CN112041335A - 用于细胞免疫治疗的组合物和方法 - Google Patents
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Abstract
本公开内容提供了包含修饰的CD28共刺激信号传导域的融合蛋白及其在免疫疗法中的用途,该疗法用于治疗癌症。在一些实施方案中,提供了表达融合蛋白并在细胞免疫疗法中用于治疗癌症和其他疾病的宿主免疫细胞。在某些实施方案中,融合蛋白包含根据本发明的嵌合抗原受体(CAR)。
Description
关于序列表的声明
与本申请相关联的序列表以文本格式代替纸质副本提供,并且据此通过引用结合到说明书中。包含序列表的文本文件的名称为360056_458WO_SEQUENCE_LISTING.txt。文本文件为274KB,其创建于2019年2月20日,正在通过EFS-Web电子提交。
政府权益声明
本发明是在美国国立卫生研究院授予CA114536的政府支持下完成的。政府拥有本发明的某些权利。
背景技术
使用针对癌症抗原的经基因工程改造的受体修饰的T细胞进行的过继免疫疗法已证明在血液系统癌症中具有临床成功,并显示出在治疗其他癌症和疾病中的潜力。工程化的受体包括嵌合抗原受体(CAR)和增强的亲和力T细胞受体(TCR)。参见,例如,Harris和Kranz,Trends Pharmacol,Sci.37(3):220(2016)。通过工程改造的受体与癌症抗原结合后,修饰的T细胞通过诱导靶肿瘤细胞的细胞溶解并释放细胞因子来刺激免疫反应,从而介导抗肿瘤反应。
除抗原驱动的刺激外,CAR和TCR还可在T细胞中表现出强直性(非抗原依赖性或组成性)或过度信号传导。高表面表达、工程受体的自我聚集特性(例如,scFv寡聚)、在γ逆转录病毒载体中的表达以及某些共刺激信号域的存在可能有助于滋补和/或过度的信号传导(Gomes-Silva等人,Cell Reports 21:17-26(2017);Long等人,Nat.Med.21:581-590(2015);Frigault等人,Cancer Immunol.Res.3:356-367(2015))。进补和/或过度信号传导可导致转导T细胞的组成性激活和增殖、加速T细胞分化、限制T细胞持久性、增加T细胞疲劳、增加T细胞凋亡、增加免疫检查点分子受体的表达(例如PD-1、TIM-3和AG-3)和/或降低T细胞的抗肿瘤活性(Gomes-Silva等,同上;Frigault等,同上;Long等,同上;Eyquem等,Nature543:113(2017))。
此外,CAR T细胞疗法还与明显的毒性相关,包括细胞因子释放综合征(CRS)和CART细胞相关性脑病综合征(CRES)。细胞因子释放综合征是指与T细胞活化和增殖相关的细胞因子水平升高引起的全身性炎症反应。CRS可能是轻度的、自限性的、伴有发烧和肌痛的症状,甚至更严重,伴有血管渗漏、低血压、呼吸和肾脏功能不全、血细胞减少、凝血病、多器官功能衰竭和神经系统毒性的症状。神经毒性可能表现为多种神经和精神症状,包括癫痫发作、神志失常、失语症和幻觉。CRES可与CRS同时或之后发生,并可能导致致命的脑水肿。
因此,在过继性细胞疗法中需要新的策略来治疗癌症。当前公开的实施例解决了这些需求并提供了其他相关的优点。
附图说明
图1A-1G显示在CD28/CD3ζ和4-1BB/CD3ζCAR的间隔区中包含(STII)标签提供了一种激活嵌合抗原受体(CAR)信号传导的精确方法。图1A:示意图STII CAR设计在细胞外铰链中包含单个STII序列。CAR包含CD19特异性FMC63scFv(SEQ ID NO:8)或ROR1特异性R12 scFv(SEQ ID NO:9)。图1B:STII CAR T细胞与涂有STII mAb的STII磁珠结合的示意图。图1C:CD19+LCL扩增后分选纯化的CD19特异性CAR T细胞的代表性FACS图。图1D和1E:代表性FACS图显示了细胞表面CAR的STII染色(D)和分选纯化的CD19特异性CD19的表型(E)或扩增后的ROR1特异性CAR T细胞。虚线:CD28/CD3ζCAR T细胞,实线:4-1BB/CD3ζCAR T细胞,阴影直方图:同种型对照。图1F:代表性的FACS图显示了CAR T细胞的DNA含量染色。门控量化了G0/G1中细胞的频率。图1G:用不同数量的STII磁珠、K562细胞或ROR1共培养45分钟后,分析从CAR T细胞(4-1BB/CD3ζ)制备的裂解物的蛋白质印迹。将裂解物印迹到PLC-γ1pY783、PLC-γ1、SLP-76pS376、SLP-76、CD247pY142和CD247。图1C-1F中的数据代表了四个独立的实验。图1G中的印迹代表两个独立的实验。
图2A-2G对CAR磷蛋白信号进行定量分析,表明CAR通过内源性T细胞信号蛋白进行信号传递。图2A和2B:CAR T细胞刺激条件和实验设计。图2C:图2C:条形图显示了在图2A和2B中所述的三种质谱(MS)实验中鉴定出的独特的磷酸丝氨酸(“pS”),磷酸苏氨酸(“pT”)和磷酸酪氨酸(“pY”)位点的总数。图2D:Venn图显示了在所有MS实验中检测到的PO4位点之间的重叠。图2E和2F:散点图显示了在CAR刺激10分钟(E)和45分钟(F)之后规范性TCR信号PO4事件的平均和log2倍变化范围。图2G:10和45分钟裂解物的蛋白质印迹分析刺激CD28/CD3ζ和4-1BB/CD3ζ+CAR T细胞。印迹代表图2A和2B中描述的三个独立实验。
图3A-3F显示CD28/CD3ζ或4-1BB/CD3ζCAR刺激以不同的动力学和强度调节相似的蛋白质磷酸化事件。图3A-3D:火山图显示已鉴定PO4位点的log2倍变化和假发现率(FDR)由MS在两个或多个实验中进行。使用Limma定义符合CAR刺激的PO4位置(火山图的左上(C,D)和右上(A,C,D)部分的灰点),满足log2倍变化和FDR截止值。图3E:散点图在45分钟的时间点比较CD28/CD3ζ和4-1BB/CD3ζCAR刺激响应的PO4位点。网格中上部和中下部的灰点(由虚线定义;包括灰点)垂直的“0”线)表示受4-1BB/CD3ζCAR刺激更强烈地调节的PO4位点。网格右下部和左上部的浅灰色点表示CD28/CD3ζCAR刺激反应性PO4位点,其被4-1BB/CD3ζCAR刺激以相反的方向调制。图3F:散点图显示了平均值刺激后,已知CD28和4-1BB信号通路成员的PO4位点的log2倍变化范围和范围。
图4显示CD28/CD3ζ或4-1BB/CD3ζCAR刺激改变了类似信号通路和细胞区室的蛋白质磷酸化。来自所有MS实验分析的CAR T细胞刺激45分钟后,差异蛋白磷酸化的选择蛋白图谱。
图5A-5C显示,与4-1BB/CD3ζCAR刺激相比,CD28/CD3ζCAR刺激在蛋白质PO4中产生更大的幅度变化。图5A:点图显示CAR刺激后20个最磷酸化位点的平均log2倍变化。图5B:点图显示在CAR刺激后,在已知KEGG TCR信号通路蛋白上检测到的每个PO4位点的绝对log2倍变化。因为在CAR激活后某些位点被去磷酸化,所以绝对log2倍变化用于量化PO4改变的幅度。图5C:用STII磁珠刺激给定时间的CAR T细胞的Western印迹分析。印迹代表3个独立实验。图5A和5B中的P值是使用未配对的两样本t检验计算的。
图6A-6K显示了CD28/CD3ζCAR信号促进了效应细胞样表型,体内抗肿瘤活性降低。图6A-6B:条形图显示了与刺激的对照CD28/CD3ζ或4-1BB/CD3ζCAR T细胞相比,基因表达的平均log2倍变化值。图6C:火山图显示将刺激的CD28/CD3ζ与4-1BB/CD3ζCAR T细胞相比,基因表达的log2倍变化和假发现率(FDR)。使用Limma定义差异表达基因(图中左上方和右上方的灰色点,如虚线所定义),满足log2倍变化和FDR截止值。图6D:条形图显示IL7R,KLF2和FOXO4的log2倍变化用qPCR测定CD28/CD3ζ和4-1BB/CD3ζCAR T细胞的表达。该图显示了在单个qPCR运行中量化的n=3个生物学重复的平均值和标准差。图6E:代表性FACS图显示了ROR1特异性CAR T细胞与K562或K562/ROR1细胞共培养5小时后细胞因子产生的细胞内染色。图6F:条形图显示了共培养ROR1特异性CAR T细胞和K562/ROR1细胞24小时后细胞因子产生倍数变化的平均值和标准偏差。n=3-4个独立实验。*表示通过Welch t检验得出的p<0.05。图6G:FACS图显示了在STII磁珠刺激后72小时通过CFSE染料稀释测量的CAR T细胞增殖。直方图线如数字键所示。图6H:Raji/ffluc植入后7天,单次注入CAR T细胞(3.0x106细胞,左或7.5x105 T细胞,右)处理的NSG小鼠的生存分析。6、9或15只小鼠的生存分析每组来自两个或三个独立的实验。图6I:代表性的生物发光图像显示了在指定时间点的小鼠总辐射(p/sec/cm2/sr)。图6J-6K:条形图显示了CAR T细胞频率(J)在骨髓或外周血中,或通过流式细胞术测量的第20天,骨髓中CAR T细胞上PD-1,Lag-3或Tim-3表达的平均荧光强度(MFI)(K)。每组n=5只小鼠。使用未配对的两尾学生t检验来比较组均值。图6G中的数据代表了四个独立的实验。图6H-6K中的数据代表两个独立的实验。
图7A-7H显示了CD28/CD3ζCAR与内源性Lck和CD28差异结合,并且CD28结构域中的酪氨酸有助于与CD28/CD3ζCAR T细胞相关的独特属性。图7A和7B:来自静止(A)或受刺激(B)的CAR T细胞的全细胞裂解物(L)和免疫沉淀级分(IP)的蛋白质印迹分析。(A)中的印迹代表3个独立的实验;(B)中的印迹代表一个实验。图7C:对CAR CD28共刺激信号域进行酪氨酸突变的示意图。图7D:FACS图显示了在与K562/ROR1细胞共培养72小时后,通过CFSE染料稀释测量的CAR T细胞增殖。直方图线显示在数字键中。图7E:条形图显示了与K562/ROR1细胞共培养24小时后细胞因子产生倍数变化的平均值和标准差。n=3个独立实验。图7F:静息CAR T细胞的全细胞裂解液(L)和免疫沉淀级分(IP)的蛋白质印迹分析。印迹代表3次独立实验。图7G:用STII磁珠刺激给定时间的CAR T细胞的蛋白质印迹。图7H:Raji/ffluc注射7天后,单次注入CAR T细胞治疗的NSG小鼠的生存分析。将每组6或15只小鼠的生存分析从两个到三个独立的实验中合并。
图8A-8B显示Jurkat细胞和原代T细胞中TCR信号的质谱分析揭示了蛋白质磷酸化的显着差异。图8A:用小鼠抗人CD3εmAb和抗小鼠IgG刺激原代CD8+T细胞或Jurkat细胞,或通过单独用抗小鼠IgG治疗而不受刺激。8B:热图,显示了近端TCR信号通路中已知PO4位点的log2倍变化。
图9显示了使用串联质量标签(TMT)试剂和多步磷酸肽富集进行肽标记的方法,可以对CAR CART细胞中蛋白质的磷酸化进行详细分析。提供了示例性磷酸肽富集策略的示意图。简而言之,将裂解物还原,烷基化,用胰蛋白酶消化,然后用TMT试剂标记。合并标记的肽,并对磷酸酪氨酸(pTyr)肽进行免疫沉淀。保留洗脱液用于LC-MS/MS。使用碱性反相液相色谱将流出物分离为13个馏分,然后使用固定的金属亲和色谱(IMAC)进行全局磷酸肽富集。然后通过LC-MS/MS分析所有14个富集的级分(1p-Tyr+13IMAC)。
图10显示了PO4 log2倍数变化摘要统计数据。直方图显示了三个实验中log2倍数变化(模拟相对于对照)值的分布。图例中标有标准偏差。
图11A-11D显示,CAR-Lck缔合部分负责与CD28/CD3ζCAR T细胞相关的信号强度增加。图11A:对CAR CD28共刺激信号结构域的突变示意图。图11B:静息CAR T细胞的全细胞裂解液(L)和免疫沉淀级分(IP)的蛋白质印迹分析。印迹代表两个独立的实验。图11C:用STII磁珠刺激给定时间的CAR T细胞的蛋白质印迹分析。印迹代表三个独立的实验。图11D:柱状图显示了与K562/ROR1细胞共培养24小时后的平均细胞因子浓度。数据代表两个独立实验。
具体实施方式
本公开总体上涉及包含修饰的CD28共刺激信号结构域的融合蛋白(例如,嵌合抗原受体、嵌合共刺激受体)。在某些方面,本公开提供了融合蛋白,其包含细胞外组分,所述细胞外组分包含特异性结合靶抗原的结合域;包含修饰的CD28共刺激信号传导域的细胞内组分;以及布置在细胞外组分和细胞内组分之间的疏水性部分,其中与包含野生型CD28共刺激信号结构域的融合蛋白相比,修饰的CD28共刺激信号结构域包含至少一个氨基酸取代,该氨基酸取代调节融合蛋白的功能活性(即,本文提供的一种或多种功能活性)。这样的融合蛋白和表达该融合蛋白的宿主细胞可以用于例如过继免疫疗法以治疗癌症的方法中。
作为背景技术,正在开发重定向T细胞特异性和/或促进所需效应子功能的合成受体作为用于癌症,自身免疫和感染的免疫治疗剂(Sadelain等人,Nature 545:423-431(2017)。能够重定向T细胞特异性和/或促进所需效应子功能的功能包括嵌合抗原受体(CARs)、嵌合趋化因子受体、嵌合共刺激受体和工程T细胞受体(TCR)。例如,一些CAR通过将细胞外抗原特异性单链变量(scFv)片段连接至模拟TCR激活的细胞内T细胞信号传导域来重定向T细胞对肿瘤细胞的特异性(van der Stegen等人,Nat Rev Drug Discov14:499–509(2015)。CAR修饰的T细胞已显示出治疗多种恶性肿瘤和癌症的希望(参见,例如Kalos等,Sci Transl Med3:95ra73(2011);Maude等,N Engl J Med371:1507–1517(2014));Davila等人,Sci Transl Med6:224ra25(2014);Lee等人,Lancet385:517-528(2015);Kochenderfer等人,J.Clin.Oncol.33:540-549(2015);Turtle等人,J.Clin.Invest.126:2123-2138(2016);Turtle等人,Sci Transl Med8,355ra116(2016);Turtle等人,J.Clin.Oncol.35:3010–3020(2017))。
由TCR参与介导的T细胞活化导致蛋白质磷酸化(PO4),是由含免疫受体酪氨酸的活化基序(ITAM)包含的CD3δ、ε、γ和ζ链引发的(Brownlie等人,Nature ReviewsImmunology13:257–269(2013))。这些事件与共刺激分子反式传递的PO4信号结合,会改变T细胞的转录程序,促进细胞因子释放并诱导增殖(Kaech和Cui,Nature ReviewsImmunology 12:749–761(2012))。CARs通常含有CD3ζ内结构域在具有来自CD28和/或4-1BB的共刺激域的单链构建体中。CD28/CD3ζ和4-1BB/CD3ζCAR T细胞均可有效治疗患者的肿瘤,但在体外和临床前小鼠模型中均表现出功能差异(Kalos等人,Sci Transl Med3,95ra73(2011);van der Stegen et al.,Nat Rev Drug Discov14:499–509(2015);Cherkassky et al.,J.Clin.Invest.126:3130-3144(2016);Kawalekaret et al.,Immunity 44:380–390(2016))。例如,据报道CD28/CD3ζCAR T细胞表现出低水平的滋补(非抗原依赖性)CD3ζ磷酸化,并且与4-1BB/CD3ζCAR T细胞相比,似乎更可能表现出滋补信号传导。补剂CAR信号传导可能对CAR T细胞产生不利影响,包括不依赖于相关抗原的结合而过度释放细胞因子、有限的持久性、疲惫、凋亡、免疫检查点分子受体表达增加或抗肿瘤活性降低(Frigaultet等,Cancer Immunol Res 3:356–367(2015);Longet等人,NatureMedicine 21:581–590(2015)。
本公开提供了融合蛋白,其包含具有在某些氨基酸残基中的一个或多个的改变的CD28共刺激信号传导域,所述某些氨基酸残基在被改变时修饰CAR的CD3ζ信号传导域的强直磷酸化和/或与内源性T细胞信号传导分子的缔合。而且,与包含野生型CD28共刺激域的那些相比,包含本公开内容的修饰的CD28共刺激信号域的CAR显示出降低的细胞因子水平,这可以导致CAR T细胞疗法的临床毒性降低(例如,细胞因子释放综合征或CAR T细胞相关性脑病综合征)。
在更详细地阐述本公开之前,提供其用于本文的某些术语的定义可能有助于其理解。贯穿本公开阐述了附加定义。
在本说明书中,任何浓度范围、百分比范围、比率范围或整数范围应理解为包括在所述范围内的任何整数的值,以及在适当时包括其分数的分数(例如,整数的十分之一和百分之一),除非另有说明。同样,除非另有说明,否则本文所叙述的涉及任何物理特征的任何数值范围,例如聚合物亚基,尺寸或厚度,应理解为包括所叙述的范围内的任何整数。如本文所用,除非另外指出,否则术语“约”是指所指示的范围、值或结构的±20%。应当理解,本文所用的术语“一个”和“一种”是指所列举的组分中的“一个或多个”。替代方案(例如“或”)的使用应理解为是指替代方案之一,两者或它们的任何组合。如本文所用,术语“包括”、“具有”和“包含”是同义使用的,其术语和变体旨在被解释为非限制性的。
另外,应当理解,本申请公开了衍生自本文所述的结构和取代基的各种组合的单个化合物或化合物组,其程度与每种化合物或化合物组分别阐述的程度相同。因此,特定结构或特定取代基的选择在本公开的范围内。
术语“基本上由...组成”并不等同于“包含”,并且是指指定的材料或步骤,或者是指不实质性影响要求保护的发明的基本特征的材料或步骤。例如,蛋白质结构域、区域、模块或盒(例如,结合结构域、铰链区、接头模块、标签盒)或蛋白质(其可具有一个或多个结构域、区域、模块或盒)“基本上由以下组成”:当结构域、区域、模块、盒或蛋白质的氨基酸序列包括延伸,缺失,突变或其组合时的特定氨基酸序列(例如,氨基酸或羧基末端或结构域之间的氨基酸)结合起来,最多贡献无畴区区域的20%(例如,最多15%、10%、8%、6%、5%、4%、3%、2%或1%),模块、盒或蛋白质,并且不会造成实质性影响(即,降低的活性不超过50%,例如不超过40%、30%、25%、20%、15%、10%、5%或1%)域、区域、模块、盒或蛋白质的活性(例如,结合蛋白或标签盒的目标结合亲和力)。
“融合蛋白”包括具有至少两个不同结构域(例如,抗原结合结构域和修饰的CD28共刺激信号传导结构域)的单链多肽,其中所述结构域不是天然一起存在于蛋白质中。可以使用PCR,重组工程等来构建编码融合蛋白的多核苷酸,或者可以合成制备此类融合蛋白。融合蛋白可以进一步包含其他组分(例如,共价结合),例如标签、接头、转导标记或生物活性分子。在某些实施方案中,融合结合蛋白是嵌合抗原受体(CAR)、基于T细胞受体的CAR(TCR-CAR)或嵌合共刺激受体(CCR)。
如本文所用,术语“嵌合抗原受体”(CAR)是指包含两个或多个不同结构域的融合蛋白,所述结构域以非天然存在的排列连接在一起,当在α-α-α表面上表达时可充当受体。细胞,并且包含:细胞外组分,其包含对抗原特异的抗原结合结构域;和任选的细胞外间隔区;疏水部分或跨膜结构域;以及包含细胞内激活域(例如,基于免疫受体酪氨酸的激活基序(ITAM)的T细胞激活基序)、细胞内共刺激域或两者的细胞内组分。在某些实施方案中,CAR的细胞内信号传导成分具有含ITAM的T细胞活化域(例如CD3ζ)和细胞内共刺激域(例如CD28)。在某些实施方案中,CAR被合成为单多肽链或由核酸分子编码为单链多肽。
“嵌合共刺激受体”(CCR)是指包含细胞外组分的融合蛋白,所述细胞外组分包含抗原结合结构域,任选的细胞外间隔子结构域,疏水部分或跨膜结构域和至少一个细胞内共刺激结构域,但不包含细胞内激活结构域(例如,基于免疫受体酪氨酸的激活基序(ITAM)包含的T细胞激活基序)。CCR可以合成为单多肽链,或者可以由核酸分子编码为单链多肽。在某些实施方案中,CCR还包含异二聚化结构域。可以将包含异二聚化结构域的CCR构建体与包含细胞内激活域(例如,基于免疫受体酪氨酸的激活基序(ITAM)的T细胞激活基序)和相应的异二聚结构域的第二种多肽在宿主细胞中共表达。异源二聚剂(例如小分子)的施用促进了CCR与包含细胞内激活结构域的多肽经由其相应的异二聚结构域的组装。这样的双多肽异二聚体构建体可以被称为“ON-switch CARs”或“split CARs”(参见,例如,Wu等人,Science350:aab4077(2015)),该构建体通过引用并入本文。
“基于T细胞受体的嵌合抗原受体”(TCR-CAR)是指异二聚体融合蛋白,异二聚体融合蛋白包含对抗原具有特异性的可溶性T细胞受体(TCR)、疏水性部分或跨膜结构域的细胞外组分、包含细胞内激活结构域细胞内组份(例如,基于免疫受体酪氨酸的激活基序(ITAM)的T细胞激活基序),以及细胞内共刺激结构域或两者的组分(参见,例如,Walseng等人,Scientific Reports 7:10713,(2017);TCR-CAR的构建体和方法全部引入作为参考。在某些实施方案中,TCR-CAR包含或由以下组成:第一多肽链,其包含细胞外组分,所述细胞外组分包含与TCRα恒定结构域或其一部分连接的TCRα链可变结构域(Vα);第二多肽链,其包含胞外成分,所述胞外成分包含与TCRβ链恒定结构域或其一部分连接的TCRβ链可变结构域(Vβ);疏水性部分或跨膜结构域,以及包含细胞内激活结构域(例如,基于免疫受体酪氨酸的活化基序(ITAM)的T细胞活化基序),细胞内共刺激域或两者的细胞内组分。在某些实施方案中,TCR-CAR的细胞内信号传导组分具有含ITAM的T细胞活化域(例如CD3ζ)和细胞内共刺激域(例如CD28)。在某些实施方案中,TCRα链恒定结构域的细胞外部分(或其一部分)和TCRβ链恒定结构域的细胞外部分(或其一部分)均被修饰以添加半胱氨酸残基以增加二聚作用。
“单链TCR”(scTCR或scTv)是指包含细胞外组分的融合蛋白,所述细胞外组分包含与TCR Vα结构域连接的TCRVα结构域,所述TCR Vβ结构域具有柔性接头(例如,与(Gly4Ser)2-5,例如SEQ ID ID:175)。将理解,可以布置scTCR,使得接头将TCR Vα结构域的C末端连接至TCR Vβ结构域的N末端,或将TCR Vα结构域的N末端连接至TCR Vβ结构域的C末端。
如本文所用,“结合结构域”(也称为“抗原结合结构域”或“结合区域”或“结合部分”)是指具有以下特征的分子,例如肽、寡肽、多肽或蛋白质与靶分子(例如病毒抗原、细菌抗原、癌症抗原、自身免疫疾病抗原、自身抗原)特异性非共价结合、联合或结合的能力。结合结构域包括任何天然存在的、合成的、半合成的或重组产生的针对生物学分子或其他目标靶标的结合伴侣。在一些实施方案中,结合结构域是抗原结合结构域,例如抗体或T细胞受体(TCR)或功能性结合结构域或其抗原结合片段。示例性的结合结构域包括单链抗体可变区(例如,域抗体、sFv、scF、Fab)、T细胞受体(TCR)的抗原结合区、例如单链TCR(scTCR)或可溶性TCR、受体胞外域、配体或为结合生物分子的特定能力而选择的合成多肽。在某些实施方案中,结合结构域不是来自CD8胞外结构域或细胞外结构域或其包含功能性IgV样结构域的任何部分的结合结构域(即,不是针对抗原肽的特异性结合结构域:来自CD8α链或CD8β的MHC复合物链)。CD8有两个亚基,即CD8α和CD8β,并且CD8共受体可以作为α同二聚体或αβ异二聚体存在。在其他实施方案中,结合域不是来自CD8α单体、CD8β单体、CD8αα同二聚体或CD8αβ异二聚体胞外域或细胞外域或其包含功能性IgV样域的任何部分的结合域。对CD8α的提及包括“规范的”人CD8α蛋白(NP_001759.3)以及剪接同工型2,该同工型2缺少一个内部区段,该内部区段包括导致分泌蛋白的跨膜结构域(RefSeq NP_741969.1),以及剪接同工型3,其中使用备用启动子和5'UTR(RefSeq NP_001139345.1)。对CD8β的提及包括“规范的”人CD8β蛋白(RefSeq NP_004922)以及同种型2-8,分别对应于RefSeq NP742099,RefSeqNP_742100,UniProt P10966-4,RefSeq NP_757362,Uniprot P10966-7,Uniprot P10966-8和参考编号NP_001171571。示例性的IgV样结构域可以在人经典CD8α蛋白的氨基酸22-135(SEQ ID NO:43)和人经典CD8β蛋白的氨基酸22-132(SEQ ID NO:44)上发现。在某些实施方案中,在TCR的背景下,CD8结合结构域与抗原肽:MHC I复合物结合,它可以是天然存在的、重组的或工程化的、或包含TCR结合域的任何其他重组结合分子(例如scTCR或基于TCR的CAR)。
如本文所用,“特异性结合”是指结合结构域或其融合结合蛋白与靶分子具有等于或大于105M-1亲和力或Ka(即,特定结合相互作用与1/M的单位的平衡缔合常数)的缔合或结合,而不会与样品中的任何其他分子或组分显着缔合或结合。结合域(或其融合结合蛋白)可以被分类为“高亲和力”结合域(或其融合结合蛋白)或“低亲和力”结合域(或其融合结合蛋白)。“高亲和力”结合结构域是指Ka至少为107M-1,至少108M-1,至少109M-1,至少1010M-1,至少1011M-1,至少1012M-1,或至少1013M-1的那些结合结构域。“低亲和力”结合结构域是指具有最高107M-1,最高106M-1,最高105M-1的Ka的那些结合结构域。或者,亲和力可定义为与M单元(例如10-5M至10-13M)的特定结合相互作用的平衡解离常数(Kd)。在某些实施方案中,结合结构域可具有“增强的亲和力”,其是指与野生型(或亲本)结合结构域相比对靶抗原具有更强结合的选择或工程化的结合结构域。例如,增强的亲和力可能是由于对靶抗原的Ka(平衡缔合常数)高于野生型结合域,或由于对靶抗原的Kd(解离常数)小于对靶抗原的Kd(解离常数)。野生型结合域,或由于靶抗原的解离速率(Koff)小于野生型结合域的解离速率。
在某些实施方案中,T细胞受体、抗体或其结合结构域或其片段可具有“增强的亲和力”,这是指与野生型(或亲本)结合相比对靶抗原具有更强结合的选择或工程化的受体或结合域。例如,亲和力增强可能是由于对靶抗原的Ka(平衡缔合常数)高于野生型结合域,而对靶抗原的Kd(解离常数)却小于对靶抗原的Kd(解离常数)。野生型结合域,因为靶抗原的解离速率(koff)小于野生型结合域的解离速率,或其组合。在某些实施方案中,本公开的融合蛋白,例如,可以对CAR或TCR(例如CAR或TCR)进行密码子优化以增强在特定宿主细胞如T细胞中的表达(Scholten等,Clin.Immunol.119:135(2006))。
已知多种测定法用于鉴定与特定靶标特异性结合的本发明的结合域,以及测定结合域或融合蛋白的亲和力,例如蛋白质印迹、ELISA、分析超速离心、光谱法、表面等离振子共振分析和MHC四聚体测定(参见,例如Scatchard等,Ann.NY Acad.Sci.51:660(1949);Wilson,Science 295:2103(2002);Wolff等,Cancer Res.53:2560(1993);Altman等人,Science 274:94-96(1996);和美国专利号5,283,173和5,468,614,或等效物)。
如本文所用,“标签盒”是指与目的蛋白固定,融合或为目的蛋白的一部分的独特肽序列,异源或非内源同源结合分子(例如,受体,配体,抗体,或其他结合伴侣)能够特异性结合,其中结合特性可用于检测、鉴定、分离或纯化、追踪、富集或靶向标记的蛋白或表达标记蛋白的细胞,特别是当标记蛋白是一部分时异质蛋白质或其他物质群体的表达,或者表达标签蛋白的细胞是异质细胞群体(例如,诸如外周血的生物学样品)的一部分。在某些实施方案中,表达标签蛋白的细胞可以与异源或非内源同源结合分子接触并诱导生物学反应,例如促进细胞活化、细胞增殖或细胞死亡。在提供的融合结合蛋白中,标签盒被同源结合分子特异性结合的能力不同于或除了结合域特异性结合靶标分子的能力之外。标签盒通常不是抗原结合分子,例如,不是抗体或TCR或其抗原结合部分。本文提供了示例性标签盒。在一些实施方案中,标签盒包含在本公开的融合蛋白的细胞外组分中,并且可以位于例如结合结构域和疏水部分之间,或位于N末端或C末端。结合结构域多肽(例如,VH、VL、TCRα、TCRβ等)的序列,或可以位于融合蛋白的结合结构域内(例如,在VH和VL之间)(或在TCRα和TCRβ之间),条件是标签不会干扰或基本不会干扰与抗原的结合。
如本文所用,“铰链区”或“铰链”是指(a)免疫球蛋白铰链序列(例如由免疫球蛋白铰链的上部和核心区域组成)或其功能片段或变体、(b)II型C-凝集素域间(茎)区域或其功能片段或变体,或(c)分化(CD)分子茎区域的簇或其功能变体。如本文所用,“野生型免疫球蛋白铰链区”是指介于CH1和CH2结构域之间并与其连接(对于IgG、IgA和IgD)或介于CH1和CH1之间并与其连接的天然存在的上部和中间铰链氨基酸序列。抗体重链中存在的CH3和CH3域(对于IgE和IgM)。在某些实施方案中,铰链区是人的,并且在特定实施方案中,包含人IgG铰链区。IgG铰链区包括IgG1铰链区、IgG2铰链区、IgG3铰链区或IgG4铰链区中的任何一个或多个。
如本文所用,“疏水部分”是指具有三维结构的氨基酸序列,该三维结构在细胞膜中是热力学稳定的,并且长度范围通常为约15个氨基酸至约30个氨基酸。疏水域的结构可以包含α螺旋、β桶、β片、β螺旋或其任何组合。在某些实施方案中,疏水部分是跨膜结构域,例如,衍生自CD8、CD28或CD27分子的跨膜结构域。
如本文所用,“基于免疫受体酪氨酸的活化基序(ITAM)T细胞活化域”是指天然或内源地存在于免疫细胞受体或细胞表面标志物上并包含至少一个基于免疫受体酪氨酸的激活基序(ITAM)的细胞内信号传导域或其功能部分。ITAM是指YXXL/I-X6-8-YXXL/I的保守基序(SEQ ID NO:42),其中X是任何氨基酸(即,在ITAM的长度上相同或不同的氨基酸)。在某些实施例中,ITAM信令域包含一个、两个、三个、四个或更多个ITAM。在抗原结合或配体结合后,ITAM信号域可以启动T细胞激活信号。ITAM信号传导域包括例如CD3γ、CD3δ、CD3ε、CD3ζ、CD79a、CD79b、FcεRI或FcγRI的γ链、FcRγ2a、FcRγ2b1、FcRγ2a1、FcRγ2b2、FcRγ3a、FcRγ3b、FcRγβ1、FcεR、杀伤细胞受体蛋白(例如DAP12)、CD5、CD16a、CD16b、CD22、CD23、CD32、CD64、CD79a、CD79b、CD89、CD278和CD66d。Paul,Fundamental Immunology 307(Wolters Kluwer;Lippincott;Wilkins&Wilkins;Seventh Ed.,2008)中描述了这些ITAM序列的示例性氨基酸序列以及来自病毒(例如BLV gp30;EBV LMP2A)的氨基酸序列。还考虑将这些ITAM及其功能片段和变体用于当前公开的融合蛋白和宿主细胞中,并通过引用并入本文。
如本文所用,“共刺激信号结构域”是指共刺激分子的细胞内信号结构域或其功能部分,其在与主要或经典(例如,ITAM驱动的)激活信号(由(例如CD3ζ细胞内信号传导域),促进或增强T细胞应答,例如T细胞活化、细胞因子产生、增殖、分化、存活、效应子功能或其组合。共刺激信号传导域包括例如CD28、CD40L、GITR、NKG2C、CARD1、CD2、CD7、CD27、CD30、CD40、CD54(ICAM)、CD83、CD134(OX-40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD223(LAG3)、CD226、CD270(HVEM)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、DAP10、LAT、LFA-1、LIGHT、SLP76、TRIM、ZAP70、CD5、BAFF-R、SLAMF7、NKp80、CD160、B7-H3、与CD83特异性结合的配体或其任何组合。
如本文所用,“CD28共刺激信号传导域”是指CD28的细胞内信号传导域或其功能部分。CD28是共刺激分子,其在所有人CD4+T细胞和约50%的人CD8+T细胞上组成性表达(Linsley等,Annu.Rev.Immunol.11:191-212(1993);June等.Immunol今日11:211-16(1990))。CD28是一种“早期”共刺激分子,已显示与TCR协同作用以降低T细胞活化的阈值,在某些情况下,仅通过TCR连接是无法达到的,从而导致存活率提高和克隆扩增和分化所需要的(例如IL-2)细胞因子产生增加(Bour-Jordan等人,Immunol.Rev.241:180-205(2011))。示例性的“野生型”或“内源”人CD28共刺激信号传导结构域包含SEQ ID NO:2的氨基酸序列。如本文所述,对人CD28共刺激信号结构域的修饰(例如氨基酸取代)可指SEQ IDNO:1所示的全长野生型人CD28多肽序列内的位置。
抗体技术领域的技术人员所理解的术语均具有本领域所获得的含义,除非本文另有明确定义。术语“抗体”是指包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链的完整抗体(尽管应当理解,缺少轻链的重链抗体仍然是术语“抗体”所涵盖的术语,以及完整抗体的具有或保留结合靶分子能力的抗原结合部分。抗体包括多克隆和单克隆抗体。抗体可以是天然存在的、重组产生的、基因工程的或修饰的,并且包括免疫球蛋白的修饰形式,例如体内抗体、肽抗体、纳米抗体、单结构域抗体和多特异性抗体(例如双特异性抗体、双抗体、三抗体)、四体、串联di-scFV、串联tri-scFv)。
来自抗体的“结合片段”、“结合部分”或“结合结构域”是指包含完整抗体的一部分并且包含抗体的抗原决定性可变区或互补决定区的“抗体片段”。抗体片段的实例包括Fab、Fab'、F(ab')2和Fv片段、Fab'-SH、F(ab')2、双抗体、线性抗体、单链抗体、scFv(即,免疫球蛋白(Ig)分子的可变重链(VH)和可变轻链(VL)区,通常与约10至约25个氨基酸的短接头肽连接)、VHH、单域抗体(例如sdAb、sdFv、纳米抗体),以及包含抗体片段的多特异性抗体。单克隆抗体或其抗原结合部分可以是非人的、嵌合的、人源化的或人的,优选地是人源化的或人的。免疫球蛋白的结构和功能在例如Harlow等编辑的《抗体:实验室手册》第14章(冷泉港实验室,冷泉港,1988)中有综述。抗体可以是任何类别或亚类,包括IgG及其亚类(IgG1、IgG2、IgG3、IgG4)、IgM、IgE、IgA和IgD。
术语“可变轻链”(VL)和“可变重链”(VH)分别是指抗体轻链和重链的可变结合区。可变结合区由离散的,定义明确的子区域组成,这些子区域称为“互补决定区”(CDR,也称为HVR(高变区))和“框架区”(FR)。CDR指的是抗体可变区内的赋予抗原特异性和/或结合亲和力的氨基酸,被FR隔开。每个抗体轻链可变区(LCDR1、LCDR2、LCDR3)中有三个CDR,每个抗体重链可变区(HCDR1、HCR2、HCDR3)中有三个CDR。
术语“CL”是指“免疫球蛋白轻链恒定区”或“轻链恒定区”,即来自抗体轻链的恒定区。
术语“CH”是指“免疫球蛋白重链恒定区”或“重链恒定区”,其根据CH1、CH2和CH3的抗体同种型(IgA、IgD、IgG)进一步可分为CH1、CH2、CH3和CH4域(IgE、IgM)。
“Fab”(片段抗原结合)是抗体与抗原结合的部分,并且包括通过链间二硫键与轻链连接的重链的可变区和CH1。
如本文所用,“Fc区部分”是指来自抗体的Fc片段的重链恒定区区段(“可结晶片段化的区域”或Fc区),其可包含一个或多个恒定域,例如CH2、CH3、CH4或其任何组合。在某些实施方案中,Fc区部分包括IgG、IgA或IgD抗体的CH2和CH3结构域或其任何组合,或IgM或IgE抗体的CH3和CH4结构域或其任何组合。在其他实施方案中,CH2CH3或CH3CH4结构具有来自相同抗体同种型的亚区域结构域并且是人的,例如人IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgD、IgE或IgM(例如,来自人IgG1或IgG4)。
作为背景,Fc区负责免疫球蛋白的效应子功能,例如ADCC(抗体依赖性细胞介导的细胞毒性)、CDC(补体依赖性细胞毒性)和补体固定,与Fc受体(例如CD16)结合(例如,CD32,FcRn),相对于缺乏Fc区,蛋白A结合甚至胎盘转移的多肽,体内半衰期更长(参见Capon等人,Nature 337:525(1989))。在某些实施方案中,在本公开的融合结合蛋白中发现的Fc区部分将能够介导一种或多种这些效应子功能,或将通过例如一种或多种途径缺乏一种或多种或所有这些活性。例如,用于修饰(例如,改善、减少或消除)Fc功能的氨基酸修饰(例如,取代)包括T250Q/M428L;M252Y/S254T/T256E;H433K/N434F;M428L/N434S;E233P/L234V/L235A/G236+A327G/A330S/P331S;E333A;S239D/A330L/I332E;P257I/Q311;K326W/E333S;S239D/I332E/G236A;N297Q;K322A;S228P;L235E+E318A/K320A/K322A;L234A/L235A;和L234A/L235A/P329G突变,这些突变被总结并在由InvivoGen(2011)出版的“工程化Fc区”中进行了注释,并可以从以下网站在线获得:
www.invivogen.com/PDF/review/review-Engineered-Fc-Regions-invivogen.pdf?utm_source=review&utm_medium=pdf&utm_campaign=review&utm_content=Engineered-Fc-Regions,在此引用作为参考。
如本文所用,“免疫系统细胞”是指源自骨髓中的造血干细胞的免疫系统的任何细胞,其产生两个主要谱系,即髓系祖细胞(其产生诸如单核细胞、巨噬细胞、树突状细胞、巨核细胞和粒细胞)和淋巴样祖细胞(会产生淋巴样细胞,例如T细胞、B细胞和自然杀伤(NK)细胞)。示例性免疫系统细胞包括CD4+T细胞、CD8+T细胞、CD4-CD8-双阴性T细胞、γδT细胞、调节性T细胞、干细胞记忆T细胞、天然杀伤细胞和树突状细胞。巨噬细胞和树突状细胞可称为“抗原呈递细胞”或“APC”,它们是当与肽复合的APC表面上的主要组织相容性复合物(MHC)受体与T细胞的表面的TCR相互作用时可以激活T细胞的专门细胞。
“T细胞”(或“T淋巴细胞”)是在胸腺中成熟并产生T细胞受体(TCR)的免疫系统细胞,可从(例如)外周血单核细胞(PBMC),在本文中称为“大量”T细胞。分离T细胞后,可以在扩增前或扩增后将细胞毒性(CD8+)和辅助(CD4+)T细胞分为幼稚,记忆和效应T细胞亚群。T细胞可以是幼稚的(不暴露于抗原;与TCM相比,CD62L,CCR7,CD28,CD3,CD127和CD45RA的表达增加,而CD45RO的表达减少),记忆性T细胞(TM)(经历过抗原且存活时间长的细胞)和效应细胞(经历过抗原的细胞毒性)。TM可以进一步分为以下子集:中央记忆T细胞(TCM,与幼稚T细胞相比,CD62L、CCR7、CD28、CD127、CD45RO和CD95的表达增加,以及CD54RA的表达减少),干细胞记忆T细胞,和效应记忆T细胞(TEM,与幼稚T细胞或TCM相比,CD62L、CCR7、CD28、CD45RA的表达降低,以及CD127的表达增加)。效应T细胞(TE)是指经历过抗原的CD8+细胞毒性T淋巴细胞,其与TCM相比,CD62L、CCR7、CD28的表达降低,并且对颗粒酶和穿孔素呈阳性。辅助性T细胞(TH)是CD4+细胞,通过释放细胞因子来影响其他免疫细胞的活性。CD4+T细胞可以激活和抑制适应性免疫反应,诱导的作用将取决于其他细胞和信号的存在。可以根据已知技术收集T细胞,并且可以通过已知技术,例如通过与抗体的亲和结合,流式细胞术或免疫磁性选择来富集或消除各种亚群或其组合。
“T细胞受体”(TCR)是指在T细胞(或T淋巴细胞)表面上发现的与CD3结合的分子,通常负责识别与主要组织相容性复合体(MHC)分子结合的抗原。在大多数T细胞中,TCR具有高度可变的α和β链的二硫键连接的异二聚体(分别称为TCRα和TCRβ)。在T细胞的一个子集中,TCR由可变的γ和δ链的异二聚体组成(分别称为TCRγ和TCRδ)。TCR的每条链都是免疫球蛋白超家族的成员,并具有一个N末端免疫球蛋白可变结构域、一个免疫球蛋白恒定结构域、一个跨膜区域和一个在C末端末端短的胞质尾巴(参见Janeway等人,Immunobiology):《免疫系统在健康和疾病中的作用》,第三版,Current Biology Publications,p.4:33,1997)。如本公开中所使用的TCR可以来自各种动物物种,包括人类、小鼠、大鼠、猫、狗、山羊、马或其他哺乳动物。TCR可以是细胞结合的(即具有跨膜区或结构域)或可溶形式。
TCRα链(Vα)和β-链(Vβ)或γδTCR的Vγ和Vδ的术语“可变区”或“可变域”是指TCR中涉及以下部分TCR与抗原的结合(例如,在肽抗原:MHC复合物中)。天然TCR的Vα和Vβ通常具有相似的结构,每个可变结构域包含四个保守的FR和三个CDR。Vα结构域由两个独立的DNA片段编码,可变基因片段和连接基因片段(V-J);Vβ结构域由三个独立的DNA片段编码,分别是可变基因片段、多样性基因片段和连接基因片段(V-D-J)。单个Vα或Vβ结构域可能足以赋予抗原结合特异性。此外,可以使用Vα或Vβ结构域从结合抗原的TCR中分离结合特定抗原的TCR,以分别筛选互补的Vα或Vβ□结构域的文库。
“主要组织相容性复合物分子”(MHC分子)是指将肽抗原递送至细胞表面的糖蛋白。MHC I类分子是由跨膜的α链(具有3个α域)和非共价结合的β2微球蛋白组成的异二聚体。MHC II类分子由两个跨膜糖蛋白α和β组成,两者都跨膜。每个链都有两个域。MHC I类分子将起源于胞质溶胶的肽传递到细胞表面,其中CD8+T细胞可识别肽:MHC复合物。MHC II类分子将起源于囊泡系统的肽传递到细胞表面,在此处被CD4+T细胞识别。MHC分子可以来自各种动物物种,包括人、小鼠、大鼠或其他哺乳动物。
如本文所用,“抗原”或“Ag”是指引起免疫应答的免疫原性分子。这种免疫应答可能涉及抗体的产生,特定免疫学上具有活性的细胞(例如,T细胞)的激活或两者。抗原(免疫原性分子)可以是例如肽,糖肽、多肽、糖多肽、多核苷酸、多糖、脂质等。显而易见,抗原可以合成、重组产生或衍生自生物样品。可以包含一种或多种抗原的示例性生物样品包括组织样品、肿瘤样品、细胞、生物液或其组合。抗原可以由经过修饰或基因工程表达抗原的细胞产生,或者内源性表达(例如,未经人为干预的修饰或基因工程)表达具有免疫原性的突变或多态性。在某些实施方案中,其中融合蛋白包含来自T细胞受体(TCR)的抗原结合区(例如,TCRVα和Vβ),抗原包含肽:MHC复合物,并且结合结构域至少接触该肽。
术语“表位”或“抗原表位”包括被关联结合分子识别并特异性结合的任何分子、结构、氨基酸序列或蛋白质决定簇,例如免疫球蛋白、T细胞受体(TCR)、嵌合抗原受体、或其他结合分子、结构域或融合蛋白。抗原决定簇通常包含分子的化学活性表面基团,例如氨基酸或糖侧链,并且可以具有特定的三维结构特征以及特定的电荷特征。
如本文所用,“核酸”或“核酸分子”是指例如通过聚合酶链反应(PCR)或DNA产生的脱氧核糖核酸(DNA)、核糖核酸(RNA)、寡核苷酸、多核苷酸,其片段中的任何一种。通过体外翻译,以及通过连接、切割、核酸内切酶作用或核酸外切酶作用产生的片段。在某些实施方案中,本公开的核酸通过PCR产生。核酸可以由天然存在的核苷酸的单体(例如脱氧核糖核苷酸和核糖核苷酸),天然存在的核苷酸的类似物(例如天然存在的核苷酸的α-对映体形式)或两者的组合组成。修饰的核苷酸可以对糖部分、嘧啶或嘌呤碱基部分进行修饰或替代。核酸单体可以通过磷酸二酯键或这种连接的类似物连接。磷酸二酯键的类似物包括硫代磷酸酯、二硫代磷酸酯、亚硒代磷酸酯、二硒代磷酸酯、苯硫代磷酸酯、苯胺基磷酸酯、氨基磷酸酯等。核酸分子可以是单链或双链的。
术语“分离的”是指将材料从其原始环境(例如,如果是天然存在的自然环境)中除去。例如,没有分离出存在于活体动物中的天然存在的核酸或多肽,而是分离了与天然系统中的一些或全部共存物质分离的相同核酸或多肽。这样的核酸可以是载体的一部分和/或这样的核酸或多肽可以是组合物(例如,细胞裂解物)的一部分,并且仍然被分离,因为这样的载体或组合物不是核酸或多肽的天然环境的一部分。术语“基因”是指与产生多肽链有关的DNA片段。它包括编码区之前和之后的区域(“前导区和尾区”)以及各个编码段(外显子)之间的插入序列(内含子)。
术语“构建体”是指含有重组核酸分子的任何多核苷酸。构建体可以存在于载体(例如细菌载体、病毒载体)中或可以整合到基因组中。
“载体”是能够转运另一种核酸的核酸分子。载体可以是例如质粒、粘粒、病毒、噬菌体、RNA载体或线性或环状DNA或RNA分子,其可以包括染色体、非染色体、半合成或合成核酸分子。示例性载体是那些能够自主复制的载体(附加载体)或表达与其连接的核酸分子的载体(表达载体)。
“逆转录病毒”是具有RNA基因组的病毒。“γ逆转录病毒”是指逆转录病毒科的属。γ逆转录病毒的例子包括小鼠干细胞病毒、鼠白血病病毒、猫白血病病毒、猫肉瘤病毒和禽网状内皮病病毒。
“慢病毒”是指能够感染分裂细胞和非分裂细胞的逆转录病毒属。慢病毒的几个例子包括HIV(人类免疫缺陷病毒:包括1型和2型HIV);马传染性贫血病毒;猫免疫缺陷病毒(FIV);牛免疫缺陷病毒(BIV);和猿猴免疫缺陷病毒(SIV)。
如本文所用,“慢病毒载体”是指用于基因递送的基于HIV的慢病毒载体,其可以是整合的或非整合的,具有相对大的包装能力,并且可以转导多种不同的细胞类型。慢病毒载体通常是在三个(包装、包膜和转移)或更多质粒瞬时转染到生产细胞后产生的。像HIV一样,慢病毒载体通过病毒表面糖蛋白与细胞表面受体的相互作用进入靶细胞。进入时,病毒RNA进行逆转录,该逆转录由病毒逆转录酶复合体介导。逆转录产物是双链线性病毒DNA,它是病毒整合到感染细胞DNA中的底物。
术语“可操作地连接”是指单个核酸片段上两个或更多个核酸分子的缔合,从而一个的功能受到另一个的影响。例如,当启动子能够影响该编码序列的表达时(即,该编码序列在该启动子的转录控制下),该启动子与该编码序列可操作地连接。“未连锁”是指相关的遗传要素彼此之间不紧密关联,并且其中一个的功能不影响另一个。
如本文所用,术语“表达”是指基于核酸分子例如基因的编码序列产生多肽的过程。该过程可以包括转录、转录后控制、转录后修饰、翻译、翻译后控制、翻译后修饰或其任何组合。
如本文所用,“表达载体”是指包含核酸分子的DNA构建体,该核酸分子可操作地连接至能够影响核酸分子在合适宿主中表达的合适控制序列。此类控制序列包括实现转录的启动子,控制此类转录的任选操纵子序列,编码合适的mRNA核糖体结合位点的序列以及控制转录和翻译终止的序列。载体可以是质粒,噬菌体颗粒,病毒或仅是潜在的基因组插入物。一旦转化为合适的宿主,载体就可以独立于宿主基因组复制和发挥作用,或者在某些情况下可以整合到基因组本身中。在本说明书中,“质粒”、“表达质粒”、“病毒”和“载体”经常互换使用。
在将核酸分子插入细胞中的上下文中的术语“引入”是指“转染”或“转化”或“转导”,并且包括提及将核酸分子掺入到真核或原核细胞中,其中可以将核酸分子掺入细胞的基因组(例如,染色体、质粒、质体或线粒体DNA)中,转化为自主复制子,或瞬时表达(例如,转染的mRNA)。
如本文所用,“异源”核酸分子,构建体或序列是指不是宿主细胞天然的但可以与核酸分子或核酸的一部分同源的核酸分子或核酸分子的一部分。来自宿主细胞的酸性分子。异源核酸分子,构建体或序列的来源可以来自不同的属或种。在某些实施方案中,通过例如缀合、转化、转染、转导、电穿孔等将异源核酸分子添加至宿主细胞或宿主基因组(即,不是内源的或天然的),其中添加的分子可以整合入宿主基因组或作为染色体外遗传物质存在(例如,作为质粒或其他形式的自我复制载体),并可以多拷贝形式存在。另外,“异源”是指由导入宿主细胞的非内源性核酸分子编码的非天然酶、蛋白质或其他活性,即使宿主细胞编码同源蛋白质或活性也是如此。
术语“同源的”或“同源的”是指在宿主细胞、物种或菌株中发现或衍生自宿主细胞、物种或菌株的分子或活性。例如,异源分子或编码该分子的基因可以分别与天然宿主或宿主细胞分子或编码该分子的基因同源,并且可以任选地具有改变的结构、序列、表达水平或其组合。
如本文所用,术语“内源的”或“天然的”是指通常存在于宿主或宿主细胞中的基因、蛋白质、化合物、分子或活性。此外,与亲本基因、蛋白质或活性相比,被突变、过度表达、改组、复制或以其他方式改变的基因、蛋白质或活性仍被认为是该特定宿主细胞的内源性或天然性。例如,来自第一基因的内源控制序列(例如,启动子、翻译减毒序列)可用于改变或调节第二天然基因或核酸分子的表达,其中第二天然基因或核酸分子的表达或调控。核酸分子不同于亲代细胞中的正常表达或调控。
如本文所用,术语“工程的”、“重组的”、“修饰的”或“非天然的”是指已经通过引入异源核酸分子而修饰的生物、微生物、细胞、核酸分子或载体,或指经过人为干预进行基因工程改造的细胞或微生物-即通过引入异源核酸分子进行修饰的细胞或微生物,或指经过改变使得内源核酸分子或基因是受控的、失控的或组成型的,其中这种改变或修饰可以通过基因工程引入。人类产生的遗传改变可以包括,例如,引入引入的核酸分子的修饰(其可以包括表达控制元件,例如启动子),该核酸分子编码一种或多种蛋白质、融合结合蛋白或酶、或其他核酸分子添加物,细胞遗传物质的缺失,替代或其他功能破坏或增加。示例性修饰包括来自参考或亲本分子的异源或同源多肽的编码区或其功能片段中的修饰。其他示例性修饰包括例如非编码调控区中的修饰,其中所述修饰改变基因或操纵子的表达。
如本文所用,“突变”是指分别与参考或野生型核酸分子或多肽分子相比,核酸分子或多肽分子的序列变化。突变可导致几种不同类型的序列变化,包括核苷酸或氨基酸的取代、插入或缺失。
如本文所用,“序列同一性”是指一个序列中的氨基酸残基(或核苷酸)与另一参考多肽(或核苷酸)序列中的氨基酸残基(或核苷酸)相同后,将序列和必要时引入缺口以实现最大的序列同一性百分比,并且不考虑将任何保守取代(对于氨基酸序列)作为序列同一性的一部分。可以使用由Altschul等人,Nucl.Acids Res.25:3389-3402(1997)定义的NCBIBLAST 2.0软件产生序列同一性百分比值,并将参数设置为默认值。
“过继细胞免疫疗法”或“过继免疫疗法”是指施用天然存在的或基因工程的疾病抗原特异性免疫细胞(例如,T细胞)。过继细胞免疫疗法可以是自体的(免疫细胞来自受体)、同种异体的(免疫细胞来自相同物种的供体)或同基因的(免疫细胞来自与受体基因上相同的供体)。
“治疗(treat)”或“治疗(treatment)”或“改善”是指对受试者(例如人类或非人类哺乳动物,例如灵长类、马、狗、小鼠、大鼠)的疾病、病症或病状的医学管理。一般而言,合适的剂量或治疗方案包括表达融合结合蛋白的宿主细胞,该融合结合蛋白包含细胞外组分和细胞内组分,所述细胞外组分包含特异性结合靶抗原,所述细胞内组分包含本发明的修饰的CD28共刺激信号结构域,并以足以引起治疗或预防益处的量施用位于细胞外组分和细胞内组分之间的疏水部分。治疗或预防/预防益处包括改善临床预后;减轻或减轻与疾病有关的症状;减少症状的发生;改善生活质量;更长的无病状态;疾病程度的减少,疾病状态的稳定;疾病进展延迟;缓解;生存延长生存期或其任何组合。
表达本发明公开内容的融合结合蛋白或细胞的“治疗有效量”或“有效量”是指足以改善被治疗疾病的一种或多种症状的化合物或细胞的量。具有统计意义的方式。当涉及单独施用的单个活性成分或表达单一活性成分的细胞时,治疗有效剂量是指该成分或单独表达该成分的细胞的作用。当提及组合时,治疗有效剂量是指活性成分或辅助活性成分与表达导致治疗效果的细胞的组合的组合量,无论是连续施用还是同时施用。另一组合可以是表达一种以上活性成分的细胞,例如两种不同的融合蛋白或其他相关的治疗剂。
如本文所用,术语“强直的”是指在免疫细胞中发生的“基础”水平或“非抗原依赖性”信号,其包括蛋白质磷酸化、激活、细胞因子表达、增殖或其组合。通过其同源TCR或融合蛋白(例如,CAR)在没有靶抗原特异性活化的情况下(例如,T细胞)。
融合蛋白
用作本文公开的过继免疫疗法组合物的融合蛋白包含修饰的功能性CD28共刺激信号传导域。修饰的功能性CD28共刺激信号传导结构域包含至少一个氨基酸取代。包含此类修饰的功能性CD28共刺激信号域的融合蛋白具有不同于包含野生型CD28共刺激域的融合蛋白的功能活性。例如,本文提供的对CD28共刺激性信号传导域的修饰可以允许定制功能活性,包括融合蛋白的活性、信号传导动力学或信号传导强度,从而提高临床功效、降低毒性(例如,当表达融合蛋白的宿主细胞时管理对象),或两者兼有。
在某些方面,本公开内容提供了一种融合蛋白,其包含细胞外组分和细胞内组分,所述细胞外组分包含特异性结合靶抗原的结合域;所述细胞内组分包含修饰的功能性CD28共刺激信号传导域,其中所述修饰的功能性CD28共刺激信号传导域至少包含一个氨基酸取代;以及位于细胞外组分和细胞内组分之间的疏水部分,其中融合蛋白的功能活性不同于包含野生型CD28共刺激信号结构域的融合蛋白。在某些实施方案中,融合蛋白由宿主细胞表达,并且功能活性包括信号传导动力学(例如,信号传导的时间、顺序、序列或速率)、信号传导强度、细胞因子产生、细胞增殖、细胞持久性、抗-抗原(例如,抗肿瘤细胞)活性、强直信号、免疫抑制成分基因的表达或其任何组合。
适用于本发明的融合结合蛋白的结合结构域可以是任何抗原结合多肽。结合结构域可以包含天然抗体、合成或重组抗体构建体或其抗原结合片段。例如,结合结构域可包含全长重链、Fab片段、Fab'、F(ab')2,可变重链结构域(VH结构域)、可变轻链结构域(VL结构域)、结构域抗体(dAb)抗体、单结构域骆驼科动物抗体(VHH)、互补决定区(CDR)或单链抗体片段(scFv),在某些实施方案中可以是多特异性的。结合结构域的其他实例包括单链T细胞受体(scTCR)、可溶性TCR,可变α链结构域(Vα)、可变β链结构域(Vβ)、受体的细胞外结合结构域、细胞表面受体/分子的配体、肿瘤结合蛋白质/肽和细胞因子。在某些实施方案中,本公开内容的融合结合蛋白的结合结构域不包含CD8的细胞外结合结构域或部分或其包含功能性IgV样结构域(即,能够结合同源配体,例如肽:MHC复合物)的任何部分。在特定实施方案中,本公开的融合结合蛋白的结合结构域不包含来自CD8α链的结合结构域、来自CD8β链的结合结构域、来自CD8α同型二聚体的结合结构域或来自CD8αβ的结合结构域。在进一步的实施方案中,本发明的融合结合蛋白的结合结构域不包含SEQ ID NO:43所示的CD8αIgV样结构域或SEQ ID NO:44所示的CD8βIgV样结构域。
在某些实施方案中,结合结构域是来自软骨鱼、嵌合的、人的或人源化的鼠科,羽扇豆、骆驼科动物。
在某些实施方案中,结合结构域包含衍生自抗CD19抗体FMC63或抗ROR1抗体R12的scFv。在一些实施方案中,结合结构域包含如SEQ ID NO:8所示的FMC63 scFv氨基酸序列或如SEQ ID NO:9所示的R12 scFv氨基酸序列。
对ROR1具有特异性的另外的示例性结合结构域包括来自例如在Yang等人,PLoSOne 6:e21018 doi:10.1371,2011。Paredes-Moscosso等,Blood 128:2052,2016;PCT公开号WO 2014/031174,WO 2016/094873和WO2017072361A1;以及美国专利/授权前公告号US2013/0251642,US 9,316,646,US 9,217,040,US 9,242,014,US 8,212,009,US 9,226,952,US 9,228,023和US 9,150,647中公开的那些抗体。这些抗体及其结合结构域,包括其氨基酸序列,通过引用并入本文。
在某些实施方案中,与ROR1抗原结合的结合结构域衍生自R12抗体、R11抗体、2A2抗体、R12抗体、UC-961抗体、D10抗体、Y31抗体或H10抗体。
融合蛋白的细胞外组分任选地包含细胞外的非信号间隔区或接头区,其例如可以使结合结构域远离宿主细胞(例如,T细胞)表面定位以实现适当的细胞/细胞接触,抗原结合(Patel等,Gene Therapy 6:412-419(1999))。融合结合蛋白的细胞外间隔区通常位于疏水部分或跨膜结构域与细胞外结合结构域之间。间隔区的长度可以根据所选的靶分子,所选的结合表位或抗原结合结构域的大小和亲和力而变化,以使抗原识别(例如,肿瘤识别)最大化(参见,例如Guest等,J.Immunother.28:203-11(2005);PCT公开号WO 2014/031687)。在某些实施方案中,间隔区包括免疫球蛋白铰链区。免疫球蛋白铰链区可以是野生型免疫球蛋白铰链区或改变的野生型免疫球蛋白铰链区。在某些实施方案中,免疫球蛋白铰链区是人免疫球蛋白铰链区。免疫球蛋白铰链区可以是IgG、IgA、IgD、IgE或IgM铰链区。IgG铰链区可以是IgG1、IgG2、IgG3或IgG4铰链区。在PCT公开号WO 2014/031687中描述了示例性的改变的IgG4铰链区,该铰链区包括其氨基酸序列,通过引用整体并入本文。在某些实施方案中,改变的IgG4铰链区包含如SEQ ID NO:12所示的氨基酸序列。本文所述的融合结合蛋白中使用的铰链区的其他实例包括存在于1型膜蛋白例如CD8α、CD4、CD28和CD7的细胞外区域中的铰链区,其可以是野生型或其变体。
在某些实施方案中,细胞外间隔区包含选自以下的Fc结构域的全部或一部分:CH1结构域、CH2结构域、CH3结构域、CH4结构域或其任何组合(参见,例如,PCT公开WO 2014/031687,其间隔区通过引用整体并入本文)。Fc结构域或其部分可以是野生型的(例如,以降低抗体效应子功能)。在某些实施方案中,细胞外组分包含位于结合结构域和疏水部分之间的免疫球蛋白铰链区、CH2结构域、CH3结构域或其任何组合。在某些实施方案中,细胞外组分包含IgG1铰链区、IgG1CH2结构域和IgG1CH3结构域。在进一步的实施方案中,IgG1CH2结构域包含(i)N297Q突变,(ii)用APPVA取代前六个氨基酸(APEFLG),或(i)和(ii)两者。在某些实施方案中,免疫球蛋白铰链区、Fc结构域或其部分或两者均为人。
在某些实施方案中,细胞外间隔区还包含标签。标签可用于确定在过继细胞疗法中使用的表达标记的融合蛋白的细胞是否已成功转移至有需要的受试者,或表达标记的融合蛋白的细胞是否在接受该治疗的受试者中增殖,持久或定位于目标位点过继细胞疗法。可以使用与标签肽特异性结合的抗体或其结合片段来检测表达标记的融合蛋白的细胞。标签还可用于从受试者或其样品(例如从全血,PBMC或肿瘤组织或部位)富集或分离表达标记的融合蛋白的细胞或细胞群。标签还可用于激活或扩展表达标签化融合蛋白的细胞或细胞群。标签可以是酶、染料、荧光标签或肽标签。示例性标签肽包括Strep-Tag(WRHPQFGG,SEQID NO:39)、Strep-Tag II(WSHPQFEK,SEQ ID NO:40)和Strep-Tag II 9-mer(NWSHPQFEK,SEQ ID NO:10),以高亲和力结合细菌蛋白链霉亲和素及其衍生物Strep-Tactin。参见,例如,美国专利号7,981,632(Strep标签,其通过引用并入本文)。包含一种或多种标签肽的标签融合蛋白(例如,嵌合抗原受体)以及检测、分离、富集、活化或扩增的方法是PCT公开号WO2015/095895中描述的标记,标记的融合蛋白及其方法在此引入作为参考。标签的其他示例包括包含发色报告酶的酶,例如辣根过氧化物酶或碱性磷酸酶、花青染料、香豆素、若丹明、黄嘌呤、荧光素或其磺化衍生物、PE、太平洋蓝、Alexa fluor、APC、FITC、荧光蛋白、Myc标签、His标签、Flag标签、Xpress标签、Avi标签、钙调蛋白标签、聚谷氨酸标签、HA标签、Nus标签、S标签、X标签、SBP标签、Softag、V5标签、CBP、GST、MBP、GFP、硫氧还蛋白标签,或其任何组合。
疏水部分或跨膜结构域位于融合蛋白的细胞外组分和细胞内组分之间。跨膜结构域是横穿并将融合蛋白锚定在宿主细胞膜(例如,T细胞)中的疏水性α螺旋。在某些实施方案中,跨膜结构域选自细胞内组分来源的相同分子,例如CD28,含ITAM的T细胞活化结构域(例如,CD3ζ、FcRγ)(如果存在),或选自另一种I型跨膜蛋白,例如,CD4、CD8、CD27。在某些实施方案中,跨膜结构域选自衍生细胞内组分的不同分子。在某些实施方案中,跨膜结构域包含CD28、CD2、CD3ε、CD3δ、CD3ζ、CD25、CD27、CD40、CD79A、CD79B、CD80、CD86、CD95(Fas)、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD200R、CD223(LAG3)、CD270(HVEM)、CD272(BTLA)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、CD279(PD-1)、CD300、CD357(GITR)、A2aR、DAP10、FcRα、FcRβ、FcRγ、Fyn、GAL9、KIR、Lck、LAT、LRP、NKG2D、NOTCH1、NOTCH2、NOTCH3、NOTCH4、PTCH2、ROR2、Ryk、Slp76、SIRPα、pTα、TCRα、TCRβ、TIM3、TRIM、LPA5或Zap70。示例性的CD28跨膜结构域包含氨基酸序列SEQ ID NO:13或SEQ ID NO:41。
作为背景,T细胞的强大活化通常涉及两个不同的信号传递事件:(1)通过T细胞受体(TCR)复合物识别抗原而提供的抗原特异性信号,该信号促进T细胞活化;和(2)由抗原呈递细胞和T细胞上表达的共刺激分子之间的相互作用或连接所提供的非抗原特异性“共刺激信号”。在没有共同刺激的情况下激活T细胞可能会导致无反应、凋亡或免疫耐受。共刺激信号结合抗原刺激T细胞,并促进T细胞增殖、分化和持久性。
细胞内组分是指融合结合蛋白的一部分,其响应于融合蛋白与靶抗原的结合而将信号转导到宿主细胞(例如,T细胞)内部,从而引发效应子功能,例如激活、细胞因子的产生、增殖、分化、持久性、细胞毒性活性、归巢、进入肿瘤的微环境或其任何组合。
本公开的融合蛋白的细胞内组分包含修饰的CD28共刺激信号传导域。CD28是幼稚T细胞的主要共刺激受体,并参与引发T细胞反应。CD28与主要在抗原呈递细胞(例如树突细胞、巨噬细胞、B细胞)上表达的CD80和CD86结合。CD28及其配体的结合,与T细胞受体信号传导结合,可促进抗原刺激的T细胞扩增并分化为效应细胞和记忆细胞。CD28信号传导增强细胞因子的产生(例如,IL-2)、上调细胞存活基因(例如,Bcl-xL)、促进能量代谢,并促进细胞周期进程。CD28共刺激信号传导域可以指CD28的全长细胞内域或细胞内信号传导域的截短部分,条件是该截短部分保留信号转导活性(例如,至少约25%、约30%、约35%、约40%、约45%、约50%、约55%、约60%、约65%、约70%、约75%、约80%、约85%、约90%、约95%、或与野生型CD28基本相似的活性)。一种示例性的野生型全长人CD28共刺激信号传导域包含氨基酸序列SEQ ID NO:2。修饰的功能性CD28共刺激信号结构域包含至少一个氨基酸取代,其中包含修饰的功能性CD28共刺激信号结构域的融合蛋白将具有不同于包含野生型CD28共刺激性信号结构域的融合蛋白的功能活性。在某些实施方案中,修饰的CD28共刺激信号传导域包含至少1、至少2、至少约3、至少约4、至少约5、至少约6、至少约7、至少约8、至少约9个、至少约10个、至少约11个、至少约12个、至少约13个、至少约14个或至少约15个氨基酸取代,条件是修饰的CD28共刺激域保留足够的信号转导活性(即,是功能变体)以促进T细胞活化。在某些实施方案中,修饰的CD28共刺激信号传导结构域包含至少约15、约16、约17、约18、约19、约20、约25或约30氨基酸取代,前提是修饰的CD28共刺激结构域保留足够的信号转导活性(即是功能性变体)以促进T细胞活化。用于测量融合蛋白功能的示例性测定包括用于测定细胞因子的测定产生(例如,细胞因子ELISA)、T细胞增殖(例如,通过FACS)、肿瘤细胞杀伤活性(例如,使用体外标记的铬释放测定(CRA)或通过体内成像肿瘤大小)、T细胞表面表达共刺激标志物的存在和响应抗原刺激(例如,通过使用可检测地标记的抗体)的T细胞衰竭标志物的存在(或不存在)。
在某些实施方案中,CD28共刺激信号结构域的氨基酸被除在野生型CD28共刺激信号结构域中该位置天然存在的氨基酸以外的任何氨基酸取代。在某些实施方案中,氨基酸被天然存在的氨基酸或非天然存在的氨基酸取代。
“保守取代”在本领域中被认为是一个氨基酸被具有相似特性的另一氨基酸(例如,另一种天然存在的或合成产生的氨基酸或其模拟物)取代。在某些实施方案中,氨基酸取代是保守氨基酸取代。示例性保守氨基酸取代包括其中氨基酸残基被具有相似侧链的氨基酸残基取代的氨基酸取代。类似的氨基酸可包括在以下类别中:具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸);具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸);具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、组氨酸);具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸);具有β支链侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香族侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸)。被认为更难以分类的脯氨酸与具有脂族侧链的氨基酸(例如亮氨酸、缬氨酸、异亮氨酸和丙氨酸)具有相同的特性。在某些情况下,可以认为谷氨酰胺代替谷氨酸或天冬酰胺代替天冬氨酸,因为谷氨酰胺和天冬酰胺分别是谷氨酸和天冬氨酸的酰胺衍生物。其他示例性的保守取代是本领域众所周知的(参见,例如,PCT公开号WO 97/09433,第10页;Lehninger,生物化学,第二版;WorthPublishers,Inc.NY,NY,第71-77页,1975);Lewin,Genes IV,New York,OxfordUniversity Press和MA,Cambridge,Cell Press,p.8,1990),这些保守性取代通过引用整体并入本文。
在某些实施方案中,尽管全长CD28分子可能不存在于融合蛋白中,但是出于参考的目的,氨基酸取代可以指全长CD28多肽内氨基酸残基的位置。在特定的实施方案中,氨基酸取代是指如UniProt:P10747(SEQ ID NO:1)中所述的全长人CD28多肽内的氨基酸残基的位置。
在某些实施方案中,修饰的CD28共刺激信号传导域包含至少一个氨基酸取代,其中:至少一个(即一个或多个)酪氨酸残基被不同的氨基酸残基取代,至少一个脯氨酸残基被α1取代。不同的氨基酸残基,或两者兼有。在一些实施方案中,至少一个、两个、三个或四个酪氨酸残基被取代。对于包含两个或多个酪氨酸取代的修饰的CD28共刺激信号结构域,每个酪氨酸取代可以相同或不同。在另外的实施方案中,至少一个酪氨酸残基被保守氨基酸取代。在一个具体的实施方案中,至少一个酪氨酸残基被苯丙氨酸残基取代。在另一个具体的实施方案中,至少一个酪氨酸残基被色氨酸残基取代。在一些实施方案中,至少一个酪氨酸残基被色氨酸残基取代,并且至少一个酪氨酸残基被苯丙氨酸残基取代。
在一些实施方案中,至少一个、两个、三个或四个脯氨酸残基被取代。对于包含两个或多个脯氨酸取代的修饰的CD28共刺激信号结构域,每个脯氨酸取代可以相同或不同。在进一步的实施方案中,至少一个脯氨酸残基被保守氨基酸取代。在一个具体的实施方案中,至少一个脯氨酸残基被丙氨酸残基取代。在一些实施方案中,至少一个脯氨酸残基被丙氨酸残基取代,并且至少一个脯氨酸残基被不同的(非丙氨酸)氨基酸取代。
在某些实施方案中,用于取代的至少一个酪氨酸残基选自位置191、206、209和218中的任何一个(位置指的是SEQ ID NO:1所示的全长野生型人CD28)。在一些实施方案中,选自位置191、206、209和218中任一个的至少两个、三个或四个酪氨酸残基被取代。在一些实施方案中,至少一个酪氨酸残基被保守氨基酸例如苯丙氨酸取代。
在某些实施方案中,用于取代的至少一个脯氨酸残基选自位置196、199、208和211中的任何一个(位置指的是SEQ ID NO:1所示的全长野生型人CD28)。在一些实施方案中,选自位置196、199、208和211中任一个的至少两个、三个或四个脯氨酸残基被取代。在一些实施方案中,至少一个脯氨酸残基被保守氨基酸例如丙氨酸取代。
在一些实施方案中,修饰的CD28共刺激信号传导域包括Y191、Y206、Y209、Y218、Y191/Y206、Y191/Y209、Y191/Y218、Y206/Y209、Y206/Y218、Y209/Y218、Y191/Y206/Y209、Y191/Y206/Y218、Y191/Y209/Y218、Y206/Y209/Y218或Y191/Y206/Y209/Y218取代。
在一些实施方案中,修饰的CD28共刺激信号传导域包括P196、P199、P208、P211、P196/P199、P196/P208、P196/P211、P199/P208、P199/P211、P208/P211、P196/P199/P208、P196/P199/P211、P196/P208/P211、P199/P208/P211或P196/P199/P208/P211取代。
在一些实施方案中,修饰的CD28共刺激域包含至少一个选自以下的酪氨酸取代:Y191、Y206、Y209、Y218、Y191/Y206、Y191/Y209、Y191/Y218、Y206/Y209、Y206/Y218、Y209/Y218、Y191/Y206/Y209、Y191/Y206/Y218、Y191/Y209/Y218、Y206/Y209/Y218或Y191/Y206/Y209/Y218;以及至少一个选自以下的脯氨酸取代:P196、P199、P208、P211、P196/P199、P196/P208、P196/P211、P199/P208、P199/P211、P208/P211、P196/P199/P208、P196/P199/P211、P196/P208/P211、P199/P208/P211或P196/P199/P208/P211。
在其他实施方案中,修饰的CD28共刺激信号传导域包括Y191F、Y206F、Y209F、Y218F、Y191F/Y206F、Y191F/Y209F、Y191F/Y218F、Y206F/Y209F、Y206F/Y218F、Y209F/Y218F、Y191F/Y206F/Y209F、Y191F/Y206F/Y218F、Y191F/Y209F/Y218F、Y206F/Y209F/Y218F或Y191F/Y206F/Y209F/Y218F取代。
在一些实施方案中,修饰的CD28共刺激信号传导域包括P196A、P199A、P208A、P211A、P196A/P199A、P196A/P208A、P196A/P211A、P199A/P208A、P199A/P211A、P208A/P211A、P196A/P199A/P208A、P196A/P199A/P211A、P196A/P208A/P211A、P199A/P208A/P211A或P196A/P199A/P208A/P211A取代。
在一些实施方案中,修饰的CD28共刺激结构域包含选自以下的至少一个的酪氨酸取代:Y191F、Y206F、Y209F、Y218F、Y191F/Y206F、Y191F/Y209F、Y191F/Y218F、Y206F/Y209F、Y206F/Y218F、Y209F/Y218F、Y191F/Y206F/Y209F、Y191F/Y206F/Y218F、Y191F/Y209F/Y218F、Y206F/Y209F/Y218F或Y191F/Y206F/Y209F/Y218F;和至少一个选自以下的脯氨酸取代:P196A、P199A、P208A、P211A、P196A/P199A、P196A/P208A、P196A/P211A、P199A/P208A、P199A/P211A、P208A/P211A、P196A/P199A/P208A、P196A/P199A/P211A、P196A/P208A/P211A、P199A/P208A/P211A或P196A/P199A/P208A/P211A。
在本文所述的任何修饰的CD28共刺激信号传导域中,所述修饰的CD28共刺激信号传导域可进一步在位置L186和L187的每个处包含取代。在一些实施方案中,修饰的CD28共刺激域包含L186G/L187G取代。已显示在位置186和187处的双亮氨酸至双甘氨酸取代增加了宿主免疫细胞中融合蛋白的表达(参见,Nguyen等人,Blood102:4320-4325(2003),其取代突变引用并入本文)。
在某些实施方案中,修饰的CD28共刺激信号传导域不包含在Y191、P208、P211或其任何组合的取代。
修饰的CD28共刺激信号传导域的示例性氨基酸序列在SEQ ID NO:4-6和45-47中提供。
与包含“野生型”CD28共刺激信号结构域的融合蛋白相比,包含在本公开中描述的修饰的CD28共刺激信号结构域的融合蛋白在表达所述融合蛋白的免疫细胞中表现出调节的功能活性。表达融合蛋白的免疫细胞功能活性的调节可包括信号传导动力学(例如信号传导的时机、顺序、序列或速率)、信号传导强度、细胞因子产生、细胞增殖、细胞持久性、抗肿瘤活性的调节、强直信号、免疫抑制成分基因的表达或其任何组合。在某些实施方案中,修饰的CD28共刺激信号结构域减少表达融合蛋白的免疫细胞中细胞因子的产生。其表达可能降低的细胞因子的实例包括IL-2和TNF-α。测量细胞因子水平的方法是本领域已知的,并且包括通过ELISA、蛋白质印迹、抗体阵列、流式细胞仪和细胞计数珠阵列的定量。
在某些实施方案中,修饰的CD28共刺激信号传导域减少表达融合蛋白的免疫细胞中的强直信号传导。进补信号传导可包括进补蛋白质磷酸化、激活、细胞因子表达、增殖或其组合。在一个具体的实施方案中,修饰的CD28共刺激信号传导域减少表达所述融合蛋白的T细胞中例如在位置Y142处的CD3ζ的强直磷酸化。
细胞内组分任选地进一步包含来自受体的细胞内激活结构域,例如含ITAM的T细胞激活结构域。在本公开的融合蛋白中使用的含ITAM的T细胞活化基序可以与免疫细胞受体的细胞内信号传导结构域或其部分或包含在其上的细胞表面标志物相同或可以是其功能变体。至少一个ITAM。通常,包含ITAM的T细胞活化域在融合蛋白的结合域与其靶抗原结合后会提供T细胞活化信号。可用于本文所述的融合蛋白中的含ITAM的细胞内激活结构域的非限制性实例包括存在于CD3γ、CD3δ、CD3ε、CD3ζ、FcRγ、CD38、CD5、CD22、CD79a、CD79b和CD66d、FcεRI或FcγRI的γ链、FcRγ2a、FcRγ2b1、FcRγ2a1、FcRγ2b2、FcRγ3a、FcRγ3b、FcRβ1、FcεR)、天然杀伤细胞受体蛋白(例如DAP12)、CD5、CD16a、CD16b、CD22、CD23、CD32、CD64、CD89、和CD278。在特定的实施方案中,本公开的融合蛋白的细胞内信号传导组分包含含有T细胞活化结构域的CD3ζITAM。包含T细胞活化域的示例性CD3ζITAM包含氨基酸序列SEQ ID NO:15。在某些实施方案中,本公开内容的融合蛋白的细胞内组分包含修饰的CD28共刺激信号传导域和包含CD3ζITAM的T细胞活化域。
细胞内组分任选地还包含除CD28共刺激信号域以外的另外的共刺激信号域。附加的共刺激信号结构域可以包含除CD28以外的共刺激分子的全长细胞内结构域或细胞内信号结构域的截短部分,条件是截短部分保留足够的信号转导活性。在某些实施方案中,所述另外的共刺激信号传导域选自CD27、CD40L、GITR、NKG2C、CARD1、CD2、CD7、CD27、CD30、CD40、CD54(ICAM)、CD83、CD134(OX-40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD223(LAG3)、CD270(HVEM)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、DAP10、LAT、NKD2CSLP76、TRIM、ZAP70、CD5、BAFF-R、SLAMF7、NKp80、CD160、B7-H3、与CD83特异性结合的配体或其组合。在某些实施方案中,本公开内容的融合蛋白的细胞内组分包含修饰的CD28共刺激信号结构域,含有CD3ζITAM的T细胞活化结构域和选自CD27、CD40L、GITR、NKG2C、CARD1,CD2,CD7,CD27,CD30,CD40,CD54(ICAM),CD83,CD134(OX-40),CD137(4-1BB),CD150(SLAMF1),CD152(CTLA4),CD223(LAG3),CD270(HVEM),CD273(PD-L2),CD274(PD-L1),CD278(ICOS),DAP 10,LAT,NKD2C SLP76,TRIM,ZAP70,CD5,BAFF-R,SLAMF7,NKp80,CD160,B7-H3,a与CD83特异性结合的配体,或其组合的另外的共刺激信号结构域。在一个具体的实施方案中,其包括修饰的CD28共刺激信号结构、含CD3ζITAM的T细胞活化结构域和4-1BB共刺激信号结构域。示例性的4-1BB共刺激信号传导结构域包含SEQ ID NO:14的氨基酸序列。
本公开的融合蛋白可以是以下形式:嵌合抗原受体(CAR)、嵌合共刺激受体(CCR)、分裂CAR或开关上CAR、与之连接的单链T细胞受体(scTCR或scTv)的形式。细胞内信号传导域、或TCR-CAR。在一些实施方案中,CAR通常具有提供激活信号的单个细胞内信号传导域(例如CD3ζ或FcγRI的细胞内信号传导域或其他含ITAM的T细胞活化域)。在一些实施方案中,CAR还包括细胞内共刺激信号传导域(例如(例如,来自内源性T细胞共刺激受体的共刺激信号域,例如CD28、4-1BB或ICOS)。在一些实施方案中,CAR进一步包括第二共刺激域。CCR在设计上与CAR相似,并且通过共刺激信号传导域提供共刺激,但不包含含ITAM的T细胞活化域。CCR还可包含异二聚化域,用于在宿主细胞中与含有以下物质的多肽共表达细胞内激活结构域和相应的异二聚结构域,用于在施用适当的异二聚试剂后进行组装(例如,分割CAR或开关CAR设计)。TCR-CAR是通常包含可溶性TCR(VαCα多肽链和VβCβ多肽链)的异二聚体融合蛋白,其中VβCβ多肽链连接至跨膜结构域和细胞内信号传导成分(例如,包含含ITAM的T细胞活化域和任选的共刺激信号传导域)。scTCR融合蛋白包含一个包含scTCR(与Vβ连接的TCR Vα)的结合结构域、一个可选的细胞外间隔子、一个跨膜结构域和一个细胞内组件,该组件包含一个提供T细胞活化信号的单个细胞内信号结构域(例如包含CD3ζITAM的T细胞活化域)和任选的共刺激信号传导域(参见Aggen等人,Gene Ther.19:365-374(2012);Stone等人,Cancer Immunol.Immunother.63:1163-76(2014))。
在某些实施方案中,本文所述的融合蛋白包含靶向来自病原体的抗原,自身免疫疾病相关抗原,癌症抗原或自身抗原的结合域。病原体相关或病原体特异性抗原的实例包括病毒抗原(例如,HIV抗原、HCV抗原、HBV抗原、CMV抗原、HPV抗原、EBV抗原、流感抗原、呼吸道合胞病毒抗原)、寄生虫抗原和细菌抗原。癌症抗原可以是临床上需要引起触发细胞介导的免疫反应从而导致癌细胞或肿瘤杀死的任何抗原。可以被融合蛋白靶向的癌症抗原的非限制性例子包括BCMA、CD3、CEACAM6、c-Met、EGFR、EGFRvIII、ErbB2、ErbB3、ErbB4、EphA2、IGF1R、GD2、O-乙酰GD2、O-乙酰基GD3、GHRHR、GHR、FLT1、KDR、FLT4、CD44v6、CD151、CA125、CEA、CTLA-4、GITR、BTLA、TGFBR2、TGFBR1、IL6R、gp130、Lewis A、Lewis Y、TNFR1、TNFR2、PD1、PD-L1、PD-L2、HVEM、MAGE-A(例如,包括MAGE-A1、MAGE-A3和MAGE-A4)、间皮素、NY-ESO-1、PSMA、RANK、ROR1、TNFRSF4、CD40、CD137、TWEAK-R、HLA、与HLA结合的肿瘤或病原体相关肽、与HLA结合的hTERT肽、与HLA结合的酪氨酸酶肽、与HLA结合的WT-1肽、LTβR、LIFRβ、LRP5、MUC1、OSMRβ、TCRα、TCRβ、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD52、CD56、CD79a、CD79b、CD80、CD81、CD86、CD123、CD171、CD276、B7H4、TLR7、TLR9、PTCH1、WT-1、HA1-H、Robo1、α-甲胎蛋白(AFP)、Frizzled、OX40、PRAME和SSX-2。
在本文提供的任何实施方案中,融合蛋白可以是“通用嵌合抗原受体”。通用CAR包含与标签而不是与疾病相关的抗原结合的结合域。可以通过施用与疾病相关抗原结合的标记蛋白(例如,与疾病相关抗原结合的标记抗体),将包含通用CAR的修饰的免疫细胞重定向至疾病相关抗原、肽、小分子或半抗原。示例性标签包括衍生自激素的肽、衍生自配体的肽、衍生自细胞因子的肽、衍生自趋化因子的肽、衍生自生长因子的肽、衍生自细胞粘附分子的肽、信号传导肽、衍生自受体的肽、细胞表面肽、异硫氰酸荧光素(FITC)、二硝基苯酚、多甲藻素叶绿素蛋白复合物、绿色荧光蛋白、生物素、藻红蛋白(PE)、组氨酸、链霉亲和素、辣根过氧化物酶、棕榈酰化、亚硝基化、碱性磷酸酶、葡萄糖氧化酶、谷胱甘肽S-转移酶、麦芽糖结合蛋白、DOTA、二硝基苯酚、醌、生物素、苯胺、阿特拉津、苯胺衍生物、邻氨基苯甲酸、对氨基苯甲酸、间氨基苯甲酸、肼苯哒嗪、氟烷、洋地黄毒苷、砷酸苯、乳糖、三硝基苯酚、半抗原、类固醇、维生素、维他命、代谢产物、抗生素、单糖、己糖、脂质、脂肪酸、核酸、生物碱、糖苷、吩嗪、聚酮化合物、萜烯、四吡咯和衍生自人核蛋白(例如人核La蛋白(E5B9))的肽。通用CAR及其制造和使用方法在本领域中是已知的,并且在例如美国专利第9,233,125号,美国专利No.PCT公开号WO2013/044225;PCT公开号WO2016/168766;PCT公开号WO2016/168773;和美国专利公开号2017/0240612,其每一个CAR和相关方法均通过引用整体并入本文。
在一些实施方案中,本公开的CAR包含细胞外组分,所述细胞外组分包含对CD19或ROR1具有特异性的结合结构域;任选地,标签(即,当融合蛋白是通用蛋白时,该标签不同于通用CAR所结合的标签的标签);免疫球蛋白铰链区;疏水部分;以及包含ITAM的细胞内组分,所述ITAM包含T细胞活化域和修饰的CD28共刺激信号传导域或其功能部分,其中所述修饰的CD28共刺激信号域或其功能部分在一个或多个位置L186、L187、Y191、Y206、Y209、Y218、P196、P199、P208和P211。在特定的实施方案中,修饰的CD28共刺激信号传导域或其功能部分在以下位置处包含取代:(a)L186、L187和Y218;(b)L186、L187、Y206、Y209和Y218;(c)L186、L187、Y191、Y206、Y209和Y218;(d)L186G、L187G和Y218F;(e)L186G、L187G、Y206F、Y209F和Y218F;或(f)L186G、L187G、Y191F、Y206F、Y209F和Y218F。在任何上述实施例中,标签包括链球菌标签II。
在某些实施方案中,本公开的CAR包含细胞外组分,所述细胞外组分包含FMC63(抗CD19)scFv、Strep标签II肽、改变的IgG4铰链区;包含CD28跨膜结构域的疏水部分;包含L186G、L187G和Y218F取代的修饰的CD28共刺激信号结构域;含有ITAM的T细胞活化域,其包含CD3ζ细胞内信号传导域。这样的CAR(例如,由CAR表达构建体编码的多肽)可以包含SEQID NO:27的氨基酸序列(包括氨基酸1-22的信号肽)或SEQ ID NO:27的氨基酸序列,而没有氨基酸1-22在进一步的实施方案中,CAR可以缺少SEQ ID NO:27的T2A和tEGFR氨基酸序列。
在进一步的实施方案中,本公开的CAR包含细胞外组分,所述细胞外组分包含FMC63(抗CD19)scFv、Strep标签II肽、改变的IgG4铰链区;和包含CD28跨膜结构域的疏水部分;修饰的CD28共刺激信号结构域,其包含L186G、L187G、Y206F、Y209F和Y218F取代;含有ITAM的T细胞活化域,其包含CD3ζ细胞内信号传导域。这样的CAR(例如,由CAR表达构建体编码的多肽)可以包含SEQ ID NO:29的氨基酸序列(包括氨基酸1-22的信号肽)或SEQ ID NO:29的氨基酸序列,而没有氨基酸1-22在进一步的实施方案中,CAR可以缺少SEQ ID NO:29的T2A和tEGFR氨基酸序列。
在另外的实施方案中,本公开的CAR包含细胞外组分,所述细胞外组分包含FMC63(抗CD19)scFv、Strep标签II肽、改变的IgG4铰链区;和包含CD28跨膜结构域的疏水部分;修饰的CD28共刺激信号域,其包含L186G、L187G、Y191F、Y206F、Y209F和Y218F取代;含有ITAM的T细胞活化域,其包含CD3ζ细胞内信号传导域。这样的CAR(例如,由CAR表达构建体编码的多肽)可以包含SEQ ID NO:35的氨基酸序列(包括氨基酸1-22的信号肽)或SEQ ID NO:35的氨基酸序列,而没有氨基酸1-22在进一步的实施方案中,CAR可以缺少SEQ ID NO:35的T2A和tEGFR氨基酸序列。
在另外的实施方案中,本公开的CAR包含细胞外组分,所述细胞外组分包含R12(抗ROR1)scFv、Strep标签II肽、改变的IgG4铰链区;和包含CD28跨膜结构域的疏水部分;包含L186G、L187G和Y218F取代的修饰的CD28共刺激信号结构域;含有ITAM的T细胞活化域,其包含CD3ζ细胞内信号传导域。这样的CAR(例如,由CAR表达构建体编码的多肽)可以包含SEQID NO:31的氨基酸序列(包括氨基酸1-22的信号肽)或SEQ ID NO:31的氨基酸序列,而没有氨基酸1-22在进一步的实施方案中,CAR可以缺少SEQ ID NO:31的T2A和tEGFR氨基酸序列。
在更多的实施方案中,本公开的CAR包含细胞外组分,所述细胞外组分包含R12(抗ROR1)scFv、Strep标签II肽、改变的IgG4铰链区;包含CD28跨膜结构域的疏水部分;修饰的CD28共刺激信号结构域,其包含L186G、L187G、Y206F、Y209F和Y218F取代;含有ITAM的T细胞活化域,其包含CD3ζ细胞内信号传导域。这样的CAR(例如,由CAR表达构建体编码的多肽)可以包含SEQ ID NO:33的氨基酸序列(包括氨基酸1-22的信号肽)或SEQ ID NO:33的氨基酸序列,而没有氨基酸1-22在进一步的实施方案中,CAR可以缺少SEQ ID NO:33的T2A和tEGFR氨基酸序列。
在更多实施方案中,本发明的CAR包含细胞外组分,所述细胞外组分包含R12(抗ROR1)scFv、Strep标签II肽、改变的IgG4铰链区;包含CD28跨膜结构域的疏水部分;修饰的CD28共刺激信号域,其包含L186G、L187G、Y191F、Y206F、Y209F和Y218F取代;含有ITAM的T细胞活化域,其包含CD3ζ细胞内信号传导域。这样的CAR(例如,由CAR表达构建体编码的多肽)可以包含SEQ ID NO:37的氨基酸序列(包括氨基酸1-22的信号肽)或SEQ ID NO:37的氨基酸序列,而没有氨基酸1-22在进一步的实施方案中,CAR可以缺乏SEQ ID NO:37的T2A和tEGFR氨基酸序列。
在一些实施方案中,本公开的CAR可以包含具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%的氨基序列,所述氨基序列与SEQ ID NO:64-69中任一个的氨基酸序列具有同一性(分别包括或不包括SEQ ID NO:64-69的氨基酸1-22处的信号肽)。
在一些实施方案中,本公开的CAR(例如,由CAR表达构建体编码的多肽)可以包含具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%,99%或100%的氨基序列,所述氨基序列与与SEQ ID NO:49、51、53、55、57或59中的任何一个的氨基酸序列(包括或不包括SEQ ID NO:49、51、53、55、57和59的1-22氨基酸信号肽)具有同一性。在进一步的实施方案中,CAR可以缺乏SEQ ID NO:49、51、53、55、57或59的T2A和tEGFR氨基酸序列。
多核苷酸、载体和宿主细胞
在某些方面,提供了编码本文所述的任何一种或多种融合蛋白的核酸分子。编码所需融合蛋白的多核苷酸可以通过使用任何合适的分子生物学工程技术来完成,包括使用限制性核酸内切酶消化、连接、转化、质粒纯化和DNA测序,例如,如Sambrook等人所述,(1989年和2001年版;Molecular Cloning:A Laboratory Manual,Cold Spring HarborLaboratory Press,纽约)和Ausubel等人,(Current Protocols in Molecular Biology,2003)。或者,可以合成产生目的序列。为了获得有效的转录和翻译,每个重组表达结构中的多核苷酸包含至少一个适当的表达控制序列(也称为调控序列),如一个先导序列,特别是一个可操作(即操作上)连接到编码免疫原的核苷酸序列的启动子序列。
本公开内容的核酸可以指任何形式的单链或双链DNA、cDNA或RNA,并且可以包括彼此互补的核酸的正链和负链,包括反义DNA、cDNA和RNA。还包括siRNA、microRNA、RNA-DNA杂交体、核酶和其他各种天然存在或合成形式的DNA或RNA。
在本文所述的任何实施方案中,可以对本公开内容的多核苷酸进行密码子优化以在包含多核苷酸的宿主细胞中有效表达(参见,例如,Scholtenet等人,Clin.Immunol.119:135-145(2006))。如本文所用,“密码子优化的”多核苷酸包含异源多核苷酸,所述异源多核苷酸具有经沉默突变修饰的密码子,所述沉默突变对应于目的宿主细胞中tRNA水平的丰度。
在某些实施方案中,编码本公开内容的融合蛋白的多核苷酸包含具有至少约75%、80%、85%、90%、91%、92%、93%、94%、95%、96%或100%的多核苷酸,多核苷酸与SEQ ID NO:18、20、22、24、26、28、30、32、34,36、48、50、52、54、56或58中任一个序列中显示的融合蛋白编码核苷酸序列具有同一性,并且任选地包含或由SEQ ID NO:18、20、22、24、26、28、30、32、34、36、48、50、52、54、56或58中任何一个所示的多核苷酸组成。
根据本文公开的任何实施方案,单个多核苷酸分子可编码一个、两个或多个融合蛋白。编码不止一个转录物的多核苷酸可包含位于每个转录物之间的用于多顺反子表达的序列(例如,病毒2A肽编码序列)。
在某些实施方案中,本公开内容的编码融合蛋白的多核苷酸可以可操作地连接至载体的一个或多个特定元件。例如,可以有效地连接实现它们所连接的编码序列的表达和加工所需的多核苷酸序列。表达控制序列可以包括适当的转录起始,终止,启动子和增强子序列;有效的RNA处理信号,例如剪接和聚腺苷酸化信号;稳定细胞质mRNA的序列;增强翻译效率的序列(即Kozak共有序列);增强蛋白质稳定性的序列;以及可能增强蛋白质分泌的序列。如果表达控制序列与目的基因和表达控制序列邻接,则它们可被有效连接,所述表达控制序列以反式或远距离作用来控制目的基因。
某些实施方案包括载体中包含的本公开的多核苷酸。示例性载体可包含能够转运与其连接的另一核酸分子或能够在宿主生物中复制的核酸分子。载体的一些实例包括质粒、病毒载体、粘粒和其他。一些载体可能能够在引入它们的宿主细胞中自主复制(例如,具有细菌复制起点的细菌载体和附加型哺乳动物载体),而其他载体可能整合到宿主细胞的基因组中或促进其整合。多核苷酸在引入宿主细胞后即插入,从而与宿主基因组(例如慢病毒载体)一起复制。另外,一些载体能够指导与其可操作连接的基因的表达(这些载体可以称为“表达载体”)。根据相关实施方案,还应理解,如果将一种或多种试剂(例如,如本文所述的编码融合蛋白的多核苷酸)共同施用给受试者,则每种试剂可存在于分开的或相同的载体中,以及多个载体(它们各自包含不同的试剂或相同的试剂)可以被引入细胞或细胞群体或被施用于受试者。
载体可以是例如质粒、粘粒、病毒、RNA载体或线性或环状DNA或RNA分子,其可以包括染色体、非染色体、半合成或合成核酸分子。示例性载体是那些能够自主复制的载体(附加载体)或表达与其连接的核酸分子的载体(表达载体)。
病毒载体包括逆转录病毒、腺病毒、细小病毒(例如腺相关病毒)、冠状病毒、负链RNA病毒(例如正粘液病毒(例如流感病毒)、弹状病毒(例如狂犬病和水疱性口炎病毒)、副粘病毒(例如麻疹和仙台)、正链RNA病毒(如小核糖核酸病毒和甲型病毒)以及双链DNA病毒、包括腺病毒、疱疹病毒(例如1型和2型单纯疱疹病毒、EB病毒、巨细胞病毒)和痘病毒(例如,牛痘、鸡痘和金丝雀痘)。例如,其他病毒包括诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、巴波病毒、肝炎病毒和肝炎病毒。逆转录病毒的例子包括禽白血病、肉瘤、哺乳动物C型、B型病毒、D型病毒、HTLV-BLV组、慢病毒、泡沫病毒(Coffin,J.M.,Retroviridae:Theviruses and their replication,In Fundamental Virology,Third Edition,B.N.Fields et al.,Eds.,Lippincott-Raven Publishers,Philadelphia,1996)。
在某些实施方案中,病毒载体可以是γ逆转录病毒,例如莫洛尼鼠碱白血病病毒(MLV)来源的载体。在其他实施方案中,病毒载体可以是更复杂的逆转录病毒来源的载体,例如慢病毒来源的载体。HIV-1衍生的载体属于这一类。其他实例包括衍生自HIV-2、FIV、马传染性贫血病毒、SIV和Maedi-Visna病毒(绵羊慢病毒)的慢病毒载体。使用逆转录病毒和慢病毒的病毒载体和包装细胞通过含有CAR转基因的病毒颗粒转导哺乳动物宿主细胞的方法在本领域是已知的,并且先前已经在例如:美国专利第8119,772号;Walchli et al.,PLoS One 6:327930(2011);Zhao et al.,J.Immunol.174:4415(2005);Engels et al.,Hum.Gene Ther.14:1155(2003);Frecha et al.,Mol.Ther.18:1748(2010);andVerhoeyen et al.,Methods Mol.Biol.506:97(2009)。逆转录病毒和慢病毒载体构建体和表达系统也是可商购的。其他病毒载体也可以用于多核苷酸递送,包括DNA病毒载体,包括例如基于腺病毒的载体和基于腺伴随病毒(AAV)的载体;源自单纯疱疹病毒(HSV)的载体,包括扩增子载体,复制缺陷型HSV和减毒的HSV(Krisky et al.,Gene Ther.5:1517(1998))。
为基因治疗用途而开发的其他载体也可以与本公开的组合物和方法一起使用。这样的载体包括衍生自杆状病毒和α-病毒的那些(Jolly,D J.1999.Emerging ViralVectors.pp.209-40in Friedmann T.ed.The Development of Human Gene Therapy.NewYork:Cold Spring Harbor Lab),或质粒载体(例如睡美人或其他转座子载体)。
为了获得有效的转录和翻译,每个重组表达构建体中的多核苷酸包括至少一个合适的表达控制序列(也称为调节序列),例如与编码免疫原的核苷酸序列可操作(即可操作地)连接的前导序列,特别是启动子。
标记有时被用来识别或监测被异源多核苷酸转导的宿主细胞的表达,或用于检测表达目的融合蛋白的细胞。在某些实施方案中,编码融合蛋白的多核苷酸还包含编码标志物的多核苷酸。标记可以是赋予抗药性的选择标记,也可以是可通过诸如流式细胞术之类的方法检测的可检测标记,例如荧光标记或细胞表面蛋白。在某些实施方案中,编码标记的多核苷酸位于编码免疫球蛋白结合蛋白或融合蛋白的多核苷酸的3'。在其他实施方案中,编码标记的多核苷酸位于编码免疫球蛋白结合蛋白或融合蛋白的多核苷酸的5'。示例性标记包括绿色荧光蛋白(GFP)、人CD2的胞外域,截短的人EGFR(huEGFRt;参见Wang等人,Blood118:1255(2011)),截短的人CD19(huCD19t),截短的人CD34(huCD34t);或截短的人类NGFR(huNGFRt)。在某些实施方案中,编码的标记物包括EGFRt、CD19t、CD34t或NGFRt。示例性的截短的人EGFR序列包含SEQ ID NO:17的氨基酸序列。
在某些实施方案中,载体可以进一步包含自杀基因,其中自杀基因的表达导致包含载体的宿主细胞死亡。例如,在某些情况下,本发明的融合蛋白的延长表达是不希望的。载体中包含自杀基因允许对受试者中融合蛋白表达的更好控制。在某些实施方案中,例如,通过使用调节自杀基因表达的诱导型启动子,可诱导自杀基因的表达。在一个具体的实施方案中,自杀基因是诱导型胱天蛋白酶9基因(参见美国授权前专利公开号US 2013/0071414,该自杀基因通过引用并入本文)。其他自杀基因包括编码以下任何一项或多项的基因:药物级抗EGFR单克隆抗体西妥昔单抗(Erbitux)的构象完整的结合表位;胱天蛋白酶多肽的EGFRt(例如iCasp9;Straathof等人,Blood 105:4247-4254,2005;Di Stasi等,N.Engl.J.Med.365:1673-1683,2011);Zhou and Brenner,Exp.Hematol.pii:S0301-472X(16)30513-6.doi:10.1016/j.exphem.2016.07.011),RQR8(Philip等人,Blood 124:1277-1287,2014),人c-myc蛋白(Myc)的10个氨基酸标签(Kieback等人,Kieback等,Natl.Acad.Sci.USA 105:623-628,2008)(如本文所讨论),和标记/安全开关多肽,例如RQR(CD20+CD34;Philip等,2014)。
当病毒载体基因组包含要在宿主细胞中表达的多个多核苷酸作为单独的转录物时,病毒载体还可以在两个(或更多个)转录物之间包含允许双顺反子或多顺反子表达的附加序列。用于病毒载体的此类序列的实例包括内部核糖体进入位点(IRES)、弗林蛋白酶切割位点、病毒2A肽或其任何组合。
在本文所述的任何实施方案中,多核苷酸可进一步包含编码自切割多肽的多核苷酸,其中编码自切割多肽的多核苷酸位于编码融合蛋白的多核苷酸和编码标记的多核苷酸之间。在某些实施方案中,自我切割的多肽包含来自猪破伤风病毒-1(P2A)、那撒那病毒(T2A)、马鼻炎病毒(E2A)、口蹄疫病毒(F2A)或变体的2A肽。在某些实施方案中,示例性的T2A肽序列包含SEQ ID NO:16的氨基酸序列。2A肽的进一步的示例性核酸和氨基酸序列在例如Kim等人给出。(PLOS One 6:e18556(2011),其中2A核酸和氨基酸序列通过引用整体并入本文)。
在某些方面,本公开的融合蛋白可以在宿主细胞的表面上表达或由宿主细胞分泌或从宿主细胞分离。宿主细胞可以包括可以接受载体或掺入核酸或表达蛋白质的任何单个细胞或细胞培养物。该术语还涵盖宿主细胞的后代,无论在遗传上还是在表型上相同或不同。合适的宿主细胞可以取决于载体,并且可以包括哺乳动物细胞、动物细胞、人细胞、猿猴细胞、昆虫细胞、酵母细胞和细菌细胞。可以通过使用病毒载体,通过磷酸钙沉淀、DEAE-葡聚糖、电穿孔、显微注射或其他方法诱导这些细胞掺入载体或其他材料。参见,例如,Sambrook等人,Molecular Cloning:A Laboratory Manual2d ed.(Cold Spring HarborLaboratory,1989)。
除载体外,某些实施方案还涉及宿主细胞,所述宿主细胞经修饰(即,基因工程改造)以包含编码融合蛋白(例如,CAR)的异源多核苷酸,或包含根据权利要求1的包含编码融合蛋白的异源多核苷酸(例如,CAR)的载体。本公开包含编码至少一种融合蛋白的异源多核苷酸的修饰的或基因工程的宿主细胞在其细胞表面上表达本发明的至少一种融合蛋白。修饰的宿主细胞可以表达本公开的单一类型的融合蛋白或两种或更多种不同类型的融合蛋白。宿主细胞可以离体或体内被修饰。宿主细胞可以包括可以接受载体或核酸或蛋白质掺入的任何单个细胞或细胞培养物,以及任何子代细胞。该术语还涵盖宿主细胞的后代,无论在遗传上还是在表型上相同或不同。合适的宿主细胞可以取决于载体,并且可以包括哺乳动物细胞、动物细胞、人细胞、猿猴细胞、昆虫细胞、酵母细胞和细菌细胞。可以通过使用病毒载体,通过磷酸钙沉淀、DEAE-葡聚糖、电穿孔、显微注射或其他方法诱导这些细胞掺入载体或其他材料。参见,例如,Sambrook等人,Molecular Cloning:A Laboratory Manual 2ded.(Cold Spring Harbor Laboratory,1989)。在任何上述实施方案中,含有编码本公开内容的融合蛋白的多核苷酸的宿主细胞由与接受修饰的宿主细胞的受试者例如在过继免疫疗法程序中自体、同种或同系的细胞组成。
在某些实施方案中,转导以表达本公开的融合蛋白的宿主细胞是造血祖细胞或人免疫系统细胞。如本文所用,“造血祖细胞”是可以衍生自造血干细胞的细胞。或胎儿组织,并且能够进一步分化成成熟细胞类型(例如免疫系统细胞)。示例性的造血祖细胞包括具有CD24Lo Lin–CD117+表型的细胞或在胸腺中发现的细胞(称为祖细胞)。
在某些实施方案中,宿主细胞是免疫系统细胞,包括例如B细胞、T细胞(例如CD4+T细胞、CD8+T细胞、CD4-CD8-双阴性T细胞、γδT细胞、调节性T细胞)、天然杀伤细胞(例如NK细胞或NK-T细胞)或树突状细胞。
在某些实施方案中,宿主细胞是T细胞。T细胞可能是幼稚T细胞、记忆T细胞(TM)、干细胞记忆T细胞、辅助T细胞(TH)、效应T细胞(TE)、γδT细胞、调节性T细胞(Treg)或其任意组合。TM可以进一步分为以下子集:中央记忆T细胞(TCM,与幼稚T细胞相比,CD62L、CCR7、CD28、CD127、CD45RO和CD95的表达增加,以及CD54RA的表达减少)和效应记忆T细胞(TEM,与幼稚T细胞或TCM相比,CD62L、CCR7、CD28、CD45RA的表达减少,而CD127的表达增加。)
可以使用已知技术收集T细胞,并且可以通过已知技术例如通过与抗体的亲和结合,流式细胞术或免疫磁选择来富集或消除各种亚群或其组合。
已经描述了用所需核酸转染/转导T细胞的方法(例如,美国专利申请公开号US2004/0087025;美国专利号6,410,319;PCT公开号WO2014/031687;Brentjens et al.,Clin.Cancer Res.13:5426(2007))具有使用期望的靶特异性的T细胞的过继转移程序(例如Schmitt et al.,Hum.Gen.20:1240(2009);Dossett et al.,Mol.Ther.17:742(2009);Till et al.,Blood 112:2261(2008);Wang et al.,Hum.Gene Ther.18:712(2007);Kuball et al.,Blood 109:2331(2007);US 2011/0243972;US 2011/0189141;Leen etal.,Ann.Rev.Immunol.25:243(2007);Kalos et al.,Sci Transl.Med.3:95ra73(2011);Porter et al.,N.Engl.J.Med.365:725-33(2011)),从而基于本文的教导,包括针对本公开内容的融合蛋白的那些,考虑了将这些方法学应用于当前公开的实施方案。
当包含根据本发明的多核苷酸,载体或蛋白质时,作为本发明的一个方面考虑的真核宿主细胞除人类免疫细胞(例如人类患者自身的免疫细胞)外,还包括VERO细胞、HeLa细胞、中国人仓鼠卵巢(CHO)细胞系(包括能够修饰表达的多价结合分子的糖基化模式的修饰的CHO细胞,请参阅美国授权前专利出版物第2003/0115614号)、COS细胞(例如COS-7)、W138、BHK、HepG2、3T3、RIN、MDCK、A549、PC12、K562、HEK293细胞、HepG2细胞、N细胞、3T3细胞、草地贪夜蛾细胞(例如Sf9细胞)、酿酒酵母细胞和任何其他已知的真核细胞用于表达和任选地分离根据本公开的蛋白质或肽的技术。还考虑了原核细胞,包括大肠杆菌、枯草芽孢杆菌、鼠伤寒沙门氏菌、链霉菌或本领域已知适合表达和任选分离根据本公开的蛋白质或肽的任何原核细胞。特别地,在从原核细胞中分离蛋白质或肽时,可以考虑使用本领域已知的从包涵体提取蛋白质的技术。考虑了糖基化本公开的融合蛋白的宿主细胞。
根据常规方法,在含有营养物质和所选宿主细胞生长所需的其他成分的培养基中培养转化或转染的宿主细胞。多种合适的介质,包括确定的介质和复合介质,在本领域中是已知的,并且通常包括碳源、氮源、必需氨基酸、维生素和矿物质。根据需要,培养基还可以包含诸如生长因子或血清的成分。生长培养基通常将通过例如药物选择或必需营养素的缺乏来选择含有异源多核苷酸的细胞,所述必需营养素由表达载体上携带的或共同转染到宿主细胞中的选择标记来补充。
在某些实施方案中,本公开的融合蛋白在宿主细胞的表面上表达,使得与靶抗原的结合引起来自宿主细胞的活性或应答。可以根据许多本领域公认的用于测定宿主细胞(例如T细胞)活性的方法来对这类表达的蛋白质进行功能表征,所述方法包括确定T细胞结合、激活或诱导,还包括确抗原特异性的定T细胞反应。实例包括确定T细胞增殖、T细胞细胞因子释放、抗原特异性T细胞刺激、MHC限制性T细胞刺激、CTL活性(例如,通过检测预载靶细胞的51Cr或Euro释放)、T细胞的变化表型标志物表达和其他T细胞功能指标。例如,可以在Lefkovits中找到进行这些和类似测定的程序(Immunology Methods Manual:TechnicalSourcebook of Techniques,1998)。另请参见Current Protocols in Immunology;Weir,Handbook of Experimental Immunology,Blackwell Scientific,Boston,MA(1986);Mishell and Shigii(eds.)Selected Methods in Cellular Immunology,FreemanPublishing,San Francisco,CA(1979);Green and Reed,Science 281:1309(1998),以及其中引用的参考文献。
可以根据本文所述和本领域中实践的方法确定细胞因子的水平,包括例如ELISA,ELISPOT,细胞内细胞因子染色,和流式细胞术及其组合(例如,细胞内细胞因子染色和流式细胞术)。可以通过分离淋巴细胞,例如外周血细胞样本中的循环淋巴细胞或淋巴结细胞中的循环淋巴细胞,用抗原刺激细胞,并测量抗原,来确定由抗原特异性引发或刺激免疫应答引起的免疫细胞增殖和克隆扩增。细胞因子的产生,细胞增殖和/或细胞活力,例如通过掺入氚化的胸腺嘧啶核苷或非放射性测定,例如MTT测定等。可以例如通过确定Th1细胞因子(例如IFN-γ、IL-12,IL-2和TNF-β)和2型细胞因子(例如IL-4,IL-5,IL-9,IL-10和IL-13)的水平来检查本文所述的免疫原对Th1免疫应答和Th2免疫应答之间的平衡的作用。
在某些实施方案中,内源基因如TCR基因、HLA基因、β2M基因、免疫抑制成分基因(例如免疫检查点分子基因)或其任何组合的表达在修饰的免疫细胞(例如,T细胞)。在某些实施方案中,TCR基因是T细胞受体α恒定(TRAC)基因,T细胞受体β恒定(TRBC)基因或两者。在某些实施方案中,HLA基因是I类HLA基因、II类HLA基因或两者。在本文提供的任何实施方案中,可以修饰修饰的免疫细胞以减少或消除涉及免疫应答的一个或多个内源基因的表达。例如,可以修饰T细胞以减少或消除HLA复合物成分或TCR或TCR复合物成分的一种或多种多肽的表达。不希望受到理论的束缚,某些内源表达的免疫细胞蛋白可能被接受修饰的免疫细胞的同种异体宿主识别为外源,这可能导致消除修饰的免疫细胞(例如,HLA等位基因),并可能介导经由内源表达的受体(例如,TCR)的移植物抗宿主疾病,可能与本公开的异源融合蛋白竞争由宿主细胞表达,或可能干扰本公开的异源表达的融合蛋白的结合活性(例如,与非肿瘤相关抗原结合并干扰与肿瘤相关抗原特异性结合的修饰的免疫细胞的抗原特异性融合蛋白的内源TCR)。因此,减少,抑制或消除这种内源基因或蛋白质的表达或活性可以改善同种异体宿主环境中修饰的免疫细胞的活性,耐受性或持久性,并且在一些实施方案中可以允许细胞的普遍施用(例如,对于任何收件人,无论HLA类型如何)。
在某些实施方案中,在修饰的免疫细胞中抑制了免疫抑制成分基因(例如,免疫检查点分子基因)的表达。如本文所用,术语“免疫抑制组分”或“免疫抑制组分”是指提供抑制信号以帮助控制或抑制免疫应答的一种或多种细胞、蛋白质、分子、化合物或复合物。例如,免疫抑制成分包括部分或全部阻断免疫刺激的分子;减少,预防或延迟免疫激活;或增加,激活或上调免疫抑制。示例性的免疫抑制成分靶标包括免疫检查点分子,例如PD-1、PD-L1、PD-L2、CD80、CD86、B7-H3、B7-H4、HVEM、腺苷、GAL9、VISTA、CEACAM-1、PVRL2、CTLA-4、BTLA、KIR、LAG3、TIM3、A2aR、CD244/2B4、CD160、TIGIT、LAIR-1、PVRIG/CD112R;代谢酶,例如精氨酸酶、吲哚胺2,3-双加氧酶(IDO);免疫抑制细胞因子,例如IL-10、IL-4、IL-1RA、IL-35;Treg细胞,或其任何组合。
在修饰的免疫细胞中,可以在基因水平,转录水平,翻译水平或两者上敲低,敲除或抑制TCR基因(例如编码TCR可变区或TCR恒定区的基因;参见例如Torikai等人,NatureSci.Rep.6:21757(2016);Torikai等人,Blood 119(24):5697(2012);和Torikai等,Blood122(8):1341(2013),其基因编辑技术、组成和过继性细胞疗法的全部内容并入本文),HLA基因(例如,可以在基因水平上敲除,敲除或抑制编码α1大球蛋白,α2大球蛋白,α3大球蛋白,β1微球蛋白或β2微球蛋白的基因),免疫抑制组分基因或其任何组合的表达。TCR,HLA或免疫抑制组分基因表达的示例性抑制剂包括抑制性核酸分子和核酸内切酶。导致染色体基因敲除的改变可包括,例如,引入的无义突变(包括过早终止密码子的形成),错义突变,基因缺失和链断裂,以及抑制性核酸分子的异源表达,抑制性核酸分子抑制宿主细胞中内源性基因的表达。
“抑制性核酸”是指短、单链或双链核酸分子,其具有与靶基因或mRNA转录物互补的序列,并且能够减少靶基因或mRNA转录物的表达,或指编码这样的分子。抑制性核酸分子包括反义寡核苷酸,双链RNA(dsRNA)分子、小干扰RNA(siRNA分子、shRNA分子和内切核糖核酸酶制备的siRNA(esiRNA)分子)。降低的表达可以通过多种过程来完成,包括阻断转录或翻译(例如位阻)、靶mRNA转录物的降解、阻断mRNA前剪接位点、阻断mRNA加工(例如加帽,聚腺苷酸化)。在某些实施方案中,抑制性核酸分子可以用于基因敲除方法。TCR,HLA和免疫抑制成分基因的基因组和mRNA序列可在国家生物技术信息中心的GenBank数据库中公开获得。制备靶向mRNA的抑制性核酸分子的方法是本领域已知的,并且例如描述于Ozcan等人,Adv.Drug Deliv.Rev.87:108-119(2016)。使用抑制性核酸分子抑制基因在免疫细胞中表达的方法在本领域中是已知的,并且例如描述于美国授权前专利公开号US 2012/0321667和US 2007/0036773;Condomines等人,PLoS ONE 10:e0130518(2015);Ohno等人,J.Immunother.Cancer 1:21(2013))。
可以使用例如内切核酸酶进行染色体编辑。如本文所用,“核酸内切酶”是指能够催化多核苷酸链内的磷酸二酯键裂解的酶。在某些实施方案中,核酸内切酶能够切割靶基因,从而失活或“敲除”靶基因。核酸内切酶可以是天然存在的、重组的、基因修饰的或融合的核酸内切酶。核酸内切酶引起的核酸链断裂通常通过同源重组或非同源末端连接(NHEJ)的不同机制修复。在同源重组期间,供体核酸分子可用于基因“敲入”以使靶基因失活。NHEJ是易于出错的修复过程,通常会导致切割位点的DNA序列发生变化,例如,取代,缺失或添加至少一个核苷酸。NHEJ可用于“敲除”靶基因。核酸内切酶的实例包括锌指核酸酶、TALE核酸酶、CRISPR-Cas核酸酶和大范围核酸酶。
如本文所用,“锌指核酸酶”(ZFN)是指包含与非特异性DNA切割结构域融合的锌指DNA结合结构域的融合蛋白,例如Fok1核酸内切酶。约30个氨基酸的每个锌指基序与约3个碱基对的DNA结合,并且某些残基上的氨基酸可以改变以改变三联体序列特异性(参见例如Desjarlais等人,Proc.Natl.Acad.Sci.90:2256-2260(1993);Wolfe等人,J.Mol.Biol.285:1917-1934(1999))。多个锌指基序可以串联连接以产生对所需DNA序列的结合特异性,例如长度范围为约9至约18个碱基对的区域。作为背景,ZFN通过催化基因组中位点特异性DNA双链断裂(DSB)的形成来介导基因组编辑,并且通过以下方式促进了转基因的靶向整合:该转基因包含与DSB位置的基因组同源的侧翼序列同源性指导修复。或者,由ZFN产生的DSB可以通过非同源末端连接(NHEJ)的修复作用导致靶基因的敲除,这是一个容易出错的细胞修复途径,会导致核苷酸在切割位点处插入或缺失。在某些实施方案中,TCR基因、HLA基因或免疫抑制组分基因敲除包含使用ZFN分子制成的插入、缺失、突变或其组合。
如本文所用,“转录激活因子样效应子核酸酶”(TALEN)是指包含TALE DNA结合结构域和DNA切割结构域的融合蛋白,例如FokI核酸内切酶。“TALE DNA结合结构域”或“TALE”由一个或多个TALE重复结构域/单元组成,每个通常具有高度保守的33-35个氨基酸序列以及不同的第12和第13个氨基酸。TALE重复结构域参与TALE与靶DNA序列的结合。不同的氨基酸残基被称为重复可变残基(RVD),与特异性核苷酸识别相关。确定了这些TALE的DNA识别的天然(规范)代码,以使12和13位的HD序列导致与胞嘧啶(C)结合,NG与T结合,NI与A结合,NN与G或A结合并且,NG与T结合并且非典型的(非典型的)RVD也是已知的(参见,例如,美国授权前专利公开号US 20110301073,该非典型的RVD通过引用整体并入本文)。TALENs可用于指导T细胞基因组中的位点特异性双链断裂(DSB)。非同源末端连接(NHEJ)从双链断裂的两侧连接DNA,在双链断裂中几乎没有或没有序列重叠用于退火,从而引入了敲除基因表达的错误。或者,同源指导的修复可以在DSB的位点引入转基因,条件是转基因中存在同源侧翼序列。在某些实施方案中,TCR基因、HLA基因或免疫抑制组分基因敲除包含使用TALEN分子制成的插入、缺失、突变或其组合。
如本文所用,“成簇的规则间隔的短回文重复序列/Cas”(CRISPR/Cas)核酸酶系统是指采用CRISPR RNA(crRNA)引导的Cas核酸酶来识别基因组内靶位点的系统(称为原型间隔子)。通过碱基配对的互补性,然后在互补靶序列的3'紧跟一个短的,保守的原间隔子相关基序(PAM)时切割DNA。根据Cas核酸酶的序列和结构,将CRISPR/Cas系统分为三种类型(即I型、II型和III型)。I型和III型的crRNA指导的监视复合体需要多个Cas亚基。研究最多的II型系统至少包含三个组件:RNA引导的Cas9核酸酶、crRNA和反式crRNA(tracrRNA)。tracrRNA包含双链体形成区。rRNA和tracrRNA形成双链体,该双链体能够与Cas9核酸酶相互作用,并通过crRNA上间隔子与在PAM上游的靶DNA上的原间隔子之间的Watson-Crick碱基配对将Cas9/crRNA:tracrRNA复合体引导至目标DNA上的特定位点。Cas9核酸酶在crRNA间隔区定义的区域内切割双链断裂。NHEJ修复导致插入和/或缺失,从而破坏了目标基因座的表达。可替代地,可以通过同源性指导的修复将具有同源侧翼序列的转基因引入DSB的位点。可以将crRNA和tracrRNA工程化成单个指导RNA(sgRNA或gRNA)(参见,例如,Jinek等人,Science 337:816-21,2012)。此外,可以改变或编程与靶位点互补的指导RNA的区域以靶向所需序列(Xie等人,PLOS One 9:e100448,2014;美国授权前专利公开号US 2014/0068797,美国授权专利公开号US 2014/0186843,美国专利号8,697,359和PCT公开号WO2015/071474;它们各自通过引用并入本文。在某些实施方案中,TCR基因、HLA基因或免疫抑制组分基因敲除包含使用CRISPR/Cas核酸酶系统制备的插入、缺失、突变或其组合。
如本文所用,“大范围核酸酶”,也称为“归巢内切核酸酶”,是指特征在于大识别位点(约12至约40个碱基对的双链DNA序列)的内脱氧核糖核酸酶。根据序列和结构基序,大范围核酸酶可分为五个家族:LAGLIDADG、GIY-YIG、HNH、His-Cys盒和PD-(D/E)XK。示例性的大范围核酸酶包括I-SceI、I-CeuI、PI-PspI、PI-Sce、I-SceIV、I-CsmI、I-PanI、I-SceII、I-PpoI、I-SceIII、I-CreI、I-TevI、I-TevII和I-TevIII,其识别序列是已知的(参见,例如,美国专利号5,420,032和6,833,252;Belfort等,Nucleic Acids Res.25:3379-3388,1997;Dubon等人,Gene 82:115-118,1989;Perler等的,Nucleic Acids Res 22:1125-1127,1994;Jasin,Trends Genet.12:224-228,1996;Gimble等人,J.Mol.Biol.263:J.Mol.Biol.280:345-353,1998;Args等人:163-180,1996;Argast等人,1998)。在某些实施方案中,天然存在的大范围核酸酶可用于促进TCR基因、HLA基因或免疫抑制组分基因的位点特异性基因组修饰。在其他实施方案中,将对TCR基因、HLA基因或免疫抑制组分基因具有新颖结合特异性的工程化大范围核酸酶用于位点特异性基因组修饰(参见例如Porteus等人,Nat.Biotechnol.23:967-73,2005;Sussman等人,J.Mol.Biol.342:31-41,2004;Epinat等人,Nucleic Acids Res.31:2952-62,2003;Chevalier等人,Molec.Cell 10:895-905,2002;Ashworth等人,Nature 441:656-659,2006;Paques等人,Curr.Gene Ther.7:49-66,2007;美国授权前专利公开号US 2007/0117128;US 2006/0206949;US 2006/0153826;US2006/0078552;以及US 2004/0002092)。
在进一步的实施方案中,通过核酸内切酶,例如锌指核酸酶、TALE核酸酶、CRISPR-Cas核酸酶或大范围核酸酶,将编码本公开内容的融合蛋白的多核苷酸插入免疫细胞(例如,T细胞)的TCR基因、HLA基因或免疫抑制组分基因位点。不希望受到理论的束缚,将融合蛋白靶向TCR基因位点可能会增强表达融合蛋白的T细胞的抗肿瘤活性(见Eyquem等人,Nature,543:113-117(2017))。
在其他方面,提供了试剂盒,其包含(a)本文所述的载体或表达构建体和用于将载体或表达构建体转导至宿主细胞中的任选试剂,(b)本文公开的分离的多核苷酸或表达载体,以及用于将多核苷酸或表达载体转导到宿主细胞中的任选试剂,或(c)本公开的宿主细胞。
治疗方法
在某些方面,本公开中提供的组合物可以用于治疗受试者的疾病的方法,其中所述方法包括向受试者施用:融合蛋白;和载体,其包含编码融合蛋白的多核苷酸;修饰的表达融合蛋白的宿主细胞;或其药物组合物,其中所述疾病与融合蛋白结合的抗原的存在有关。在某些实施方案中,该疾病是病毒感染、细菌感染、癌症、炎性疾病或自身免疫性疾病。
如本文所用,术语“癌症”包括实体瘤和血液系统恶性肿瘤(例如,白血病)。可以治疗的示例性癌症包括黑素瘤;非小细胞肺癌;肾细胞癌;肾癌;血液学癌症;前列腺癌;去势抵抗性前列腺癌;结肠癌;直肠癌;胃癌;食道癌;膀胱癌;头颈癌;甲状腺癌;乳腺癌;三阴性乳腺癌;卵巢癌;子宫颈癌;肺癌;尿路上皮癌;胰腺癌;胶质母细胞瘤;肝细胞癌;脑癌;中枢神经系统癌;恶性神经胶质瘤,肉瘤和癌,包括软骨肉瘤;纤维肉瘤(成纤维细胞肉瘤);隆突性皮肤皮肤肉瘤(DFSP);骨肉瘤横纹肌肉瘤尤因氏肉瘤;胃肠道间质瘤;平滑肌肉瘤;血管肉瘤(血管肉瘤);卡波济肉瘤;脂肪肉瘤多形性肉瘤滑膜肉瘤PNET;恶性血管内皮瘤;恶性神经鞘瘤骨肉瘤肺泡软部分肉瘤;血管肉瘤膀胱肉瘤叶状;胶质瘤增生性小圆形细胞瘤;上皮样肉瘤骨骼外软骨肉瘤;骨骼外骨肉瘤;血管周细胞瘤;血管肉瘤;淋巴管肉瘤;淋巴肉瘤;未分化的多形性肉瘤;恶性周围神经鞘瘤(MPNST);神经纤维肉瘤横纹肌肉瘤未分化的多形性肉瘤;鳞状细胞癌;腺癌;腺鳞癌;间变性癌大细胞癌;小细胞癌;乳腺癌(例如,原位导管癌(非侵入性),原位小叶癌(非侵入性),侵袭性导管癌,侵袭性小叶癌,非侵入性癌);肝癌(例如,肝细胞癌,胆管癌或胆管癌);肺癌(例如,腺癌,鳞状细胞癌(类鳞癌)、大细胞未分化癌、支气管肺泡癌);卵巢癌(例如表面上皮间质瘤(腺癌)或卵巢上皮癌(包括浆液性肿瘤、子宫内膜样肿瘤和粘液性囊腺癌)、表皮样(鳞状细胞癌)、胚胎癌和绒毛膜癌(生殖细胞肿瘤));肾癌(例如,肾腺癌、肾炎、移行细胞癌(肾盂)、鳞状细胞癌、贝里尼导管癌、透明细胞腺癌、移行细胞癌、肾盂类癌);肾上腺癌(例如,肾上腺皮质癌)、睾丸癌(例如,生殖细胞癌(精原细胞瘤、绒毛膜癌、胚胎性癌、畸胎瘤)、浆液性癌);胃癌(例如腺癌);肠癌(例如十二指肠腺癌);大肠癌;或皮肤癌(例如基底细胞癌、鳞状细胞癌);基底细胞癌、腺癌;鼻炎塑料;病毒瘤胆管癌;肝细胞癌;腺样囊性癌肾细胞癌;Grawitz肿瘤,室管膜瘤;星形细胞瘤少突胶质细胞瘤脑干神经胶质瘤视神经胶质瘤;卵巢癌、卵巢上皮癌、宫颈腺癌或小细胞癌、胰腺癌、结直肠癌(例如,腺癌或鳞状细胞癌)、肺癌、乳腺导管癌、腺癌前列腺、软骨肉瘤;纤维肉瘤(成纤维细胞肉瘤);隆突性皮肤皮肤肉瘤(DFSP);骨肉瘤横纹肌肉瘤多形性肉瘤或滑膜肉瘤;肺癌(例如,腺癌、鳞状细胞癌(类鳞癌);鳞状细胞癌;腺癌;腺鳞癌;间变性癌;大细胞癌;小细胞癌;乳腺癌(例如,原位导管癌)(非侵入性)、原位小叶癌、非侵袭性导管癌、非侵袭性小叶癌、非侵袭性癌;肝癌(如肝细胞癌、胆管癌或胆管癌)、大细胞未分化癌、支气管肺泡癌;卵巢癌(例如表面上皮间质瘤(腺癌)或卵巢上皮癌(包括浆液性肿瘤、子宫内膜样肿瘤和粘液性囊腺癌)、表皮样(鳞状细胞癌)、胚胎癌和绒毛膜癌(生殖细胞肿瘤));肾癌(例如,肾腺癌、肾炎、移行细胞癌(肾盂)、鳞状细胞癌、贝里尼导管癌、透明细胞腺癌、移行细胞癌、肾盂类癌);肾上腺癌(例如,肾上腺皮质癌)、睾丸癌(例如,生殖细胞癌(精原细胞瘤,绒毛膜癌,胚胎性癌,畸胎瘤),浆液性癌);胃癌(例如腺癌);肠癌(例如十二指肠腺癌);大肠癌;或皮肤癌(例如基底细胞癌、鳞状细胞癌);卵巢癌,卵巢上皮癌、子宫颈腺癌或小细胞癌、胰腺癌、结直肠癌(例如,腺癌或鳞状细胞癌)、肺癌、乳腺导管癌或前列腺腺癌。
典型的血液系统恶性肿瘤包括急性淋巴细胞白血病(ALL);急性髓细胞性白血病(AML);慢性粒细胞性白血病(CML);慢性嗜酸性粒细胞白血病(CEL);骨髓增生异常综合症(MDS);霍奇金淋巴瘤、非霍奇金淋巴瘤(NHL)(例如滤泡性淋巴瘤、弥漫性大B细胞淋巴瘤或慢性淋巴细胞性白血病);骨髓瘤;多发性骨髓瘤(MM);浆细胞瘤浆细胞白血病的巨球蛋白血症;B细胞淋巴瘤;淋巴浆细胞性淋巴瘤伯基特氏淋巴瘤;小淋巴细胞淋巴瘤(SLL);滤泡性淋巴瘤免疫母细胞大细胞淋巴瘤;前体B淋巴母细胞淋巴瘤;和套细胞淋巴瘤;CD37+树突状细胞淋巴瘤;淋巴浆细胞性淋巴瘤脾边缘区淋巴瘤;黏膜相关(MALT)淋巴组织的结外边缘区B细胞淋巴瘤;淋巴结边缘区B细胞淋巴瘤;纵隔(胸腺)大B细胞淋巴瘤;血管内大B细胞淋巴瘤;原发性积液性淋巴瘤成人T细胞淋巴瘤;结外NK/T细胞淋巴瘤;鼻型肠病相关的T细胞淋巴瘤;肝脾T细胞淋巴瘤;NK细胞淋巴瘤Sezary综合征、血管免疫母细胞T细胞淋巴瘤;间变性大细胞淋巴瘤。
使用本文提供的组合物可以治疗的其他疾病包括病原微生物的感染,包括病毒(例如,HIV、BK多瘤病毒、腺病毒、丙型肝炎病毒(HCV)、乙型肝炎病毒(HBV)、巨细胞病毒(CMV)、爱泼斯坦-巴尔病毒(EBV)、细菌和寄生虫;另一方面,提供了用于治疗自身免疫疾病的方法,包括系统性红斑狼疮、糖尿病、类风湿性关节炎、反应性关节炎、多发性硬化、寻常性天疱疮、乳糜泻、克罗恩病、炎症性肠病疾病、溃疡性结肠炎和自身免疫性甲状腺疾病。
在某些实施方案中,所述受试者是人类或非人类动物,例如非人类灵长类动物、牛、马、绵羊、猪、猫、狗、山羊、小鼠、大鼠、兔、豚鼠。在一个实施方案中,受试者是人,例如成人、青少年、儿童或婴儿。
在某些实施方案中,给予受试者的修饰的宿主细胞是自体的、同种异体的或同基因的。
在某些实施方案中,用本公开内容提供的组合物治疗的受试者表现出缓慢或轻度的细胞因子释放综合征(CRS)、低或轻度的CAR T细胞相关性脑病综合征(CRES)或两者。与已经施用参考宿主细胞或组合物的受试者(即,参考受试者或同一受试者)相比,本公开提供的组合物表现出降低的细胞因子释放综合征细胞相关性脑病综合征,或两者兼有。蛋白包含野生型CD28共刺激信号结构域。
来自CD19特异性CAR T细胞临床应用的数据表明,与包含4-1BB/CD3ζ信号域的CAR相比,包含CD28/CD3ζ信号域的CAR在患者中更可能诱导严重CRS(Davila等人,Sci.TranslMed.6:224ra25(2014);Turtle等人,J.Clin.Invest.126:2123-2138(2016))。根据本公开的CD28共刺激信号传导域的修饰可以降低CRS、CRES或两者的发生率或严重性,在某些实施方案中,可以通过降低信号传导和细胞因子产生的强度同时保留抗肿瘤细胞毒性来达到此目的。不受理论的束缚,CRS和CRES是由于深层T细胞增殖和细胞因子释放而导致过度炎症的结果而发展的。CRS的症状包括发烧、不适、肌痛、厌食、恶心、心动过速、毛细血管渗漏、心脏功能障碍、肾功能不全、肝功能衰竭、弥散性血管内凝血、低血压、缺氧,并可影响身体的任何器官系统。CRES是一种毒性脑病,其特征是混乱、精神错乱、癫痫发作和脑水肿。CRS可演变为暴发性吞噬淋巴细胞组织细胞增生症(HLH),其特征是严重的免疫激活,淋巴细胞组织细胞浸润和免疫介导的多器官功能衰竭。包括干扰素-γ;IL-6;IL-8;sIL-2Rα;可溶性糖蛋白130(gp130);sIL-6R;IL-15;IL-8;IL-10;单核细胞趋化蛋白(MCP1);巨噬细胞炎性蛋白(MIP1)-α;MIP1-β和粒细胞巨噬细胞集落刺激因子(GM-CSF)的高血浆细胞因子,特别是在CAR T细胞治疗早期,与严重CRS高度相关。根据发烧、低血压、缺氧和器官毒性的存在,CRS分级为1至4级,其中严重CRS分级为≥3,并且表现出器官毒性和/或可能危及生命,需要积极的临床干预。1级CRS可表现为发烧和1级器官毒性,而对于2-4级,除发烧外,任何一项标准均已足够。根据神经系统评估评分和颅内压升高,癫痫发作或运动无力的情况,CRES评分为1-4级。评估CRS和CRES的方法是本领域已知的(参见,例如,Neelapu等人,Nat.Rev.Clin.Onc.15:47-62(2018);Lee等人,Blood(2014);CTCAE v4.03);Porter等.J.Hematol.Oncol.11:35(2018);以及Liu和Zhao,J.Hematol。Oncol.11:121(2018)),其方法和分级系统通过引用并入本文。在某些实施方案中,用本公开内容提供的组合物治疗的受试者此后不表现出≥3级的CRS或CRES。在某些实施例中,低水平或轻度的CRS或CRS是指等级小于3、等级2或等级1的CRS或CRES。分级可以根据任何本领域接受的方法、规模、或专栏,例如本文所述的那些。
抗原特异性T细胞应答可通过根据本文所述的任何T细胞功能参数(例如,增殖、细胞因子释放、CTL活性、改变的细胞表面标志物表型等)根据观察到的T细胞应答进行比较来确定。在适当情况下暴露于同源抗原的T细胞(例如,当由免疫相容性抗原呈递细胞呈递时用于引发或激活T细胞的抗原)与来自相同来源群体的T细胞之间产生的结构上不同或无关的对照抗原。具有统计学显着性的对同源抗原的应答大于对对照抗原的应答表示抗原特异性。
可以从受试者获得生物学样品,用于确定免疫反应的存在和水平,例如表达CAR或TCR的T细胞释放的细胞因子。如本文所用,“生物样品”可以是血液样品(可以从其制备血清或血浆)、活检样品;体液(例如肺灌洗液、腹水、粘膜洗涤液、滑液);骨髓;淋巴结;和淋巴结。组织外植体;器官培养物;或来自受试者或生物来源的任何其他组织或细胞制剂。也可以在接受任何免疫原性组合物之前从受试者获得生物样品,该生物样品可用作建立基线(即免疫前)数据的对照。
在一些实施方案中,可以通过本发明治疗的受试者是人类和其他灵长类受试者,例如用于兽医学目的的猴子和猿。在任何上述实施方式中,受试者可以是人类受试者。受试者可以是男性或女性,并且可以是任何合适的年龄,包括婴儿、少年、青少年、成人和老年受试者。如本领域技术人员所确定的,可以以适合于待治疗的疾病,病症或失调的方式施用根据本发明的细胞。在任何上述实施方案中,将包含本文所述细胞的细胞静脉内、腹膜内、肿瘤内地给予骨髓、淋巴结或脑脊髓液。组合物的合适剂量,合适的持续时间和给药频率将由诸如患者的年龄、大小、性别和状况等因素来确定;疾病、状况或失调的类型和严重性;活性成分的特定形式;和管理方法。
本文所述的融合蛋白,多核苷酸,载体或修饰的宿主细胞可以药学上或生理学上可接受或合适的赋形剂或载体的形式施用于受试者。药学上可接受的赋形剂是生物相容性载体,例如生理盐水,其在本文中更详细地描述,其适合于施用于人类或其他非人类哺乳动物受试者。在过继细胞疗法的上下文中,治疗有效剂量是能够产生临床上期望的结果(例如,细胞毒性T细胞)的过继转移中使用的宿主细胞(表达根据本发明的结合蛋白)的量在人类或非人类哺乳动物中以统计学上显着的方式反应)。如医学领域所公知的,任何一位患者的剂量取决于许多因素,包括患者的身材、体重、体表面积、年龄、所要施用的特定疗法、性别、施用时间和途径、总体健康状况,以及其他同时使用的药物。剂量将变化,但是用于施用包含本文所述的重组表达载体的宿主细胞的优选剂量为约105细胞/m2、约5x 105细胞/m2、约106细胞/m2、约5x 106细胞/m2、大约107细胞/m2、大约5x 107细胞/m2、大约108细胞/m2、大约5x 108细胞/m2、大约109细胞/m2、大约5x 109细胞/m2、大约1010细胞/m2、大约5个x1010细胞/m2、或大约1011细胞/m2。
细胞的数量将取决于组合物所预期的最终用途以及其中所含细胞的类型。例如,在某些实施方案中,经修饰以包含融合蛋白的细胞将包含含有至少30%、35%、40%、45%、50%、55%、60%、65%、70%、75%,80%,85%,90%,95%或100%的此类细胞的细胞群。对于本文提供的用途,细胞的体积通常为1升或更小、500ml或更小、250ml或更小或100ml或更小。在实施方案中,所需细胞的密度通常大于104细胞/ml,并且通常大于107细胞/ml,通常为108细胞/ml或更高。可以在一段时间内以单次输注或多次输注的形式给予细胞。临床相关数量的免疫细胞可以分配给多次输注,这些输注累计等于或超过105、106、107、108、109、1010或1011细胞。
在某些实施方案中,单位剂量包括(i)包含至少约30%、至少约40%、至少约50%、至少约60%、至少约70%、至少约80%、至少约85%、至少约90%或至少约95%的修饰或未修饰的CD4+T细胞,与(ii)包含至少约30%、至少约40%、至少约50%的、至少约60%,至少约70%,至少约80%,至少约85%,至少约90%或至少约95%的修饰或未修饰的CD8+T细胞的组合物组合约1:1的比例,其中单位剂量包含减少的量或基本上没有幼稚的T细胞(即,与具有相当数量的PBMC的患者样品相比,具有以单位剂量存在的幼稚T细胞的数量约小于约50%、小于约40%、小于约30%、小于约20%、小于约10%、5%、或少于约1%)。
在一些实施方案中,单位剂量包括(i)包含至少约50%的经修饰或未修饰的CD4+T细胞的组合物,以及(ii)包含至少约50%的经修饰或未经修饰的CD8+T细胞的1:1比例组合物,其中单位剂量包含的T细胞数量减少或基本没有。在进一步的实施方案中,单位剂量包含(i)包含至少约60%的经修饰或未修饰的CD4+T细胞的组合物,以及(ii)包含至少约60%的经修饰或未经修饰的CD8+T细胞的1:1比例组合物,其中单位剂量包含的T细胞数量减少或基本没有。在另外的实施方案中,单位剂量包含(i)包含至少约70%修饰或未修饰的CD4+T细胞的组合物,与(ii)包含至少约70%修饰或未修饰的CD8+T细胞的1:1的比例的组合物,其中单位剂量包含的T细胞数量减少或基本没有。在一些实施方案中,单位剂量包含(i)包含至少约80%的经修饰或未修饰的CD4+T细胞的组合物,以及(ii)包含至少约80%的经修饰或未经修饰的CD8+T细胞的1:1比例组合物,其中单位剂量包含的T细胞数量减少或基本没有。在一些实施方案中,单位剂量包含(i)包含至少约85%的经修饰或未修饰的CD4+T细胞的组合物,以及(ii)包含至少约85%的经修饰或未经修饰的CD8+T细胞的1:1比例组合物,其中单位剂量包含的T细胞数量减少或基本没有。在一些实施方案中,单位剂量包括(i)包含至少约90%的经修饰或未修饰的CD4+T细胞的组合物,以及(ii)包含至少约90%的经修饰或未经修饰的CD8+T细胞的1:1比例组合物,其中单位剂量包含的T细胞数量减少或基本没有。
在本文所述的任何实施方案中,单位剂量包含相等或近似相等数目的修饰或未修饰的CD45RA-CD3+CD8+和修饰或未修饰的CD45RA-CD3+CD4+TM细胞。
还考虑了药物组合物,其包含融合蛋白或表达如本文公开的融合蛋白的细胞和药学上可接受的载体、稀释剂或赋形剂。合适的赋形剂包括水、盐水、右旋糖、甘油等及其组合。在实施方案中,包含本文公开的融合蛋白或宿主细胞的组合物还包含合适的输注介质。合适的输注介质可以是任何等渗介质制剂,通常是生理盐水、Normosol R(Abbott)或Plasma-Lyte A(Baxter)、5%葡萄糖水溶液,可以使用林格氏乳酸盐。输注介质可以补充人血清白蛋白或其他人血清成分。
如医学领域技术人员所确定的,可以以适合于待治疗(或预防)的疾病或状况的方式施用药物组合物。组合物的合适剂量以及合适的持续时间和施用频率将由诸如患者的健康状况、患者的大小(即体重、质量或身体面积)、药物的类型和严重性等因素来确定。患者的疾病、有效成分的特殊形式以及给药方法。通常,合适的剂量和治疗方案以足以提供治疗和/或预防益处的量提供组合物(例如本文所述,包括改善的临床结果,例如更频繁的完全或部分缓解,或更长时间)。无疾病和/或总体生存率,或症状严重程度的减轻)。为了预防使用,剂量应足以预防、延迟与疾病或病症相关的疾病的发作或减轻其严重性。可以通过进行临床前(包括体外和体内动物研究)和临床研究,并分析通过适当的统计,生物学和临床方法及技术从中获得的数据,来确定根据本文所述方法施用的免疫原性组合物的预防益处,所有这些都可以由本领域技术人员容易地实践。
本文描述的药物组合物可以存在于单位剂量或多剂量容器中,例如密封的安瓿或小瓶中。这样的容器可以冷冻直到保持制剂的稳定性。在某些实施方案中,单位剂量包含本文所述的重组宿主细胞,其剂量为约105个细胞/m2至约1011个细胞/m2。开发用于在各种治疗方案中使用本文所述的特定组合物的合适的剂量和治疗方案,包括例如肠胃外或静脉内给药或制剂。
如果本发明的组合物肠胃外给药,则该组合物还可包含无菌的水性或油性溶液或悬浮液。合适的无毒胃肠外可接受的稀释剂或溶剂包括水、林格氏溶液、等渗盐溶液、1,3-丁二醇、乙醇、丙二醇或聚乙二醇与水的混合物。水溶液或悬浮液可进一步包含一种或多种缓冲剂,例如乙酸钠、柠檬酸钠、硼酸钠或酒石酸钠。当然,用于制备任何剂量单位制剂的任何材料应该是药学上纯的,并且在使用量上基本上是无毒的。另外,可以将活性化合物掺入缓释制剂和制剂中。如本文所用,剂量单位形式是指物理上离散的单位,其适合于作为待治疗对象的单位剂量。每个单元可以包含预定量的重组细胞或活性化合物,所述重组细胞或活性化合物经计算可与合适的药物载体结合产生期望的治疗效果。
通常,适当的剂量和治疗方案以足以提供治疗或预防益处的量提供活性分子或细胞。与未治疗的受试者相比,可以通过在治疗的受试者中建立改善的临床结局(例如,更频繁的缓解,完全或部分或更长的无病生存期)来监测这种反应。预先存在的针对肿瘤蛋白的免疫反应的增加通常与改善的临床结果相关。通常可以使用标准增殖,细胞毒性或细胞因子测定法来评估这种免疫应答,其可以使用在治疗之前和之后从受试者获得的样品来进行。
在某些实施方案中,治疗疾病的方法包括与一种或多种其他试剂组合施用修饰的免疫细胞。
在某些实施方案中,将本公开的修饰的免疫细胞与免疫抑制组分的抑制剂一起施用于受试者。
如本文所用,术语“免疫抑制组分”或“免疫抑制组分”是指提供抑制信号以帮助控制或抑制免疫应答的一种或多种细胞、蛋白质、分子、化合物或复合物。例如,免疫抑制成分包括部分或全部阻断免疫刺激的分子;减少,预防或延迟免疫激活;或增加,激活或上调免疫抑制。示例性免疫抑制组分靶标在本文中进一步详细描述,包括免疫检查点分子,例如PD-1、PD-L1、PD-L2、CD80、CD86、B7-H3、B7-H4、HVEM、腺苷、GAL9、VISTA、CEACAM-1、PVRL2、CTLA-4、BTLA、KIR、LAG3、TIM3、A2aR、CD244/2B4、CD160、TIGIT、LAIR-1、PVRIG/CD112R;代谢酶,例如精氨酸酶、吲哚胺2,3-双加氧酶(IDO);免疫抑制细胞因子,例如IL-10、IL-4、IL-1RA、IL-35;Treg细胞,或其任何组合。
免疫抑制成分的抑制剂可以是化合物、抗体、抗体片段或融合多肽(例如Fc融合物,例如CTLA4-Fc或LAG3-Fc)、反义分子、核酶或RNAi分子、或低分子量有机分子。在本文公开的任何实施方案中,方法可以包括将修饰的免疫细胞与以下免疫抑制组分中的任一种的一种或多种抑制剂单独或以任何组合给予。
在某些实施方案中,修饰的免疫细胞与PD-1抑制剂例如PD-1特异性抗体或其结合片段组合使用,所述抗体例如是pidilizumab、nivolumab(Keytruda,以前是MDX-1106),pembrolizumab(Opdivo,以前称为MK-3475),MEDI0680(以前称为AMP-514),AMP-224,BMS-936558或其任意组合。
在某些实施方案中,修饰的免疫细胞与PD-L1特异性抗体或其结合片段组合使用,例如BMS-936559、durvalumab(MEDI4736)、atezolizumab(RG7446)、avelumab(MSB0010718C)、MPDL3280A或任何组合其。
在某些实施方案中,修饰的免疫细胞与LAG3抑制剂例如LAG525、IMP321、IMP701、9H12、BMS-986016或其任何组合组合使用。
在某些实施方案中,修饰的免疫细胞与CTLA4的抑制剂组合使用。在特定的实施方案中,修饰的免疫细胞与CTLA4特异性抗体或其结合片段结合使用,所述抗体或其结合片段例如ipilimumab、tremelimumab、CTLA4-Ig融合蛋白(例如,ababatcept、belatacept)或其任何组合。
在某些实施方案中,修饰的免疫细胞与B7-H3特异性抗体或其抗原结合片段,例如依诺妥珠单抗(MGA271)、376.96或两者结合使用。
在某些实施方案中,修饰的免疫细胞与B7-H4特异性抗体或其结合片段,例如scFv或其融合蛋白结合使用,如例如Dangaj等人,Cancer Res.73:4820,2013,以及美国专利第9,574,000号和PCT专利公开号WO 2016/40724和WO 2013/025779中所述的那些。
在一些实施方案中,修饰的免疫细胞与CD244的抑制剂组合使用。
在某些实施方案中,修饰的免疫细胞与BLTA、HVEM、CD160或其任何抑制剂的抑制剂组合使用。抗CD160抗体描述于例如PCT公开号WO 2010/084158。
在更多的实施方案中,修饰的免疫细胞与TIM3的抑制剂组合使用。
在更多实施方案中,修饰的免疫细胞与Gal9的抑制剂组合使用。
在某些实施方案中,修饰的免疫细胞与腺苷信号传导抑制剂如诱饵腺苷受体联合使用。
在某些实施方案中,修饰的免疫细胞与A2aR抑制剂组合使用。
在某些实施方案中,修饰的免疫细胞与KIR抑制剂,例如lirilumab(BMS-986015)组合使用。
在某些实施方案中,修饰的免疫细胞与抑制性细胞因子(通常是除TGFβ以外的细胞因子)或Treg发育或活性的抑制剂组合使用。
在某些实施方案中,将修饰的免疫细胞与IDO抑制剂例如左-1-甲基色氨酸,依帕考定(INCB024360;Liu等人,Blood 115:3520-30,2010)、ebselen(Terentis等人),Biochem.49:591-600,2010)、吲哚莫德、NLG919(Mautino等人,美国癌症研究协会第104届年会,2013年4月6-10日)、1-甲基色氨酸(1-MT)-替拉扎明或其任何组合组合使用。
在某些实施方案中,修饰的免疫细胞与精氨酸酶抑制剂组合使用,所述精氨酸酶抑制剂例如N(ω)-硝基-L-精氨酸甲酯(L-NAME)、N-ω-羟基-nor-l-精氨酸(nor-NOHA)、L-NOHA、2(S)-氨基-6-硼代己酸(ABH)、S-(2-硼代乙基)-L-半胱氨酸(BEC)或其任意组合。
在某些实施方案中,修饰的免疫细胞与VISTA的抑制剂例如CA-170(Curis,Lexington,MA)组合使用。
在某些实施方案中,修饰的免疫细胞与LAIR1抑制剂组合使用。
在某些实施方案中,修饰的免疫细胞与CEACAM-1、CEACAM-3、CEACAM-5或其任何组合的抑制剂组合使用。
在某些实施方案中,修饰的免疫细胞与增加刺激性免疫检查点分子的活性的试剂(即,激动剂)组合使用。例如,修饰的免疫细胞可以与CD137(4-1BB)激动剂(例如urelumab)、CD134(OX-40)激动剂(例如MEDI6469、MEDI6383、或MEDI0562)、来那度胺、泊马利度胺、CD27激动剂(例如CDX-1127)、CD28激动剂(例如TGN1412、CD80或CD86)、CD40激动剂(例如,CP-870,893、rhuCD40L或SGN-40)、CD122激动剂(例如IL-2)、GITR激动剂(例如PCT专利公开号WO 2016/054638中所述的人源化单克隆抗体)或ICOS(CD278)的激动剂(例如,GSK3359609、mAb 88.2、JTX-2011、Icos 145-1或Icos 314-8),或其任意组合。在本文公开的任何实施方案中,一种方法可以包括将修饰的免疫细胞与一种或多种刺激性免疫检查点分子的激动剂一起施用,包括上述任何一种,或者以任何组合的形式。
在其他实施方案中,本公开的方法进一步包括给予包括以下中的一种或多种的次级疗法:对由所靶向的实体瘤表达的癌症抗原具有特异性的抗体或抗原结合片段;和化学治疗剂;手术;放射治疗;细胞因子;RNA干扰疗法,或其任何组合。
可用于癌症疗法的示例性单克隆抗体包括,例如,在Galluzzi等人,Oncotarget5(24):12472-12508(2014)中描述的单克隆抗体,该抗体通过引用整体并入本文。
在某些实施方案中,联合治疗方法包括给予修饰的免疫细胞并进一步对受试者进行放射治疗或手术。放射疗法包括X射线疗法(例如γ射线)和放射性药物疗法。适用于治疗给定的癌症或非发炎的实体瘤的手术和外科技术可以与本公开的修饰的免疫细胞组合用于受试者。
在某些实施方案中,组合疗法方法包括将修饰的免疫细胞和化学治疗剂给予受试者。化学治疗剂包括但不限于染色质功能抑制剂、拓扑异构酶抑制剂、微管抑制药物、DNA损伤剂、抗代谢物(例如叶酸拮抗剂、嘧啶类似物、嘌呤类似物和糖修饰的类似物)、DNA合成抑制剂、DNA交互作用剂(例如嵌入剂)和DNA修复抑制剂。示例性化学治疗剂包括但不限于以下组:抗代谢物/抗癌剂、例如嘧啶类似物(5-氟尿嘧啶、氟尿苷、卡培他滨、吉西他滨和阿糖胞苷)和嘌呤类似物、叶酸拮抗剂和相关抑制剂(巯基嘌呤、硫鸟嘌呤、喷司他丁和2-氯脱氧腺苷(克拉屈滨));抗增殖/抗有丝分裂剂包括长春花生物碱(长春碱、长春新碱和长春瑞滨)、微管干扰物、如紫杉烷(紫杉醇、多西紫杉醇)、长春新碱、长春花碱、诺考达唑、埃博霉素和萘韦尔滨、表鬼臼毒素(鬼臼乙叉甙、依托泊苷、氨苄青霉素、蒽环类、博来霉素、白消安、喜树碱、卡铂、苯丁酸氮芥、顺铂、环磷酰胺、癌得星、放线菌素、柔红霉素、阿霉素、表柔比星、六甲基三聚氰胺草酸铂、异磷酰胺、美育素、三聚氰胺、三聚氰胺、三聚氰胺、四氢呋喃、四氢呋喃替莫唑胺、替尼泊苷、三亚乙基硫代磷酰胺和依托泊苷(VP 16));抗生素,如放线菌素(放线菌素D)、柔红霉素、阿霉素(阿霉素)、伊达比星、蒽环类、米托蒽醌、博来霉素、普卡霉素(线霉素)和丝裂霉素;酶(L天冬酰胺酶可全身性代谢L-天冬酰胺,并剥夺没有能力合成其自身天冬酰胺的细胞);抗血小板药;抗增殖/抗有丝分裂的烷基化剂,例如氮芥子碱(甲氯乙胺、环磷酰胺及其类似物、美法仑、苯丁酸氮芥)、亚乙基亚胺和甲基三聚氰胺(六甲基三聚氰胺和噻替帕)、烷基磺酸盐-白硫丹、亚硝基脲(卡莫司汀(卡莫汀)和类似物、链金星霉素)(DTIC);抗增殖/抗有丝分裂抗代谢物,例如叶酸类似物(甲氨蝶呤);铂配位配合物(顺铂、卡铂)、丙卡巴嗪、羟基脲、米托坦、氨基谷氨酰胺;激素、激素类似物(雌激素、他莫昔芬、戈舍瑞林、比卡鲁胺、尼鲁米特)和芳香酶抑制剂(来曲唑、阿那曲唑);抗凝剂(肝素、合成肝素盐和其他凝血酶抑制剂);纤维蛋白溶解剂(例如组织纤溶酶原激活剂、链激酶和尿激酶)、阿司匹林、双嘧达莫、噻氯匹定、氯吡格雷、阿昔单抗;反移民代理;抗分泌剂(breveldin);免疫抑制剂(环孢霉素、他克莫司(FK-506)、西罗莫司(雷帕霉素)、硫唑嘌呤、霉酚酸酯);抗血管生成化合物(TNP470、染料木黄酮)和生长因子抑制剂(血管内皮生长因子(VEGF)抑制剂、成纤维细胞生长因子(FGF)抑制剂);血管紧张素受体阻滞剂;一氧化氮供体;反义寡核苷酸;抗体(曲妥珠单抗、利妥昔单抗);嵌合抗原受体;细胞周期抑制剂和分化诱导剂(维甲酸);mTOR抑制剂、拓扑异构酶抑制剂(阿霉素、阿霉素、喜树碱、喜树碱、柔红霉素、放线菌素、依诺泊苷、表柔比星、依托泊苷、伊达柔比星、伊立替康(CPT-11)和米托蒽醌、托泊替康、甲基氢吗啡酮、氢化可的松、依地替康、依地美可松、可可甜、泼尼松和泼尼松);生长因子信号转导激酶抑制剂线粒体功能障碍诱导剂、霍乱毒素、蓖麻毒蛋白、假单胞菌外毒素,百日咳博德特氏菌腺苷酸环化酶毒素或白喉毒素等毒素,以及胱天蛋白酶激活剂;和染色质破坏剂。
细胞因子可用于操纵宿主针对抗癌活性的免疫反应。参见例如Floros andTarhini,Semin.Oncol.42:539,2015.可用于促进抗癌或抗肿瘤反应的细胞因子包括,例如IFN-α、IL-2、IL-3、IL-4、IL-10、IL-12、IL-13、IL-15、IL-16、IL-17、IL-18、IL-21、IL-24和GM-CSF单独或以任何组合使用。
另一种癌症治疗方法涉及减少癌基因以及癌细胞生长、维持、增殖和免疫逃避所需的其他基因的表达。RNA干扰,尤其是使用microRNA(miRNA),小型抑制性RNA(siRNA)提供了一种降低癌症基因表达的方法。参见,例如,Larsson等,Cancer Treat.Rev.16:128,2017。
在本文公开的任何实施方案中,任何治疗剂(例如,修饰的免疫细胞、免疫抑制成分的抑制剂、刺激性免疫检查点分子的激动剂、化学治疗剂、放射疗法、外科手术,细胞因子或抑制性RNA)可以在治疗过程中一次或多次施用于受试者,并且可以以任何顺序(例如,同时、同时或以任何顺序)组合施用于受试者)或任何组合。组合物的合适剂量、合适的持续时间和施用频率将由诸如患者的状况等因素来确定。肿瘤或癌症的大小、类型、扩散、生长和严重性;活性成分的特定形式;和管理方法。
在某些实施方案中,向受试者施用多种剂量的本文所述的修饰的免疫细胞,其可以在约两周至约四周的施用之间的间隔内施用。在进一步的实施方案中,顺序给予细胞因子(例如IL-2、IL-15、IL-21),条件是在给予细胞因子之前至少向受试者给予重组宿主细胞至少三或四次。在某些实施方案中,细胞因子与宿主细胞同时施用。在某些实施方案中,皮下施用细胞因子。
在另外的实施方案中,被治疗的受试者进一步接受免疫抑制疗法,例如钙调神经磷酸酶抑制剂、皮质类固醇、微管抑制剂、低剂量的霉酚酸前药或其任何组合。在另外的实施方案中,被治疗的受试者已经接受了非清髓性或造血性造血细胞移植,其中可以在所述非清髓性的造血细胞移植后至少两至至少三个月施用所述治疗。
如本文所述,治疗或药物组合物的有效量是指在所需剂量和所需时间段内足以达到所需临床结果或有益治疗的量。有效量可以一次或多次给药方式递送。如果对已知或已确认患有某种疾病或疾病状态的受试者给药,则术语“治疗量”可以用于治疗,而“预防有效量”可以用于描述给药有效量的方法。作为预防性过程易感或有患疾病或疾病状态(例如复发)的风险的受试者。
示例
示例1
材料与方法
从健康供体获得外周血T细胞
健康成年人(>18岁)参加了机构审查委员会批准的外周血收集研究。所有参与者均已获得知情同意。研究人员对所有与研究参与者有关的个人身份信息一无所知,仅提供了捐赠者的年龄和无描述的捐赠者ID号。通过静脉穿刺收集400cc的外周血,并使用淋巴细胞分离培养基(Corning Cat#25-072-CV)通过密度梯度分离出单核细胞(PBMC)。使用EasySep人CD8+T细胞分离试剂盒(Stem Cell Technologies目录号#s17952和17953)分离CD4+和CD8+T细胞。通过用CD62L-PE(ThermoFisher目录号12-0629-42)和EasySep人PE选择试剂盒(Stem Cell Technologies目录号18551)染色,进一步富集CD8+CD62L+T细胞。按照制造商的说明进行分离。
细胞培养
在补充有10%胎牛血清,1mM L-谷氨酰胺(Gibco)和100U/mL青霉素/链霉素(Gibco)的DMEM(Gibco)中培养293T LentiX细胞(Clontech Cat#632180)。从ATCC获得K562(CCL-243)和Jurkat(TIB-152)细胞,并在补充有5%胎牛血清和100U/mL青霉素/链霉素的RPMI-1640(Gibco)中培养。将人类原代T细胞在CTL培养基中培养,该培养基由RPMI-1640补充,其中RPMI-1640补充了10%的人血清,2mM L-谷氨酰胺,100U/mL青霉素/链霉素,50μMβ-巯基乙醇(Sigma)和50U/mL IL-2(Prometheus Proleukin/Aldesleukin)。所有细胞均在37℃和5%CO2下培养,并使用MycoAlert支原体检测试剂盒(Lonza Cat#LT07-318)每两个月测试一次是否存在支原体。
嵌合抗原受体(CARs)和重组慢病毒载体的产生
先前已经描述了CD19特异性和ROR1特异性CAR构建体(Hudecek等人,Clin.CancerRes.19:3153-3164(2013);Sommermeyer等人,Leukemia 30:492-500(2016)(图1A在FMC63(SEQ ID NO:8)或R12单链可变片段(SEQ ID NO:9)和IgG4铰链序列之间插入了一个单一的Strep-tag II序列(SEQ ID NO:10)和两个G4S接头(SEQ ID NO:12)(Liu等人,Nat.Biotechnol.34:430–434(2016))。它们与人CD28的27个氨基酸跨膜结构域相连(UniProt:P10747;SEQ ID NO:13)并包含一个信号模块,该模块包含(i)人CD28的41个氨基酸的胞质结构域,具有LL→GG取代,位于天然CD28蛋白的186-187位(Nguyen等,Blood 102:4320–4325(2003)(SEQ ID NO:3)或(ii)人4-1BB的42个氨基酸的胞质结构域(UniProt:Q07011;SEQ ID NO:14),每个都连接到4-1BB的112个氨基酸的胞质结构域人CD3ζ的同工型3(UniProt:P20963-3;SEQ ID NO:15)。通过定点诱变产生在CD28 UniProt位置206、209和218上具有酪氨酸至苯丙氨酸取代的突变CD28/CD3ζCAR。所有CAR基因构建体通过编码T2A的序列(SEQ ID NO:16)与编码截短的表皮生长因子受体(EGFRt;SEQ ID NO:17)的序列连接,经过密码子优化,并克隆到HIV7慢病毒载体中。为了制备抗原阳性的K562细胞,将人CD19的氨基酸1-325(UniProt:P15391)克隆到HIV7慢病毒载体中,将人ROR1的氨基酸1-937(UniProt:Q01973)克隆到mp71逆转录病毒载体中。所有克隆均通过PCR,酶切和/或Gibson组装进行。通过毛细管测序和限制酶切消化验证质粒。
慢病毒的制备和转导
为了制备CAR T细胞,用HIV7 CAR载体以及psPAX2(Addgene目录号12260)和pMD2.G(Addgene目录号12259)包装质粒瞬时转染LentiX细胞。一天后(第1天),使用Dynabeads人T激活剂CD3/CD28(ThermoFisher目录号11132D)激活原代T细胞,并在添加了50U/mL IL-2的CTL中培养。第二天(第2天),从LentiX细胞中收获慢病毒上清液,使用0.45μmPES注射器过滤器(Millipore Cat#SLHP033RB)过滤,然后添加到活化的T细胞中。加入聚乙烯(Millipore目录号TR-1003-G)至终浓度为4.4μg/mL,并将细胞在800×g和32℃旋转接种90分钟。病毒上清液在8小时后用补充了50U/mL IL-2的新鲜CTL代替。然后每隔48小时使用补充了50U/mL IL-2的CTL进行半培养基更换。在第6天去除了Dynabeads;在第9天,将CD8+EGFRt+转导的T细胞在FACSAriaII(BD Biosciences)上进行FACS分选。
为了制备K562/CD19细胞,用psPAX2,pMD2.G和编码CD19的HIV7慢病毒载体瞬时转染LentiX细胞。为了制备K562/ROR1细胞,用MLV g/p,10A1和编码ROR1的mp71逆转录病毒载体瞬时转染LentiX细胞(Uckertet等人,Hum.Gene Ther.11:1005-1014(2000))。两天后,用0.45μmPES针筒式过滤器过滤病毒上清液,并添加到K562细胞中。五天后,将转导的K562细胞对CD19(Biolegend Cat#302212)或ROR1(Miltenyi Biotec Cat#130-098-317)特异性染色的单克隆抗体,并在FACSAria II上进行FACS分选。
T细胞扩展用于质谱和功能分析
在MS和/或功能分析之前,将FACS分选的CD8+EGFRt+细胞在单个刺激周期内扩增。通过与CD19+淋巴母细胞细胞系(LCL)共培养,以1:7(T细胞:LCL)的比例扩增CD19特异性CAR-T细胞,并在刺激后8天进行检测。使用包含纯化的OKT3,LCL,辐照的PBMC的快速扩增方案扩增ROR1特异性CAR-T细胞,并在刺激后11天进行测定。在扩增过程中,每2-3天将新鲜的CTL培养基(含50U/mL IL-2)喂入培养物。
流式细胞仪和细胞表型
T细胞用从BD Biosciences,ThermoFisher或Biolegend购买的荧光团偶联单克隆抗体的1:100稀释液染色。使用EZ-Link Sulfo-NHS-Biotin试剂盒(ThermoFisher Cat#21217)对西妥昔单抗(抗EGFR,Bristol Myers Squibb)和3E8(抗STII,FHCRC)mAb进行生物素化处理,然后使用Zeba旋转脱盐柱(ThermoFisher)进行纯化Cat#89882),并与链霉亲和素APC(ThermoFisher Cat#17-4317-82)一起对T细胞染色。通过用70%的冰冷乙醇固定T细胞,用1%的Triton-X渗透细胞(Sigma Cat#T8787),用100μg/mL RNAse A降解RNA(ThermoFisher Cat#EN0531),对DNA进行染色。含20μg/mL碘化丙啶(ThermoFisher目录号P3566)。所有数据均在FACSCanto II(BD Biosciences)上收集,并使用FlowJo版本9(Treestar)进行分析。
抗STII和对照珠的制备
用台式磁体在补充了100U/mL青霉素/链霉素(PBS+P/S)的过量1PBS中洗涤1mL涂有链霉亲和素的磁性颗粒(Spherotech目录号SVMS-30-10)一次。通过将珠沉淀重悬于1mLPBS+P/S中来制备抗STII珠。涡旋时,每1mL磁珠添加16.67μg抗STII mAb(GenScript目录号A01737)。将珠子和抗STII抗体在3D轨道振荡器上孵育过夜,使用台式磁体用过量的PBS+P/S洗涤3次,然后重悬于4mL PBS+P/S。为了制备对照珠,使用台式磁体将1mL涂有链霉亲和素的磁性颗粒洗涤一次,并将珠粒重悬于4mL PBS+P/S中。所有珠粒均储存在4℃下。
细胞刺激,蛋白质裂解物和RNA分离
洗涤CAR T或Jurkat细胞,并重悬于温热的CTL培养基中。对于OKT3刺激,将细胞与1.25μg/mL OKT3(Biolegend Cat#317303)在冰上孵育5分钟,然后与5μg/mL抗小鼠IgG(ThermoFisher Cat#A16068)在冰上孵育10分钟。将细胞转移至37℃水浴中以启动信号传导。对于基于K562细胞的刺激,在37℃水浴中将CAR T细胞与K562或K562/CD19细胞以效应子与靶标比率为4:1共培养。对于基于珠子的刺激,将T细胞与STII或对照微珠在37℃水浴中以每1×106个细胞30μL珠的比例孵育(图1B)。在分配的时间后,将细胞用冰冷的PBS快速洗涤两次,然后在6M尿素,25mM Tris(pH 8.0),1mM EDTA,1mM EGTA溶液中裂解,并添加蛋白酶(Sigma Cat#P8340-1ML)和磷酸酶抑制剂(Sigma Cat#s P5726-1ML和P0044-1ML)以1:100的稀释度稀释,在此称为裂解缓冲液。超声处理裂解液15秒钟,然后在10,000×g和4℃下离心10分钟。在裂解液清除过程中去除了珠子。刺激6小时后,根据制造商的说明使用NucleoSpin RNA试剂盒(Macherey-Nagel Cat#740955)进行RNA分离。在使用台式磁铁进行细胞裂解之前,先去除珠子。
蛋白质消化,TMT标记和磷酸酪氨酸(pTyr)肽免疫沉淀
通过Micro BCA分析(ThermoFisher Cat#23235)对裂解物中的蛋白质进行定量,然后使用裂解缓冲液将裂解物稀释至2mg/mL。在37℃摇动下,在24mM TCEP(ThermoFisherCat#77720)中将裂解液还原30分钟,然后在黑暗中于室温在室温下与48mM碘乙酰胺(SigmaCat#A3221)烷基化30分钟。然后将溶胞产物用200mM Tris(pH 8.0)稀释至2M尿素浓度。将Lys-C(Wako Cat#125-05061)以200ug/mL的浓度溶于25mM Tris(pH 8.0)中,并以1:100(酶:蛋白质)的质量比添加至裂解液中,并在37℃下孵育2小时并振动。样品进一步用200mMTris(pH 8.0)稀释至尿素浓度为1M,然后以1:50的胰蛋白酶:蛋白质比例添加胰蛋白酶。2小时后,以1:100的胰蛋白酶:蛋白质比例加入第二胰蛋白酶等分试样。在37℃振荡下消化过夜。16小时后,将反应用甲酸淬灭至终浓度为1体积%。使用Oasis HLB 96孔板(Waters目录号WAT058951)和正压歧管(Waters目录号186005521)对样品进行脱盐。用3x 400μL的50%MeCN/0.1%FA洗涤平板孔,然后用4x 400μL的0.1%FA平衡。将消化液加到孔中,然后用4x 400μL 0.1%FA洗涤,然后用3x 400μL 50%MeCN/0.1%FA逐滴洗脱。将洗脱液冻干,然后在-80℃下保存直至使用。对于TMT标记(ThermoFisher,目录号90406),基于起始蛋白质质量,将脱盐的肽以1mg/mL的浓度重悬于50mM HEPES中。将TMT试剂重悬于257μL MeCN中,并转移至肽样品中。将样品在室温下混合孵育1小时。通过添加50μL 5%羟基胺(SigmaCat#438227)淬灭标记反应,并在室温下混合孵育15分钟。然后将独立的标记反应汇集在一起并冻干。如上所述将标记的肽脱盐,然后冻干并保存在-80℃。然后按照制造商的说明进行pTyr肽的免疫沉淀(Cell Signaling目录号8803)。使用C18 Spin Tip(ThermoFisherCat#84850)纯化富集的pTyr肽级分,冻干并保存在80℃直至分析。将流通部分脱盐,冻干并储存在80℃下。
碱性(高pH)反相液相色谱
通过高pH反相(RP)液相色谱分离脱盐和pTyr肽耗尽的流通液。将4mg的蛋白质消化物上样到LC系统,该系统由Agilent 1200HPLC组成,流动相为5mM NH4HCO3(pH 10)(A)和5mM NH4HCO3的90%MeCN(pH 10)(B)。下列时间表在96分钟内以4.6mm x 250mm ZorbaxExtend-C18、3.5μm色谱柱(Agilent Cat#770953-902)分离肽,流速为1.0mL/min:将0%B保持9分钟,从0到10%B持续4分钟,从10到28.5%B持续50分钟,从28.5到34%B持续5.5分钟,从34至60%B持续13分钟,在60%B保持8.5分钟,60到0%B持续1分钟,然后在0%B重新平衡5分钟。在1mL深孔板(Thermo Cat#95040450)中,通过最短路径逐段收集0-96分钟内的1分钟级分。从第15分钟开始,通过每隔一个板柱将高pH RP馏分合并为24个样品(例如:样品1包含来自B10,D10,F10等孔的馏分)。合并剩余的馏分,以便将12至14分钟的馏分添加到样品1中,将86分钟后的所有馏分添加到样品24中,并将0到11分钟的所有馏分合并到样品“A”中。将24个样品中每12个馏分中的95%(1,13;2,14;...)合并,再生成12个样品,将其干燥并保存在-80℃,然后通过固定的金属亲和力富集磷酸肽层析。
固定金属亲和层析(IMAC)
使用剥去EDTA的Ni-NTA-琼脂糖珠(Qiagen Cat#36113)进行IMAC富集,并在10mMFeCl3溶液中孵育以制备Fe3+-NTA-琼脂糖珠。分馏的裂解物在80%MeCN中的200μL0.1%TFA中重构,并与100μL 5%珠悬浮液一起孵育30分钟,同时在室温下混合。孵育后,将小珠用300μL的80%MeCN中的0.1%TFA洗涤3次。在室温搅拌下,使用200μL70%ACN,1%氢氧化铵从珠子上洗脱磷酸化的肽1分钟。将样品转移到装有60uL 10%FA的新鲜试管中,干燥并重悬于0.1%FA,3%MeCN中。将样品冷冻在-80℃直至分析。
纳米液相色谱-串联质谱
通过LC-MS/MS在Easy-nLC 1000(ThermoFisher)和以正离子模式运行的LTQ-Orbitrap Fusion质谱仪(ThermoFisher)上对富含磷酸肽的样品进行分析。LC系统以通风形式配置,包括一个内部装有ReproSil-Pur C18-AQ,3μm的熔融石英纳米喷雾针(PicoTipTM发射器,50μmID x 20cm,New Objective)和一个捕集阱(IntegraFritTM毛细管),100μm内径x2厘米,新物镜)所含树脂与分析柱中的树脂相同,流动相在水中的0.1%FA(A)和在MeCN中的0.1%FA(B)。将肽样品在20μL的0.1%FA,3%MeCN中稀释,并将8.5μL上样到色谱柱上,并以300nL/min的流速在210分钟内分离,梯度为5至7%B,持续2分钟,7至35%B持续150分钟,35至50%B 1分钟,保持50%B 9分钟,50至95%B 2分钟,保持95%B 7分钟,95至5%B 1分钟,在5%B下重新平衡38分钟。将2000V的喷雾电压施加至纳米喷雾头。MS/MS分析发生在3秒的周期内,包括1个分辨率为120,000的350-1500m/z的全扫描MS,然后是使用HCD激活的数据相关MS/MS扫描,其中27%的归一化碰撞能量为最丰富离子。重复计数1后,动态排除选定离子45秒钟。
蛋白质印迹
将等质量的5-13.3μg蛋白质裂解物装入4-12%Bis-Tris NuPAGE凝胶(ThermoFisher)或3-8%Tris-醋酸NuPAGE凝胶(ThermoFisher)中。将蛋白质转移到硝酸纤维素膜(ThermoFisher目录号LC2001)上后,用Western封闭试剂(Sigma目录号11921673001)封闭膜。膜用在添加了0.1%Tween的SuperBlock中稀释的一级和二级mAb染色。在该研究中使用了以下抗体:抗人CD247(BD Biosciences目录号551034),抗人CD247pY142(BD Biosciences目录号558402),抗ZAP-70pY319(Cell Signaling Cat#2717S),抗SLP-76pS376(Cell Signaling Cat#14745S),抗PLC-g1 pY783(Cell Signaling Cat#14008S)和anti-DAPP1 pY139(Cell Signaling Cat#13703S),抗鼠标HRP(Cell SignalingCat#7076S),以及抗兔HRP(Cell Signaling Cat#7074S)。典型的抗体稀释度范围为1/10,000至1/2500。使用CL-XPosure胶片(ThermoFisher目录号34091)或ChemiDoc XRS+系统(Bio-Rad)显影印迹。
体外功能测定
将CAR T细胞与K562,K562/CD19或K562/ROR1细胞以T细胞与肿瘤细胞的比例为2:1共培养。在某些实验中,CAR T细胞还与对照或抗STII珠以每百万个细胞30μL珠的比率孵育。刺激后24小时,通过ELISA(ThermoFisher目录号88-7316-88、88-7025-88、88-7346-88)定量细胞上清液中的细胞因子浓度。对于细胞内细胞因子染色,在测定设置期间添加了GolgiPlug(BD Bioscience目录号555029),并在刺激5小时后收获细胞并进行了染色。通过用0.2μM的羧基荧光素琥珀酰亚胺酯(CFSE)染料溶液(ThermoFisher Cat#C34554)对CAR T细胞进行染色并将CAR T细胞与K562/CD19,K562/ROR1细胞,对照珠或抗STII珠粒持续72小时。
RNA测序(RNA-seq)
从三个供体的24个样品中提取RNA。使用Agilent 4200TapeStation(安捷伦科技公司)检查总RNA完整性,并使用Trinean DropSense96分光光度计(Caliper LifeSciences)进行定量。使用TruSeq RNA样品制备试剂盒v2(Illumina目录号RS-122-2001)和Sciclone NGSx工作站(PerkinElmer)从总RNA中制备RNA-seq文库。库大小分布已使用Agilent 4200TapeStation进行了验证。使用Qubit 2.0荧光计(ThermoFisher)进行附加的库质量控制,合并索引库的混合以及聚类优化。汇集RNA-seq文库(6-8重),并聚簇到流通池泳道上。使用Illumina HiSeq 2500在快速模式下进行测序,采用配对末端50碱基读取长度(PE50)测序策略。
定量PCR
提取RNA,并用SuperScript III(ThermoFisher Cat#11752-250)制备500ngcDNA。将15ng cDNA添加到以下TaqMan测定探针之一的反应中:Hs00953178_m1(EPHA4),Hs00172973_m1(FOXO4),Hs00902234_m1(IL7R),Hs00360439_g1(KLF2)或Hs99999907_m1(B2M)。反应在一个384孔板上进行一式两份或一式三份。ΔCt值通过将来自EPHA4,FOXO4,IL7R和KLF2探针的技术三联体的平均Ct除以管家基因β2微球蛋白(B2M)的平均Ct来计算。计算每个供体的CD28/CD3ζ,M1或M3与4-1BB/CD3ζ之比,并进行log2转化。
NOD/SCID/γc-/-(NSG)鼠标模型
通过尾静脉注射向6至8周大的雄性NSG小鼠移植5×105Raji/ffluc细胞。一周后,静脉注射PBS或已用CD19+爱泼斯坦巴尔病毒淋巴母细胞扩增9天的CD8+和CD4+CAR T细胞混合物(9x105细胞)。如Sommermeyer等人(Leukemia 30:492-500(2016))所述进行生物发光成像。对于预期个体小鼠之间存在差异的实验,每个实验组至少使用五只小鼠进行数据分析,以提供81%的能力检测1.75的效应量,基于t检验的单侧显着性水平为0.05。小鼠处理者不知道组分配。治疗组如下:PBS/Sham(n=6只小鼠);和PBS。4-1BB/CD3ζCAR T细胞(n=15小鼠);CD28/CD3ζCAR T细胞(n=15小鼠);CD28/CD3ζY1 CAR T细胞(n=15小鼠);CD28/CD3ζY3 CAR T细胞(n=15只小鼠)。
弹枪质谱数据分析
使用MaxQuant/Andromeda搜索引擎1.6.0.1版,将每个重复实验的原始MS/MS谱图与已审查的人类通用蛋白质资源(UniProt)序列数据库(2016_01版)以及常见实验室污染物一起进行搜索(Cox et al.,Nat.Biotechnol.26:1367–1372(2008)。使用胰蛋白酶限制了多达两个缺失的裂解,进行了搜索,其中氧化甲硫氨酸,磷酸化丝氨酸,磷酸化苏氨酸和磷酸化酪氨酸进行了可变修饰,将氨基甲酰甲基化的半胱氨酸设置为静态修饰:肽MH+的质量公差设置为20ppm,使用反向数据库目标诱饵方法将总FDR设置为≤1%。
对于这三个TMT实验,通过MaxQuant确定磷酸肽位点的定位,并使用Perseus版本1.6.0.7将其转换为磷酸化位点(Tyanova等人,Nat.Methods13:731–740(2016))。在此步骤中,将反向命中率和潜在污染物排除在进一步分析之外。数据归一化是通过将每个TMT通道缩放到通道中位数来执行的,然后进行log2转换。通过从刺激通道中减去适当的控制通道来计算刺激与控制比率。由于MS采样不完全,仅在一次或两次重复实验中发现了一些磷酸化位点(特征),而在每个TMT通道中均未发现更小的少数位点(<1%)。
使用limma统计框架和相关的R包对PO4位点进行差异表达分析(G.K.Smyth,StatAppl Genet Mol Biol 3:Article3(2004);Ritchie等人,Nucleic Acids Res.43:e47(2015))。对于这些分析,仅保留那些至少在两个实验中具有值且所有TMT通道均具有值的特征,从而保留14490个量化的磷酸化位点。将线性模型拟合到每个PO4位点,并使用经验贝叶斯调节的t统计量来评估表达/丰度的差异。测试了对比刺激与对照治疗的对比。鉴于ROR1特异性和CD19特异性CAR在两个供体上均表现出相似的表型,并且以与配体无关的方式受到相同的刺激(图1D-1F),因此将相应的测量结果视为重复。使用limma程序包的duplicateCorrelation函数估计类内相关性,以说明源自相同患者和相同抗原的测量值(Smyth等人,Bioinformatics 21:2067-2075(2005))。绝对对数变化截止值(刺激相对于对照)为0.7,错误发现率(FDR)截止值为5%,用于确定差异表达的PO4位点。使用StringDB进行信号网络和KEGG通路的分析。
RNA测序数据分析
使用Illumina的Real Time Analysis v1.18软件执行图像分析和碱基检出,然后使用Illumina的bcl2fastq转换软件v1.8.4对索引读取进行“多路分解”并生成FASTQ文件。使用STAR将RNA-seq数据与人类基因组(UCSC人类基因组组装GRCh38参考)比对,并使用RSEM(Dobin等人,Bioinformatics 29:15–21(2013);Li&Dewey,BMC)进行基因定量生物信息学12:323(2011))。丢弃少于10个非零读取计数(考虑到技术重复)的基因,剩下18,498个表达的基因。所有文库均通过了质量控制标准(具有200,000多个读数,12,000个检测到的基因以及外显子范围>60%的图书馆)。将原始计数数据导入R.edgeR用于计算归一化因子以缩放原始文库大小,然后从limma Bioconductor软件包进行voom转换(Robinson&Oshlack,Genome Biol.11:R25(2010);Law等等,Genome Biol.15:R29(2014))。它将计数数据转换为每百万log2counts,并估计均值-方差关系以计算适当的观察级权重。如上文在Shotgun质谱数据分析中所述,将具有对象随机效应的线性模型再次用于差异基因表达分析。测试了比较治疗(对照与刺激)或CAR(CD28/CD3ζ与4-1BB/CD3ζ)的对比。绝对对数变化临界值1和FDR临界值1%用于确定差异表达的基因。
实施例2
临床相关的原发性T细胞模型
用于CAR信号分析
已经对通过抗CD3单克隆抗体(mAb)刺激的转化Jurkat T细胞进行了LC-MS/MS分析,研究了TCR信号传导(Mayyaet等人,Sci Signal 2:ra46–ra46(2009);Nguyenet等人,Mol.Cell Proteomic 8:2418-2431(2009);Saleket等人,PLoS ONE8:e77423(2013))。选择Jurkat细胞进行信号转导研究是因为它们可以使用常规分子生物学技术轻松生长和操纵。考虑将CAR引入Jurkat细胞以分析信号传导,但在抗CD3 mAb刺激后通过LC-MS/MS比较已知的近端TCR信号分子的PO4显示,永生的Jurkat和培养的原代人T细胞表现出不同的蛋白质PO4模式(图8A-8B)。与永生化的Jurkat细胞相反,原代T细胞在刺激后显示CD3链和ZAP-70的PO4延长,并且CD28,LAT,LCK,PAK2,SHP1,SOS1和VAV1磷酸化程度不同。因此,在原代人T细胞中研究了CAR信号传导,以获得临床相关数据。
实施例3
标记CAR的设计
用于CAR信号分析
可以将表达CAR抗原的肿瘤细胞用于刺激原代CAR T细胞以进行LC-MS/MS,但是这种方法需要长时间培养带有同位素标记氨基酸(SILAC)的肿瘤细胞和T细胞,以便能够区分磷酸肽每个单元格类型肿瘤细胞还可以与T细胞上的辅助分子结合,从而促进信号蛋白的磷酸化。为了避免基于细胞的刺激的这些缺点,开发了一种无细胞的激活CAR信号传导的方法(图1A-1B)。编码CD19特异性和ROR1特异性CAR的慢病毒载体,其在间隔区中包含CD28/CD3ζ或4-1BB/CD3ζ信号域和九个氨基酸的Strep-tag II(STII;SEQ ID NO:10)序列设计区域(SEQ ID NO:19、21、23和25)(图1A)。所有构建体都在T2A元件下游包含一个截短的EGFR(EGFRt)转导标记,用于纯化表达CAR的T细胞(Wang等人,Blood 118:1255–1263(2011)。先前已证明包含STII序列不会干扰CAR T细胞的识别或功能,STII CD28/CD3ζ或4-1BB/CD3ζCAR T细胞可以通过抗STII mAb的模拟在体外有效激活和扩增(((Liu et al.,Nat.Biotechnol.34,430–434(2016))。
用四种慢病毒载体分别转导原代CD8+T细胞,分选EGFRt+表达,并在单个刺激周期内扩增至>1.6x108个细胞以进行后续分析(图1C)。CD28/CD3ζ和4-1BB/CD3ζCAR每个scFv特异性在细胞表面的表达水平相似(图1D)。CAR T细胞也表达相似水平的CD45RO,CD62L,CD27和CD28(图1E),尽管CD19 4-1BB/CD3ζCAR T细胞的频率稍高一些表达CD62L。PD-1和Tim3在任何CAR T细胞群体中均未表达,>85%的CD28/CD3ζ和4-1BB/CD3ζCAR T细胞处于G0/G1细胞周期阶段,这与缺乏明显的补品一致(图1E-1F)。
在准备用于MS分析的裂解物之前,先评估由STII连接诱导的典型T细胞信号转导事件。将ROR1特异的4-1BB/CD3ζCAR T细胞与各种抗STII mAb包被的微珠孵育45分钟,并通过Western blot检测CD3ζ、Y142、SLP-76Y376和PLC-γ1Y783。为了进行比较,用ROR1转导的K562(K562/ROR1)肿瘤细胞刺激CAR T细胞。在最高的珠与细胞比率下,抗STII珠刺激可将CD3ζ、SLP-76和PLC-γ1的PO4增至与K562/ROR1细胞刺激的CAR T细胞裂解物中观察到的水平相似,该珠与细胞的比率用于所有后续实验(图1G)。这些底物的PO4在用K562/CD19或抗STII mAb包被的珠刺激的CD19 CAR T细胞中也相似。因此,抗STII mAb包被的磁珠提供了一种精确的方法来选择性激活原代T细胞中的CAR信号传导。
实施例4
载有T细胞的蛋白质磷酸化分析
具有不同的协同刺激域
进行了三个独立的实验,其中将CD28/CD3ζ或4-1BB/CD3ζCART细胞与涂有抗STIImAb的或未覆盖的(对照)磁珠孵育10或45分钟,以捕获早期和晚期PO4事件。CAR刺激(图2A)。从两个不同的供体产生的CD19特异性CAR T细胞用于两个实验。第三个实验利用了来自两个供体之一的ROR1特异性CAR T细胞(图2B)。为了在每个实验中提供磷酸肽的相对定量,每个胰蛋白酶消化的裂解物都用独特的等压串联质谱标签(TMT)进行标记,并通过抗磷酸酪氨酸免疫沉淀法对磷酸肽进行富集,然后对剩余洗脱液进行固定化金属亲和层析(Thompsonet等,Anal.Chem.75:1895-1904(2003))(图9)。
使用这些技术,在三个实验中共检测到26,804个PO4位点,对应于4,849个基因产物。PO4位点中,磷酸酪氨酸为571个(2.13%),磷酸苏氨酸为4,647个(17.33%),磷酸丝氨酸为21,586个(80.53%)图2C)。重复实验之间捕获的磷酸化蛋白质组有相当多的重叠(图2D)。每个实验中检测到的PO4位点的99%都存在于未刺激和刺激的T细胞裂解物中,从而能够定量分析CAR激活诱导的变化(Navarroet等,Nat.Immunol.12:352-361(2011);van Oers等,Mol Cell Biol 13:5771-5780(1993))。
实施例5
规范T细胞信号中间体的CAR磷酸化
首先在MS数据集中分析描述充分的TCR刺激诱导的PO4事件,以确定这些位点是否也对CAR刺激有反应(Brownlie和Zamoyska,Nature Reviews Immunology 13:257-269(2013))。通过将刺激的样品与适当的对照(即CD28/CD3ζCAR 10分钟刺激与CD28/CD3ζCAR10分钟对照)进行比较,计算出每个重复实验中每个PO4位点的log2倍变化(log2FC)值。在第10分钟时,在受刺激的CAR T细胞中发现Y83,Y111和Y142的CD3ζ以及Y493的ZAP-70的PO4增加(图2E)。值得注意的是,CD28/CD3ζ和4-1BB/CD3ζCART细胞在Y206和Y209处显示CD28的PO4增加,表明4-1BB/CD3ζCAR的连接导致内源CD28的PO4。刺激45分钟后,观察到PO4扩散到下游TCR信号传导节点,包括在S1248和BCL10在S138的PO4增加(图2F)(另请参见Ruedaet等人,J Immunol 178:4373-4384(2007);Ishiguroet等,Mol.Immunol.44:2095-2100(2007))。对于大多数这些已知位点,在10分钟和45分钟时,与4-1BB/CD3ζCAR相比,对CD28/CD3ζCAR的刺激引发的log2FC幅度更大。在任何一个时间点都未观察到CDδ、ε、或γITAM的可再现变化,这些变化已知在T细胞中被抗CD3刺激后磷酸化。
为了验证MS数据集中CD28/CD3ζ和4-1BB/CD3ζCAR T细胞之间观察到的大小差异,评估了细胞裂解液的三种CAR激活诱导的磷蛋白信号转导事件–CD3ζpY142;ZAP-70pY319;和1pY783-通过Western blot。如图2G所示,CD28/CD3ζ或4-1BB/CD3ζCAR刺激增加了CAR CD3ζ结构域,ZAP-70和PLC-γ1的PO4。与MS数据一致,在刺激的CD28/CD3ζCART细胞中观察到这些蛋白的PO4强度更高。值得注意的是,在未刺激的CD28/CD3ζCD19和ROR1 CAR T细胞中检测到低水平的基础CAR CD3ζPO4,而这两种特异性都不存在于4-1BB/CD3ζCART细胞中。已经描述了几种CARs的组成型CAR CD3ζ结构域PO4,并且可以与不良T细胞表型和T细胞衰竭标志物的上调相关(见Frigault等人,Cancer Immunol Res 3:356-367(2015);Long等人,Nature Medicine 21:581–590(2015)。然而,未观察到未刺激的CD28/CD3ζ或4-1BB/CD3ζCAR T细胞的细胞周期或PD-1或Tim3的表达差异(图1E-1F)。因此,低水平的基础PO4在未刺激的CD28/CD3ζCART细胞中检测到的信号不同于在某些具有不同scFv特异性的CAR中观察到的更极端的强直信号。
实施例6
具有不同动力学和突变率的离散共刺激域诱导相似蛋白磷酸化事件活化T细胞
弹枪MS的一个优点是它可以定量测量数千个PO4事件,而这些事件都没有已知的经过实验验证的抗体。使用边缘统计框架和相关的R包来鉴定在CD28/CD3ζ和4-1BB/CD3ζCAR连接后被调节的PO4位点(G.K.Smyth,Stat Appl Genet Mol Biol3:Article3(2004))。如果在三个实验中的至少两个实验中检测到PO4位点,则将其鉴定为对CAR刺激有反应的PO4位点,在10或45分钟的刺激和未刺激条件之间显示平均|log2FC|≥0.7,并满足5%FDR截止值的要求。选择log2FC截止值为0.7,因为这代表了log2FC值分布的大约两个标准偏差(图10)。
使用这些严格的临界值,在10分钟的时间点,CD28/CD3ζCAR T细胞中22种蛋白质的26个PO4位点被鉴定为刺激响应性的。这些差异磷酸化的蛋白质富含KEGG TCR信号通路的成员,包括在S64处的p21活化激酶2(PAK2),在S231处的CD8 alpha(CD8A)的PO4升高,在S370处的蛋白激酶Cθ(PKCT)和原-致癌基因vav(VAV1)位于S748和T749(图3A和表1A-1B)。相反,在10分钟的时间点,没有4-1BB/CD3ζCART细胞的位点满足log2FC和FDR标准(图3B)。74个PO4位点符合log2FC截止值,但不满足FDR截止点,其中,S4903处的神经母细胞分化相关蛋白(AHNAK)的PO4升高,而S315和S319处的钙/钙调蛋白依赖性蛋白激酶II型亚基δ(CAMK2D)升高。检测到了指示Ca2+信号转导的信号(表1A-1B)。4-1BB/CD3ζCAR诱导的早期蛋白PO4缺乏强健的改变是出乎意料的,但与Western blot数据一致,在10分钟时显示CARCD3ζ和PLC-γ1的最低PO4(图2G)。
刺激45分钟后,发生了更强烈的蛋白质PO4,并且有1289个PO4位点从CD28/CD3ζ或4-1BB/CD3ζCAR样品中遇到了log2FC和FDR截止值。这些包括CD28/CD3ζCAR T细胞中743个基因产物的1,279个PO4位点和4-1BB/CD3ζCAR T细胞中346个基因产物的522个位点(图3C-3D)。因此,刺激CD28/CD3ζCAR继续导致更多PO4位点的改变。然而,令人惊讶的是,观察到刺激反应性PO4位点之间存在很强的相关性,从而几乎所有位点都通过CD28/CD3ζ和4-1BB/CD3ζCAR刺激以相似的方式进行调节(图3E)。在CD28/CD3ζCAR样品中达到临界值的1,289个位点中,只有12个(0.93%)受4-1BB/CD3ζCAR刺激的差异调节,而只有43个(3.3%)位点在经过CD28/CD3□CAR激发后出现了更大的log2FC 4-1BB/CD3ζCAR刺激,与先前的数据一致,表明CD28/CD3ζCAR的下游信号更强(图3E)。此外,这些12和43个站点的子集未映射到当前定义的4-1BB信令网络。
在刺激CD28/CD3ζ和4-1BB/CD3ζCAR后发现蛋白质PO4事件几乎没有差异,这出乎意料,并引发了一个问题,即如何影响经典T细胞共刺激信号通路中涉及的蛋白质。与早期发现4-1BB/CD3ζCAR激活内源性CD28一致,CD28信号传导中间体VAV1,PKCT和PIK3C2A在任一CAR刺激后显示PO4升高(图3F)(Acuto和Michel,Nature Reviews Immunology3:939-951(2003))。在4-1BB信号传导途径中,LSP-1(4-1BB和TRAF2信号传导的直接靶标)均由两种CAR调节(Sabbagh等人,J.Leukoc.Biol.93:713-721(2013))。尽管有这些相似性,但CD28/CD3ζCAR调节的每个PO4位点(包括4-1BB信号通路中的那些)比4-1BB/CD3ζCAR的调节幅度更大。因此,CD28/CD3ζ和4-1BB/CD3ζCAR的连接并没有激活受体中不同的共刺激结构域所预期的发散性信号网络,而是引起了细胞内蛋白PO4高度相似的变化,其中包括规范的CD28和4-1BB信号中间体。
图解说明受CD28/CD3ζ和4-1BB/CD3ζCAR刺激影响的主要途径和单个蛋白质磷酸化事件的图谱,包括参与规范的TCR信号传导和丝裂原激活的蛋白激酶(MAPK)信号传导通路的蛋白质(图4和表1A)。
实施例7
CAR结扎后CAR信号保持力的差异
CD28/CD3ζ和4-1BB/CD3ζCAR刺激介导的蛋白PO4高度相似,但在绝大多数PO4位点强度不同。有理由认为,量化CAR刺激后蛋白质PO4的变化可以提供CAR信号强度的整体度量。通过减少log2FC排列CD28/CD3ζ和4-1BB/CD3ζCAR样品在45分钟时的刺激响应PO4位点。与先前结果一致的结果表明,两种CAR均以相似的方式调节蛋白PO4,刺激后20个最磷酸化的位点中有15个在CD28/CD3ζ和4-1BB/CD3ζCAR T细胞之间共享(表2A-2B)。但是,在CD28/CD3ζCAR样品中,前20个位点的PO4平均增加11.15倍,而在4-1BB/CD3ζCAR样品中,平均PO4仅增加5.8倍(图5A)。通过信号通路对CAR刺激应答PO4位点进行分层,进一步显示KEGG TCR信号通路内的平均PO4位点在CD28/CD3ζCAR样品中被2.52倍调节,但在4-1BB/CD3ζCAR样品中仅1.69倍被调节(图5B)。
为了确定4-1BB/CD3ζCAR信号在以后的时间是否达到与CD28/CD3ζCAR相似的强度,将CAR T细胞刺激60、120或180分钟,然后将经典的和新近鉴定的信号中间体的PO4刺激。如图5C所示,CD28/CD3ζCAR刺激导致所有样品中SLP-76,和DAPP1的PO4强度更高,表明4-1BB/CD3ζCAR刺激从未与之竞争在此时间段内,CD28/CD3ζCAR信号强度。
实施例8
CAR信号强度与无反应性细胞表型呈正相关,体内抗肿瘤活性降低
T细胞活化和信号转导的强度导致调节效应细胞分化和记忆形成的转录差异(Kaech和Cui,Nature Reviews Immunology12:749–761(2012))。在STII磁珠刺激6小时后,使用RNA-Seq分析CD28/CD3ζ或4-1BB/CD3ζCAR T细胞中的转录程序,并鉴定满足|log2FC|>1.0和FDR<1%的差异表达基因。与更快,更强烈的磷蛋白信号一致,CD28/CD3ζCAR刺激引发了更明显的早期转录变化。使用limma比较刺激和未刺激的CD28/CD3ζCAR-T细胞可鉴定出4,894个差异表达的基因,而4-1BB/CD3ζCAR刺激可产生197个差异表达的基因。T细胞活化标记CD69被CD28□/CD3ζ或4-1BB/CD3ζCAR刺激上调至相似程度(图6A),但与4-1BB/CD3ζCAR T细胞相比,在激活的CD28/CD3ζCAR T细胞中观察到效应分子颗粒酶B(GZMB)、干扰素-γ(IFNG)、白介素2(IL2)、肿瘤坏死因子-1(TNF)、白介素6(IL6)、巨噬细胞炎性蛋白1α(CCL3)和巨噬细胞炎性蛋白1β(CCL4)(图6B)。
直接比较刺激的CD28/CD3ζ和4-1BB/CD3ζ样品,鉴定出1,673个差异表达的基因(表3)。其中,Krüppel样因子2(KLF2),白介素7受体(IL7R)和与Rho家族相互作用的细胞极化调节剂2(RIPOR2,以前称为FAM65B)在CD28/CD3ζCAR T细胞样本中的表达水平较低(图6C)。KLF2和IL7R与记忆T细胞形成有关,并且是转录因子FOXO家族的靶标(Rougerie等人,JImmunol 190:748-755(2013);Kaech等人,Nat Immunol 4:1191-1198(2003);Kerdiles等,Nat Immunol 10:176-184(2009))。沿着这些路线,受刺激的CD28/CD3ζCART细胞中FOXO4表达降低。qPCR证实了这些表达差异,并表明这些T细胞记忆相关基因在未刺激的CD28/CD3ζ或4-1BB/CD3ζCAR T细胞中未差异表达(图6D)。因此,CD28/CD3ζCAR活化优先诱导转录谱,其特征是效应分子表达增加和FOXO家族基因靶点的丢失。
假设像TCR信号一样,CD28/CD3ζ和4-1BB/CD3ζCAR之间的信号量差异也会影响T细胞功能。为了测试这一点,用表达CAR抗原的K562细胞或STII珠激活了CD28/CD3ζ和4-1BB/CD3ζCAR T细胞,并在不同时间点测量了细胞因子的产生和增殖。5小时后,较大比例的CD28/CD3ζCAR T细胞产生IFN-γ、IL-2和TNF-α(图6E),而24小时后,4-1BB/CD3βCAR T细胞相比,CD28/CD3ζCAR T细胞分泌的IFN-γ、、IL-2和TNF-α明显更多(图6F)。72小时后,与4-1BB/CD3ζCAR T细胞相比,更多的CD28/CD3ζCAR T细胞已经分裂并且经历了更多的分裂(图6G)。
尽管具有优异的体外效应器功能,但事实证明CD28/CD3ζCAR T细胞在体内肿瘤控制中效果较差。当将3×106个CAR T细胞过继转移到带有已建立的CD19+Raji淋巴瘤异种移植物的NOD/SCID/γc-/-(NSG)小鼠中时,CD28/CD3ζ和4-1BB/CD3ζCAR T细胞均介导了肿瘤的完全消退(图6H)。但是,输注减少的(7.5-8×105)细胞剂量表明,CD28/CD3ζCAR T细胞的效力远低于4-1BB/CD3ζCAR T细胞,并且所有CD28/CD3ζCAR T细胞均经过处理小鼠在40天内死于肿瘤进展。尽管在涉及肿瘤的骨髓中CAR T细胞积累到较高频率,但以较低CD28/CD3ζCAR T细胞剂量治疗的小鼠仍发生肿瘤进展(图6I)。骨髓中的CD28/CD3ζCAR T细胞表达较高水平的PD-1、Lag-3和Tim-3(图6J),与疲惫表型的获得一致。综上所述,这些数据表明,尽管体外细胞因子产生和T细胞增殖增加,但CD28/CD3ζCAR激活介导的快速而强烈的信号转导导致疲惫的T细胞表型具有降低的抗肿瘤活性。
实施例9
CD28/CD3Z和4-1BB/CD3Z CAR与内源性CD28和LCK不同关联
为了探究CD28/CD3ζCAR信号动力学和强度增加的可能原因,从CD8+T细胞中免疫沉淀出CAR复合物,并探测基础状态下相关T细胞信号蛋白之间的差异。Western印迹证实有效的CAR下拉,并显示内源性CD28和Lck与CD28/CD3ζCAR缔合,但仅有最小的CD28和Lck与4-1BB/CD3ζCAR缔合(图7A)。在CAR刺激45分钟后,来自激活的4-1BB/CD3ζCAR T细胞的免疫沉淀也未能检测到内源性CD28(图7B)。由于CD28共刺激结构域的存在赋予了基础CAR磷酸化作用,并且CAR刺激后Y206,Y209和Y218被强烈磷酸化(MS数据),因此构建了在这些残基上具有酪氨酸至苯丙氨酸突变的CD28/CD3□CAR(图7B)。具有Y218F取代的CD19特异性CAR和具有Y206F、Y209F和Y218F取代的CD19 CAR分别包含SEQ ID NO:27和29所示的氨基酸序列。具有Y218F取代的ROR1特异性CAR和具有Y206F、Y209F和Y218F取代的ROR1 CAR分别包含SEQID NO:31和33所示的氨基酸序列。
具有突变一个(Y1)或所有三个酪氨酸(Y3)的CD19和ROR1特异性CAR在T细胞中有效表达,并通过增殖和产生干扰素(IFN-γ)与ROR1+或CD19+肿瘤细胞共培养而在体外发挥作用(图7C-7D)。值得注意的是,酪氨酸突变以分级的方式减少了IL-2和TNF-α的分泌。免疫沉淀表明,尽管内源性CD28与Y3 CAR无关,但Y1和Y3突变并未消除Lck的结合(图7F)。在突变型CAR中观察到了部分CAR(Y1)或完全(Y3)消除基础CAR CD3ζPO4(图7G),但Y1和Y3 CAR仍使SLP-76和PLC-γ1磷酸化,动力学和强度与野生型CD28/CD3ζCAR相似,STII磁珠刺激后(图7G)。与Western印迹数据显示Y1和Y3信号强度增加一致,用Y1或Y3 CAR T细胞治疗的小鼠的中位生存期与用野生型CD28/CD3ζCAR T细胞治疗的小鼠相似,数量少于4-1BB/CD3ζCAR T细胞的数量(图7H)。这些结果共同表明,基础CAR CD3ζ磷酸化和内源性CD28缔合都不是CD28/CD3ζCAR信号动力学和强度增加的原因。
Lck的组成性激活可促进T细胞效应子的功能(Tavanoet等人,J Immunol 173:5392-5397(2004))。为检查Lck与CD28/CD3ζCAR的缔合是否介导CAR激活后脯氨酸信号传导中间体的快速和强健的磷酸化,脯氨酸-丙氨酸突变在CD28的Lck结合位点产生(图11A)。免疫沉淀和野生型CD28/CD3ζCARs的免疫沉淀表明,脯氨酸-丙氨酸突变的CAR中不存在Lck关联(图11B)。信号分析表明,仅CD28P CAR中脯氨酸残基的突变部分消除了基础CAR CD3ζ磷酸化,但并未降低信号强度(图11C)。相反,Y3P CAR中脯氨酸和酪氨酸残基的同时突变完全消除了基础CAR的磷酸化,并降低了刺激后SLP-76和PLC-γ1磷酸化的幅度。值得注意的是,脯氨酸到丙氨酸的突变减少了IFN-γ、IL-2和TNF-α的分泌,因此细胞因子的总体产生水平类似于4-1BB/CD3ζCAR T细胞(图11D)。表明CD28/CD3ζCAR和4-1BB/CD3ζCAR之间的CAR信号强度差异部分与和CD28/CD3ζCAR的更大Lck关联有关。此外,可以通过突变CD28信号域中的酪氨酸和脯氨酸残基来改变CAR信号强度和促炎细胞因子的产生。
实施例10
使用修饰的CD28-CAR T细胞治疗癌症
从患有实体或血液癌症的患者的PBMC中分离自体T细胞,并进行分类以分离效应T细胞和辅助T细胞。用抗CD3/抗CD28珠和IL-2培养T细胞级分,并用编码嵌合抗原受体(CAR)的慢病毒构建体进行体外转导,所述嵌合抗原受体包括对感兴趣的肿瘤抗原,间隔子,跨膜结构域,包含Y206F、Y209F和Y218F取代的修饰的CD28共刺激结构域以及细胞内信号传导结构域。该构建体还编码用于转导的细胞表面标记。响应于表达抗原的肿瘤细胞的刺激,体外检查转导的T细胞的功能性(增殖,细胞因子释放)。此后,CAR T细胞在体外扩增。
患者接受去淋巴细胞化疗,然后通过静脉输注给予临床相关剂量的CAR T细胞。过继转移后,CAR T细胞在患者体内扩增,并通过流式细胞仪和q-PCR测定载体序列。在体内扩增的高峰期从接受治疗的患者中采集样品,以测量表型,评估功能并确定抗肿瘤持久性。进行流式细胞仪和基因表达谱分析以表征细胞中的基因表达。通过MRI监测肿瘤大小、数量和分布的减少。
可以将上述各种实施例组合以提供其他实施例。本说明书中提及和/或在应用数据表中列出的所有美国专利、美国专利申请出版物、美国专利申请、外国专利、外国专利申请和非专利出版物、包括美国临时专利申请第62/635,450号于2018年2月26日提交的美国临时专利申请、于2018年5月24日提交的美国临时专利申请号62/676,787和于2018年10月1日提交的美国临时专利申请号62/739,792均通过引用全部并入本文。如果需要采用各种专利,申请和出版物的概念以提供进一步的实施例,则可以修改实施例的各方面。
可以根据以上详细描述对实施例进行这些和其他改变。通常,在以下权利要求书中,所使用的术语不应解释为将权利要求书限制为说明书和权利要求书中公开的特定实施例,而是应解释为包括所有可能的实施例以及权利要求所要求的等同物的全部范围。因此,权利要求不受公开内容的限制。
表
表1A:在CD28/CD3z
CAR样品中鉴定出26个对CAR刺激有反应的PO4位点
表1B:4-1BB/CD3z CAR刺激后74个PO4位点达到log2FC截止
表2A:45分钟时CD28/CD3z
CAR刺激上调的20个PO4位点
Log2FC排列 | UniProt功能 | 基因功能 | Log2FC |
1 | O75582_S381_1 | RPS6KA5_S381 | 4.884 |
2 | O94768_S10_1 | STK17B_S10 | 4.690 |
3 | O75582_S376_1 | RPS6KA5_S376 | 4.436 |
4 | Q14241_S163_1 | TCEB3_S163 | 3.851 |
5 | O94804_S13_2 | STK10_S13 | 3.631 |
6 | O94804_T14_2 | STK10_T14 | 3.631 |
7 | Q8NFC6_S800_1 | BOD1L1_S800 | 3.568 |
8 | Q6ZSZ5_S1160_1 | ARHGEF18_S1160 | 3.496 |
9 | P29590_S480_1 | PML_S480 | 3.364 |
10 | Q02880-2_S1447_1 | top2B_S1452 | 3.248 |
11 | Q9UN19-2_Y139_1 | DAPP1_Y139 | 3.185 |
12 | P62753_S240_2 | RPS6_S240 | 3.168 |
13 | O75937_S35_1 | DNAJC8_S35 | 3.126 |
14 | P14316-2_T336_1 | IRF2_T338 | 3.121 |
15 | O75152_S290_1 | ZC3H11A_S290 | 3.107 |
16 | Q9Y4F9_S175_1 | FAM65B_S175 | 3.094 |
17 | Q5TDH0_S194_1 | DDI2_S194 | 3.085 |
18 | P19338_T583_1 | NCL_T583 | 2.993 |
19 | O75152_S295_1 | ZC3H11A_S295 | 2.981 |
20 | Q14005-3_S144_1 | IL16_S845 | 2.944 |
表2B:45分钟时4-1BB/CD3z
CAR刺激上调的20个PO4位点
表3:受刺激的CD28/CD3z和4个1BB/CD3z
CAR
T细胞之间的1,673个差异表达基因
序列表
<110> 弗雷德哈钦森癌症研究中心
A·索尔特
S·里德尔
<120> 用于细胞免疫治疗的组合物和方法
<130> 360056.458WO
<140> PCT
<141> 2019-02-21
<150> US 62/739,792
<151> 2018-10-01
<150> US 62/676,787
<151> 2018-05-25
<150> US 62/635,450
<151> 2018-02-26
<160> 175
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 220
<212> PRT
<213> 智人
<220>
<223> 人CD28
<400> 1
Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val
1 5 10 15
Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr
20 25 30
Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser
35 40 45
Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu
50 55 60
Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser
65 70 75 80
Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr
85 90 95
Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys
100 105 110
Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser
115 120 125
Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro
130 135 140
Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly
145 150 155 160
Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
165 170 175
Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
180 185 190
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
195 200 205
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
210 215 220
<210> 2
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人CD28细胞内共刺激
结构域
<400> 2
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 3
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人CD28细胞内共刺激
结构域(L186G, L187G)]
<400> 3
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 4
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人CD28细胞内共刺激
结构域(Y218F)
<400> 4
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Phe Arg Ser
35 40
<210> 5
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> 合成序列CD28细胞内共刺激
结构域(Y191F,Y206F, Y209F, Y218F)
<400> 5
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Phe Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Phe Gln Pro Phe Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Phe Arg Ser
35 40
<210> 6
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> 合成序列CD28细胞内共刺激
结构域(Y206F, Y209F, Y218F)
<400> 6
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Phe Gln Pro Phe Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Phe Arg Ser
35 40
<210> 7
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人GM-CSF受体信号肽
<400> 7
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro
20
<210> 8
<211> 245
<212> PRT
<213> 人工序列
<220>
<223> 合成序列FMC63 (抗CD19) scFv
<400> 8
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser
130 135 140
Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu
180 185 190
Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn
195 200 205
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
225 230 235 240
Val Thr Val Ser Ser
245
<210> 9
<211> 248
<212> PRT
<213> 人工序列
<220>
<223> 合成序列R12 (抗ROR1) scFv
<400> 9
Gln Glu Gln Leu Val Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly Phe Asp Phe Ser Ala Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr Tyr Tyr Ala Thr Trp Val
50 55 60
Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Gln Asn Thr Val Asp
65 70 75 80
Leu Gln Met Asn Ser Leu Thr Ala Ala Asp Arg Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala Leu Phe Asn Ile Trp Gly
100 105 110
Pro Gly Thr Leu Val Thr Ile Ser Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Leu Thr Gln Ser Pro
130 135 140
Ser Val Ser Ala Ala Leu Gly Ser Pro Ala Lys Ile Thr Cys Thr Leu
145 150 155 160
Ser Ser Ala His Lys Thr Asp Thr Ile Asp Trp Tyr Gln Gln Leu Gln
165 170 175
Gly Glu Ala Pro Arg Tyr Leu Met Gln Val Gln Ser Asp Gly Ser Tyr
180 185 190
Thr Lys Arg Pro Gly Val Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly
195 200 205
Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val Gln Ala Asp Asp Glu Ala
210 215 220
Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly Gly Tyr Val Phe Gly Gly
225 230 235 240
Gly Thr Gln Leu Thr Val Thr Gly
245
<210> 10
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 合成序列Strep tag II肽, 9-mer
<400> 10
Asn Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 11
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 合成序列(G4S)2接头
<400> 11
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 12
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人IgG4铰链
<400> 12
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
1 5 10
<210> 13
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人CD28跨膜结构域
<400> 13
Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
1 5 10 15
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 14
<211> 42
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人4-1BB共刺激
信号结构域
<400> 14
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 15
<211> 112
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人CD3ζ信号结构域
<400> 15
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 16
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> 合成序列T2A病毒肽
<400> 16
Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp
1 5 10 15
Val Glu Glu Asn Pro Gly Pro Arg
20
<210> 17
<211> 335
<212> PRT
<213> 人工序列
<220>
<223> 合成序列截断的人EGFR (tEGFR)
多肽,缺少信号肽
<400> 17
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
1 5 10 15
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile
20 25 30
Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe
35 40 45
Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
50 55 60
Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn
65 70 75 80
Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
85 90 95
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile
100 105 110
Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val
115 120 125
Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp
130 135 140
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn
145 150 155 160
Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
165 170 175
Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser
180 185 190
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
195 200 205
Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
210 215 220
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly
225 230 235 240
Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
245 250 255
His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr
260 265 270
Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
275 280 285
Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
290 295 300
Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala
305 310 315 320
Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
325 330 335
<210> 18
<211> 2586
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A042 CAR (FMC63 scFv_STII_IgG4
铰链_CD28tm_41BB_CD3z_T2A_tEGR)表达
盒
<400> 18
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60
atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120
gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180
aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240
cctagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300
gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360
ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420
ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480
cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540
gtgagctgga tcaggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600
agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660
agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720
tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780
accagcgtga ccgtgagcag caattggagc cacccgcagt tcgaaaaagg aggtggaggt 840
tcaggtggtg gaggctctga gagcaagtac ggaccgccct gccccccttg ccctatgttc 900
tgggtgctgg tggtggtcgg aggcgtgctg gcctgctaca gcctgctggt caccgtggcc 960
ttcatcatct tttgggtgaa acggggcaga aagaaactcc tgtatatatt caaacaacca 1020
tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 1080
gaagaagaag gaggatgtga actgcgggtg aagttcagca gaagcgccga cgcccctgcc 1140
taccagcagg gccagaatca gctgtacaac gagctgaacc tgggcagaag ggaagagtac 1200
gacgtcctgg ataagcggag aggccgggac cctgagatgg gcggcaagcc tcggcggaag 1260
aacccccagg aaggcctgta taacgaactg cagaaagaca agatggccga ggcctacagc 1320
gagatcggca tgaagggcga gcggaggcgg ggcaagggcc acgacggcct gtatcagggc 1380
ctgtccaccg ccaccaagga tacctacgac gccctgcaca tgcaggccct gcccccaagg 1440
ctcgagggcg gcggagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat 1500
cccggcccta ggatgcttct cctggtgaca agccttctgc tctgtgagtt accacaccca 1560
gcattcctcc tgatcccacg caaagtgtgt aacggaatag gtattggtga atttaaagac 1620
tcactctcca taaatgctac gaatattaaa cacttcaaaa actgcacctc catcagtggc 1680
gatctccaca tcctgccggt ggcatttagg ggtgactcct tcacacatac tcctcctctg 1740
gatccacagg aactggatat tctgaaaacc gtaaaggaaa tcacagggtt tttgctgatt 1800
caggcttggc ctgaaaacag gacggacctc catgcctttg agaacctaga aatcatacgc 1860
ggcaggacca agcaacatgg tcagttttct cttgcagtcg tcagcctgaa cataacatcc 1920
ttgggattac gctccctcaa ggagataagt gatggagatg tgataatttc aggaaacaaa 1980
aatttgtgct atgcaaatac aataaactgg aaaaaactgt ttgggacctc cggtcagaaa 2040
accaaaatta taagcaacag aggtgaaaac agctgcaagg ccacaggcca ggtctgccat 2100
gccttgtgct cccccgaggg ctgctggggc ccggagccca gggactgcgt ctcttgccgg 2160
aatgtcagcc gaggcaggga atgcgtggac aagtgcaacc ttctggaggg tgagccaagg 2220
gagtttgtgg agaactctga gtgcatacag tgccacccag agtgcctgcc tcaggccatg 2280
aacatcacct gcacaggacg gggaccagac aactgtatcc agtgtgccca ctacattgac 2340
ggcccccact gcgtcaagac ctgcccggca ggagtcatgg gagaaaacaa caccctggtc 2400
tggaagtacg cagacgccgg ccatgtgtgc cacctgtgcc atccaaactg cacctacgga 2460
tgcactgggc caggtcttga aggctgtcca acgaatgggc ctaagatccc gtccatcgcc 2520
actgggatgg tgggggccct cctcttgctg ctggtggtgg ccctggggat cggcctcttc 2580
atgtga 2586
<210> 19
<211> 861
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A042 CAR盒蛋白
序列
<400> 19
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
325 330 335
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
340 345 350
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
355 360 365
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
370 375 380
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
385 390 395 400
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
405 410 415
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
420 425 430
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
435 440 445
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
450 455 460
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475 480
Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp
485 490 495
Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu
500 505 510
Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys
515 520 525
Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile
530 535 540
Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly
545 550 555 560
Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His
565 570 575
Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys
580 585 590
Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr
595 600 605
Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys
610 615 620
Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser
625 630 635 640
Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile
645 650 655
Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys
660 665 670
Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly
675 680 685
Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser
690 695 700
Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg
705 710 715 720
Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu
725 730 735
Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His
740 745 750
Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly
755 760 765
Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys
770 775 780
Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val
785 790 795 800
Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn
805 810 815
Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn
820 825 830
Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu
835 840 845
Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 20
<211> 2583
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A043CAR (FMC63 scFv_STII_IgG4
铰链_CD28tm_CD28icd_CD3z_T2A_tEGFR)表达
盒
<400> 20
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60
atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120
gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180
aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240
cctagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300
gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360
ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420
ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480
cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540
gtgagctgga tcaggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600
agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660
agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720
tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780
accagcgtga ccgtgagcag caattggagc cacccgcagt tcgaaaaagg aggtggaggt 840
tcaggtggtg gaggctctga gagcaagtac ggaccgccct gccccccttg ccctatgttc 900
tgggtgctgg tggtggtcgg aggcgtgctg gcctgctaca gcctgctggt caccgtggcc 960
ttcatcatct tttgggtccg cagcaagcgg agcagaggcg gccacagcga ctacatgaac 1020
atgaccccta gacggcctgg ccccaccaga aagcactacc agccctacgc ccctccccgg 1080
gactttgccg cctacagaag ccgggtgaag ttcagcagaa gcgccgacgc ccctgcctac 1140
cagcagggcc agaatcagct gtacaacgag ctgaacctgg gcagaaggga agagtacgac 1200
gtcctggata agcggagagg ccgggaccct gagatgggcg gcaagcctcg gcggaagaac 1260
ccccaggaag gcctgtataa cgaactgcag aaagacaaga tggccgaggc ctacagcgag 1320
atcggcatga agggcgagcg gaggcggggc aagggccacg acggcctgta tcagggcctg 1380
tccaccgcca ccaaggatac ctacgacgcc ctgcacatgc aggccctgcc cccaaggctc 1440
gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 1500
ggccctagga tgcttctcct ggtgacaagc cttctgctct gtgagttacc acacccagca 1560
ttcctcctga tcccacgcaa agtgtgtaac ggaataggta ttggtgaatt taaagactca 1620
ctctccataa atgctacgaa tattaaacac ttcaaaaact gcacctccat cagtggcgat 1680
ctccacatcc tgccggtggc atttaggggt gactccttca cacatactcc tcctctggat 1740
ccacaggaac tggatattct gaaaaccgta aaggaaatca cagggttttt gctgattcag 1800
gcttggcctg aaaacaggac ggacctccat gcctttgaga acctagaaat catacgcggc 1860
aggaccaagc aacatggtca gttttctctt gcagtcgtca gcctgaacat aacatccttg 1920
ggattacgct ccctcaagga gataagtgat ggagatgtga taatttcagg aaacaaaaat 1980
ttgtgctatg caaatacaat aaactggaaa aaactgtttg ggacctccgg tcagaaaacc 2040
aaaattataa gcaacagagg tgaaaacagc tgcaaggcca caggccaggt ctgccatgcc 2100
ttgtgctccc ccgagggctg ctggggcccg gagcccaggg actgcgtctc ttgccggaat 2160
gtcagccgag gcagggaatg cgtggacaag tgcaaccttc tggagggtga gccaagggag 2220
tttgtggaga actctgagtg catacagtgc cacccagagt gcctgcctca ggccatgaac 2280
atcacctgca caggacgggg accagacaac tgtatccagt gtgcccacta cattgacggc 2340
ccccactgcg tcaagacctg cccggcagga gtcatgggag aaaacaacac cctggtctgg 2400
aagtacgcag acgccggcca tgtgtgccac ctgtgccatc caaactgcac ctacggatgc 2460
actgggccag gtcttgaagg ctgtccaacg aatgggccta agatcccgtc catcgccact 2520
gggatggtgg gggccctcct cttgctgctg gtggtggccc tggggatcgg cctcttcatg 2580
tga 2583
<210> 21
<211> 860
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A043 CAR盒蛋白
序列
<400> 21
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu
465 470 475 480
Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val
485 490 495
Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu
500 505 510
Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val
515 520 525
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
530 535 540
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
545 550 555 560
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
565 570 575
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
580 585 590
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
595 600 605
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
610 615 620
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
625 630 635 640
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
645 650 655
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
660 665 670
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
675 680 685
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
690 695 700
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
705 710 715 720
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
725 730 735
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
740 745 750
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
755 760 765
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
770 775 780
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
785 790 795 800
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
805 810 815
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
820 825 830
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
835 840 845
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 22
<211> 2595
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A044 CAR (R12 scFv_STII_IgG4
铰链_CD28tm_41BB_CD3z_T2A_tEGFR)表达
盒
<400> 22
atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60
atcccccagg aacagctcgt cgaaagcggc ggcagactgg tgacacctgg cggcagcctg 120
accctgagct gcaaggccag cggcttcgac ttcagcgcct actacatgag ctgggtccgc 180
caggcccctg gcaagggact ggaatggatc gccaccatct accccagcag cggcaagacc 240
tactacgcca cctgggtgaa cggacggttc accatctcca gcgacaacgc ccagaacacc 300
gtggacctgc agatgaacag cctgacagcc gccgaccggg ccacctactt ttgcgccaga 360
gacagctacg ccgacgacgg cgccctgttc aacatctggg gccctggcac cctggtgaca 420
atctctagcg gcggaggcgg atctggtggc ggaggaagtg gcggcggagg atctgagctg 480
gtgctgaccc agagcccctc tgtgtctgct gccctgggaa gccctgccaa gatcacctgt 540
accctgagca gcgcccacaa gaccgacacc atcgactggt atcagcagct gcagggcgag 600
gcccccagat acctgatgca ggtgcagagc gacggcagct acaccaagag gccaggcgtg 660
cccgaccggt tcagcggatc tagctctggc gccgaccgct acctgatcat ccccagcgtg 720
caggccgatg acgaggccga ttactactgt ggcgccgact acatcggcgg ctacgtgttc 780
ggcggaggca cccagctgac cgtgaccggc aattggagcc acccgcagtt cgaaaaagga 840
ggtggaggtt caggtggtgg aggctctgag agcaagtacg gaccgccctg ccccccttgc 900
cctatgttct gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc 960
accgtggcct tcatcatctt ttgggtgaaa cggggcagaa agaaactcct gtatatattc 1020
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1080
tttccagaag aagaagaagg aggatgtgaa ctgcgggtga agttcagcag aagcgccgac 1140
gcccctgcct accagcaggg ccagaatcag ctgtacaacg agctgaacct gggcagaagg 1200
gaagagtacg acgtcctgga taagcggaga ggccgggacc ctgagatggg cggcaagcct 1260
cggcggaaga acccccagga aggcctgtat aacgaactgc agaaagacaa gatggccgag 1320
gcctacagcg agatcggcat gaagggcgag cggaggcggg gcaagggcca cgacggcctg 1380
tatcagggcc tgtccaccgc caccaaggat acctacgacg ccctgcacat gcaggccctg 1440
cccccaaggc tcgagggcgg cggagagggc agaggaagtc ttctaacatg cggtgacgtg 1500
gaggagaatc ccggccctag gatgcttctc ctggtgacaa gccttctgct ctgtgagtta 1560
ccacacccag cattcctcct gatcccacgc aaagtgtgta acggaatagg tattggtgaa 1620
tttaaagact cactctccat aaatgctacg aatattaaac acttcaaaaa ctgcacctcc 1680
atcagtggcg atctccacat cctgccggtg gcatttaggg gtgactcctt cacacatact 1740
cctcctctgg atccacagga actggatatt ctgaaaaccg taaaggaaat cacagggttt 1800
ttgctgattc aggcttggcc tgaaaacagg acggacctcc atgcctttga gaacctagaa 1860
atcatacgcg gcaggaccaa gcaacatggt cagttttctc ttgcagtcgt cagcctgaac 1920
ataacatcct tgggattacg ctccctcaag gagataagtg atggagatgt gataatttca 1980
ggaaacaaaa atttgtgcta tgcaaataca ataaactgga aaaaactgtt tgggacctcc 2040
ggtcagaaaa ccaaaattat aagcaacaga ggtgaaaaca gctgcaaggc cacaggccag 2100
gtctgccatg ccttgtgctc ccccgagggc tgctggggcc cggagcccag ggactgcgtc 2160
tcttgccgga atgtcagccg aggcagggaa tgcgtggaca agtgcaacct tctggagggt 2220
gagccaaggg agtttgtgga gaactctgag tgcatacagt gccacccaga gtgcctgcct 2280
caggccatga acatcacctg cacaggacgg ggaccagaca actgtatcca gtgtgcccac 2340
tacattgacg gcccccactg cgtcaagacc tgcccggcag gagtcatggg agaaaacaac 2400
accctggtct ggaagtacgc agacgccggc catgtgtgcc acctgtgcca tccaaactgc 2460
acctacggat gcactgggcc aggtcttgaa ggctgtccaa cgaatgggcc taagatcccg 2520
tccatcgcca ctgggatggt gggggccctc ctcttgctgc tggtggtggc cctggggatc 2580
ggcctcttca tgtga 2595
<210> 23
<211> 864
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A044 CAR盒蛋白
序列
<400> 23
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu
325 330 335
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
340 345 350
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
355 360 365
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
370 375 380
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
385 390 395 400
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
405 410 415
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
420 425 430
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
435 440 445
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
450 455 460
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
465 470 475 480
Pro Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr
485 490 495
Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val
500 505 510
Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile
515 520 525
Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser
530 535 540
Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser
545 550 555 560
Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser
565 570 575
Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys
580 585 590
Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu
595 600 605
Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly
610 615 620
Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn
625 630 635 640
Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp
645 650 655
Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn
660 665 670
Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser
675 680 685
Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala
690 695 700
Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val
705 710 715 720
Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn
725 730 735
Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile
740 745 750
Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr
755 760 765
Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly
770 775 780
Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn
785 790 795 800
Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys
805 810 815
His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys
820 825 830
Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly
835 840 845
Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 24
<211> 2592
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A045 CAR (R12 scFv_STII_IgG4
铰链_CD28tm_CD28icd_CD3z_T2A_tEGFR)表达
盒
<400> 24
atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60
atcccccagg aacagctcgt cgaaagcggc ggcagactgg tgacacctgg cggcagcctg 120
accctgagct gcaaggccag cggcttcgac ttcagcgcct actacatgag ctgggtccgc 180
caggcccctg gcaagggact ggaatggatc gccaccatct accccagcag cggcaagacc 240
tactacgcca cctgggtgaa cggacggttc accatctcca gcgacaacgc ccagaacacc 300
gtggacctgc agatgaacag cctgacagcc gccgaccggg ccacctactt ttgcgccaga 360
gacagctacg ccgacgacgg cgccctgttc aacatctggg gccctggcac cctggtgaca 420
atctctagcg gcggaggcgg atctggtggc ggaggaagtg gcggcggagg atctgagctg 480
gtgctgaccc agagcccctc tgtgtctgct gccctgggaa gccctgccaa gatcacctgt 540
accctgagca gcgcccacaa gaccgacacc atcgactggt atcagcagct gcagggcgag 600
gcccccagat acctgatgca ggtgcagagc gacggcagct acaccaagag gccaggcgtg 660
cccgaccggt tcagcggatc tagctctggc gccgaccgct acctgatcat ccccagcgtg 720
caggccgatg acgaggccga ttactactgt ggcgccgact acatcggcgg ctacgtgttc 780
ggcggaggca cccagctgac cgtgaccggc aattggagcc acccgcagtt cgaaaaagga 840
ggtggaggtt caggtggtgg aggctctgag agcaagtacg gaccgccctg ccccccttgc 900
cctatgttct gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc 960
accgtggcct tcatcatctt ttgggtccgc agcaagcgga gcagaggcgg ccacagcgac 1020
tacatgaaca tgacccctag acggcctggc cccaccagaa agcactacca gccctacgcc 1080
cctccccggg actttgccgc ctacagaagc cgggtgaagt tcagcagaag cgccgacgcc 1140
cctgcctacc agcagggcca gaatcagctg tacaacgagc tgaacctggg cagaagggaa 1200
gagtacgacg tcctggataa gcggagaggc cgggaccctg agatgggcgg caagcctcgg 1260
cggaagaacc cccaggaagg cctgtataac gaactgcaga aagacaagat ggccgaggcc 1320
tacagcgaga tcggcatgaa gggcgagcgg aggcggggca agggccacga cggcctgtat 1380
cagggcctgt ccaccgccac caaggatacc tacgacgccc tgcacatgca ggccctgccc 1440
ccaaggctcg agggcggcgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag 1500
gagaatcccg gccctaggat gcttctcctg gtgacaagcc ttctgctctg tgagttacca 1560
cacccagcat tcctcctgat cccacgcaaa gtgtgtaacg gaataggtat tggtgaattt 1620
aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 1680
agtggcgatc tccacatcct gccggtggca tttaggggtg actccttcac acatactcct 1740
cctctggatc cacaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1800
ctgattcagg cttggcctga aaacaggacg gacctccatg cctttgagaa cctagaaatc 1860
atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1920
acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1980
aacaaaaatt tgtgctatgc aaatacaata aactggaaaa aactgtttgg gacctccggt 2040
cagaaaacca aaattataag caacagaggt gaaaacagct gcaaggccac aggccaggtc 2100
tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 2160
tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 2220
ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 2280
gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 2340
attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 2400
ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 2460
tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 2520
atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 2580
ctcttcatgt ga 2592
<210> 25
<211> 863
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A045 CAR盒蛋白
序列
<400> 25
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys
485 490 495
Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr
500 505 510
Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro
515 520 525
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
530 535 540
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile
545 550 555 560
Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe
565 570 575
Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
580 585 590
Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn
595 600 605
Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
610 615 620
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile
625 630 635 640
Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val
645 650 655
Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp
660 665 670
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn
675 680 685
Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
690 695 700
Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser
705 710 715 720
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
725 730 735
Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
740 745 750
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly
755 760 765
Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
770 775 780
His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr
785 790 795 800
Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
805 810 815
Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
820 825 830
Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala
835 840 845
Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 26
<211> 2583
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A142 CAR (FMC63 scFv_STII_IgG4
铰链_CD28tm_CD28icd (Y218F)_CD3z_T2A_tEGFR)
表达盒
<400> 26
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60
atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120
gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180
aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240
cctagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300
gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360
ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420
ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480
cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540
gtgagctgga tcaggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600
agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660
agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720
tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780
accagcgtga ccgtgagcag caattggagc cacccgcagt tcgaaaaagg aggtggaggt 840
tcaggtggtg gaggctctga gagcaagtac ggaccgccct gccccccttg ccctatgttc 900
tgggtgctgg tggtggtcgg aggcgtgctg gcctgctaca gcctgctggt caccgtggcc 960
ttcatcatct tttgggtccg cagcaagcgg agcagaggcg gccacagcga ctacatgaac 1020
atgaccccta gacggcctgg ccccaccaga aagcactacc agccctacgc ccctccccgg 1080
gactttgccg ccttcagaag ccgggtgaag ttcagcagaa gcgccgacgc ccctgcctac 1140
cagcagggcc agaatcagct gtacaacgag ctgaacctgg gcagaaggga agagtacgac 1200
gtcctggata agcggagagg ccgggaccct gagatgggcg gcaagcctcg gcggaagaac 1260
ccccaggaag gcctgtataa cgaactgcag aaagacaaga tggccgaggc ctacagcgag 1320
atcggcatga agggcgagcg gaggcggggc aagggccacg acggcctgta tcagggcctg 1380
tccaccgcca ccaaggatac ctacgacgcc ctgcacatgc aggccctgcc cccaaggctc 1440
gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 1500
ggccctagga tgcttctcct ggtgacaagc cttctgctct gtgagttacc acacccagca 1560
ttcctcctga tcccacgcaa agtgtgtaac ggaataggta ttggtgaatt taaagactca 1620
ctctccataa atgctacgaa tattaaacac ttcaaaaact gcacctccat cagtggcgat 1680
ctccacatcc tgccggtggc atttaggggt gactccttca cacatactcc tcctctggat 1740
ccacaggaac tggatattct gaaaaccgta aaggaaatca cagggttttt gctgattcag 1800
gcttggcctg aaaacaggac ggacctccat gcctttgaga acctagaaat catacgcggc 1860
aggaccaagc aacatggtca gttttctctt gcagtcgtca gcctgaacat aacatccttg 1920
ggattacgct ccctcaagga gataagtgat ggagatgtga taatttcagg aaacaaaaat 1980
ttgtgctatg caaatacaat aaactggaaa aaactgtttg ggacctccgg tcagaaaacc 2040
aaaattataa gcaacagagg tgaaaacagc tgcaaggcca caggccaggt ctgccatgcc 2100
ttgtgctccc ccgagggctg ctggggcccg gagcccaggg actgcgtctc ttgccggaat 2160
gtcagccgag gcagggaatg cgtggacaag tgcaaccttc tggagggtga gccaagggag 2220
tttgtggaga actctgagtg catacagtgc cacccagagt gcctgcctca ggccatgaac 2280
atcacctgca caggacgggg accagacaac tgtatccagt gtgcccacta cattgacggc 2340
ccccactgcg tcaagacctg cccggcagga gtcatgggag aaaacaacac cctggtctgg 2400
aagtacgcag acgccggcca tgtgtgccac ctgtgccatc caaactgcac ctacggatgc 2460
actgggccag gtcttgaagg ctgtccaacg aatgggccta agatcccgtc catcgccact 2520
gggatggtgg gggccctcct cttgctgctg gtggtggccc tggggatcgg cctcttcatg 2580
tga 2583
<210> 27
<211> 860
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A142 CAR盒蛋白
序列
<400> 27
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu
465 470 475 480
Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val
485 490 495
Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu
500 505 510
Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val
515 520 525
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
530 535 540
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
545 550 555 560
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
565 570 575
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
580 585 590
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
595 600 605
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
610 615 620
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
625 630 635 640
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
645 650 655
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
660 665 670
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
675 680 685
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
690 695 700
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
705 710 715 720
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
725 730 735
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
740 745 750
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
755 760 765
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
770 775 780
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
785 790 795 800
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
805 810 815
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
820 825 830
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
835 840 845
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 28
<211> 2583
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A143 CAR (FMC63 scFv_STII_IgG4
铰链_CD28tm_CD28icd (Y206F, Y209F,
Y218F)_CD3z_T2A_tEGFR)表达盒
<400> 28
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60
atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120
gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180
aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240
cctagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300
gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360
ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420
ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480
cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540
gtgagctgga tcaggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600
agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660
agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720
tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780
accagcgtga ccgtgagcag caattggagc cacccgcagt tcgaaaaagg aggtggaggt 840
tcaggtggtg gaggctctga gagcaagtac ggaccgccct gccccccttg ccctatgttc 900
tgggtgctgg tggtggtcgg aggcgtgctg gcctgctaca gcctgctggt caccgtggcc 960
ttcatcatct tttgggtccg cagcaagcgg agcagaggcg gccacagcga ctacatgaac 1020
atgaccccta gacggcctgg ccccaccaga aagcacttcc agcccttcgc ccctccccgg 1080
gactttgccg ccttcagaag ccgggtgaag ttcagcagaa gcgccgacgc ccctgcctac 1140
cagcagggcc agaatcagct gtacaacgag ctgaacctgg gcagaaggga agagtacgac 1200
gtcctggata agcggagagg ccgggaccct gagatgggcg gcaagcctcg gcggaagaac 1260
ccccaggaag gcctgtataa cgaactgcag aaagacaaga tggccgaggc ctacagcgag 1320
atcggcatga agggcgagcg gaggcggggc aagggccacg acggcctgta tcagggcctg 1380
tccaccgcca ccaaggatac ctacgacgcc ctgcacatgc aggccctgcc cccaaggctc 1440
gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 1500
ggccctagga tgcttctcct ggtgacaagc cttctgctct gtgagttacc acacccagca 1560
ttcctcctga tcccacgcaa agtgtgtaac ggaataggta ttggtgaatt taaagactca 1620
ctctccataa atgctacgaa tattaaacac ttcaaaaact gcacctccat cagtggcgat 1680
ctccacatcc tgccggtggc atttaggggt gactccttca cacatactcc tcctctggat 1740
ccacaggaac tggatattct gaaaaccgta aaggaaatca cagggttttt gctgattcag 1800
gcttggcctg aaaacaggac ggacctccat gcctttgaga acctagaaat catacgcggc 1860
aggaccaagc aacatggtca gttttctctt gcagtcgtca gcctgaacat aacatccttg 1920
ggattacgct ccctcaagga gataagtgat ggagatgtga taatttcagg aaacaaaaat 1980
ttgtgctatg caaatacaat aaactggaaa aaactgtttg ggacctccgg tcagaaaacc 2040
aaaattataa gcaacagagg tgaaaacagc tgcaaggcca caggccaggt ctgccatgcc 2100
ttgtgctccc ccgagggctg ctggggcccg gagcccaggg actgcgtctc ttgccggaat 2160
gtcagccgag gcagggaatg cgtggacaag tgcaaccttc tggagggtga gccaagggag 2220
tttgtggaga actctgagtg catacagtgc cacccagagt gcctgcctca ggccatgaac 2280
atcacctgca caggacgggg accagacaac tgtatccagt gtgcccacta cattgacggc 2340
ccccactgcg tcaagacctg cccggcagga gtcatgggag aaaacaacac cctggtctgg 2400
aagtacgcag acgccggcca tgtgtgccac ctgtgccatc caaactgcac ctacggatgc 2460
actgggccag gtcttgaagg ctgtccaacg aatgggccta agatcccgtc catcgccact 2520
gggatggtgg gggccctcct cttgctgctg gtggtggccc tggggatcgg cctcttcatg 2580
tga 2583
<210> 29
<211> 860
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A143 CAR盒蛋白
序列
<400> 29
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Phe Gln Pro Phe Ala Pro Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu
465 470 475 480
Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val
485 490 495
Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu
500 505 510
Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val
515 520 525
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
530 535 540
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
545 550 555 560
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
565 570 575
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
580 585 590
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
595 600 605
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
610 615 620
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
625 630 635 640
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
645 650 655
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
660 665 670
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
675 680 685
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
690 695 700
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
705 710 715 720
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
725 730 735
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
740 745 750
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
755 760 765
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
770 775 780
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
785 790 795 800
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
805 810 815
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
820 825 830
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
835 840 845
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 30
<211> 2592
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A144 CAR (R12 scFv_STII_IgG4
铰链_CD28tm_CD28icd (Y218F)_CD3z_T2A_tEGFR)
表达盒
<400> 30
atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60
atcccccagg aacagctcgt cgaaagcggc ggcagactgg tgacacctgg cggcagcctg 120
accctgagct gcaaggccag cggcttcgac ttcagcgcct actacatgag ctgggtccgc 180
caggcccctg gcaagggact ggaatggatc gccaccatct accccagcag cggcaagacc 240
tactacgcca cctgggtgaa cggacggttc accatctcca gcgacaacgc ccagaacacc 300
gtggacctgc agatgaacag cctgacagcc gccgaccggg ccacctactt ttgcgccaga 360
gacagctacg ccgacgacgg cgccctgttc aacatctggg gccctggcac cctggtgaca 420
atctctagcg gcggaggcgg atctggtggc ggaggaagtg gcggcggagg atctgagctg 480
gtgctgaccc agagcccctc tgtgtctgct gccctgggaa gccctgccaa gatcacctgt 540
accctgagca gcgcccacaa gaccgacacc atcgactggt atcagcagct gcagggcgag 600
gcccccagat acctgatgca ggtgcagagc gacggcagct acaccaagag gccaggcgtg 660
cccgaccggt tcagcggatc tagctctggc gccgaccgct acctgatcat ccccagcgtg 720
caggccgatg acgaggccga ttactactgt ggcgccgact acatcggcgg ctacgtgttc 780
ggcggaggca cccagctgac cgtgaccggc aattggagcc acccgcagtt cgaaaaagga 840
ggtggaggtt caggtggtgg aggctctgag agcaagtacg gaccgccctg ccccccttgc 900
cctatgttct gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc 960
accgtggcct tcatcatctt ttgggtccgc agcaagcgga gcagaggcgg ccacagcgac 1020
tacatgaaca tgacccctag acggcctggc cccaccagaa agcactacca gccctacgcc 1080
cctccccggg actttgccgc cttcagaagc cgggtgaagt tcagcagaag cgccgacgcc 1140
cctgcctacc agcagggcca gaatcagctg tacaacgagc tgaacctggg cagaagggaa 1200
gagtacgacg tcctggataa gcggagaggc cgggaccctg agatgggcgg caagcctcgg 1260
cggaagaacc cccaggaagg cctgtataac gaactgcaga aagacaagat ggccgaggcc 1320
tacagcgaga tcggcatgaa gggcgagcgg aggcggggca agggccacga cggcctgtat 1380
cagggcctgt ccaccgccac caaggatacc tacgacgccc tgcacatgca ggccctgccc 1440
ccaaggctcg agggcggcgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag 1500
gagaatcccg gccctaggat gcttctcctg gtgacaagcc ttctgctctg tgagttacca 1560
cacccagcat tcctcctgat cccacgcaaa gtgtgtaacg gaataggtat tggtgaattt 1620
aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 1680
agtggcgatc tccacatcct gccggtggca tttaggggtg actccttcac acatactcct 1740
cctctggatc cacaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1800
ctgattcagg cttggcctga aaacaggacg gacctccatg cctttgagaa cctagaaatc 1860
atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1920
acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1980
aacaaaaatt tgtgctatgc aaatacaata aactggaaaa aactgtttgg gacctccggt 2040
cagaaaacca aaattataag caacagaggt gaaaacagct gcaaggccac aggccaggtc 2100
tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 2160
tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 2220
ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 2280
gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 2340
attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 2400
ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 2460
tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 2520
atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 2580
ctcttcatgt ga 2592
<210> 31
<211> 863
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A144 CAR盒蛋白
序列
<400> 31
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys
485 490 495
Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr
500 505 510
Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro
515 520 525
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
530 535 540
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile
545 550 555 560
Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe
565 570 575
Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
580 585 590
Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn
595 600 605
Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
610 615 620
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile
625 630 635 640
Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val
645 650 655
Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp
660 665 670
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn
675 680 685
Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
690 695 700
Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser
705 710 715 720
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
725 730 735
Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
740 745 750
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly
755 760 765
Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
770 775 780
His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr
785 790 795 800
Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
805 810 815
Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
820 825 830
Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala
835 840 845
Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 32
<211> 2592
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A145 (R12 scFv_STII_IgG4
铰链_CD28tm_CD28icd (Y206F, Y209F,
Y218F)_CD3z_T2A_tEGFR)表达盒
<400> 32
atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60
atcccccagg aacagctcgt cgaaagcggc ggcagactgg tgacacctgg cggcagcctg 120
accctgagct gcaaggccag cggcttcgac ttcagcgcct actacatgag ctgggtccgc 180
caggcccctg gcaagggact ggaatggatc gccaccatct accccagcag cggcaagacc 240
tactacgcca cctgggtgaa cggacggttc accatctcca gcgacaacgc ccagaacacc 300
gtggacctgc agatgaacag cctgacagcc gccgaccggg ccacctactt ttgcgccaga 360
gacagctacg ccgacgacgg cgccctgttc aacatctggg gccctggcac cctggtgaca 420
atctctagcg gcggaggcgg atctggtggc ggaggaagtg gcggcggagg atctgagctg 480
gtgctgaccc agagcccctc tgtgtctgct gccctgggaa gccctgccaa gatcacctgt 540
accctgagca gcgcccacaa gaccgacacc atcgactggt atcagcagct gcagggcgag 600
gcccccagat acctgatgca ggtgcagagc gacggcagct acaccaagag gccaggcgtg 660
cccgaccggt tcagcggatc tagctctggc gccgaccgct acctgatcat ccccagcgtg 720
caggccgatg acgaggccga ttactactgt ggcgccgact acatcggcgg ctacgtgttc 780
ggcggaggca cccagctgac cgtgaccggc aattggagcc acccgcagtt cgaaaaagga 840
ggtggaggtt caggtggtgg aggctctgag agcaagtacg gaccgccctg ccccccttgc 900
cctatgttct gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc 960
accgtggcct tcatcatctt ttgggtccgc agcaagcgga gcagaggcgg ccacagcgac 1020
tacatgaaca tgacccctag acggcctggc cccaccagaa agcacttcca gcccttcgcc 1080
cctccccggg actttgccgc cttcagaagc cgggtgaagt tcagcagaag cgccgacgcc 1140
cctgcctacc agcagggcca gaatcagctg tacaacgagc tgaacctggg cagaagggaa 1200
gagtacgacg tcctggataa gcggagaggc cgggaccctg agatgggcgg caagcctcgg 1260
cggaagaacc cccaggaagg cctgtataac gaactgcaga aagacaagat ggccgaggcc 1320
tacagcgaga tcggcatgaa gggcgagcgg aggcggggca agggccacga cggcctgtat 1380
cagggcctgt ccaccgccac caaggatacc tacgacgccc tgcacatgca ggccctgccc 1440
ccaaggctcg agggcggcgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag 1500
gagaatcccg gccctaggat gcttctcctg gtgacaagcc ttctgctctg tgagttacca 1560
cacccagcat tcctcctgat cccacgcaaa gtgtgtaacg gaataggtat tggtgaattt 1620
aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 1680
agtggcgatc tccacatcct gccggtggca tttaggggtg actccttcac acatactcct 1740
cctctggatc cacaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1800
ctgattcagg cttggcctga aaacaggacg gacctccatg cctttgagaa cctagaaatc 1860
atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1920
acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1980
aacaaaaatt tgtgctatgc aaatacaata aactggaaaa aactgtttgg gacctccggt 2040
cagaaaacca aaattataag caacagaggt gaaaacagct gcaaggccac aggccaggtc 2100
tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 2160
tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 2220
ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 2280
gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 2340
attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 2400
ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 2460
tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 2520
atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 2580
ctcttcatgt ga 2592
<210> 33
<211> 863
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A145 CAR盒蛋白
序列
<400> 33
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Phe Gln Pro Phe Ala Pro Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys
485 490 495
Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr
500 505 510
Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro
515 520 525
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
530 535 540
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile
545 550 555 560
Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe
565 570 575
Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
580 585 590
Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn
595 600 605
Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
610 615 620
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile
625 630 635 640
Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val
645 650 655
Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp
660 665 670
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn
675 680 685
Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
690 695 700
Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser
705 710 715 720
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
725 730 735
Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
740 745 750
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly
755 760 765
Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
770 775 780
His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr
785 790 795 800
Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
805 810 815
Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
820 825 830
Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala
835 840 845
Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 34
<211> 2583
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A175 CAR (FMC63 scFv_STII_IgG4
铰链_CD28tm_CD28icd (Y191F, Y206F, Y209F,
Y218F)_CD3z_T2A_tEGFR)表达盒
<400> 34
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60
atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120
gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180
aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240
cctagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300
gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360
ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420
ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480
cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540
gtgagctgga tcaggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600
agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660
agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720
tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780
accagcgtga ccgtgagcag caattggagc cacccgcagt tcgaaaaagg aggtggaggt 840
tcaggtggtg gaggctctga gagcaagtac ggaccgccct gccccccttg ccctatgttc 900
tgggtgctgg tggtggtcgg aggcgtgctg gcctgctaca gcctgctggt caccgtggcc 960
ttcatcatct tttgggtccg cagcaagcgg agcagaggcg gccacagcga cttcatgaac 1020
atgaccccta gacggcctgg ccccaccaga aagcacttcc agcccttcgc ccctccccgg 1080
gactttgccg ccttcagaag ccgggtgaag ttcagcagaa gcgccgacgc ccctgcctac 1140
cagcagggcc agaatcagct gtacaacgag ctgaacctgg gcagaaggga agagtacgac 1200
gtcctggata agcggagagg ccgggaccct gagatgggcg gcaagcctcg gcggaagaac 1260
ccccaggaag gcctgtataa cgaactgcag aaagacaaga tggccgaggc ctacagcgag 1320
atcggcatga agggcgagcg gaggcggggc aagggccacg acggcctgta tcagggcctg 1380
tccaccgcca ccaaggatac ctacgacgcc ctgcacatgc aggccctgcc cccaaggctc 1440
gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 1500
ggccctagga tgcttctcct ggtgacaagc cttctgctct gtgagttacc acacccagca 1560
ttcctcctga tcccacgcaa agtgtgtaac ggaataggta ttggtgaatt taaagactca 1620
ctctccataa atgctacgaa tattaaacac ttcaaaaact gcacctccat cagtggcgat 1680
ctccacatcc tgccggtggc atttaggggt gactccttca cacatactcc tcctctggat 1740
ccacaggaac tggatattct gaaaaccgta aaggaaatca cagggttttt gctgattcag 1800
gcttggcctg aaaacaggac ggacctccat gcctttgaga acctagaaat catacgcggc 1860
aggaccaagc aacatggtca gttttctctt gcagtcgtca gcctgaacat aacatccttg 1920
ggattacgct ccctcaagga gataagtgat ggagatgtga taatttcagg aaacaaaaat 1980
ttgtgctatg caaatacaat aaactggaaa aaactgtttg ggacctccgg tcagaaaacc 2040
aaaattataa gcaacagagg tgaaaacagc tgcaaggcca caggccaggt ctgccatgcc 2100
ttgtgctccc ccgagggctg ctggggcccg gagcccaggg actgcgtctc ttgccggaat 2160
gtcagccgag gcagggaatg cgtggacaag tgcaaccttc tggagggtga gccaagggag 2220
tttgtggaga actctgagtg catacagtgc cacccagagt gcctgcctca ggccatgaac 2280
atcacctgca caggacgggg accagacaac tgtatccagt gtgcccacta cattgacggc 2340
ccccactgcg tcaagacctg cccggcagga gtcatgggag aaaacaacac cctggtctgg 2400
aagtacgcag acgccggcca tgtgtgccac ctgtgccatc caaactgcac ctacggatgc 2460
actgggccag gtcttgaagg ctgtccaacg aatgggccta agatcccgtc catcgccact 2520
gggatggtgg gggccctcct cttgctgctg gtggtggccc tggggatcgg cctcttcatg 2580
tga 2583
<210> 35
<211> 860
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A175 CAR盒蛋白
序列
<400> 35
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Phe Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Phe Gln Pro Phe Ala Pro Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu
465 470 475 480
Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val
485 490 495
Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu
500 505 510
Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val
515 520 525
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
530 535 540
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
545 550 555 560
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
565 570 575
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
580 585 590
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
595 600 605
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
610 615 620
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
625 630 635 640
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
645 650 655
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
660 665 670
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
675 680 685
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
690 695 700
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
705 710 715 720
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
725 730 735
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
740 745 750
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
755 760 765
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
770 775 780
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
785 790 795 800
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
805 810 815
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
820 825 830
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
835 840 845
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 36
<211> 2592
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A176 CAR (R12
scFv_STII_IgG4_CD28tm_CD28icd (Y191F, Y206F,
Y209F, Y218F)_CD3z_T2A_tEGFR)表达盒
<400> 36
atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60
atcccccagg aacagctcgt cgaaagcggc ggcagactgg tgacacctgg cggcagcctg 120
accctgagct gcaaggccag cggcttcgac ttcagcgcct actacatgag ctgggtccgc 180
caggcccctg gcaagggact ggaatggatc gccaccatct accccagcag cggcaagacc 240
tactacgcca cctgggtgaa cggacggttc accatctcca gcgacaacgc ccagaacacc 300
gtggacctgc agatgaacag cctgacagcc gccgaccggg ccacctactt ttgcgccaga 360
gacagctacg ccgacgacgg cgccctgttc aacatctggg gccctggcac cctggtgaca 420
atctctagcg gcggaggcgg atctggtggc ggaggaagtg gcggcggagg atctgagctg 480
gtgctgaccc agagcccctc tgtgtctgct gccctgggaa gccctgccaa gatcacctgt 540
accctgagca gcgcccacaa gaccgacacc atcgactggt atcagcagct gcagggcgag 600
gcccccagat acctgatgca ggtgcagagc gacggcagct acaccaagag gccaggcgtg 660
cccgaccggt tcagcggatc tagctctggc gccgaccgct acctgatcat ccccagcgtg 720
caggccgatg acgaggccga ttactactgt ggcgccgact acatcggcgg ctacgtgttc 780
ggcggaggca cccagctgac cgtgaccggc aattggagcc acccgcagtt cgaaaaagga 840
ggtggaggtt caggtggtgg aggctctgag agcaagtacg gaccgccctg ccccccttgc 900
cctatgttct gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc 960
accgtggcct tcatcatctt ttgggtccgc agcaagcgga gcagaggcgg ccacagcgac 1020
ttcatgaaca tgacccctag acggcctggc cccaccagaa agcacttcca gcccttcgcc 1080
cctccccggg actttgccgc cttcagaagc cgggtgaagt tcagcagaag cgccgacgcc 1140
cctgcctacc agcagggcca gaatcagctg tacaacgagc tgaacctggg cagaagggaa 1200
gagtacgacg tcctggataa gcggagaggc cgggaccctg agatgggcgg caagcctcgg 1260
cggaagaacc cccaggaagg cctgtataac gaactgcaga aagacaagat ggccgaggcc 1320
tacagcgaga tcggcatgaa gggcgagcgg aggcggggca agggccacga cggcctgtat 1380
cagggcctgt ccaccgccac caaggatacc tacgacgccc tgcacatgca ggccctgccc 1440
ccaaggctcg agggcggcgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag 1500
gagaatcccg gccctaggat gcttctcctg gtgacaagcc ttctgctctg tgagttacca 1560
cacccagcat tcctcctgat cccacgcaaa gtgtgtaacg gaataggtat tggtgaattt 1620
aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 1680
agtggcgatc tccacatcct gccggtggca tttaggggtg actccttcac acatactcct 1740
cctctggatc cacaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1800
ctgattcagg cttggcctga aaacaggacg gacctccatg cctttgagaa cctagaaatc 1860
atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1920
acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1980
aacaaaaatt tgtgctatgc aaatacaata aactggaaaa aactgtttgg gacctccggt 2040
cagaaaacca aaattataag caacagaggt gaaaacagct gcaaggccac aggccaggtc 2100
tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 2160
tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 2220
ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 2280
gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 2340
attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 2400
ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 2460
tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 2520
atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 2580
ctcttcatgt ga 2592
<210> 37
<211> 863
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A176 CAR盒蛋白
序列
<400> 37
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Phe Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Phe Gln Pro Phe Ala Pro Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys
485 490 495
Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr
500 505 510
Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro
515 520 525
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
530 535 540
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile
545 550 555 560
Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe
565 570 575
Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
580 585 590
Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn
595 600 605
Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
610 615 620
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile
625 630 635 640
Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val
645 650 655
Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp
660 665 670
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn
675 680 685
Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
690 695 700
Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser
705 710 715 720
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
725 730 735
Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
740 745 750
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly
755 760 765
Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
770 775 780
His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr
785 790 795 800
Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
805 810 815
Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
820 825 830
Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala
835 840 845
Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 38
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人 CD28细胞内
共刺激结构域(L186G/L187G)
<400> 38
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 39
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 合成序列Strep-Tag肽
<400> 39
Trp Arg His Pro Gln Phe Gly Gly
1 5
<210> 40
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 合成序列Strep-Tag II肽, 8-mer
<400> 40
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 41
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> 合成序列野生型人CD28
跨膜结构域
<400> 41
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 42
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 合成序列ITAM基序
<220>
<221> 变体
<222> (2)...(3)
<223> Xaa =任何氨基酸
<220>
<221> 变体
<222> 4
<223> Xaa = L或I
<220>
<221> 变体
<222> (5)...(15)
<223> Xaa =任何氨基酸
<220>
<221> 变体
<222> 16
<223> Xaa = L或I
<220>
<221> 变体
<222> (10)...(12)
<223> 氨基酸10-12中的任何一个或全部可以存在或不存在。
<400> 42
Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa
1 5 10 15
<210> 43
<211> 114
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人CD8α IgV样结构域
<400> 43
Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr Trp Asn Leu Gly Glu
1 5 10 15
Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser Asn Pro Thr Ser Gly
20 25 30
Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala Ala Ser Pro Thr Phe
35 40 45
Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala Ala Glu Gly Leu Asp
50 55 60
Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp Thr Phe Val Leu Thr
65 70 75 80
Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr Tyr Phe Cys Ser Ala
85 90 95
Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu
100 105 110
Pro Ala
<210> 44
<211> 111
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人CD8β IgV样结构域
<400> 44
Leu Gln Gln Thr Pro Ala Tyr Ile Lys Val Gln Thr Asn Lys Met Val
1 5 10 15
Met Leu Ser Cys Glu Ala Lys Ile Ser Leu Ser Asn Met Arg Ile Tyr
20 25 30
Trp Leu Arg Gln Arg Gln Ala Pro Ser Ser Asp Ser His His Glu Phe
35 40 45
Leu Ala Leu Trp Asp Ser Ala Lys Gly Thr Ile His Gly Glu Glu Val
50 55 60
Glu Gln Glu Lys Ile Ala Val Phe Arg Asp Ala Ser Arg Phe Ile Leu
65 70 75 80
Asn Leu Thr Ser Val Lys Pro Glu Asp Ser Gly Ile Tyr Phe Cys Met
85 90 95
Ile Val Gly Ser Pro Glu Leu Thr Phe Gly Lys Gly Thr Gln Leu
100 105 110
<210> 45
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人CD28细胞内
共刺激结构域(P208A, P211A)
<400> 45
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Ala Tyr Ala Ala
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 46
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> 合成序列人CD28细胞内
共刺激结构域(P208A, P211A, Y218F)
<400> 46
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Ala Tyr Ala Ala
20 25 30
Pro Arg Asp Phe Ala Ala Phe Arg Ser
35 40
<210> 47
<211> 41
<212> PRT
<213> 人工序列
<220>
<223> 合成序列CD28细胞内
共刺激结构域(Y206F, P208A, Y209F, P211A,
Y218F)
<400> 47
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Phe Gln Ala Phe Ala Ala
20 25 30
Pro Arg Asp Phe Ala Ala Phe Arg Ser
35 40
<210> 48
<211> 2583
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A192 CAR (FMC63 scFv_STII_IgG4
铰链_CD28tm_CD28icd (P208A,
P211A)_CD3z_T2A_tEGFR)表达盒
<400> 48
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60
atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120
gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180
aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240
cctagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300
gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360
ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420
ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480
cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540
gtgagctgga tcaggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600
agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660
agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720
tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780
accagcgtga ccgtgagcag caattggagc cacccgcagt tcgaaaaagg aggtggaggt 840
tcaggtggtg gaggctctga gagcaagtac ggaccgccct gccccccttg ccctatgttc 900
tgggtgctgg tggtggtcgg aggcgtgctg gcctgctaca gcctgctggt caccgtggcc 960
ttcatcatct tttgggtccg cagcaagcgg agcagaggcg gccacagcga ctacatgaac 1020
atgaccccta gacggcctgg ccccaccaga aagcactacc aggcctacgc cgctccccgg 1080
gactttgccg cctacagaag ccgggtgaag ttcagcagaa gcgccgacgc ccctgcctac 1140
cagcagggcc agaatcagct gtacaacgag ctgaacctgg gcagaaggga agagtacgac 1200
gtcctggata agcggagagg ccgggaccct gagatgggcg gcaagcctcg gcggaagaac 1260
ccccaggaag gcctgtataa cgaactgcag aaagacaaga tggccgaggc ctacagcgag 1320
atcggcatga agggcgagcg gaggcggggc aagggccacg acggcctgta tcagggcctg 1380
tccaccgcca ccaaggatac ctacgacgcc ctgcacatgc aggccctgcc cccaaggctc 1440
gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 1500
ggccctagga tgcttctcct ggtgacaagc cttctgctct gtgagttacc acacccagca 1560
ttcctcctga tcccacgcaa agtgtgtaac ggaataggta ttggtgaatt taaagactca 1620
ctctccataa atgctacgaa tattaaacac ttcaaaaact gcacctccat cagtggcgat 1680
ctccacatcc tgccggtggc atttaggggt gactccttca cacatactcc tcctctggat 1740
ccacaggaac tggatattct gaaaaccgta aaggaaatca cagggttttt gctgattcag 1800
gcttggcctg aaaacaggac ggacctccat gcctttgaga acctagaaat catacgcggc 1860
aggaccaagc aacatggtca gttttctctt gcagtcgtca gcctgaacat aacatccttg 1920
ggattacgct ccctcaagga gataagtgat ggagatgtga taatttcagg aaacaaaaat 1980
ttgtgctatg caaatacaat aaactggaaa aaactgtttg ggacctccgg tcagaaaacc 2040
aaaattataa gcaacagagg tgaaaacagc tgcaaggcca caggccaggt ctgccatgcc 2100
ttgtgctccc ccgagggctg ctggggcccg gagcccaggg actgcgtctc ttgccggaat 2160
gtcagccgag gcagggaatg cgtggacaag tgcaaccttc tggagggtga gccaagggag 2220
tttgtggaga actctgagtg catacagtgc cacccagagt gcctgcctca ggccatgaac 2280
atcacctgca caggacgggg accagacaac tgtatccagt gtgcccacta cattgacggc 2340
ccccactgcg tcaagacctg cccggcagga gtcatgggag aaaacaacac cctggtctgg 2400
aagtacgcag acgccggcca tgtgtgccac ctgtgccatc caaactgcac ctacggatgc 2460
actgggccag gtcttgaagg ctgtccaacg aatgggccta agatcccgtc catcgccact 2520
gggatggtgg gggccctcct cttgctgctg gtggtggccc tggggatcgg cctcttcatg 2580
tga 2583
<210> 49
<211> 859
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A192 CAR盒蛋白
序列
<400> 49
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Thr Lys Leu Glu Ile Thr
115 120 125
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
130 135 140
Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro
145 150 155 160
Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro
165 170 175
Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu
180 185 190
Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala
195 200 205
Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val
210 215 220
Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr
225 230 235 240
Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp
245 250 255
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro Gln
260 265 270
Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys
275 280 285
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val
290 295 300
Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe
305 310 315 320
Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp
325 330 335
Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
340 345 350
Gln Ala Tyr Ala Ala Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val
355 360 365
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
370 375 380
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
385 390 395 400
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
405 410 415
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
420 425 430
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
435 440 445
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
450 455 460
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu Glu
465 470 475 480
Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu Leu
500 505 510
Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys
515 520 525
Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala
530 535 540
Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu
545 550 555 560
His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro
565 570 575
Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile
580 585 590
Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu
595 600 605
His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His
610 615 620
Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly
625 630 635 640
Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly
645 650 655
Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe
660 665 670
Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn
675 680 685
Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu
690 695 700
Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val
705 710 715 720
Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu
725 730 735
Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu
740 745 750
Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp
755 760 765
Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys
770 775 780
Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys
785 790 795 800
Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr
805 810 815
Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro
820 825 830
Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu
835 840 845
Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855
<210> 50
<211> 2583
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A193 CAR (FMC63 scFv_STII_IgG4
铰链_CD28tm_CD28icd (P208A, P211A,
Y218F)_CD3z_T2A_tEGFR)表达盒
<400> 50
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60
atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120
gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180
aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240
cctagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300
gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360
ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420
ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480
cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540
gtgagctgga tcaggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600
agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660
agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720
tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780
accagcgtga ccgtgagcag caattggagc cacccgcagt tcgaaaaagg aggtggaggt 840
tcaggtggtg gaggctctga gagcaagtac ggaccgccct gccccccttg ccctatgttc 900
tgggtgctgg tggtggtcgg aggcgtgctg gcctgctaca gcctgctggt caccgtggcc 960
ttcatcatct tttgggtccg cagcaagcgg agcagaggcg gccacagcga ctacatgaac 1020
atgaccccta gacggcctgg ccccaccaga aagcactacc aggcctacgc cgctccccgg 1080
gactttgccg ccttcagaag ccgggtgaag ttcagcagaa gcgccgacgc ccctgcctac 1140
cagcagggcc agaatcagct gtacaacgag ctgaacctgg gcagaaggga agagtacgac 1200
gtcctggata agcggagagg ccgggaccct gagatgggcg gcaagcctcg gcggaagaac 1260
ccccaggaag gcctgtataa cgaactgcag aaagacaaga tggccgaggc ctacagcgag 1320
atcggcatga agggcgagcg gaggcggggc aagggccacg acggcctgta tcagggcctg 1380
tccaccgcca ccaaggatac ctacgacgcc ctgcacatgc aggccctgcc cccaaggctc 1440
gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 1500
ggccctagga tgcttctcct ggtgacaagc cttctgctct gtgagttacc acacccagca 1560
ttcctcctga tcccacgcaa agtgtgtaac ggaataggta ttggtgaatt taaagactca 1620
ctctccataa atgctacgaa tattaaacac ttcaaaaact gcacctccat cagtggcgat 1680
ctccacatcc tgccggtggc atttaggggt gactccttca cacatactcc tcctctggat 1740
ccacaggaac tggatattct gaaaaccgta aaggaaatca cagggttttt gctgattcag 1800
gcttggcctg aaaacaggac ggacctccat gcctttgaga acctagaaat catacgcggc 1860
aggaccaagc aacatggtca gttttctctt gcagtcgtca gcctgaacat aacatccttg 1920
ggattacgct ccctcaagga gataagtgat ggagatgtga taatttcagg aaacaaaaat 1980
ttgtgctatg caaatacaat aaactggaaa aaactgtttg ggacctccgg tcagaaaacc 2040
aaaattataa gcaacagagg tgaaaacagc tgcaaggcca caggccaggt ctgccatgcc 2100
ttgtgctccc ccgagggctg ctggggcccg gagcccaggg actgcgtctc ttgccggaat 2160
gtcagccgag gcagggaatg cgtggacaag tgcaaccttc tggagggtga gccaagggag 2220
tttgtggaga actctgagtg catacagtgc cacccagagt gcctgcctca ggccatgaac 2280
atcacctgca caggacgggg accagacaac tgtatccagt gtgcccacta cattgacggc 2340
ccccactgcg tcaagacctg cccggcagga gtcatgggag aaaacaacac cctggtctgg 2400
aagtacgcag acgccggcca tgtgtgccac ctgtgccatc caaactgcac ctacggatgc 2460
actgggccag gtcttgaagg ctgtccaacg aatgggccta agatcccgtc catcgccact 2520
gggatggtgg gggccctcct cttgctgctg gtggtggccc tggggatcgg cctcttcatg 2580
tga 2583
<210> 51
<211> 860
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A193 CAR盒蛋白
序列
<400> 51
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Tyr Gln Ala Tyr Ala Ala Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu
465 470 475 480
Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val
485 490 495
Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu
500 505 510
Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val
515 520 525
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
530 535 540
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
545 550 555 560
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
565 570 575
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
580 585 590
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
595 600 605
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
610 615 620
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
625 630 635 640
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
645 650 655
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
660 665 670
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
675 680 685
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
690 695 700
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
705 710 715 720
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
725 730 735
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
740 745 750
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
755 760 765
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
770 775 780
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
785 790 795 800
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
805 810 815
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
820 825 830
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
835 840 845
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 52
<211> 2583
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A194 CAR (FMC63 scFv_STII_IgG4
铰链_CD28tm_CD28icd (Y206F, P208A, Y209F, P211A,
Y218F)_CD3z_T2A_tEGFR)表达盒
<400> 52
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60
atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120
gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180
aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240
cctagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300
gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360
ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420
ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480
cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540
gtgagctgga tcaggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600
agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660
agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720
tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780
accagcgtga ccgtgagcag caattggagc cacccgcagt tcgaaaaagg aggtggaggt 840
tcaggtggtg gaggctctga gagcaagtac ggaccgccct gccccccttg ccctatgttc 900
tgggtgctgg tggtggtcgg aggcgtgctg gcctgctaca gcctgctggt caccgtggcc 960
ttcatcatct tttgggtccg cagcaagcgg agcagaggcg gccacagcga ctacatgaac 1020
atgaccccta gacggcctgg ccccaccaga aagcacttcc aggccttcgc cgctccccgg 1080
gactttgccg ccttcagaag ccgggtgaag ttcagcagaa gcgccgacgc ccctgcctac 1140
cagcagggcc agaatcagct gtacaacgag ctgaacctgg gcagaaggga agagtacgac 1200
gtcctggata agcggagagg ccgggaccct gagatgggcg gcaagcctcg gcggaagaac 1260
ccccaggaag gcctgtataa cgaactgcag aaagacaaga tggccgaggc ctacagcgag 1320
atcggcatga agggcgagcg gaggcggggc aagggccacg acggcctgta tcagggcctg 1380
tccaccgcca ccaaggatac ctacgacgcc ctgcacatgc aggccctgcc cccaaggctc 1440
gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 1500
ggccctagga tgcttctcct ggtgacaagc cttctgctct gtgagttacc acacccagca 1560
ttcctcctga tcccacgcaa agtgtgtaac ggaataggta ttggtgaatt taaagactca 1620
ctctccataa atgctacgaa tattaaacac ttcaaaaact gcacctccat cagtggcgat 1680
ctccacatcc tgccggtggc atttaggggt gactccttca cacatactcc tcctctggat 1740
ccacaggaac tggatattct gaaaaccgta aaggaaatca cagggttttt gctgattcag 1800
gcttggcctg aaaacaggac ggacctccat gcctttgaga acctagaaat catacgcggc 1860
aggaccaagc aacatggtca gttttctctt gcagtcgtca gcctgaacat aacatccttg 1920
ggattacgct ccctcaagga gataagtgat ggagatgtga taatttcagg aaacaaaaat 1980
ttgtgctatg caaatacaat aaactggaaa aaactgtttg ggacctccgg tcagaaaacc 2040
aaaattataa gcaacagagg tgaaaacagc tgcaaggcca caggccaggt ctgccatgcc 2100
ttgtgctccc ccgagggctg ctggggcccg gagcccaggg actgcgtctc ttgccggaat 2160
gtcagccgag gcagggaatg cgtggacaag tgcaaccttc tggagggtga gccaagggag 2220
tttgtggaga actctgagtg catacagtgc cacccagagt gcctgcctca ggccatgaac 2280
atcacctgca caggacgggg accagacaac tgtatccagt gtgcccacta cattgacggc 2340
ccccactgcg tcaagacctg cccggcagga gtcatgggag aaaacaacac cctggtctgg 2400
aagtacgcag acgccggcca tgtgtgccac ctgtgccatc caaactgcac ctacggatgc 2460
actgggccag gtcttgaagg ctgtccaacg aatgggccta agatcccgtc catcgccact 2520
gggatggtgg gggccctcct cttgctgctg gtggtggccc tggggatcgg cctcttcatg 2580
tga 2583
<210> 53
<211> 860
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A194 CAR盒蛋白
序列
<400> 53
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Phe Gln Ala Phe Ala Ala Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu
465 470 475 480
Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val
485 490 495
Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu
500 505 510
Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val
515 520 525
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
530 535 540
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
545 550 555 560
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
565 570 575
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
580 585 590
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
595 600 605
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
610 615 620
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
625 630 635 640
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
645 650 655
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
660 665 670
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
675 680 685
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
690 695 700
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
705 710 715 720
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
725 730 735
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
740 745 750
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
755 760 765
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
770 775 780
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
785 790 795 800
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
805 810 815
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
820 825 830
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
835 840 845
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 54
<211> 2592
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A195 CAR (R12 scFv_STII_IgG4
铰链_CD28tm_CD28icd (P208A,
P211A)_CD3z_T2A_tEGFR)表达盒
<400> 54
atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60
atcccccagg aacagctcgt cgaaagcggc ggcagactgg tgacacctgg cggcagcctg 120
accctgagct gcaaggccag cggcttcgac ttcagcgcct actacatgag ctgggtccgc 180
caggcccctg gcaagggact ggaatggatc gccaccatct accccagcag cggcaagacc 240
tactacgcca cctgggtgaa cggacggttc accatctcca gcgacaacgc ccagaacacc 300
gtggacctgc agatgaacag cctgacagcc gccgaccggg ccacctactt ttgcgccaga 360
gacagctacg ccgacgacgg cgccctgttc aacatctggg gccctggcac cctggtgaca 420
atctctagcg gcggaggcgg atctggtggc ggaggaagtg gcggcggagg atctgagctg 480
gtgctgaccc agagcccctc tgtgtctgct gccctgggaa gccctgccaa gatcacctgt 540
accctgagca gcgcccacaa gaccgacacc atcgactggt atcagcagct gcagggcgag 600
gcccccagat acctgatgca ggtgcagagc gacggcagct acaccaagag gccaggcgtg 660
cccgaccggt tcagcggatc tagctctggc gccgaccgct acctgatcat ccccagcgtg 720
caggccgatg acgaggccga ttactactgt ggcgccgact acatcggcgg ctacgtgttc 780
ggcggaggca cccagctgac cgtgaccggc aattggagcc acccgcagtt cgaaaaagga 840
ggtggaggtt caggtggtgg aggctctgag agcaagtacg gaccgccctg ccccccttgc 900
cctatgttct gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc 960
accgtggcct tcatcatctt ttgggtccgc agcaagcgga gcagaggcgg ccacagcgac 1020
tacatgaaca tgacccctag acggcctggc cccaccagaa agcactacca ggcctacgcc 1080
gctccccggg actttgccgc ctacagaagc cgggtgaagt tcagcagaag cgccgacgcc 1140
cctgcctacc agcagggcca gaatcagctg tacaacgagc tgaacctggg cagaagggaa 1200
gagtacgacg tcctggataa gcggagaggc cgggaccctg agatgggcgg caagcctcgg 1260
cggaagaacc cccaggaagg cctgtataac gaactgcaga aagacaagat ggccgaggcc 1320
tacagcgaga tcggcatgaa gggcgagcgg aggcggggca agggccacga cggcctgtat 1380
cagggcctgt ccaccgccac caaggatacc tacgacgccc tgcacatgca ggccctgccc 1440
ccaaggctcg agggcggcgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag 1500
gagaatcccg gccctaggat gcttctcctg gtgacaagcc ttctgctctg tgagttacca 1560
cacccagcat tcctcctgat cccacgcaaa gtgtgtaacg gaataggtat tggtgaattt 1620
aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 1680
agtggcgatc tccacatcct gccggtggca tttaggggtg actccttcac acatactcct 1740
cctctggatc cacaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1800
ctgattcagg cttggcctga aaacaggacg gacctccatg cctttgagaa cctagaaatc 1860
atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1920
acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1980
aacaaaaatt tgtgctatgc aaatacaata aactggaaaa aactgtttgg gacctccggt 2040
cagaaaacca aaattataag caacagaggt gaaaacagct gcaaggccac aggccaggtc 2100
tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 2160
tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 2220
ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 2280
gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 2340
attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 2400
ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 2460
tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 2520
atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 2580
ctcttcatgt ga 2592
<210> 55
<211> 863
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A195 CAR盒蛋白
序列
<400> 55
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Tyr Gln Ala Tyr Ala Ala Pro Arg Asp Phe Ala Ala Tyr
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys
485 490 495
Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr
500 505 510
Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro
515 520 525
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
530 535 540
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile
545 550 555 560
Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe
565 570 575
Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
580 585 590
Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn
595 600 605
Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
610 615 620
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile
625 630 635 640
Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val
645 650 655
Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp
660 665 670
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn
675 680 685
Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
690 695 700
Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser
705 710 715 720
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
725 730 735
Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
740 745 750
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly
755 760 765
Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
770 775 780
His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr
785 790 795 800
Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
805 810 815
Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
820 825 830
Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala
835 840 845
Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 56
<211> 2592
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A196 CAR (R12 scFv_STII_IgG4
铰链_CD28tm_CD28icd (P208A, P211A,
Y218F)_CD3z_T2A_tEGFR)表达盒
<400> 56
atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60
atcccccagg aacagctcgt cgaaagcggc ggcagactgg tgacacctgg cggcagcctg 120
accctgagct gcaaggccag cggcttcgac ttcagcgcct actacatgag ctgggtccgc 180
caggcccctg gcaagggact ggaatggatc gccaccatct accccagcag cggcaagacc 240
tactacgcca cctgggtgaa cggacggttc accatctcca gcgacaacgc ccagaacacc 300
gtggacctgc agatgaacag cctgacagcc gccgaccggg ccacctactt ttgcgccaga 360
gacagctacg ccgacgacgg cgccctgttc aacatctggg gccctggcac cctggtgaca 420
atctctagcg gcggaggcgg atctggtggc ggaggaagtg gcggcggagg atctgagctg 480
gtgctgaccc agagcccctc tgtgtctgct gccctgggaa gccctgccaa gatcacctgt 540
accctgagca gcgcccacaa gaccgacacc atcgactggt atcagcagct gcagggcgag 600
gcccccagat acctgatgca ggtgcagagc gacggcagct acaccaagag gccaggcgtg 660
cccgaccggt tcagcggatc tagctctggc gccgaccgct acctgatcat ccccagcgtg 720
caggccgatg acgaggccga ttactactgt ggcgccgact acatcggcgg ctacgtgttc 780
ggcggaggca cccagctgac cgtgaccggc aattggagcc acccgcagtt cgaaaaagga 840
ggtggaggtt caggtggtgg aggctctgag agcaagtacg gaccgccctg ccccccttgc 900
cctatgttct gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc 960
accgtggcct tcatcatctt ttgggtccgc agcaagcgga gcagaggcgg ccacagcgac 1020
tacatgaaca tgacccctag acggcctggc cccaccagaa agcactacca ggcctacgcc 1080
gctccccggg actttgccgc cttcagaagc cgggtgaagt tcagcagaag cgccgacgcc 1140
cctgcctacc agcagggcca gaatcagctg tacaacgagc tgaacctggg cagaagggaa 1200
gagtacgacg tcctggataa gcggagaggc cgggaccctg agatgggcgg caagcctcgg 1260
cggaagaacc cccaggaagg cctgtataac gaactgcaga aagacaagat ggccgaggcc 1320
tacagcgaga tcggcatgaa gggcgagcgg aggcggggca agggccacga cggcctgtat 1380
cagggcctgt ccaccgccac caaggatacc tacgacgccc tgcacatgca ggccctgccc 1440
ccaaggctcg agggcggcgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag 1500
gagaatcccg gccctaggat gcttctcctg gtgacaagcc ttctgctctg tgagttacca 1560
cacccagcat tcctcctgat cccacgcaaa gtgtgtaacg gaataggtat tggtgaattt 1620
aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 1680
agtggcgatc tccacatcct gccggtggca tttaggggtg actccttcac acatactcct 1740
cctctggatc cacaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1800
ctgattcagg cttggcctga aaacaggacg gacctccatg cctttgagaa cctagaaatc 1860
atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1920
acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1980
aacaaaaatt tgtgctatgc aaatacaata aactggaaaa aactgtttgg gacctccggt 2040
cagaaaacca aaattataag caacagaggt gaaaacagct gcaaggccac aggccaggtc 2100
tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 2160
tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 2220
ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 2280
gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 2340
attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 2400
ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 2460
tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 2520
atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 2580
ctcttcatgt ga 2592
<210> 57
<211> 863
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A196 CAR盒蛋白
序列
<400> 57
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Tyr Gln Ala Tyr Ala Ala Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys
485 490 495
Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr
500 505 510
Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro
515 520 525
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
530 535 540
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile
545 550 555 560
Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe
565 570 575
Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
580 585 590
Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn
595 600 605
Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
610 615 620
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile
625 630 635 640
Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val
645 650 655
Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp
660 665 670
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn
675 680 685
Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
690 695 700
Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser
705 710 715 720
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
725 730 735
Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
740 745 750
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly
755 760 765
Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
770 775 780
His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr
785 790 795 800
Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
805 810 815
Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
820 825 830
Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala
835 840 845
Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 58
<211> 2592
<212> DNA
<213> 人工序列
<220>
<223> 合成序列A1974 CAR (R12FMC63
scFv_STII_IgG4 铰链_CD28tm_CD28icd (Y206F, P208A,
Y209F, P211A, Y218F)_CD3z_T2A_tEGFR)表达
盒
<400> 58
atgctgctgc tggtgacaag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60
atcccccagg aacagctcgt cgaaagcggc ggcagactgg tgacacctgg cggcagcctg 120
accctgagct gcaaggccag cggcttcgac ttcagcgcct actacatgag ctgggtccgc 180
caggcccctg gcaagggact ggaatggatc gccaccatct accccagcag cggcaagacc 240
tactacgcca cctgggtgaa cggacggttc accatctcca gcgacaacgc ccagaacacc 300
gtggacctgc agatgaacag cctgacagcc gccgaccggg ccacctactt ttgcgccaga 360
gacagctacg ccgacgacgg cgccctgttc aacatctggg gccctggcac cctggtgaca 420
atctctagcg gcggaggcgg atctggtggc ggaggaagtg gcggcggagg atctgagctg 480
gtgctgaccc agagcccctc tgtgtctgct gccctgggaa gccctgccaa gatcacctgt 540
accctgagca gcgcccacaa gaccgacacc atcgactggt atcagcagct gcagggcgag 600
gcccccagat acctgatgca ggtgcagagc gacggcagct acaccaagag gccaggcgtg 660
cccgaccggt tcagcggatc tagctctggc gccgaccgct acctgatcat ccccagcgtg 720
caggccgatg acgaggccga ttactactgt ggcgccgact acatcggcgg ctacgtgttc 780
ggcggaggca cccagctgac cgtgaccggc aattggagcc acccgcagtt cgaaaaagga 840
ggtggaggtt caggtggtgg aggctctgag agcaagtacg gaccgccctg ccccccttgc 900
cctatgttct gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc 960
accgtggcct tcatcatctt ttgggtccgc agcaagcgga gcagaggcgg ccacagcgac 1020
tacatgaaca tgacccctag acggcctggc cccaccagaa agcacttcca ggccttcgcc 1080
gctccccggg actttgccgc cttcagaagc cgggtgaagt tcagcagaag cgccgacgcc 1140
cctgcctacc agcagggcca gaatcagctg tacaacgagc tgaacctggg cagaagggaa 1200
gagtacgacg tcctggataa gcggagaggc cgggaccctg agatgggcgg caagcctcgg 1260
cggaagaacc cccaggaagg cctgtataac gaactgcaga aagacaagat ggccgaggcc 1320
tacagcgaga tcggcatgaa gggcgagcgg aggcggggca agggccacga cggcctgtat 1380
cagggcctgt ccaccgccac caaggatacc tacgacgccc tgcacatgca ggccctgccc 1440
ccaaggctcg agggcggcgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag 1500
gagaatcccg gccctaggat gcttctcctg gtgacaagcc ttctgctctg tgagttacca 1560
cacccagcat tcctcctgat cccacgcaaa gtgtgtaacg gaataggtat tggtgaattt 1620
aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 1680
agtggcgatc tccacatcct gccggtggca tttaggggtg actccttcac acatactcct 1740
cctctggatc cacaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1800
ctgattcagg cttggcctga aaacaggacg gacctccatg cctttgagaa cctagaaatc 1860
atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1920
acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1980
aacaaaaatt tgtgctatgc aaatacaata aactggaaaa aactgtttgg gacctccggt 2040
cagaaaacca aaattataag caacagaggt gaaaacagct gcaaggccac aggccaggtc 2100
tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 2160
tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 2220
ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 2280
gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 2340
attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 2400
ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 2460
tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 2520
atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 2580
ctcttcatgt ga 2592
<210> 59
<211> 863
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A197 CAR盒蛋白
序列
<400> 59
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Phe Gln Ala Phe Ala Ala Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys
485 490 495
Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr
500 505 510
Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro
515 520 525
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
530 535 540
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile
545 550 555 560
Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe
565 570 575
Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
580 585 590
Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn
595 600 605
Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
610 615 620
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile
625 630 635 640
Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val
645 650 655
Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp
660 665 670
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn
675 680 685
Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
690 695 700
Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser
705 710 715 720
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
725 730 735
Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
740 745 750
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly
755 760 765
Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
770 775 780
His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr
785 790 795 800
Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
805 810 815
Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
820 825 830
Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala
835 840 845
Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
850 855 860
<210> 60
<211> 480
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A042 CAR蛋白
序列
<400> 60
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
325 330 335
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
340 345 350
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
355 360 365
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
370 375 380
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
385 390 395 400
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
405 410 415
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
420 425 430
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
435 440 445
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
450 455 460
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475 480
<210> 61
<211> 479
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A043 CAR蛋白
序列
<400> 61
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475
<210> 62
<211> 483
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A044 CAR蛋白
序列
<400> 62
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu
325 330 335
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
340 345 350
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
355 360 365
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
370 375 380
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
385 390 395 400
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
405 410 415
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
420 425 430
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
435 440 445
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
450 455 460
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
465 470 475 480
Pro Pro Arg
<210> 63
<211> 482
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A045 CAR蛋白
序列
<400> 63
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg
<210> 64
<211> 479
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A142 CAR蛋白
序列
<400> 64
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475
<210> 65
<211> 479
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A143 CAR蛋白
序列
<400> 65
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Phe Gln Pro Phe Ala Pro Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475
<210> 66
<211> 482
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A144 CAR蛋白
序列
<400> 66
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg
<210> 67
<211> 482
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A145 CAR蛋白
序列
<400> 67
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Phe Gln Pro Phe Ala Pro Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg
<210> 68
<211> 479
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A175 CAR蛋白
序列
<400> 68
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Phe Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Phe Gln Pro Phe Ala Pro Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475
<210> 69
<211> 482
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A176 CAR蛋白
序列
<400> 69
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Phe Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Phe Gln Pro Phe Ala Pro Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg
<210> 70
<211> 478
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A192 CAR蛋白
序列
<400> 70
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Thr Lys Leu Glu Ile Thr
115 120 125
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
130 135 140
Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro
145 150 155 160
Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro
165 170 175
Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu
180 185 190
Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala
195 200 205
Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val
210 215 220
Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr
225 230 235 240
Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp
245 250 255
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro Gln
260 265 270
Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys
275 280 285
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val
290 295 300
Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe
305 310 315 320
Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp
325 330 335
Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
340 345 350
Gln Ala Tyr Ala Ala Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val
355 360 365
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
370 375 380
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
385 390 395 400
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
405 410 415
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
420 425 430
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
435 440 445
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
450 455 460
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475
<210> 71
<211> 479
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A193 CAR蛋白
序列
<400> 71
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Tyr Gln Ala Tyr Ala Ala Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475
<210> 72
<211> 479
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A194 CAR蛋白
序列
<400> 72
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
130 135 140
Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala
145 150 155 160
Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu
165 170 175
Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
180 185 190
Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
195 200 205
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln
210 215 220
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
245 250 255
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asn Trp Ser His Pro
260 265 270
Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser
275 280 285
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val
290 295 300
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
305 310 315 320
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser
325 330 335
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
340 345 350
Phe Gln Ala Phe Ala Ala Pro Arg Asp Phe Ala Ala Phe Arg Ser Arg
355 360 365
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
370 375 380
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
385 390 395 400
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
405 410 415
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
420 425 430
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
435 440 445
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
450 455 460
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475
<210> 73
<211> 482
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A195 CAR蛋白
序列
<400> 73
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Tyr Gln Ala Tyr Ala Ala Pro Arg Asp Phe Ala Ala Tyr
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg
<210> 74
<211> 482
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A196 CAR蛋白
序列
<400> 74
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Tyr Gln Ala Tyr Ala Ala Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg
<210> 75
<211> 482
<212> PRT
<213> 人工序列
<220>
<223> 合成序列A197 CAR蛋白
序列
<400> 75
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Glu Gln Leu Val Glu Ser Gly Gly Arg
20 25 30
Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Ser Cys Lys Ala Ser Gly
35 40 45
Phe Asp Phe Ser Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Ile Ala Thr Ile Tyr Pro Ser Ser Gly Lys Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Val Asn Gly Arg Phe Thr Ile Ser Ser Asp Asn
85 90 95
Ala Gln Asn Thr Val Asp Leu Gln Met Asn Ser Leu Thr Ala Ala Asp
100 105 110
Arg Ala Thr Tyr Phe Cys Ala Arg Asp Ser Tyr Ala Asp Asp Gly Ala
115 120 125
Leu Phe Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Ile 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 Leu Thr Gln Ser Pro Ser Val Ser Ala Ala Leu Gly Ser Pro Ala
165 170 175
Lys Ile Thr Cys Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp
180 185 190
Trp Tyr Gln Gln Leu Gln Gly Glu Ala Pro Arg Tyr Leu Met Gln Val
195 200 205
Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ile Ile Pro Ser Val
225 230 235 240
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Tyr Ile Gly
245 250 255
Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Thr Gly Asn Trp
260 265 270
Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
275 280 285
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp
290 295 300
Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
305 310 315 320
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
325 330 335
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
340 345 350
Arg Lys His Phe Gln Ala Phe Ala Ala Pro Arg Asp Phe Ala Ala Phe
355 360 365
Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
370 375 380
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
385 390 395 400
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
405 410 415
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
420 425 430
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
435 440 445
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
450 455 460
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
465 470 475 480
Pro Arg
<210> 76
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 76
Ser Pro Ala Arg Leu Gly Ser Gln His Ser Pro Gly Arg Thr Ala Ser
1 5 10 15
Leu Asn Gln Arg Pro Arg Thr His Ser Gly Ser Ser Gly Gly Ser
20 25 30
<210> 77
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 77
Asp Leu Ser Thr Gln Leu Ser Arg Thr Gly Thr Leu Ser Arg Lys Ser
1 5 10 15
Ile Lys Ala Pro Ala Thr Pro Ala Ser Ala Thr Leu Gly Arg Pro
20 25 30
<210> 78
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 78
Asp Asp Glu Thr Gly Lys Leu Gln Gly Ser Gly Val Ser Leu Ala Ser
1 5 10 15
Lys Lys Ser Arg Leu Ser Ser Ser Ser Ser Asn Asp Ser Gly Asn
20 25 30
<210> 79
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 79
Glu Ala Arg Gly Gly Leu Gly Ala Pro Pro Leu Gln Ser Ala Arg Ser
1 5 10 15
Leu Pro Gly Pro Ala Pro Cys Leu Lys His Phe Pro Leu Asp Leu
20 25 30
<210> 80
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 80
Lys Cys Cys Ser Ile Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser Ser
1 5 10 15
Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val Gly Leu
20 25 30
<210> 81
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 81
Lys Cys Pro Arg Pro Val Val Lys Ser Gly Asp Lys Pro Ser Leu Ser
1 5 10 15
Ala Arg Tyr Val
20
<210> 82
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 82
Met Ser Phe Arg Gly Lys Val Phe Lys Arg Glu Pro Ser Glu Phe Trp
1 5 10 15
Lys Lys Arg Arg Thr Val Arg Arg Val Asn Gln Glu
20 25
<210> 83
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 83
Glu Ala Asn Thr Ser Pro Arg Leu Ser Gln Thr Phe Leu Gln Leu Ser
1 5 10 15
Asp Gly Asp Lys Lys Thr Leu Thr Arg Lys Lys Val Asn Gln Phe
20 25 30
<210> 84
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 84
Ile Pro Gly Leu Asp Glu Ala Asn Thr Ser Pro Arg Leu Ser Gln Thr
1 5 10 15
Phe Leu Gln Leu Ser Asp Gly Asp Lys Lys Thr Leu Thr Arg Lys
20 25 30
<210> 85
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 85
Asn Lys Glu Arg Thr Ser Glu Ser Arg Gly Leu Ser Arg Leu Phe Ser
1 5 10 15
Ser Phe Leu Lys Arg Pro Lys Ser Gln Val Ser Glu Glu Glu Gly
20 25 30
<210> 86
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 86
Lys Glu Arg Thr Ser Glu Ser Arg Gly Leu Ser Arg Leu Phe Ser Ser
1 5 10 15
Phe Leu Lys Arg Pro Lys Ser Gln Val Ser Glu Glu Glu Gly Lys
20 25 30
<210> 87
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 87
Thr Lys Lys Gly Thr Ser Ser Lys Lys Val Ile Tyr Ser Gln Pro Ser
1 5 10 15
Ala Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu Val
20 25 30
<210> 88
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 88
Gly Ser Ser Thr Glu Thr Ser Asp Ser His Leu Thr Lys Ala Leu Ser
1 5 10 15
Thr Phe Ile His Ala Glu Asp Phe Ala Arg Arg Arg Lys Arg Ser
20 25 30
<210> 89
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 89
Ser Ser Arg Pro Asp Arg Glu Thr Arg Ala Ser Val Ile Lys Lys Thr
1 5 10 15
Ser Asp Ile Thr Gln Ala Arg Val Lys Ser Cys
20 25
<210> 90
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 90
Arg Gln Gln Gln Gln Ser Gly Ala Trp Gly Ala Pro Arg Lys Asp Ser
1 5 10 15
Leu Leu Lys Pro Gly Leu Arg Ala Val Val Gly Gly Ala Ala Ala
20 25 30
<210> 91
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 91
His Ser Gln Gln Pro Pro Pro Pro Gln Gln Glu Arg Ser Lys Pro Ser
1 5 10 15
Phe His Ala Pro Glu Pro Lys Ala His Tyr Glu Pro Ala Asp Arg
20 25 30
<210> 92
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 92
Pro Arg His Lys Ile Ile Ser Ile Phe Ser Gly Thr Glu Lys Gly Ser
1 5 10 15
Lys Lys Lys Glu Lys Glu Arg Pro Glu Ile Ser Pro Pro Ser Asp
20 25 30
<210> 93
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 93
Lys Met Cys His Leu Pro Glu Pro Glu Leu Asn Lys Glu Arg Pro Ser
1 5 10 15
Leu Gln Ile Lys Leu Lys Ile Glu Asp Phe Ile Leu His Lys Met
20 25 30
<210> 94
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 94
Leu Glu Ser His Arg Ala Val Tyr Ile Gly Val His Val Pro Phe Ser
1 5 10 15
Lys Glu Ser Arg Arg Arg His Arg His Arg Gly His Lys His His
20 25 30
<210> 95
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 95
Gly Lys Arg Glu Lys Asp Lys Glu Lys Asp Lys Glu Lys Arg Phe Ser
1 5 10 15
Leu Phe Gly Lys Lys Lys
20
<210> 96
<211> 29
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 96
Met Ala Phe Ala Asn Phe Arg Arg Ile Leu Arg Leu Ser Thr Phe Glu
1 5 10 15
Lys Arg Lys Ser Arg Glu Tyr Glu His Val Arg Arg Asp
20 25
<210> 97
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 97
Ile Thr Lys Lys Leu Glu Asp Val Lys Asn Ser Pro Thr Phe Lys Ser
1 5 10 15
Phe Glu Glu Lys Val Glu Asn Leu Lys Ser Lys Val Gly Gly Thr
20 25 30
<210> 98
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 98
Met Ala Ala Gly Glu Thr Gln Leu Tyr Ala Lys Val Ser Asn Lys Leu
1 5 10 15
Lys Ser Arg Ser Ser
20
<210> 99
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 99
Phe Lys Glu Pro Glu Lys Arg Thr Ile Ser Arg Pro Ala Val Gly Ser
1 5 10 15
Thr Lys Tyr Phe Gly Thr Ala Lys Ala Arg Tyr Asp Phe Cys Ala
20 25 30
<210> 100
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 100
Lys Glu Pro Glu Lys Arg Thr Ile Ser Arg Pro Ala Val Gly Ser Thr
1 5 10 15
Lys Tyr Phe Gly Thr Ala Lys Ala Arg Tyr Asp Phe Cys Ala Arg
20 25 30
<210> 101
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 101
Pro Ala Ala Leu Pro Gln Ser Ser Glu Lys Leu Phe Gln Gly Tyr Ser
1 5 10 15
Phe Val Ala Pro Ser Ile Leu Phe Lys Arg Asn Ala Ala Val Ile
20 25 30
<210> 102
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 102
Met Ala Phe Ala Asn Phe Arg Arg Ile Leu Arg Leu Ser Thr Phe Glu
1 5 10 15
Lys Arg Lys Ser Arg Glu Tyr Glu His Val Arg Arg
20 25
<210> 103
<211> 29
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 103
Met Ala Phe Ala Asn Phe Arg Arg Ile Leu Arg Leu Ser Thr Phe Glu
1 5 10 15
Lys Arg Lys Ser Arg Glu Tyr Glu His Val Arg Arg Asp
20 25
<210> 104
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 104
Val Thr Gly Ile Arg Leu Leu Ser Leu Gly Ala Gly Glu Phe Lys Ser
1 5 10 15
Gln Glu His Ala Lys His Lys Gly Pro Lys Val Glu Arg Asp Gln
20 25 30
<210> 105
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 105
Gln Ser Ser Glu Lys Leu Phe Gln Gly Tyr Ser Phe Val Ala Pro Ser
1 5 10 15
Ile Leu Phe Lys Arg Asn Ala Ala Val Ile Asp Pro Leu Gln Phe
20 25 30
<210> 106
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 106
Met Ser Arg Arg Arg Phe Asp Cys Arg Ser Ile Ser Gly Leu Leu Thr
1 5 10 15
Thr Thr Pro Gln Ile Pro Ile Lys Met
20 25
<210> 107
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 107
Arg Ser Tyr Ser Pro Asp His Arg Gln Lys Lys His Arg Lys Leu Ser
1 5 10 15
Glu Leu Glu Arg Pro His Lys Val Ser His Gly His Glu Arg Arg
20 25 30
<210> 108
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 108
Tyr Phe Ile Asp Phe Val Ala Arg Glu Thr Thr Cys Ser Lys Glu Ser
1 5 10 15
Asn Glu Glu Leu Thr Glu Ser Cys Glu Thr Lys Lys Leu Gly Gln
20 25 30
<210> 109
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 109
Arg Ser Arg Ser Arg Thr Ser Pro Val Thr Arg Arg Arg Ser Arg Ser
1 5 10 15
Arg Thr Pro Pro Ala Ile Arg Arg Arg Ser Arg Ser Arg Thr Pro
20 25 30
<210> 110
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 110
Arg Ser Arg Thr Ser Pro Val Thr Arg Arg Arg Ser Arg Ser Arg Thr
1 5 10 15
Pro Pro Ala Ile Arg Arg Arg Ser Arg Ser Arg Thr Pro Leu Leu
20 25 30
<210> 111
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 111
Val Lys Lys Glu Ala Arg Thr Leu Ser Asp Phe Asn Ser Leu Ile Ser
1 5 10 15
Ser Pro His Leu Gly Arg Glu Lys Lys Lys Val Lys Ser Gln Lys
20 25 30
<210> 112
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 112
Gln Glu Gln Ile Ala Lys Arg Arg Arg Leu Ser Ser Leu Arg Ala Ser
1 5 10 15
Thr Ser Lys Ser Glu Ser Ser Gln Lys
20 25
<210> 113
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 113
Asn Pro Asn Arg Phe Ile Thr Leu Leu Leu Pro Gly Gly Ala Gln Thr
1 5 10 15
Ala Val Arg Pro Gly Ser Pro Ser Thr Ser Thr Met Arg Leu Asp
20 25 30
<210> 114
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 114
Ala Thr Val Thr Ala Phe Ser Phe Glu Asp Asp Thr Val Pro Leu Ser
1 5 10 15
Pro Leu Lys Tyr Met Ala Gln Arg Gln Gln Arg Glu Lys Thr Arg
20 25 30
<210> 115
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 115
Leu Lys Gly Ala Ile Leu Thr Thr Met Leu Ala Thr Arg Asn Phe Ser
1 5 10 15
Ala Ala Lys Ser Leu Leu Lys Lys Pro Asp Gly Val Lys Glu Ser
20 25 30
<210> 116
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 116
Ile Leu Thr Thr Met Leu Ala Thr Arg Asn Phe Ser Ala Ala Lys Ser
1 5 10 15
Leu Leu Lys Lys Pro Asp Gly Val Lys Glu Ser Thr Glu Ser Ser
20 25 30
<210> 117
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 117
Val Leu Lys His Pro Tyr Pro Arg Lys Val Glu Glu Pro Ser Ile Tyr
1 5 10 15
Glu Ser Val Arg Val His Thr Ala Met Gln Thr Gly Arg Thr Glu
20 25 30
<210> 118
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 118
Thr Ala Val Leu Ala Pro Trp Pro Arg Pro Pro Pro Arg Arg Phe Ser
1 5 10 15
Pro Pro Arg Arg Met Leu Pro Pro Pro Pro Met Trp Arg Arg Ser
20 25 30
<210> 119
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 119
Leu Ser Pro Gly Glu Thr Pro Glu Arg Ser Leu Arg Leu Ala Glu Ser
1 5 10 15
Arg Glu Gln Ser Pro Arg Arg Lys Glu Val Glu Ser Arg Leu Ser
20 25 30
<210> 120
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 120
Glu Thr Pro Glu Arg Ser Leu Arg Leu Ala Glu Ser Arg Glu Gln Ser
1 5 10 15
Pro Arg Arg Lys Glu Val Glu Ser Arg Leu Ser Pro Gly Glu Ser
20 25 30
<210> 121
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 121
Ala Gln Pro Leu Glu Leu Asn Gln His Ser Arg Phe Ile Ile Gly Ser
1 5 10 15
Val Ser Glu Asp Asn Ser Glu Asp Glu Ile Ser Asn Leu Val Lys
20 25 30
<210> 122
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 122
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
1 5 10 15
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
20 25
<210> 123
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 123
Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val Ser
1 5 10 15
Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile
20 25 30
<210> 124
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 124
His His Asp Ile Val Arg Leu Leu Asp Glu Tyr Asn Val Thr Pro Ser
1 5 10 15
Pro Pro Gly Thr Val Leu Thr Ser Ala Leu Ser Pro Val Ile Cys
20 25 30
<210> 125
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 125
Met Asp Asn Tyr Ala Asp Leu Ser Asp Thr Glu Leu Thr Thr Leu Leu
1 5 10 15
Arg Arg Tyr Asn Ile Pro His
20
<210> 126
<211> 62
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 126
Asp Leu Asp Val Ser Asn Thr Thr Thr Ala Gln Lys Arg Lys Cys Ser
1 5 10 15
Gln Thr Gln Cys Pro Arg Lys Val Ile Lys Met Glu Ser Glu Glu Gly
20 25 30
Glu Asp Val Ser Asn Thr Thr Thr Ala Gln Lys Arg Lys Cys Ser Gln
35 40 45
Thr Gln Cys Pro Arg Lys Val Ile Lys Met Glu Ser Glu Glu
50 55 60
<210> 127
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 127
Ile Arg Ala Ser Ser Ser Ser Ser Ser Ile Arg Gln Arg Ile Ser Ser
1 5 10 15
Phe Glu Thr Phe Gly Ser Ser Gln Leu Pro Asp Lys Gly Ala Gln
20 25 30
<210> 128
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 128
Leu Val Arg Leu Ser Leu Thr Glu Arg Leu Gly Lys Arg Lys Phe Ser
1 5 10 15
Ala Gly Gly Asp Ser Asp Pro Pro Leu Lys Arg Ser Leu Ala Gln
20 25 30
<210> 129
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 129
Ala Gln Gln Ala Ala Ser Ser Ser Gly Gln Gly Gln Gln Ala Gln Thr
1 5 10 15
Pro Thr Gly Lys Gln Thr Asp Lys Thr Lys Ser Asn Met Lys Gly
20 25 30
<210> 130
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 130
Ser Ser Lys Val Lys Tyr Ser Arg Leu Ser Ser Thr Asp Asp Gly Tyr
1 5 10 15
Ile Asp Leu Gln Phe Lys Lys Thr Pro Pro Lys Ile Pro Tyr Lys
20 25 30
<210> 131
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 131
Arg Lys Glu Arg Glu Leu Arg Glu Arg Leu Leu Ser Ile Leu Leu Ser
1 5 10 15
Lys Lys Pro Asp Asp Ser His Thr His Asp Glu Leu Gly Val Ala
20 25 30
<210> 132
<211> 26
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 132
Leu Leu Gln Asp Leu Ser Glu Val Ser Ala Pro Pro Leu Pro Pro Thr
1 5 10 15
Ser Pro Gly Arg Asp Val Ala Gln Asp Pro
20 25
<210> 133
<211> 29
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 133
Leu Pro Gly Pro Pro Ala Pro Ser Pro Leu Pro Ala Thr Pro Leu Ser
1 5 10 15
Ala Lys Glu Asp Ala Ser Lys Glu Asp Val Ile Phe Phe
20 25
<210> 134
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 134
Met Asp Gly Ala Ile Ala Ser Gly Val Ser Lys Phe Ala Thr Leu Ser
1 5 10 15
Leu His Asp Arg Lys Glu Arg His His Glu Lys Asp His Lys Arg
20 25 30
<210> 135
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 135
Ser Ser Arg Pro Asp Arg Glu Thr Arg Ala Ser Val Ile Lys Lys Thr
1 5 10 15
Ser Asp Ile Thr Gln Ala Arg Val Lys Ser Cys
20 25
<210> 136
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 136
Tyr Ala Tyr Arg His Pro Leu Ile Arg Glu Lys Pro Arg His Lys Ser
1 5 10 15
Asp Val Glu Ile Pro Ala Thr Val Thr Ala Phe Ser Phe Glu Asp
20 25 30
<210> 137
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 137
Leu Thr Glu Arg Leu Gly Lys Arg Lys Phe Ser Ala Gly Gly Asp Ser
1 5 10 15
Asp Pro Pro Leu Lys Arg Ser Leu Ala Gln Arg Leu Gly Lys Lys
20 25 30
<210> 138
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 138
Leu Ala Met Ala Ala Lys Arg Lys Ala Glu Asn Pro Ser Pro Arg Ser
1 5 10 15
Pro Ser Ser Gln Thr Pro Asn Ser Arg Arg Gln Ser Gly Lys Lys
20 25 30
<210> 139
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 139
Asn Val Thr Pro Ser Pro Pro Gly Thr Val Leu Thr Ser Ala Leu Ser
1 5 10 15
Pro Val Ile Cys Gly Pro Asn Arg Ser Phe Leu Ser Leu Lys His
20 25 30
<210> 140
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 140
His Asp Lys Lys Ser Gln Asp Phe Gly Asn Leu Phe Ser Phe Pro Ser
1 5 10 15
Tyr Ser Gln Lys Ser Glu Asp Asp Ser Ala Lys Phe Asp Ser Asn
20 25 30
<210> 141
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 141
Lys Glu Ala Arg Leu Ala Arg Ser Ser Pro Glu Gln Pro Arg Pro Ser
1 5 10 15
Thr Ser Lys Ala Val Ser Pro Pro His Leu Asp Gly Pro Pro Ser
20 25 30
<210> 142
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 142
Ser Leu Val Ala Pro Ala Leu Asn Lys Pro Lys Lys Pro Leu Thr Ser
1 5 10 15
Ser Ser Ala Ala Pro Gln Arg Pro Ile Ser Thr Gln Arg Thr Ala
20 25 30
<210> 143
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 143
Ala Glu Ile Glu Lys Phe Asp Lys Ser Lys Leu Lys Lys Thr Glu Thr
1 5 10 15
Gln Glu Lys Asn Pro Leu Pro Ser Lys Glu Thr Ile Glu Gln Glu
20 25 30
<210> 144
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 144
Ile Glu Lys Tyr Arg Met Glu Arg Pro Lys Ile Gln Gln Gln Phe Ser
1 5 10 15
Asp Leu Lys Arg Lys Leu Ala Glu Val Thr Glu Glu Glu Trp Leu
20 25 30
<210> 145
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 145
Pro Pro Thr His Gln Ala Ser Val Gly Leu Leu Asp Thr Pro Arg Ser
1 5 10 15
Arg Glu Arg Ser Pro Ser Pro Leu Arg Gly Asn Val Val Pro Ser
20 25 30
<210> 146
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 146
Ala Asp Gly Ala Pro Ser Ala Ala Pro Pro Asp Gly Leu Leu Ala Ser
1 5 10 15
Pro Asp Leu Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg
20 25 30
<210> 147
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 147
Thr Arg Pro Lys Gln Glu Lys Ala Phe Ser Leu Lys Thr Ile Ser Thr
1 5 10 15
Ser Asp Pro Ala Glu Val Leu Val Lys Asn Ser Gln Pro Ile Lys
20 25 30
<210> 148
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 148
Asp Asp Gly Val Gly Asn Gln Leu Gly Ala Leu Val His Gln Arg Thr
1 5 10 15
Val Ile Thr Glu Glu Phe Lys Val Pro Asp Lys Met Val Gly Phe
20 25 30
<210> 149
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 149
His Thr Asp Arg Glu Ala Thr Pro Asp Gly Gly Glu Asp Ser Asp Ser
1 5 10 15
<210> 150
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 150
Pro Arg Leu Asp Phe Glu Gly Pro Asp Ala Lys Leu Ser Gly Pro Ser
1 5 10 15
Leu Lys Met Pro Ser Leu Glu Ile Ser Ala Pro Lys Val Thr Ala
20 25 30
<210> 151
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 151
Lys His Glu Pro Asp Val Ser Ser Lys Ser Ser Ser Val Leu Asp Ser
1 5 10 15
Ser Leu Asp His Arg His Lys Gln Lys Val Leu His Asp Thr Met
20 25 30
<210> 152
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 152
Ser Glu Gly Ser Leu Gln Lys Gly Thr Glu Pro Ser Pro Gly Gly Thr
1 5 10 15
Pro Gln Pro Ser Arg Pro Val Ser Pro Ala Gly Pro Pro Glu Gly
20 25 30
<210> 153
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 153
Leu Gln Lys Gly Thr Glu Pro Ser Pro Gly Gly Thr Pro Gln Pro Ser
1 5 10 15
Arg Pro Val Ser Pro Ala Gly Pro Pro Glu Gly Val Pro Glu Glu
20 25 30
<210> 154
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 154
Thr Glu Pro Ser Pro Gly Gly Thr Pro Gln Pro Ser Arg Pro Val Ser
1 5 10 15
Pro Ala Gly Pro Pro Glu Gly Val Pro Glu Glu Ala Gln Pro Pro
20 25 30
<210> 155
<211> 30
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 155
Ser Ile His Glu Ala Leu Arg Arg Ile Lys Glu Ala Ser Pro Glu Ser
1 5 10 15
Glu Asp Glu Glu Glu Ala Leu Pro Cys Thr Asp Trp Glu Asn
20 25 30
<210> 156
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 156
Lys Gln Ala Phe Ala Thr Ser Pro Ala Ser Lys Ala Ala Arg Glu Ser
1 5 10 15
Leu Thr Glu Ile Asn Arg Ser Phe Lys Glu Tyr Thr Glu Asn Met
20 25 30
<210> 157
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 157
His Ile Met Gln His His Val Leu Pro Ile Gln Ala Arg Leu Gly Ser
1 5 10 15
Ile Ala Glu Ile Asp Leu Gly Val Pro Pro Pro Val Met Lys Thr
20 25 30
<210> 158
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 158
Lys His Lys Lys Lys Gly Arg Gln Ser Arg Pro Ala Asn Lys Gln Ser
1 5 10 15
Pro Ser Pro Ser Glu Val Ser Gln Ser Pro Gly Arg Glu Val Met
20 25 30
<210> 159
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 159
Tyr Lys Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser
1 5 10 15
Ser Glu Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu
20 25 30
<210> 160
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 160
Lys Phe Arg Ser Leu Phe Phe Gly Ser Ile Leu Ala Pro Val Arg Ser
1 5 10 15
Pro Gln Gly Pro Ser Pro Val Leu Ala Glu Asp Ser Glu Gly Glu
20 25 30
<210> 161
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 161
Lys Lys Lys Gly Arg Gln Ser Arg Pro Ala Asn Lys Gln Ser Pro Ser
1 5 10 15
Pro Ser Glu Val Ser Gln Ser Pro Gly Arg Glu Val Met Ser Tyr
20 25 30
<210> 162
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 162
Arg Arg His Glu Arg Arg His Glu Ala Gly Leu Lys Arg Lys Pro Ser
1 5 10 15
Gln Thr Leu Gln Pro Ser Glu Asp Leu Ala Asp Gly Lys Ala Ser
20 25 30
<210> 163
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 163
Phe Ser Gln Glu Asn Lys Ala Pro Phe Glu Ala Val Lys Arg Phe Ser
1 5 10 15
Leu Asp Glu Arg Ser Leu Ala Cys Arg Gln Asp Ser Asp Ser Ser
20 25 30
<210> 164
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 164
Met Ala Glu Thr Pro Pro Pro Pro Thr Ala Gly Ala Glu Ser Cys Ser
1 5 10 15
Glu Glu Pro
<210> 165
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 165
Arg Ser Arg Ser Arg Thr Ser Pro Ile Thr Arg Arg Arg Ser Arg Ser
1 5 10 15
Arg Thr Ser Pro Val Thr Arg Arg Arg Ser Arg Ser Arg Thr Ser
20 25 30
<210> 166
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 166
Ser Arg Tyr Arg Thr Thr Ser Ser Ala Asn Asn Pro Asn Leu Met Tyr
1 5 10 15
Gln Asp Glu Cys Asp Arg Arg Leu Arg Gly Val Lys Asp Gly Gly
20 25 30
<210> 167
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 167
Gly Ile Pro Pro Ser Ala Gly Ala His Gln Leu Phe Arg Gly Phe Ser
1 5 10 15
Phe Val Ala Thr Gly Leu Met Glu Asp Asp Gly Lys Pro Arg Ala
20 25 30
<210> 168
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 168
Asp Glu Lys Thr Thr Lys Ile Ala Cys Lys Ser Pro Pro Pro Glu Ser
1 5 10 15
Val Asp Thr Pro Thr Ser Thr Lys Gln Trp Pro Lys Arg Ser Leu
20 25 30
<210> 169
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 169
Leu Gln Asp Leu Ser Glu Val Ser Ala Pro Pro Leu Pro Pro Thr Ser
1 5 10 15
Pro Gly Arg Asp Val Ala Gln Asp Pro
20 25
<210> 170
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 170
Leu Pro Trp Arg Pro Arg Gly Leu Arg Asn Leu Pro Arg Ser Arg Ser
1 5 10 15
Gln Pro Cys Asp Leu Asp Ala Arg Lys Thr Gly Val Lys Arg Arg
20 25 30
<210> 171
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 171
Met Ser Arg Arg Arg Phe Asp Cys Arg Ser Ile Ser Gly Leu Leu Thr
1 5 10 15
Thr Thr Pro Gln Ile Pro Ile Lys Met Glu Asn
20 25
<210> 172
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 172
Pro Glu Glu Val Leu Pro Ser Pro Thr Leu Gln Ser Leu Ala Thr Ser
1 5 10 15
Pro Arg Ala Ile Leu Gly Ser Tyr Arg Lys Lys Arg Lys Asn Ser
20 25 30
<210> 173
<211> 31
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 173
Glu Ala Arg Gly Gly Leu Gly Ala Pro Pro Leu Gln Ser Ala Arg Ser
1 5 10 15
Leu Pro Gly Pro Ala Pro Cys Leu Lys His Phe Pro Leu Asp Leu
20 25 30
<210> 174
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 合成序列Strep-Tag II peptide
<400> 174
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 175
<211> 50
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<220>
<221> 变体
<222> (6)...(50)
<223> 氨基酸6-50中的任何一个或全部可以存在或不存在。
<400> 175
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
20 25 30
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45
Gly Ser
50
Claims (70)
1.一种融合蛋白,其包含:
(a)包含特异性结合靶抗原的结合域的细胞外组分;
(b)包含修饰的功能性CD28共刺激信号结构域的细胞内组分,其中所述修饰的功能性CD28共刺激信号结构域包含至少一个氨基酸取代;和
(c)位于细胞外组分和细胞内组分之间的疏水部分,
其中所述融合蛋白具有与包含野生型CD28共刺激信号结构域的融合蛋白不同的一种或多种功能活性。
2.权利要求1的融合蛋白,其中CD28共刺激信号传导域中的至少一个酪氨酸残基由不同的氨基酸残基取代。
3.权利要求2的融合蛋白,其中所述至少一个酪氨酸残基选自位置191、206、209和218中的任何一个。
4.权利要求2的融合蛋白,其中选自位置191、206、209和218中任一个的至少两个酪氨酸残基各自由不同的氨基酸残基取代。
5.权利要求2的融合蛋白,其中选自位置191、206、209和218中任一个的至少三个酪氨酸残基各自由不同的氨基酸残基取代。
6.权利要求2的融合蛋白,其中在位置191、206、209和218的四个酪氨酸残基由不同的氨基酸残基取代。
7.权利要求2-6中任一项的融合蛋白,其中每个酪氨酸残基独立地由色氨酸残基或苯丙氨酸残基取代。
8.权利要求2-6中任一项的融合蛋白,其中每个酪氨酸残基由苯丙氨酸残基取代。
9.权利要求2-6中任一项的融合蛋白,其中每个酪氨酸残基由色氨酸残基取代。
10.权利要求2-7中任一项的融合蛋白,其中所述修饰的CD28共刺激信号传导结构域包含Y218F取代。
11.权利要求2-7中任一项的融合蛋白,其中所述修饰的CD28共刺激信号传导域包含Y206F、Y209F和Y218F取代。
12.权利要求2-7中任一项的融合蛋白,其中所述修饰的CD28共刺激信号传导域包含Y191F、Y206F、Y209F和Y218F取代。
13.权利要求1-12中任一项的融合蛋白,其中所述CD28共刺激信号域中的至少一个脯氨酸残基由不同的氨基酸残基取代。
14.权利要求13的融合蛋白,其中所述至少一个脯氨酸残基选自位置196、199、208和211中的任何一个。
15.权利要求13的融合蛋白,其中选自位置196、199、208和211中的任一个的至少两个脯氨酸残基各自由不同的氨基酸残基取代。
16.权利要求13的融合蛋白,其中选自位置196、199、208和211中任一个的至少三个脯氨酸残基各自由不同的氨基酸残基取代。
17.权利要求13的融合蛋白,其中在196、199、208和211位的四个脯氨酸残基由不同的氨基酸残基取代。
18.权利要求13-17中任一项的融合蛋白,其中每个脯氨酸残基由丙氨酸残基取代。
19.权利要求1-18中任一项的融合蛋白,其中所述修饰的CD28共刺激信号传导域还包含L186和L187取代。
20.权利要求19的融合蛋白,其中所述L186取代为L186G,所述L187取代为L187G。
21.权利要求1-11、13-15和18-20中任一项的融合蛋白,其中所述修饰的CD28共刺激信号结构域在Y191、P208、P211或其任何组合上不包含取代。
22.根据权利要求1-21中任一项所述的融合蛋白,其中所述结合结构域是scFv、scTCR、受体胞外域或配体。
23.权利要求1-22中任一项的融合蛋白,其中所述结合结构域不包含CD8的细胞外结合结构域或部分或其包含功能性IgV样结构域的任何部分。
24.根据权利要求23所述的融合蛋白,其中所述结合结构域不包含来自CD8α链的结合结构域、来自CD8β链的结合结构域、来自CD8α同二聚体的结合结构域,或来自CD8αβ异二聚体的结合结构域。
25.根据权利要求1至24中任一项所述的融合蛋白,其中所述结合结构域是嵌合的、人的或人源化的。
26.根据权利要求1至25中任一项所述的融合蛋白,其中所述细胞外组分还包括位于所述结合结构域和所述疏水性部分之间的间隔子。
27.根据权利要求26所述的融合蛋白,其中所述间隔物包含免疫球蛋白铰链区,CH2结构域、CH3结构域或其任何组合。
28.根据权利要求27所述的融合蛋白,其中所述免疫球蛋白铰链区是IgG4铰链区。
29.根据权利要求1-28中任一项所述的融合蛋白,其中所述细胞外组分还包含位于所述结合结构域和所述疏水性部分之间的标签。
30.根据权利要求29所述的融合蛋白,其中所述标签具有Trp-Ser-His-Pro-Gln-Phe-Glu-Lys的氨基酸序列(SEQ ID NO:40)。
31.根据权利要求1至30中任一项所述的融合蛋白,其中所述细胞内组分还包含含ITAM的T细胞活化域。
32.权利要求31的融合蛋白,其中所述含ITAM的T细胞活化结构域包含CD3γ、CD3δ、CD3ε、CD3ζ、FcεRI的γ链或FcγRI的γ链的细胞内信号传导域。
33.根据权利要求32所述的融合蛋白,其中所述细胞内组分还包含CD3ζ细胞内信号传导结构域。
34.根据权利要求1-33中任一项所述的融合蛋白,其中所述细胞内组分还包含至少一个另外的共刺激信号传导域。
35.根据权利要求34所述的融合蛋白,其中所述至少一个另外的共刺激信号传导结构域选自CD27、CD40L、GITR、NKG2C、CARD1、CD2、CD7、CD27、CD30、CD40、CD54(ICAM)、CD83、CD134(OX-40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD223(LAG3)、CD270(HVEM)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、DAP10、LAT、NKD2C SLP76、TRIM、ZAP70、CD5、BAFF-R、SLAMF7、NKp80、CD160、B7-H3、与CD83特异性结合的配体或其组合。
36.根据权利要求1-35中任一项所述的融合蛋白,其中所述疏水部分是跨膜结构域。
37.根据权利要求36所述的融合蛋白,其中所述跨膜结构域包含CD28、CD2、CD3ε、CD3δ、CD3ζ、CD25、CD27、CD40、CD79A、CD79B、CD80、CD86、CD95(Fas)、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD200R、CD223(LAG3)、CD270(HVEM)、CD272(BTLA)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、CD279(PD-1)、CD300、CD357(GITR)、A2aR、DAP10、FcRα、FcRβ、FcRγ、Fyn、GAL9、KIR、Lck、LAT、LRP、NKG2D、NOTCH1、NOTCH2、NOTCH3、NOTCH4、PTCH2、ROR2、Ryk、Slp76、SIRPα、pTα、TCRα、TCRβ、TIM3、TRIM、LPA5或Zap70。
38.根据权利要求1-37中任一项所述的融合蛋白,其中所述靶抗原是癌症抗原、病毒抗原、细菌抗原或自身抗原。
39.根据权利要求36所述的融合蛋白,其中所述靶抗原是选自以下的癌症抗原:BCMA、CD3、CEACAM6、c-Met、EGFR、EGFRvIII、ErbB2、ErbB3、ErbB4、EphA2、IGF1R、GD2、O-乙酰GD2、O-乙酰基GD3、GHRHR、GHR、FLT1、KDR、FLT4、CD44v6、CD151、CA125、CEA、CTLA-4、GITR、BTLA、TGFBR2、TGFBR1、IL6R、gp130、Lewis A、Lewis Y、TNFR1、TNFR2、PD1、PD-L1、PD-L2、HVEM、MAGE-A、间皮素、NY-ESO-1、PSMA、RANK、ROR1、TNFRSF4、CD40、CD137、TWEAK-R、HLA、与HLA结合的肿瘤或病原体相关肽与HLA结合的hTERT肽、与HLA结合的酪氨酸酶肽、与HLA结合的WT-1肽、LTβR、LIFRβ、LRP5、MUC1、OSMRβ、TCRα、TCRβ、CD19、CD20、CD22、CD25、CD28、CD30、CD33、CD52、CD56、CD79a、CD79b、CD80、CD81、CD86、CD123、CD171、CD276、B7H4、TLR7、TLR9、PTCH1、WT-1、HA1-H、Robo1、α-甲胎蛋白(AFP)、Frizzled、OX40、PRAME和SSX-2。
40.编码权利要求1-39中任一项的融合蛋白的多核苷酸。
41.根据权利要求40所述的多核苷酸,其中所述核酸分子是密码子优化的。
42.根据权利要求40或41所述的多核苷酸,其包含与SEQ ID NO:18、20、22、24、26、28、30、32、34、36、48、50、52、54、56或58中任一项所示的编码融合蛋白的核苷酸序列具有至少约75%同一性的多核苷酸。
43.根据权利要求40-42中任一项所述的多核苷酸,其进一步包含编码转导标记,自杀基因或两者的多核苷酸。
44.根据权利要求43所述的多核苷酸,其中,所述转导标记是截短的EGFR分子。
45.根据权利要求40-44中任一项所述的多核苷酸,其中所述多核苷酸包含与SEQ IDNO:18、20、22、24、26、28、30、32、34、36、48、50、52、54、56或58中的任何一个具有至少约75%同一性的多核苷酸或由其组成。
46.一种载体,其包含权利要求40-45中任一项的多核苷酸。
47.根据权利要求46所述的载体,其中,所述载体是病毒载体。
48.根据权利要求47所述的载体,其中,所述病毒载体是慢病毒或逆转录病毒载体。
49.一种宿主细胞,其包含权利要求1-39中任一项的融合蛋白、权利要求40-45中任一项的多核苷酸,或权利要求46-48中任一项的载体。
50.根据权利要求49所述的宿主细胞,其中所述宿主细胞是免疫系统细胞。
51.根据权利要求50所述的宿主细胞,其中,所述免疫系统细胞是T细胞。
52.根据权利要求51所述的宿主细胞,其中所述T细胞是CD4+T细胞或CD8+T细胞。
53.根据权利要求51或52所述的宿主细胞,其中所述T细胞是记忆T细胞。
54.根据权利要求49-53中任一项所述的宿主细胞,其中所述宿主细胞是人细胞。
55.根据权利要求49-54中任一项所述的宿主细胞,其中所述内源基因的表达在所述宿主细胞中被抑制,其中所述被抑制的内源基因选自TCR基因、HLA基因、免疫抑制成分基因或其任何组合。
56.根据权利要求55所述的宿主细胞,其中所述TCR基因是T细胞受体α恒定(TRAC)基因、T细胞受体β恒定(TRBC)基因或两者。
57.权利要求55或56的宿主细胞,其中经由核酸内切酶系统将权利要求40-45中任一项的多核苷酸或权利要求46-48中任一项的载体靶向TCR基因或HLA基因。
58.根据权利要求57所述的宿主细胞,其中,所述内切核酸酶系统是CRISPR/Cas核酸酶系统、锌指核酸酶(ZFN)系统或转录激活因子样效应子核酸酶(TALEN)系统。
59.根据权利要求49-58中任一项所述的宿主细胞,其中所述宿主细胞表现出融合蛋白的强直磷酸化降低、融合蛋白的信号传导降低、细胞因子表达降低、持久性增强、抗原特异性溶细胞活性增强或任何它们的组合。
60.根据权利要求49-59中任一项所述的宿主细胞,其中与包含融合蛋白的宿主细胞相比,所述宿主细胞表达水平降低的靶标抗原诱导的IL-2、TNF-α或两者,其中融合蛋白包括野生型CD28共刺激信号域。
61.药物组合物,其包含权利要求40-45中任一项的多核苷酸、权利要求46-48中任一项的载体或权利要求49-60中任一项的宿主细胞、以及药学上可接受的载体、稀释剂、或赋形剂。
62.一种治疗受试者的疾病的方法,其包括向所述受试者施用权利要求49-60中任一项的宿主细胞或权利要求61的药物组合物,其中所述疾病与融合蛋白结合的抗原的存在有关。
63.根据权利要求62所述的方法,其中所述疾病是病毒感染、细菌感染、癌症、炎性疾病或自身免疫性疾病。
64.根据权利要求62或63所述的方法,其中所述受试者是人类。
65.根据权利要求62-64中任一项所述的方法,其中所述宿主细胞对于所述受试者是同种异体的或自体的。
66.根据权利要求62-65中任一项所述的方法,其中,与已施用参照宿主细胞或组合物的参照受试者相比,所述受试者在所述治疗之后患有低水平或轻度细胞因子释放综合征、低水平或轻度细胞相关性脑病综合征或两者,和/或具有降低的细胞因子释放综合征、细胞相关性脑病综合征或两者,其中所述融合蛋白包含野生型CD28共刺激信号结构域。
67.根据权利要求62-66中任一项所述的方法,其中所述疾病是癌症。
68.根据权利要求67所述的方法,其中所述癌症是实体瘤、黑素瘤、非小细胞肺癌、肾细胞癌、肾癌、血液癌、前列腺癌、去势抵抗性前列腺癌、结肠癌、直肠癌、胃癌、食道癌、膀胱癌、头颈癌、甲状腺癌、乳腺癌、三阴性乳腺癌、卵巢癌、宫颈癌、肺癌、尿路上皮癌、胰腺癌、成胶质细胞瘤、肝细胞癌、骨髓瘤、多发性骨髓瘤、白血病、霍奇金淋巴瘤、非霍奇金淋巴瘤、骨髓增生异常综合症、脑癌、中枢神经系统癌症或恶性神经胶质瘤。
69.根据权利要求62-68中任一项所述的方法,其进一步包括施用免疫抑制成分的化学治疗剂或抑制剂。
70.一种套件,包括:
(a)权利要求46-48中任一项的载体或表达构建体,和用于将载体或表达构建体转导至宿主细胞中的任选试剂;
(b)权利要求40-45中任一项的分离的多核苷酸,和用于将多核苷酸转导到宿主细胞中的任选试剂;和/或
(c)权利要求49-60中任一项的宿主细胞。
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