CN108795877B - 一种嵌合抗原受体成纤维细胞及其建立方法和应用 - Google Patents

一种嵌合抗原受体成纤维细胞及其建立方法和应用 Download PDF

Info

Publication number
CN108795877B
CN108795877B CN201810731610.7A CN201810731610A CN108795877B CN 108795877 B CN108795877 B CN 108795877B CN 201810731610 A CN201810731610 A CN 201810731610A CN 108795877 B CN108795877 B CN 108795877B
Authority
CN
China
Prior art keywords
gly
ser
leu
thr
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201810731610.7A
Other languages
English (en)
Other versions
CN108795877A (zh
Inventor
孙志坚
康平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Ke Ke Medical Science And Technology Co ltd
Original Assignee
Zhejiang K2oncology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang K2oncology Co ltd filed Critical Zhejiang K2oncology Co ltd
Priority to CN201810731610.7A priority Critical patent/CN108795877B/zh
Publication of CN108795877A publication Critical patent/CN108795877A/zh
Application granted granted Critical
Publication of CN108795877B publication Critical patent/CN108795877B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Rheumatology (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明提供了一种工程化改造的成纤维细胞及其建立方法和应用。本发明工程改造细胞是在自人或者小鼠中分离建立的原代成纤维细胞或者细胞系的基础上，经细胞工程改造后，细胞膜表面表达一种嵌合抗原受体蛋白的成纤维细胞或细胞系，所述嵌合抗原受体蛋白胞外区表达抗特定靶点的单链抗体。本发明所述工程化改造的成纤维细胞命名为“CAR‑F”，CAR‑F可结合肿瘤上皮细胞，应用于肿瘤疾病的治疗或体外检测。

Description

一种嵌合抗原受体成纤维细胞及其建立方法和应用
技术领域
本发明属于基因工程、药物和生物医学医疗技术领域，具体涉及一种工程化改造的嵌合抗原受体成纤维细胞，包含嵌合抗原受体成纤维细胞的药物组合物和试剂盒，以及嵌合抗原受体成纤维细胞、嵌合抗原受体蛋白在诊断和治疗肿瘤疾病中的应用。
背景技术
成纤维细胞(fibroblast)，也称为纤维母细胞，是结缔组织中最常见的细胞，由胚胎时期的间充质细胞(mesenchymal cell)分化而来。成纤维细胞胞体大，为多突的纺锤形或星形的扁平细胞，细胞核呈规则的卵圆形，细胞轮廓不清。在结缔组织中，成纤维细胞还以其成熟状态--纤维细胞(fibrocyte)的形式存在，二者在一定条件下可以互相转变。不同类型的结缔组织含成纤维细胞的数量不同。成纤维细胞制造胶原蛋白、糖胺聚糖、网状和弹性纤维、糖蛋白(存在于细胞外基质中)和细胞因子TSLP。组织损伤会刺激纤维细胞并诱导成纤维细胞的有丝分裂，在伤口愈合中起关键作用(1；2；3)。
成纤维细胞在肿瘤中也起到多样性的关键作用。肿瘤中的成纤维细胞被称为肿瘤相关成纤维细胞(Cancer-Associated Fibroblasts，CAF)。肿瘤相关成纤维细胞分泌大量细胞外基质(ECM)组分和调节因子，在肿瘤内的免疫抑制反应、炎症反应、肿瘤转移等方面起到关键作用。肿瘤相关成纤维细胞(CAF)分泌的细胞外基质组分引起肿瘤微环境的改变称为肿瘤细胞外基质重塑(ECM remodeling)。肿瘤细胞外基质重塑可导致降解细胞外基质的酶活性激活。蛋白酶如基质金属蛋白酶(MMPs)裂解细胞外基质，这些蛋白酶来自成纤维细胞。在许多肿瘤类型中，尤其是与上皮细胞相关的肿瘤类型，肿瘤细胞外基质重塑是常见的，在肿瘤转移和免疫调节中，细胞外基质重塑起到重要作用(4；5)。
肿瘤从原发位置发生转移，进入血液循环系统或者淋巴循环系统，形成循环肿瘤细胞(Circulating Tumor Cells，CTC)，肿瘤细胞由原发灶进入循环系统，循环肿瘤细胞面临缺乏贴壁生存信号(细胞-细胞间接触信号)、免疫系统攻击、流体剪切力等因素，循环系统的环境并不适宜肿瘤生存。研究者发现大量凋亡循环肿瘤细胞(Apoptotic CTCs)。凋亡循环肿瘤细胞是正在经历细胞凋亡(细胞死亡)的循环肿瘤细胞，特征为核碎裂或细胞质起泡等。肿瘤组织形成循环肿瘤细胞团簇(CTC clusters)可以增加肿瘤细胞在循环系统中的生存能力。循环肿瘤细胞团簇(CTC clusters)是由包含循环肿瘤细胞和多种非肿瘤细胞组成，包括间充质细胞、免疫细胞、血小板和肿瘤相关成纤维细胞。肿瘤相关成纤维细胞增加循环肿瘤细胞团簇内肿瘤细胞的活力，并促进了转移灶的形成。如果去除肿瘤相关成纤维细胞，肿瘤转移的数目明显减少(6；7；8)。
人上皮细胞黏附分子(epithelial cell adhesion molecule，EpCAM)是最早在结肠癌中发现的一种Ⅰ型跨膜糖蛋白，分子质量为40kDa，由314个氨基酸残基构成，分子结构包括细胞外结构域(EpEX)、单次跨膜结构域和细胞内结构域(EpICD)，可介导非Ca2+依赖性同源细胞间的黏附。EpCAM广泛表达于上皮来源的正常组织，并在多种上皮源性癌组织中不同程度的过表达，已被多项研究证实促进细胞周期和细胞增殖，并与肿瘤的发生、进展、侵袭及转移等相关。近年来，EpCAM被多项研究证实为循环肿瘤细胞和肿瘤干细胞的标志物(9；10)。
CAR-T细胞疗法是Chimeric Antigen ReceptorT-Cell Immunotherapy的缩写，通过外源基因转导技术，把识别肿瘤相关特异性抗原的单链抗体片段(single-chainantibody fragment，scFv)和T细胞活化序列的融合蛋白表达到T细胞表面，能够以非MHC限制性的模式表现出较强的抗肿瘤作用(11；12)。
现有技术中还没有使用嵌合抗原受体(CAR)工程化改造成纤维细胞的相关研究报道。
参考文献：
1.Weissman-Shomer P,Fry M(1975)Chick embryo fibroblasts senscence invitro:pattern of cell division and life span as a function of celldensity.Mechanisms of ageing and development 4,159-166.
2.Smith RS,Smith TJ,Blieden TM et al.(1997)Fibroblasts as sentinelcells.Synthesis of chemokines and regulation of inflammation.The Americanjournal of pathology 151,317-322.
3.Bonnans C,Chou J,Werb Z(2014)Remodelling the extracellular matrixin development and disease.Nature reviews Molecular cell biology 15,786-801.
4.Dave JM,Bayless KJ(2014)Vimentin as an integral regulator of celladhesion and endothelial sprouting.Microcirculation 21,333-344.
5.Cirri P,Chiarugi P(2012)Cancer-associated-fibroblasts and tumourcells:a diabolic liaison driving cancer progression.Cancer metastasis reviews31,195-208.
6.McCarthy JB,El-Ashry D,Turley EA(2018)Hyaluronan,Cancer-AssociatedFibroblasts and the Tumor Microenvironment in Malignant Progression.Frontiersin cell and developmental biology 6,48.
7.De Vlieghere E,Verset L,Demetter P et al.(2015)Cancer-associatedfibroblasts as target and tool in cancer therapeutics anddiagnostics.Virchows Archiv:an international journal of pathology 467,367-382.
8.Gunaydin G,Kesikli SA,Guc D(2015)Cancer associated fibroblasts havephenotypic and functional characteristics similar to the fibrocytes thatrepresent a novel MDSC subset.Oncoimmunology 4,e1034918.
9.Litvinov SV,Velders MP,Bakker HA et al.(1994)Ep-CAM:a humanepithelial antigen is a homophilic cell-cell adhesion molecule.The Journal ofcell biology 125,437-446.
10.Ao Z,Shah SH,Machlin LM et al.(2015)Identification of Cancer-Associated Fibroblasts in Circulating Blood from Patients with MetastaticBreast Cancer.Cancer research 75,4681-4687.
11.Li F,Zhang T,Cao L et al.(2018)Chimeric Antigen Receptor T CellBased Immunotherapy for Cancer.Current stem cell research&therapy 13,327-335.
12.Newick K,O'Brien S,Moon E et al.(2017)CAR T Cell Therapy for SolidTumors.Annual review of medicine 68,139-152.
13.Shirasu N,Yamada H,Shibaguchi H et al.(2012)Molecularcharacterization of a fully human chimeric T-cell antigen receptor for tumor-associated antigen EpCAM.Journal of biomedicine&biotechnology 2012,853879.
14.Bezverbnaya K,Mathews A,Sidhu J et al.(2017)Tumor-targetingdomains for chimeric antigen receptor T cells.Immunotherapy 9,33-46.
15.Melenhorst JJ,Lacey SF,Bedoya F(2017)Chimeric Antigen Receptor TCells:Self-Replicating Drugs for Cancer.Current drug targets 18,332-340.
16.Luo H,Tu G,Liu Z et al.(2015)Cancer-associated fibroblasts:amultifaceted driver of breast cancer progression.Cancer letters 361,155-163.
17.Zhi K,Shen X,Zhang H et al.(2010)Cancer-associated fibroblasts arepositively correlated with metastatic potential of human gastriccancers.Journal of experimental&clinical cancer research:CR 29,66.
发明内容
基于上述现有技术的空白，本发明提供一种嵌合抗原受体成纤维细胞(CAR-F)，CAR-F细胞是Chimeric Antigen Receptor Fibroblast-Cell的缩写，通过外源基因转染技术或者载体病毒感染，把识别特定抗原的单链抗体片段(single-chain antibodyfragment，scFv)和跨膜区域相连，表达在成纤维细胞表面，能够介导成纤维细胞识别特定细胞，例如肿瘤细胞，并与之相互作用，发挥特定生物学效应。本发明工作具有创新性。
本发明的具体技术方案如下：
本发明的一个目的是提供一种工程化改造的成纤维细胞CAR-F，所述CAR-F细胞表面表达一种嵌合抗原受体蛋白。
在本发明的一个实施例中，所述嵌合抗原受体蛋白包含一个单链抗体区域和一个跨细胞膜区域；优选的，所述单链抗体区域包含信号肽和单链抗体；优选的，所述跨细胞膜区域包含包括跨膜序列的连接肽，和/或胞内片段。
在本发明的另一个实施例中，所述嵌合抗原受体(CAR)针对如下抗原中的一种或多种：CD19、CD20、CEA、GD2(又称B4GALNT1、b1,4-乙酰基-氨基半乳糖基转移酶1)、FR(Flavin还原酶)、PSMA(前列腺特异性膜抗原)、PMEL(前黑素小体蛋白)、CA9(碳酸酐酶IX)、CD171/Ll-CAM、IL-13Ra2、MART-l(又称粘蛋白-A)、ERBB2、NY-ESO-l(又称CTAG1B，癌/睾丸抗原IB)、MAGE(黑素瘤相关抗原El)家族蛋白、BAGE(B黑素瘤抗原家族)家族蛋白、GAGE(生长激素释放因子)家族蛋白、AFP(a-胎蛋白)、MUC1(mucin1，细胞表面相关)、CD22、CD23、CD30、CD33、CD44v7/8、CD70、VEGFR1、VEGFR2、IL-llREGP-2、EGP-40、FBP、GD3(又称ST8SIA1，ST8a-N-乙酰基-神经酰胺a-2，8-唾液酸转换酶1)、PSCA(前列腺干细胞抗原)、FSA(又称KIAA1109)、PSA(又称KLK3，激肽释放酶相关的肽酶3)、HMGA2，胎儿型乙酰胆碱受体、LeY(又称FUT3)、EpCAM、MSLN(间皮素)、IGFR1、EGFR、EGFRvIILERBB3、ERBB4、CA125(又称MUC16，mucin16，细胞表面相关)、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、SCC(又称SERPINB3)、AFU(又称FUCA1)、EBV-VCA、POA(又称VDR，维生素D(l，25-二氢维生素D3)受体)、p2-MG(b-2-微球蛋白)和PROGRP(GRP胃泌素释放肽)。优选地，所述CAR为针对EpCAM的嵌合抗原受体(13；14；15)。
在某些实施方案中，所述用于工程化改造的成纤维细胞来自如下一种或多种：
Ku80+/+；L Cells、MEF(DR4)、Ku80-/-、53Bp1-/-、MEF(CF-1)、STO、SNL76/7、BALB/3T3 clone A31、NIH/3T3、3T3-Swiss albino、SNLP 76/7-4、CT26.WT、MB352、GCN2-KO-DR、3T3 MEFs KO、McCoy、PEA 10、SYF；3T3 MEFs WT、DR-Wildtype、GP+E-86；PEA13、SYF+c-Src、Wt MEFs、Bax Bak DKO SV40 MEF、WT SV40 MEF、Mfn2-null MEFs、Bid KO SV40 MEF、BakKO SV40 MEF、GP+envAM-12、AFT024、OPA1-null MEFs、Mfn1-null MEFs、Bcl-2 KO SV40MEF、Mfn1/Mfn2-null MEFs、BAX KO SV40 MEF Bad KO SV40 MEF、MEF(C57BL/6)[MEF-BL/6-1]、CHOP-KO-DR、MEF(CF-1)MITC、MEF(C57BL/6)IRR、AFT024 IRR、MEF(CF-1)IRR、F19、TIN2 F/F、TRF2 F/-Rosa26-CreERT2、ES-D3[D3]、ES-E14TG2a、MEF-1、C3H/10T1/2、RAG、NCTC clone 2472、NCTC clone 929[L cell，L-929，derivative of Strain L]、A9(APRTand HPRT negative derivative of Strain L)、HGF、HLF、HDFn、hTERT、Lung Fibroblast、HDFa、IMR-90、ATCC-CYS0105、Hs 895.Sk、Malme-3M、Hs27、Hs 746T、MRC-9、Detroit 551、CCD-1112Sk、L Wnt-3A、L Wnt-5A、CCD-16Lu、MRC-5、WI-38、HYS0103、BJ、LL 29(AnHa)、COLO829、PHM1-41、CCD-1070Sk、MC-IXC、HEL 299、LL 47(MaDo)、CCD-986Sk、Hs68、CCD-1064Sk、LL 97A(AlMy)、CCD-33Lu、LL 24CCD-19Lu、CCD-27Sk、BJ-5ta、hTERT PF179T CAF、ATCC-DYP0730、CCD-1059Sk、CCD-1079Sk、Malme-3、WS1、WPMY-1、ATCC-DYP0250、ATCC-HYR0103、CCD-13Lu、CCD-1090Sk、HFL1、C 211、CHON-002、HFF-1、WI-38、HFF-1IRR、IMR-32、9TR#1、BRL3A、Rat2、CFSE、RFL-6和OP9。优选地，所述成纤维细胞为NIH/3T3细胞；优选地，所述成纤维细胞为3T3-Swiss albino细胞；优选地，所述成纤维细胞为OP9细胞；优选地，所述成纤维细胞为MEF细胞。
在本发明的另一个实施例中，所述单链抗体为针对抗原EpCAM的单链抗体；更优选的，所述单链抗体的氨基酸序列如SEQ ID NO.12、14、16、18、20、或22所示。
在本发明的另一个实施例中，所述跨细胞膜区域包含的胞内片段优选小鼠CD8细胞内片段或GFP；更优选的，所述胞内片段的氨基酸序列如SEQ ID NO.6或10所示。
在本发明的另一个实施例中，所述嵌合抗原受体蛋白的氨基酸序列如SEQ IDNO.24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、或58所示。
本发明的另一个目的是提供一种包含抗人上皮细胞黏附分子(EpCAM)的单链抗体的嵌合抗原受体蛋白，所述嵌合抗原受体蛋白包含一个抗人上皮细胞黏附分子(EpCAM)的单链抗体区域和一个跨细胞膜区域；优选的，所述单链抗体区域包含信号肽和单链抗体；优选的，所述跨细胞膜区域包含包括跨膜序列的连接肽，和/或胞内片段。
在本发明的一个实施例中，所述单链抗体的氨基酸序列如SEQ ID NO.12、14、16、18、20、或22所示。
在本发明的另一个实施例中，该嵌合抗原受体蛋白的氨基酸序列如SEQ IDNO.24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、或58所示。
本发明的另一个目的是提供一种载体，所述载体包含编码所述嵌合抗原受体蛋白的核酸分子；优选的，所述核酸分子的核苷酸序列如SEQ ID NO.23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、或57所示。
本发明的另一个目的是提供一种非人细胞，所述细胞包含权利要求6所述嵌合抗原受体蛋白或权利要求7所述载体；优选的，所述非人细胞为病毒；优选的，所述非人细胞用于CAR-F的生产制备。
本发明的另一个目的是提供一项本发明所述工程化改造的成纤维细胞CAR-F、所述嵌合抗原受体蛋白、所述载体、和/或所述非人细胞在制备体外检测试剂盒中的应用，所述试剂盒优选为用于循环肿瘤细胞捕获和培养的试剂盒。
本发明的另一个目的是提供一项本发明所述工程化改造的成纤维细胞CAR-F、所述嵌合抗原受体蛋白、所述载体、和/或所述非人细胞在体外构建肿瘤类器官中的用途。本发明所述工程化改造的成纤维细胞CAR-F促进肿瘤上皮细胞的生长，促进肿瘤类器官的形成和生长。
本发明的另一个目的是提供一项本发明所述工程化改造的成纤维细胞CAR-F、所述嵌合抗原受体蛋白、所述载体、和/或所述非人细胞在制备肿瘤免疫制剂中的应用，所述肿瘤免疫制剂优选为肿瘤疫苗。
本发明的另一个目的是提供一种药物组合物或试剂盒，其特征在于，所述药物组合物或试剂盒包含本发明所述工程化改造的成纤维细胞CAR-F、所述嵌合抗原受体蛋白、所述载体、和/或所述非人细胞。
术语及定义
除非特别说明，本申请中所使用的术语和定义均是本领域中惯常使用的含义并且为本领域技术人员所知晓。
如本申请中所使用的，术语“肿瘤位点”是指含有或被怀疑含有肿瘤细胞的体内或离体位置。所述肿瘤位点包括固体肿瘤以及接近或邻近肿瘤生长处的位置。
术语“抗体”涵盖例如，单克隆抗体、多克隆抗体、单链抗体、抗体片段(其显示出所需的生物学或免疫学活性)。在本申请中，术语“免疫球蛋白”(Ig)与抗体可互换地使用。所述抗体可特异性地靶向肿瘤抗原，例如表面肿瘤抗原，例如EGFR，CD4，CD8、Neu等。
本发明所述“肿瘤”可选自肺癌、支气管癌、结肠直肠癌、前列腺癌、乳腺癌、胰腺癌、胃癌、卵巢癌、膀胱癌、脑或中枢神经系统癌症、外周神经系统癌症、食道癌、宫颈癌、黑素瘤、子宫或子宫内膜癌、口腔癌或喉癌、肝癌、肾癌、胆管癌、小肠癌或阑尾癌、唾液腺癌、胸腺癌、肾上腺癌、骨肉瘤、软骨肉瘤、脂肪瘤、睾丸癌以及恶性纤维组织细胞瘤。
本发明所述“肿瘤细胞”可选自肺癌、支气管癌、结肠直肠癌、前列腺癌、乳腺癌、胰腺癌、胃癌、卵巢癌、膀胱癌、脑或中枢神经系统癌症、外周神经系统癌症、食道癌、宫颈癌、黑素瘤、子宫或子宫内膜癌、口腔癌或喉癌、肝癌、肾癌、胆管癌、小肠癌或阑尾癌、唾液腺癌、胸腺癌、肾上腺癌、骨肉瘤、软骨肉瘤、脂肪瘤、睾丸癌以及恶性纤维组织细胞瘤的癌症产生的细胞。
本发明所述“应用”或“用途”，既可表示以疾病治疗和疾病治疗方案检测为目的的应用，也可表示非治疗目的的应用，例如，科学研究等。
本发明的有益效果：
1)本发明采用改造成纤维细胞，利用基因工程技术，在细胞表面表达抗EpCAM单链抗体的嵌合抗原受体蛋白。经过工程化改造的成纤维细胞，可以识别表达EpCAM的肿瘤细胞，并与之结合，形成细胞团簇。成纤维细胞分泌各种生长因子，提供细胞-细胞间信号，提高肿瘤细胞活力。
2)本发明设计了一组细胞膜表达抗EpCAM的单链抗体的分子结构。验证了这些分子结构的生物学活性。
3)本发明设计试验证明了，工程化改造的表达嵌合抗原受体蛋白的成纤维细胞(CAR-F)，可以促进肿瘤细胞形成三维培养的类器官(organoids)结构。
附图说明
图1.包含抗人上皮细胞黏附分子(EpCAM)的单链抗体的嵌合抗原受体蛋白结构示意图。示意图(由左至右为1-6)展示了几种细胞膜表达嵌合抗原受体的模式：1.EpHL-V(自N端至C端，以下相同)：信号肽-单链抗体重链-连接肽-单链抗体轻链-连接肽-跨膜区域；2.EpLH-V:信号肽-单链抗体轻链-连接肽-单链抗体重链-连接肽-跨膜区域；3.EpHL-C8:信号肽-单链抗体重链-连接肽-单链抗体轻链-连接肽-跨膜区域-CD8胞内序列；4.EpLH-C8:信号肽-单链抗体轻链-连接肽-单链抗体重链-连接肽-跨膜区域-CD8胞内序列；5.EpHL-GP:信号肽-单链抗体重链-连接肽-单链抗体轻链-连接肽-跨膜区域-绿色荧光蛋白序列；6.EpLH-GP:信号肽-单链抗体轻链-连接肽-单链抗体重链-连接肽-跨膜区域-绿色荧光蛋白序列。
图2.pLVX-IRES-Puro载体结构图。
图3.pLVX-IRES-Puro-CAR阳性克隆鉴定。
图4.慢病毒活力和滴度定性测定。
图5.NIH-3T3和OP9细胞中稳定表达嵌合抗原受体蛋白：通过绿色荧光指示成纤维细胞表达融合GFP嵌合抗原受体蛋白。
图6.WI-38被带有各种序列的嵌合抗原受体蛋白载体的病毒感染，感染48小时后，在荧光显微镜下观察绿色荧光蛋白的表达。
图7.NIH-3T3细胞稳定表达各种嵌合抗原受体蛋白的鉴定：NIH-3T3细胞加入慢病毒(核心载体分别是pLVX-IRES-Puro-EpHL-GP、pLVX-IRES-Puro-EpLH-GP、pLVX-IRES-Puro-EpHL-V、pLVX-IRES-Puro-EpLH-V、pLVX-IRES-Puro-EpHL-C8、pLVX-IRES-Puro-EpLH-C8)2天后，加入嘌呤霉素筛选2星期后，得到稳定表达株。稳定表达株提取蛋白，标记为：3T3-EpHL-V、3T3-EpLH-V、3T3-EpHL-C8、3T3-EpLH-C8、3T3-EpHL-GP、3T3-EpLH-GP。阳性对照为在Hek293细胞中瞬时转染pLVX-IRES-Puro-EpHL-V，细胞裂解后提取的蛋白，Hek293-EpHL-V。阴性对照为Hek293细胞瞬时转染pLVX-IRES-Puro空载体质粒后提取的蛋白Hek293-Velcle。抗HA标签的抗体用来检测外源表达的蛋白。
图8.CTL-F和CAR-F结合重组人EPCAM蛋白染色：CAR-F细胞表面表达抗人EpCAM单链抗体的嵌合抗原受体蛋白，能够结合重组的人EPCAM蛋白。图A和B为绿色FITC信号，图C和D为蓝色DAPI信号。图A和C为NIH-3T3成纤维细胞(CTL-F)，图B和D为表达嵌合蛋白抗体的NIH-3T3成纤维细胞(CAR-F)。表达嵌合抗体蛋白的成纤维细胞(CAR-F)可以结合加入的重组人EpCAM/TROP1Fc蛋白，绿色信号的强度代表结合EpCAM蛋白的能力。表明CAR-F表面表达的嵌合抗体，具有结合EpCAM的功能活性。
图9.对比CTL-F和多种CAR-F的上皮细胞捕获能力：A-B，短时间细胞粘附实验结果；C，捕获上皮细胞后，全体细胞中检测上皮细胞标志物的qRT-PCR实验结果；D，捕获上皮细胞后，全体细胞中检测上皮细胞标志物的Westernblot实验结果。
图10.肿瘤上皮细胞和CAR-F混合培养体系：表达各种嵌合抗原受体的成纤维细胞促进类器官生长的能力对比。
图11.肿瘤和CAR-F比例影响类器官形成的大小和数目。
具体实施方式
通过以下实施例对本发明作进一步的详细描述，但应理解本发明并不受以下内容所限制。
实施例1：包含抗人EpCAM单链抗体的嵌合抗原受体蛋白的质粒的构建
包含抗人上皮细胞黏附分子(EpCAM)的单链抗体的嵌合抗原受体蛋白自N端至C端顺序包含：1)信号肽序列；2)抗人EpCAM单链抗体序列；3)包括跨膜序列的连接序列；4)小鼠CD8细胞内序列或GFP序列；5)HAtag序列(图1)。所涉及的序列信息详见下表：
Figure GDA0002522437280000081
Figure GDA0002522437280000091
信号肽的核苷酸和氨基酸序列如SEQ ID NO.1、2所示；抗人EpCAM单链抗体的编码核苷酸和氨基酸序列如SEQ ID NO.11-22所示；包括跨膜序列的连接序列的核苷酸和氨基酸序列如SEQ ID NO.3、4所示；小鼠CD8细胞内序列的编码核苷酸和氨基酸序列如SEQ IDNO.5、6所示；绿色荧光蛋白的编码核苷酸和氨基酸序列如SEQ ID NO.9、10所示；HAtag序列的编码核苷酸和氨基酸序列如SEQ ID NO.7、8所示。包含抗人EpCAM单链抗体的嵌合抗原受体蛋白的编码核苷酸和氨基酸序列如SEQ ID NO.23-58所示。
一组包含抗人EpCAM单链抗体的嵌合抗原受体蛋白的编码核苷酸序列(SEQ IDNO.23、25、27、29、31、33、35、37、39、41、43、45、47、49、51、53、55、57)采用全序列基因合成的方式合成(通用生物系统(安徽)有限公司)目标DNA序列，序列合成后接入pLVX-IRES-Puro(Clontech公司，货号CatalogNo.632183)。形成质粒“pLVX-IRES-Puro-EpHL-GP”、“pLVX-IRES-Puro-EpLH-GP”、“pLVX-IRES-Puro-EpHL-V”、“pLVX-IRES-Puro-EpLH-V”、“pLVX-IRES-Puro-EpHL-C8”、“pLVX-IRES-Puro-EpLH-C8”，统一称为pLVX-IRES-Puro-EpCAR质粒。用XhoI和NotI双酶切pLVX-IRES-Puro-EpCAR鉴定阳性克隆(图3)。不同结构的CAR在图3中从1-11道标注分别为EpHL-V-31、EpHL-V-510、EpHL-V-47，EpLH-V-47、EpHL-C8-31、EpHL-C8-510、EpHL-C8-47、EpLH-C8-47、EpHL-GP-31、EpHL-GP-510和EpHL-GP-47。
采用低内毒素质粒大提试剂盒(品牌：Qiagen货号：#12362)制备高纯度pLVX-IRES-Puro-CAR质粒：将阳性克隆的表达菌株用LP液体培养基培养，37℃，200rpm转速，过夜培养后，6000g离心15分钟，收获菌株沉淀；按照试剂盒说明书加入裂解缓冲液，中和缓冲液，离心后收集上清；上清液加入柱子，加入清洗缓冲液后，加入洗脱缓冲液洗脱质粒DNA，通过异丙醇沉淀，15000g离心30分钟收集沉淀，乙醇清洗后晾干，加入纯水溶解DNA，定量后冻存、或用于转染细胞、或作为骨架质粒包装慢病毒。
实施例2：慢病毒包装、浓缩和滴度测定
1、实验材料:
1)病毒包装质粒：pCMVΔR.89质粒(Pcg41)、pVSV-Gplasmid质粒(Pcg10)。
2)包装细胞：Hek-293T细胞(DuBridge,et.al.1987，Pear，etal1993)。
3)病毒包装培养基：DMEM完全培养基(Dulbecco's Modified Eagle Medium，Invitrogen，catalog#11995)；胎牛血清(FBS，Hyclone，货号：SH30084.03)；非必需氨基酸(Gibcocatalog#1140-050)；抗生素(PenStrep，Gibic，货号：15070063)。
4)细胞转染试剂：Lipofectamine2000(Invitrogene，货号：11668-019)；无血清培养基Opti-MEM(Gibco，catalog#11058)；
5)病毒纯化和感染：0.45μM过滤器(Corning，catalog#430314)；蔗糖(Sigma，货号：S7903)；聚凝胺(Polybrene，Sigma，货号：H9268)。
6)检测抗体：HAtag抗体(金斯瑞，货号：A01244)。
2、病毒包装：
1)Hek-293T细胞培养：在10cm细胞培养皿中，加入含有3x106细胞的10ml完全培养基(DMEM+10％FBS+非必需氨基酸)，在37℃，5％CO2培养箱内培养过夜，第二天细胞汇合度达到80％。
2)准备转染复合物：DNA：脂质体(Lipofectamine2000)比例按照1:3。在Opti-MEM中分别稀释DNA和脂质体。在1.5ml无菌离心管中加入587.4μl Opti-MEM，滴入12.6μl脂质体，涡轮震荡，充分混匀。室温静置5分钟后，向脂质体稀释液中，加入2.0μg骨架蛋白、2.0μgPcg41和0.2μgPcg10。轻轻震荡后，室温静置20分钟。
3)转染细胞：向Hek-293T培养皿中滴加转染复合物，一边滴加，一般轻轻晃动培养皿，使转染复合液迅速分散。
4)培养和保存：在37℃，5％CO2培养箱内培养12h后，换成6ml新鲜培养基(DMEM+10％FBS)。继续培养2天后，向培养皿中加入6ml新鲜培养液，继续培养2天后，向培养皿中加入6ml新鲜培养液。再过48h后，收集约15ml上清液到50ml离心管中。上清液4℃，1000rpm离心5分钟，去除细胞碎片，用0.45微米过滤器过滤上清，直接感染细胞或者分装冻存在-80℃冰箱，或者用蔗糖梯度离心进一步浓缩纯化病毒颗粒后分装冻存。分装按照每次估计的用量，避免反复冻融。
3、慢病毒浓缩纯化：
取6个Beckman超速离心管，70％乙醇清洗后在生物安全柜中风干。每管加入30ml病毒上清，然后将2ml的20％蔗糖溶液(PBS配制)轻轻加入上清液的底部，形成一个蔗糖垫。务必确定离心管没有裂缝且两两平衡。4℃离心，25000rpm，2h。离心完毕后小心取出超速离心管，小心弃去上清液，留下管底沉淀。管底可见少量的半透明或白色沉淀。将超速离心管倒置在吸水纸上晾干，并用无菌枪头吸去管壁上多余的液体。100ul冷培养基重悬，并轻轻吹打溶解沉淀。分装10ul/管，-80℃冻存。
4、慢病毒活力和滴度定性测定：
通过病毒载体表达的GFP，可以通过在荧光显微镜下拍摄照片，计数被成功感染的表达GFP细胞的数目，推算活性病毒的滴度。在本实施例中，如图4所示，SEQ ID NO.55和SEQID NO.57核苷酸序列所对应的CAR序列EpHL-GP-510和EpLH-GP-510被装载在PLVX-IRES-Puro质粒中，制备pLVX-IRES-Puro-EpHL-GP和pLVX-IRES-Puro-EpLH-GP，并作为病毒核心质粒，进行病毒包装、浓缩后，感染Hek293T细胞。具体步骤如下：
绿色荧光蛋白标记法测定病毒浓缩液从100，10-2到10-7稀释，以无血清培养基梯度稀释重组腺病毒。取对数生长期Hek-293T细胞，胰酶消化、计数，铺24孔板，每孔500μl(5×105细胞/ml)。每个病毒梯度设2个复孔，每孔滴加50μl待测病毒样品。37℃，5％CO2培养箱培养48h。直接荧光显微镜下计数每视野荧光细胞数(图4)。每孔计数3个视野，每滴度计数3个复孔。以每视野荧光细胞数10～50个为最佳。测得病毒滴度以绿色荧光形成单位/ml(PFU/ml)表示，每个荧光细胞对应一个绿色荧光形成单位。计算病毒滴度(PFU/ml)＝[(每个视野中绿色荧光阳性细胞数)×(每孔视野数)]/[病毒体积(ml)×稀释倍数]。浓缩后表达EpHL-GP-510和EpLH-GP-510两个CAR的慢病毒的滴度分别为7x108PFU/ml和5x108PFU/ml。
在本实施例中，表达CAR的慢病毒可以包装，包装、浓缩后的慢病毒，经多滴度测定(图4)，证明具有感染活性强，滴度高，满足后续感染靶细胞的要求。
实施例3：靶细胞感染和嵌合抗原受体稳定表达细胞株的筛选
1、小鼠胚胎成纤维细胞(Mouse embryonic fibroblast，MEF)制备
1)性成熟母小鼠与种公鼠合笼，观察小鼠阴道口见有冻胶状物当天上午定为怀孕的0.5天。
2)处死怀孕12.5～14.5天母鼠，取出带有胎膜的胚胎，置于无菌平皿，PBS洗涤弃除红细胞。剥离去除胎膜，取出胎鼠，用PBS洗涤三次。去除胚胎头部、内脏和四肢，将躯干部置于无菌平皿内，用PBS至少洗涤三次，弃除红细胞。将鼠胚躯干剪成1mm3以下的碎块，吸置于离心管内。
3)加适量胰酶，37℃，孵育酶解10-20分钟，取出离心管，移液管反复吹打，加足量带有血清的培养液终止消化酶解，1000rpm，离心5分钟，弃掉上清，加适量培养液，反复吹打20次，37℃、5％CO2、培养箱培养3-7天。细胞互汇合度大于90％后按照1：5传代。传到第3代后，当细胞90％-100％汇合度时，用于病毒转染或者程序降温冻存，至于液氮长期保存。
2、病毒感染建立嵌合抗原受体蛋白稳定表达的3T3、OP9和MEF细胞：
1)3T3、OP9、WI-38或MEF细胞复苏或者传代后，细胞密度调整到3x104细胞/ml，种2ml细胞悬液到6孔培养板中，培养过夜，保持细胞汇合度在20％～30％。
2)接种12h后，取出培养液，加入1ml完全细胞培养液，加入polybrene，终浓度为5μg/ml。加入1ml未浓缩病毒液或者10ul浓缩病毒液。
3)在37℃，5％CO2培养箱内培养12h后，换成新鲜的完全培养基。
4)48h后，在荧光显微镜下，在病毒表达蛋白结构带有融合绿色荧光蛋白的被感染靶细胞中，观察绿色荧光信号。当细胞汇合度达到80％～100％，从6孔板传代细胞到10cm培养皿中。
5)有绿色荧光的靶细胞，通过FACS无菌分选有绿色荧光的细胞群，获得稳定表达外源嵌合抗原受体蛋白的细胞群CAR-F，命名为“3T3-EpHL-GP”、“3T3-EpLH-GP”、“MEF-EpHL-GP”、“MEF-EpLH-GP”、“OP9-EpHL-GP”、“OP9-EpLH-GP”(图5，使用EpHL-GP-510和EpLH-GP-510)。
如图5所示，在NIH-3T3、OP9和MEF细胞中，加入表达CAR的慢病毒感染，慢病毒表达EpHL-GP-510和EpLH-GP-510，加入病毒48小时后，在荧光显微镜下观察荧光蛋白(GPF)的水平，证明表达CAR的慢病毒可以在靶成纤维细胞中高效表达。
在某些案例中人肺成纤维细胞WI-38被带有各种序列的嵌合抗原受体蛋白载体的病毒感染，感染48小时后，WI-38细胞在荧光显微镜下观察绿色荧光蛋白的表达。如图6所示，A为明场照片，B-G分别为感染表达如下不同序列的嵌合抗原受体蛋白的细胞荧光照片：EpHL-GP-31、EpHL-GP-510、EpHL-GP-47、EpLH-GP-31、EpLH-GP-510和EpLH-GP-47。在该案例中，不同序列的嵌合抗原受体蛋白均有很好的表达。
在某些应用中，病毒不带有绿色荧光蛋白，该类病毒感染靶细胞，从6孔板消化的细胞，每孔分到6个10cm培养皿中，第二天加入100ng/ml～1μg/ml的嘌呤霉素(Puromycin，浓度根据不同靶细胞确定)，筛选7天后，换带有嘌呤霉素的新鲜培养液继续培养7天。挑取细胞团转移到24孔板中，进行放大培养，用蛋白裂解液裂解挑取的单克隆，用免疫印迹(western blot)鉴定带有HA标签的外源蛋白的表达(图7，使用EpHL-V-510和EpLH-V-510，EpHL-C8-510和EpLH-C8-510，EpHL-GP-510和EpLH-GP-510)。稳定表达CAR-F细胞命名为：“3T3-EpHL-V”、“3T3-EpLH-V”、“3T3-EpHL-C8”、“3T3-EpLH-C8”、“MEF-EpHL-V”、“MEF-EpLH-V”、“MEF-EpHL-C8”和“MEF-EpLH-C8”、“OP9-EpHL-V”、“OP9-EpLH-V”、“OP9-EpHL-C8”和“OP9-EpLH-C8”。
如图7所示，通过HA标签检测外源表达的蛋白，可以证明，嵌合抗原受体蛋白在成纤维细胞中可以高效的表达。
实施例4：CAR-F表达嵌合抗体蛋白识别并结合EpCAM的功能检测
1、EpCAM亲和染色实验：
1)对照成纤维细胞(CTL-F)和工程化改造的成纤维细胞(CAR-F)按照5000细胞/孔，培养于96孔培养板中(Corning，#3603)中。在37℃，5％CO2细胞培养箱中培养24h。
2)取出微孔板，移除细胞培养液，加入100ul4％多聚甲醛固定15分钟。
3)移除多聚甲醛后，用PBS清洗2遍，然后加入3％BSA，37℃孵育1h。
4)用PBST清洗3遍后，加入100ng/ml重组人EpCAM/TROP1Fc蛋白(R&DSystems，#960-EP-050)，37℃孵育1h。
5)移除重组蛋白，用PBST清洗3次后，加入1:1000稀释的FITC标记的抗人IgG1抗体(Sigma-ALDRICH，#F0767)，37℃孵育30分钟。
6)移除二抗，用PBST清洗3次，加入5μg/mlDAPI(Sigma-ALDRICH，#D9542)，室温孵育10分钟。
7)移除DAPI，用PBST清洗三次后，加入100ulPBS到微孔中。在荧光显微镜下观察拍摄绿色荧光和蓝色荧光。
2、实验结果：请见图8，图A和B为绿色FITC信号，图C和D为蓝色DAPI信号。图A和C为感染对照病毒颗粒的NIH-3T3成纤维细胞(CTL-F，表达空载体)，图B和D为表达嵌合蛋白抗体的NIH-3T3成纤维细胞(CAR-F，表达嵌合抗原受体蛋白EpHL-C8-510)。表达嵌合抗体蛋白的成纤维细胞(CAR-F)可以结合加入的重组人EpCAM/TROP1Fc蛋白，绿色信号的强度代表结合EpCAM蛋白的能力。表明CAR-F表面表达的嵌合抗体，具有结合EpCAM的功能活性。
实施例5：CAR-F捕获肿瘤上皮细胞能力测试
成纤维细胞不表达人钙黏蛋白，只有捕获的上皮细胞(HepG2)表达人钙黏蛋白，通过检测捕获、洗脱后孔中细胞人钙黏蛋白的水平，可以定量评价对照组成纤维细胞(CTL-F)和工程改造的成纤维细胞(CAR-F)对上皮细胞的亲和和捕获能力。
1、上皮细胞捕获实验：
1)未经工程化改造的对照细胞(CTL-F)和工程化改造成纤维细胞(CAR-F)细胞分别生长到90％聚合度后，加入10μg/ml丝裂霉素处理3h，用PBST清洗2次，然后加入胰酶消化后，离心收集细胞沉淀，用完全培养基重悬后，细胞计数，调整浓度到2x105细胞/ml，重新在6孔板每孔中加入1mlCTL-F或者CAR-F细胞悬液中，每孔中种植入2x105细胞，放在37℃，5％CO2的细胞培养箱中培养过夜。
2)肝癌上皮细胞HepG2细胞，培养汇合度超过80％以上，用胰酶消化细胞，离心单细胞悬液收集细胞沉淀，用PBS清洗后，离心收集细胞沉淀，用含有2％胎牛血清的PBS重悬细胞，调节细胞浓度在1x105细胞/ml。
3)用37℃孵育的PBS清洗前述6孔板中的CTL-F或者CAR-F细胞2次，移除PBS，加入2ml含有HepG2的细胞悬液到上述孔中。放置在细胞培养箱中孵育10分钟。
4)取出细胞培养板，移除培养液，小心加入2ml37℃孵育的PBS，轻轻震荡后移除PBS，重复PBS清洗3次后，加入2ml完全培养基。
5)在相差显微镜下拍照，记录成纤维细胞(CTL-F或者CAR-F)和粘附的上皮细胞(HepG2)。
短时间细胞粘附实验表明，CAR-F细胞比CTL-F细胞捕获更多的HepG2细胞(图9，A、B)。
2、荧光定量PCR测定捕获上皮细胞的人钙黏蛋白的RNA水平：
1)RNA抽提采用商品化的试剂盒(QIAGEN，RNeasay kit，#74104)：
用0.7ml细胞裂解液(BufferRLT，QIAGEN)，裂解细胞，加入0.7ml70％乙醇，充分混匀后加入RNeasy mini column亲和分离柱中，8000g离心15秒，丢弃废液，加入0.7mlRW1缓冲液。8000g离心15秒，丢弃废液，加入0.5mlRPE缓冲液清洗2遍，用纯水洗脱RNA。
2)通过逆转录PCR(Reverse Transcription-Polymerase Chain Reaction，RT-PCR)以总RNA中的mRNA作为模板，采用Oligo(dT)利用逆转录酶反转录成cDNA。采用商品化试剂盒(品牌Invitrogen货号18080051)实现反转录获得cDNA。试验步骤遵循试剂盒说明书。除总RNA外，Oligo(dT)，dNTP，10xRT缓冲液，25mMMgCl2，0.1MDTT，RNA酶H(RNaseH)，RNase OUT(40U/μl)和逆转录酶均为试剂盒中提供组分，具体步骤为，根据总RNA的量，在200μl PCR管中，加入3μg总RNA，用无RNA酶的纯水补充到8ul，加入1μlOligo(dT)引物，加入1μl的dNTP，轻轻震荡后离心，在PCR仪中65℃孵育5分钟，放置在冰上1分钟加入10μl逆转录PCR混合物，该10μl混合物中含有2μl10x逆转录缓冲液，4μl氯化镁(MgCl2)，2μlDTT，1μlRNase OUT和1μl逆转录酶。用枪头轻轻混匀后，放置于PCR仪中，50℃孵育50分钟后，85℃孵育5分钟加入1μl RNaseH，在37℃孵育20分钟。获得的cDNA保存在-20℃或者用于PCR扩增。
3)PCR扩增引物序列为：
hCDH1-F：GAACAGCACGTACACAGCCCT；
hCDH1-R：GCAGAAGTGTCCCTGTTCCAG；
hGAPDH-F:GAAGGTGAAGGTCGGAGTC；
hGAPDH-R:GAAGATGGTGATGGGATTTC。
每条引物用纯水稀释到6uM。
荧光定量PCR采用的试剂为SYBR Green PCR Master Mix试剂盒(AppliedBiosystems，#4368706)，荧光定量PCR仪器型号为ABI-7900HT。在10ul反应体系中，加入5ulSYBR Green PCR Master Mix，0.5ul正向引物，0.5ul反向引物，10ngcDNA，其余用水补足10ul。经过95℃加热5分钟后，循环35次(95℃加热15秒，60℃加热30秒)。通过Ct值，计算总RNA中表达hCDH1的mRNA的相对水平。
3、Westernblot免疫印记实验检测总蛋白中人钙黏蛋白(CDH1，E-cadherin)的水平：
1)移除培养液，用1mlRIPA裂解细胞，提取总蛋白。
2)总蛋白用Bradford方法定量，用RIPA调整蛋白浓度。
3)总蛋白通过10％SDS-PAGE凝胶电泳分离后，转到PVDF膜上，用10％脱脂奶粉孵育30ml，抗人钙黏素蛋白抗体(#14472)和微管蛋白抗体(#3700)均购买自CST公司。
此处用NIH-3T3细胞构建的不同CAR-F细胞，来捕获肿瘤上皮细胞，通过检测肿瘤上皮细胞的标志物：E-Cadherin(CDH1)，来定量CAR-F捕获上皮细胞的能力，在图9中C图检测CDH1 mRNA的水平，在图9中D图中，检测CDH1蛋白的水平。在本实验中，不同CAR在NIH-3T3捕获肿瘤上皮细胞HepG2的能力存在差异，该差异源自CAR结合EpCAM能力及NIH-3T3表达不同CAR蛋白的能力的综合结果(图9，使用EpHL-V-510和EpLH-V-510，EpHL-C8-510和EpLH-C8-510，EpHL-GP-510和EpLH-GP-510)。
实施例6：CAR-F促进肿瘤类器官的形成和生长
肿瘤存在一个复杂的微环境系统，其中肿瘤中的成纤维细胞被称为CAFs(CancerAssocicated Fibroblasts)，CAFs能促进肿瘤细胞的生长，转移以及在循环系统的生存能力。CAFs通过分泌生长因子、趋化因子和膜蛋白促进肿瘤细胞的生长、转移和浸润，这些因子包括白介素6(IL-6)、肝细胞生长因子(Hepatocyte Growth Factor，HGF)、成纤维细胞分泌蛋白1(Fibroblast Secreted Protein-1，FSP1)等(16；17)。在体外类器官模型中，加入工程化改造的成纤维细胞，可能促进肿瘤上皮细胞的自组装活力和提供更接近体内的生长环境，从而促进类器官的形成和生长。
在某些案例中，从非小细胞肺癌建立的PDX(Patient Derived Xenograft)模型或者从非小细胞肺癌外科手术中获得切除的肿瘤组织。蛋白酶酶解后，通过细胞过滤网，离心获取肿瘤上皮细胞。
肿瘤细胞分离合纯化的具体具体步骤如下：用蛋白酶稀释液制备得到蛋白酶复合工作液，50ul蛋白酶混合液(K2Oncology，#K2O-CRCEM-200L)加入9950ul蛋白酶稀释液(K2Oncology，#K2O-EDB)中。将组织块放入平皿中，加入4ml磷酸盐缓冲液清洗。吸出磷酸盐缓冲液，加入0.5ml，蛋白酶复合工作液。用剪刀将组织剪至匀浆状。加入4ml蛋白酶复合工作液，转移到24孔低贴壁细胞板中，放置于37℃孵育1h，显微镜下观察组织块酶解的程度。当观察到大量单细胞和寡细胞团时，用5ml移液管吸取组织匀浆，加入70微米尼龙网过滤到50ml离心管内，加入Advanced DMEM细胞培养液(Gibico，#12491-015)，一并收集入细胞滤网。用5ml注射器的活塞，研磨滤网中的组织块，加入10-20ml细胞培养液A(#K2O-TWB)，直到大部分组织通过滤网。离心200g，3分钟，加入30ml细胞培养液A(#K2O-TWB)，再200g，3分钟离心收沉淀，用5-10ml细胞培养液A(#K2O-TWB)重悬。细胞计数和活力镜检，取10ul细胞悬液，加入10ul台盼蓝，观察活细胞比例，计算细胞密度。
在某些案例中，肿瘤上皮细胞和成纤维细胞，CTL-F(3T3-vehlcle)或者CAR-F细胞按照1:1比例混合，在基质胶中生长成肿瘤类器官。如图10中A所示，肿瘤细胞和对照组成纤维细胞混合形成3D肿瘤类器官培养结构，当表达不同嵌合抗原受体蛋白的成纤维细胞(CAR-Fs，此案例中为“3T3-EpHL-V”、“3T3-EpLH-V”、“3T3-EpHL-GP”、“3T3-EpLH-GP”、“3T3-EpHL-C8”、“3T3-EpLH-C8”)和肿瘤细胞按照1:1培养后，比对照组的成纤维细胞形成更多的肿瘤类器官细胞团，并且可以看到细胞生长速度更快。实验证明，CAR-F可以提高类器官模型培养的成功率和培养速度(图10，使用EpHL-V-510和EpLH-V-510，EpHL-C8-510和EpLH-C8-510，EpHL-GP-510和EpLH-GP-510)。
在某些案例中，原代肿瘤上皮细胞和工程化改造的成纤维细胞(CAR-F，此案例中为3T3-EpHL-C8)按照细胞数1:0.1、1:0.2、1:0.5、1:0.6、1:0.8、1:1、1:2、1:3、1:4、1:5混合基质胶进行3D肿瘤类器官培养。观察3D培养中，形成肿瘤类器官的数目和大小。实验证明，CAR-F可以提高类器官模型培养的成功率和培养速度，肿瘤细胞和CAR-F细胞混合比例在1:0.1～1:5之间。(图11，使用EpHL-V-510和EpLH-V-510，EpHL-C8-510和EpLH-C8-510，EpHL-GP-510和EpLH-GP-510)
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 浙江科途医学科技有限公司
<120> 一种嵌合抗原受体成纤维细胞及其建立方法和应用
<130> CP11801134C
<160> 62
<170> PatentIn version 3.3
<210> 1
<211> 57
<212> DNA
<213> 信号肽编码核酸序列
<400> 1
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactcc 57
<210> 2
<211> 19
<212> PRT
<213> 信号肽氨基酸序列
<400> 2
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser
<210> 3
<211> 234
<212> DNA
<213> 包括跨膜序列的连接序列的编码核酸序列
<400> 3
agttctgtcg tgccagtcct tcagaaagtg aactctacta ctaccaagcc agtgctgcga 60
actccctcac ctgtgcaccc taccgggaca tctcagcccc agagaccaga agattgtcgg 120
ccccgtggct cagtgaaggg gaccggattg gacttcgcct gtgatattta catctgggca 180
cccttggccg gaatctgcgt ggcccttctg ctgtccttga tcatcactct catc 234
<210> 4
<211> 78
<212> PRT
<213> 包括跨膜序列的连接序列的氨基酸序列
<400> 4
Ser Ser Val Val Pro Val Leu Gln Lys Val Asn Ser Thr Thr Thr Lys
1 5 10 15
Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln
20 25 30
Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr
35 40 45
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
50 55 60
Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu Ile
65 70 75
<210> 5
<211> 90
<212> DNA
<213> 小鼠CD8细胞内序列的编码核酸序列
<400> 5
tgctaccaca ggagccgaaa gcgtgtttgc aaatgtccca ggccgctagt cagacaggaa 60
ggcaagccca gaccttcaga gaaaattgtg 90
<210> 6
<211> 30
<212> PRT
<213> 小鼠CD8细胞内序列的氨基酸序列
<400> 6
Cys Tyr His Arg Ser Arg Lys Arg Val Cys Lys Cys Pro Arg Pro Leu
1 5 10 15
Val Arg Gln Glu Gly Lys Pro Arg Pro Ser Glu Lys Ile Val
20 25 30
<210> 7
<211> 27
<212> DNA
<213> HA标签的编码核酸序列
<400> 7
tacccatacg atgttccaga ttacgct 27
<210> 8
<211> 9
<212> PRT
<213> HA标签的氨基酸序列
<400> 8
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 9
<211> 717
<212> DNA
<213> 绿色荧光蛋白标签的编码核酸序列
<400> 9
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717
<210> 10
<211> 239
<212> PRT
<213> 绿色荧光蛋白标签的氨基酸序列
<400> 10
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 11
<211> 726
<212> DNA
<213> Anti-EpCAM ScfvVH-VL-31编码核酸序列
<400> 11
gctcgtcatg acccagtctc catcttatct tgctgcatct cctggagaaa ccattactat 60
taattgcagg gcaagtaaga gcattagcaa atatttagcc tggtatcaag agaaacctgg 120
gaaaactaat aagcttctta tctactctgg atccactttg caatctggaa ttccatcaag 180
gttcagtggc agtggatctg gtacagattt cactctcacc atcagtagcc tggagcctga 240
agattttgca atgtattact gtcaacagca taatgaatat ccgtacacgt tcggaggggg 300
gaccaagctt gagatcaaag gtggtggtgg ttctggcggc ggcggctccg gtggtggtgg 360
ttctgagagg tgcagctgct cgagcagtct ggagctgagc tggtgaaacc tggggcctca 420
gtgaagatat cctgcaaggc ttctggatac gccttcacta actactggct aggttgggta 480
aagcagaggc ctggacatgg acttgagtgg attggagatc ttttccctgg aagtggtaat 540
actcactaca atgagaggtt caggggcaaa gccacactga ctgcagacaa atcctcgagc 600
acagccttta tgcagctcag tagcctgaca tctgaggact ctgctgtcta tttctgtgca 660
agattgagga actgggacga ggctatggac tactggggcc aagggaccac ggtcaccgtc 720
tcctca 726
<210> 12
<211> 242
<212> PRT
<213> Anti-EpCAM ScfvVH-VL-31 氨基酸序列
<400> 12
Glu Leu Val Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly
1 5 10 15
Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu
115 120 125
Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser
130 135 140
Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val
145 150 155 160
Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp Leu Phe Pro
165 170 175
Gly Ser Gly Asn Thr His Tyr Asn Glu Arg Phe Arg Gly Lys Ala Thr
180 185 190
Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe Met Gln Leu Ser Ser
195 200 205
Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn
210 215 220
Trp Asp Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val
225 230 235 240
Ser Ser
<210> 13
<211> 726
<212> DNA
<213> Anti-EpCAM ScfvVL-VH-31的编码核酸序列
<400> 13
gaggtgcagc tgctcgagca gtctggagct gagctggtga aacctggggc ctcagtgaag 60
atatcctgca aggcttctgg atacgccttc actaactact ggctaggttg ggtaaagcag 120
aggcctggac atggacttga gtggattgga gatcttttcc ctggaagtgg taatactcac 180
tacaatgaga ggttcagggg caaagccaca ctgactgcag acaaatcctc gagcacagcc 240
tttatgcagc tcagtagcct gacatctgag gactctgctg tctatttctg tgcaagattg 300
aggaactggg acgaggctat ggactactgg ggccaaggga ccacggtcac cgtctcctca 360
ggtggtggtg gttctggcgg cggcggctcc ggtggtggtg gttctgagct cgtcatgacc 420
cagtctccat cttatcttgc tgcatctcct ggagaaacca ttactattaa ttgcagggca 480
agtaagagca ttagcaaata tttagcctgg tatcaagaga aacctgggaa aactaataag 540
cttcttatct actctggatc cactttgcaa tctggaattc catcaaggtt cagtggcagt 600
ggatctggta cagatttcac tctcaccatc agtagcctgg agcctgaaga ttttgcaatg 660
tattactgtc aacagcataa tgaatatccg tacacgttcg gaggggggac caagcttgag 720
atcaaa 726
<210> 14
<211> 242
<212> PRT
<213> Anti-EpCAM ScfvVL-VH-31氨基酸序列
<400> 14
Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Lys Pro Gly
1 5 10 15
Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn
20 25 30
Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp
35 40 45
Ile Gly Asp Leu Phe Pro Gly Ser Gly Asn Thr His Tyr Asn Glu Arg
50 55 60
Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
65 70 75 80
Phe Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
85 90 95
Cys Ala Arg Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln Ser Pro Ser
130 135 140
Tyr Leu Ala Ala Ser Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala
145 150 155 160
Ser Lys Ser Ile Ser Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly
165 170 175
Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly
180 185 190
Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln
210 215 220
Gln His Asn Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys
<210> 15
<211> 620
<212> DNA
<213> Anti-EpCAM ScfvVH-VL-47编码核酸序列
<400> 15
gctcgtgatg acccagactc cactctccct gcctgtcagt cttggagatc aagcctccat 60
ctcttgcaga tctagtcaga gccttgtaca cagtaatgga aacacctatt tacattggta 120
cctgcagaag ccaggccagt ctccaaagct cctgatctac aaagtttcca accgattttc 180
tggggtccca gacaggttca gtggcagtgg atcagggaca gatttcacac tcaagatcag 240
cagagtggag gctgaggatc tgggagttta tttctgctct caaagtacac atgttccgta 300
cacgttcgga ggggggacca agcttgagat caaaggtggt ggtggttctg gcggcggcgg 360
ctccggtggt ggtggttctg atccttgagt ggattggaga ggtttatcct agaattggta 420
atgcttacta caatgagaag ttcaagggca aggccacact gactgcagac aaatcctcca 480
gcacagcgtc catggagctc cgcagcctga cctctgagga ctctgcggtc tatttctgtg 540
caagacgggg atcctacgat actaactacg actggtactt cgatgtctgg ggccaaggga 600
ccacggtcac cgtctcctca 620
<210> 16
<211> 251
<212> PRT
<213> Anti-EpCAM ScfvVH-VL-47氨基酸序列
<400> 16
Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
115 120 125
Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
130 135 140
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
145 150 155 160
Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln Val Leu Glu Trp Ile
165 170 175
Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr Asn Glu Lys Phe
180 185 190
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Ser
195 200 205
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
210 215 220
Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp Trp Tyr Phe Asp Val
225 230 235 240
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
245 250
<210> 17
<211> 620
<212> DNA
<213> Anti-EpCAM ScfvVL-VH-47编码核酸序列
<400> 17
tccttgagtg gattggagag gtttatccta gaattggtaa tgcttactac aatgagaagt 60
tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcgtcc atggagctcc 120
gcagcctgac ctctgaggac tctgcggtct atttctgtgc aagacgggga tcctacgata 180
ctaactacga ctggtacttc gatgtctggg gccaagggac cacggtcacc gtctcctcag 240
gtggtggtgg ttctggcggc ggcggctccg gtggtggtgg ttctgagctc gtgatgaccc 300
agactccact ctccctgcct gtcagtcttg gagatcaagc ctccatctct tgcagatcta 360
gtcagagcct tgtacacagt aatggaaaca cctatttaca ttggtacctg cagaagccag 420
gccagtctcc aaagctcctg atctacaaag tttccaaccg attttctggg gtcccagaca 480
ggttcagtgg cagtggatca gggacagatt tcacactcaa gatcagcaga gtggaggctg 540
aggatctggg agtttatttc tgctctcaaa gtacacatgt tccgtacacg ttcggagggg 600
ggaccaagct tgagatcaaa 620
<210> 18
<211> 251
<212> PRT
<213> Anti-EpCAM ScfvVL-VH-47 氨基酸序列
<400> 18
Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly
1 5 10 15
Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
20 25 30
Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln Val Leu Glu Trp
35 40 45
Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr Asn Glu Lys
50 55 60
Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
65 70 75 80
Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
85 90 95
Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp Trp Tyr Phe Asp
100 105 110
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr
130 135 140
Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile
145 150 155 160
Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr
165 170 175
Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
180 185 190
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly
195 200 205
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
210 215 220
Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr
225 230 235 240
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
245 250
<210> 19
<211> 738
<212> DNA
<213> Anti-EpCAM ScfvVH-VL-510的编码核酸序列
<400> 19
gaggtgcagc tgctcgagca gtctggagct gagctggtaa ggcctgggac ttcagtgaag 60
atatcctgca aggcttctgg atacgccttc actaactact ggctaggttg ggtaaagcag 120
aggcctggac atggacttga gtggattgga gatattttcc ctggaagtgg taatatccac 180
tacaatgaga agttcaaggg caaagccaca ctgactgcag acaaatcttc gagcacagcc 240
tatatgcagc tcagtagcct gacatttgag gactctgctg tctatttctg tgcaagactg 300
aggaactggg acgagcctat ggactactgg ggccaaggga ccacggtcac cgtcggtggt 360
ggtggttctg gcggcggcgg ctccggtggt ggtggttctg agctcgtgat gacacagtct 420
ccatcctccc tgactgtgac agcaggagag aaggtcacta tgagctgcaa gtccagtcag 480
agtctgttaa acagtggaaa tcaaaagaac tacttgacct ggtaccagca gaaaccaggg 540
cagcctccta aactgttgat ctactgggca tccactaggg aatctggggt ccctgatcgc 600
ttcacaggca gtggatctgg aacagatttc actctcacca tcagcagtgt gcaggctgaa 660
gacctggcag tttattactg tcagaatgat tatagttatc cgctcacgtt cggtgctggg 720
accaagcttg agatcaaa 738
<210> 20
<211> 246
<212> PRT
<213> Anti-EpCAM ScfvVH-VL-510的氨基酸序列
<400> 20
Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly
1 5 10 15
Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn
20 25 30
Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp
35 40 45
Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys
50 55 60
Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
65 70 75 80
Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe
85 90 95
Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu
130 135 140
Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln
145 150 155 160
Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
180 185 190
Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val
210 215 220
Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly
225 230 235 240
Thr Lys Leu Glu Ile Lys
245
<210> 21
<211> 738
<212> DNA
<213> Anti-EpCAM ScfvVL-VH-510的编码核酸序列
<400> 21
gagctcgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60
atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120
tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 180
gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240
atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300
ccgctcacgt tcggtgctgg gaccaagctt gagatcaaag gtggtggtgg ttctggcggc 360
ggcggctccg gtggtggtgg ttctgaggtg cagctgctcg agcagtctgg agctgagctg 420
gtaaggcctg ggacttcagt gaagatatcc tgcaaggctt ctggatacgc cttcactaac 480
tactggctag gttgggtaaa gcagaggcct ggacatggac ttgagtggat tggagatatt 540
ttccctggaa gtggtaatat ccactacaat gagaagttca agggcaaagc cacactgact 600
gcagacaaat cttcgagcac agcctatatg cagctcagta gcctgacatt tgaggactct 660
gctgtctatt tctgtgcaag actgaggaac tgggacgagc ctatggacta ctggggccaa 720
gggaccacgg tcaccgtc 738
<210> 22
<211> 246
<212> PRT
<213> Anti-EpCAM ScfvVL-VH-510的氨基酸序列
<400> 22
Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly
130 135 140
Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn
145 150 155 160
Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp
165 170 175
Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys
180 185 190
Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
195 200 205
Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe
210 215 220
Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln
225 230 235 240
Gly Thr Thr Val Thr Val
245
<210> 23
<211> 1047
<212> DNA
<213> 嵌合抗原受体蛋白EpHL-V-31编码核酸序列
<400> 23
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgct 60
cgtcatgacc cagtctccat cttatcttgc tgcatctcct ggagaaacca ttactattaa 120
ttgcagggca agtaagagca ttagcaaata tttagcctgg tatcaagaga aacctgggaa 180
aactaataag cttcttatct actctggatc cactttgcaa tctggaattc catcaaggtt 240
cagtggcagt ggatctggta cagatttcac tctcaccatc agtagcctgg agcctgaaga 300
ttttgcaatg tattactgtc aacagcataa tgaatatccg tacacgttcg gaggggggac 360
caagcttgag atcaaaggtg gtggtggttc tggcggcggc ggctccggtg gtggtggttc 420
tgagaggtgc agctgctcga gcagtctgga gctgagctgg tgaaacctgg ggcctcagtg 480
aagatatcct gcaaggcttc tggatacgcc ttcactaact actggctagg ttgggtaaag 540
cagaggcctg gacatggact tgagtggatt ggagatcttt tccctggaag tggtaatact 600
cactacaatg agaggttcag gggcaaagcc acactgactg cagacaaatc ctcgagcaca 660
gcctttatgc agctcagtag cctgacatct gaggactctg ctgtctattt ctgtgcaaga 720
ttgaggaact gggacgaggc tatggactac tggggccaag ggaccacggt caccgtctcc 780
tcaagttctg tcgtgccagt ccttcagaaa gtgaactcta ctactaccaa gccagtgctg 840
cgaactccct cacctgtgca ccctaccggg acatctcagc cccagagacc agaagattgt 900
cggccccgtg gctcagtgaa ggggaccgga ttggacttcg cctgtgatat ttacatctgg 960
gcacccttgg ccggaatctg cgtggccctt ctgctgtcct tgatcatcac tctcatctac 1020
ccatacgatg ttccagatta cgcttaa 1047
<210> 24
<211> 348
<212> PRT
<213> 嵌合抗原受体蛋白EpHL-V-31氨基酸序列
<400> 24
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala
20 25 30
Ser Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile
35 40 45
Ser Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys
50 55 60
Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg
65 70 75 80
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
85 90 95
Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu
100 105 110
Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
130 135 140
Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val
145 150 155 160
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu
165 170 175
Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp
180 185 190
Leu Phe Pro Gly Ser Gly Asn Thr His Tyr Asn Glu Arg Phe Arg Gly
195 200 205
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe Met Gln
210 215 220
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg
225 230 235 240
Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr
245 250 255
Val Thr Val Ser Ser Ser Ser Val Val Pro Val Leu Gln Lys Val Asn
260 265 270
Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro
275 280 285
Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly
290 295 300
Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile
325 330 335
Thr Leu Ile Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
340 345
<210> 25
<211> 1047
<212> DNA
<213> 嵌合抗原受体蛋白EpLH-V-31编码核酸序列
<400> 25
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60
gtgcagctgc tcgagcagtc tggagctgag ctggtgaaac ctggggcctc agtgaagata 120
tcctgcaagg cttctggata cgccttcact aactactggc taggttgggt aaagcagagg 180
cctggacatg gacttgagtg gattggagat cttttccctg gaagtggtaa tactcactac 240
aatgagaggt tcaggggcaa agccacactg actgcagaca aatcctcgag cacagccttt 300
atgcagctca gtagcctgac atctgaggac tctgctgtct atttctgtgc aagattgagg 360
aactgggacg aggctatgga ctactggggc caagggacca cggtcaccgt ctcctcaggt 420
ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgagctcgt catgacccag 480
tctccatctt atcttgctgc atctcctgga gaaaccatta ctattaattg cagggcaagt 540
aagagcatta gcaaatattt agcctggtat caagagaaac ctgggaaaac taataagctt 600
cttatctact ctggatccac tttgcaatct ggaattccat caaggttcag tggcagtgga 660
tctggtacag atttcactct caccatcagt agcctggagc ctgaagattt tgcaatgtat 720
tactgtcaac agcataatga atatccgtac acgttcggag gggggaccaa gcttgagatc 780
aaaagttctg tcgtgccagt ccttcagaaa gtgaactcta ctactaccaa gccagtgctg 840
cgaactccct cacctgtgca ccctaccggg acatctcagc cccagagacc agaagattgt 900
cggccccgtg gctcagtgaa ggggaccgga ttggacttcg cctgtgatat ttacatctgg 960
gcacccttgg ccggaatctg cgtggccctt ctgctgtcct tgatcatcac tctcatctac 1020
ccatacgatg ttccagatta cgcttaa 1047
<210> 26
<211> 348
<212> PRT
<213> 嵌合抗原受体蛋白EpLH-V-31氨基酸序列
<400> 26
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val
20 25 30
Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
35 40 45
Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
50 55 60
Leu Glu Trp Ile Gly Asp Leu Phe Pro Gly Ser Gly Asn Thr His Tyr
65 70 75 80
Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
85 90 95
Ser Thr Ala Phe Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln
145 150 155 160
Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly Glu Thr Ile Thr Ile Asn
165 170 175
Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr Leu Ala Trp Tyr Gln Glu
180 185 190
Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu
195 200 205
Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 215 220
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr
225 230 235 240
Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr
245 250 255
Lys Leu Glu Ile Lys Ser Ser Val Val Pro Val Leu Gln Lys Val Asn
260 265 270
Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro
275 280 285
Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly
290 295 300
Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile
325 330 335
Thr Leu Ile Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
340 345
<210> 27
<211> 941
<212> DNA
<213> 嵌合抗原受体蛋白EpHL-V-47编码核酸序列
<400> 27
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgct 60
cgtgatgacc cagactccac tctccctgcc tgtcagtctt ggagatcaag cctccatctc 120
ttgcagatct agtcagagcc ttgtacacag taatggaaac acctatttac attggtacct 180
gcagaagcca ggccagtctc caaagctcct gatctacaaa gtttccaacc gattttctgg 240
ggtcccagac aggttcagtg gcagtggatc agggacagat ttcacactca agatcagcag 300
agtggaggct gaggatctgg gagtttattt ctgctctcaa agtacacatg ttccgtacac 360
gttcggaggg gggaccaagc ttgagatcaa aggtggtggt ggttctggcg gcggcggctc 420
cggtggtggt ggttctgatc cttgagtgga ttggagaggt ttatcctaga attggtaatg 480
cttactacaa tgagaagttc aagggcaagg ccacactgac tgcagacaaa tcctccagca 540
cagcgtccat ggagctccgc agcctgacct ctgaggactc tgcggtctat ttctgtgcaa 600
gacggggatc ctacgatact aactacgact ggtacttcga tgtctggggc caagggacca 660
cggtcaccgt ctcctcaagt tctgtcgtgc cagtccttca gaaagtgaac tctactacta 720
ccaagccagt gctgcgaact ccctcacctg tgcaccctac cgggacatct cagccccaga 780
gaccagaaga ttgtcggccc cgtggctcag tgaaggggac cggattggac ttcgcctgtg 840
atatttacat ctgggcaccc ttggccggaa tctgcgtggc ccttctgctg tccttgatca 900
tcactctcat ctacccatac gatgttccag attacgctta a 941
<210> 28
<211> 357
<212> PRT
<213> 嵌合抗原受体蛋白EpHL-V-47氨基酸序列
<400> 28
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45
Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Ala Arg
145 150 155 160
Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
165 170 175
Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln Val Leu
180 185 190
Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr Asn
195 200 205
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
210 215 220
Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
225 230 235 240
Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp Trp Tyr
245 250 255
Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Ser
260 265 270
Val Val Pro Val Leu Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val
275 280 285
Leu Arg Thr Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln
290 295 300
Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu
305 310 315 320
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys
325 330 335
Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu Ile Tyr Pro Tyr Asp
340 345 350
Val Pro Asp Tyr Ala
355
<210> 29
<211> 941
<212> DNA
<213> 嵌合抗原受体蛋白EpLH-V-47编码核酸序列
<400> 29
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactcctcc 60
ttgagtggat tggagaggtt tatcctagaa ttggtaatgc ttactacaat gagaagttca 120
agggcaaggc cacactgact gcagacaaat cctccagcac agcgtccatg gagctccgca 180
gcctgacctc tgaggactct gcggtctatt tctgtgcaag acggggatcc tacgatacta 240
actacgactg gtacttcgat gtctggggcc aagggaccac ggtcaccgtc tcctcaggtg 300
gtggtggttc tggcggcggc ggctccggtg gtggtggttc tgagctcgtg atgacccaga 360
ctccactctc cctgcctgtc agtcttggag atcaagcctc catctcttgc agatctagtc 420
agagccttgt acacagtaat ggaaacacct atttacattg gtacctgcag aagccaggcc 480
agtctccaaa gctcctgatc tacaaagttt ccaaccgatt ttctggggtc ccagacaggt 540
tcagtggcag tggatcaggg acagatttca cactcaagat cagcagagtg gaggctgagg 600
atctgggagt ttatttctgc tctcaaagta cacatgttcc gtacacgttc ggagggggga 660
ccaagcttga gatcaaaagt tctgtcgtgc cagtccttca gaaagtgaac tctactacta 720
ccaagccagt gctgcgaact ccctcacctg tgcaccctac cgggacatct cagccccaga 780
gaccagaaga ttgtcggccc cgtggctcag tgaaggggac cggattggac ttcgcctgtg 840
atatttacat ctgggcaccc ttggccggaa tctgcgtggc ccttctgctg tccttgatca 900
tcactctcat ctacccatac gatgttccag attacgctta a 941
<210> 30
<211> 357
<212> PRT
<213> 嵌合抗原受体蛋白EpLH-V-47氨基酸序列
<400> 30
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Ala
20 25 30
Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr
35 40 45
Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln Val
50 55 60
Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr
65 70 75 80
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
85 90 95
Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp Trp
115 120 125
Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu
145 150 155 160
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln
165 170 175
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly
180 185 190
Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys
195 200 205
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg
210 215 220
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg
225 230 235 240
Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His
245 250 255
Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser
260 265 270
Val Val Pro Val Leu Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val
275 280 285
Leu Arg Thr Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln
290 295 300
Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu
305 310 315 320
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys
325 330 335
Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu Ile Tyr Pro Tyr Asp
340 345 350
Val Pro Asp Tyr Ala
355
<210> 31
<211> 1059
<212> DNA
<213> 嵌合抗原受体蛋白EpHL-V-510编码核酸序列
<400> 31
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60
gtgcagctgc tcgagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaagata 120
tcctgcaagg cttctggata cgccttcact aactactggc taggttgggt aaagcagagg 180
cctggacatg gacttgagtg gattggagat attttccctg gaagtggtaa tatccactac 240
aatgagaagt tcaagggcaa agccacactg actgcagaca aatcttcgag cacagcctat 300
atgcagctca gtagcctgac atttgaggac tctgctgtct atttctgtgc aagactgagg 360
aactgggacg agcctatgga ctactggggc caagggacca cggtcaccgt cggtggtggt 420
ggttctggcg gcggcggctc cggtggtggt ggttctgagc tcgtgatgac acagtctcca 480
tcctccctga ctgtgacagc aggagagaag gtcactatga gctgcaagtc cagtcagagt 540
ctgttaaaca gtggaaatca aaagaactac ttgacctggt accagcagaa accagggcag 600
cctcctaaac tgttgatcta ctgggcatcc actagggaat ctggggtccc tgatcgcttc 660
acaggcagtg gatctggaac agatttcact ctcaccatca gcagtgtgca ggctgaagac 720
ctggcagttt attactgtca gaatgattat agttatccgc tcacgttcgg tgctgggacc 780
aagcttgaga tcaaaagttc tgtcgtgcca gtccttcaga aagtgaactc tactactacc 840
aagccagtgc tgcgaactcc ctcacctgtg caccctaccg ggacatctca gccccagaga 900
ccagaagatt gtcggccccg tggctcagtg aaggggaccg gattggactt cgcctgtgat 960
atttacatct gggcaccctt ggccggaatc tgcgtggccc ttctgctgtc cttgatcatc 1020
actctcatct acccatacga tgttccagat tacgcttaa 1059
<210> 32
<211> 352
<212> PRT
<213> 嵌合抗原受体蛋白EpHL-V-510氨基酸序列
<400> 32
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val
20 25 30
Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
35 40 45
Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
50 55 60
Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr
65 70 75 80
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
85 90 95
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Thr Val Thr Val Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln Ser Pro
145 150 155 160
Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys
165 170 175
Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr
180 185 190
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp
195 200 205
Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
210 215 220
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp
225 230 235 240
Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe
245 250 255
Gly Ala Gly Thr Lys Leu Glu Ile Lys Ser Ser Val Val Pro Val Leu
260 265 270
Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser
275 280 285
Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys
290 295 300
Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp
305 310 315 320
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu
325 330 335
Ser Leu Ile Ile Thr Leu Ile Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
340 345 350
<210> 33
<211> 1059
<212> DNA
<213> 嵌合抗原受体蛋白EpLH-V-510编码核酸序列
<400> 33
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60
ctcgtgatga cacagtctcc atcctccctg actgtgacag caggagagaa ggtcactatg 120
agctgcaagt ccagtcagag tctgttaaac agtggaaatc aaaagaacta cttgacctgg 180
taccagcaga aaccagggca gcctcctaaa ctgttgatct actgggcatc cactagggaa 240
tctggggtcc ctgatcgctt cacaggcagt ggatctggaa cagatttcac tctcaccatc 300
agcagtgtgc aggctgaaga cctggcagtt tattactgtc agaatgatta tagttatccg 360
ctcacgttcg gtgctgggac caagcttgag atcaaaggtg gtggtggttc tggcggcggc 420
ggctccggtg gtggtggttc tgaggtgcag ctgctcgagc agtctggagc tgagctggta 480
aggcctggga cttcagtgaa gatatcctgc aaggcttctg gatacgcctt cactaactac 540
tggctaggtt gggtaaagca gaggcctgga catggacttg agtggattgg agatattttc 600
cctggaagtg gtaatatcca ctacaatgag aagttcaagg gcaaagccac actgactgca 660
gacaaatctt cgagcacagc ctatatgcag ctcagtagcc tgacatttga ggactctgct 720
gtctatttct gtgcaagact gaggaactgg gacgagccta tggactactg gggccaaggg 780
accacggtca ccgtcagttc tgtcgtgcca gtccttcaga aagtgaactc tactactacc 840
aagccagtgc tgcgaactcc ctcacctgtg caccctaccg ggacatctca gccccagaga 900
ccagaagatt gtcggccccg tggctcagtg aaggggaccg gattggactt cgcctgtgat 960
atttacatct gggcaccctt ggccggaatc tgcgtggccc ttctgctgtc cttgatcatc 1020
actctcatct acccatacga tgttccagat tacgcttaa 1059
<210> 34
<211> 352
<212> PRT
<213> 嵌合抗原受体蛋白EpLH-V-510氨基酸序列
<400> 34
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val
20 25 30
Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu
35 40 45
Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys
50 55 60
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
65 70 75 80
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
85 90 95
Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr
100 105 110
Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys
115 120 125
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val
145 150 155 160
Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
165 170 175
Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
180 185 190
Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr
195 200 205
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
210 215 220
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala
225 230 235 240
Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Val Pro Val Leu
260 265 270
Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser
275 280 285
Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys
290 295 300
Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp
305 310 315 320
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu
325 330 335
Ser Leu Ile Ile Thr Leu Ile Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
340 345 350
<210> 35
<211> 1137
<212> DNA
<213> 嵌合抗原受体蛋白EpHL-C8-31编码核酸序列
<400> 35
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgct 60
cgtcatgacc cagtctccat cttatcttgc tgcatctcct ggagaaacca ttactattaa 120
ttgcagggca agtaagagca ttagcaaata tttagcctgg tatcaagaga aacctgggaa 180
aactaataag cttcttatct actctggatc cactttgcaa tctggaattc catcaaggtt 240
cagtggcagt ggatctggta cagatttcac tctcaccatc agtagcctgg agcctgaaga 300
ttttgcaatg tattactgtc aacagcataa tgaatatccg tacacgttcg gaggggggac 360
caagcttgag atcaaaggtg gtggtggttc tggcggcggc ggctccggtg gtggtggttc 420
tgagaggtgc agctgctcga gcagtctgga gctgagctgg tgaaacctgg ggcctcagtg 480
aagatatcct gcaaggcttc tggatacgcc ttcactaact actggctagg ttgggtaaag 540
cagaggcctg gacatggact tgagtggatt ggagatcttt tccctggaag tggtaatact 600
cactacaatg agaggttcag gggcaaagcc acactgactg cagacaaatc ctcgagcaca 660
gcctttatgc agctcagtag cctgacatct gaggactctg ctgtctattt ctgtgcaaga 720
ttgaggaact gggacgaggc tatggactac tggggccaag ggaccacggt caccgtctcc 780
tcaagttctg tcgtgccagt ccttcagaaa gtgaactcta ctactaccaa gccagtgctg 840
cgaactccct cacctgtgca ccctaccggg acatctcagc cccagagacc agaagattgt 900
cggccccgtg gctcagtgaa ggggaccgga ttggacttcg cctgtgatat ttacatctgg 960
gcacccttgg ccggaatctg cgtggccctt ctgctgtcct tgatcatcac tctcatctgc 1020
taccacagga gccgaaagcg tgtttgcaaa tgtcccaggc cgctagtcag acaggaaggc 1080
aagcccagac cttcagagaa aattgtgtac ccatacgatg ttccagatta cgcttaa 1137
<210> 36
<211> 378
<212> PRT
<213> 嵌合抗原受体蛋白EpHL-C8-31氨基酸序列
<400> 36
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala
20 25 30
Ser Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile
35 40 45
Ser Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys
50 55 60
Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg
65 70 75 80
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
85 90 95
Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu
100 105 110
Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
130 135 140
Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val
145 150 155 160
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu
165 170 175
Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp
180 185 190
Leu Phe Pro Gly Ser Gly Asn Thr His Tyr Asn Glu Arg Phe Arg Gly
195 200 205
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe Met Gln
210 215 220
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg
225 230 235 240
Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr
245 250 255
Val Thr Val Ser Ser Ser Ser Val Val Pro Val Leu Gln Lys Val Asn
260 265 270
Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro
275 280 285
Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly
290 295 300
Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile
325 330 335
Thr Leu Ile Cys Tyr His Arg Ser Arg Lys Arg Val Cys Lys Cys Pro
340 345 350
Arg Pro Leu Val Arg Gln Glu Gly Lys Pro Arg Pro Ser Glu Lys Ile
355 360 365
Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
370 375
<210> 37
<211> 1137
<212> DNA
<213> 嵌合抗原受体蛋白EpLH-C8-31编码核酸序列
<400> 37
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60
gtgcagctgc tcgagcagtc tggagctgag ctggtgaaac ctggggcctc agtgaagata 120
tcctgcaagg cttctggata cgccttcact aactactggc taggttgggt aaagcagagg 180
cctggacatg gacttgagtg gattggagat cttttccctg gaagtggtaa tactcactac 240
aatgagaggt tcaggggcaa agccacactg actgcagaca aatcctcgag cacagccttt 300
atgcagctca gtagcctgac atctgaggac tctgctgtct atttctgtgc aagattgagg 360
aactgggacg aggctatgga ctactggggc caagggacca cggtcaccgt ctcctcaggt 420
ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgagctcgt catgacccag 480
tctccatctt atcttgctgc atctcctgga gaaaccatta ctattaattg cagggcaagt 540
aagagcatta gcaaatattt agcctggtat caagagaaac ctgggaaaac taataagctt 600
cttatctact ctggatccac tttgcaatct ggaattccat caaggttcag tggcagtgga 660
tctggtacag atttcactct caccatcagt agcctggagc ctgaagattt tgcaatgtat 720
tactgtcaac agcataatga atatccgtac acgttcggag gggggaccaa gcttgagatc 780
aaaagttctg tcgtgccagt ccttcagaaa gtgaactcta ctactaccaa gccagtgctg 840
cgaactccct cacctgtgca ccctaccggg acatctcagc cccagagacc agaagattgt 900
cggccccgtg gctcagtgaa ggggaccgga ttggacttcg cctgtgatat ttacatctgg 960
gcacccttgg ccggaatctg cgtggccctt ctgctgtcct tgatcatcac tctcatctgc 1020
taccacagga gccgaaagcg tgtttgcaaa tgtcccaggc cgctagtcag acaggaaggc 1080
aagcccagac cttcagagaa aattgtgtac ccatacgatg ttccagatta cgcttaa 1137
<210> 38
<211> 378
<212> PRT
<213> 嵌合抗原受体蛋白EpLH-C8-31氨基酸序列
<400> 38
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val
20 25 30
Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
35 40 45
Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
50 55 60
Leu Glu Trp Ile Gly Asp Leu Phe Pro Gly Ser Gly Asn Thr His Tyr
65 70 75 80
Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
85 90 95
Ser Thr Ala Phe Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln
145 150 155 160
Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly Glu Thr Ile Thr Ile Asn
165 170 175
Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr Leu Ala Trp Tyr Gln Glu
180 185 190
Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu
195 200 205
Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 215 220
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr
225 230 235 240
Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr
245 250 255
Lys Leu Glu Ile Lys Ser Ser Val Val Pro Val Leu Gln Lys Val Asn
260 265 270
Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro
275 280 285
Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly
290 295 300
Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
305 310 315 320
Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile
325 330 335
Thr Leu Ile Cys Tyr His Arg Ser Arg Lys Arg Val Cys Lys Cys Pro
340 345 350
Arg Pro Leu Val Arg Gln Glu Gly Lys Pro Arg Pro Ser Glu Lys Ile
355 360 365
Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
370 375
<210> 39
<211> 1031
<212> DNA
<213> 嵌合抗原受体蛋白EpHL-C8-47编码核酸序列
<400> 39
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgct 60
cgtgatgacc cagactccac tctccctgcc tgtcagtctt ggagatcaag cctccatctc 120
ttgcagatct agtcagagcc ttgtacacag taatggaaac acctatttac attggtacct 180
gcagaagcca ggccagtctc caaagctcct gatctacaaa gtttccaacc gattttctgg 240
ggtcccagac aggttcagtg gcagtggatc agggacagat ttcacactca agatcagcag 300
agtggaggct gaggatctgg gagtttattt ctgctctcaa agtacacatg ttccgtacac 360
gttcggaggg gggaccaagc ttgagatcaa aggtggtggt ggttctggcg gcggcggctc 420
cggtggtggt ggttctgatc cttgagtgga ttggagaggt ttatcctaga attggtaatg 480
cttactacaa tgagaagttc aagggcaagg ccacactgac tgcagacaaa tcctccagca 540
cagcgtccat ggagctccgc agcctgacct ctgaggactc tgcggtctat ttctgtgcaa 600
gacggggatc ctacgatact aactacgact ggtacttcga tgtctggggc caagggacca 660
cggtcaccgt ctcctcaagt tctgtcgtgc cagtccttca gaaagtgaac tctactacta 720
ccaagccagt gctgcgaact ccctcacctg tgcaccctac cgggacatct cagccccaga 780
gaccagaaga ttgtcggccc cgtggctcag tgaaggggac cggattggac ttcgcctgtg 840
atatttacat ctgggcaccc ttggccggaa tctgcgtggc ccttctgctg tccttgatca 900
tcactctcat ctgctaccac aggagccgaa agcgtgtttg caaatgtccc aggccgctag 960
tcagacagga aggcaagccc agaccttcag agaaaattgt gtacccatac gatgttccag 1020
attacgctta a 1031
<210> 40
<211> 387
<212> PRT
<213> 嵌合抗原受体蛋白EpHL-C8-47氨基酸序列
<400> 40
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45
Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Ala Arg
145 150 155 160
Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
165 170 175
Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln Val Leu
180 185 190
Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr Asn
195 200 205
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
210 215 220
Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
225 230 235 240
Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp Trp Tyr
245 250 255
Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Ser
260 265 270
Val Val Pro Val Leu Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val
275 280 285
Leu Arg Thr Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln
290 295 300
Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu
305 310 315 320
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys
325 330 335
Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr His Arg
340 345 350
Ser Arg Lys Arg Val Cys Lys Cys Pro Arg Pro Leu Val Arg Gln Glu
355 360 365
Gly Lys Pro Arg Pro Ser Glu Lys Ile Val Tyr Pro Tyr Asp Val Pro
370 375 380
Asp Tyr Ala
385
<210> 41
<211> 1031
<212> DNA
<213> 嵌合抗原受体蛋白EpLH-C8-47编码核酸序列
<400> 41
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactcctcc 60
ttgagtggat tggagaggtt tatcctagaa ttggtaatgc ttactacaat gagaagttca 120
agggcaaggc cacactgact gcagacaaat cctccagcac agcgtccatg gagctccgca 180
gcctgacctc tgaggactct gcggtctatt tctgtgcaag acggggatcc tacgatacta 240
actacgactg gtacttcgat gtctggggcc aagggaccac ggtcaccgtc tcctcaggtg 300
gtggtggttc tggcggcggc ggctccggtg gtggtggttc tgagctcgtg atgacccaga 360
ctccactctc cctgcctgtc agtcttggag atcaagcctc catctcttgc agatctagtc 420
agagccttgt acacagtaat ggaaacacct atttacattg gtacctgcag aagccaggcc 480
agtctccaaa gctcctgatc tacaaagttt ccaaccgatt ttctggggtc ccagacaggt 540
tcagtggcag tggatcaggg acagatttca cactcaagat cagcagagtg gaggctgagg 600
atctgggagt ttatttctgc tctcaaagta cacatgttcc gtacacgttc ggagggggga 660
ccaagcttga gatcaaaagt tctgtcgtgc cagtccttca gaaagtgaac tctactacta 720
ccaagccagt gctgcgaact ccctcacctg tgcaccctac cgggacatct cagccccaga 780
gaccagaaga ttgtcggccc cgtggctcag tgaaggggac cggattggac ttcgcctgtg 840
atatttacat ctgggcaccc ttggccggaa tctgcgtggc ccttctgctg tccttgatca 900
tcactctcat ctgctaccac aggagccgaa agcgtgtttg caaatgtccc aggccgctag 960
tcagacagga aggcaagccc agaccttcag agaaaattgt gtacccatac gatgttccag 1020
attacgctta a 1031
<210> 42
<211> 387
<212> PRT
<213> 嵌合抗原受体蛋白EpLH-C8-47氨基酸序列
<400> 42
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Ala
20 25 30
Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr
35 40 45
Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln Val
50 55 60
Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr
65 70 75 80
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
85 90 95
Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp Trp
115 120 125
Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu
145 150 155 160
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln
165 170 175
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly
180 185 190
Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys
195 200 205
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg
210 215 220
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg
225 230 235 240
Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His
245 250 255
Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser
260 265 270
Val Val Pro Val Leu Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val
275 280 285
Leu Arg Thr Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln
290 295 300
Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu
305 310 315 320
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys
325 330 335
Val Ala Leu Leu Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr His Arg
340 345 350
Ser Arg Lys Arg Val Cys Lys Cys Pro Arg Pro Leu Val Arg Gln Glu
355 360 365
Gly Lys Pro Arg Pro Ser Glu Lys Ile Val Tyr Pro Tyr Asp Val Pro
370 375 380
Asp Tyr Ala
385
<210> 43
<211> 1149
<212> DNA
<213> 嵌合抗原受体蛋白EpHL-C8-510编码核酸序列
<400> 43
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60
gtgcagctgc tcgagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaagata 120
tcctgcaagg cttctggata cgccttcact aactactggc taggttgggt aaagcagagg 180
cctggacatg gacttgagtg gattggagat attttccctg gaagtggtaa tatccactac 240
aatgagaagt tcaagggcaa agccacactg actgcagaca aatcttcgag cacagcctat 300
atgcagctca gtagcctgac atttgaggac tctgctgtct atttctgtgc aagactgagg 360
aactgggacg agcctatgga ctactggggc caagggacca cggtcaccgt cggtggtggt 420
ggttctggcg gcggcggctc cggtggtggt ggttctgagc tcgtgatgac acagtctcca 480
tcctccctga ctgtgacagc aggagagaag gtcactatga gctgcaagtc cagtcagagt 540
ctgttaaaca gtggaaatca aaagaactac ttgacctggt accagcagaa accagggcag 600
cctcctaaac tgttgatcta ctgggcatcc actagggaat ctggggtccc tgatcgcttc 660
acaggcagtg gatctggaac agatttcact ctcaccatca gcagtgtgca ggctgaagac 720
ctggcagttt attactgtca gaatgattat agttatccgc tcacgttcgg tgctgggacc 780
aagcttgaga tcaaaagttc tgtcgtgcca gtccttcaga aagtgaactc tactactacc 840
aagccagtgc tgcgaactcc ctcacctgtg caccctaccg ggacatctca gccccagaga 900
ccagaagatt gtcggccccg tggctcagtg aaggggaccg gattggactt cgcctgtgat 960
atttacatct gggcaccctt ggccggaatc tgcgtggccc ttctgctgtc cttgatcatc 1020
actctcatct gctaccacag gagccgaaag cgtgtttgca aatgtcccag gccgctagtc 1080
agacaggaag gcaagcccag accttcagag aaaattgtgt acccatacga tgttccagat 1140
tacgcttaa 1149
<210> 44
<211> 382
<212> PRT
<213> 嵌合抗原受体蛋白EpHL-C8-510氨基酸序列
<400> 44
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val
20 25 30
Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
35 40 45
Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
50 55 60
Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr
65 70 75 80
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
85 90 95
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Thr Val Thr Val Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln Ser Pro
145 150 155 160
Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys
165 170 175
Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr
180 185 190
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp
195 200 205
Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
210 215 220
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp
225 230 235 240
Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe
245 250 255
Gly Ala Gly Thr Lys Leu Glu Ile Lys Ser Ser Val Val Pro Val Leu
260 265 270
Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser
275 280 285
Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys
290 295 300
Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp
305 310 315 320
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu
325 330 335
Ser Leu Ile Ile Thr Leu Ile Cys Tyr His Arg Ser Arg Lys Arg Val
340 345 350
Cys Lys Cys Pro Arg Pro Leu Val Arg Gln Glu Gly Lys Pro Arg Pro
355 360 365
Ser Glu Lys Ile Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
370 375 380
<210> 45
<211> 1149
<212> DNA
<213> 嵌合抗原受体蛋白EpLH-C8-510编码核酸序列
<400> 45
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60
ctcgtgatga cacagtctcc atcctccctg actgtgacag caggagagaa ggtcactatg 120
agctgcaagt ccagtcagag tctgttaaac agtggaaatc aaaagaacta cttgacctgg 180
taccagcaga aaccagggca gcctcctaaa ctgttgatct actgggcatc cactagggaa 240
tctggggtcc ctgatcgctt cacaggcagt ggatctggaa cagatttcac tctcaccatc 300
agcagtgtgc aggctgaaga cctggcagtt tattactgtc agaatgatta tagttatccg 360
ctcacgttcg gtgctgggac caagcttgag atcaaaggtg gtggtggttc tggcggcggc 420
ggctccggtg gtggtggttc tgaggtgcag ctgctcgagc agtctggagc tgagctggta 480
aggcctggga cttcagtgaa gatatcctgc aaggcttctg gatacgcctt cactaactac 540
tggctaggtt gggtaaagca gaggcctgga catggacttg agtggattgg agatattttc 600
cctggaagtg gtaatatcca ctacaatgag aagttcaagg gcaaagccac actgactgca 660
gacaaatctt cgagcacagc ctatatgcag ctcagtagcc tgacatttga ggactctgct 720
gtctatttct gtgcaagact gaggaactgg gacgagccta tggactactg gggccaaggg 780
accacggtca ccgtcagttc tgtcgtgcca gtccttcaga aagtgaactc tactactacc 840
aagccagtgc tgcgaactcc ctcacctgtg caccctaccg ggacatctca gccccagaga 900
ccagaagatt gtcggccccg tggctcagtg aaggggaccg gattggactt cgcctgtgat 960
atttacatct gggcaccctt ggccggaatc tgcgtggccc ttctgctgtc cttgatcatc 1020
actctcatct gctaccacag gagccgaaag cgtgtttgca aatgtcccag gccgctagtc 1080
agacaggaag gcaagcccag accttcagag aaaattgtgt acccatacga tgttccagat 1140
tacgcttaa 1149
<210> 46
<211> 382
<212> PRT
<213> 嵌合抗原受体蛋白EpLH-C8-510氨基酸序列
<400> 46
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val
20 25 30
Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu
35 40 45
Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys
50 55 60
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
65 70 75 80
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
85 90 95
Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr
100 105 110
Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys
115 120 125
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val
145 150 155 160
Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
165 170 175
Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
180 185 190
Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr
195 200 205
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
210 215 220
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala
225 230 235 240
Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Val Pro Val Leu
260 265 270
Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser
275 280 285
Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys
290 295 300
Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp
305 310 315 320
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu
325 330 335
Ser Leu Ile Ile Thr Leu Ile Cys Tyr His Arg Ser Arg Lys Arg Val
340 345 350
Cys Lys Cys Pro Arg Pro Leu Val Arg Gln Glu Gly Lys Pro Arg Pro
355 360 365
Ser Glu Lys Ile Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
370 375 380
<210> 47
<211> 1530
<212> DNA
<213> 嵌合抗原受体蛋白EpHL-GP-31编码核酸序列
<400> 47
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgct 60
cgtcatgacc cagtctccat cttatcttgc tgcatctcct ggagaaacca ttactattaa 120
ttgcagggca agtaagagca ttagcaaata tttagcctgg tatcaagaga aacctgggaa 180
aactaataag cttcttatct actctggatc cactttgcaa tctggaattc catcaaggtt 240
cagtggcagt ggatctggta cagatttcac tctcaccatc agtagcctgg agcctgaaga 300
ttttgcaatg tattactgtc aacagcataa tgaatatccg tacacgttcg gaggggggac 360
caagcttgag atcaaaggtg gtggtggttc tggcggcggc ggctccggtg gtggtggttc 420
tgagaggtgc agctgctcga gcagtctgga gctgagctgg tgaaacctgg ggcctcagtg 480
aagatatcct gcaaggcttc tggatacgcc ttcactaact actggctagg ttgggtaaag 540
cagaggcctg gacatggact tgagtggatt ggagatcttt tccctggaag tggtaatact 600
cactacaatg agaggttcag gggcaaagcc acactgactg cagacaaatc ctcgagcaca 660
gcctttatgc agctcagtag cctgacatct gaggactctg ctgtctattt ctgtgcaaga 720
ttgaggaact gggacgaggc tatggactac tggggccaag ggaccacggt caccgtctcc 780
tcaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 840
gacggcgacg taaacggcca caagttcagc gtgtctggcg agggcgaggg cgatgccacc 900
tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 960
accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 1020
aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 1080
ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 1140
ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 1200
cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 1260
aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 1320
gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 1380
cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 1440
gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 1500
tacccatacg atgttccaga ttacgcttaa 1530
<210> 48
<211> 509
<212> PRT
<213> 嵌合抗原受体蛋白EpHL-GP-31氨基酸序列
<400> 48
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala
20 25 30
Ser Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile
35 40 45
Ser Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys
50 55 60
Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg
65 70 75 80
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
85 90 95
Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu
100 105 110
Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
130 135 140
Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val
145 150 155 160
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu
165 170 175
Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp
180 185 190
Leu Phe Pro Gly Ser Gly Asn Thr His Tyr Asn Glu Arg Phe Arg Gly
195 200 205
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe Met Gln
210 215 220
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg
225 230 235 240
Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr
245 250 255
Val Thr Val Ser Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly
260 265 270
Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys
275 280 285
Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
290 295 300
Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
305 310 315 320
Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr
325 330 335
Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu
340 345 350
Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
355 360 365
Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
370 375 380
Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
385 390 395 400
His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala
405 410 415
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
420 425 430
Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
435 440 445
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
450 455 460
Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
465 470 475 480
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
485 490 495
Glu Leu Tyr Lys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
500 505
<210> 49
<211> 1530
<212> DNA
<213> 嵌合抗原受体蛋白EpLH-GP-31编码核酸序列
<400> 49
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60
gtgcagctgc tcgagcagtc tggagctgag ctggtgaaac ctggggcctc agtgaagata 120
tcctgcaagg cttctggata cgccttcact aactactggc taggttgggt aaagcagagg 180
cctggacatg gacttgagtg gattggagat cttttccctg gaagtggtaa tactcactac 240
aatgagaggt tcaggggcaa agccacactg actgcagaca aatcctcgag cacagccttt 300
atgcagctca gtagcctgac atctgaggac tctgctgtct atttctgtgc aagattgagg 360
aactgggacg aggctatgga ctactggggc caagggacca cggtcaccgt ctcctcaggt 420
ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgagctcgt catgacccag 480
tctccatctt atcttgctgc atctcctgga gaaaccatta ctattaattg cagggcaagt 540
aagagcatta gcaaatattt agcctggtat caagagaaac ctgggaaaac taataagctt 600
cttatctact ctggatccac tttgcaatct ggaattccat caaggttcag tggcagtgga 660
tctggtacag atttcactct caccatcagt agcctggagc ctgaagattt tgcaatgtat 720
tactgtcaac agcataatga atatccgtac acgttcggag gggggaccaa gcttgagatc 780
aaaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 840
gacggcgacg taaacggcca caagttcagc gtgtctggcg agggcgaggg cgatgccacc 900
tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 960
accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 1020
aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 1080
ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 1140
ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 1200
cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 1260
aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 1320
gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 1380
cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 1440
gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 1500
tacccatacg atgttccaga ttacgcttaa 1530
<210> 50
<211> 509
<212> PRT
<213> 嵌合抗原受体蛋白EpLH-GP-31氨基酸序列
<400> 50
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val
20 25 30
Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
35 40 45
Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
50 55 60
Leu Glu Trp Ile Gly Asp Leu Phe Pro Gly Ser Gly Asn Thr His Tyr
65 70 75 80
Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
85 90 95
Ser Thr Ala Phe Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln
145 150 155 160
Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly Glu Thr Ile Thr Ile Asn
165 170 175
Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr Leu Ala Trp Tyr Gln Glu
180 185 190
Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu
195 200 205
Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 215 220
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr
225 230 235 240
Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr
245 250 255
Lys Leu Glu Ile Lys Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly
260 265 270
Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys
275 280 285
Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
290 295 300
Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
305 310 315 320
Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr
325 330 335
Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu
340 345 350
Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
355 360 365
Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
370 375 380
Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
385 390 395 400
His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala
405 410 415
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
420 425 430
Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
435 440 445
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
450 455 460
Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
465 470 475 480
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
485 490 495
Glu Leu Tyr Lys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
500 505
<210> 51
<211> 1424
<212> DNA
<213> 嵌合抗原受体蛋白EpHL-GP-47编码核酸序列
<400> 51
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgct 60
cgtgatgacc cagactccac tctccctgcc tgtcagtctt ggagatcaag cctccatctc 120
ttgcagatct agtcagagcc ttgtacacag taatggaaac acctatttac attggtacct 180
gcagaagcca ggccagtctc caaagctcct gatctacaaa gtttccaacc gattttctgg 240
ggtcccagac aggttcagtg gcagtggatc agggacagat ttcacactca agatcagcag 300
agtggaggct gaggatctgg gagtttattt ctgctctcaa agtacacatg ttccgtacac 360
gttcggaggg gggaccaagc ttgagatcaa aggtggtggt ggttctggcg gcggcggctc 420
cggtggtggt ggttctgatc cttgagtgga ttggagaggt ttatcctaga attggtaatg 480
cttactacaa tgagaagttc aagggcaagg ccacactgac tgcagacaaa tcctccagca 540
cagcgtccat ggagctccgc agcctgacct ctgaggactc tgcggtctat ttctgtgcaa 600
gacggggatc ctacgatact aactacgact ggtacttcga tgtctggggc caagggacca 660
cggtcaccgt ctcctcaatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 720
tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtct ggcgagggcg 780
agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 840
ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 900
accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 960
aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 1020
tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 1080
gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 1140
ccgacaagca gaagaacggc atcaaggcga acttcaagat ccgccacaac atcgaggacg 1200
gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 1260
tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 1320
agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 1380
acgagctgta caagtaccca tacgatgttc cagattacgc ttaa 1424
<210> 52
<211> 518
<212> PRT
<213> 嵌合抗原受体蛋白EpHL-GP-47氨基酸序列
<400> 52
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45
Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Ala Arg
145 150 155 160
Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
165 170 175
Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln Val Leu
180 185 190
Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr Asn
195 200 205
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
210 215 220
Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
225 230 235 240
Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp Trp Tyr
245 250 255
Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Met Val
260 265 270
Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu
275 280 285
Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
290 295 300
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr
305 310 315 320
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr
325 330 335
Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His
340 345 350
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
355 360 365
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys
370 375 380
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp
385 390 395 400
Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr
405 410 415
Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile
420 425 430
Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln
435 440 445
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val
450 455 460
Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys
465 470 475 480
Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr
485 490 495
Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Tyr Pro Tyr
500 505 510
Asp Val Pro Asp Tyr Ala
515
<210> 53
<211> 1424
<212> DNA
<213> 嵌合抗原受体蛋白EpLH-GP-47编码核酸序列
<400> 53
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactcctcc 60
ttgagtggat tggagaggtt tatcctagaa ttggtaatgc ttactacaat gagaagttca 120
agggcaaggc cacactgact gcagacaaat cctccagcac agcgtccatg gagctccgca 180
gcctgacctc tgaggactct gcggtctatt tctgtgcaag acggggatcc tacgatacta 240
actacgactg gtacttcgat gtctggggcc aagggaccac ggtcaccgtc tcctcaggtg 300
gtggtggttc tggcggcggc ggctccggtg gtggtggttc tgagctcgtg atgacccaga 360
ctccactctc cctgcctgtc agtcttggag atcaagcctc catctcttgc agatctagtc 420
agagccttgt acacagtaat ggaaacacct atttacattg gtacctgcag aagccaggcc 480
agtctccaaa gctcctgatc tacaaagttt ccaaccgatt ttctggggtc ccagacaggt 540
tcagtggcag tggatcaggg acagatttca cactcaagat cagcagagtg gaggctgagg 600
atctgggagt ttatttctgc tctcaaagta cacatgttcc gtacacgttc ggagggggga 660
ccaagcttga gatcaaaatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 720
tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtct ggcgagggcg 780
agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 840
ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 900
accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 960
aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 1020
tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 1080
gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 1140
ccgacaagca gaagaacggc atcaaggcga acttcaagat ccgccacaac atcgaggacg 1200
gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 1260
tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 1320
agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 1380
acgagctgta caagtaccca tacgatgttc cagattacgc ttaa 1424
<210> 54
<211> 518
<212> PRT
<213> 嵌合抗原受体蛋白EpLH-GP-47氨基酸序列
<400> 54
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Ala
20 25 30
Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr
35 40 45
Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln Val
50 55 60
Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr
65 70 75 80
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
85 90 95
Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp Trp
115 120 125
Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu
145 150 155 160
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln
165 170 175
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly
180 185 190
Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys
195 200 205
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg
210 215 220
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg
225 230 235 240
Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His
245 250 255
Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Met Val
260 265 270
Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu
275 280 285
Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly
290 295 300
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr
305 310 315 320
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr
325 330 335
Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His
340 345 350
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr
355 360 365
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys
370 375 380
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp
385 390 395 400
Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr
405 410 415
Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile
420 425 430
Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln
435 440 445
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val
450 455 460
Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys
465 470 475 480
Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr
485 490 495
Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Tyr Pro Tyr
500 505 510
Asp Val Pro Asp Tyr Ala
515
<210> 55
<211> 1542
<212> DNA
<213> 嵌合抗原受体蛋白EpHL-GP-510编码核酸序列
<400> 55
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60
gtgcagctgc tcgagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaagata 120
tcctgcaagg cttctggata cgccttcact aactactggc taggttgggt aaagcagagg 180
cctggacatg gacttgagtg gattggagat attttccctg gaagtggtaa tatccactac 240
aatgagaagt tcaagggcaa agccacactg actgcagaca aatcttcgag cacagcctat 300
atgcagctca gtagcctgac atttgaggac tctgctgtct atttctgtgc aagactgagg 360
aactgggacg agcctatgga ctactggggc caagggacca cggtcaccgt cggtggtggt 420
ggttctggcg gcggcggctc cggtggtggt ggttctgagc tcgtgatgac acagtctcca 480
tcctccctga ctgtgacagc aggagagaag gtcactatga gctgcaagtc cagtcagagt 540
ctgttaaaca gtggaaatca aaagaactac ttgacctggt accagcagaa accagggcag 600
cctcctaaac tgttgatcta ctgggcatcc actagggaat ctggggtccc tgatcgcttc 660
acaggcagtg gatctggaac agatttcact ctcaccatca gcagtgtgca ggctgaagac 720
ctggcagttt attactgtca gaatgattat agttatccgc tcacgttcgg tgctgggacc 780
aagcttgaga tcaaaatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 840
ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtctgg cgagggcgag 900
ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 960
gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 1020
cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 1080
gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 1140
gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1200
aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 1260
gacaagcaga agaacggcat caaggcgaac ttcaagatcc gccacaacat cgaggacggc 1320
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 1380
ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 1440
cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 1500
gagctgtaca agtacccata cgatgttcca gattacgctt aa 1542
<210> 56
<211> 513
<212> PRT
<213> 嵌合抗原受体蛋白EpHL-GP-510氨基酸序列
<400> 56
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val
20 25 30
Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
35 40 45
Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
50 55 60
Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr
65 70 75 80
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
85 90 95
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala
100 105 110
Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Thr Val Thr Val Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln Ser Pro
145 150 155 160
Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys
165 170 175
Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr
180 185 190
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp
195 200 205
Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
210 215 220
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp
225 230 235 240
Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe
245 250 255
Gly Ala Gly Thr Lys Leu Glu Ile Lys Met Val Ser Lys Gly Glu Glu
260 265 270
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
275 280 285
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
290 295 300
Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro
305 310 315 320
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys
325 330 335
Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
340 345 350
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
355 360 365
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
370 375 380
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly
385 390 395 400
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val
405 410 415
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys
420 425 430
Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
435 440 445
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
450 455 460
His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys
465 470 475 480
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
485 490 495
Leu Gly Met Asp Glu Leu Tyr Lys Tyr Pro Tyr Asp Val Pro Asp Tyr
500 505 510
Ala
<210> 57
<211> 1542
<212> DNA
<213> 嵌合抗原受体蛋白EpLH-GP-510编码核酸序列
<400> 57
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60
ctcgtgatga cacagtctcc atcctccctg actgtgacag caggagagaa ggtcactatg 120
agctgcaagt ccagtcagag tctgttaaac agtggaaatc aaaagaacta cttgacctgg 180
taccagcaga aaccagggca gcctcctaaa ctgttgatct actgggcatc cactagggaa 240
tctggggtcc ctgatcgctt cacaggcagt ggatctggaa cagatttcac tctcaccatc 300
agcagtgtgc aggctgaaga cctggcagtt tattactgtc agaatgatta tagttatccg 360
ctcacgttcg gtgctgggac caagcttgag atcaaaggtg gtggtggttc tggcggcggc 420
ggctccggtg gtggtggttc tgaggtgcag ctgctcgagc agtctggagc tgagctggta 480
aggcctggga cttcagtgaa gatatcctgc aaggcttctg gatacgcctt cactaactac 540
tggctaggtt gggtaaagca gaggcctgga catggacttg agtggattgg agatattttc 600
cctggaagtg gtaatatcca ctacaatgag aagttcaagg gcaaagccac actgactgca 660
gacaaatctt cgagcacagc ctatatgcag ctcagtagcc tgacatttga ggactctgct 720
gtctatttct gtgcaagact gaggaactgg gacgagccta tggactactg gggccaaggg 780
accacggtca ccgtcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 840
ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtctgg cgagggcgag 900
ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 960
gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 1020
cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 1080
gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 1140
gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1200
aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 1260
gacaagcaga agaacggcat caaggcgaac ttcaagatcc gccacaacat cgaggacggc 1320
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 1380
ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 1440
cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 1500
gagctgtaca agtacccata cgatgttcca gattacgctt aa 1542
<210> 58
<211> 513
<212> PRT
<213> 嵌合抗原受体蛋白EpLH-GP-510氨基酸序列
<400> 58
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val
20 25 30
Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu
35 40 45
Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys
50 55 60
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
65 70 75 80
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
85 90 95
Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr
100 105 110
Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys
115 120 125
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val
145 150 155 160
Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala
165 170 175
Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly
180 185 190
Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr
195 200 205
Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
210 215 220
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala
225 230 235 240
Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Thr Val Thr Val Met Val Ser Lys Gly Glu Glu
260 265 270
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
275 280 285
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
290 295 300
Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro
305 310 315 320
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys
325 330 335
Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
340 345 350
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
355 360 365
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
370 375 380
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly
385 390 395 400
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val
405 410 415
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys
420 425 430
Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
435 440 445
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
450 455 460
His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys
465 470 475 480
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
485 490 495
Leu Gly Met Asp Glu Leu Tyr Lys Tyr Pro Tyr Asp Val Pro Asp Tyr
500 505 510
Ala
<210> 59
<211> 21
<212> DNA
<213> hCDH1-F荧光定量PCR引物
<400> 59
gaacagcacg tacacagccc t 21
<210> 60
<211> 21
<212> DNA
<213> hCDH1-R荧光定量PCR引物
<400> 60
gcagaagtgt ccctgttcca g 21
<210> 61
<211> 19
<212> DNA
<213> hGAPDH-F荧光定量PCR引物
<400> 61
gaaggtgaag gtcggagtc 19
<210> 62
<211> 20
<212> DNA
<213> hGAPDH-R荧光定量PCR引物
<400> 62
gaagatggtg atgggatttc 20

Claims (3)

1.工程化改造的成纤维细胞在构建肿瘤类器官中的用途，其中，所述工程化改造的成纤维细胞表面表达有嵌合抗原受体蛋白，所述嵌合抗原受体蛋白中含有抗人上皮细胞黏附分子(EpCAM)的单链抗体，所述嵌合抗原受体蛋白的氨基酸序列如SEQ ID NO.24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、或58所示。
2.根据权利要求1所述的用途，其特征在于，所述成纤维细胞来自从人或者小鼠中分离的原代成纤维细胞或者成纤维细胞系。
3.根据权利要求2所述的用途，其特征在于，所述成纤维细胞选自小鼠胚胎成纤维细胞(Mouse Embryo Fibroblast，MEF)、NIH-3T3或OP9。
CN201810731610.7A 2018-07-05 2018-07-05 一种嵌合抗原受体成纤维细胞及其建立方法和应用 Active CN108795877B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810731610.7A CN108795877B (zh) 2018-07-05 2018-07-05 一种嵌合抗原受体成纤维细胞及其建立方法和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810731610.7A CN108795877B (zh) 2018-07-05 2018-07-05 一种嵌合抗原受体成纤维细胞及其建立方法和应用

Publications (2)

Publication Number Publication Date
CN108795877A CN108795877A (zh) 2018-11-13
CN108795877B true CN108795877B (zh) 2021-07-09

Family

ID=64075133

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810731610.7A Active CN108795877B (zh) 2018-07-05 2018-07-05 一种嵌合抗原受体成纤维细胞及其建立方法和应用

Country Status (1)

Country Link
CN (1) CN108795877B (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111876386B (zh) * 2020-08-10 2023-05-30 上海市第一人民医院 一种乳腺癌类器官的培养方法以及和肿瘤相关成纤维细胞的共培养方法
CN114574444B (zh) * 2020-12-01 2024-05-03 达达生物技术(北京)有限公司 自体纤维母细胞在制备抗类风湿关节炎药物中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105949323A (zh) * 2016-06-24 2016-09-21 安徽未名细胞治疗有限公司 一种EpCAM特异性嵌合抗原受体及其编码基因、应用
WO2020046910A1 (en) * 2018-08-27 2020-03-05 Figene, Llc Chimeric antigen receptor fibroblast cells for treatment of cancer

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104126009B (zh) * 2011-10-07 2019-05-10 国立大学法人三重大学 嵌合抗原受体

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105949323A (zh) * 2016-06-24 2016-09-21 安徽未名细胞治疗有限公司 一种EpCAM特异性嵌合抗原受体及其编码基因、应用
WO2020046910A1 (en) * 2018-08-27 2020-03-05 Figene, Llc Chimeric antigen receptor fibroblast cells for treatment of cancer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Chimeric antigen receptor T cells with dissociated signaling domain exhibit focused anti-tumor activity with reduced potential for toxicity in vivo;Evripidis Lanitis, et al.;《Cancer Immunnol Res》;20130701;Results部分第1段 *

Also Published As

Publication number Publication date
CN108795877A (zh) 2018-11-13

Similar Documents

Publication Publication Date Title
CN107827990B (zh) 一种多肽、编码其的核酸、其修饰的t淋巴细胞及其应用
CN110248669B (zh) 工程化天然杀伤细胞及其用途
WO2020125277A1 (zh) 基因工程修饰的双靶点嵌合抗原受体及其用途
KR20210099573A (ko) 개선된 종양 반응성 t-세포의 선택
WO2017088269A1 (zh) 一种嵌合抗原受体hCD87-CAR及载有hCD87-CAR基因结构的慢病毒及质粒及其应用
CN108795877B (zh) 一种嵌合抗原受体成纤维细胞及其建立方法和应用
CN111051349A (zh) Strep-tag特异性嵌合受体及其用途
CN106047817A (zh) Car‑t细胞及其制备方法与应用
US11215618B2 (en) Articles and methods directed to personalized therapy of cancer
WO2022076606A1 (en) Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
EP4225331A1 (en) Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
CN111303287B (zh) 一种anti-CD19的全人源抗体或抗体片段及其嵌合抗原受体和应用
CN114276454B (zh) 一种抗间皮素的纳米抗体及其应用
KR101593049B1 (ko) 3차원 콜라겐 겔 환경에서 라이실-ｔＲＮＡ 합성효소가 발현하거나 발현하지 않도록 조절된 세포 또는 회전타원체로 덩어리진 세포 배양을 이용한 암 전이 억제제의 스크리닝 방법
EP3946435B1 (en) Enhancement of cytolytic t-cell activity by inhibiting ebag9
CN115125272A (zh) 一种car-t治疗载体及其构建方法和应用
CN110325551B (zh) 一种嵌合抗原受体及其应用
Lee et al. A functionally robust phenotypic screen that identifies drug resistance-associated genes using 3D cell culture
JP5924741B2 (ja) 人工癌幹細胞の作製方法及びその分化誘導方法
EP4377446A1 (en) Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with kras inhibitors
CN114191556A (zh) 敲降rbms1的试剂在制备治疗三阴性乳腺癌的药物中的应用
EP4015625A1 (en) Universal car-t cell and preparation and use thereof
CN113004423B (zh) 一种特异性靶向活化肝星状细胞的car-t细胞及其制备方法和应用
CN114075548A (zh) 一种靶向axl的car-t细胞及其制备方法和应用
WO2024098027A1 (en) Methods for tumor infiltrating lymphocyte (til) expansion related to cd39/cd103 selection

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20240407

Address after: 101111, 5th Floor, Building 3, Zone 3, No. 88 Jinghai Fifth Road, Beijing Economic and Technological Development Zone, Daxing District, Beijing

Patentee after: Beijing Ke Ke medical science and Technology Co.,Ltd.

Country or region after: China

Address before: 313099 1001, 10th floor, building 6, No. 1366, Hongfeng Road, Huzhou City, Zhejiang Province

Patentee before: ZHEJIANG K2ONCOLOGY Co.,Ltd.

Country or region before: China

TR01 Transfer of patent right