来源于自体T淋巴细胞、异体T淋巴细胞或多能干细胞诱导的T淋巴细胞。上述来源的多种T淋巴细胞中，来自患者自体T淋巴细胞为首选，这有利于降低免疫反应的发生率和副作用。由于骨肉瘤等恶性肿瘤细胞表面HER2抗原高表达，而正常组织细胞表面HER2抗原表达量很低，因此靶向HER2抗原的CAR-T细胞可以特异性的识别和杀伤恶性肿瘤细胞，从而发挥抗肿瘤作用，而又避免正常组织受到攻击。
本发明提供了一种治疗骨肉瘤、食管癌、胃癌、胰腺癌、乳腺癌和卵巢癌等多种恶性肿瘤的药物，所述药物成分包括芦荟苷和HER2-CAR-T细胞，该药物使用方法为静脉注射。芦荟苷与HER2-CAR-T细胞可以单独应用，也可以混合应用。具体而言，HER2-CAR-T细胞的常见给药方式为注射，包括静脉注射、皮下注射、皮内注射、鞘内注射或肌内注射，常用方法是将CAR-T细胞溶于生理盐水或者葡萄糖注射液中静脉注射。芦荟苷的常见给药方式为静脉注射，芦荟苷可通过静脉注射，通过静脉注射可以使药物分布广泛，可以到达心、骨、肺、肝。联合制剂药物中含有药学上可搭配载体物质，如水、生理盐水、葡萄糖水溶液以及其他非水性溶剂等至少一种稀释剂，以及稳定剂中的至少一种，但不限于此。
给药方法一：先静脉输注芦荟苷注射液，再静脉输入HER2-CAR-T细胞；给药方法二：芦荟苷与HER2-CAR-T细胞混合液溶于生理盐水后，同时静脉输注。芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物的好处如下：第一，芦荟苷增加骨肉瘤细胞表面的HER2抗原表达的数量，有利于提高HER2-CAR-T细胞对骨肉瘤细胞的识别、结合以及杀伤效果；第二，减少HER2-CAR-T细胞的应用剂量，避免HER2-CAR-T细胞引起的不良反应；第三，联合制剂的治疗效果优于单一用药的治疗效果；第四，降低患者医疗费用。
总之，芦荟苷与HER2-CAR-T细胞联合制剂能够加强对骨肉瘤等高表达HER2抗原的恶性肿瘤的治疗效果，发挥协同增效作用，在保证肿瘤杀伤效能前提下，降低了HER2-CAR-T细胞的使用剂量、减少毒副作用和减少医疗费用。
附图说明
图1为HER2-CAR的结构示意图；
图2为Alo，HER2-CAR-T以及Alo+HER2-CAR-T对U2OS细胞裂解作用的柱状图；
图3为Alo，HER2-CAR-T以及Alo+HER2-CAR-T为杀伤U2OS细胞过程中产生肿瘤细胞杀伤因子TNF-γ、IL-2的柱状图；
图4为Alo，HER2-CAR-T以及Alo+HER2-CAR-T为杀伤U2OS细胞过程中产生肿瘤细胞杀伤因子穿孔素颗粒酶B的柱状图；
其中，Alo代表芦荟苷，HER2-CAR-T代表靶向HER2抗原的嵌合抗原受体T淋巴细胞，U2OS细胞为人骨肉瘤细胞。
具体实施方式
本发明提供了芦荟苷与HER2-CAR-T细胞联合制剂在制备治疗恶性肿瘤药物中的用途。本专利申请人发现，芦荟苷能够上调恶性肿瘤表达HER2抗原的数量，进而明显增强HER2-CAR-T细胞对肿瘤细胞的识别、结合以及杀伤效果。两者的联用表现出协同效应，可达到良好的治疗肿瘤效果，并可使HER2-CAR-T淋巴细胞的用量降低、降低单独的CAR-T淋巴细胞治疗导致的副作用。此外，芦荟苷为小分子有机化合物，价格便宜，能够降低CAR-T细胞的免疫治疗成本。
下面通过具体的实施例验证芦荟苷与HER2-CAR-T细胞联合制剂用于治疗骨肉瘤的协同作用。如图1所示，HER2-CAR序列的信号肽选择了CD8α，胞外识别区为HER2单链抗体，之后顺次连接了CD8α的胞外区和CD8α的跨膜区作为连接结构，然后顺次连接CD28、CD137、CD3ζ的胞内区。
实施例1
构建质粒：本专利申请构建了第三代嵌合抗原受体质粒，即CD8α-HER2scFv-CD8-CD28-CA137-CD3ζ。其中，CD8α为信号肽，对应的核苷酸序列为5’-atg gcc tta cca gtg acc gcc ttg ctcctg ccg ctg gcc ttg ctg ctc cac gcc gcc agg ccg-3’（核苷酸序列为SEQ ID No.2），连接HER2scFv胞外识别区（核苷酸序列为SEQ ID No.3），顺次连接CD8α铰链区和CD8α跨膜区（核苷酸序列为SEQ ID No.4）以及CD28（核苷酸序列为SEQ ID No.5）、CD137（核苷酸序列为SEQ ID No.6）和CD3ζ（核苷酸序列为SEQ ID No.7）的胞内区共刺激信号。本发明专利所述的第三代嵌合抗原受体HER2-CAR的核苷酸序列为SEQ ID No.1。第三代CAR-T细胞相比于第一代和第二代CAR-T细胞，增加了胞内区的共刺激结构域，能够增加肿瘤细胞杀伤因子的分泌数量，提高肿瘤杀伤效果。
构建慢病毒载体和包装质粒：将嵌合抗原受体HER2-CAR的编码基因片段（核苷酸序列为SEQ ID No.1）插入到带有酶切位点AtsI和BstXI的慢病毒真核表达载体pLV[Exp]中，经过酶切、连接、鉴定等步骤确保基因片段的准确性，从而得到HER2-CAR重组质粒pLV[Exp]-HER2-CAR；计算核酸纯度及核酸含量，最后分装保存于-20℃备用。
T淋巴细胞慢病毒转染：采集并分离得到健康人的外周血单个核细胞，加入CD3/CD28免疫磁珠进行孵育，筛选得到CD3阳性T淋巴细胞；向其中加入上述重组慢病毒进行培养，获得HER2-CAR-T淋巴细胞，转染成功后，采用流式细胞仪、免疫荧光等方法检测转染效率≥70％，这表明成功制备得到靶向HER2抗原的CAR-T细胞，保存在回输专用的细胞冻存液中，以供患者回输。
实施例2
芦荟苷和HER2-CAR-T细胞联合制剂对肿瘤细胞的体外杀伤作用，靶细胞选择表达HER2的U2OS细胞（人骨肉瘤细胞），将其在含胎牛血清的DMEM高糖培养基中传代培养。将U2OS细胞接种于96孔细胞培养板，细胞数为1×104/孔，培养24小时。根据不同处理方法为3个实验组，即芦荟苷组（芦荟苷注射剂3mL）、HER2-CAR-T细胞组（1×106/mL细胞悬液）和芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物组（1×106/mL细胞悬液+5%芦荟苷注射剂3mL）。
乳酸脱氢酶释放实验评估药物对肿瘤细胞杀伤效果，乳酸脱氢酶释放法利用乳酸脱氢酶释放存在于细胞质中，当细胞受损细胞膜破裂，乳酸脱氢酶释放到细胞外的培养基中，测定乳酸脱氢酶释放的量评估细胞损害的程度；应用乳酸脱氢酶释放细胞毒性检测试剂盒检测细胞培养上清中的乳酸脱氢酶释放活性，判定药物对肿瘤细胞杀伤效果。将不同效应细胞分别加入U2OS细胞中进行共培养。24小时后，在未加淋巴细胞肿瘤细胞组加入试剂盒提供的细胞裂解液20μl，37℃作用1h；用多孔板离心机进行离心，400g，5min。取上清液120μl加入到一个新96孔板中；用乳酸溶液20μl、INT溶液（1X） 20μl及酶溶液 20μl，共计每孔体系60μl，完成乳酸脱氢酶检测工作液配置；将配置好的乳酸脱氢酶检测工作液60μl加至含有细胞培养上清的96孔板中；混匀，室温，约25℃，避光条件下孵育30min；490nm处测吸光度；单纯肿瘤细胞孔上清设为样品对照孔，裂解的肿瘤细胞孔上清设为最大酶活性对照孔；细胞杀伤效率计算：（作用样品孔（OD值）-样本空白孔（OD值）/（最大酶活性对照孔（（OD值）-样品对照孔（（OD值））×100%。不同治疗方法对肿瘤细胞的裂解作用，如图2。
实施例3
通过细胞因子检测揭示芦荟苷与HER2-CAR-T细胞制剂发挥强效肿瘤杀伤作用的药理，将表达HER2抗原的U2OS细胞以每以100000个/孔，接种于96孔板，将不同组别的药物与靶细胞混合，以总体积200μl的培养液（RPMI 1640培养基+10％FBS）在CO2培养箱中共孵育24小时，温度为37℃。每组3个复孔。接下来，离心后取10μl上清，用Perkin Elmer的Alpha LISA assay试剂盒检测TNF-γ以及IL-2的细胞因子浓度水平。如图3所示，芦荟苷与HER2-CAR-T细胞联合制剂中检测出的TNF-γ、IL-2细胞因子含量高于单独HER2-CAR-T细胞组和单独芦荟苷组，这阐明了二者联合应用增强了对肿瘤细胞杀伤作用的原因。
实施例4
通过ELISA法检测颗粒酶B，收集上清后，将其用试剂检测增强剂稀释5倍；向标准品管内加入500μl样本稀释液；配置成浓度为220ng/ml。进行倍比稀释成10000pg/ml，5000pg/ml，2500pg/ml，1250pg/ml，625pg/ml，313pg/ml，156pg/ml，0pg/ml；向检测版内分别加入标准品及样本上清50μl，加入抗体混合物50μl，室温震荡孵育1h；去上清，用350μl洗液，洗三次，每次2分钟。最后一次清洗后，用吸水纸拍板，洗去剩余液体；加入100μl TMB显色液，室温孵育10min，加100μl 终止液，混匀；利用酶标仪的450nm波长，测各孔吸光度值；制标准曲线，利用回归方程式，将各样品吸光度值代入后，得出的结果浓度需乘以稀释倍数（5倍），确定待测样品浓度。图4所示，芦荟苷与HER2-CAR-T细胞联合制剂中检测出的颗粒酶B的含量高于单独HER2-CAR-T细胞组和单独芦荟苷组，这阐明了二者联合应用增强了对肿瘤细胞杀伤作用的原因。
序列表信息:
DTD版本: V1_3
文件名: 序列表 DNA1.xml
软件名称: WIPO Sequence
软件版本: 2.3.0
生成日期: 2023-08-02
基本信息:
当前申请 / 申请人档案名: Jilin University
申请人姓名或名称: 吉林大学
申请人姓名或名称 / 语言: zh
申请人姓名或名称 / 拉丁名称: Jilin University
发明人姓名: 杨利丽
发明人姓名 / 语言: zh
发明人姓名 / 拉丁名称: LILI YANG
发明人姓名: 于同
发明人姓名 / 语言: zh
发明人姓名 / 拉丁名称: TONG YU
发明人姓名: 矫健航
发明人姓名 / 语言: zh
发明人姓名 / 拉丁名称: JIANHANG JIAO
发明名称: 芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物 ( zh )
序列总量: 1
序列:
序列号 (ID): 1
长度: 12270
分子类型: DNA
特征 位置/限定符:
- source, 1..12270
>mol_type, unassigned DNA
>organism, DNA
残基:
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 (9)
1.一种芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物，其特征在于：包括芦荟苷和HER2-CAR-T细胞；芦荟苷的CAS 号为1415-73-2，分子式C21H22O9，分子量：418.394，结构式如下：
。
2.如权利要求1所述芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物，其特征在于：HER2-CAR结构，核苷酸序列为SEQ ID No.1；HER2-CAR结构包括胞外结构域、跨膜区和胞内结构域。
3.如权利要求2所述芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物，其特征在于：胞外结构域由CD8α Leader、HER2 scFv单链抗体以及CD8α铰链区组成；跨膜区由CD8α构成；胞内结构域由CD28共刺激结构域、CD137共刺激结构域以及CD3ζ胞内信号区共同组成。
4.如权利要求3所述芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物，其特征在于：CD8α Leader为HER2-CAR中的嵌合受体信号肽，核苷酸序列为SEQ ID No.2。
5.如权利要求3所述芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物，其特征在于：HER2 scFv为HER2-CAR中的靶向恶性肿瘤细胞表面HER2抗原的人源化单链抗体，核苷酸序列为SEQ ID No.3。
6.如权利要求3所述芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物，其特征在于：CD8α-hinge为HER2-CAR中的铰链区，CD8α TM为HER2-CAR中的跨膜区，核苷酸序列为SEQID No.4。
7.如权利要求3所述芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物，其特征在于：CD28为HER2-CAR中的胞内的共刺激结构域，用于抗原提呈、促进T淋巴细胞活化和分泌抗肿瘤的细胞因子，核苷酸序列为SEQ ID No.5。
8.如权利要求3所述芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物，其特征在于：HER2-CAR中的CD137为胞内的另一个共刺激结构域，与CD28共刺激结构域相似，用于抗原提呈、促进T淋巴细胞活化和分泌抗肿瘤的细胞因子，核苷酸序列为SEQ ID No.6。
9.如权利要求3所述芦荟苷与HER2-CAR-T细胞联合制备治疗骨肉瘤的药物，其特征在于：HER2-CAR中的CD3ζ为胞内信号区，用于提呈信号活化T淋巴细胞，核苷酸序列为SEQ IDNo.7；HER2-CAR及其载体的核苷酸序列为SEQ ID No.8。
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