CN116600820A - 嵌合抗原受体(car)间隔子修饰增强car t细胞功能 - Google Patents

嵌合抗原受体(car)间隔子修饰增强car t细胞功能 Download PDF

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CN116600820A
CN116600820A CN202180084760.3A CN202180084760A CN116600820A CN 116600820 A CN116600820 A CN 116600820A CN 202180084760 A CN202180084760 A CN 202180084760A CN 116600820 A CN116600820 A CN 116600820A
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M·科霍宁
J·科斯基
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Orion Oyj
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Abstract

本发明涉及包含惰性和可修饰的间隔子的嵌合抗原受体(CAR)，该间隔子在CAR T细胞疗法中避免了Fc受体(FcR)表达细胞的非靶结合。间隔子基于信号调节蛋白α的Ig样C1结构域。

Description

嵌合抗原受体(CAR)间隔子修饰增强CAR T细胞功能
技术领域
本发明涉及包含惰性和可修饰的间隔子的嵌合抗原受体(CAR)，该间隔子在CAR T细胞疗法中避免了Fc受体(FcR)表达细胞的非靶结合。间隔子基于信号调节蛋白α的Ig样C1结构域。
背景技术
基于嵌合抗原受体(CAR)的T细胞疗法是血液癌症的新型治疗方式，在治疗难治性和复发性急性淋巴细胞白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)和非霍奇金淋巴瘤患者中表现出显著效果。然而，在不断发展的疗法中，当前的CAR需要改进，以通过防止先前确定的和可能尚未确定的副作用来实现高疗效，但可耐受的细胞毒性。微调CAR以避免间隔子相关的与非靶(off-target)细胞的相互作用，并比较最佳间隔子修饰尚未得到广泛研究，需要更准确的洞察力来调整细胞毒性反应。
在细胞膜和抗原结合结构域之间具有其结构功能的间隔子在微调CAR相关的抗原非依赖性或依赖性信号传导中具有重要作用。常用的CAR具有间隔子，其由免疫球蛋白G(IgG)恒定结构域、CD8-α或CD28的细胞外结构域、NGFR(Casucci等人，2018)或NKG2D(Sentman等人，2014)的细胞外部分组成。传统的基于IgG1的CAR(IgG1-CAR)中Fc区的IgG1-CH2结构域与表达FcR的髓系细胞(通常是单核细胞或巨噬细胞)或与NK细胞相互作用，这可导致髓系细胞激活和炎症(等人，2015)。FcR与CAR的结合可导致CAR T细胞激活和表达FcR的髓系细胞破坏、CAR T细胞在肺中的隔离、激活诱导的细胞死亡(AICD)和CAR T细胞活性的整体降低(/>等人，2015；Hombach等人，2010；Hudecek等人，2015)。必须避免与非靶细胞不必要的相互作用和可能的副作用，以获得功能性治疗性CAR T细胞。
信号调节蛋白(SIRP)家族(也称为，例如，SHPS、CD172)成员是参与白细胞功能调节的膜蛋白(van Beek等人，2005)。SIRP家族成员的细胞外区通常由单个Ig样V型结构域和两个Ig样C1型结构域组成。SIRP-α(也称为SHPS-1、BIT、MFR、CD172a、p84)是SIRP家族成员，其具有典型的细胞外区，由单个Ig样V型结构域、Ig样C1-1型结构域和Ig样C1-2型结构域组成(van Beek等人，2005)。已知SIRP-α的细胞外区在细胞外仅通过其N-末端的V型Ig样结构域结合靶配体CD47(Hatherley D等人，2009)，而目前SIRP-α的Ig样C1型结构域被称为惰性骨架。
发明内容
本发明涉及包含细胞外间隔子的嵌合抗原受体(CAR)，该细胞外间隔子包含至少一个信号调节蛋白α(SIRP-α)的Ig样C1结构域或其片段或其变体。
在一些实施方案中，SIRP-α的Ig样C1结构域选自(i)根据SEQ ID NO 1的1型结构域或其片段或其变体；或(ii)根据SEQ ID NO 2的2型结构域或其片段或其变体。
在一些实施方案中，细胞外间隔子包含SIRP-α的Ig样C1-1型结构域和Ig样C1-2型结构域。
在一些实施方案中，细胞外间隔子还包含至少一个多聚化结构域，其中所述多聚化结构域选自或多个多聚化结构域选自IgG铰链区，所述IgG铰链区选自根据SEQ ID NO 4或SEQ ID NO 80的IgG1铰链区、根据SEQ ID NO 81的IgG2铰链区、根据SEQ ID NO 82的IgG3铰链区、根据SEQ ID NO 83的IgG4铰链区和/或根据SEQ ID NO 3的CD28细胞外结构域和/或其片段和变体。在一些实施方案中，所述多聚化结构域选自或多个多聚化结构域选自根据SEQ ID NO 4的IgG1铰链区或其片段和/或根据SEQ ID NO 3的CD28细胞外结构域或其片段。在一些实施方案中，所述多聚化结构域选自或多个多聚化结构域选自根据SEQ ID NO83的IgG4铰链区或其片段和/或根据SEQ ID NO 3的CD28细胞外结构域或其片段。
在一些实施方案中，细胞外间隔子位于跨膜结构域和抗原结合结构域之间。在一些实施方案中，抗原结合结构域是单链可变区(scFv)。
在一些实施方案中，细胞外间隔子至少通过一个二硫键使CAR二聚化。CD28细胞外包含一个二硫键。IgG铰链区包含两个二硫键。在一些实施方案中，CAR通过一个二硫键、两个二硫键或三个二硫键二聚化。
本发明还涉及包含细胞外间隔子的CAR，该细胞外间隔子包含根据SEQ ID NO 10、SEQ ID NO 11、SEQ ID NO 12、SEQ ID NO 13、SEQ ID NO 14、SEQ ID NO 15、SEQ ID NO16、SEQ ID NO 17、SEQ ID NO 18、SEQ ID NO 56、SEQ ID NO 57、SEQ ID NO 58、SEQ ID NO59、SEQ ID NO 60或SEQ ID NO 61的氨基酸序列。
在一些实施方案中，CAR包含任何先前的细胞外间隔子结构域、抗原结合结构域、跨膜结构域、细胞内信号传导结构域和任选的共刺激结构域。
在一些实施方案中，CAR的抗原结合结构域包含抗体或其片段。
在一些实施方案中，CAR的抗原结合结构域包含单链可变区片段(scFv)。
在一些实施方案中，CAR的抗原结合结构域靶向肿瘤抗原或癌抗原。肿瘤抗原可以选自CD19、HER-2、BCMA、CD22、CS1、CD38、CD33、CD20、CD30、CD38、CD123、TAA、GD2、MSLN、EGFR、EBV、GPC3、MUC1、PSMA、NY-ESO-1，在Yu等人2020和Townsend等人2018中进行了综述。本发明的CAR靶向的肿瘤抗原优选选自CD19或HER-2。
在一些实施方案中，CAR的跨膜结构域选自膜蛋白的跨膜结构域。跨膜结构域可以选自CD28、CD8、CD8α、OX40L受体(也称为CD134)、4-1BB(也称为CD137)、CD3、CD3δ、CD3γ、CD3ε或CD3ζ或其片段。在一个优选的实施方案中，CAR的跨膜结构域包含根据SEQ ID NO 23的CD28跨膜结构域或其片段。
CAR的细胞内信号传导结构域可以选自CD3ζ、CD3δ、CD3γ、CD3ε、CD28、FcγRIII、FcR胞质尾或酪氨酸激酶的细胞内结构域或其片段。在优选的实施方案中，细胞内信号传导结构域包含根据SEQ ID NO 25的CD3ζ细胞内结构域或其片段。
CAR的共刺激结构域可以选自CD28、CD8、CD8α、OX40L受体(也称为CD134)、4-1BB(也称为CD137)、KIR2DS2、ICOS、CD27、MYD88-D40或其片段或其变体。CAR的共刺激结构域优选包含根据SEQ ID NO 24的细胞内CD28或其片段。
本发明还涉及嵌合抗原受体(CAR)，其包含
i.单链可变区片段(scFv)；
ii.IgG铰链结构域；
iii.信号调节蛋白α-1的Ig样C1型1和/或Ig样C1-2型结构域；
iv.CD3ζ；
v.CD28跨膜结构域；
vi.任选地CD28细胞外结构域和/或CD28细胞内结构域。
本发明还涉及CAR，其包含或由以下氨基酸序列组成：根据SEQ ID NO 26,SEQ IDNO 27,SEQ ID NO 28,SEQ ID NO 29SEQ ID NO 30,SEQ ID NO 31,SEQ ID NO 32,SEQ IDNO 33,SEQ ID NO 34,SEQ ID NO 54,SEQ ID NO 62,SEQ ID NO 63,SEQ ID NO 64,SEQ IDNO 65,SEQ ID NO 66或SEQ ID NO 67的氨基酸序列。
本发明进一步涉及多核苷酸，其编码任何先前描述的CAR。
本发明还涉及载体，其包含编码任何先前描述的CAR的多核苷酸。
本发明还涉及细胞，其包含任何先前描述的CAR或编码它们的任何多核苷酸。在一些实施方案中，细胞是T细胞。
本发明还涉及一种通过选择至少两个结构域构成间隔子结构域来调整CAR长度的方法，所述结构域从(i)IgG铰链结构域、(ii)信号调节蛋白α-1的Ig样C1-1型结构域、(iii)信号调节蛋白α-1的Ig样C1-2型结构域、或(iv)CD28细胞外片段，导致嵌合抗原受体具有不同的长度。
在一些实施方案中，细胞外间隔子结构域不结合Fc受体或具有降低的对Fc受体的结合亲和力。
附图说明
图1间隔子修饰的CAR和T细胞扩增动力学的示意图(n＝3)。A)示意图模型中的CAR结构域和设计的结构。CAR 1S和CAR X1S没有出现在图中。CAR 1S和CAR X1S分别对应CAR2S和CAR X2S，除了SIRP-αIg样C1-2型结构域是SIRP-αIg样C1-1型结构域。B)在细胞传代培养之前的第2、3、6、8和10天，使用台盼蓝评估T细胞活力并使用Bio-Rad TC20自动细胞计数器计数。结果显示为具有标准偏差的平均值。C)每2-3天对基于传代培养的倍数扩增进行计数，并评估传代培养之间的倍数扩增。线条代表不同CAR的平均值(SD)。D)第13天通过流式细胞术分析CAR表达。结果显示单个数据点和平均值(线条)。
图2扩增后的细胞表型。扩增T细胞产物(n＝3)13天，并通过流式细胞术分析其表型。结果显示为带有平均值的单个数据点。A)使用以下抗体组合确定细胞表型：T细胞CD3+CD56-；NKT细胞CD3+CD56+；NK细胞CD3-CD56+和其他细胞CD3-CD56-。B)和C)T细胞和NKT细胞群中CD4和CD8阳性细胞的比例。
图3不同记忆表型、耗竭和末端分化的T细胞和NKT细胞的百分比。结果(通过流式细胞术测量)表示为具有最小值和最大值的平均值(图A)或具有平均值的单个数据点(图B和C)。A)在扩增的第13天，分析细胞的记忆表型。B)SCM、SCM样和CM记忆表型被归类为“早期记忆表型”组，EM和Eff被归类为“效应表型”组。C)分析细胞的耗竭(PD-1阳性)和末端分化(CD57阳性)组。
图4针对CD19阳性Nalm-6细胞的T细胞反应和细胞毒性。显示了平均值(黑色水平线)和单个数据点(图A和B)；A)CAR T细胞与Nalm-6细胞以1:1的E:T比例共培养18小时。使用基于流式细胞术的CBA阵列从共培养上清液中分析细胞因子。B)通过在存在GolgiStop蛋白转运抑制剂的情况下共培养4小时后对T细胞中的CD107a进行染色，分析响应CD19阳性Nalm-6细胞的T细胞脱颗粒。结果表示T细胞中表达CD107a的细胞的百分比值，以及从这些值得出的CD4和CD8阳性细胞的百分比。C)测量荧光素酶活性以分析CAR T细胞在不同E:T比率下对表达荧光素酶的CD19+Nalm-6细胞的体外细胞毒性。显示了平均值+/-SD。
图5CAR T细胞与表达FcR的THP-1单核细胞的相互作用。CAR T细胞与单核细胞以1：1(效应细胞：非靶细胞)比率共培养。通过染色细胞表面激活标志物(图5A：CD25、CD69；流式细胞术)并通过使用基于流式细胞术的CBA阵列测量CAR T细胞和单核细胞激活诱导的细胞因子(图5B：CAR T细胞：IFN-γ和IL-2；图5C：单核细胞：IL-1β)测量CAR T细胞的激活。
图6编码不同长度CAR的Jurkat T细胞的CAR表达和细胞毒性效力。A)在转导(模拟、CAR 2S和IgG CAR)后或在转导和阳性选择(CAR M、CARXM、CAR L和CAR XL)后，通过流式细胞术测量CAR表达。结果显示在等高线图中。B)通过测量不同E:T比率下CD19 Nalm-6-luc细胞的荧光素酶活性来评估体外细胞毒性。结果表示为平均值+/-SD(n＝3)。
图7CAR对HER-2阳性SKBR-3乳腺癌细胞的细胞毒性，所述CAR具有HER-2靶向抗原结合结构域CAR M。测量荧光素酶活性以定量HER-2靶向CAR T细胞在不同E:T比率下对表达荧光素酶的HER-2阳性SKBR-3细胞的体外细胞毒性。结果显示平均值+/-SD。
图8表达HER-2靶向CAR M的T细胞对HER-2阳性SKBR-3乳腺癌细胞的细胞毒性。测量荧光素酶活性以定量HER-2靶向CAR T细胞在不同E:T比率下对表达荧光素酶的HER-2阳性SKBR-3细胞的体外细胞毒性。
图9具有修饰的多聚化结构域的CAR构建体的细胞扩增、CAR表达和细胞毒性。A)扩增来自同一供体的T细胞，所述供体用具有CAR构建体CAR M、CAR XM、CAR M1、CAR XM2、CARXM3、CAR M4、CAR2S5和CAR M6的慢病毒转导。扩增倍数与实验开始时的T细胞数量相关。在第1、3、6、8和10天测量扩增倍数。B)CAR构建体的嵌合抗原受体表达。使用抗体从细胞表面检测T细胞的嵌合抗原受体表达。使用定量PCR从分离的基因组DNA测量载体拷贝数。显示了CAR构建体CAR M、CAR XM、CAR M1、CAR XM2、CAR XM3、CAR M4、CAR2S5和CAR M6的活细胞百分比和载体拷贝数。C)CAR-T效应细胞(CAR构建体CAR M、CAR XM、CAR M1、CAR XM2、CARXM3、CAR M4、CAR2S5和CAR M6)与NALM-6靶细胞以不同比率共培养24小时的细胞毒性。使用4:1、2:1、1:1、0.5:1、0.25:1、0.125:1和0.0625:1的效靶(E:T)比率。测量靶特异性转基因(荧光素酶)的量并仅确定相对于靶细胞的杀伤百分比。
具体实施方式
本发明的特征和实施方案在本公开中通过非限制性示例进行描述。本公开不应被视为对本公开中描述的特定化合物、组合物、方法、用途的限制。应当理解，技术人员可以对本发明和实施方案进行明显的修改和变化。本申请中使用的单数形式a、an、the是指一个或多个。
为了实践本发明和实施方案，技术人员可以采用生物学、分子生物学、微生物学、化学、生物化学、免疫学和肿瘤学的常用技术和方法。常用技术和方法在文献中有描述，例如在实验室手册和实验室规程中。此类文献例如，细胞生物学的最新方案(CurrentProtocols in Cell Biology)、免疫学的最新方案(Current Protocols in Immunology)、分子生物学的最新方案(Current Protocols in Molecular Biology)、微生物学的最新方案(Current Protocols in Microbiology)、分子克隆：实验室手册(Molecular cloning:ALaboratory Manual)。所使用的技术和科学术语具有技术人员根据科学文献和技术词典通常理解的含义。
嵌合抗原受体(CAR)或(CARs)是指与特定抗原结合并参与细胞激活的受体蛋白。CAR包含抗原结合结构域、间隔子结构域、跨膜结构域、细胞内信号传导结构域和任选的共刺激结构域。表达CAR的细胞能够结合特定抗原，从而激活细胞。CAR细胞优选为T细胞、幼稚T细胞、记忆T细胞、效应T细胞。
间隔子结构域是CAR的细胞外结构域。其位于跨膜结构域和抗原结合结构域之间并将其连接。间隔子结构域在微调CAR的信号传导中发挥作用。
基于免疫球蛋白(Ig)的间隔子结构域源自免疫球蛋白Fc区或包括来自免疫球蛋白Fc区的片段。免疫球蛋白Fc区可源自IgG、IgM、IgA或IgE。IgG的Fc区可源自IgG1、IgG2、IgG3或IgG4。基于IgG的间隔子结构域包含来自IgG Fc区的CH2和CH3结构域。例如，在Hombach等人，2010中描述了具有IgG恒定区CH2和CH3的基于IgG的间隔子结构域。
信号调节蛋白(SIRP)家族(也称为，例如，SHPS、CD172)成员是参与白细胞功能调节的膜蛋白(van Beek等人，2005)。SIRP家族成员的细胞外区通常由单个Ig样V型结构域和两个Ig样C1型结构域组成。SIRP-α(也称为SHPS-1、BIT、MFR、CD172a、p84)是SIRP家族成员，其具有典型的细胞外区，所述细胞外区具有单个Ig样V型结构域、Ig样C1-1型结构域和Ig样C1-2型结构域(van Beek等人，2005)。已知SIRP-α的细胞外区在细胞外仅通过其N-末端的V型Ig样结构域结合靶配体CD47(Hatherley D等人，2009)，而目前SIRP-α的Ig样C1型结构域被称为惰性骨架。Ig样结构域的尺寸通常约为4×2.5×2.5nm。SIRP-α的氨基酸序列存在于UniProt数据库中，登录号为P78324。
细胞外间隔子结构域
本发明的间隔子结构域包含信号调节蛋白α(SIRP-α)的至少一个Ig样C1结构域。信号调节蛋白α在整个申请中缩写为SIRP-α。SIRP-αIg样C1结构域选自1型结构域(SEQ IDNO 1)和/或2型结构域(SEQ ID NO 2)。在一个实施方案中，间隔子包含SIRP-αIg样C1-1型结构域。在另一个实施方案中，间隔子包含SIRP-αIg样C1-2型结构域。在另一个实施方案中，间隔子包含SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域。间隔子可包含多个SIRP-αIg样C1-1型结构域和/或SIRP-αIg样C1-2型结构域。
间隔子可包含多聚化结构域。多聚化结构域使CAR单体多聚化。在多聚化中，CAR可以从CAR单体形成二聚体、三聚体、四聚体、五聚体或多聚体。优选地，CAR形成由两个CAR单体形成的二聚体。多聚化结构域能够在CAR的单体之间形成链接。优选单体之间的链接是二硫键。优选地，多聚化结构域在单体之间形成至少一个、两个或三个二硫键。在本发明的一些实施方案中，间隔子的多聚化结构域选自：IgG1铰链区、IgG2铰链区、IgG3铰链区、IgG4铰链区、CD28细胞外结构域或其片段或变体。在一些实施方案中，间隔子包含多聚化结构域，所述多聚化结构域含有IgGl铰链区或其片段。在一些实施方案中，间隔子包含多聚化结构域，所述多聚化结构域含有IgG4铰链区或其片段。在一个优选的实施方案中，多聚化结构域包含根据SEQ ID NO 4的氨基酸序列。在优选的实施方案中，多聚化结构域包含根据SEQ IDNO 80或SEQ ID NO 83的氨基酸序列。IgG1铰链区或其片段从一端结合SIRP-αIg样C1型结构域，从另一端结合CAR的抗原结合结构域。IgG4铰链区或其片段从一端结合SIRP-αIg样C1型结构域，从另一端结合CAR的抗原结合结构域。额外的接头序列可用于结合。在另一个实施方案中，间隔子包含多聚化结构域，所述多聚化结构域含有CD28细胞外结构域或其片段。在一个优选的实施方案中，多聚化结构域包含根据SEQ ID NO 3的氨基酸序列。CD28细胞外结构域或其片段从一端结合SIRP-αIG样C1型结构域，从另一端结合跨膜结构域，例如结合CD28的跨膜结构域(SEQ ID NO 23)。额外的接头序列可用于结合。间隔子可包含多个多聚化结构域。间隔子可包含多个不同的多聚化结构域。在一些实施方案中，间隔子包含IgGl铰链区和CD28细胞外结构域。在一些实施方案中，间隔子包含IgG4铰链区和CD28细胞外结构域。
间隔子结构域位于跨膜结构域和抗原结合结构域之间并将其连接起来。间隔子结构域在微调CAR的抗原信号传导中起作用。在本发明中，间隔子的长度可通过在间隔子中使用不同的结构域及其组合来调节。这导致不同的间隔子长度和CAR与其抗原的最佳结合。在一些实施方案中，间隔子中的结构域可以选自SIRP-α的Ig样C1-1型结构域、SIRP-α的Ig样C1-2型结构域、CD28细胞外结构域和/或IgG铰链区和/或其片段或变体。表1显示了不同CAR间隔子的氨基酸序列，其包含导致间隔子不同长度的选定结构域(SEQ ID NO 10-18、56-61)。
在基于免疫球蛋白(Ig)的CAR中，CH2结构域与髓系细胞的Fc受体(FcR)相互作用。表达FcR的髓系细胞是例如单核细胞、巨噬细胞和NK细胞。FcR与CAR的结合可导致CAR T细胞激活和表达FcR的髓系细胞破坏、CAR T细胞在肺中的隔离、激活诱导的细胞死亡(AICD)和CAR T细胞活性的整体降低(等人，2015；Hombach等人，2010；Hudecek等人，2015)。必须避免与非靶细胞不必要的相互作用和可能的副作用，以获得功能性治疗性CART细胞。
在本发明中，间隔子结构域包含信号调节蛋白α的至少一个Ig样C1结构域或其片段。Ig样C1结构域选自1型结构域和/或2型结构域。优选地，间隔子包含Ig样C1-1型结构域和Ig样C1-2型结构域。本发明的间隔子结构域不与导致功能效应的髓系细胞的FcR相互作用。具有本发明的CAR的T细胞不影响由非靶结合引起的CAR T细胞激活、表达FcR的髓系细胞的破坏、CAR T细胞在肺中的隔离、激活诱导的细胞死亡(AICD)和CAR T细胞活性的整体降低。
在本发明的优选实施方案中，间隔子结构域包含氨基酸序列SEQ ID NO 10、SEQID NO 11、SEQ ID NO 12、SEQ ID NO 13、SEQ ID NO 14、SEQ ID NO 15、SEQ ID NO 16、SEQID NO 17或SEQ ID NO 18或其变体或片段。其变体与SEQ ID NO 10-18中的任何一个具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性。表1总结了间隔子结构域的氨基酸序列。
在本发明的优选实施方案中，间隔子结构域包含氨基酸序列SEQ ID NO 56、SEQID NO 57、SEQ ID NO 58、SEQ ID NO 59、SEQ ID NO 60或SEQ ID NO 61或其变体或片段。其变体与SEQ ID NO 56-61中的任何一个具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性。表1总结了间隔子结构域的氨基酸序列。
根据SEQ ID NO 10的CAR间隔子XS包含IgGl铰链区和CD28细胞外片段。
根据SEQ ID NO 11的CAR间隔子1S包含IgG1铰链区和SIRP-αIg样C1-1型结构域。
根据SEQ ID NO 12的CAR间隔子2S包含IgG1铰链区和SIRP-αIg样C1-2型结构域。
根据SEQ ID NO 13的CAR间隔子X1S包含IgG1铰链区、SIRP-αIg样C1-1型结构域和CD28细胞外片段。
根据SEQ ID NO 14的CAR间隔子X2S包含IgG1铰链区、SIRP-αIg样C1-2型结构域和CD28细胞外片段。
根据SEQ ID NO 15的CAR间隔子M包含IgGl铰链区、SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域。
根据SEQ ID NO 16的CAR间隔子XM包含IgGl铰链区、SIRP-αIg样C1-1型结构域、SIRP-αIg样C1-2型结构域和CD28细胞外片段。
根据SEQ ID NO 17的CAR间隔子L包含IgGl铰链区、SIRP-αIg样C1-2型结构域、SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域。
根据SEQ ID NO 18的CAR间隔子XL包含IgGl铰链区、SIRP-αIg样C1-2型结构域、SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域和CD28细胞外片段。
根据SEQ ID NO 56的CAR间隔子M1包含IgG4铰链区、SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域。
根据SEQ ID NO 57的CAR间隔子XM2包含IgG4铰链区、SIRP-αIg样C1-1型结构域、SIRP-αIg样C1-2型结构域和CD28细胞外片段。
根据SEQ ID NO 58的CAR间隔子XM3包含IgG4铰链区、SIRP-αIg样C1-1型结构域、IgG4铰链区、SIRP-αIg样C1-2型结构域和CD28细胞外片段。
根据SEQ ID NO 59的CAR间隔子M4包含IgG4铰链区、SIRP-αIg样C1-1型结构域、IgG4铰链区、SIRP-αIg样C1-2型结构域和IgG4铰链区。
根据SEQ ID NO 60的CAR间隔子2S5包含IgG4铰链区、SIRP-αIg样C1-2型结构域和IgG4铰链区。
根据SEQ ID NO 61的CAR间隔子M6包含SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域。
所有上述CAR间隔子可包含将结构域彼此结合的接头序列。所有CAR间隔子及其氨基酸序列总结在表1中。
抗原结合结构域
嵌合抗原受体的抗原结合结构域识别抗原。CAR的抗原结合结构域结合所述抗原的表位。抗原结合结构域可包含与抗原结合的蛋白质、肽或其模拟物。在一些实施方案中，抗原结合结构域是抗体或其功能片段。抗体是指特异性结合抗原表位的免疫球蛋白。抗体可以是单克隆抗体或多克隆抗体。抗体或其功能片段包括但不限于嵌合抗体、人源化抗体、双特异性抗体、纳米抗体、骆驼抗体、抗原结合片段(Fab)、二价Fab区(F(ab')2)、单链抗体片段(scAb)Fv、单链可变区片段(scFv)、二价scFv(sc(Fv)2)。在一些实施方案中，抗原结合结构域包含单链可变区片段(scFv)。scFv包含可变轻链(VL)和可变重链(VH)。
已知多种抗原与癌症相关。癌症相关抗原可以是由癌细胞表达的抗原。癌症相关抗原可被癌细胞过表达。癌症相关抗原可以是基因的突变产物，或正常基因的产物，其在癌细胞上的表达量使得能够通过使用CAR靶向。癌症相关抗原可以是蛋白质、肽、碳水化合物、糖蛋白、糖脂、蛋白多糖、蛋白脂质或其任何组合。Townsend等人，2018和Yu等人，2020综述了一些癌症相关抗原。
在一些实施方案中，CAR的抗原结合结构域结合癌症相关抗原。癌症相关抗原可以选自例如已知的癌症相关抗原。Townsend等人2018、Yu等人2020综述了此类抗原。在一些实施方案中，抗原结合结构域结合CD19。在一些实施方案中，结合CD19的抗原结合结构域是单链可变区片段(scFv)。在一些实施方案中，结合CD19的抗原结合结构域是scFV，其包含SEQID NO 22或与SEQ ID NO 22具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的其变体。在一些实施方案中，抗原结合结构域结合HER-2。在一些实施方案中，结合HER-2。的抗原结合结构域是单链可变区片段(scFv)。在一些实施方案中，结合HER-2的抗原结合结构域是scFV，其包含SEQ ID NO 53或与SEQ ID NO 53具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的其变体。
跨膜结构域
CAR的跨膜结构域可以选自或衍生自膜蛋白的任何跨膜结构域。CAR的跨膜结构域可以是例如CD28、CD8、CD8α、OX40L受体(也称为CD134)、4-1BB(也称为CD137)、CD3、CD3δ、CD3γ、CD3ε、CD3ζ的跨膜结构域。在一些实施方案中，CAR的跨膜结构域是CD28的跨膜结构域或其片段或其变体。在一些实施方案中，CAR的跨膜结构域包含根据SEQ ID NO 23的氨基酸序列。
信号传导结构域
CAR可以包含细胞内信号传导结构域。细胞内信号传导结构域可以是细胞质的。CAR的细胞内信号传导结构域介导信号，从而在表达CAR的细胞中产生效应子功能。CAR的细胞内信号传导结构域可以例如介导T细胞激活的CAR信号。细胞内信号传导结构域可以选自CD3ζ、CD3δ、CD3γ、CD3ε、CD28、FcγRIII、FcR细胞质尾、酪氨酸激酶的细胞内结构域。在一些实施方案中，细胞内信号传导结构域包含CD3ζ的细胞内结构域或其片段。在一些实施方案中，细胞内信号传导结构域包含根据SEQ ID NO 25的氨基酸序列或其片段。
共刺激结构域
CAR可以任选地包含一个或多个共刺激结构域。共刺激结构域是细胞质的，并且可以影响细胞增殖、表型分化。CAR的共刺激结构域可以选自，例如CD28、CD8、CD8α、OX40L受体(也称为CD134)、4-1BB(也称为CD137)、KIR2DS2、ICOS、CD27、MYD88-D40或其片段或其变体。在一些实施方案中，CAR的共刺激结构域包含细胞内CD28或其片段或其变体。在一些实施方案中，CAR的共刺激结构域包含根据SEQ ID NO 24的氨基酸序列。
在一些实施方案中，CAR的细胞内或细胞质区包含细胞内信号传导结构域和共刺激结构域。在一些实施方案中，CAR的细胞内区包含CD3ζ或其片段和细胞内CD28结构域或其片段。在一些实施方案中，CAR的细胞质区包含根据SEQ ID NO 24的氨基酸序列或其片段和根据SEQ ID NO 25的氨基酸序列或其片段。
CAR
CAR包含抗原结合结构域、间隔子结构域、跨膜结构域、细胞内信号传导结构域和任选的共刺激结构域。本发明的CAR可以选自根据SEQ ID NO 26,SEQ ID NO 27,SEQ ID NO28,SEQ ID NO 28,SEQ ID NO 29,SEQ ID NO 30,SEQ ID NO 31,SEQ ID NO 32,SEQ ID NO33,SEQ ID NO 34或SEQ ID NO 54的氨基酸序列或其变体或其片段。其变体与SEQ ID NO26-34或SEQ ID NO 54中的任何具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性。CAR结构和氨基酸序列总结在表1中。
本发明的CAR可以选自根据SEQ ID NO 62、SEQ ID NO 63、SEQ ID NO 64、SEQ IDNO 65、SEQ ID NO 66或SEQ ID NO 67的氨基酸序列或其变体或其片段。其变体与SEQ IDNO 62-67中的任何一个具有至少80％、85％、90％、95％、96％、97％、98％、99％的序列同一性。CAR结构和氨基酸序列总结在表1中。
根据SEQ ID NO 26的CAR XS包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG1铰链区和CD28细胞外片段、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 27的CAR 1S包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG1铰链区和SIRP-αIg样C1-1型结构域、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 28的CAR 2S包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG1铰链区和SIRP-αIg样C1-2型结构域、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 29的CAR X1S包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG1铰链区、SIRP-αIg样C1-1型结构域和CD28细胞外片段、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 30的CAR X2S包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG1铰链区、SIRP-αIg样C1-2型结构域和CD28细胞外片段、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 31的CAR M包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG1铰链区、SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 32的CAR XM包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG1铰链区、SIRP-αIg样C1-1型结构域、SIRP-αIg样C1-2型结构域和CD28细胞外片段、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 33的CAR L包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG1铰链区、SIRP-αIg样C1-2型结构域、SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 34的CAR XL包含作为抗原结合结构域的结合CD19的scFv、作为间隔子片段的IgG1铰链区、SIRP-αIg样C1-2型结构域、SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域和CD28细胞外片段、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 54的HER-2CAR M包含作为抗原结合结构域的结合HER-2的scFv、作为间隔子结构域的IgG1铰链区、SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 62的CAR M包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG4铰链区、SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 63的CAR XM2包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG4铰链区、SIRP-αIg样C1-1型结构域、SIRP-αIg样C1-2型结构域和CD28细胞外片段、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 64的CAR XM3包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG4铰链区、SIRP-αIg样C1-1型结构域、IgG4铰链区、SIRP-αIg样C1-2型结构域和CD28细胞外片段、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 65的CAR M4包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG4铰链区、SIRP-αIg样C1-1型结构域、IgG4铰链区、SIRP-αIg样C1-2型结构域和IgG4铰链区、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 66的CAR 2S5包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的IgG4铰链区、SIRP-αIg样C1-2型结构域和IgG4铰链区、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
根据SEQ ID NO 67的CAR M6包含作为抗原结合结构域的结合CD19的scFv、作为间隔子结构域的SIRP-αIg样C1-1型结构域和SIRP-αIg样C1-2型结构域、作为跨膜结构域的CD28片段、作为共刺激结构域的CD28细胞内片段和作为细胞内信号传导结构域的CD3ζ片段。
所有上述CAR可包含将结构域彼此结合的接头序列。所有CAR及其氨基酸序列总结在表1中。
本发明的CAR具有基于信号调节蛋白α(SIRP-α)的骨架，以为CAR T细胞和在避免与表达Fc受体(FcR)的细胞非靶结合的其他细胞疗法中提供惰性和可修饰的通用间隔子。通过FcR与髓系细胞的非靶结合导致CAR T细胞功能受阻、细胞因子产生过多和CAR T细胞整体受损。
所有具有SIRP-α骨架的新的CAR在CD4:CD8比率上都有微小的变化，有利于CD4+群，然而，与传统的基于IgG的CAR相比，具有相同的细胞毒性和功能。
携带基于SIRP-α的CAR的T细胞在与THP-1单核细胞共培养后未显示激活水平增加，这与具有基于hIgG-CH2CH3的CAR的T细胞相反，后者表达高水平的早期激活标志物CD69和IL-2以及IFN-γ。与具有基于IgG的CAR的T细胞相反，在SIRP-αCAR T细胞中也避免了通过产生IL-1β所测量的单核细胞激活。
多核苷酸和载体
本发明涉及编码本发明的嵌合抗原受体的多核苷酸。多核苷酸可以是DNA或RNA或修饰的DNA或修饰的RNA或核酸类似物。多核苷酸可以是单链或双链的。本发明的多核苷酸可以通过技术人员可用的任何方法分离、纯化、重组产生或合成。多核苷酸的核苷可以被化学修饰。核酸类似物是结构上与DNA和RNA类似的化合物。核酸类似物可以是例如肽核酸(PNA)、锁核酸(LNA)、桥接核酸(BNA)、吗啉代。多核苷酸可包含一种或多种核苷类似物。
还应当理解，相似的氨基酸序列可以由替代的多核苷酸序列编码。本发明中的密码子优化是使用智人密码子通过基于内源性受体中频率分布的估计概率来进行的。在本发明的一些实施方案中，编码CAR间隔子的多核苷酸序列可以选自SEQ ID NO 35、SEQ ID NO36、SEQ ID NO 37、SEQ ID NO 38、SEQ ID NO 39、SEQ ID NO 40、SEQ ID NO 41、SEQ ID NO42或SEQ ID NO 43。在本发明的一些实施方案中，编码CAR间隔子的多核苷酸序列可以选自SEQ ID NO 68、SEQ ID NO 69、SEQ ID NO 70、SEQ ID NO 71、SEQ ID NO 72或SEQ ID NO73。在本发明的一些实施方案中，编码CAR的多核苷酸序列可以选自SEQ ID NO 44、SEQ IDNO 45、SEQ ID NO 46、SEQ ID NO 47、SEQ ID NO 48、SEQ ID NO 49、SEQ ID NO 50、SEQ IDNO 51、SEQ ID NO 52或SEQ ID NO 55。在本发明的一些实施方案中，编码CAR的多核苷酸序列可以选自SEQ ID NO 74、SEQ ID NO 75、SEQ ID NO 76、SEQ ID NO 77、SEQ ID NO 78或SEQ ID NO 79。
编码本发明的CAR的多核苷酸可以形成表达盒。所述表达盒包含编码本发明的CAR的遗传信息。表达盒包含编码本发明的CAR的多核苷酸序列。所述表达盒可包含抗原结合结构域、间隔子结构域、跨膜结构域、细胞内细胞信号传导结构域和任选的共刺激结构域的编码序列。除了编码序列之外，所述表达盒还可以包含选自以下的序列：启动子序列、增强子序列、翻译终止序列和转录终止序列。可以用病毒或非病毒方法将编码本发明的CAR的表达盒引入宿主细胞。
在非病毒方法中，使用基于打开靶细胞的脂质膜(例如用电流)和/或将多核苷酸与脂质包膜偶联的方法将CAR编码多核苷酸引入宿主细胞。表达盒可以在编码CAR的质粒中或作为编码CAR的mRNA。表达盒可包含能够整合至宿主细胞的部分。技术人员可以选择任何可用的非病毒基因递送方法。这样的方法例如是转染和核转染方法，使用脂质体、阳离子试剂和电穿孔。非病毒方法及其用途由Harris等人2020和Riedl等人2018综述。
使用病毒方法，使用病毒载体将本发明的CAR编码多核苷酸引入宿主细胞。病毒载体可以是例如逆转录病毒载体、慢病毒载体或腺病毒载体。可以使用含有表达盒的质粒来产生病毒载体，所述表达盒包含CAR编码物质、包装物质和包膜相关物质。质粒可以选自例如pRRL.SIN-19、RSV-rev、pMDLg/pRRE和pMD.G。其他表达盒物质可以选自嵌合5'LTR-包装信号-REV-反应元件-启动子-转基因盒、REV表达质粒、基质和衣壳和核衣壳的前体蛋白以及逆转录酶和整合酶组分的前体的表达载体、包膜蛋白例如VSV-G的表达载体。此类质粒将被引入宿主细胞，导致产生含有CAR表达盒插入的自身失活的病毒颗粒。这样的载体可以将盒整合到受体细胞基因组中。技术人员可以使用任何可用的基于病毒的方法将编码本发明的CAR的多核苷酸引入宿主细胞。病毒载体和相关方法在例如Dull等人1998、Levine等人2016的参考文献中描述。
细胞
本发明的宿主细胞是指表达本发明的CAR的细胞。可以通过病毒或非病毒方法将编码本发明的CAR的多核苷酸引入宿主细胞。宿主细胞可以是真核细胞或原核细胞。原核细胞可以是例如细菌细胞。真核细胞可以是例如动物细胞、植物细胞、真菌细胞、昆虫细胞。宿主细胞可以是培养的细胞系。这样的细胞系可以是例如NK92或Jurkat T细胞。宿主细胞可以分离自生物体，例如动物、植物、真菌、昆虫。优选地，宿主细胞分离自人类。宿主细胞可以是例如血细胞、神经元细胞、上皮细胞、内皮细胞、肝细胞。优选地，宿主细胞是血细胞，更优选白细胞。宿主细胞可以是选自嗜中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、淋巴细胞、单核细胞的白细胞。宿主细胞可以是选自自然杀伤细胞(NK)、T淋巴细胞(T细胞)和/或B淋巴细胞(B细胞)或浆细胞的淋巴细胞。优选地，本发明的宿主细胞是T细胞。T细胞可以是T辅助细胞(TH)细胞、细胞毒性T(TC)细胞、调节性T(Treg)细胞、自然杀伤性T(NKT)细胞。T细胞可以表达特定的细胞表面分子，例如T细胞CD3、TH细胞CD4、TC细胞CD8。不同的记忆表型是幼稚T细胞、T记忆干细胞样(TSCM样)细胞、T中央记忆(TCM)细胞、T记忆干细胞(TSCM)细胞、T效应(Teff)细胞、T效应记忆(TEM)细胞。可以基于细胞表面分子表达例如CD95、CD45RO、CD45RA、CD27来鉴定记忆表型。表2总结了记忆T细胞及其表面标志物。记忆T细胞可以表达CD4或CD8。宿主细胞可包含单一细胞类型或不同细胞类型的群体，优选宿主细胞是特定T细胞类型或特定NK细胞类型，或包含多种T细胞类型和/或NK细胞类型的群体。在本发明中，宿主细胞可以是不同细胞类型的细胞群，例如从血液样品中分离的外周血单个核细胞的细胞群。宿主细胞可以是从外周血单个核细胞分离的T细胞。T细胞应该理解为在其表面表达CD3的细胞。细胞还可以包含自然杀伤T(NKT)细胞、不同的T细胞表型、记忆T细胞、T辅助细胞、T效应细胞、NK细胞。细胞可以特异性表达例如细胞表面标志物，如CD3、CD4和/或CD8。细胞群中不同细胞类型的比例可以不同。
细胞群可包含T细胞和NKT细胞。优选地，宿主细胞群包含超过80％、86％或90％的T细胞。优选地，宿主细胞群包含少于15％、13％或9％的NKT细胞。在优选的实施方案中，宿主细胞群包含多于86％的T细胞和少于13％的NKT细胞。宿主细胞的T细胞可以包括例如CD4阳性和CD8阳性细胞。宿主细胞群可以包含T细胞，其中少于40％的细胞是CD57阳性的和/或PD-1阳性的。
本发明的CAR、编码间隔子修饰的CAR的多核苷酸、包含编码间隔子修饰的CAR的多核苷酸的载体和/或表达本发明的CAR的细胞可用于治疗与抗原相关的疾病，所述抗原被CAR的抗原结合结构域靶向。CAR与抗原的结合导致表达抗原的靶细胞的细胞毒性。表达本发明的CAR的细胞可用于癌症疾病的细胞疗法，优选地用于治疗患有难治性和复发性血液恶性肿瘤、急性淋巴细胞性白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)和非-霍金氏淋巴瘤的患者。表达CAR的细胞(优选T细胞)的靶抗原可以是例如CD19、HER-2和其他癌症相关靶抗原，例如选自Townsend等人2018和Yu等人2020综述的癌症相关抗原。治疗性CAR T细胞可用于癌症免疫疗法。治疗性CAR T细胞可以是自体的或同种异体的。从患者分离自体细胞，通过载体将编码CAR的多核苷酸引入细胞，并将表达CAR的细胞施用回患者。同种异体细胞分离自不同的个体，但在遗传上与患者的细胞相似。
表达CAR的细胞，优选T细胞，可以在药物组合物中施用于患者。除了表达CAR的细胞之外，药物组合物还可以包含其他药物活性剂、防腐剂和/或缓冲物质。
实施例
实施例1材料和方法
CAR的设计
对FMC63抗体克隆可变区(Genbank：免疫球蛋白轻链，可变区；CAA74660.1，和免疫球蛋白重链，可变区；CAA74659.1)的序列进行修饰，以设计靶向CD19的单链可变区片段(scFv)。使用四个规范的GGGGS接头连接可变轻链和可变重链。来自IgG1-CH1结构域的铰链区用于将间隔子与CD19结合域连接。抗原结合结构域和细胞膜之间的间隔子由SIRP-αIg样C1-1型和/或C1-2型结构域构建。SIRP-α一级结构获自Uniprot数据库(P78324)，并通过基于频率分布的估计概率使用智人密码子进行反向翻译。构建了一些间隔子结构以包括T细胞特异性表面糖蛋白CD28的额外细胞外片段。跨膜(TM)和细胞内(IC)序列来自T细胞特异性表面糖蛋白CD28和T细胞受体的细胞内T淋巴细胞激活结构域(TCR、CD3ζ链、UniprotP20963-3、CD28、Uniprot P10747)。表1总结了不同CAR的氨基酸序列。
人Ab4D5(Carter等人，1992)抗体克隆用于设计HER-2靶向单链可变区片段。在HER-2靶向CAR构建体中，CAR的其他结构域与CD19靶向CAR M中的相同。与CD19靶向CAR类似地制备HER-2靶向CAR。
基于IgG1的CAR(FMC63 scFv、IgG1-CH2-CH3间隔子、CD28跨膜和细胞内结构域、以及CD3ζ信号传导结构域)用作阳性对照。不含FcR结合位点的对照是基于CD28的CAR(CARXS；FMC63 scFv、IgG铰链区、来自CD28的细胞外、跨膜和细胞内序列以及来自CD3ζ信号传导结构域的细胞内序列)。为了评估(CAR-)T细胞在转导后与靶细胞的特异性相互作用，使用了阴性转导对照，一个空的pLV载体(模拟)。
T细胞扩增
CAR T细胞由分离自血沉棕黄层的外周血单个核细胞制造，如前所述(Kaartinen等人，2017)。在T细胞培养中，使用补充有5％人AB血清(Seralab,Oviedo,Spain)和100U/mlIL-2(Proleukin，Novartis，Basel，Switzerland)的X-VIVO(Lonza，Basel，Switzerland)培养基。在第0-2天，T细胞密度调整为1x106个细胞/ml，在第3天，洗掉载体后，通过加入新鲜培养基将T细胞密度调整为0.5x106个细胞/ml。在第2天，使用包含编码不同CAR结构的序列的第三代慢病毒载体(Koponen等人，2003)或模拟载体转导T细胞。CAR T细胞培养至第10天，然后冷冻以等待对细胞功能的进一步分析。为了评估CAR T细胞的功能，将第10天CAR T细胞解冻，调整为0.5x106个细胞/ml的细胞密度，并培养至第13天后进行分析。对于记忆表型分析，CAR T细胞在不冷冻的情况下培养至第13天。
细胞系
NALM-6(CD19+B谱系，急性淋巴细胞性白血病，ALL)细胞、THP-1(FcR+单核细胞，急性单核细胞性白血病)细胞和E6.1 Jurkat T细胞在RPMI-1640培养基(Thermo FisherScientific，Waltham，USA)中培养，所述RPMI-1640培养基补充有10％胎牛血清(ThermoFisher Scientific)、100IU/mL青霉素和100μg/mL链霉素(Thermo Fisher Scientific)。此外，对于Jurkat T细胞，还添加了2mM L-谷氨酰胺。NALM-6-luc细胞系是按照Dufva等人2019中的描述生成的。
流式细胞术
在用抗人抗体染色之前，细胞用1％多聚甲醛固定(10分钟，+4℃)。作为对照荧光减一(FMO)和/或使用适当的同种型对照。样品在BD FACSAria IIu细胞仪(BDBiosciences，Franklin Lakes，USA)上运行，结果使用FlowJo(版本10.5.3，BDBiosciences)软件进行分析。
CAR T细胞的记忆表型分型
扩增后，使用BD Biosciences的以下抗人抗体对T细胞亚型和残留的NK-和NKT细胞(表2)进行染色：CD3(克隆UCHT1)-异硫氰酸荧光素(FITC)、CD4(克隆SK3)-BDHorizonTMBrilliant VioletTM510(BV510)、CD8(RPA-T8)-BD HorizonTMBrilliantVioletTM421(BV421)、CD56(克隆B159)-别藻蓝蛋(APC)。使用与花青素5.5(Cy 5.5)缀合的CD27(克隆M-T271)-哌啶宁-叶绿素蛋白(PerCP)、CD45RA(克隆HI100)-APC、与花青7(Cy7)缀合的CD45RO(克隆UCHL1)-藻红蛋白(PE)和CD95(克隆DX2)-PE鉴定记忆T细胞表型。
T细胞记忆表型是使用表2中所示的CD4和CD8亚群表达标志物定义的。为了将T细胞成熟指定为末端效应表型和耗竭，使用了CD57(克隆NK-1)-BD HorizonTMBrilliantVioletTM421(BV421)和CD279(克隆MIH4)-AF647的抗体。在CD95+CD27+/-CD45RO+/-群体中评估程序性细胞死亡蛋白1(CD279)和T细胞末端效应诱导标志物CD57的表达。使用与Alexa647(Jackson Immunoresearch,Inc West Grove,USA.)缀合的F(ab')2片段山羊抗人免疫球蛋白(Ig)G(H+L)测量CAR的表达。
细胞毒性测定法
为了评估间隔子修饰的CAR的细胞毒性效力，将细胞与Luc+NALM-6细胞在各种T细胞：B细胞比率(效应子：靶标比率，E:T)下共培养18小时。在共培养结束时，添加荧光素(ONE-Glo荧光素酶试剂，Promega)并根据制造商的说明使用CLARIOstar Plus多模式微孔板读数器(BMG Labtech)定量存在的活靶细胞。
脱颗粒测定法
为了测量靶细胞诱导的T细胞脱颗粒，在溶酶体相关膜蛋白1(CD107a)抗体(PE缀合,克隆H4A3,BD Biosciences)和GolgiStopTM蛋白转运抑制剂(BD Biosciences)存在的情况下，将细胞与NALM-6靶细胞以1:1(E:T)的比率共培养4小时。脱颗粒被评估为共培养物中细胞表面表达CD107a+T细胞与总T细胞的比例，使用流式细胞术测量。
证明CAR T细胞与单核细胞相互作用的分析
为了分析与单核细胞结合的CAR T细胞的作用，将T细胞与THP-1单核细胞以1:1的比率在+37℃下共培养18小时。使用流式细胞术测量T细胞上的细胞表面激活标志物CD25(克隆BC96，BioLegend)和CD69(克隆FN50，BD Biosciences)，并收集细胞培养基以进一步分析激活诱导的细胞因子(单核细胞：IL-1β和CAR T细胞：IFN-γ和IL-2)。
细胞因子测定法
为了从细胞毒性测定法(IFN-γ和IL-2)和证明CAR T细胞与单核细胞相互作用的分析(IFN-γ、IL-2和IL-1β)中定量激活诱导的细胞因子，根据制造商的说明分析细胞培养基(效应子：靶标比率；1:1)的细胞计数珠阵列(CBA人可溶性蛋白主缓冲液试剂盒以及IL-2,IFN-γ和IL-1βCBA Flex Sets,BD Biosciences)。使用FCAP阵列软件第3.0版(BDBiosciences)分析结果。
CAR阳性Jurkat T细胞的抗体缀合和磁微珠选择
根据制造商的说明使用LYNX Rapid Plus Cy5抗体缀合试剂盒(Bio-Rad,Hercules,USA)使SIRP-α结合抗体(SE12B6；Seiffert等人2001)与青色素5(Cy5)荧光染料缀合。根据制造商的说明，利用单细胞分离(抗Cy5/抗Alexa Fluor 647微珠，MiltenyiBiotec)选择Jurkat T细胞，并通过流式细胞术确认表达。
实施例2T细胞扩增及CAR表达
CAR构建体CAR XS、CAR XM和CAR M包含来自单克隆抗体FMC63的scFv部分、来自SIRP-α的Ig样C1-1型和Ig样C1-2型结构域的细胞外间隔子、IgG铰链区和/或CD28、来自CD28的跨膜结构域和来自CD28和CD3ζ的细胞内结构域(图1A)。使用慢病毒载体(pLV)在hPGK启动子下将CAR转导到T细胞中(Koponen JK等人，2003)。
不同的CAR转导的T细胞在13天内扩增了48-260倍(图1B)。扩增率没有显著差异，但CAR XM转导的细胞显示出生长较慢的趋势。尽管在早期阶段可以看到生长数据的差异，但与IgG1-CAR不同，CAR M和XM在第10天解冻细胞后似乎具有特征性的第二个生长高峰(图1C)。
在扩增的第二天，T细胞被携带CAR基因的慢病毒或模拟载体稳定转导。制造细胞后，在第13天，我们分析了细胞的CAR表达，如通过减去空载体转导的T细胞的CAR抗体结合结果(模拟13.25±5.2)所测量的，在25.3％至88.8％的细胞中检测到CAR表达(平均值±SD；IgG1-CAR 88.8±5.6，CAR M 45.0±22.6，CAR XM 60.6±22.6和CAR XS 25.3±14.3)(图1D)。
所有CAR均在T细胞上成功表达，但在培养第6天后，CAR XS、CAR M和CAR XM T细胞的扩增率似乎略低于IgG-CAR和模拟T细胞(图1C)。
实施例3不同表达CAR的T细胞之间T细胞表型和成熟的表征
扩增13天后，大部分细胞(86％-90％)是T细胞(CD3+CD56-)，包括9-13％NKT细胞(CD3+CD56+)，NK细胞(CD3-Cd56+)或剩余CD3-CD56-细胞提供非常少的额外贡献(图2A)。除了细胞表型外，我们还评估了T细胞记忆表型。早些时候，我们报道了CAR T细胞扩增过程中IL-2的浓度影响T细胞记忆表型(Kaartinen T等人，2017)。因此，我们在培养物中使用100U/ml IL-2以防止T细胞过度分化。T细胞记忆表型的全部内容如图3A所示。为了更容易区分扩增过程和各种CAR对T细胞记忆表型的影响，我们将T细胞记忆亚群分为早期记忆(＝Tscm、Tscm样和Tcm)和效应子(＝Tem和Teff)组(图3B)。到扩增第13天，带有基于SIRP-α的CAR M和CAR XM的T细胞倾向于有利于向CD4+细胞分化(图2B)，其中效应T细胞的比例倾向于高于CD8细胞，表明早期记忆细胞的优势更强。然而，由于不同供体之间的差异，未检测到与各种CAR相关的T记忆细胞分化中的统计学显著差异。除此之外，记忆表型以及使用PD-1表面表达所测量的耗竭水平和T细胞末端效应成熟相关的标志物CD57的增殖能力保持相同。13天扩增后，大多数CD4和CD8细胞对耗竭标志物CD57和PD-1(66.2-79.9％)呈阴性，少数表达一种或两种表面标志物(图3C)。同样，CAR不会对T细胞中的耗竭标志物表达产生差异性影响。
实施例4CAR T细胞的激活和细胞毒性活性
CAR T细胞与携带靶抗原的细胞相互作用诱导T细胞激活和靶细胞杀伤。在确定可以成功生成携带间隔子修饰的CAR构建体的T细胞后，我们接下来分析了CAR T细胞响应靶标依赖性激活的功能特征。为了分析响应CD19+靶细胞的T细胞激活中的CAR功能，我们使用1:1的效应子：靶细胞比率测量了过夜共培养的细胞因子产量(图4A)。携带不同CAR的所有T细胞产生相似量的IL-2，携带CAR M的细胞具有更高的IL-2产量(约1.3倍以上)，具有非显著的趋势。未检测到IFN-γ产量的差异。
然后，我们通过测量CD107a细胞表面表达的出现，研究了T细胞响应与CD19+靶细胞共培养4小时的脱颗粒能力。表达CAR的细胞的比例与响应靶细胞的脱颗粒细胞组分直接相关(图4B)，证实了表达CAR的细胞的功能。尽管具有IgG1-CAR的CAR表达水平高于CAR M、CAR XM或CAR XS，但CD4+细胞的CD107a表达相似。相比之下，在CD8+细胞中，IgG1-CAR和CARXS显示比CAR M和CAR XM更高的CD107a表达。
尽管CAR表达和CD8+细胞脱颗粒水平不同，但所有CAR T细胞对NALM-6细胞靶标都显示出非常相似的细胞毒性效力(图4C)。在与CD19+靶细胞进行的18小时共培养实验中，所有CAR T细胞系在2:1(E:T)的比率下均表现出100％的杀伤效力，在较低的E:T比率下也表现出相似的效率。
实施例5间隔子修饰的CAR T细胞未显示“非靶”髓系细胞的激活
设计基于SIRP-α的FiCAR，以规避与表达Fc受体的髓系细胞的相互作用。我们通过将CAR T细胞与THP-1单核细胞以1:1(效应细胞：非靶细胞；E:OT)的比率共培养来评估CART细胞与髓系细胞的相互作用。CAR T细胞激活是通过染色细胞表面激活标志物CD25和CD69(CD25指示长期激活，CD69指示短期激活)(图5A)、并通过测量响应于CAR相关激活的T细胞(图5B：CAR T细胞:IFN-γ和IL-2)和单核细胞(图5C：单核细胞：IL-1β)产生的细胞因子来测量的。所有的CAR T细胞在有或没有THP-1单核细胞的情况下都表达高水平和同等水平的CD25激活标志物。此外，在CAR T细胞和THP-1单核细胞的共培养中，含有Fc区的CAR，即IgG1-CAR，表达高水平的细胞表面早期激活标志物CD69。相比之下，具有间隔子修饰的CAR构建体(即CAR XS、CAR M和CAR XM)的T细胞不显示与模拟T细胞结合的CD69表达。在细胞因子产量中可以看到类似的调整，其中除了THP-1产生激活诱导的细胞因子IL-1β之外，IgG-CAR还产生激活诱导的细胞因子IL-2和IFN-γ。同样，在含有或不含THP-1单核细胞的情况下，间隔子修饰的CAR T细胞产生低水平的IL-2和IFN-γ，其均与模拟转导的对照T细胞相同。此外，THP-1单核细胞与间隔子修饰的T细胞共培养，THP-1单核细胞产生低水平的IL-1β，该IL-1β水平与THP-1细胞单独或与模拟转导的对照T细胞一起时相同。综上所述，这些数据表明，间隔子修饰的CAR T细胞与携带FcR的单核细胞共培养不导致T细胞或单核细胞的不当激活。
实施例6修饰SIRP-α间隔子长度
为了进一步研究是否可以修饰CAR骨架结构以更好地结合靶细胞上的膜近端或膜远端抗原，我们设计了各种长度的CAR以靶向CD19。通过利用SIRP-α的不同Ig样C1结构域调整间隔子长度，我们通过从CAR M或CAR XM中去除另一个Ig样C1结构域或通过向CAR M和CAR XM添加额外的Ig样C1结构域来设计长度调整的CAR。
首先，为了保证不同长度CAR的高表达，使用单细胞微珠分离来选择表达CAR的Jurkat T细胞。然后，为了测量表达，使用生物素化的抗人CD19 CAR检测试剂(MiltenyiBiotec)和作为二抗的与APC(Miltenyi Biotec)缀合的生物素抗体对不同长度的CAR进行染色。根据制造商的说明进行染色。所有转导的Jurkat T细胞培养物都显示出不同CAR的高表达水平(图6A：CAR 2S 90,6％,CAR L88,1％,CAR XL 95,4％)，相反，空载体转导的模拟Jurkat T细胞未显示抗体的非特异性结合。
此外，为了评估各种长度CAR的功能，我们测试了CAR转导的Jurkat T细胞在几种E:T比率下对CD19阳性Nalm-6-luc细胞的细胞毒性效力(图6B)。表达各种CAR(CAR 2S、CARL和CAR XL)中的每一种的Jurkat T细胞都显示出与配备CAR M、CAR XM和基于IgG1的对照CAR的Jurkat T细胞相似的杀伤效力；从0-20％到66.8-82％，取决于E:T比率。作为杀伤的阳性对照，我们使用了在不同E:T比率下表现出卓越杀伤效力(48-94,7％)的原代T细胞，并且作为阴性对照使用了模拟转导的Jurkat T细胞，在不同E:T比率下显示出0-15.4％的非CAR相关杀伤效力。
实施例7用基于SIRPα骨架的CAR靶向HER-2。
在证明间隔子长度可以调整后，通过用靶向HER-2的ScFv结构域替换之前CAR M结构中靶向CD19的scFv结构域，我们设计了一种新的靶向HER-2的CAR。为了证明靶向HER-2的CAR M的功能，将CAR转导到原代T细胞中。扩增后，将靶向HER2的CAR T细胞与HER-2阳性SKBR-3-eGFP-luc乳腺癌细胞以各种效应子-靶标(E:T)比率共培养(图7)。在18小时细胞毒性初步测试(n＝1)中，与模拟转导的T细胞相比，用靶向HER-2的CAR M转导的T细胞显示出更高的杀伤效力，模拟转导的T细胞本身表现出轻微的CAR非依赖性细胞杀伤。
实施例8表达具有靶向HER-2的scFv的CAR M的T细胞的细胞毒性。
从健康供体血沉棕黄层中分离出T细胞，使用携带HER-2CAR M基因构建体的慢病毒载体、使用不同的感染复数(MOI)1.25、2.5和5进行转导，并扩增11天。将表达含有备选靶向HER-2的scFv的HER-2CAR M的T细胞(效应细胞)与表达萤火虫荧光素酶的HER-2+SKBR3乳腺癌细胞(靶细胞)在效应子-靶标(E:T)比率4:1、2:1、1:1、1:2、1:4和1:8下一起孵育。24小时后，添加荧光素并定量活的靶细胞，与空载体(模拟)转导的T细胞相比，在所有不同的E:T比率下显示出高的杀伤效力。
实施例9具有修饰的多聚化结构域的CAR构建体的细胞扩增、CAR表达和细胞毒性。
用磁珠(Miltenyi Biotec)从外周血单个核细胞中纯化CD4+和CD8+T细胞。用编码CAR构建体(CAR M、CAR,XM、CAR M1、CAR XM2、CAR XM3、CAR M4、CAR 2S5、CAR M6)的慢病毒载体转导纯化的CD4+和CD8+T细胞，并在含有12.5ng/ml的IL-7和IL-15的培养基(MiltenyiBiotec)中扩增。在扩增过程中测量细胞数量和活力。研究不同的CAR构建体对扩增的影响直到第10天(图9A)。不同的构建体对细胞扩增没有明显影响，所有构建体都达到了20倍以上的扩增。
还使用流式细胞术研究了细胞的CAR表达。通过生物素标记的抗体检测CAR构建体，该抗体检测所有CAR构建体中存在的特定结构域(图9B)。通过分离基因组DNA并用转基因特异性引物检测整合基因来研究载体拷贝数(VCN)(图9B)。细胞群中的VCN大约为1，超过50％的细胞在细胞表面表达CAR转基因。
CAR-T细胞(解冻后)与CD19+NALM-6靶细胞以不同的效应细胞(CAR-T)和靶细胞(癌细胞)比率共培养24小时。此时，细胞被裂解并测量靶细胞特异性(转)基因活性(图9C)。在杀伤测定法中，CAR M、XM、M1和M6显示出比其他CAR构建体具有更高杀伤效力的趋势，但与未转导的或空载体(模拟)转导的T细胞相比，所有构建体都显示出显著提高的靶细胞杀伤效力。
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表1氨基酸序列和核酸序列总结
缩写(T)序列类型；(a)包含蛋白质或肽序列的氨基酸；(n)包含多核苷酸序列的核酸；(SP)物种；(h)智人；(a)人工
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表2
表1.用于细胞表型和T细胞记忆表型分析的细胞表面标志物表达模式
1早期记忆2效应子
序列表
<110>奥赖恩公司
<120>嵌合抗原受体(CAR)间隔子修饰增强CAR T细胞功能
<130> CAR SPACER
<150> FI20206315
<151> 2020-12-16
<160> 84
<170> BiSSAP 1.3.6
<210> 1
<211> 100
<212> PRT
<213> 智人
<400> 1
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
1 5 10 15
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
20 25 30
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
35 40 45
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
50 55 60
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
65 70 75 80
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
85 90 95
Ala Asn Leu Ser
100
<210> 2
<211> 95
<212> PRT
<213> 智人
<400> 2
Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val
1 5 10 15
Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu
20 25 30
Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr
35 40 45
Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu
50 55 60
Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val
65 70 75 80
Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys
85 90 95
<210> 3
<211> 17
<212> PRT
<213> 智人
<400> 3
Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys
1 5 10 15
Pro
<210> 4
<211> 25
<212> PRT
<213> 智人
<400> 4
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro
20 25
<210> 5
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 5
Gly Gly Gly Gly Ser
1 5
<210> 6
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 6
Gly Gly Gly Gly Ser Val Pro
1 5
<210> 7
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 7
Gly Gly Gly Gly Ser Ala Lys
1 5
<210> 8
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 8
Val Ser Gly Gly Gly Gly Ser
1 5
<210> 9
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 接头 C1
<400> 9
Glu Thr Ile Arg Val Pro
1 5
<210> 10
<211> 42
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子XS
<400> 10
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro Lys Gly Lys His Leu Cys Pro
20 25 30
Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro
35 40
<210> 11
<211> 132
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子1S
<400> 11
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys
20 25 30
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
35 40 45
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
50 55 60
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
65 70 75 80
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
85 90 95
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
100 105 110
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
115 120 125
Ala Asn Leu Ser
130
<210> 12
<211> 129
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子2S
<400> 12
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro
20 25 30
Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val
35 40 45
Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu
50 55 60
Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr
65 70 75 80
Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu
85 90 95
Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val
100 105 110
Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val
115 120 125
Ser
<210> 13
<211> 154
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子X1S
<400> 13
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys
20 25 30
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
35 40 45
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
50 55 60
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
65 70 75 80
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
85 90 95
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
100 105 110
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
115 120 125
Ala Asn Leu Ser Gly Gly Gly Gly Ser Lys Gly Lys His Leu Cys Pro
130 135 140
Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro
145 150
<210> 14
<211> 146
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子X2S
<400> 14
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro
20 25 30
Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val
35 40 45
Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu
50 55 60
Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr
65 70 75 80
Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu
85 90 95
Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val
100 105 110
Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val
115 120 125
Ser Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser
130 135 140
Lys Pro
145
<210> 15
<211> 235
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子M
<400> 15
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys
20 25 30
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
35 40 45
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
50 55 60
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
65 70 75 80
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
85 90 95
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
100 105 110
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
115 120 125
Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr
130 135 140
Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val
145 150 155 160
Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly
165 170 175
Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp
180 185 190
Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala His
195 200 205
Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro
210 215 220
Ala Val Ser Lys Ser His Asp Leu Lys Val Ser
225 230 235
<210> 16
<211> 252
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子XM
<400> 16
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys
20 25 30
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
35 40 45
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
50 55 60
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
65 70 75 80
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
85 90 95
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
100 105 110
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
115 120 125
Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr
130 135 140
Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val
145 150 155 160
Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly
165 170 175
Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp
180 185 190
Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala His
195 200 205
Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro
210 215 220
Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Lys Gly Lys His Leu
225 230 235 240
Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro
245 250
<210> 17
<211> 339
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子L
<400> 17
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro
20 25 30
Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val
35 40 45
Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu
50 55 60
Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr
65 70 75 80
Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu
85 90 95
Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val
100 105 110
Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val
115 120 125
Ser Gly Gly Gly Gly Ser Ala Lys Pro Ser Ala Pro Val Val Ser Gly
130 135 140
Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu
145 150 155 160
Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn
165 170 175
Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu
180 185 190
Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg
195 200 205
Glu Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu
210 215 220
Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg
225 230 235 240
Val Pro Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn
245 250 255
Gln Val Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu
260 265 270
Gln Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala
275 280 285
Ser Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp
290 295 300
Leu Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys
305 310 315 320
Gln Val Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu
325 330 335
Lys Val Ser
<210> 18
<211> 356
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子XL
<400> 18
Tyr Val Thr Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp
1 5 10 15
Lys Thr His Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro
20 25 30
Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val
35 40 45
Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu
50 55 60
Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr
65 70 75 80
Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu
85 90 95
Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val
100 105 110
Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val
115 120 125
Ser Gly Gly Gly Gly Ser Ala Lys Pro Ser Ala Pro Val Val Ser Gly
130 135 140
Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu
145 150 155 160
Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn
165 170 175
Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu
180 185 190
Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg
195 200 205
Glu Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu
210 215 220
Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg
225 230 235 240
Val Pro Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn
245 250 255
Gln Val Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu
260 265 270
Gln Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala
275 280 285
Ser Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp
290 295 300
Leu Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys
305 310 315 320
Gln Val Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu
325 330 335
Lys Val Ser Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly
340 345 350
Pro Ser Lys Pro
355
<210> 19
<211> 108
<212> PRT
<213> 智人
<400> 19
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 Leu Lys Arg
100 105
<210> 20
<211> 120
<212> PRT
<213> 智人
<400> 20
Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 21
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 接头
<400> 21
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 22
<211> 248
<212> PRT
<213> 人工序列
<220>
<223> scFV抗-CD19
<400> 22
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 Leu Lys Arg Gly Gly Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
130 135 140
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
145 150 155 160
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
165 170 175
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
180 185 190
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
195 200 205
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
210 215 220
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
225 230 235 240
Gly Thr Thr Val Thr Val Ser Ser
245
<210> 23
<211> 27
<212> PRT
<213> 智人
<400> 23
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> 24
<211> 41
<212> PRT
<213> 智人
<400> 24
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> 25
<211> 112
<212> PRT
<213> 智人
<400> 25
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> 26
<211> 491
<212> PRT
<213> 人工序列
<220>
<223> CAR XS
<400> 26
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Lys Gly Lys His Leu Cys Pro Ser Pro Leu
290 295 300
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
305 310 315 320
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
325 330 335
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
340 345 350
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
355 360 365
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser
370 375 380
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
385 390 395 400
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
405 410 415
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
420 425 430
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
435 440 445
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
465 470 475 480
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 27
<211> 586
<212> PRT
<213> 人工序列
<220>
<223> CAR 1S
<400> 27
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys Pro Ser Ala
290 295 300
Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val
305 310 315 320
Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu
325 330 335
Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val
340 345 350
Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys
355 360 365
Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile Cys Glu Val
370 375 380
Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu
385 390 395 400
Ser Gly Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val
405 410 415
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
420 425 430
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
435 440 445
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
450 455 460
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg
465 470 475 480
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
485 490 495
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
500 505 510
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
515 520 525
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
530 535 540
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
545 550 555 560
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
565 570 575
Ala Leu His Met Gln Ala Leu Pro Pro Arg
580 585
<210> 28
<211> 583
<212> PRT
<213> 人工序列
<220>
<223> CAR 2S
<400> 28
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro Pro Thr Leu
290 295 300
Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
305 310 315 320
Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu
325 330 335
Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
340 345 350
Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
355 360 365
Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
370 375 380
Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Gly Gly
385 390 395 400
Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
405 410 415
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
420 425 430
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
435 440 445
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
450 455 460
Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp
465 470 475 480
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
485 490 495
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
500 505 510
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
515 520 525
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
530 535 540
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
545 550 555 560
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
565 570 575
Met Gln Ala Leu Pro Pro Arg
580
<210> 29
<211> 603
<212> PRT
<213> 人工序列
<220>
<223> CAR X1S
<400> 29
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys Pro Ser Ala
290 295 300
Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val
305 310 315 320
Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu
325 330 335
Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val
340 345 350
Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys
355 360 365
Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile Cys Glu Val
370 375 380
Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu
385 390 395 400
Ser Gly Gly Gly Gly Ser Lys Gly Lys His Leu Cys Pro Ser Pro Leu
405 410 415
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
420 425 430
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
435 440 445
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
450 455 460
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
465 470 475 480
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser
485 490 495
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
500 505 510
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
515 520 525
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
530 535 540
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
545 550 555 560
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
565 570 575
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
580 585 590
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
595 600
<210> 30
<211> 595
<212> PRT
<213> 人工序列
<220>
<223> CAR X2S
<400> 30
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro Pro Thr Leu
290 295 300
Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
305 310 315 320
Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu
325 330 335
Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
340 345 350
Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
355 360 365
Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
370 375 380
Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Lys Gly
385 390 395 400
Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe
405 410 415
Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu
420 425 430
Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg
435 440 445
Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro
450 455 460
Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala
465 470 475 480
Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
485 490 495
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
500 505 510
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
515 520 525
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
530 535 540
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
545 550 555 560
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
565 570 575
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
580 585 590
Pro Pro Arg
595
<210> 31
<211> 689
<212> PRT
<213> 人工序列
<220>
<223> CAR M
<400> 31
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys Pro Ser Ala
290 295 300
Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val
305 310 315 320
Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu
325 330 335
Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val
340 345 350
Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys
355 360 365
Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile Cys Glu Val
370 375 380
Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu
385 390 395 400
Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr Gln Gln Pro
405 410 415
Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val Arg Lys Phe
420 425 430
Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly Asn Val Ser
435 440 445
Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr
450 455 460
Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala His Arg Asp Asp
465 470 475 480
Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro Ala Val Ser
485 490 495
Lys Ser His Asp Leu Lys Val Ser Gly Gly Gly Gly Ser Phe Trp Val
500 505 510
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
515 520 525
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
530 535 540
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
545 550 555 560
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
565 570 575
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
580 585 590
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
595 600 605
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
610 615 620
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
625 630 635 640
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
645 650 655
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
660 665 670
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
675 680 685
Arg
<210> 32
<211> 701
<212> PRT
<213> 人工序列
<220>
<223> CAR XM
<400> 32
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys Pro Ser Ala
290 295 300
Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val
305 310 315 320
Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu
325 330 335
Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val
340 345 350
Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys
355 360 365
Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile Cys Glu Val
370 375 380
Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu
385 390 395 400
Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr Gln Gln Pro
405 410 415
Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val Arg Lys Phe
420 425 430
Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly Asn Val Ser
435 440 445
Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr
450 455 460
Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala His Arg Asp Asp
465 470 475 480
Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro Ala Val Ser
485 490 495
Lys Ser His Asp Leu Lys Val Ser Lys Gly Lys His Leu Cys Pro Ser
500 505 510
Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val
515 520 525
Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile
530 535 540
Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr
545 550 555 560
Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
565 570 575
Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys
580 585 590
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
595 600 605
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
610 615 620
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
625 630 635 640
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
645 650 655
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
660 665 670
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
675 680 685
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
690 695 700
<210> 33
<211> 793
<212> PRT
<213> 人工序列
<220>
<223> CAR L
<400> 33
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro Pro Thr Leu
290 295 300
Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
305 310 315 320
Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu
325 330 335
Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
340 345 350
Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
355 360 365
Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
370 375 380
Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Gly Gly
385 390 395 400
Gly Gly Ser Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala
405 410 415
Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly
420 425 430
Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu
435 440 445
Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser
450 455 460
Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val
465 470 475 480
His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp
485 490 495
Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro
500 505 510
Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn
515 520 525
Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
530 535 540
Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val
545 550 555 560
Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val
565 570 575
Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu
580 585 590
His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser
595 600 605
Gly Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu
610 615 620
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
625 630 635 640
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
645 650 655
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
660 665 670
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser
675 680 685
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu
690 695 700
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
705 710 715 720
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
725 730 735
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
740 745 750
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
755 760 765
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
770 775 780
Leu His Met Gln Ala Leu Pro Pro Arg
785 790
<210> 34
<211> 805
<212> PRT
<213> 人工序列
<220>
<223> CAR XL
<400> 34
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro Pro Thr Leu
290 295 300
Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
305 310 315 320
Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu
325 330 335
Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
340 345 350
Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
355 360 365
Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
370 375 380
Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Gly Gly
385 390 395 400
Gly Gly Ser Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala
405 410 415
Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly
420 425 430
Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu
435 440 445
Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser
450 455 460
Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val
465 470 475 480
His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp
485 490 495
Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro
500 505 510
Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn
515 520 525
Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
530 535 540
Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val
545 550 555 560
Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val
565 570 575
Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu
580 585 590
His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser
595 600 605
Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys
610 615 620
Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
625 630 635 640
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg
645 650 655
Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
660 665 670
Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe
675 680 685
Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
690 695 700
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
705 710 715 720
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
725 730 735
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
740 745 750
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
755 760 765
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
770 775 780
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
785 790 795 800
Ala Leu Pro Pro Arg
805
<210> 35
<211> 129
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子XS
<400> 35
agctacgtga ccgtgagcag ccaggacccc gccgagccca agagccccga caagacccac 60
acctgccccc cctgccccaa gggcaagcac ctgtgcccca gccccctgtt ccccggcccc 120
agcaagccc 129
<210> 36
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子1S
<400> 36
agctacgtga ccgtgagcag ccaggacccc gccgagccca agagccccga caagacccac 60
acctgccccc cctgccccgg aggaggagga tctgccaagc ccagcgcccc cgtggtgagc 120
ggccccgccg ccagggccac cccccagcac accgtgagct tcacctgcga gagccacggc 180
ttcagcccca gggacatcac cctgaagtgg ttcaagaacg gcaacgagct gagcgacttc 240
cagaccaacg tggaccccgt gggcgagagc gtgagctaca gcatccacag caccgccaag 300
gtggtgctga ccagggagga cgtgcacagc caggtgatct gcgaggtggc ccacgtgacc 360
ctgcagggcg accccctgag gggcaccgcc aacctgagc 399
<210> 37
<211> 390
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子2S
<400> 37
agctacgtga ccgtgagcag ccaggacccc gccgagccca agagccccga caagacccac 60
acctgccccc cctgccccgg aggaggagga tctgtgcccc ccaccctgga ggtgacccag 120
cagcccgtga gggccgagaa ccaggtgaac gtgacctgcc aggtgaggaa gttctacccc 180
cagaggctgc agctgacctg gctggagaac ggcaacgtga gcaggaccga gaccgccagc 240
accgtgaccg agaacaagga cggcacctac aactggatga gctggctgct ggtgaacgtg 300
agcgcccaca gggacgacgt gaagctgacc tgccaggtgg agcacgacgg ccagcccgcc 360
gtgagcaaga gccacgacct gaaggtgagc 390
<210> 38
<211> 465
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子X1S
<400> 38
agctacgtga ccgtgagcag ccaggacccc gccgagccca agagccccga caagacccac 60
acctgccccc cctgccccgg aggaggagga tctgccaagc ccagcgcccc cgtggtgagc 120
ggccccgccg ccagggccac cccccagcac accgtgagct tcacctgcga gagccacggc 180
ttcagcccca gggacatcac cctgaagtgg ttcaagaacg gcaacgagct gagcgacttc 240
cagaccaacg tggaccccgt gggcgagagc gtgagctaca gcatccacag caccgccaag 300
gtggtgctga ccagggagga cgtgcacagc caggtgatct gcgaggtggc ccacgtgacc 360
ctgcagggcg accccctgag gggcaccgcc aacctgagcg gaggaggagg atctaagggc 420
aagcacctgt gccccagccc cctgttcccc ggccccagca agccc 465
<210> 39
<211> 441
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子X2S
<400> 39
agctacgtga ccgtgagcag ccaggacccc gccgagccca agagccccga caagacccac 60
acctgccccc cctgccccgg aggaggagga tctgtgcccc ccaccctgga ggtgacccag 120
cagcccgtga gggccgagaa ccaggtgaac gtgacctgcc aggtgaggaa gttctacccc 180
cagaggctgc agctgacctg gctggagaac ggcaacgtga gcaggaccga gaccgccagc 240
accgtgaccg agaacaagga cggcacctac aactggatga gctggctgct ggtgaacgtg 300
agcgcccaca gggacgacgt gaagctgacc tgccaggtgg agcacgacgg ccagcccgcc 360
gtgagcaaga gccacgacct gaaggtgagc aagggcaagc acctgtgccc cagccccctg 420
ttccccggcc ccagcaagcc c 441
<210> 40
<211> 707
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子M
<400> 40
agctacgtga ccgtgagcag ccaggacccc gccgagccca agagccccga caagacccac 60
acctgccccc cctgccccgg aggaggagga tctgccaagc ccagcgcccc cgtggtgagc 120
ggccccgccg ccagggccac cccccagcac accgtgagct tcacctgcga gagccacggc 180
ttcagcccca gggacatcac cctgaagtgg ttcaagaacg gcaacgagct gagcgacttc 240
cagaccaacg tggaccccgt gggcgagagc gtgagctaca gcatccacag caccgccaag 300
gtggtgctga ccagggagga cgtgcacagc caggtgatct gcgaggtggc ccacgtgacc 360
ctgcagggcg accccctgag gggcaccgcc aacctgagcg agaccatcag ggtcccccca 420
ccctggaggt gacccagcag cccgtgaggg ccgagaacca ggtgaacgtg acctgccagg 480
tgaggaagtt ctacccccag aggctgcagc tgacctggct ggagaacggc aacgtgagca 540
ggaccgagac cgccagcacc gtgaccgaga acaaggacgg cacctacaac tggatgagct 600
ggctgctggt gaacgtgagc gcccacaggg acgacgtgaa gctgacctgc caggtggagc 660
acgacggcca gcccgccgtg agcaagagcc acgacctgaa ggtgagc 707
<210> 41
<211> 759
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子XM
<400> 41
agctacgtga ccgtgagcag ccaggacccc gccgagccca agagccccga caagacccac 60
acctgccccc cctgccccgg aggaggagga tctgccaagc ccagcgcccc cgtggtgagc 120
ggccccgccg ccagggccac cccccagcac accgtgagct tcacctgcga gagccacggc 180
ttcagcccca gggacatcac cctgaagtgg ttcaagaacg gcaacgagct gagcgacttc 240
cagaccaacg tggaccccgt gggcgagagc gtgagctaca gcatccacag caccgccaag 300
gtggtgctga ccagggagga cgtgcacagc caggtgatct gcgaggtggc ccacgtgacc 360
ctgcagggcg accccctgag gggcaccgcc aacctgagcg agaccatcag ggtgcccccc 420
accctggagg tgacccagca gcccgtgagg gccgagaacc aggtgaacgt gacctgccag 480
gtgaggaagt tctaccccca gaggctgcag ctgacctggc tggagaacgg caacgtgagc 540
aggaccgaga ccgccagcac cgtgaccgag aacaaggacg gcacctacaa ctggatgagc 600
tggctgctgg tgaacgtgag cgcccacagg gacgacgtga agctgacctg ccaggtggag 660
cacgacggcc agcccgccgt gagcaagagc cacgacctga aggtgagcaa gggcaagcac 720
ctgtgcccca gccccctgtt ccccggcccc agcaagccc 759
<210> 42
<211> 1020
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子L
<400> 42
agctacgtga ccgtgagcag ccaggacccc gccgagccca agagccccga caagacccac 60
acctgccccc cctgccccgg tggcggtgga agtgtgcccc ccaccctgga ggtgacccag 120
cagcccgtga gagccgagaa ccaggtgaac gtgacctgcc aggtgagaaa gttctacccc 180
cagagactgc agctgacctg gctggagaac ggcaacgtga gcagaaccga gaccgccagc 240
accgtgaccg agaacaagga cggcacctac aactggatga gctggctgct ggtgaacgtg 300
agcgcccaca gagacgacgt gaagctgacc tgccaggtgg agcacgacgg ccagcccgcc 360
gtgagcaaga gccacgacct gaaggtgagc ggcggaggcg ggagtgccaa gcccagcgcc 420
cccgtggtga gcggccccgc cgccagggcc accccccagc acaccgtgag cttcacctgc 480
gagagccacg gcttcagccc cagggacatc accctgaagt ggttcaagaa cggcaacgag 540
ctgagcgact tccagaccaa cgtggacccc gtgggcgaga gcgtgagcta cagcatccac 600
agcaccgcca aggtggtgct gaccagggag gacgtgcaca gccaggtgat ctgcgaggtg 660
gcccacgtga ccctgcaggg cgaccccctg aggggcaccg ccaacctgag cgagaccatc 720
agggtgcccc ccaccctgga ggtgacccag cagcccgtga gggccgagaa ccaggtgaac 780
gtgacctgcc aggtgaggaa gttctacccc cagaggctgc agctgacctg gctggagaac 840
ggcaacgtga gcaggaccga gaccgccagc accgtgaccg agaacaagga cggcacctac 900
aactggatga gctggctgct ggtgaacgtg agcgcccaca gggacgacgt gaagctgacc 960
tgccaggtgg agcacgacgg ccagcccgcc gtgagcaaga gccacgacct gaaggtgagc 1020
<210> 43
<211> 1071
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子XL
<400> 43
agctacgtga ccgtgagcag ccaggacccc gccgagccca agagccccga caagacccac 60
acctgccccc cctgccccgg tggcggtgga agtgtgcccc ccaccctgga ggtgacccag 120
cagcccgtga gagccgagaa ccaggtgaac gtgacctgcc aggtgagaaa gttctacccc 180
cagagactgc agctgacctg gctggagaac ggcaacgtga gcagaaccga gaccgccagc 240
accgtgaccg agaacaagga cggcacctac aactggatga gctggctgct ggtgaacgtg 300
agcgcccaca gagacgacgt gaagctgacc tgccaggtgg agcacgacgg ccagcccgcc 360
gtgagcaaga gccacgacct gaaggtgagc ggcggaggcg ggagtgccaa gcccagcgcc 420
cccgtggtga gcggccccgc cgccagggcc accccccagc acaccgtgag cttcacctgc 480
gagagccacg gcttcagccc cagggacatc accctgaagt ggttcaagaa cggcaacgag 540
ctgagcgact tccagaccaa cgtggacccc gtgggcgaga gcgtgagcta cagcatccac 600
agcaccgcca aggtggtgct gaccagggag gacgtgcaca gccaggtgat ctgcgaggtg 660
gcccacgtga ccctgcaggg cgaccccctg aggggcaccg ccaacctgag cgagaccatc 720
agggtgcccc ccaccctgga ggtgacccag cagcccgtga gggccgagaa ccaggtgaac 780
gtgacctgcc aggtgaggaa gttctacccc cagaggctgc agctgacctg gctggagaac 840
ggcaacgtga gcaggaccga gaccgccagc accgtgaccg agaacaagga cggcacctac 900
aactggatga gctggctgct ggtgaacgtg agcgcccaca gggacgacgt gaagctgacc 960
tgccaggtgg agcacgacgg ccagcccgcc gtgagcaaga gccacgacct gaaggtgagc 1020
aagggcaagc acctgtgccc cagccccctg ttccccggcc ccagcaagcc c 1071
<210> 44
<211> 1476
<212> DNA
<213> 人工序列
<220>
<223> CAR XS
<400> 44
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgacagggtg 120
accatcagct gcagggccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180
cccgacggca ccgtgaagct gctgatctac cacaccagca ggctgcacag cggcgtgccc 240
agcaggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300
caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcggc 360
ggcggcacca agctggagct gaagaggggt ggtggtggtt ctggtggtgg tggttctggc 420
ggcggcggct ccggtggtgg tgggtccgag gtgcagctgc agcagagcgg ccccggcctg 480
gtggccccca gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540
tacggcgtga gctggatcag gcagcccccc aggaagggcc tggagtggct gggcgtgatc 600
tggggcagcg agaccaccta ctacaacagc gccctgaaga gcaggctgac catcatcaag 660
gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720
atctactact gcgccaagca ctactactac ggcggcagct acgccatgga ctactggggc 780
cagggcacca ccgtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccaagggcaa gcacctgtgc 900
cccagccccc tgttccccgg ccccagcaag cccttctggg tgctggtggt ggtgggcggc 960
gtgctggcct gctacagcct gctggtgacc gtggccttca tcatcttctg ggtgaggagc 1020
aagaggagca ggctgctgca cagcgactac atgaacatga cccccaggag gcccggcccc 1080
accaggaagc actaccagcc ctacgccccc cccagggact tcgccgccta caggagcagg 1140
gtgaagttca gcaggagcgc cgacgccccc gcctaccagc agggccagaa ccagctgtac 1200
aacgagctga acctgggcag gagggaggag tacgacgtgc tggacaagag gaggggcagg 1260
gaccccgaga tgggcggcaa gcccaggagg aagaaccccc aggagggcct gtacaacgag 1320
ctgcagaagg acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagaggagg 1380
aggggcaagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggacacctac 1440
gacgccctgc acatgcaggc cctgcccccc aggtaa 1476
<210> 45
<211> 1761
<212> DNA
<213> 人工序列
<220>
<223> CAR 1S
<400> 45
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgacagggtg 120
accatcagct gcagggccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180
cccgacggca ccgtgaagct gctgatctac cacaccagca ggctgcacag cggcgtgccc 240
agcaggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300
caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcggc 360
ggcggcacca agctggagct gaagaggggt ggtggtggtt ctggtggtgg tggttctggc 420
ggcggcggct ccggtggtgg tgggtccgag gtgcagctgc agcagagcgg ccccggcctg 480
gtggccccca gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540
tacggcgtga gctggatcag gcagcccccc aggaagggcc tggagtggct gggcgtgatc 600
tggggcagcg agaccaccta ctacaacagc gccctgaaga gcaggctgac catcatcaag 660
gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720
atctactact gcgccaagca ctactactac ggcggcagct acgccatgga ctactggggc 780
cagggcacca ccgtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccggaggagg aggatctgcc 900
aagcccagcg cccccgtggt gagcggcccc gccgccaggg ccacccccca gcacaccgtg 960
agcttcacct gcgagagcca cggcttcagc cccagggaca tcaccctgaa gtggttcaag 1020
aacggcaacg agctgagcga cttccagacc aacgtggacc ccgtgggcga gagcgtgagc 1080
tacagcatcc acagcaccgc caaggtggtg ctgaccaggg aggacgtgca cagccaggtg 1140
atctgcgagg tggcccacgt gaccctgcag ggcgaccccc tgaggggcac cgccaacctg 1200
agcggtggtg gtggttcctt ctgggtgctg gtggtggtgg gcggcgtgct ggcctgctac 1260
agcctgctgg tgaccgtggc cttcatcatc ttctgggtga ggagcaagag gagcaggctg 1320
ctgcacagcg actacatgaa catgaccccc aggaggcccg gccccaccag gaagcactac 1380
cagccctacg ccccccccag ggacttcgcc gcctacagga gcagggtgaa gttcagcagg 1440
agcgccgacg cccccgccta ccagcagggc cagaaccagc tgtacaacga gctgaacctg 1500
ggcaggaggg aggagtacga cgtgctggac aagaggaggg gcagggaccc cgagatgggc 1560
ggcaagccca ggaggaagaa cccccaggag ggcctgtaca acgagctgca gaaggacaag 1620
atggccgagg cctacagcga gatcggcatg aagggcgaga ggaggagggg caagggccac 1680
gacggcctgt accagggcct gagcaccgcc accaaggaca cctacgacgc cctgcacatg 1740
caggccctgc cccccaggta a 1761
<210> 46
<211> 1752
<212> DNA
<213> 人工序列
<220>
<223> CAR 2S
<400> 46
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgacagggtg 120
accatcagct gcagggccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180
cccgacggca ccgtgaagct gctgatctac cacaccagca ggctgcacag cggcgtgccc 240
agcaggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300
caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcggc 360
ggcggcacca agctggagct gaagaggggt ggtggtggtt ctggtggtgg tggttctggc 420
ggcggcggct ccggtggtgg tgggtccgag gtgcagctgc agcagagcgg ccccggcctg 480
gtggccccca gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540
tacggcgtga gctggatcag gcagcccccc aggaagggcc tggagtggct gggcgtgatc 600
tggggcagcg agaccaccta ctacaacagc gccctgaaga gcaggctgac catcatcaag 660
gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720
atctactact gcgccaagca ctactactac ggcggcagct acgccatgga ctactggggc 780
cagggcacca ccgtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccggaggagg aggatctgtg 900
ccccccaccc tggaggtgac ccagcagccc gtgagggccg agaaccaggt gaacgtgacc 960
tgccaggtga ggaagttcta cccccagagg ctgcagctga cctggctgga gaacggcaac 1020
gtgagcagga ccgagaccgc cagcaccgtg accgagaaca aggacggcac ctacaactgg 1080
atgagctggc tgctggtgaa cgtgagcgcc cacagggacg acgtgaagct gacctgccag 1140
gtggagcacg acggccagcc cgccgtgagc aagagccacg acctgaaggt gagcggtggt 1200
ggtggttcct tctgggtgct ggtggtggtg ggcggcgtgc tggcctgcta cagcctgctg 1260
gtgaccgtgg ccttcatcat cttctgggtg aggagcaaga ggagcaggct gctgcacagc 1320
gactacatga acatgacccc caggaggccc ggccccacca ggaagcacta ccagccctac 1380
gcccccccca gggacttcgc cgcctacagg agcagggtga agttcagcag gagcgccgac 1440
gcccccgcct accagcaggg ccagaaccag ctgtacaacg agctgaacct gggcaggagg 1500
gaggagtacg acgtgctgga caagaggagg ggcagggacc ccgagatggg cggcaagccc 1560
aggaggaaga acccccagga gggcctgtac aacgagctgc agaaggacaa gatggccgag 1620
gcctacagcg agatcggcat gaagggcgag aggaggaggg gcaagggcca cgacggcctg 1680
taccagggcc tgagcaccgc caccaaggac acctacgacg ccctgcacat gcaggccctg 1740
ccccccaggt aa 1752
<210> 47
<211> 1812
<212> DNA
<213> 人工序列
<220>
<223> CAR X1S
<400> 47
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgacagggtg 120
accatcagct gcagggccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180
cccgacggca ccgtgaagct gctgatctac cacaccagca ggctgcacag cggcgtgccc 240
agcaggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300
caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcggc 360
ggcggcacca agctggagct gaagaggggt ggtggtggtt ctggtggtgg tggttctggc 420
ggcggcggct ccggtggtgg tgggtccgag gtgcagctgc agcagagcgg ccccggcctg 480
gtggccccca gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540
tacggcgtga gctggatcag gcagcccccc aggaagggcc tggagtggct gggcgtgatc 600
tggggcagcg agaccaccta ctacaacagc gccctgaaga gcaggctgac catcatcaag 660
gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720
atctactact gcgccaagca ctactactac ggcggcagct acgccatgga ctactggggc 780
cagggcacca ccgtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccggaggagg aggatctgcc 900
aagcccagcg cccccgtggt gagcggcccc gccgccaggg ccacccccca gcacaccgtg 960
agcttcacct gcgagagcca cggcttcagc cccagggaca tcaccctgaa gtggttcaag 1020
aacggcaacg agctgagcga cttccagacc aacgtggacc ccgtgggcga gagcgtgagc 1080
tacagcatcc acagcaccgc caaggtggtg ctgaccaggg aggacgtgca cagccaggtg 1140
atctgcgagg tggcccacgt gaccctgcag ggcgaccccc tgaggggcac cgccaacctg 1200
agcggaggag gaggatctaa gggcaagcac ctgtgcccca gccccctgtt ccccggcccc 1260
agcaagccct tctgggtgct ggtggtggtg ggcggcgtgc tggcctgcta cagcctgctg 1320
gtgaccgtgg ccttcatcat cttctgggtg aggagcaaga ggagcaggct gctgcacagc 1380
gactacatga acatgacccc caggaggccc ggccccacca ggaagcacta ccagccctac 1440
gcccccccca gggacttcgc cgcctacagg agcagggtga agttcagcag gagcgccgac 1500
gcccccgcct accagcaggg ccagaaccag ctgtacaacg agctgaacct gggcaggagg 1560
gaggagtacg acgtgctgga caagaggagg ggcagggacc ccgagatggg cggcaagccc 1620
aggaggaaga acccccagga gggcctgtac aacgagctgc agaaggacaa gatggccgag 1680
gcctacagcg agatcggcat gaagggcgag aggaggaggg gcaagggcca cgacggcctg 1740
taccagggcc tgagcaccgc caccaaggac acctacgacg ccctgcacat gcaggccctg 1800
ccccccaggt aa 1812
<210> 48
<211> 1788
<212> DNA
<213> 人工序列
<220>
<223> CAR X2S
<400> 48
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgacagggtg 120
accatcagct gcagggccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180
cccgacggca ccgtgaagct gctgatctac cacaccagca ggctgcacag cggcgtgccc 240
agcaggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300
caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcggc 360
ggcggcacca agctggagct gaagaggggt ggtggtggtt ctggtggtgg tggttctggc 420
ggcggcggct ccggtggtgg tgggtccgag gtgcagctgc agcagagcgg ccccggcctg 480
gtggccccca gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540
tacggcgtga gctggatcag gcagcccccc aggaagggcc tggagtggct gggcgtgatc 600
tggggcagcg agaccaccta ctacaacagc gccctgaaga gcaggctgac catcatcaag 660
gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720
atctactact gcgccaagca ctactactac ggcggcagct acgccatgga ctactggggc 780
cagggcacca ccgtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccggaggagg aggatctgtg 900
ccccccaccc tggaggtgac ccagcagccc gtgagggccg agaaccaggt gaacgtgacc 960
tgccaggtga ggaagttcta cccccagagg ctgcagctga cctggctgga gaacggcaac 1020
gtgagcagga ccgagaccgc cagcaccgtg accgagaaca aggacggcac ctacaactgg 1080
atgagctggc tgctggtgaa cgtgagcgcc cacagggacg acgtgaagct gacctgccag 1140
gtggagcacg acggccagcc cgccgtgagc aagagccacg acctgaaggt gagcaagggc 1200
aagcacctgt gccccagccc cctgttcccc ggccccagca agcccttctg ggtgctggtg 1260
gtggtgggcg gcgtgctggc ctgctacagc ctgctggtga ccgtggcctt catcatcttc 1320
tgggtgagga gcaagaggag caggctgctg cacagcgact acatgaacat gacccccagg 1380
aggcccggcc ccaccaggaa gcactaccag ccctacgccc cccccaggga cttcgccgcc 1440
tacaggagca gggtgaagtt cagcaggagc gccgacgccc ccgcctacca gcagggccag 1500
aaccagctgt acaacgagct gaacctgggc aggagggagg agtacgacgt gctggacaag 1560
aggaggggca gggaccccga gatgggcggc aagcccagga ggaagaaccc ccaggagggc 1620
ctgtacaacg agctgcagaa ggacaagatg gccgaggcct acagcgagat cggcatgaag 1680
ggcgagagga ggaggggcaa gggccacgac ggcctgtacc agggcctgag caccgccacc 1740
aaggacacct acgacgccct gcacatgcag gccctgcccc ccaggtaa 1788
<210> 49
<211> 2070
<212> DNA
<213> 人工序列
<220>
<223> CAR M
<400> 49
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgacagggtg 120
accatcagct gcagggccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180
cccgacggca ccgtgaagct gctgatctac cacaccagca ggctgcacag cggcgtgccc 240
agcaggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300
caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcggc 360
ggcggcacca agctggagct gaagaggggt ggtggtggtt ctggtggtgg tggttctggc 420
ggcggcggct ccggtggtgg tgggtccgag gtgcagctgc agcagagcgg ccccggcctg 480
gtggccccca gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540
tacggcgtga gctggatcag gcagcccccc aggaagggcc tggagtggct gggcgtgatc 600
tggggcagcg agaccaccta ctacaacagc gccctgaaga gcaggctgac catcatcaag 660
gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720
atctactact gcgccaagca ctactactac ggcggcagct acgccatgga ctactggggc 780
cagggcacca ccgtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccggaggagg aggatctgcc 900
aagcccagcg cccccgtggt gagcggcccc gccgccaggg ccacccccca gcacaccgtg 960
agcttcacct gcgagagcca cggcttcagc cccagggaca tcaccctgaa gtggttcaag 1020
aacggcaacg agctgagcga cttccagacc aacgtggacc ccgtgggcga gagcgtgagc 1080
tacagcatcc acagcaccgc caaggtggtg ctgaccaggg aggacgtgca cagccaggtg 1140
atctgcgagg tggcccacgt gaccctgcag ggcgaccccc tgaggggcac cgccaacctg 1200
agcgagacca tcagggtgcc ccccaccctg gaggtgaccc agcagcccgt gagggccgag 1260
aaccaggtga acgtgacctg ccaggtgagg aagttctacc cccagaggct gcagctgacc 1320
tggctggaga acggcaacgt gagcaggacc gagaccgcca gcaccgtgac cgagaacaag 1380
gacggcacct acaactggat gagctggctg ctggtgaacg tgagcgccca cagggacgac 1440
gtgaagctga cctgccaggt ggagcacgac ggccagcccg ccgtgagcaa gagccacgac 1500
ctgaaggtga gcggtggtgg tggttccttc tgggtgctgg tggtggtggg cggcgtgctg 1560
gcctgctaca gcctgctggt gaccgtggcc ttcatcatct tctgggtgag gagcaagagg 1620
agcaggctgc tgcacagcga ctacatgaac atgaccccca ggaggcccgg ccccaccagg 1680
aagcactacc agccctacgc cccccccagg gacttcgccg cctacaggag cagggtgaag 1740
ttcagcagga gcgccgacgc ccccgcctac cagcagggcc agaaccagct gtacaacgag 1800
ctgaacctgg gcaggaggga ggagtacgac gtgctggaca agaggagggg cagggacccc 1860
gagatgggcg gcaagcccag gaggaagaac ccccaggagg gcctgtacaa cgagctgcag 1920
aaggacaaga tggccgaggc ctacagcgag atcggcatga agggcgagag gaggaggggc 1980
aagggccacg acggcctgta ccagggcctg agcaccgcca ccaaggacac ctacgacgcc 2040
ctgcacatgc aggccctgcc ccccaggtaa 2070
<210> 50
<211> 2106
<212> DNA
<213> 人工序列
<220>
<223> CAR XM
<400> 50
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgacagggtg 120
accatcagct gcagggccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180
cccgacggca ccgtgaagct gctgatctac cacaccagca ggctgcacag cggcgtgccc 240
agcaggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300
caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcggc 360
ggcggcacca agctggagct gaagaggggt ggtggtggtt ctggtggtgg tggttctggc 420
ggcggcggct ccggtggtgg tgggtccgag gtgcagctgc agcagagcgg ccccggcctg 480
gtggccccca gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540
tacggcgtga gctggatcag gcagcccccc aggaagggcc tggagtggct gggcgtgatc 600
tggggcagcg agaccaccta ctacaacagc gccctgaaga gcaggctgac catcatcaag 660
gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720
atctactact gcgccaagca ctactactac ggcggcagct acgccatgga ctactggggc 780
cagggcacca ccgtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccggaggagg aggatctgcc 900
aagcccagcg cccccgtggt gagcggcccc gccgccaggg ccacccccca gcacaccgtg 960
agcttcacct gcgagagcca cggcttcagc cccagggaca tcaccctgaa gtggttcaag 1020
aacggcaacg agctgagcga cttccagacc aacgtggacc ccgtgggcga gagcgtgagc 1080
tacagcatcc acagcaccgc caaggtggtg ctgaccaggg aggacgtgca cagccaggtg 1140
atctgcgagg tggcccacgt gaccctgcag ggcgaccccc tgaggggcac cgccaacctg 1200
agcgagacca tcagggtgcc ccccaccctg gaggtgaccc agcagcccgt gagggccgag 1260
aaccaggtga acgtgacctg ccaggtgagg aagttctacc cccagaggct gcagctgacc 1320
tggctggaga acggcaacgt gagcaggacc gagaccgcca gcaccgtgac cgagaacaag 1380
gacggcacct acaactggat gagctggctg ctggtgaacg tgagcgccca cagggacgac 1440
gtgaagctga cctgccaggt ggagcacgac ggccagcccg ccgtgagcaa gagccacgac 1500
ctgaaggtga gcaagggcaa gcacctgtgc cccagccccc tgttccccgg ccccagcaag 1560
cccttctggg tgctggtggt ggtgggcggc gtgctggcct gctacagcct gctggtgacc 1620
gtggccttca tcatcttctg ggtgaggagc aagaggagca ggctgctgca cagcgactac 1680
atgaacatga cccccaggag gcccggcccc accaggaagc actaccagcc ctacgccccc 1740
cccagggact tcgccgccta caggagcagg gtgaagttca gcaggagcgc cgacgccccc 1800
gcctaccagc agggccagaa ccagctgtac aacgagctga acctgggcag gagggaggag 1860
tacgacgtgc tggacaagag gaggggcagg gaccccgaga tgggcggcaa gcccaggagg 1920
aagaaccccc aggagggcct gtacaacgag ctgcagaagg acaagatggc cgaggcctac 1980
agcgagatcg gcatgaaggg cgagaggagg aggggcaagg gccacgacgg cctgtaccag 2040
ggcctgagca ccgccaccaa ggacacctac gacgccctgc acatgcaggc cctgcccccc 2100
aggtaa 2106
<210> 51
<211> 2382
<212> DNA
<213> 人工序列
<220>
<223> CAR L
<400> 51
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgacagggtg 120
accatcagct gcagggccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180
cccgacggca ccgtgaagct gctgatctac cacaccagca ggctgcacag cggcgtgccc 240
agcaggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300
caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcggc 360
ggcggcacca agctggagct gaagaggggc ggtggaggtt ccggcggtgg cggttccgga 420
ggcggtgggt caggaggtgg aggctccgag gtgcagctgc agcagagcgg ccccggcctg 480
gtggccccca gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540
tacggcgtga gctggatcag gcagcccccc aggaagggcc tggagtggct gggcgtgatc 600
tggggcagcg agaccaccta ctacaacagc gccctgaaga gcaggctgac catcatcaag 660
gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720
atctactact gcgccaagca ctactactac ggcggcagct acgccatgga ctactggggc 780
cagggcacca ccgtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccggtggcgg tggaagtgtg 900
ccccccaccc tggaggtgac ccagcagccc gtgagagccg agaaccaggt gaacgtgacc 960
tgccaggtga gaaagttcta cccccagaga ctgcagctga cctggctgga gaacggcaac 1020
gtgagcagaa ccgagaccgc cagcaccgtg accgagaaca aggacggcac ctacaactgg 1080
atgagctggc tgctggtgaa cgtgagcgcc cacagagacg acgtgaagct gacctgccag 1140
gtggagcacg acggccagcc cgccgtgagc aagagccacg acctgaaggt gagcggcgga 1200
ggcgggagtg ccaagcccag cgcccccgtg gtgagcggcc ccgccgccag ggccaccccc 1260
cagcacaccg tgagcttcac ctgcgagagc cacggcttca gccccaggga catcaccctg 1320
aagtggttca agaacggcaa cgagctgagc gacttccaga ccaacgtgga ccccgtgggc 1380
gagagcgtga gctacagcat ccacagcacc gccaaggtgg tgctgaccag ggaggacgtg 1440
cacagccagg tgatctgcga ggtggcccac gtgaccctgc agggcgaccc cctgaggggc 1500
accgccaacc tgagcgagac catcagggtg ccccccaccc tggaggtgac ccagcagccc 1560
gtgagggccg agaaccaggt gaacgtgacc tgccaggtga ggaagttcta cccccagagg 1620
ctgcagctga cctggctgga gaacggcaac gtgagcagga ccgagaccgc cagcaccgtg 1680
accgagaaca aggacggcac ctacaactgg atgagctggc tgctggtgaa cgtgagcgcc 1740
cacagggacg acgtgaagct gacctgccag gtggagcacg acggccagcc cgccgtgagc 1800
aagagccacg acctgaaggt gagcggcggt ggcggcagct tctgggtgct ggtggtggtg 1860
ggcggcgtgc tggcctgcta cagcctgctg gtgaccgtgg ccttcatcat cttctgggtg 1920
aggagcaaga ggagcaggct gctgcacagc gactacatga acatgacccc caggaggccc 1980
ggccccacca ggaagcacta ccagccctac gcccccccca gggacttcgc cgcctacagg 2040
agcagggtga agttcagcag gagcgccgac gcccccgcct accagcaggg ccagaaccag 2100
ctgtacaacg agctgaacct gggcaggagg gaggagtacg acgtgctgga caagaggagg 2160
ggcagggacc ccgagatggg cggcaagccc aggaggaaga acccccagga gggcctgtac 2220
aacgagctgc agaaggacaa gatggccgag gcctacagcg agatcggcat gaagggcgag 2280
aggaggaggg gcaagggcca cgacggcctg taccagggcc tgagcaccgc caccaaggac 2340
acctacgacg ccctgcacat gcaggccctg ccccccaggt aa 2382
<210> 52
<211> 2418
<212> DNA
<213> 人工序列
<220>
<223> CAR XL
<400> 52
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgacagggtg 120
accatcagct gcagggccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180
cccgacggca ccgtgaagct gctgatctac cacaccagca ggctgcacag cggcgtgccc 240
agcaggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300
caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcggc 360
ggcggcacca agctggagct gaagaggggc ggtggaggtt ccggcggtgg cggttccgga 420
ggcggtgggt caggaggtgg aggctccgag gtgcagctgc agcagagcgg ccccggcctg 480
gtggccccca gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540
tacggcgtga gctggatcag gcagcccccc aggaagggcc tggagtggct gggcgtgatc 600
tggggcagcg agaccaccta ctacaacagc gccctgaaga gcaggctgac catcatcaag 660
gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720
atctactact gcgccaagca ctactactac ggcggcagct acgccatgga ctactggggc 780
cagggcacca ccgtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccggtggcgg tggaagtgtg 900
ccccccaccc tggaggtgac ccagcagccc gtgagagccg agaaccaggt gaacgtgacc 960
tgccaggtga gaaagttcta cccccagaga ctgcagctga cctggctgga gaacggcaac 1020
gtgagcagaa ccgagaccgc cagcaccgtg accgagaaca aggacggcac ctacaactgg 1080
atgagctggc tgctggtgaa cgtgagcgcc cacagagacg acgtgaagct gacctgccag 1140
gtggagcacg acggccagcc cgccgtgagc aagagccacg acctgaaggt gagcggcgga 1200
ggcgggagtg ccaagcccag cgcccccgtg gtgagcggcc ccgccgccag ggccaccccc 1260
cagcacaccg tgagcttcac ctgcgagagc cacggcttca gccccaggga catcaccctg 1320
aagtggttca agaacggcaa cgagctgagc gacttccaga ccaacgtgga ccccgtgggc 1380
gagagcgtga gctacagcat ccacagcacc gccaaggtgg tgctgaccag ggaggacgtg 1440
cacagccagg tgatctgcga ggtggcccac gtgaccctgc agggcgaccc cctgaggggc 1500
accgccaacc tgagcgagac catcagggtg ccccccaccc tggaggtgac ccagcagccc 1560
gtgagggccg agaaccaggt gaacgtgacc tgccaggtga ggaagttcta cccccagagg 1620
ctgcagctga cctggctgga gaacggcaac gtgagcagga ccgagaccgc cagcaccgtg 1680
accgagaaca aggacggcac ctacaactgg atgagctggc tgctggtgaa cgtgagcgcc 1740
cacagggacg acgtgaagct gacctgccag gtggagcacg acggccagcc cgccgtgagc 1800
aagagccacg acctgaaggt gagcaagggc aagcacctgt gccccagccc cctgttcccc 1860
ggccccagca agcccttctg ggtgctggtg gtggtgggcg gcgtgctggc ctgctacagc 1920
ctgctggtga ccgtggcctt catcatcttc tgggtgagga gcaagaggag caggctgctg 1980
cacagcgact acatgaacat gacccccagg aggcccggcc ccaccaggaa gcactaccag 2040
ccctacgccc cccccaggga cttcgccgcc tacaggagca gggtgaagtt cagcaggagc 2100
gccgacgccc ccgcctacca gcagggccag aaccagctgt acaacgagct gaacctgggc 2160
aggagggagg agtacgacgt gctggacaag aggaggggca gggaccccga gatgggcggc 2220
aagcccagga ggaagaaccc ccaggagggc ctgtacaacg agctgcagaa ggacaagatg 2280
gccgaggcct acagcgagat cggcatgaag ggcgagagga ggaggggcaa gggccacgac 2340
ggcctgtacc agggcctgag caccgccacc aaggacacct acgacgccct gcacatgcag 2400
gccctgcccc ccaggtaa 2418
<210> 53
<211> 247
<212> PRT
<213> 智人
<220>
<223> HER-2 scFv
<400> 53
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
145 150 155 160
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
165 170 175
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
180 185 190
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
225 230 235 240
Gly Thr Leu Val Thr Val Ser
245
<210> 54
<211> 689
<212> PRT
<213> 人工序列
<220>
<223> Her2 CAR M
<400> 54
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
35 40 45
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
50 55 60
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
100 105 110
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
145 150 155 160
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
165 170 175
Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
180 185 190
Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg
195 200 205
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
210 215 220
Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
225 230 235 240
Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Tyr Val Thr
260 265 270
Val Ser Ser Gln Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His
275 280 285
Thr Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys Pro Ser Ala
290 295 300
Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val
305 310 315 320
Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu
325 330 335
Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val
340 345 350
Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys
355 360 365
Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile Cys Glu Val
370 375 380
Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu
385 390 395 400
Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr Gln Gln Pro
405 410 415
Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val Arg Lys Phe
420 425 430
Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly Asn Val Ser
435 440 445
Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr
450 455 460
Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala His Arg Asp Asp
465 470 475 480
Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro Ala Val Ser
485 490 495
Lys Ser His Asp Leu Lys Val Ser Gly Gly Gly Gly Ser Phe Trp Val
500 505 510
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
515 520 525
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
530 535 540
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
545 550 555 560
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
565 570 575
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
580 585 590
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
595 600 605
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
610 615 620
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
625 630 635 640
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
645 650 655
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
660 665 670
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
675 680 685
Arg
<210> 55
<211> 2070
<212> DNA
<213> 人工序列
<220>
<223> HER-2 CAR M
<400> 55
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgcagc 60
agggacatcc agatgaccca gagccccagc agcctgagcg ccagcgtggg cgacagggtg 120
accatcacct gcagggccag ccaggacgtg aacaccgccg tggcctggta ccagcagaag 180
cccggcaagg cccccaagct gctgatctac agcgccagct tcctgtacag cggcgtgccc 240
agcaggttca gcggcagcag gagcggcacc gacttcaccc tgaccatcag cagcctgcag 300
cccgaggact tcgccaccta ctactgccag cagcactaca ccaccccccc caccttcggc 360
cagggcacca aggtggagat caagaggggc ggtggaggtt ccggcggtgg cggttccgga 420
ggcggtgggt caggaggtgg aggctccgag gtgcagctgg tggagagcgg cggcggcctg 480
gtgcagcccg gcggcagcct gaggctgagc tgcgccgcca gcggcttcaa catcaaggac 540
acctacatcc actgggtgag gcaggccccc ggcaagggcc tggagtgggt ggccaggatc 600
taccccacca acggctacac caggtacgcc gacagcgtga agggcaggtt caccatcagc 660
gccgacacca gcaagaacac cgcctacctg cagatgaaca gcctgagggc cgaggacacc 720
gccgtgtact actgcagcag gtggggcggc gacggcttct acgccatgga ctactggggc 780
cagggcaccc tggtgaccgt gagcagctac gtgaccgtga gcagccagga ccccgccgag 840
cccaagagcc ccgacaagac ccacacctgc cccccctgcc ccggtggcgg tggaagtgcc 900
aagcccagcg cccccgtggt gagcggcccc gccgccaggg ccacccccca gcacaccgtg 960
agcttcacct gcgagagcca cggcttcagc cccagggaca tcaccctgaa gtggttcaag 1020
aacggcaacg agctgagcga cttccagacc aacgtggacc ccgtgggcga gagcgtgagc 1080
tacagcatcc acagcaccgc caaggtggtg ctgaccaggg aggacgtgca cagccaggtg 1140
atctgcgagg tggcccacgt gaccctgcag ggcgaccccc tgaggggcac cgccaacctg 1200
agcgagacca tcagggtgcc ccccaccctg gaggtgaccc agcagcccgt gagggccgag 1260
aaccaggtga acgtgacctg ccaggtgagg aagttctacc cccagaggct gcagctgacc 1320
tggctggaga acggcaacgt gagcaggacc gagaccgcca gcaccgtgac cgagaacaag 1380
gacggcacct acaactggat gagctggctg ctggtgaacg tgagcgccca cagggacgac 1440
gtgaagctga cctgccaggt ggagcacgac ggccagcccg ccgtgagcaa gagccacgac 1500
ctgaaggtga gcggcggtgg cggcagcttc tgggtgctgg tggtggtggg cggcgtgctg 1560
gcctgctaca gcctgctggt gaccgtggcc ttcatcatct tctgggtgag gagcaagagg 1620
agcaggctgc tgcacagcga ctacatgaac atgaccccca ggaggcccgg ccccaccagg 1680
aagcactacc agccctacgc cccccccagg gacttcgccg cctacaggag cagggtgaag 1740
ttcagcagga gcgccgacgc ccccgcctac cagcagggcc agaaccagct gtacaacgag 1800
ctgaacctgg gcaggaggga ggagtacgac gtgctggaca agaggagggg cagggacccc 1860
gagatgggcg gcaagcccag gaggaagaac ccccaggagg gcctgtacaa cgagctgcag 1920
aaggacaaga tggccgaggc ctacagcgag atcggcatga agggcgagag gaggaggggc 1980
aagggccacg acggcctgta ccagggcctg agcaccgcca ccaaggacac ctacgacgcc 2040
ctgcacatgc aggccctgcc ccccaggtaa 2070
<210> 56
<211> 220
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子M1
<400> 56
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly
1 5 10 15
Ser Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala
20 25 30
Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser
35 40 45
Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
50 55 60
Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser
65 70 75 80
Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser
85 90 95
Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
100 105 110
Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu
115 120 125
Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
130 135 140
Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu
145 150 155 160
Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
165 170 175
Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
180 185 190
Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
195 200 205
Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys
210 215 220
<210> 57
<211> 239
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子XM2
<400> 57
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly
1 5 10 15
Ser Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala
20 25 30
Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser
35 40 45
Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
50 55 60
Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser
65 70 75 80
Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser
85 90 95
Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
100 105 110
Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu
115 120 125
Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
130 135 140
Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu
145 150 155 160
Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
165 170 175
Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val
180 185 190
Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
195 200 205
Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Lys Gly
210 215 220
Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro
225 230 235
<210> 58
<211> 256
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子XM3
<400> 58
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly
1 5 10 15
Ser Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala
20 25 30
Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser
35 40 45
Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
50 55 60
Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser
65 70 75 80
Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser
85 90 95
Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
100 105 110
Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Glu Ser Lys Tyr Gly
115 120 125
Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro Pro Thr
130 135 140
Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val
145 150 155 160
Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp
165 170 175
Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr
180 185 190
Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn
195 200 205
Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His
210 215 220
Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Lys
225 230 235 240
Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro
245 250 255
<210> 59
<211> 256
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子M4
<400> 59
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly
1 5 10 15
Ser Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala
20 25 30
Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser
35 40 45
Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
50 55 60
Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser
65 70 75 80
Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser
85 90 95
Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
100 105 110
Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Glu Ser Lys Tyr Gly
115 120 125
Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro Pro Thr
130 135 140
Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val
145 150 155 160
Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp
165 170 175
Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr
180 185 190
Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn
195 200 205
Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His
210 215 220
Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Glu
225 230 235 240
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser
245 250 255
<210> 60
<211> 133
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子2S5
<400> 60
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly
1 5 10 15
Ser Val Pro Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu
20 25 30
Asn Gln Val Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg
35 40 45
Leu Gln Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr
50 55 60
Ala Ser Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser
65 70 75 80
Trp Leu Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr
85 90 95
Cys Gln Val Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp
100 105 110
Leu Lys Val Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
115 120 125
Gly Gly Gly Gly Ser
130
<210> 61
<211> 208
<212> PRT
<213> 人工序列
<220>
<223> CAR间隔子M6
<400> 61
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
1 5 10 15
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
20 25 30
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
35 40 45
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
50 55 60
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
65 70 75 80
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
85 90 95
Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr
100 105 110
Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val
115 120 125
Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly
130 135 140
Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp
145 150 155 160
Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala His
165 170 175
Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro
180 185 190
Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Gly Gly Gly Gly Ser
195 200 205
<210> 62
<211> 676
<212> PRT
<213> 人工序列
<220>
<223> CAR M1
<400> 62
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu Ser Lys
260 265 270
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys
275 280 285
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
290 295 300
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
305 310 315 320
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
325 330 335
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
340 345 350
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
355 360 365
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
370 375 380
Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr
385 390 395 400
Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val
405 410 415
Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly
420 425 430
Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp
435 440 445
Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala His
450 455 460
Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro
465 470 475 480
Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Gly Gly Gly Gly Ser
485 490 495
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
500 505 510
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
515 520 525
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
530 535 540
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
545 550 555 560
Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
565 570 575
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
580 585 590
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
595 600 605
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
610 615 620
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
625 630 635 640
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
645 650 655
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
660 665 670
Leu Pro Pro Arg
675
<210> 63
<211> 688
<212> PRT
<213> 人工序列
<220>
<223> CAR XM2
<400> 63
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu Ser Lys
260 265 270
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys
275 280 285
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
290 295 300
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
305 310 315 320
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
325 330 335
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
340 345 350
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
355 360 365
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
370 375 380
Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr
385 390 395 400
Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val
405 410 415
Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly
420 425 430
Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp
435 440 445
Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala His
450 455 460
Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro
465 470 475 480
Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Lys Gly Lys His Leu
485 490 495
Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu
500 505 510
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
515 520 525
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
530 535 540
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
545 550 555 560
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
565 570 575
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
580 585 590
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
595 600 605
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
610 615 620
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
625 630 635 640
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
645 650 655
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
660 665 670
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
675 680 685
<210> 64
<211> 705
<212> PRT
<213> 人工序列
<220>
<223> CAR XM3
<400> 64
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu Ser Lys
260 265 270
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys
275 280 285
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
290 295 300
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
305 310 315 320
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
325 330 335
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
340 345 350
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
355 360 365
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
370 375 380
Ala Asn Leu Ser Glu Thr Ile Arg Glu Ser Lys Tyr Gly Pro Pro Cys
385 390 395 400
Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro Pro Thr Leu Glu Val
405 410 415
Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln
420 425 430
Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn
435 440 445
Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys
450 455 460
Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala
465 470 475 480
His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln
485 490 495
Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Lys Gly Lys His
500 505 510
Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val
515 520 525
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
530 535 540
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
545 550 555 560
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
565 570 575
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
580 585 590
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
595 600 605
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
610 615 620
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
625 630 635 640
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
645 650 655
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
660 665 670
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
675 680 685
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
690 695 700
Arg
705
<210> 65
<211> 705
<212> PRT
<213> 人工序列
<220>
<223> CAR M4
<400> 65
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu Ser Lys
260 265 270
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Ala Lys
275 280 285
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln
290 295 300
His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp
305 310 315 320
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln
325 330 335
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser
340 345 350
Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile
355 360 365
Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr
370 375 380
Ala Asn Leu Ser Glu Thr Ile Arg Glu Ser Lys Tyr Gly Pro Pro Cys
385 390 395 400
Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro Pro Thr Leu Glu Val
405 410 415
Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln
420 425 430
Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn
435 440 445
Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys
450 455 460
Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala
465 470 475 480
His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln
485 490 495
Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Glu Ser Lys Tyr
500 505 510
Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Phe Trp Val
515 520 525
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
530 535 540
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
545 550 555 560
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
565 570 575
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
580 585 590
Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
595 600 605
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
610 615 620
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
625 630 635 640
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
645 650 655
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
660 665 670
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
675 680 685
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
690 695 700
Arg
705
<210> 66
<211> 582
<212> PRT
<213> 人工序列
<220>
<223> CAR 2S5
<400> 66
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu Ser Lys
260 265 270
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Val Pro
275 280 285
Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val
290 295 300
Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu
305 310 315 320
Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr
325 330 335
Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu
340 345 350
Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val
355 360 365
Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val
370 375 380
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly
385 390 395 400
Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr
405 410 415
Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys
420 425 430
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg
435 440 445
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
450 455 460
Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
465 470 475 480
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
485 490 495
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
500 505 510
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
515 520 525
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
530 535 540
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
545 550 555 560
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
565 570 575
Gln Ala Leu Pro Pro Arg
580
<210> 67
<211> 664
<212> PRT
<213> 人工序列
<220>
<223> CAR M6
<400> 67
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
145 150 155 160
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
165 170 175
Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys
180 185 190
Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
195 200 205
Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
210 215 220
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala
225 230 235 240
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met
245 250 255
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
260 265 270
Gly Ser Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg
275 280 285
Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe
290 295 300
Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu
305 310 315 320
Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr
325 330 335
Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His
340 345 350
Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
355 360 365
Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr
370 375 380
Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val
385 390 395 400
Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp
405 410 415
Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr
420 425 430
Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn
435 440 445
Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His
450 455 460
Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Gly
465 470 475 480
Gly Gly Gly Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala
485 490 495
Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg
500 505 510
Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro
515 520 525
Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
530 535 540
Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala
545 550 555 560
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
565 570 575
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
580 585 590
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
595 600 605
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
610 615 620
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
625 630 635 640
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
645 650 655
His Met Gln Ala Leu Pro Pro Arg
660
<210> 68
<211> 666
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子M1
<400> 68
gagagcaagt acggcccccc ctgccccccc tgccccggtg gcggtggaag tgccaagccc 60
agcgcccccg tggtgagcgg ccccgccgcc agggccaccc cccagcacac cgtgagcttc 120
acctgcgaga gccacggctt cagccccagg gacatcaccc tgaagtggtt caagaacggc 180
aacgagctga gcgacttcca gaccaacgtg gaccccgtgg gcgagagcgt gagctacagc 240
atccacagca ccgccaaggt ggtgctgacc agggaggacg tgcacagcca ggtgatctgc 300
gaggtggccc acgtgaccct gcagggcgac cccctgaggg gcaccgccaa cctgagcgag 360
accatcaggg tgccccccac cctggaggtg acccagcagc ccgtgagggc cgagaaccag 420
gtgaacgtga cctgccaggt gaggaagttc tacccccaga ggctgcagct gacctggctg 480
gagaacggca acgtgagcag gaccgagacc gccagcaccg tgaccgagaa caaggacggc 540
acctacaact ggatgagctg gctgctggtg aacgtgagcg cccacaggga cgacgtgaag 600
ctgacctgcc aggtggagca cgacggccag cccgccgtga gcaagagcca cgacctgaag 660
gtgagc 666
<210> 69
<211> 717
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子XM2
<400> 69
gagagcaagt acggcccccc ctgccccccc tgccccggtg gcggtggaag tgccaagccc 60
agcgcccccg tggtgagcgg ccccgccgcc agggccaccc cccagcacac cgtgagcttc 120
acctgcgaga gccacggctt cagccccagg gacatcaccc tgaagtggtt caagaacggc 180
aacgagctga gcgacttcca gaccaacgtg gaccccgtgg gcgagagcgt gagctacagc 240
atccacagca ccgccaaggt ggtgctgacc agggaggacg tgcacagcca ggtgatctgc 300
gaggtggccc acgtgaccct gcagggcgac cccctgaggg gcaccgccaa cctgagcgag 360
accatcaggg tgccccccac cctggaggtg acccagcagc ccgtgagggc cgagaaccag 420
gtgaacgtga cctgccaggt gaggaagttc tacccccaga ggctgcagct gacctggctg 480
gagaacggca acgtgagcag gaccgagacc gccagcaccg tgaccgagaa caaggacggc 540
acctacaact ggatgagctg gctgctggtg aacgtgagcg cccacaggga cgacgtgaag 600
ctgacctgcc aggtggagca cgacggccag cccgccgtga gcaagagcca cgacctgaag 660
gtgagcaagg gcaagcacct gtgccccagc cccctgttcc ccggccccag caagccc 717
<210> 70
<211> 768
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子XM3
<400> 70
gagagcaagt acggcccccc ctgccccccc tgccccggtg gcggtggaag tgccaagccc 60
agcgcccccg tggtgagcgg ccccgccgcc agggccaccc cccagcacac cgtgagcttc 120
acctgcgaga gccacggctt cagccccagg gacatcaccc tgaagtggtt caagaacggc 180
aacgagctga gcgacttcca gaccaacgtg gaccccgtgg gcgagagcgt gagctacagc 240
atccacagca ccgccaaggt ggtgctgacc agggaggacg tgcacagcca ggtgatctgc 300
gaggtggccc acgtgaccct gcagggcgac cccctgaggg gcaccgccaa cctgagcgag 360
accatcaggg aatccaaata cggaccacca tgcccaccat gcccaggcgg aggcggtagt 420
gtgcccccca ccctggaggt gacccagcag cccgtgaggg ccgagaacca ggtgaacgtg 480
acctgccagg tgaggaagtt ctacccccag aggctgcagc tgacctggct ggagaacggc 540
aacgtgagca ggaccgagac cgccagcacc gtgaccgaga acaaggacgg cacctacaac 600
tggatgagct ggctgctggt gaacgtgagc gcccacaggg acgacgtgaa gctgacctgc 660
caggtggagc acgacggcca gcccgccgtg agcaagagcc acgacctgaa ggtgagcaag 720
ggcaagcacc tgtgccccag ccccctgttc cccggcccca gcaagccc 768
<210> 71
<211> 768
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子M4
<400> 71
gagagcaagt acggcccccc ctgccccccc tgccccggtg gcggtggaag tgccaagccc 60
agcgcccccg tggtgagcgg ccccgccgcc agggccaccc cccagcacac cgtgagcttc 120
acctgcgaga gccacggctt cagccccagg gacatcaccc tgaagtggtt caagaacggc 180
aacgagctga gcgacttcca gaccaacgtg gaccccgtgg gcgagagcgt gagctacagc 240
atccacagca ccgccaaggt ggtgctgacc agggaggacg tgcacagcca ggtgatctgc 300
gaggtggccc acgtgaccct gcagggcgac cccctgaggg gcaccgccaa cctgagcgag 360
accatcaggg aatccaaata cggaccacca tgcccaccat gcccaggagg tggcggaagt 420
gtgcccccca ccctggaggt gacccagcag cccgtgaggg ccgagaacca ggtgaacgtg 480
acctgccagg tgaggaagtt ctacccccag aggctgcagc tgacctggct ggagaacggc 540
aacgtgagca ggaccgagac cgccagcacc gtgaccgaga acaaggacgg cacctacaac 600
tggatgagct ggctgctggt gaacgtgagc gcccacaggg acgacgtgaa gctgacctgc 660
caggtggagc acgacggcca gcccgccgtg agcaagagcc acgacctgaa ggtgagcgaa 720
tccaaatacg gaccaccatg cccaccatgc ccaggcggtg gcggcagc 768
<210> 72
<211> 399
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子2S5
<400> 72
gagagcaagt acggcccccc ctgccccccc tgccccggtg gcggtggaag tgtgcccccc 60
accctggagg tgacccagca gcccgtgagg gccgagaacc aggtgaacgt gacctgccag 120
gtgaggaagt tctaccccca gaggctgcag ctgacctggc tggagaacgg caacgtgagc 180
aggaccgaga ccgccagcac cgtgaccgag aacaaggacg gcacctacaa ctggatgagc 240
tggctgctgg tgaacgtgag cgcccacagg gacgacgtga agctgacctg ccaggtggag 300
cacgacggcc agcccgccgt gagcaagagc cacgacctga aggtgagcga atccaaatac 360
ggaccaccat gcccaccatg cccaggcggt ggcggcagc 399
<210> 73
<211> 645
<212> DNA
<213> 人工序列
<220>
<223> CAR间隔子M6
<400> 73
ggtggcggtg gaagtgccaa gcccagcgcc cccgtggtga gcggccccgc cgccagggcc 60
accccccagc acaccgtgag cttcacctgc gagagccacg gcttcagccc cagggacatc 120
accctgaagt ggttcaagaa cggcaacgag ctgagcgact tccagaccaa cgtggacccc 180
gtgggcgaga gcgtgagcta cagcatccac agcaccgcca aggtggtgct gaccagggag 240
gacgtgcaca gccaggtgat ctgcgaggtg gcccacgtga ccctgcaggg cgaccccctg 300
aggggcaccg ccaacctgag cgagaccatc agggtgcccc ccaccctgga ggtgacccag 360
cagcccgtga gggccgagaa ccaggtgaac gtgacctgcc aggtgaggaa gttctacccc 420
cagaggctgc agctgacctg gctggagaac ggcaacgtga gcaggaccga gaccgccagc 480
accgtgaccg agaacaagga cggcacctac aactggatga gctggctgct ggtgaacgtg 540
agcgcccaca gggacgacgt gaagctgacc tgccaggtgg agcacgacgg ccagcccgcc 600
gtgagcaaga gccacgacct gaaggtgagc ggcggtggcg gcagc 645
<210> 74
<211> 2037
<212> DNA
<213> 人工序列
<220>
<223> CAR M1
<400> 74
gccaccatgg agttcggcct gagctggctg ttcctggtgg ccatcctgaa gggcgtgcag 60
tgcagcaggg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120
agggtgacca tcagctgcag ggccagccag gacatcagca agtacctgaa ctggtaccag 180
cagaagcccg acggcaccgt gaagctgctg atctaccaca ccagcaggct gcacagcggc 240
gtgcccagca ggttcagcgg cagcggcagc ggcaccgact acagcctgac catcagcaac 300
ctggagcagg aggacatcgc cacctacttc tgccagcagg gcaacaccct gccctacacc 360
ttcggcggcg gcaccaagct ggagctgaag aggggcggtg gaggttccgg cggtggcggt 420
tccggaggcg gtgggtcagg aggtggaggc tccgaggtgc agctgcagca gagcggcccc 480
ggcctggtgg cccccagcca gagcctgagc gtgacctgca ccgtgagcgg cgtgagcctg 540
cccgactacg gcgtgagctg gatcaggcag ccccccagga agggcctgga gtggctgggc 600
gtgatctggg gcagcgagac cacctactac aacagcgccc tgaagagcag gctgaccatc 660
atcaaggaca acagcaagag ccaggtgttc ctgaagatga acagcctgca gaccgacgac 720
accgccatct actactgcgc caagcactac tactacggcg gcagctacgc catggactac 780
tggggccagg gcaccaccgt gaccgtgagc agcgagagca agtacggccc cccctgcccc 840
ccctgccccg gtggcggtgg aagtgccaag cccagcgccc ccgtggtgag cggccccgcc 900
gccagggcca ccccccagca caccgtgagc ttcacctgcg agagccacgg cttcagcccc 960
agggacatca ccctgaagtg gttcaagaac ggcaacgagc tgagcgactt ccagaccaac 1020
gtggaccccg tgggcgagag cgtgagctac agcatccaca gcaccgccaa ggtggtgctg 1080
accagggagg acgtgcacag ccaggtgatc tgcgaggtgg cccacgtgac cctgcagggc 1140
gaccccctga ggggcaccgc caacctgagc gagaccatca gggtgccccc caccctggag 1200
gtgacccagc agcccgtgag ggccgagaac caggtgaacg tgacctgcca ggtgaggaag 1260
ttctaccccc agaggctgca gctgacctgg ctggagaacg gcaacgtgag caggaccgag 1320
accgccagca ccgtgaccga gaacaaggac ggcacctaca actggatgag ctggctgctg 1380
gtgaacgtga gcgcccacag ggacgacgtg aagctgacct gccaggtgga gcacgacggc 1440
cagcccgccg tgagcaagag ccacgacctg aaggtgagcg gcggtggcgg cagcttctgg 1500
gtgctggtgg tggtgggcgg cgtgctggcc tgctacagcc tgctggtgac cgtggccttc 1560
atcatcttct gggtgaggag caagaggagc aggctgctgc acagcgacta catgaacatg 1620
acccccagga ggcccggccc caccaggaag cactaccagc cctacgcccc ccccagggac 1680
ttcgccgcct acaggagcag ggtgaagttc agcaggagcg ccgacgcccc cgcctaccag 1740
cagggccaga accagctgta caacgagctg aacctgggca ggagggagga gtacgacgtg 1800
ctggacaaga ggaggggcag ggaccccgag atgggcggca agcccaggag gaagaacccc 1860
caggagggcc tgtacaacga gctgcagaag gacaagatgg ccgaggccta cagcgagatc 1920
ggcatgaagg gcgagaggag gaggggcaag ggccacgacg gcctgtacca gggcctgagc 1980
accgccacca aggacaccta cgacgccctg cacatgcagg ccctgccccc caggtaa 2037
<210> 75
<211> 2073
<212> DNA
<213> 人工序列
<220>
<223> CAR XM2
<400> 75
gccaccatgg agttcggcct gagctggctg ttcctggtgg ccatcctgaa gggcgtgcag 60
tgcagcaggg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120
agggtgacca tcagctgcag ggccagccag gacatcagca agtacctgaa ctggtaccag 180
cagaagcccg acggcaccgt gaagctgctg atctaccaca ccagcaggct gcacagcggc 240
gtgcccagca ggttcagcgg cagcggcagc ggcaccgact acagcctgac catcagcaac 300
ctggagcagg aggacatcgc cacctacttc tgccagcagg gcaacaccct gccctacacc 360
ttcggcggcg gcaccaagct ggagctgaag aggggcggtg gaggttccgg cggtggcggt 420
tccggaggcg gtgggtcagg aggtggaggc tccgaggtgc agctgcagca gagcggcccc 480
ggcctggtgg cccccagcca gagcctgagc gtgacctgca ccgtgagcgg cgtgagcctg 540
cccgactacg gcgtgagctg gatcaggcag ccccccagga agggcctgga gtggctgggc 600
gtgatctggg gcagcgagac cacctactac aacagcgccc tgaagagcag gctgaccatc 660
atcaaggaca acagcaagag ccaggtgttc ctgaagatga acagcctgca gaccgacgac 720
accgccatct actactgcgc caagcactac tactacggcg gcagctacgc catggactac 780
tggggccagg gcaccaccgt gaccgtgagc agcgagagca agtacggccc cccctgcccc 840
ccctgccccg gtggcggtgg aagtgccaag cccagcgccc ccgtggtgag cggccccgcc 900
gccagggcca ccccccagca caccgtgagc ttcacctgcg agagccacgg cttcagcccc 960
agggacatca ccctgaagtg gttcaagaac ggcaacgagc tgagcgactt ccagaccaac 1020
gtggaccccg tgggcgagag cgtgagctac agcatccaca gcaccgccaa ggtggtgctg 1080
accagggagg acgtgcacag ccaggtgatc tgcgaggtgg cccacgtgac cctgcagggc 1140
gaccccctga ggggcaccgc caacctgagc gagaccatca gggtgccccc caccctggag 1200
gtgacccagc agcccgtgag ggccgagaac caggtgaacg tgacctgcca ggtgaggaag 1260
ttctaccccc agaggctgca gctgacctgg ctggagaacg gcaacgtgag caggaccgag 1320
accgccagca ccgtgaccga gaacaaggac ggcacctaca actggatgag ctggctgctg 1380
gtgaacgtga gcgcccacag ggacgacgtg aagctgacct gccaggtgga gcacgacggc 1440
cagcccgccg tgagcaagag ccacgacctg aaggtgagca agggcaagca cctgtgcccc 1500
agccccctgt tccccggccc cagcaagccc ttctgggtgc tggtggtggt gggcggcgtg 1560
ctggcctgct acagcctgct ggtgaccgtg gccttcatca tcttctgggt gaggagcaag 1620
aggagcaggc tgctgcacag cgactacatg aacatgaccc ccaggaggcc cggccccacc 1680
aggaagcact accagcccta cgcccccccc agggacttcg ccgcctacag gagcagggtg 1740
aagttcagca ggagcgccga cgcccccgcc taccagcagg gccagaacca gctgtacaac 1800
gagctgaacc tgggcaggag ggaggagtac gacgtgctgg acaagaggag gggcagggac 1860
cccgagatgg gcggcaagcc caggaggaag aacccccagg agggcctgta caacgagctg 1920
cagaaggaca agatggccga ggcctacagc gagatcggca tgaagggcga gaggaggagg 1980
ggcaagggcc acgacggcct gtaccagggc ctgagcaccg ccaccaagga cacctacgac 2040
gccctgcaca tgcaggccct gccccccagg taa 2073
<210> 76
<211> 2124
<212> DNA
<213> 人工序列
<220>
<223> CAR XM3
<400> 76
gccaccatgg agttcggcct gagctggctg ttcctggtgg ccatcctgaa gggcgtgcag 60
tgcagcaggg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120
agggtgacca tcagctgcag ggccagccag gacatcagca agtacctgaa ctggtaccag 180
cagaagcccg acggcaccgt gaagctgctg atctaccaca ccagcaggct gcacagcggc 240
gtgcccagca ggttcagcgg cagcggcagc ggcaccgact acagcctgac catcagcaac 300
ctggagcagg aggacatcgc cacctacttc tgccagcagg gcaacaccct gccctacacc 360
ttcggcggcg gcaccaagct ggagctgaag aggggcggtg gaggttccgg cggtggcggt 420
tccggaggcg gtgggtcagg aggtggaggc tccgaggtgc agctgcagca gagcggcccc 480
ggcctggtgg cccccagcca gagcctgagc gtgacctgca ccgtgagcgg cgtgagcctg 540
cccgactacg gcgtgagctg gatcaggcag ccccccagga agggcctgga gtggctgggc 600
gtgatctggg gcagcgagac cacctactac aacagcgccc tgaagagcag gctgaccatc 660
atcaaggaca acagcaagag ccaggtgttc ctgaagatga acagcctgca gaccgacgac 720
accgccatct actactgcgc caagcactac tactacggcg gcagctacgc catggactac 780
tggggccagg gcaccaccgt gaccgtgagc agcgagagca agtacggccc cccctgcccc 840
ccctgccccg gtggcggtgg aagtgccaag cccagcgccc ccgtggtgag cggccccgcc 900
gccagggcca ccccccagca caccgtgagc ttcacctgcg agagccacgg cttcagcccc 960
agggacatca ccctgaagtg gttcaagaac ggcaacgagc tgagcgactt ccagaccaac 1020
gtggaccccg tgggcgagag cgtgagctac agcatccaca gcaccgccaa ggtggtgctg 1080
accagggagg acgtgcacag ccaggtgatc tgcgaggtgg cccacgtgac cctgcagggc 1140
gaccccctga ggggcaccgc caacctgagc gagaccatca gggaatccaa atacggacca 1200
ccatgcccac catgcccagg cggaggcggt agtgtgcccc ccaccctgga ggtgacccag 1260
cagcccgtga gggccgagaa ccaggtgaac gtgacctgcc aggtgaggaa gttctacccc 1320
cagaggctgc agctgacctg gctggagaac ggcaacgtga gcaggaccga gaccgccagc 1380
accgtgaccg agaacaagga cggcacctac aactggatga gctggctgct ggtgaacgtg 1440
agcgcccaca gggacgacgt gaagctgacc tgccaggtgg agcacgacgg ccagcccgcc 1500
gtgagcaaga gccacgacct gaaggtgagc aagggcaagc acctgtgccc cagccccctg 1560
ttccccggcc ccagcaagcc cttctgggtg ctggtggtgg tgggcggcgt gctggcctgc 1620
tacagcctgc tggtgaccgt ggccttcatc atcttctggg tgaggagcaa gaggagcagg 1680
ctgctgcaca gcgactacat gaacatgacc cccaggaggc ccggccccac caggaagcac 1740
taccagccct acgccccccc cagggacttc gccgcctaca ggagcagggt gaagttcagc 1800
aggagcgccg acgcccccgc ctaccagcag ggccagaacc agctgtacaa cgagctgaac 1860
ctgggcagga gggaggagta cgacgtgctg gacaagagga ggggcaggga ccccgagatg 1920
ggcggcaagc ccaggaggaa gaacccccag gagggcctgt acaacgagct gcagaaggac 1980
aagatggccg aggcctacag cgagatcggc atgaagggcg agaggaggag gggcaagggc 2040
cacgacggcc tgtaccaggg cctgagcacc gccaccaagg acacctacga cgccctgcac 2100
atgcaggccc tgccccccag gtaa 2124
<210> 77
<211> 2124
<212> DNA
<213> 人工序列
<220>
<223> CAR M4
<400> 77
gccaccatgg agttcggcct gagctggctg ttcctggtgg ccatcctgaa gggcgtgcag 60
tgcagcaggg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120
agggtgacca tcagctgcag ggccagccag gacatcagca agtacctgaa ctggtaccag 180
cagaagcccg acggcaccgt gaagctgctg atctaccaca ccagcaggct gcacagcggc 240
gtgcccagca ggttcagcgg cagcggcagc ggcaccgact acagcctgac catcagcaac 300
ctggagcagg aggacatcgc cacctacttc tgccagcagg gcaacaccct gccctacacc 360
ttcggcggcg gcaccaagct ggagctgaag aggggcggtg gaggttccgg cggtggcggt 420
tccggaggcg gtgggtcagg aggtggaggc tccgaggtgc agctgcagca gagcggcccc 480
ggcctggtgg cccccagcca gagcctgagc gtgacctgca ccgtgagcgg cgtgagcctg 540
cccgactacg gcgtgagctg gatcaggcag ccccccagga agggcctgga gtggctgggc 600
gtgatctggg gcagcgagac cacctactac aacagcgccc tgaagagcag gctgaccatc 660
atcaaggaca acagcaagag ccaggtgttc ctgaagatga acagcctgca gaccgacgac 720
accgccatct actactgcgc caagcactac tactacggcg gcagctacgc catggactac 780
tggggccagg gcaccaccgt gaccgtgagc agcgagagca agtacggccc cccctgcccc 840
ccctgccccg gtggcggtgg aagtgccaag cccagcgccc ccgtggtgag cggccccgcc 900
gccagggcca ccccccagca caccgtgagc ttcacctgcg agagccacgg cttcagcccc 960
agggacatca ccctgaagtg gttcaagaac ggcaacgagc tgagcgactt ccagaccaac 1020
gtggaccccg tgggcgagag cgtgagctac agcatccaca gcaccgccaa ggtggtgctg 1080
accagggagg acgtgcacag ccaggtgatc tgcgaggtgg cccacgtgac cctgcagggc 1140
gaccccctga ggggcaccgc caacctgagc gagaccatca gggaatccaa atacggacca 1200
ccatgcccac catgcccagg aggtggcgga agtgtgcccc ccaccctgga ggtgacccag 1260
cagcccgtga gggccgagaa ccaggtgaac gtgacctgcc aggtgaggaa gttctacccc 1320
cagaggctgc agctgacctg gctggagaac ggcaacgtga gcaggaccga gaccgccagc 1380
accgtgaccg agaacaagga cggcacctac aactggatga gctggctgct ggtgaacgtg 1440
agcgcccaca gggacgacgt gaagctgacc tgccaggtgg agcacgacgg ccagcccgcc 1500
gtgagcaaga gccacgacct gaaggtgagc gaatccaaat acggaccacc atgcccacca 1560
tgcccaggcg gtggcggcag cttctgggtg ctggtggtgg tgggcggcgt gctggcctgc 1620
tacagcctgc tggtgaccgt ggccttcatc atcttctggg tgaggagcaa gaggagcagg 1680
ctgctgcaca gcgactacat gaacatgacc cccaggaggc ccggccccac caggaagcac 1740
taccagccct acgccccccc cagggacttc gccgcctaca ggagcagggt gaagttcagc 1800
aggagcgccg acgcccccgc ctaccagcag ggccagaacc agctgtacaa cgagctgaac 1860
ctgggcagga gggaggagta cgacgtgctg gacaagagga ggggcaggga ccccgagatg 1920
ggcggcaagc ccaggaggaa gaacccccag gagggcctgt acaacgagct gcagaaggac 1980
aagatggccg aggcctacag cgagatcggc atgaagggcg agaggaggag gggcaagggc 2040
cacgacggcc tgtaccaggg cctgagcacc gccaccaagg acacctacga cgccctgcac 2100
atgcaggccc tgccccccag gtaa 2124
<210> 78
<211> 1755
<212> DNA
<213> 人工序列
<220>
<223> CAR 2S5
<400> 78
gccaccatgg agttcggcct gagctggctg ttcctggtgg ccatcctgaa gggcgtgcag 60
tgcagcaggg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120
agggtgacca tcagctgcag ggccagccag gacatcagca agtacctgaa ctggtaccag 180
cagaagcccg acggcaccgt gaagctgctg atctaccaca ccagcaggct gcacagcggc 240
gtgcccagca ggttcagcgg cagcggcagc ggcaccgact acagcctgac catcagcaac 300
ctggagcagg aggacatcgc cacctacttc tgccagcagg gcaacaccct gccctacacc 360
ttcggcggcg gcaccaagct ggagctgaag aggggcggtg gaggttccgg cggtggcggt 420
tccggaggcg gtgggtcagg aggtggaggc tccgaggtgc agctgcagca gagcggcccc 480
ggcctggtgg cccccagcca gagcctgagc gtgacctgca ccgtgagcgg cgtgagcctg 540
cccgactacg gcgtgagctg gatcaggcag ccccccagga agggcctgga gtggctgggc 600
gtgatctggg gcagcgagac cacctactac aacagcgccc tgaagagcag gctgaccatc 660
atcaaggaca acagcaagag ccaggtgttc ctgaagatga acagcctgca gaccgacgac 720
accgccatct actactgcgc caagcactac tactacggcg gcagctacgc catggactac 780
tggggccagg gcaccaccgt gaccgtgagc agcgagagca agtacggccc cccctgcccc 840
ccctgccccg gtggcggtgg aagtgtgccc cccaccctgg aggtgaccca gcagcccgtg 900
agggccgaga accaggtgaa cgtgacctgc caggtgagga agttctaccc ccagaggctg 960
cagctgacct ggctggagaa cggcaacgtg agcaggaccg agaccgccag caccgtgacc 1020
gagaacaagg acggcaccta caactggatg agctggctgc tggtgaacgt gagcgcccac 1080
agggacgacg tgaagctgac ctgccaggtg gagcacgacg gccagcccgc cgtgagcaag 1140
agccacgacc tgaaggtgag cgaatccaaa tacggaccac catgcccacc atgcccaggc 1200
ggtggcggca gcttctgggt gctggtggtg gtgggcggcg tgctggcctg ctacagcctg 1260
ctggtgaccg tggccttcat catcttctgg gtgaggagca agaggagcag gctgctgcac 1320
agcgactaca tgaacatgac ccccaggagg cccggcccca ccaggaagca ctaccagccc 1380
tacgcccccc ccagggactt cgccgcctac aggagcaggg tgaagttcag caggagcgcc 1440
gacgcccccg cctaccagca gggccagaac cagctgtaca acgagctgaa cctgggcagg 1500
agggaggagt acgacgtgct ggacaagagg aggggcaggg accccgagat gggcggcaag 1560
cccaggagga agaaccccca ggagggcctg tacaacgagc tgcagaagga caagatggcc 1620
gaggcctaca gcgagatcgg catgaagggc gagaggagga ggggcaaggg ccacgacggc 1680
ctgtaccagg gcctgagcac cgccaccaag gacacctacg acgccctgca catgcaggcc 1740
ctgcccccca ggtaa 1755
<210> 79
<211> 2001
<212> DNA
<213> 人工序列
<220>
<223> CAR M6
<400> 79
gccaccatgg agttcggcct gagctggctg ttcctggtgg ccatcctgaa gggcgtgcag 60
tgcagcaggg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac 120
agggtgacca tcagctgcag ggccagccag gacatcagca agtacctgaa ctggtaccag 180
cagaagcccg acggcaccgt gaagctgctg atctaccaca ccagcaggct gcacagcggc 240
gtgcccagca ggttcagcgg cagcggcagc ggcaccgact acagcctgac catcagcaac 300
ctggagcagg aggacatcgc cacctacttc tgccagcagg gcaacaccct gccctacacc 360
ttcggcggcg gcaccaagct ggagctgaag aggggcggtg gaggttccgg cggtggcggt 420
tccggaggcg gtgggtcagg aggtggaggc tccgaggtgc agctgcagca gagcggcccc 480
ggcctggtgg cccccagcca gagcctgagc gtgacctgca ccgtgagcgg cgtgagcctg 540
cccgactacg gcgtgagctg gatcaggcag ccccccagga agggcctgga gtggctgggc 600
gtgatctggg gcagcgagac cacctactac aacagcgccc tgaagagcag gctgaccatc 660
atcaaggaca acagcaagag ccaggtgttc ctgaagatga acagcctgca gaccgacgac 720
accgccatct actactgcgc caagcactac tactacggcg gcagctacgc catggactac 780
tggggccagg gcaccaccgt gaccgtgagc agcggtggcg gtggaagtgc caagcccagc 840
gcccccgtgg tgagcggccc cgccgccagg gccacccccc agcacaccgt gagcttcacc 900
tgcgagagcc acggcttcag ccccagggac atcaccctga agtggttcaa gaacggcaac 960
gagctgagcg acttccagac caacgtggac cccgtgggcg agagcgtgag ctacagcatc 1020
cacagcaccg ccaaggtggt gctgaccagg gaggacgtgc acagccaggt gatctgcgag 1080
gtggcccacg tgaccctgca gggcgacccc ctgaggggca ccgccaacct gagcgagacc 1140
atcagggtgc cccccaccct ggaggtgacc cagcagcccg tgagggccga gaaccaggtg 1200
aacgtgacct gccaggtgag gaagttctac ccccagaggc tgcagctgac ctggctggag 1260
aacggcaacg tgagcaggac cgagaccgcc agcaccgtga ccgagaacaa ggacggcacc 1320
tacaactgga tgagctggct gctggtgaac gtgagcgccc acagggacga cgtgaagctg 1380
acctgccagg tggagcacga cggccagccc gccgtgagca agagccacga cctgaaggtg 1440
agcggcggtg gcggcagctt ctgggtgctg gtggtggtgg gcggcgtgct ggcctgctac 1500
agcctgctgg tgaccgtggc cttcatcatc ttctgggtga ggagcaagag gagcaggctg 1560
ctgcacagcg actacatgaa catgaccccc aggaggcccg gccccaccag gaagcactac 1620
cagccctacg ccccccccag ggacttcgcc gcctacagga gcagggtgaa gttcagcagg 1680
agcgccgacg cccccgccta ccagcagggc cagaaccagc tgtacaacga gctgaacctg 1740
ggcaggaggg aggagtacga cgtgctggac aagaggaggg gcagggaccc cgagatgggc 1800
ggcaagccca ggaggaagaa cccccaggag ggcctgtaca acgagctgca gaaggacaag 1860
atggccgagg cctacagcga gatcggcatg aagggcgaga ggaggagggg caagggccac 1920
gacggcctgt accagggcct gagcaccgcc accaaggaca cctacgacgc cctgcacatg 1980
caggccctgc cccccaggta a 2001
<210> 80
<211> 15
<212> PRT
<213> 智人
<400> 80
Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
<210> 81
<211> 12
<212> PRT
<213> 智人
<400> 81
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro
1 5 10
<210> 82
<211> 16
<212> PRT
<213> 智人
<400> 82
Glu Leu Lys Thr Pro Leu Gly Asp Thr His Thr Cys Pro Arg Cys Pro
1 5 10 15
<210> 83
<211> 12
<212> PRT
<213> 智人
<400> 83
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
1 5 10
<210> 84
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> 接头 C2
<400> 84
Glu Thr Ile Arg Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
1 5 10 15
Gly Gly Gly Gly Ser Val Pro
20

Claims (26)

1.一种包含细胞外间隔子的嵌合抗原受体(CAR)，所述细胞外间隔子包含至少一个信号调节蛋白α(SIRP-α)的Ig样C1结构域或其片段或其变体。
2.根据权利要求1所述的CAR，其中所述SIRP-α的Ig样C1结构域选自(i)根据SEQ ID NO1的1型结构域或其片段或其变体；或(ii)根据SEQ ID NO 2的2型结构域或其片段或其变体。
3.根据权利要求1或2所述的CAR，其中所述细胞外间隔子包含SIRP-α的Ig样C1-1型结构域和Ig样C1-2型结构域。
4.根据前述权利要求中任一项所述的CAR，其中所述细胞外间隔子还包含至少一个多聚化结构域。
5.根据权利要求4所述的CAR，其中所述多聚化结构域或多个多聚化结构域选自IgG铰链区和/或根据SEQ ID NO 3的CD28细胞外结构域和/或其片段和变体，所述IgG铰链区选自根据SEQ ID NO 4或SEQ ID NO 80的IgG1铰链区、根据SEQ ID NO 81的IgG2铰链区、根据SEQ ID NO 82的IgG3铰链区、根据SEQ ID NO 83的IgG4铰链区。
6.根据权利要求4所述的CAR，其中所述多聚化结构域或多个多聚化结构域选自根据SEQ ID NO 4的IgG1铰链区或其片段和/或根据SEQ ID NO 3的CD28细胞外结构域或其片段。
7.根据权利要求4所述的CAR，其中所述多聚化结构域或多个多聚化结构域选自根据SEQ ID NO 83的IgG4铰链区或其片段和/或根据SEQ ID NO 3的CD28细胞外结构域或其片段。
8.根据前述权利要求中任一项所述的CAR，其中所述细胞外间隔子位于跨膜结构域和抗原结合结构域之间并将其连接起来。
9.根据权利要求8所述的CAR，其中所述抗原结合结构域包含单链可变区片段(scFv)。
10.根据前述权利要求中任一项所述的CAR，其中所述间隔子至少通过一个二硫键使CAR二聚化。
11.一种包含细胞外间隔子的CAR，所述细胞外间隔子包含根据SEQ ID NO 10、SEQ IDNO 11、SEQ ID NO 12、SEQ ID NO 13、SEQ ID NO 14、SEQ ID NO 15、SEQ ID NO 16、SEQ IDNO 17、SEQ ID NO 18、SEQ ID NO 56、SEQ ID NO 57、SEQ ID NO 58、SEQ ID NO 59、SEQ IDNO 60或SEQ ID NO61的氨基酸序列。
12.一种嵌合抗原受体(CAR)，其包含(i)根据权利要求1-11中任一项所述的细胞外间隔子，(ii)抗原结合结构域，(iii)跨膜结构域，(iv)细胞内信号传导结构域，(v)任选地共刺激结构域。
13.根据权利要求12所述的CAR，其中所述抗原结合结构域包含抗体或其片段。
14.根据权利要求12的任一项所述的CAR，其中所述抗原结合结构域包含单链可变区片段(scFv)。
15.根据权利要求12至14中任一项所述的CAR，其中所述抗原结合结构域靶向肿瘤抗原。
16.根据权利要求15所述的CAR，其中所述肿瘤抗原选自CD19或HER-2。
17.根据权利要求12至16中任一项所述的CAR，其中所述跨膜结构域包含根据SEQ IDNO 23的CD28的跨膜结构域。
18.根据权利要求12至17中任一项所述的CAR，其中所述细胞内信号传导结构域和/或共刺激结构域包含根据SEQ ID NO 25的CD3ζ的细胞内结构域或其片段和/或根据SEQ IDNO 24的CD28的细胞内结构域或其片段。
19.一种嵌合抗原受体(CAR)，其包含
(i)单链可变区片段(scFv)；
(ii)IgG铰链结构域；
(iii)信号调节蛋白α-1的Ig样C1-1型和/或Ig样C1-2型结构域；
(iv)CD3ζ；
(v)CD28跨膜结构域；
(vi)任选地CD28细胞外结构域和/或CD28细胞内结构域。
20.一种CAR，其包含根据SEQ ID NO 26、SEQ ID NO 27、SEQ ID NO 28、SEQ ID NO 29、SEQ ID NO 30、SEQ ID NO 31、SEQ ID NO 32、SEQ ID NO 33、SEQ ID NO 34、SEQ ID NO54、SEQ ID NO 62、SEQ ID NO 63、SEQ ID NO 64、SEQ ID NO 65、SEQ ID NO 66或SEQ IDNO 67的氨基酸序列，或由其组成。
21.一种多核苷酸，其编码权利要求1至20中任一项所述的CAR。
22.一种载体，其包含权利要求21所述的多核苷酸。
23.一种细胞，其包含根据权利要求1至20中任一项所述的CAR或根据权利要求21所述的多核苷酸。
24.根据权利要求23所述的细胞，其中所述细胞是T细胞
25.一种通过从以下组中选择至少两个结构域构成间隔子结构域来调整嵌合抗原受体(CAR)的长度的方法：(i)IgG铰链结构域、(ii)信号调节蛋白α-1的Ig样C1-1型结构域、(iii)信号调节蛋白α-1的Ig样C1-2型结构域、或(iv)CD28细胞外片段，导致嵌合抗原受体具有不同的长度。
26.根据权利要求25所述的方法，其中所述间隔子结构域不结合Fc受体或具有降低的对Fc受体的结合亲和力。
CN202180084760.3A 2020-12-16 2021-12-14 嵌合抗原受体(car)间隔子修饰增强car t细胞功能 Pending CN116600820A (zh)

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