CN115197314A - 一种pd1变体及其用途 - Google Patents
一种pd1变体及其用途 Download PDFInfo
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- CN115197314A CN115197314A CN202110398389.XA CN202110398389A CN115197314A CN 115197314 A CN115197314 A CN 115197314A CN 202110398389 A CN202110398389 A CN 202110398389A CN 115197314 A CN115197314 A CN 115197314A
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Abstract
本发明提供一种PD1变体，所述PD1变体含有ITSM基序突变；1）所述PD1变体的ITSM基序为活化型ITSM基序；或，2）所述PD1变体的ITSM基序含有活化型ITSM基序的部分氨基酸残基或其片段。野生型PD1介导下游抑制信号，不利于CAR‑T或TCR‑T肿瘤治疗；PD1敲除仅仅减少PD1介导的抑制信号，本发明的PD1变体在EAT‑2的共表达情况下能够增强CAR‑T或TCR‑T的抗肿瘤功能。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种PD1变体及其用途。
背景技术
程序性死亡受体1(PD1)是T细胞中重要的抑制性免疫受体。T细胞活化过程上调PD1的表达，PD1介导的下游信号通路能够抑制活化相关的信号通路(如TCR和CD28信号通路)，从而抑制T细胞的活化。在肿瘤微环境中，肿瘤细胞常常高表达PD1配体PDL1并通过激活T细胞的PD1信号来抑制T细胞抗肿瘤活性。临床上一般通过PD1或PDL1抗体阻断PD1与PDL1的结合能够恢复肿瘤微环境中T细胞的抗肿瘤活性，从而达到肿瘤治疗的效果。尚未有通过突变PD1改变其抑制性从而恢复T细胞抗肿瘤活性，或者进一步活化T细胞的报道。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供PD1变体及其用途。
为实现上述目的及其他相关目的，本发明第一方面提供一种PD1变体，所述PD1变体含有ITSM基序突变：
1)所述PD1变体的ITSM基序为活化型ITSM基序；或，
2)所述PD1变体的ITSM基序含有活化型ITSM基序的部分氨基酸残基或其片段。
本发明第二方面提供一种多核苷酸，编码前述的PD1变体。
本发明第三方面提供一种核酸构建物，所述核酸构建物含有前述的多核苷酸，能够表达所述PD1变体。
本发明第四方面提供一种慢病毒载体系统，所述慢病毒载体系统含有前述的核酸构建物及慢病毒载体辅助成分。
本发明第五方面提供一种T细胞，所述T细胞表达前述的PD1变体。
前述PD1变体或多核苷酸，或核酸构建物，或慢病毒载体系统在制备以下任一种或多种用途产品中的应用：(1)制备T细胞；(2)降低T细胞在肿瘤微环境中受到PD-L1/PD1信号通路的抑制；(3)增强T细胞的抗肿瘤功能。
前述的PD1变体或多核苷酸，或核酸构建物，或慢病毒载体系统，或T细胞在制备肿瘤治疗产品或抗慢性感染产品中的应用。
如上所述，本发明的PD1变体及其用途，具有以下有益效果：
野生型PD1介导下游抑制信号，不利于CAR-T或TCR-T肿瘤治疗；PD1敲除仅仅减少PD1介导的抑制信号，本发明的PD1变体在EAT-2的共表达情况下能够增强CAR-T或TCR-T的抗肿瘤功能。
考虑到Immunoreceptor Tyrosine Switch Motif(ITSM)是PD1介导下游抑制信号的主要基序，ITSM的酪氨酸磷酸化能够招募SHP2，后者作为磷酸酶能够去磷酸化TCR或CD28下游信号分子，进而抑制T细胞的免疫功能。本发明尝试了将其它免疫受体(主要为SLAM家族)的ITSM基序替换PD1的ITSM基序，意外发现，活化型ITSM基序全部或部分替代PD1的ITSM基序，可改变PD1的免疫抑制性，甚至反转其免疫抑制性使其成为具备一定活化性的免疫受体，使其具备在EAT2表达的情况下可介导下游活化信号，进一步激活T细胞，从而达到抗肿瘤的目的。
附图说明
图1：SEE(30ng/ml)孵育过的Raji细胞刺激过表达载体对照、野生型PD1、PD1变体的Jurkat细胞，24小时后通过ELISA试剂盒检测上清中IL-2的浓度。根据不表达PD-L1的Raji细胞刺激下IL-2产生的量对表达PD-L1的Raji细胞刺激产生的IL-2进行归一化，得到的IL-2产生相对量可以表征PD1及变体的抑制能力。图中vector control为表达载体对照的Jurkat细胞，其它为表达野生型PD1及不同PD1变体的Jurkat细胞。
图2：检测表达PD1-WT、PD1-tailless和PD1-M2的Jurkart细胞在PD-L1作用下的相对IL-2产生。
图3：检测表达PD1-WT的ITSM基序部分突变获得的PD1变体的Jurkart细胞在PD-L1作用下的相对IL-2产生。A图检测PD1 ITSM左边(PD1-M2_L)或右边(PD1-M2_R)4个氨基酸残基替换为SLAMF1的第二个ITSM中对应的氨基酸残基后Jurkat细胞在PD-L1作用下的相对IL-2产生。B图为进一步替换左边非保守位点中任意两个氨基酸残基后Jurkat细胞在PD-L1作用下的相对IL-2产生。
图4：检测在共表达载体对照(VC)和EAT-2的情况下，表达载体对照(vectorcontrol)、PD1-WT、PD1-M1和PD1-M2的Jurkart细胞在PD-L1作用下的相对IL-2产生。
图5：A为PD-L1的作用下，Jurkat细胞中PD1-WT、PD1-M1和PD1-M2对SHP2的招募。B为PD-L1作用下，共表达EAT-2的Jurkat细胞在不同时间点PD1-WT和PD1-M2对SHP2和EAT-2的招募。C为共表达EAT-2的Jurkat细胞中，表达载体对照(VC)、PD1-WT和PD1-M2对下游信号分子磷酸化的影响。
附图中，*p<0.05,**p<0.01,***p<0.001,****p<0.0001,ns无显著性差异。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
本发明一实施例的PD1变体，所述PD1变体含有ITSM基序突变：
1)所述PD1变体的ITSM基序为活化型ITSM基序；或，
2)所述PD1变体的ITSM基序含有活化型ITSM基序的部分氨基酸残基或其片段。
活化型ITSM基序的来源可以为NTRK2、CXADR、CD244、SLAMF1、CD84、SLAMF6、SLAMF7或KDR。
所述活化型ITSM基序的氨基酸序列如SEQ ID NO：2-16任一所示。
具体的如表1所示：
表1
所述PD1变体为人源且具有PD-L1结合活性。
所述PD1变体相比野生型PD1，使免疫细胞经PD-L1刺激后产生的IL-2分泌抑制作用减弱；或者，所述PD1变体使免疫细胞经PD-L1刺激后促进IL-2分泌。
在一种实施方式中，所述PD1变体的氨基酸序列如SEQ ID NO：18-32任一所示。
PD1-NTRK2(SEQ ID NO：18)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPYSTDYYRVGFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-CXADR(SEQ ID NO：19)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPSKTQYNQVPFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-CD244-ITSM1(SEQ ID NO：20)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPFLTIYEDVKFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-CD244-ITSM2(SEQ ID NO：21)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPGSTIYSMIQFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-CD244-ITSM3(SEQ ID NO：22)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPAYTLYSLIQFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-CD244-ITSM4(SEQ ID NO：23)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPNSTIYEVIGFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-SLAMF1-ITSM1(SEQ ID NO：24)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPSLTIYAQVQFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-SLAMF1-ITSM2(SEQ ID NO：25)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPSITVYASVTFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-CD84-ITSM1(SEQ ID NO：26)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPKKTIYTYIMFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-CD84-ITSM2(SEQ ID NO：27)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPVNTVYSEVQFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-SLAMF6-ITSM1(SEQ ID NO：28)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPNNTVYASVTFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-SLAMF6-ITSM2(SEQ ID NO：29)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPTITIYSTINFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-SLAMF7-ITSM1(SEQ ID NO：30)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPENTEYDTIPFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-SLAMF7-ITSM2(SEQ ID NO：31)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPANTVYSTVEFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-KDR(SEQ ID NO：32)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPLKTGYLSIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
进一步的，所述方案2)中，以野生型PD1序列为基准，所述PD1变体的ITSM基序的突变位点选自以下位点中的一个或多个位点：244、245、246、247、249、250、251、252。
野生型PD1的ITSM基序如SEQ ID NO：1所示。具体的，EQTEYATIV。
进一步的，所述野生型PD1的氨基酸序列如SEQ ID NO：17所示。具体的，
PD1-WT：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL。
具体的，野生型PD1序列的ITSM基序如SEQ ID NO：1所示，为：EQTEYATIV。为野生型PD1的氨基酸序列的244-252位。
进一步的，
所述244位点的突变选自：E244A、E244R、E244N、E244D、E244C、E244Q、E244G、E244H、E244I、E244L、E244K、E244M、E244F、E244P、E244S、E244T、E244W、E244Y、E244V之任一。
所述245位点的突变选自：Q245A、Q245R、Q245N、Q245D、Q245C、Q245E、Q245G、Q245H、Q245I、Q245L、Q245K、Q245M、Q245F、Q245P、Q245S、Q245T、Q245W、Q245Y、Q245V之任一。
所述246位点的突变选自：T246A、T246R、T246N、T246D、T246C、T246Q、T246G、T246H、T246I、T246L、T246K、T246M、T246F、T246P、T246S、T246E、T246W、T246Y、T246V之任一。
所述247位点的突变选自：E247A、E247R、E247N、E247D、E247C、E247Q、E247G、E247H、E247I、E247L、E247K、E247M、E247F、E247P、E247S、E247T、E247W、E247Y、E247V之任一。
所述249位点的突变选自：A249E、A249R、A249N、A249D、A249C、A249Q、A249G、A249H、A249I、A249L、A249K、A249M、A249F、A249P、A249S、A249T、A249W、A249Y、A249V之任一。
所述250位点的突变选自：T250A、T250R、T250N、T250D、T250C、T250Q、T250G、T250H、T250I、T250L、T250K、T250M、T250F、T250P、T250S、T250E、T250W、T250Y、T250V之任一。
所述251位点的突变选自：I251A、I251R、I251N、I251D、I251C、I251Q、I251G、I251H、I251E、I251L、I251K、I251M、I251F、I251P、I251S、I251T、I251W、I251Y、I251V之任一。
所述252位点的突变选自：V252A、V252R、V252N、V252D、V252C、V252Q、V252G、V252H、V252I、V252L、V252K、V252M、V252F、V252P、V252S、V252T、V252W、V252Y、V252E之任一。
在优选的实施方式中，所述突变至少包括Q245I和E247V的突变。
在一种实施方式中，所述PD1变体的氨基酸序列如SEQ ID NO：33-34任一所示。
具体的：
PD1-M2_L(SEQ ID NO：33)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPSITVYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
PD1-Q245I/E247V(SEQ ID NO：34)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEITVYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL。
本发明一实施例的多核苷酸，编码前述的PD1变体。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明也包括编码融合蛋白的多核苷酸序列的简并变异体，即编码相同的氨基酸序列但核苷酸序列有所不同的核苷酸序列。
本文所述的多核苷酸通常可以用PCR扩增法获得。具体而言，可根据本文所公开的核苷酸，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
本发明一实施例的核酸构建物，含有前述的多核苷酸，能够表达所述PD1变体。
所述核酸构建物还包括与前述的多核苷酸序列操作性连接的一个或多个调控序列。本发明所述的PD1变体的编码序列可以多种方式被操作以保证所述蛋白的表达。在将核酸构建物插入载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。
调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。
调控序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。
调控序列也可以是合适的前导序列，对宿主细胞翻译重要的mRNA的非翻译区。前导序列与编码该多肽的核苷酸序列的5′末端可操作连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。
优选的，所述核酸构建物为载体。
通常通过可操作地连接编码PD1变体的多核苷酸至启动子，并将构建体并入表达载体，实现编码PD1变体的多核苷酸的表达。该载体对于复制和整合真核细胞可为合适的。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、起始序列和启动子。
编码本发明PD1变体的多核苷酸序列可被克隆入许多类型的载体。例如，可被克隆入质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。进一步地，载体是表达载体。表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。
更优选的，所述核酸构建物为慢病毒载体含有复制起始位点、3’LTR、5’LTR以及前述多核苷酸。
合适的启动子的一个例子为早期巨细胞病毒(CMV)启动子序列。该启动子序列是能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、EB病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，也可考虑使用诱导型启动子。诱导型启动子的使用提供了分子开关，其能够在期限表达时打开可操作地连接诱导型启动子的多核苷酸序列的表达，而在当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估PD1变体或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两个，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色荧光蛋白基因的基因。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。
将多核苷酸引入宿主细胞的生物学方法包括使用病毒载体，特别是慢病毒载体，这已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。已经开发了许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，慢病毒提供了用于基因传递系统的方便的平台。可利用本领域中已知的技术将选择的基因插入载体并包装入慢病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多反转录病毒系统在本领域中是已知的。在一些实施方案中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方案中，使用慢病毒载体。
前述的核酸构建物，还含有编码EAT-2蛋白的多核苷酸，能够同时表达所述PD1变体和EAT-2蛋白。
可以通过内部核糖体进入位点(IRES)连接两个序列，在一个载体中同时表达两个蛋白。
编码EAT-2蛋白的多核苷酸序列如SEQ ID NO：35所示；具体的：
atggatctgccttactaccatggacgtctgaccaagcaagactgtgagaccttgctgctcaaggaaggggtggatggcaactttcttttaagagacagcgagtcgataccaggagtcctgtgcctctgtgtctcgtttaaaaatattgtctacacataccgaatcttcagagagaaacacgggtattacaggatacagactgcagaaggttctccaaaacaggtctttccaagcctaaaggaactgatctccaaatttgaaaaaccaaatcaggggatggtggttcaccttttaaagccaataaagagaaccagccccagcttgagatggagaggattgaaattagagttggaaacatttgtgaacagtaacagcgattatgtggatgtcttgccttga。
本发明一实施例的慢病毒载体系统，含有前述的核酸构建物及慢病毒载体辅助成分。
所述慢病毒辅助成分包括慢病毒包装质粒和细胞系。
所述慢病毒载体系统是前述核酸构建物在慢病毒包装质粒、细胞系的辅助下，经过病毒包装而成。所述慢病毒载体系统构建方法为本领域常用方法。
本发明一实施例的T细胞，表达前述的PD1变体。
可选的，所述T细胞含有前述的多核苷酸，或含有前述的核酸构建物，或感染了前述慢病毒载体系统。
前述的PD1变体或多核苷酸，或核酸构建物，或慢病毒载体系统在制备以下任一种或多种用途产品中的应用：(1)制备T细胞；(2)降低T细胞在肿瘤微环境中受到PD-L1/PD1信号通路的抑制；(3)增强T细胞的抗肿瘤功能。
前述的PD1变体或多核苷酸，或核酸构建物，或慢病毒载体系统，或T细胞在制备肿瘤治疗产品或抗慢性感染产品中的应用。
本发明还提供一种治疗肿瘤的方法，为向对象施用前述的PD1变体或多核苷酸，或核酸构建物，或慢病毒载体系统，或T细胞。
所述的对象可以为哺乳动物或哺乳动物的癌细胞。所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述灵长目动物优选为猴、猿或人。所述癌细胞可以为离体癌细胞。
所述对象可以是罹患癌症的患者或者期待治疗的癌症的个体。或者所述对象为癌症患者或者期待治疗癌症的个体的离体癌细胞。
前述的PD1变体或多核苷酸，或核酸构建物，或慢病毒载体系统，或T细胞可以在接受肺癌治疗前、中、后向对象施用。
本发明还提供一种抗慢性感染的方法，为向对象施用前述的PD1变体或多核苷酸，或核酸构建物，或慢病毒载体系统，或T细胞。
所述的对象可以为哺乳动物或哺乳动物的感染细胞。所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述灵长目动物优选为猴、猿或人。所述感染细胞可以为离体感染细胞。
所述对象可以是罹患慢性感染的患者或者期待治疗的慢性感染的个体。或者所述对象为慢性感染患者或者期待治疗慢性感染的个体的离体感染细胞。
前述的PD1变体或多核苷酸，或核酸构建物，或慢病毒载体系统，或T细胞可以在接受慢性感染治疗前、中、后向对象施用。
本发明所述的T细胞可以为CAR-T细胞或TCR-T细胞。
本发明的基因修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如相关的细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的基因修饰的T细胞，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的基因修饰的T细胞的药物组合物可以以104至109个细胞/kg体重的剂量，优选105至106个细胞/kg体重的剂量。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内注射或腹膜内施用给患者。在一个实施方案中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方案中，本发明的T细胞组合物优选通过静脉注射施用。T细胞的组合物可被直接注入肿瘤、淋巴结或感染位置。
在本发明的一些实施方案中，本发明的基因修饰的T细胞或其组合物可与本领域已知的其它疗法结合。所述疗法包括但不限于化疗、放疗和免疫抑制剂。例如，可结合各种放疗制剂进行治疗，这些放疗制剂包括：环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯、FK506、氟达拉滨、雷帕霉素和麦考酚酸等。在进一步的实施方案中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺或抗体诸如OKT3或CAMPATH的T细胞烧蚀疗法结合(例如，之前、同时或之后)而施用给患者。
本文中，“肿瘤治疗”指一种生物学效应，其可由肿瘤体积的减少、肿瘤细胞数的减少、转移数的减少、预期寿命的增加或与癌相关的各种生理症状的改善表示。所述肿瘤选自结肠癌、直肠癌、肺癌、肝癌、卵巢癌、黑色素瘤、肾癌、胰腺癌、皮肤癌、胶质瘤、前列腺癌、膀胱癌、胃肠道癌、乳腺癌、脑癌和白血病等。
本文中，“抗慢性感染”是指一种生物学效应，可由为降低体内慢性病毒载量、缓解慢性病毒感染导致的机体损伤、与慢性病毒感染相关的各种生理症状的改善表示。慢性感染相关的疾病可以为人类乙肝病毒(HBV)、人类丙肝病毒(HCV)和人类免疫缺陷病毒(HIV)等。
“患者”、“对象”、“个体”等等在本文中可交换使用，指可引起免疫应答的活有机体，如哺乳动物。例子包括但不限于人、狗、猫、小鼠、大鼠和其转基因物种。
实施例1构建PD1变体的表达载体
以SLAMF1的第二个ITSM基序为例(SEQ ID NO：9)，通过基因合成技术获得PD1变体，其开放编码框序列如SEQ ID NO：36所示(PD1-SLAMF1-ITSM2)。与野生型PD1的ITSM基序9个氨基酸(以酪氨酸为中心前后各延伸4个氨基酸残基，EQTEYATIV；SEQ ID NO：1)相比，该变体的ITSM基序为SLAMF1的第二个ITSM基序(SEQ ID NO：9)。进一步通过酶切(NheI和BamHI位点)酶连将得到的PD1变体序列插入到pHAGE表达载体(pHAGE-fullEF1a-MCS-IRES-ZsGreen)中。
ATGGTACCGTGCACGCTGCTCCTGCTGTTGGCGGCCGCCCTGGCTCCGACTCAGACCCGCGCGAAGTACCCCTACGACGTGCCCGACTACGCCAGCCTGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTTCGATCACGGTGTATGCCAGCGTTACCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA(SEQ ID NO：36)。
其他PD1变体的构建方法与PD1-SLAMF1-ITSM2相同，区别仅在于，与野生型PD1的ITSM基序9个氨基酸(以酪氨酸为中心前后各延伸4个氨基酸残基，SEQ ID NO：1)相比，该变体的ITSM基序为表1中的其他的活化型ITSM基序，获得能够表达SEQ ID NO：18-32所述的各个PD1变体。
实施例2构建改造PD1与EAT-2共表达载体
通过逆转录和PCR扩增得到人源EAT-2开放阅读框序列(SEQ ID NO：35)，将EAT-2开放阅读框序列构建到实施例1获得的PD1变体(PD1-SLAMF1-ITSM2)的表达载体中，通过pHAGE载体中的内部核糖体进入位点(IRES)连接两个序列，在一个载体中同时表达两个蛋白。
实施例3在T细胞系Jurkat中验证PD1变体对Jurkat细胞功能的影响
将野生型PD1和SEQ ID NO：18-32所述的各个PD1变体构建到pHAGE载体中，将PD1表达质粒与包装质粒pSPAX2和pMD2G分别按照10：7.5：3.5的比例混合后用磷酸钙转染的方法转入293FT细胞以生产慢病毒颗粒。使用慢病毒上清感染Jurkat细胞系，获得表达改造PD1的Jurkat细胞系。
通过分选获得细胞表面PD1表达量接近的Jurkat细胞系，进行Jurkat细胞功能实验。将10万表达PD1的Jurkat与5万预先孵育超抗原SEE的表达PD-L1或载体对照的Raji细胞在圆底96孔板中共培养24小时，收取细胞上清，用IL-2ELISA试剂盒检测上清中IL-2的浓度。根据不表达PD-L1的Raji细胞刺激下IL-2产生的量对表达PD-L1的Raji细胞刺激产生的IL-2进行归一化，得到的IL-2产生相对量可以表征PD1及变体的抑制能力。(IL-2产生相对量越高，表明PD1及变体的抑制能力越弱)结果如图1所示，将野生型的PD1(WT)的ITSM基序替换为不同ITSM基序之后，PD-L1对Jurkat细胞分泌IL-2的功能的抑制有不同程度的变弱，其中改造为SLAMF1的第二个ITSM序列的PD1变体(PD1-SLAMF1-ITSM2)的抑制功能减弱最显著。
为了进一步检验改造为SLAMF1第二个ITSM的PD1变体(SEQ ID NO：25，图中标识为PD1-M2)的抑制功能减弱程度，采用本实施例前述的方法，以无功能的胞内段截短PD1(PD1-tailless)(SEQ ID NO：37)为对照，检测表达PD1-WT、PD1-tailless和PD1-M2的Jurkart细胞在PD-L1作用下的相对IL-2产生；如图2结果所示，PD1-M2对Jurkat细胞IL-2分泌的抑制能够与无功能的PD1-tailless对照几乎一致。
PD1-tailless(SEQ ID NO：37)：
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIG。
采用本实施例前述的方法，本发明还对PD1-M2中影响抑制功能的关键氨基酸残基进行了定位，以酪氨酸为0点，检测PD1 ITSM左边或右边4个氨基酸残基替换为SLAMF1的第二个ITSM中对应的氨基酸残基，获得PD1-M2_L，(SEQ ID NO：33)和PD1-M2_R，(即将野生型PD1做以下突变：T250S、I251V、V252T)，然后检测Jurkat细胞在PD-L1作用下的相对IL-2产生；结果如图3A所示，PD1-M2_L会显著降低抑制功能。
采用本实施例前述的方法，本发明还将野生型PD1的ITSM基序中的位点进行突变，以酪氨酸为0点，将野生型PD1的ITSM基序的左边非保守位点中任意两个氨基酸残基替换为SLAMF1的第二个ITSM中对应的氨基酸残基，分别获得PD1-E244S/Q245I、PD1-E244S/E247V、PD1-Q245I/E247V，然后检测Jurkat细胞在PD-L1作用下的相对IL-2产生。结果如图3B所示，将野生型PD1的ITSM基序中-3和-1位点(以酪氨酸为0点)替换为SLAMF1的第二个ITSM中对应的氨基酸残基，获得的PD1变体PD1-Q245I/E247V会显著降低抑制功能。
实施例4在T细胞系Jurkat中验证改造后PD1与EAT-2共表达对Jurkat细胞功能的影响
将EAT-2，和PD1表达质粒与包装质粒pSPAX2和pMD2G分别按照10：7.5：3.5的比例混合后用磷酸钙转染的方法转入293FT细胞以生产慢病毒颗粒。使用慢病毒上清感染Jurkat细胞系，获得共表达EAT-2和PD1变体的Jurkat细胞系。
通过分选获得细胞表面PD1表达量接近的Jurkat细胞系，进行Jurkat细胞功能实验。将10万Jurkat与5万预先孵育超抗原SEE的表达PD-L1或载体对照的Raji细胞在圆底96孔板中共培养24小时，收取细胞上清，用IL-2ELISA试剂盒检测上清中IL-2的浓度。结果如图图4所示，将野生型PD1 ITSM基序替换为SLAMF1的第一个ITSM(图中为PD1-M1，即SEQ IDNO：24)或第二个ITSM序列(图中为PD1-M2，即SEQ ID NO：25)能够减弱PD1对Jurkat细胞IL-2分泌的抑制，而加入EAT-2的表达能够将PD1变体对Jurkat细胞IL-2分泌的抑制转变为增强。
为了研究PD1-M2抑制功能降低以及在EAT-2共表达下介导正向信号的机制，本发明通过免疫共沉淀和免疫印迹来检测PD1-M2的SHP2和EAT-2的招募功能以及下游信号通路。将1千万Jurkat细胞和1千万预先孵育超抗原SEE的表达PD-L1的Raji细胞在EP管中混匀，通过离心使两种细胞充分接触，然后在37摄氏度水浴中刺激不同时间点，裂解细胞后进行免疫共沉淀和免疫印迹实验。结果如图5A所示，在没有EAT-2共表达的情况下PD1-M2招募SHP2的能力相对PD1-WT显著下降。而在共表达EAT-2的情况下，PD1-M2招募EAT-2而PD1-WT招募SHP2(图5B)。进一步检测下游信号分子，发现在共表达EAT-2的情况下，PD1-WT显著降低PLCγ1的磷酸化，而PD1-M2则显著增强PLCγ1的磷酸化(图5C)。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。
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<110> 中国科学院分子细胞科学卓越创新中心
<120> 一种PD1变体及其用途
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115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Ala Tyr Thr Leu Tyr Ser Leu Ile Gln Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 23
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Asn Ser Thr Ile Tyr Glu Val Ile Gly Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 24
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Ser Leu Thr Ile Tyr Ala Gln Val Gln Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 25
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Ser Ile Thr Val Tyr Ala Ser Val Thr Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 26
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Lys Lys Thr Ile Tyr Thr Tyr Ile Met Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 27
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Val Asn Thr Val Tyr Ser Glu Val Gln Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 28
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Asn Asn Thr Val Tyr Ala Ser Val Thr Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 29
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Thr Ile Thr Ile Tyr Ser Thr Ile Asn Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 30
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Glu Asn Thr Glu Tyr Asp Thr Ile Pro Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 31
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Ala Asn Thr Val Tyr Ser Thr Val Glu Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 32
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Leu Lys Thr Gly Tyr Leu Ser Ile Val Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 33
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Ser Ile Thr Val Tyr Ala Thr Ile Val Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 34
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205
Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
210 215 220
Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240
Cys Val Pro Glu Ile Thr Val Tyr Ala Thr Ile Val Phe Pro Ser Gly
245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285
<210> 35
<211> 399
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
atggatctgc cttactacca tggacgtctg accaagcaag actgtgagac cttgctgctc 60
aaggaagggg tggatggcaa ctttctttta agagacagcg agtcgatacc aggagtcctg 120
tgcctctgtg tctcgtttaa aaatattgtc tacacatacc gaatcttcag agagaaacac 180
gggtattaca ggatacagac tgcagaaggt tctccaaaac aggtctttcc aagcctaaag 240
gaactgatct ccaaatttga aaaaccaaat caggggatgg tggttcacct tttaaagcca 300
ataaagagaa ccagccccag cttgagatgg agaggattga aattagagtt ggaaacattt 360
gtgaacagta acagcgatta tgtggatgtc ttgccttga 399
<210> 36
<211> 906
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
atggtaccgt gcacgctgct cctgctgttg gcggccgccc tggctccgac tcagacccgc 60
gcgaagtacc cctacgacgt gcccgactac gccagcctgc caggatggtt cttagactcc 120
ccagacaggc cctggaaccc ccccaccttc tccccagccc tgctcgtggt gaccgaaggg 180
gacaacgcca ccttcacctg cagcttctcc aacacatcgg agagcttcgt gctaaactgg 240
taccgcatga gccccagcaa ccagacggac aagctggccg ccttccccga ggaccgcagc 300
cagcccggcc aggactgccg cttccgtgtc acacaactgc ccaacgggcg tgacttccac 360
atgagcgtgg tcagggcccg gcgcaatgac agcggcacct acctctgtgg ggccatctcc 420
ctggccccca aggcgcagat caaagagagc ctgcgggcag agctcagggt gacagagaga 480
agggcagaag tgcccacagc ccaccccagc ccctcaccca ggccagccgg ccagttccaa 540
accctggtgg ttggtgtcgt gggcggcctg ctgggcagcc tggtgctgct agtctgggtc 600
ctggccgtca tctgctcccg ggccgcacga gggacaatag gagccaggcg caccggccag 660
cccctgaagg aggacccctc agccgtgcct gtgttctctg tggactatgg ggagctggat 720
ttccagtggc gagagaagac cccggagccc cccgtgccct gtgtcccttc gatcacggtg 780
tatgccagcg ttacctttcc tagcggaatg ggcacctcat cccccgcccg caggggctca 840
gctgacggcc ctcggagtgc ccagccactg aggcctgagg atggacactg ctcttggccc 900
ctctga 906
<210> 37
<211> 201
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly
195 200
Claims (16)
1.一种PD1变体，其特征在于，所述PD1变体含有ITSM基序突变：
1)所述PD1变体的ITSM基序为活化型ITSM基序；或，
2)所述PD1变体的ITSM基序含有活化型ITSM基序的部分氨基酸残基或其片段。
2.如权利要求1所述的PD1变体，其特征在于，还包括以下特征中的一项或多项：
a)所述活化型ITSM基序的氨基酸序列如SEQ ID NO：2-16任一所示；
b)所述PD1变体为人源且具有PD-L1结合活性；
c)所述PD1变体相比野生型PD1，使免疫细胞经PD-L1刺激后产生的IL-2分泌抑制作用减弱；或者，所述PD1变体使免疫细胞经PD-L1刺激后促进IL-2分泌；
d)所述PD1变体的氨基酸序列如SEQ ID NO：18-32任一所示。
3.如权利要求1所述的PD1变体，其特征在于，所述方案2)中，以野生型PD1序列为基准，所述PD1变体的ITSM基序的突变位点选自以下位点中的一个或多个位点：244、245、246、247、249、250、251、252，所述野生型PD1的氨基酸序列如SEQ ID NO：17所示。
4.如权利要求3所述的PD1变体，其特征在于，
所述244位点的突变选自：E244A、E244R、E244N、E244D、E244C、E244Q、E244G、E244H、E244I、E244L、E244K、E244M、E244F、E244P、E244S、E244T、E244W、E244Y、E244V之任一；
所述245位点的突变选自：Q245A、Q245R、Q245N、Q245D、Q245C、Q245E、Q245G、Q245H、Q245I、Q245L、Q245K、Q245M、Q245F、Q245P、Q245S、Q245T、Q245W、Q245Y、Q245V之任一；
所述246位点的突变选自：T246A、T246R、T246N、T246D、T246C、T246Q、T246G、T246H、T246I、T246L、T246K、T246M、T246F、T246P、T246S、T246E、T246W、T246Y、T246V之任一；
所述247位点的突变选自：E247A、E247R、E247N、E247D、E247C、E247Q、E247G、E247H、E247I、E247L、E247K、E247M、E247F、E247P、E247S、E247T、E247W、E247Y、E247V之任一；
所述249位点的突变选自：A249E、A249R、A249N、A249D、A249C、A249Q、A249G、A249H、A249I、A249L、A249K、A249M、A249F、A249P、A249S、A249T、A249W、A249Y、A249V之任一；
所述250位点的突变选自：T250A、T250R、T250N、T250D、T250C、T250Q、T250G、T250H、T250I、T250L、T250K、T250M、T250F、T250P、T250S、T250E、T250W、T250Y、T250V之任一；
所述251位点的突变选自：I251A、I251R、I251N、I251D、I251C、I251Q、I251G、I251H、I251E、I251L、I251K、I251M、I251F、I251P、I251S、I251T、I251W、I251Y、I251V之任一；
所述252位点的突变选自：V252A、V252R、V252N、V252D、V252C、V252Q、V252G、V252H、V252I、V252L、V252K、V252M、V252F、V252P、V252S、V252T、V252W、V252Y、V252E之任一。
5.如权利要求4所述的PD1变体，其特征在于，还包括以下特征中的一项或多项：
a)所述突变至少包括Q245I和E247V的突变；
b)述PD1变体的氨基酸序列如SEQ ID NO：33-34任一所示。
6.一种多核苷酸，编码权利要求1-5任一所述的PD1变体。
7.一种核酸构建物，所述核酸构建物含有权利要求6所述的多核苷酸，能够表达所述PD1变体。
8.如权利要求7所述的核酸构建物，其特征在于，所述核酸构建物还含有编码EAT-2蛋白的多核苷酸，能够同时表达所述PD1变体和EAT-2蛋白。
9.如权利要求8所述的核酸构建物，其特征在于，还包括以下特征中的一项或多项：
编码EAT-2蛋白的多核苷酸序列如SEQ ID NO：35所示。
10.一种慢病毒载体系统，所述慢病毒载体系统含有权利要求7-9任一所述的核酸构建物及慢病毒载体辅助成分。
11.一种T细胞，其特征在于，所述T细胞表达权利要求1-5中任一项所述的PD1变体。
12.如权利要求11所述的T细胞，其特征在于，所述T细胞含有权利要求6所述的多核苷酸，或含有权利要求7-9任一所述的核酸构建物，或感染了权利要求10所述的慢病毒载体系统。
13.如权利要求11或12所述的T细胞，其特征在于，所述T细胞共表达EAT-2蛋白。
14.如权利要求13所述的T细胞，其特征在于，所述T细胞经PD-L1刺激后增强IL-2分泌。
15.权利要求1-5中任一项所述的PD1变体或权利要求6所述的多核苷酸，或权利要求7-9任一所述的核酸构建物，或权利要求10所述的慢病毒载体系统在制备以下任一种或多种用途产品中的应用：(1)制备T细胞；(2)降低T细胞在肿瘤微环境中受到PD-L1/PD1信号通路的抑制；(3)增强T细胞的抗肿瘤功能。
16.权利要求1-5中任一项所述的PD1变体或权利要求6所述的多核苷酸，或权利要求7-9任一所述的核酸构建物，或权利要求10所述的慢病毒载体系统，或权利要求11-14任一所述的T细胞在制备肿瘤治疗产品或抗慢性感染产品中的应用。
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| WO2020123806A1 (en) * | 2018-12-14 | 2020-06-18 | Beth Israel Deaconess Medical Center. Inc. | Modulation of pd-1 |
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