CN114206928A - Cd33靶向免疫疗法 - Google Patents
Cd33靶向免疫疗法 Download PDFInfo
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Abstract
本公开提供了用于过继性T细胞疗法的改进的CD33靶向多肽和组合物,用于治疗、预防或改善癌症、传染病、自身免疫性疾病、炎性疾病和免疫缺陷的至少一种症状或与其相关的病状。
Description
相关申请的交叉引用
本申请根据35U.S.C.§119(e)要求2019年9月10日提交的美国临时申请第62/898,392号以及2019年5月8日提交的美国临时申请第62/845,304号的权益,以上申请各自通过引用整体并入本文。
关于序列表的声明
与本申请相关联的序列表是以文本格式提供以代替纸质副本,并以引用的方式并入本说明书中。含有序列表的文本文件的名称是BLBD_119_02WO_ST25.txt。文本文件为302KB,创建于2020年5月5日,并在本说明书提交的同时,经由EFS-Web以电子方式提交。
背景技术
技术领域
本公开涉及针对CD33的改进的过继性细胞疗法。更具体地,本公开涉及含抗CD33VHH的化学调节信号传导分子、含抗CD33 VHH的嵌合抗原受体、细胞以及使用它们的相关治疗方法。
现有技术
全球癌症负担在1975年与2000年间翻倍。癌症是世界范围内导致发病率和死亡率的第二大主导原因,并且在2012年间有约1410万例新增病例和820万例癌症相关死亡。最常见的癌症是乳癌、肺和支气管癌、前列腺癌、结肠和直肠癌症、膀胱癌、皮肤黑素瘤、非霍奇金氏淋巴瘤(non-Hodgkin lymphoma)、甲状腺癌、肾脏和肾盂癌、子宫内膜癌、白血病以及胰腺癌。预计在接下来的二十年里新增癌症病例的数量将增加至2200万例。
过继性细胞疗法正在成为一种用于递送复杂生物信号以治疗癌症的强大范例。与小分子和生物药物组合物相比,过继性细胞疗法由于其极大数量的感觉和应答程序以及日益明确的遗传控制机制而具有执行独特治疗任务的潜力。现有方法主要集中在基于scFv的嵌合抗原受体(CAR)。CART细胞疗法由于CAR表达差、CART细胞的体内扩增、输注后细胞快速消失、令人失望的临床活性和抗原逃逸而获得有限的成功。
需要用改进的CAR架构(CAR结构)和/或改进的机制来改造免疫效应细胞,以用于感测和整合与局部生理环境相关联的化学和/或生物学信息。
发明内容
本公开总体上部分涉及针对CD33的基于VHH的二聚化剂调节的免疫受体复合物(DARIC)和基于VHH的嵌合抗原受体(CAR)、编码它们的多核苷酸、它们的组合物以及制备和使用它们治疗癌症的方法。
在特定实施方案中,VHH DARIC或VHH CAR结合全长CD33。在特定实施方案中,VHHDARIC或VHH CAR结合CD33剪接变体。在某些实施方案中,CD33剪接变体缺乏由人CD33基因的外显子2编码的124个氨基酸(CD33 C2变体)。在某些实施方案中,CD33剪接变体缺乏由驻留于外显子7a中的早期翻译停止信号导致的54个羧基末端氨基酸。在某些实施方案中,CD33剪接变体缺乏由外显子2编码的124个氨基酸和由驻留于外显子7a中的早期翻译停止信号导致的54个羧基末端氨基酸。
在特定实施方案中,VHH DARIC或VHH CAR结合全长CD33和CD33剪接变体两者。
在各种实施方案中,非天然细胞包含:第一多肽,该第一多肽包含:FRB多聚化结构域多肽或其变体,CD8α跨膜结构域或CD4跨膜结构域,CD137共刺激结构域,和/或CD3ζ初级信号传导结构域;和第二多肽,该第二多肽包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33VHH抗体,FKBP多聚化结构域多肽或其变体,和CD4跨膜结构域或CD8α跨膜结构域;其中桥接因子促进在非天然细胞的表面上形成多肽复合物,该桥接因子与第一多肽和第二多肽的多聚化结构域缔合并安置在多聚化结构域之间。
在特定实施方案中,抗CD33VHH抗体具有SEQ ID NO:10中所示的氨基酸序列。
在特定实施方案中,抗CD33VHH抗体具有SEQ ID NO:20中所示的氨基酸序列。
在特定实施方案中,FKBP多聚化结构域是FKBP12。
在一些实施方案中,FRB多肽是FRB T2098L。
在某些实施方案中,桥接因子选自由以下组成的组:AP21967、西罗莫司、依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。
在各种实施方案中,第一多肽包含信号肽、CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域。
在特定实施方案中,第二多肽包含信号肽和CD4跨膜结构域。
在另外的实施方案中,第二多肽包含共刺激结构域。
在一些实施方案中,第二多肽的共刺激结构域选自由以下组成的组的共刺激分子:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNFRS14、TNFRS18、TNRFS25和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
在另外的实施方案中,第二多肽的共刺激结构域是从OX40或TNFR2中分离的共刺激结构域。
在另外的实施方案中,第二多肽包含SEQ ID NO:22-31中任一者所示的序列。
在特定实施方案中,第二多肽包含SEQ ID NO:30中所示的序列。
在优选的实施方案中,第一多肽包含SEQ ID NO 82中所示的氨基酸序列。
在各种实施方案中,非天然细胞包含多肽复合物,该复合物包含:第一多肽,该第一多肽包含:FRB多聚化结构域多肽或其变体,CD8α跨膜结构域或CD4跨膜结构域,CD137共刺激结构域,和/或CD3ζ初级信号传导结构域;第二多肽,该第二多肽包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体,FKBP多聚化结构域多肽或其变体,和CD4跨膜结构域或CD8α跨膜结构域;以及桥接因子,该桥接因子与第一多肽和第二多肽的多聚化结构域缔合并安置在多聚化结构域之间。
在特定实施方案中,抗CD33 VHH抗体具有SEQ ID NO:10中所示的氨基酸序列。
在特定实施方案中,抗CD33 VHH抗体具有SEQ ID NO:20中所示的氨基酸序列。
在特定实施方案中,FKBP多聚化结构域是FKBP12。
在某些实施方案中,FRB多肽是FRB T2098L。
在一些实施方案中,桥接因子选自由以下组成的组:AP21967、西罗莫司、依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。
在另外的实施方案中,第一多肽包含信号肽、CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域。
在特定实施方案中,第二多肽包含信号肽和CD4跨膜结构域。
在一些实施方案中,第二多肽包含共刺激结构域。
在各种实施方案中,第二多肽的共刺激结构域选自由以下组成的组的共刺激分子:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNFRS14、TNFRS18、TNRFS25和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
在另外的实施方案中,第二多肽的共刺激结构域是从OX40或TNFR2中分离的共刺激结构域。
在另外的实施方案中,第二多肽包含SEQ ID NO:22-31中任一者所示的序列。
在特定实施方案中,第二多肽包含SEQ ID NO:30中所示的序列。
在优选的实施方案中,第一多肽包含SEQ ID NO 82中所示的氨基酸序列。
在某些实施方案中,细胞是造血细胞。
在特定实施方案中,细胞是T细胞、αβ T细胞或γδ T细胞。
在另外的实施方案中,细胞是CD3+、CD4+和/或CD8+细胞。
在各种实施方案中,细胞是免疫效应细胞。
在一些实施方案中,细胞是细胞毒性T淋巴细胞、肿瘤浸润淋巴细胞或辅助T细胞。
在附加的实施方案中,细胞是自然杀伤(NK)细胞或自然杀伤T(NKT)细胞。
在各种实施方案中,细胞的来源是外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织或肿瘤。
在特定实施方案中,当第一多肽和第二多肽被表达时,FRB多聚化结构域和FKBP多聚化结构域在细胞外定位。
在一些实施方案中,融合多肽包含:第一多肽,该第一多肽包含:FRB多聚化结构域多肽或其变体,CD8α跨膜结构域或CD4跨膜结构域,CD137共刺激结构域,和/或CD3ζ初级信号传导结构域,多肽裂解信号;和第二多肽,该第二多肽包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体,FKBP多聚化结构域多肽或其变体,和CD4跨膜结构域或CD8α跨膜结构域。
在特定实施方案中,抗CD33 VHH抗体具有SEQ ID NO:10中所示的氨基酸序列。
在特定实施方案中,FKBP多聚化结构域是FKBP12。
在某些实施方案中,FRB多肽是FRB T2098L。
在一些实施方案中,桥接因子选自由以下组成的组:AP21967、西罗莫司、依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。
在另外的实施方案中,第一多肽包含信号肽、CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域。
在特定实施方案中,第二多肽包含信号肽和CD4跨膜结构域。
在某些实施方案中,融合多肽包含SEQ ID NO:32-41中任一者所示的序列。
在某些实施方案中,融合多肽包含SEQ ID NO:40中所示的序列。
在另外的实施方案中,第二多肽包含共刺激结构域。
在各种实施方案中,第二多肽的共刺激结构域选自由以下组成的组的共刺激分子:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNFRS14、TNFRS18、TNRFS25和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
在另外的实施方案中,第二多肽的共刺激结构域是从OX40或TNFR2中分离的共刺激结构域。
在某些实施方案中,多肽裂解信号是病毒自裂解多肽。
在特定实施方案中,多肽裂解信号是病毒自裂解2A多肽。
在各种实施方案中,多肽裂解信号是选自由以下组成的组的病毒自裂解多肽:口蹄疫病毒(FMDV)(F2A)肽、马A型鼻炎病毒(ERAV)(E2A)肽、明脉扁刺蛾β四体病毒(TaV)(T2A)肽、猪捷申病毒-1(PTV-1)(P2A)肽、泰勒病毒2A肽以及脑心肌炎病毒2A肽。
在一些实施方案中,融合多肽包含SEQ ID NO:42-61中任一者所示的序列。
在一些实施方案中,融合多肽包含SEQ ID NO:50或60中任一者所示的序列。
在另外的实施方案中,当第一多肽和第二多肽被表达时,FRB多聚化结构域和FKBP多聚化结构域在细胞外定位。
在各种实施方案中,多肽复合物包含:第一多肽,该第一多肽包含:FRB多聚化结构域多肽或其变体,CD8α跨膜结构域或CD4跨膜结构域,CD137共刺激结构域,和/或CD3ζ初级信号传导结构域;第二多肽,该第二多肽包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体,FKBP多聚化结构域多肽或其变体,和CD4跨膜结构域或CD8α跨膜结构域;以及桥接因子,该桥接因子与第一多肽和第二多肽的多聚化结构域缔合并安置在多聚化结构域之间。
在特定实施方案中,抗CD33 VHH抗体具有SEQ ID NO:10中所示的氨基酸序列。
在特定实施方案中,FKBP多聚化结构域是FKBP12。
在另外的实施方案中,FRB多肽是FRB T2098L。
在特定实施方案中,桥接因子选自由以下组成的组:AP21967、西罗莫司、依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。
在某些实施方案中,第一多肽包含CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域。
在各种实施方案中,第二多肽包含CD4跨膜结构域。
在另外的实施方案中,第二多肽包含共刺激结构域。
在一些实施方案中,第二多肽的共刺激结构域选自由以下组成的组的共刺激分子:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNFRS14、TNFRS18、TNRFS25和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
在特定实施方案中,第二多肽的共刺激结构域是从OX40或TNFR2中分离的共刺激结构域。
在某些实施方案中,细胞是造血细胞。
在各种实施方案中,细胞是T细胞、αβT细胞或γδT细胞。
在各种实施方案中,细胞是CD3+、CD4+和/或CD8+细胞。
在另外的实施方案中,细胞是免疫效应细胞。
在一些实施方案中,细胞是细胞毒性T淋巴细胞、肿瘤浸润淋巴细胞或辅助T细胞。
在特定实施方案中,细胞是自然杀伤(NK)细胞或自然杀伤T(NKT)细胞。
在另外的实施方案中,细胞的来源是外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织或肿瘤。
在另外的实施方案中,当第一多肽和第二多肽被表达时,FRB多聚化结构域和FKBP多聚化结构域在细胞外定位。
在优选的实施方案中,第一多肽包含SEQ ID NO 82中所示的氨基酸序列。
在特定实施方案中,嵌合抗原受体(CAR)包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33VHH抗体;铰链结构域;跨膜结构域;一个或多个细胞内共刺激信号传导域;和/或初级信号传导结构域。
在特定实施方案中,抗CD33 VHH抗体具有SEQ ID NO:10中所示的氨基酸序列。
在特定实施方案中,抗CD33 VHH抗体具有SEQ ID NO:20中所示的氨基酸序列。
在各种实施方案中,CAR从5′至3′包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体;铰链结构域;跨膜结构域;一个或多个细胞内共刺激信号传导域;和/或初级信号传导结构域。
在特定实施方案中,抗CD33 VHH抗体具有SEQ ID NO:10中所示的氨基酸序列。
在特定实施方案中,抗CD33 VHH抗体具有SEQ ID NO:20中所示的氨基酸序列。
在某些实施方案中,铰链结构域和跨膜结构域从CD8α、CD27、CD28、CD33、CD37、CD45、CD64、CD71、CD80、CD86、CD 134、CD137、CD152、CD154、AMN和PD1中分离。
在另外的实施方案中,一个或多个共刺激信号传导结构域从选自由以下组成的组的共刺激分子中分离:CD28、CD134、CD137和CD278。
在特定实施方案中,CAR包含CD8α信号肽、CD8α铰链和跨膜结构域、CD134共刺激结构域和CD3ζ初级信号传导结构域。
在另外的实施方案中,CAR包含SEQ ID NO:62-81中任一者所示的氨基酸序列。
在另外的实施方案中,CAR包含SEQ ID NO:70或80中任一者所示的氨基酸序列。
在一些实施方案中,提供了编码本文所涵盖的第一多肽或第二多肽、融合多肽或CAR的多核苷酸。
在各种实施方案中,提供了编码本文所涵盖的第一多肽或第二多肽、融合多肽或CAR的cDNA。
在特定实施方案中,提供了编码本文所涵盖的第一多肽或第二多肽、融合多肽或CAR的RNA。
在另外的实施方案中,提供了包含本文所涵盖的多核苷酸的载体。
在某些实施方案中,载体是表达载体。
在某些实施方案中,载体是转座子。
在另外的实施方案中,载体是piggyBAC转座子或Sleeping Beauty转座子。
在特定实施方案中,载体是病毒载体。
在特定实施方案中,载体是腺病毒载体、腺相关病毒(AAV)载体、疱疹病毒载体、痘苗病毒载体或逆转录病毒载体。
在另外的实施方案中,逆转录病毒载体是慢病毒载体。
在各种实施方案中,慢病毒载体选自由以下组成的组:人免疫缺陷病毒1(HIV-1);人免疫缺陷病毒2(HIV-2)、维斯纳-梅迪病毒(VMV)病毒;山羊关节炎-脑炎病毒(CAEV);马传染性贫血病毒(EIAV);猫免疫缺陷病毒(FIV);牛免疫缺陷病毒(BIV);以及猿猴免疫缺陷病毒(SIV)。
在另外的实施方案中,提供了包含本文所涵盖的第一多肽和第二多肽、融合多肽或CAR的细胞。
在特定实施方案中,细胞是造血细胞。
在某些实施方案中,细胞是免疫效应细胞。
在各种实施方案中,细胞是T细胞、αβ T细胞或γδ T细胞。
在一些实施方案中,细胞表达CD3+、CD4+、CD8+或它们的组合。
在特定实施方案中,细胞是细胞毒性T淋巴细胞(CTL)、肿瘤浸润淋巴细胞(TIL)或辅助T细胞。
在另外的实施方案中,细胞是自然杀伤(NK)细胞或自然杀伤T(NKT)细胞。
在某些实施方案中,组合物包含本文所涵盖的细胞。
在特定实施方案中,组合物包含生理上可接受的载体和本文所涵盖的细胞。
在另外的实施方案中,提供了治疗有需要的受试者的方法,所述方法包括向所述受试者施用有效量的本文所涵盖的组合物。
在特定实施方案中,一种治疗、预防或改善癌症、传染病、自身免疫性疾病、炎性疾病和免疫缺陷的至少一种症状或与其相关的病状的方法包括向受试者施用有效量的本文所涵盖的组合物。
在一些实施方案中,一种治疗实体癌的方法,所述方法包括向受试者施用有效量的本文所涵盖的组合物。
在各种实施方案中,实体癌选自由以下组成的组:肺癌、肝癌、胃癌、结直肠癌、头颈癌、尿路上皮癌、前列腺癌、睾丸癌、子宫内膜癌、胰腺癌、乳腺癌、宫颈癌、卵巢癌、皮肤癌和黑色素瘤。
在某些实施方案中,一种治疗血液恶性肿瘤的方法包括向受试者施用有效量的本文所涵盖的组合物。
在各种实施方案中,血液恶性肿瘤是白血病、淋巴瘤或多发性骨髓瘤。
在特定实施方案中,恶性血液病是急性骨髓性白血病(AML)。
附图说明
图1A示出了VHH-DARIC多肽复合物的草图。
图1B示出了CD33 VHH DARIC架构的草图。
图2A示出了如通过抗VHH染色(顶行)和通过CD33-Fc结合(底行)检测的CD33VHH1-5 DARIC在转导的T细胞中的表达。
图2B示出了如通过CD33-Fc结合检测的CD33 VHH9-10 DARIC在转导的T细胞中的表达。
图3A示出了用CD33 VHH1-5 DARIC或对照转导的T细胞的表型。
图3B示出了用CD33 VHH9-10 DARIC或对照转导的T细胞的表型。
图4A示出了来自与CD33+THP-1细胞以1∶1的E∶T比率在存在或不存在AP21967的情况下一起培养24小时的CD33 VHH1-5 DARIC或对照细胞的IFNγ分泌。
图4B示出了来自与CD33+THP-1细胞以1∶1的E∶T比率在存在或不存在AP21967的情况下一起培养24小时的CD33 VHH9-10 DARIC或对照细胞的IFNγ分泌。
图4C示出了来自与表达全长CD33(CD33M)或CD33剪接变体(CD33m,C2)的经过修饰的293T细胞以1∶1的E∶T比率在存在或不存在AP21967的情况下一起培养24小时的CD33VHH9-10 DARIC或对照细胞的IFNγ分泌。
图5A示出了在MV4-11细胞、经工程改造以敲除CD33基因的MV4-11细胞(CD33-KO细胞)和未染色对照中的CD33表达。
图5B示出了来自与MV4-11细胞或CD33-KO细胞以1∶1的E∶T比率在存在或不存在AP21967的情况下共培养24小时的抗CD33 VHH9 DARIC T细胞或UTD T细胞的IFNγ分泌。
图5C示出了来自与MV4-11细胞(左图)或经工程改造以表达CD33m剪接变体的CD33-KO细胞(CD33-KO-C2细胞;右图)以1∶1的E∶T比率在存在或不存在AP21967的情况下共培养24小时的UTD T细胞、抗CD33 CAR T细胞或抗CD33VHH DARIC T细胞的IFNγ分泌。
图6示出了来自与CD33+THP-1细胞以1∶1的E∶T比率在存在或不存在可溶性CD33(CD33-Fc)和AP21967的情况下共培养24小时的抗CD33 VHH DARIC T细胞的IFNγ分泌。
图7示出了来自与用不同量的编码CD33的mRNA转染的CD33neg 293T细胞以1∶1的E∶T比率在存在或不存在AP21967的情况下共培养24小时的抗CD33 VHH DARIC T细胞的IFNγ分泌。
图8A示出了在用表达荧光素酶报告基因的HL60 AML肿瘤细胞接种并在接种后10天(第0天)用UTD T细胞或抗CD33 VHH DARIC T细胞在不存在雷帕霉素的情况下治疗的免疫缺陷NSG小鼠中作为发光的函数测量的肿瘤生长。
图8B示出了在用表达荧光素酶报告基因的HL60 AML肿瘤细胞接种并在接种后10天(第0天)用UTD T细胞或抗CD33 VHH DARIC T细胞和0.1mg/kg雷帕霉素治疗的免疫缺陷NSG小鼠中作为发光测量的肿瘤生长。
序列标识符简述
SEQ ID NO:1列出了全长人CD33的氨基酸序列。
SEQ ID NO:2-21列出了抗CD33 VHH结构域的氨基酸序列。
SEQ ID NO:22-31列出了抗CD33 VHH DARIC结合组分的氨基酸序列。
SEQ ID NO:32-41列出了抗CD33 VHH DARIC融合蛋白的氨基酸序列。
SEQ ID NO:42-51列出了抗CD33VHH DARIC.OX40融合蛋白的氨基酸序列。
SEQ ID NO:52-61列出了抗CD33 VHH DARIC.TNFR2融合蛋白的氨基酸序列。
SEQ ID NO:62-81列出了抗CD33VHH CAR的氨基酸序列。
SEQ ID NO:82列出了抗CD33 VHH DARIC信号传导组分的氨基酸序列。
SEQ ID NO:83列出了Kozak序列的多核苷酸序列。
SEQ ID NO:84-94列出了各种接头的氨基酸序列。
SEQ ID NO:95-119列出了蛋白酶裂解位点和自裂解多肽裂解位点的氨基酸序列。
在前述序列中,Xaa,如果存在,可指任何氨基酸或不存在某一氨基酸。在优选的实施方案中,XaaXaa是指氨基酸序列SS或KP。
具体实施方案
A.概述
癌症是世界范围内死亡的主要原因之一。约10%的癌症是血液恶性肿瘤,其包括白血病、淋巴瘤和骨髓瘤。急性髓性白血病(AML)是成人中最常见的和致命的血液恶性肿瘤。尽管在过去的四十年中有了重大的科学发现和新的疗法,但AML的治疗结果,尤其是在成年患者群体中,仍然是令人沮丧的。标准化学疗法可在所选择的患者中诱导完全缓解;然而,大多数患者最终复发并死于该疾病。2012年,AML的全球发病率为约351,965人,并且约265,461人死于AML。
CD33在大多数急性髓性白血病(AML)白血病母细胞和可能的白血病干细胞上表达。CD33由于其低表达和缓慢内化而成为具有挑战性的靶标;这些特征限制了抗体依赖性细胞介导的细胞毒性和细胞内药物累积,并且因此限制了未标记的和携带毒素的抗体的活性。
本公开总体上涉及用于使用结合CD33的二聚化剂调节的免疫受体复合物(DARIC)来调节过继性细胞疗法的空间和时间控制的改进的组合物和方法。不希望受到任何特定理论的束缚,本文所涵盖的DARIC组合物和方法提供了优于本领域中现有的CAR T细胞疗法的许多优点,包括但不限于对免疫效应细胞信号转导结合和信号传导活性的空间和时间两者的控制。DARIC时间控制通过桥接因子介导的DARIC结合组分与DARIC信号传导组分之间的缔合来引发信号传导的DARIC机制。DARIC空间控制通过DARIC结合组分的DARIC结合结构域识别CD33来参与信号传导机制。以此方式,当表达CD33的靶细胞和桥接因子两者存在时,DARIC免疫效应细胞被活化。
本公开还涉及克服现有CAR T疗法的潜在限制的改进的抗CD33 CAR架构,这些潜在限制包括但不限于强直信号传导或抗原依赖性信号传导、弱表达和/或亚治疗活性。
在各种实施方案中,本公开涵盖针对癌症例如表达CD33(例如全长CD33和/或CD33剪接变体)的AML生成抗癌应答的抗CD33 VHH DARIC或抗CD33VHH CAR。
在特定实施方案中,DARIC包括多肽(DARIC信号传导组分),该多肽包含多聚化结构域多肽或其变体、跨膜结构域、共刺激结构域和/或初级信号传导结构域;以及多肽(DARIC结合组分),该多肽包含抗CD33 VHH、多聚化结构域多肽或其变体、跨膜结构域和任选的共刺激结构域。在存在桥接因子的情况下,DARIC结合和信号传导组分通过桥接因子相互缔合,以形成靶向表达CD33的细胞的具有功能活性的DARIC。
在特定实施方案中,DARIC结合组分和DARIC信号传导组分的多聚化结构域在细胞外定位。相比于细胞内定位,多聚化结构域的细胞外位置提供许多优点,包括但不限于抗CD33 VHH结构域的更有效定位、对桥接因子调节的更高时间敏感性以及由于使用非免疫抑制剂量的特定桥接因子的能力而毒性较小。
本文涵盖了对DARIC、DARIC结合组分和DARIC信号传导组分进行编码的多核苷酸;DARIC结合组分、DARIC信号传导组分、DARIC蛋白复合物、DARIC融合蛋白;包括对DARIC、DARIC结合组分和DARIC信号传导组分进行编码和/或表达其的多核苷酸的细胞;以及使用其治疗免疫病症的方法。
用于重组(即,工程改造)DNA、肽和寡核苷酸合成的技术、免疫分析、组织培养、转化(例如电穿孔、脂质体转染)、酶促反应、纯化以及相关技术和程序一般可以如本说明书通篇引用和论述的微生物学、分子生物学、生物化学、分子遗传学、细胞生物学、病毒学及免疫学中的各种通用且更特定的参考文献中所描述来执行。参见,例如,Sambrook等人,Molecular Cloning:A Laboratory Manual,第3版,Cold Spring Harbor LaboratoryPress,Cold Spring Harbor,N.Y.;Current Protocols in Molecular Biology(JohnWiley and Sons,2008年7月更新);Short Protocols in Molecular Biology:ACompendium of Methods from Current Protocols in Molecular Biology,GreenePub.Associates and Wiley-Interscience;Glover,DNA Cloning:A PracticalApproach,vol.I&II(IRL Press,Oxford Univ.Press USA,1985);Current Protocols inImmunology(编辑:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,EthanM.Shevach,Warren Strober 2001 John Wiley&Sons,NY,NY);Real-Time PCR:CurrentTechnology and Applications,编辑:Julie Logan,Kirstin Edwards和Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis ofComplex Genomes,(Academic Press,New York,1992);Guthrie和Fink,Guide to YeastGenetics and Molecular Biology(Academic Press,New York,1991);OligonucleotideSynthesis(N.Gait编,1984);Nucleic Acid The Hybridization(B.Hames&S.Higgins编,1985);Transcription and Translation(B.Hames&S.Higgins编,1984);Animal CellCulture(R.Freshney编,1986);Perbal,APractical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park编,第3版,2010Humana Press);ImmobilizedCells And Enzymes(IRL Press,1986);the treatise,Methods In Enzymology(AcademicPress,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Calos编,1987,Cold Spring Harbor Laboratory);Harlow和Lane,Antibodies,(ColdSpring HarborLaboratory Press,Cold Spring Harbor,N.Y.,1998);ImmunochemicalMethods InCellAndMolecular Biology(Mayer和Walker编,Academic Press,London,1987);Handbook Of Experimental Immunology,Volumes I-IV(D.M.Weir和CCBlackwell编,1986);Roitt,Essential Immunology,第6版,(Blackwell ScientificPublications,Oxford,1988);Current Protocols in Immunology(Q.E.Coligan,A.M.Kruisbeek,D.H.Margulies,E.M.Shevach和W.Strober编,1991);Annual Review of Immunology;以及诸如Advances in Immunology的期刊中的专题论文。
B.定义
在更加详细地阐述本公开之前,提供本文要使用的某些术语的定义可能有助于理解本公开。
除非另外定义,否则本文所使用的所有技术和科学术语都具有与本发明所属领域的一般技术人员通常所了解相同的含义。尽管可以使用与本文所描述的方法和材料类似或相当的任何方法和材料来实践或测试特定实施方案,但本文中描述组合物、方法和材料的优选实施方案。出于本公开的目的,下文定义以下术语。
本文使用的冠词“一(a/an)”是指一个(种)或超过一个(种)(即至少一个(种)或一个(种)或多个(种))该冠词的语法对象。作为实例,“一个元件”意思指一个元件或一个或多个元件。
替代选择(例如“或”)的使用应理解为意思指替代选择中的一种、两种或其任何组合。
术语“和/或”应理解为意思指替代选择中的一种或两种。
如本文所使用,术语“约”或“大约”是指相对于参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度变化高达15%、10%、9%、8%、7%、6%、5%、4%、3%、2%或1%的数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度。在一个实施方案中,术语“约”或“大约”是指数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度在大致参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度±15%、±10%、±9%、±8%、±7%、±6%、±5%、±4%、±3%、±2%或±1%的范围内。
在一个实施方案中,范围,例如1至5、约1至5或约1至约5是指所述范围所涵盖的每个数字值。例如,在一个非限制性且仅说明性实施方案中,范围“1至5”相当于表述1、2、3、4、5;或1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5或5.0;或1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9或5.0。
如本文所使用,术语“基本上”是指数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度是参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度的80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高百分比。在一个实施方案中,“基本上相同”是指数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度产生的作用,例如生理作用与参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度大致相同。
在本说明书通篇,除非上下文另外要求,否则“包含(comprise/comprises/comprising)”一词应理解为暗指包括所述步骤或要素或一组步骤或要素,但不排除任何其它步骤或要素或任何其它组步骤或要素。“由……组成”意图包括且限于短语“由……组成”之后的事物。因此,短语“由……组成”指示所列要素是必不可少或必需的,并且不能存在其他要素。“基本上由……组成”意图包括短语后所列的任何要素,并且限于不干扰或影响关于所列要素的公开内容中所说明的活性或作用的其它要素。因此,短语“基本上由……组成”指示所列要素是必不可少的或必需的,而且不存在实质上影响所列要素的活性或作用的其它要素。
本说明书通篇提到的“一个实施方案(one embodiment/an embodiment)”、“一个特定实施方案”、“一个相关实施方案”、“某一实施方案”、“一个另外的实施方案”或“另一实施方案”或其组合意味着,结合所述实施方案描述的特定特征、结构或特性被包括在至少一个实施方案中。因此,在本说明书通篇各处出现的前述短语未必都参考同一实施方案。此外,特定特征、结构或特性可以通过任何适合方式组合于一个或多个实施方案中。还应理解,在一个实施方案中关于一个特征的正面叙述用作一个特定实施方案中排除所述特征的基础。
“抗原(Ag)”是指可以在动物中刺激抗体产生或T细胞应答的化合物、组合物或物质,包含注射或吸收到动物内的组合物(如包含癌症特异性蛋白的组合物)。示例性抗原包括但不限于脂质、碳水化合物、多糖、糖蛋白、肽或核酸。抗原与具有特定体液或细胞免疫的产物反应,包含由如公开的抗原等异源抗原诱导的产物。
“靶抗原”或“所关注的靶抗原”是指本文所涵盖的结合结构域被设计成与其结合的CD33的一部分。在特定实施方案中,靶抗原是SEQ ID NO:1中所示的氨基酸序列的表位。
“CD33”是指细胞表面受体,也称为唾液酸结合免疫球蛋白样凝集素3(SIGLEC-3)或GP67。CD33基因位于染色体19上,并且产生约67kD的糖基化蛋白。CD33具有两个Ig样结构域,一个V-set结构域和一个C2-set结构域。CD33在介导细胞-细胞相互作用和维持免疫细胞处于静息状态中起作用。CD33识别并结合具有α-2,3-连接的唾液酸的聚糖,并且更强烈地识别并结合具有α-2,6-连接的唾液酸的聚糖。在接合配体诸如C1q或唾液酸化糖蛋白时,位于CD33胞质尾部中的两个免疫受体酪氨酸基抑制基序(ITIM)被Src样激酶诸如LCK磷酸化。这些磷酸化为蛋白质-酪氨酸磷酸酶PTPN6/SHP-1和PTPN11/SHP-2的募集和活化提供了对接位点。CD33还具有至少三种经鉴定的剪接变体。CD33ΔE2剪接变体缺乏由人CD33基因的外显子2编码的氨基酸序列(全长CD33的氨基酸13-139;例如NP_001076087.1,C2)。CD337a剪接变体缺乏由驻留于外显子7a中的早期翻译终止信号(例如,NP_001171079.1)导致的54个羧基末端氨基酸。CD33ΔE2/7a缺乏由外显子2编码的氨基酸以及54个羧基末端氨基酸。CD33通常在正常B细胞和活化T细胞和自然杀伤细胞的亚群上表达,但不在造血干细胞上或造血系统外部表达。全长CD33和/或CD33剪接变体两者也在大多数AML患者中的急性髓性白血病(AML)母细胞中表达。
“抗体”是指属于包括至少轻链或重链免疫球蛋白可变区的多肽的结合剂,所述轻链或重链免疫球蛋白可变区特异性地识别并结合靶标的表位,如脂质、碳水化合物、多糖、糖蛋白、肽或含有抗原决定簇的核酸,如通过免疫细胞识别的那些。
提及“VH”或“VH”是指免疫球蛋白重链或其抗原结合片段的可变区。
“重链抗体”指含有两个VH结构域且无轻链的抗体(Riechmann L.等人,J.Immunol.Methods 231:25-38(1999);WO94/04678;WO94/25591;美国专利6,005,079)。“骆驼科抗体”是指含有两个VH结构域且无轻链的从骆驼、羊驼或美洲驼分离的抗体。“人源化VHH”或“人源化骆驼科抗体”是指已经历人源化以降低抗体在人接受者中的潜在免疫原性的非人VHH或骆驼科抗体。
如本文所用,“VHH”、“VHH抗体”或“VHH结构域”是指含有重链抗体可变区的最小已知抗原结合单元的抗体片段(Koch-Nolte等人,FASEB J.,21:3490-3498(2007))。
“接头”是指为了分子的适当间隔和构象而添加的各个多肽结构域之间的多个氨基酸残基。在特定实施方案中,接头将一个或多个VHH结构域、铰链结构域、多聚化结构域、跨膜结构域、共刺激结构域和/或初级信号传导结构域分开。
适于在本文所涵盖的特定实施方案中使用的接头的说明性实例包括但不限于以下氨基酸序列:GGG;DGGGS(SEQ ID NO:84);TGEKP(SEQ ID NO:85)(参见,例如,Liu等人,PNAS 5525-5530(1997));GGRR(SEQ ID NO:86)(Pomerantz等人1995,supra);(GGGGS)n,其中n=1、2、3、4或5(SEQ ID NO:87)(Kim等人,PNAS 93,1156-1160(1996.));EGKSSGSGSESKVD(SEQ ID NO:88)(Chaudhary等人,1990,Proc.Natl.Acad.Sci.U.S.A.87:1066-1070);KESGSVSSEQLAQFRSLD(SEQ ID NO:89)(Bird等人,1988,Science 242:423-426),GGRRGGGS(SEQ ID NO:90);LRQRDGERP(SEQ ID NO:91);LRQKDGGGSERP(SEQ ID NO:92);LRQKD(GGGS)2ERP(SEQ ID NO:93)。另选地,可以使用能够对DNA结合位点和肽本身建模的计算机程序(Desjarlais&Berg,PNAS 90:2256-2260(1993),PNAS 91:11099-11103(1994))或通过噬菌体展示方法合理地设计柔性接头。在一个实施方案中,接头包含以下氨基酸序列:GSTSGSGKPGSGEGSTKG(SEQ ID NO:94)(Cooper等人,Blood,101(4):1637-1644(2003))。
“间隔子结构域”是指将两个结构域分开的多肽。在一个实施方案中,间隔子结构域使VHH结构域移动远离效应细胞表面以实现适当的细胞/细胞接触、抗原结合和活化(Patel等人,Gene Therapy,1999;6:412-419)。在特定实施方案中,间隔子结构域将一个或多个VHH结构域、多聚化结构域、跨膜结构域、共刺激结构域和/或初级信号传导结构域分开。间隔子结构域可来源于天然、合成、半合成或重组来源。在某些实施方案中,间隔子结构域是免疫球蛋白的一部分,包括但不限于一个或多个重链恒定区,例如CH2和CH3。间隔子结构域可以包括天然存在的免疫球蛋白铰链区或改变的免疫球蛋白铰链区的氨基酸序列。
“铰链结构域”是指在将抗原结合结构域定位成远离效应细胞表面以实现适当的细胞/细胞接触、抗原结合和活化中起作用的多肽。在特定实施方案中,多肽可以包括在结合结构域与多聚化结构域之间、在结合结构域与跨膜结构域(TM)之间或在多聚化结构域与跨膜结构域之间的一个或多个铰链结构域。铰链结构域可以来源于天然、合成、半合成或重组来源。铰链结构域可以包括天然存在的免疫球蛋白铰链区或改变的免疫球蛋白铰链区的氨基酸序列。
如本文所使用,“多聚化结构域”是指直接或经由桥连分子优先与另一个不同的多肽相互作用或缔合的多肽,例如,化学可诱导的二聚体,其中不同多聚化结构域的相互作用实质上有助于或高效地促进了多聚化(即,二聚体、三聚体或多分复合物的形成,所述二聚体、所述三聚体或所述多分复合物可以是同源二聚体、异源二聚体、同源三聚体、异源三聚体、同源多聚体、异多聚体)。多聚化结构域可以源白天然的、合成的、半合成的或重组的来源。
适于在本文所涵盖的特定实施方案中使用的多聚化结构域的说明性实例包含FK506结合蛋白(FKBP)多肽或其变体、FKBP-雷帕霉素结合(FRB)多肽或其变体、钙调神经磷酸酶多肽或其变体、亲环蛋白多肽或其变体、细菌二氢叶酸还原酶(DHFR)多肽或其变体、PYR1样1(PYL1)多肽或其变体、脱落酸不敏感1(ABI1)多肽或其变体、GIB1多肽或其变体或GAI多肽或其变体。
如本文所使用,术语“FKBP-雷帕霉素结合多肽”是指FRB多肽。在特定实施方案中,FRB多肽是FKBP12-雷帕霉素结合多肽。适于在本文所涵盖的特定实施方案中使用的FRB多肽通常含有至少约85个到约100个氨基酸残基。在某些实施方案中,参考GenBank登录号L34075.1,FRB多肽包含93个氨基酸序列Ile-2021至Lys-2113以及T2098L的突变。本文所涵盖的FRB多肽通过桥接因子与FKBP多肽结合,由此形成三元复合物。
如本文所使用,术语“FK506结合蛋白”是指FKBP多肽。在特定实施方案中,FKBP多肽是FKBP12多肽或包括F36V突变的FKBP12多肽。在某些实施方案中,FKBP结构域也可以被称为“雷帕霉素结合结构域”。有关各种FKBP种类的核苷酸序列、克隆和其它方面的信息是本领域已知的(参见,Staendart等人,Nature 346:671,1990(human FKBP12);Kay,Biochem.J.314:361,1996)。本文所涵盖的FKBP多肽通过桥接因子与FRB多肽结合,由此形成三元复合物。
“桥接因子”是指与两个或更多个多聚化结构域缔合并且置于两个或更多个多聚化结构域之间的分子。在特定实施方案中,多聚化结构域仅在桥接因子存在的情况下实质上有助于或高效地促进多肽复合物的形成。在特定实施方案中,多聚化结构域在不存在桥接因子的情况下不会有助于或不会高效地促进多肽复合物的形成。适于在本文所涵盖的特定实施方案中使用的桥接因子的说明性实例包括但不限于AP21967、雷帕霉素(西罗莫司)或雷帕霉素类似物、库马霉素或其衍生物、赤霉素或其衍生物、脱落酸(ABA)或其衍生物、甲氨蝶呤或其衍生物、环孢菌素A或其衍生物、FKCsA或其衍生物、甲氧苄啶(Tmp)-FKBP合成配体(SLF)或其衍生物、或其任何组合。
雷帕霉素类似物(rapalog)包括但不限于美国专利6,649,595号中公开的那些,所述rapalog结构以引用方式整体并入本文。在某些实施方案中,桥接因子是相较于雷帕霉素,具有明显较低的免疫抑制作用的雷帕霉素类似物。在优选实施方案中,雷帕霉素类似物是AP21967(也被称为C-16-(S)-7-甲基吲哚雷帕霉素,IC50=10nM,经过化学修饰的非免疫抑制雷帕霉素类似物)。适于在本文所涵盖的特定实施方案中使用的其它说明性雷帕霉素类似物包括但不限于依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。
“显著降低的免疫抑制作用”是指是针对在临床上或在人类免疫抑制活性的适当的体外(例如,抑制T细胞增殖)或体内替代物中测量的同一剂量观察或预期的免疫抑制作用的至少小于0.1倍到0.005倍。
“跨膜结构域”或“TM结构域”是将多肽与细胞的质膜锚定的结构域。TM结构域可以来源于天然、合成、半合成或重组来源。
术语“效应子功能”或“效应细胞功能”是指免疫效应细胞的专门功能。效应子功能包括但不限于活化、细胞因子产生、增殖和细胞毒活性,包含细胞毒性因子的释放,或通过与免疫效应细胞上表达的受体进行抗原结合引发的其它细胞应答。
“细胞内信号传导结构域”或“细胞内结构域”是指蛋白质的一部分,所述部分转导效应子功能信号并引导细胞执行专门功能。尽管通常可采用整个细胞内信号传导结构域,但在许多情况下不必使用整个结构域。在使用细胞内信号传导结构域的截短部分的程度上,可以使用这种截短部分代替整个结构域,只要其转导效应子功能信号即可。术语细胞内信号传导结构域意指包含细胞内信号传导结构域的必需或足以转导效应子功能信号的任何截短部分。
已知的是,单独通过TCR产生的信号不足以完全活化T细胞,并且还需要次级信号或共刺激信号。因此,可以说T细胞活化由两种不同类别的细胞内信号传导结构域介导:初级信号传导结构域,其通过TCR(例如,TCR/CD3复合物)启动抗原依赖性初级活化;以及共刺激信号传导结构域,其是以抗原非依赖性方式起作用以提供次级信号或共刺激信号。
“初级信号传导结构域”是指以刺激方式或以抑制方式调节TCR复合物的初级活化的细胞内信号传导结构域。以刺激性方式起作用的初级信号传导结构域可含有信号传导基元,所述信号传导基元称为免疫受体酪氨酸活化基元或ITAM。适于在特定实施方案中使用的含ITAM初级信号传导结构域的说明性实例包括但不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。
如本文所使用,术语“共刺激信号传导结构域”或“共刺激结构域”是指共刺激分子的细胞内信号传导结构域。共刺激分子是除抗原受体或Fc受体之外的细胞表面分子,所述细胞表面分子提供在与抗原结合后使T淋巴细胞有效活化和起作用所需的第二信号。可以从其中分离共刺激结构域的这种共刺激分子的说明性实例包括但不限于:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNF受体超家族成员14(TNFRS14;HVEM)、TNF受体超家族成员18(TNFRS18;GITR)、TNF受体超家族成员25(TNFRS25;DR3)和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
“免疫病症”是指引起免疫系统应答的疾病。在特定实施方案中,术语“免疫病症”是指癌症、自身免疫疾病或免疫缺陷。
如本文所使用,术语“癌症”通常涉及异常细胞在没有控制的情况下分裂并且可能侵入附近组织的一类疾病或病状。
如本文所使用,术语“恶性”是指一组肿瘤细胞显示出不受控制的生长(即,超出正常限度的分裂)、侵入(即,侵入和破坏相邻组织)和转移(即,经由淋巴或血液扩散到体内的其他位置)中的一种或多种的癌症。如本文所使用,术语“转移”是指癌症从身体的一个部位扩散到另一个部位。由已经扩散的细胞形成的肿瘤被称为“转移性肿瘤”或“转移”。转移性肿瘤含有与原始(原发性)肿瘤中的细胞相似的细胞。
如本文所使用,术语“良性”或“非恶性”是指可以长得更大但不扩散到身体的其他部位的肿瘤。良性肿瘤是自限性的并且通常不会侵入或转移。
“癌细胞”是指癌性生长或组织的单个细胞。癌细胞包含实体癌和液体癌。“肿瘤”或“肿瘤细胞”通常是指由细胞的异常生长形成的肿胀或病变,所述肿胀或病变可以是良性的、恶化前的(pre-malignant)或恶性的。大多数癌症形成肿瘤,但液体癌例如白血病不一定形成肿瘤。对于形成肿瘤的那些癌症,术语癌症(细胞)和肿瘤(细胞)可互换使用。个体中肿瘤的量是可以测量为肿瘤的数目、体积或重量的“肿瘤负荷”。
术语“复发”是指经过一段时间的改进或缓解后诊断出癌症重现或重现体征和症状。
“缓解”也被称为“临床缓解”并且包含部分缓解和完全缓解两者。在部分缓解中,一些但并非所有癌症体征和症状已经消失。在完全缓解中,所有癌症体征和症状均已消失,尽管癌可能仍在体内。
“难治”是指癌症对用特定治疗剂进行的疗法具有抗性或无应答。癌症可以在治疗开始时是难治的(即,对初始暴露于治疗剂无应答),或者由于在第一治疗期内或在随后的治疗期期间对治疗剂产生抗性而是难治的。
如本文所使用,术语“个体”和“受试者”经常可互换使用并且是指展现出可以用本文中其它地方所涵盖的组合物和方法治疗的癌症或其它免疫病症的症状的任何动物。适合的受试者(例如,患者)包含实验用动物(如小鼠、大鼠、兔或豚鼠)、农场动物和家养动物或宠物(如猫或狗)。包含非人灵长类和优选地人类患者。典型的受试者包含患有、已经被诊断为患有或有风险患有癌症或另一种免疫病状的人类患者。
如本文所使用,术语“患者”是指已经被诊断为患有可以用本文中的其它地方所公开的组合物和方法治疗的癌症或另一种免疫病症的受试者。
如本文所使用,“治疗(treatment或treating)”包含对疾病或病理状况的症状或病理的任何有益的或期望的效果并且甚至可以包含正在治疗的疾病或病症的一个或多个可测量标志物的最低限度的减少。任选地,治疗可以涉及疾病或病状减少或者疾病或病状的进展延迟,例如延迟肿瘤生长。“治疗”不一定指示完全根除或治愈疾病或病症或其相关症状。
如本文所使用,“预防(prevent)”和如“预防(prevented/preventing)”等类似词语指示用于预防、抑制或减少疾病或病症发生或复发的可能性。预防还是指延迟疾病或病症的发作或复发或者延迟疾病或病症的症状的发生或复发。如本文所使用,“预防(prevention)”和类似词语还包含在疾病或病症发作或复发之前减少疾病或病症的强度、效果、症状和/或负担。
如本文所使用,短语“减轻……的至少一种症状”是指减少正在治疗的受试者的疾病或病症的一种或多种症状。在特定实施方案中,被治疗的疾病或病状是癌症,其中所减轻的所述一种或多种症状包括但不限于虚弱、疲劳、呼吸短促、容易挫伤和出血、频繁感染、淋巴结肿大、腹部肿胀或疼痛(由于腹部器官肿大)、骨骼或关节疼痛、骨折、意外体重减轻、食欲不振、盗汗、持续性轻度发烧以及排尿减少(由于肾功能受损)。
“增强”或“促进”或者“增加”或“扩增”通常是指与由媒介物或对照分子/组合物引起的应答相比,本文所涵盖的组合物能够产生、引发或引起更大的生理应答(即,下游效应)。可测量的生理应答可以包含T细胞扩增、活化、持续、细胞因子分泌的增加和/或癌细胞杀伤能力的增加以及从本领域的理解和本文中的描述中显而易见的其它方面。“增加的”或“增强的”量通常是“统计上显著的”量并且可以包含是由媒介物或对照组合物产生的应答的1.1倍、1.2倍、1.5倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、15倍、20倍、30倍或更多倍(例如,500倍、1000倍)(包含其间且在1以上的所有整数和小数点,例如1.5、1.6、1.7、1.8等)的增加。
“减小”或“减弱”或“变少”或“减少”或“减轻”通常是指与由媒介物或对照分子/组合物引起的应答相比,本文所涵盖的组合物能够产生、引发或引起更少的应答(即,生理应答)。“减小的”或“减少的”量通常是“统计上显著”的量并且可以包含是由媒介物、对照组合物或特定细胞谱系中的应答产生的应答(参考应答)的1.1倍、1.2倍、1.5倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、15倍、20倍、30倍或更多倍(例如,500倍、1000倍)(包含其间且在1以上的所有整数和小数点,如1.5、1.6、1.7、1.8等)的减小。
“维持(maintain或maintenance)”或“保持”或“无变化”或“无实质变化”或“无实质减小”通常是指与由媒介物、对照分子/组合物或特定细胞谱系中的应答引起的应答相比,本文所涵盖的组合物能够在细胞中产生、引发或引起基本上类似的或相当的生理应答(即,下游效应)。相当的反应是与参考反应并无显著不同或可测量不同的反应。
在本公开通篇阐述了另外的定义。
C.CD33 VHH DARIC
在特定实施方案中,涵盖了包含抗CD33 VHH结构域的DARIC受体,其将免疫效应细胞的细胞毒性重定向为朝向表达CD33的癌细胞。如本文所用,术语“CD33 VHH DARIC受体”、“抗CD33 VHH DARIC受体”、“CD33VHH DARIC”或“抗CD33 VHH DARIC”可互换使用,并且是指一种或多种非天然存在的多肽,其在暴露于表达全长CD33或CD33剪接变体的靶细胞和多聚化剂或桥接因子时,在免疫效应细胞中转导免疫刺激信号,例如刺激免疫效应细胞活性和功能、增加促炎性细胞因子的产生和/或分泌。在优选的实施方案中,CD33 VHH DARIC是多链嵌合受体,其包含DARIC信号传导组分和包含识别全长CD33和/或CD33剪接变体的VHH结构域的DARIC结合组分。
在一个实施方案中,从同一细胞表达DARIC信号传导组分和DARIC结合组分。在另一个实施方案中,从不同细胞表达DARIC信号传导组分和DARIC结合组分。在特定实施方案中,从细胞表达DARIC信号传导组分,并且外源供应DARIC结合组分作为多肽。在一个实施方案中,将预先装载有桥接因子的DARIC结合组分外源供应给表达DARIC信号传导组分的细胞。
1.CD33 DARIC信号传导组分
术语“DARIC信号传导组分”、“CD33 DARIC信号传导组分”、“DARIC信号传导多肽”或“DARIC信号传导多肽”可互换使用,是指包含一个或多个多聚化结构域、跨膜结构域和一个或多个细胞内信号传导结构域的多肽。在特定实施方案中,DARIC信号传导组分包括多聚化结构域、跨膜结构域、共刺激结构域和/或初级信号传导结构域。在特定实施方案中,DARIC信号传导组分包括第一多聚化结构域、第一跨膜结构域、第一共刺激结构域和/或初级信号传导结构域。
在特定实施方案中,DARIC信号传导组分包括一个或多个多聚化结构域。
适于在本文所涵盖的特定CD33 DARIC信号传导组分中使用的多聚化结构域的说明性实例包括但不限于FK506结合蛋白(FKBP)多肽或其变体、FKBP-雷帕霉素结合(FRB)多肽或其变体、钙调神经磷酸酶多肽或其变体、亲环蛋白多肽或其变体、细菌二氢叶酸还原酶(DHFR)多肽或其变体、PYR1样1(PYL1)多肽或其变体和脱落酸不敏感1(ABI1)多肽或其变体。
在特定实施方案中,CD33 DARIC信号传导组分包括FRB多肽。
在特别优选的实施方案中,CD33 DARIC信号传导组分包括包含T2098L突变的FRB多肽或其变体。在某些优选的实施方案中,CD33 DARIC信号传导组分包括FKBP12多肽或其变体。
在一些实施方案中,CD33 VHH DARIC信号传导组分包括铰链结构域。
适于在本文所述的CD33 VHH DARIC信号传导组分中使用的说明性铰链结构域包括源自1型膜蛋白诸如CD28、CD8α和CD4的细胞外区域的铰链区,其可以是来自这些分子的野生型铰链区或可以被改变。
在特定实施方案中,DARIC信号传导组分包括跨膜结构域。
在特定实施方案中,DARIC信号传导组分包括铰链结构域和跨膜结构域。
适于在本文所涵盖的特定CD33 DARIC信号传导组分中使用的跨膜结构域的说明性实例包括但不限于T细胞受体的α链、β链、γ链或δ链的跨膜区、CD3ε、CD3ζ、CD4、CD5、CD8α、CD9、CD 16、CD22、CD27、CD28、CD33、CD37、CD45、CD64、CD71、CD80、CD86、CD 134、CD137、CD152、CD 154、无羊膜蛋白(AMN)和程序性细胞死亡1(PDCD1)。在优选的实施方案中,CD33DARIC信号传导组分包括CD4跨膜结构域。在优选的实施方案中,CD33 DARIC信号传导组分包括CD8α跨膜结构域。
在特定实施方案中,DARIC信号传导组分包括接头,其将跨膜结构域的C-末端与细胞内信号传导结构域的N-末端连接。在各种优选实施方案中,优选地长度在1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸之间的短寡肽接头或多肽接头连接跨膜结构域和细胞内信号传导结构域。基于甘氨酸-丝氨酸的接头提供了特别适合的接头。
本文特定实施方案中所涵盖的DARIC信号传导组分包括一个或多个细胞内信号传导结构域。在一个实施方案中,CD33 DARIC信号传导组分包括一个或多个共刺激信号传导结构域和/或初级信号传导结构域。在一个实施方案中,细胞内信号传导结构域包括免疫受体酪氨酸活化基序(ITAM)。
适于在本文所涵盖的特定CD33 DARIC信号传导组分中使用的含ITAM初级信号传导结构域的说明性实例包括但不限于源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。在优选的实施方案中,CD33 DARIC信号传导组分包括CD3ζ初级信号传导结构域和一个或多个共刺激信号传导结构域。初级信号传导结构域和共刺激信号传导结构域可以按任何顺序与跨膜结构域的羧基端串联连接。
适于在本文所涵盖的特定CD33 DARIC信号传导组分中使用的共刺激结构域的说明性实例包括但不限于从以下共刺激分子中分离的那些结构域:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNF受体超家族成员14(TNFRS14;HVEM)、TNF受体超家族成员18(TNFRS18;GITR)、TNF受体超家族成员25(TNFRS25;DR3)和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
在特定实施方案中,本文所涵盖的CD33 DARIC信号传导组分包括信号肽。适于在特定CD33 DARIC信号传导组分中使用的信号肽的说明性实例包括但不限于IgG1重链信号多肽、Igκ轻链信号多肽、CD8α信号多肽或人GM-CSF受体α信号多肽。在各种优选实施方案中,CD33 DARIC信号传导组分包括CD8α信号多肽。
在特定实施方案中,CD33 DARIC信号传导组分包括选自由CD28、CD137和CD134组成的组的一个或多个共刺激信号传导结构域。在特定实施方案中,CD33 DARIC信号传导组分包括选自由CD28、CD137和CD134组成的组的一个或多个共刺激信号传导结构域以及CD3ζ初级信号传导结构域。在特定实施方案中,CD33 DARIC信号传导组分包括CD137共刺激结构域和CD3ζ初级信号传导结构域。
在优选实施方案中,CD33 DARIC信号传导组分包括FRB T2098L多聚化结构域、CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域。
在优选的实施方案中,CD33 VHH DARIC信号传导组分包含SEQ ID NO:82中所示的氨基酸序列。
2.CD33 DARIC结合组分
“DARIC结合组分”、“DARIC结合多肽”、“CD33 VHH DARIC结合组分”或“CD33 VHHDARIC结合多肽”可互换使用,并且是指包含抗CD33 VHH结构域和一个或多个多聚化结构域的多肽。在特定实施方案中,CD33 VHH DARIC结合组分包括抗CD33 VHH结构域、多聚化结构域和跨膜结构域。在特定实施方案中,CD33 VHH DARIC结合组分包括抗CD33 VHH结构域、第二多聚化结构域和第二跨膜结构域。在其他特定实施方案中,CD33 VHH DARIC结合组分包括抗CD33 VHH结构域、多聚化结构域、跨膜结构域和一个或多个细胞内信号传导结构域。在特定实施方案中,CD33 VHH DARIC结合组分包括抗CD33 VHH结构域、第二多聚化结构域、第二跨膜结构域和第二共刺激结构域。
在特定实施方案中,CD33 VHH DARIC结合组分包括一个或多个抗CD33 VHH结构域。
在特别优选的实施方案中,抗CD33 VHH结构域是人源化的骆驼科VHH。在特定实施方案中,抗CD33 VHH结构域是人源化的骆驼科VHH,其结合全长CD33(例如SEQ ID NO:1)的一个或多个表位或CD33剪接变体的一个或多个表位。在特定实施方案中,抗CD33 VHH结构域是人源化的骆驼科VHH,其结合在全长CD33和CD33剪接变体两者上显示的相同的一个或多个表位。
在特别优选的实施方案中,抗CD33 VHH结构域是包含SEQ ID NO:3-6、10-11、13-16和20-21中任一者所示的氨基酸序列的人源化骆驼科VHH。在某些优选的实施方案中,抗CD33 VHH结构域是包含SEQ ID NO:10中所示的氨基酸序列的人源化骆驼科VHH。在某些优选的实施方案中,抗CD33VHH结构域是包含SEQ ID NO:20中所示的氨基酸序列的人源化骆驼科VHH。
在特定实施方案中,DARIC结合组分包括一个或多个多聚化结构域。
适于在本文所涵盖的特定CD33 VHHDARIC结合组分中使用的多聚化结构域的说明性实例包括但不限于FKBP多肽或其变体、FRB多肽或其变体、钙调神经磷酸酶多肽或其变体、亲环蛋白多肽或其变体、DHFR多肽或其变体、PYL1多肽或其变体和ABI1多肽或其变体。
在特定实施方案中,CD33 VHH DARIC结合组分包括FRB多肽或其变体,并且DARIC信号传导组分包括FKBP多肽或其变体。在优选的实施方案中,CD33 VHH DARIC结合组分包括包含T2098L突变的FRB多肽或其变体,并且DARIC信号传导组分包括FKBP12多肽或其变体。
在特定实施方案中,CD33 VHH DARIC结合组分包括FKBP多肽或其变体,并且DARIC信号传导组分包括FRB多肽或其变体。在优选的实施方案中,CD33 VHH DARIC结合组分包括FKBP12多肽或其变体,并且DARIC信号传导组分包括包含T2098L突变的FRB多肽或其变体。
在一些实施方案中,CD33 VHH DARIC结合组分包括铰链结构域。
适于在本文所述的CD33 VHH DARIC结合组分中使用的说明性铰链结构域包括源自1型膜蛋白诸如CD28、CD8α和CD4的细胞外区域的铰链区,其可以是来自这些分子的野生型铰链区或可以被改变。
在特定实施方案中,DARIC结合组分包括跨膜结构域。在特定实施方案中,DARIC结合组分包括铰链结构域和跨膜结构域。在一个实施方案中,跨膜结构域可以与DARIC信号传导组分中使用的跨膜结构域相同。在一个实施方案中,跨膜结构域可以不同于DARIC信号传导组分中使用的跨膜结构域。
适于在本文所涵盖的特定CD33 VHH DARIC结合组分中使用的跨膜结构域的说明性实例包括但不限于T细胞受体的α链、β链、γ链或δ链的跨膜区、CD3ε、CD3ζ、CD4、CD5、CD8α、CD9、CD 16、CD22、CD27、CD28、CD33、CD37、CD45、CD64、CD71、CD80、CD86、CD 134、CD137、CD152、CD 154、无羊膜蛋白(AMN)和程序性细胞死亡1(PDCD1)。在优选的实施方案中,CD33DARIC结合组分包括CD8α跨膜结构域。在优选的实施方案中,CD33 VHH DARIC结合组分包括CD4跨膜结构域。
在各种优选实施方案中,优选地长度在1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸之间的短寡肽接头或多肽接头连接跨膜结构域和细胞内信号传导结构域。基于甘氨酸-丝氨酸的接头提供了特别适合的接头。
本文特定实施方案中所涵盖的DARIC结合组分不包含一个或多个细胞内信号传导结构域。
在其他特定实施方案中,本文所涵盖的CD33 VHH DARIC结合组分包括一个或多个细胞内信号传导结构域。在优选的实施方案中,其中CD33VHH DARIC结合组分包括一个或多个细胞内信号传导结构域,那些结构域不同于同源CD33 DARIC信号传导组分中存在的细胞内信号传导结构域。在一个实施方案中,CD33 VHH DARIC结合组分包括共刺激信号传导结构域。
适于在本文所涵盖的特定CD33 VHH DARIC结合组分中使用的共刺激结构域的说明性实例包括但不限于从以下共刺激分子中分离的那些结构域:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNF受体超家族成员14(TNFRS14;HVEM)、TNF受体超家族成员18(TNFRS18;GITR)、TNF受体超家族成员25(TNFRS25;DR3)和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。在优选的实施方案中,从TNFR2或OX40衍生、获得或分离共刺激结构域。
在特定实施方案中,本文所涵盖的DARIC结合组分包括信号肽。适于在特定CD33VHH DARIC结合组分中使用的信号肽的说明性实例包括但不限于IgG1重链信号多肽、Igκ轻链信号多肽、CD8α信号多肽或人GM-CSF受体α信号多肽。在各种优选的实施方案中,CD33VHH DARIC结合组分包括CD8α信号多肽。
在特定实施方案中,CD33 VHH DARIC结合组分包括结合CD33的VHH结构域、FKBP12多聚化结构域、CD4跨膜结构域和任选的共刺激结构域。
在某些实施方案中,CD33 VHH DARIC结合组分包括结合CD33的VHH以及FKBP12多聚化结构域。
在一些实施方案中,CD33 VHH DARIC结合组分包括包含SEQ ID NO:2-21中任一者所示的氨基酸序列的VHH结构域、FKBP12多聚化结构域、CD4跨膜结构域和任选的共刺激结构域。
在一些实施方案中,CD33 VHH DARIC结合组分包括包含SEQ ID NO:10或SEQ IDNO:20中所示的氨基酸序列的VHH结构域、FKBP12多聚化结构域、CD4跨膜结构域和任选的共刺激结构域。
在一些实施方案中,CD33 VHH DARIC结合组分包括包含SEQ ID NO:2-21中任一者所示的氨基酸序列的VHH结构域和FKBP12多聚化结构域。
在一些实施方案中,CD33 VHH DARIC结合组分包括包含SEQ ID NO:10或SEQ IDNO:20中所示的氨基酸序列的VHH结构域和FKBP12多聚化结构域。
在一些实施方案中,CD33 VHH DARIC结合组分包含SEQ ID NO:22-31中任一者所示的氨基酸序列。
在一些实施方案中,CD33 VHH DARIC结合组分包含SEQ ID NO:30中所示的氨基酸序列。
3.桥接因子
本文的特定实施方案中所涵盖的桥接因子介导或促进CD33 DARIC信号传导组分与CD33 VHHDARIC结合组分通过各自组分中的多聚化结构域的缔合。桥接因子与多聚化结构域缔合并置于所述多聚化结构域之间,以促进CD33 DARIC信号传导组分与CD33 VHHDARIC结合组分的缔合。在存在桥接因子的情况下,当CD33 VHH DARIC结合组分结合到靶细胞上表达的CD33时,CD33 VHH DARIC结合组分与CD33 DARIC信号传导组分缔合并引发针对靶细胞的免疫效应细胞活性。在不存在桥接因子的情况下,CD33VHH DARIC结合组分不与CD33 DARIC信号传导组分缔合,并且CD33 VHHDARIC是无活性的。
在特定实施方案中,CD33 DARIC信号传导组分和CD33 VHH DARIC结合组分包括选自由以下组成的组的多聚化结构域的同源对:FKBP和FKBP12-雷帕霉素结合(FRB)、FKBP和钙调磷酸酶、FKBP和亲环蛋白、FKBP和细菌二氢叶酸还原酶(DHFR)、钙调磷酸酶和亲环蛋白、和PYR1-样l(PYL1)和脱落酸不敏感1(ABI1)。
在某些实施方案中,CD33 VHH DARIC信号传导和结合组分的多聚化结构域与选自由以下组成的组的桥接因子缔合:雷帕霉素或雷帕霉素类似物、库马霉素或其衍生物、赤霉素或其衍生物、脱落酸(ABA)或其衍生物、甲氨蝶呤或其衍生物、环孢菌素A或其衍生物、FK506/环孢菌素A(FKCsA)或其衍生物、甲氧苄啶(Tmp)-FK506结合蛋白(FKBP)合成配体(SLF)或其衍生物。
在特定实施方案中,CD33 DARIC信号传导组分和CD33 VHH DARIC结合组分包括一个或多个FRB和/或FKBP多聚化结构域或其变体。在某些实施方案中,CD33 DARIC信号传导组分包括FRB多聚化结构域或其变体,并且CD33 VHH DARIC结合组分包括FKBP多聚化结构域或其变体。在特别优选的实施方案中,CD33 DARIC信号传导组分包括FRB T2098L多聚化结构域或其变体,并且CD33 VHH DARIC结合组分包括FKBP12或FKBP12 F36V多聚化结构域或其变体。
适于在本文所涵盖的特定实施方案中使用的桥接因子的说明性实例包括但不限于AP1903、AP20187、AP21967(也称为C-16-(S)-7-甲基吲哚雷帕霉素)、依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。在特定的优选实施方案中,桥接因子是AP21967。在某些优选的实施方案中,桥接因子是非免疫抑制剂量的西罗莫司(雷帕霉素)。
D.抗CD33嵌合抗原受体
在特定实施方案中,本文所涵盖的免疫效应细胞包含抗CD33 VHH CAR。嵌合抗原受体(CAR)是将抗体针对靶抗原(例如肿瘤抗原)的特异性与T细胞受体活化细胞内结构域相组合以产生展现特异性抗肿瘤细胞免疫活性的嵌合蛋白的分子。如本文所使用,术语“嵌合”描述由来自不同来源的不同蛋白质或DNA部分构成。
在特定实施方案中,通过引入编码抗CD33 VHH CAR的多核苷酸来工程改造T细胞。
在特定实施方案中,通过引入包含编码抗CD33 VHH CAR的多核苷酸的载体来工程改造T细胞。
在各种实施方案中,抗CD33 CAR包括结合CD33的VHH结构域、跨膜结构域和一个或多个细胞内信号结构域。CAR的主要特性是其能够利用单克隆抗体、可溶性配体或细胞特异性共同受体的细胞特异性靶向能力来重新定向免疫效应子细胞特异性,从而触发增殖、细胞因子产生、吞噬作用或可以以主要组织相容性(MHC)非依赖方式介导靶抗原表达细胞的细胞死亡的分子的产生。
在一些实施方案中,抗CD33 VHH CAR包含间隔子结构域。在特定实施方案中,间隔子结构域包含IgG1、IgG4或IgD的CH2和CH3。
适于在本文所述的抗CD33 VHH CAR中使用的说明性铰链结构域包括源自1型膜蛋白诸如CD28、CD8α和CD4的细胞外区域的铰链区,其可以是来自这些分子的野生型铰链区或可以被改变。在另一个实施方案中,铰链结构域包含CD8α铰链区。
CAR的“跨膜(TM)结构域”融合细胞外结合部分和细胞内信号传导结构域并且将CAR锚定到免疫效应细胞的质膜上。TM结构域可以来源于天然、合成、半合成或重组来源。
说明性TM结构域可以源自(即至少包括以下的一个或多个跨膜区):T细胞受体的α链、β链、γ链或δ链、CD3ε、CD3ζ、CD4、CD5、CD8α、CD9、CD 16、CD22、CD27、CD28、CD33、CD37、CD45、CD64、CD71、CD80、CD86、CD 134、CD137、CD152、CD 154、AMN以及PDCD1。
在一个实施方案中,抗CD33VHH CAR包含源自CD8α的TM结构域。在另一个实施方案中,本文所涵盖的CAR包含来源于CD8α的TM结构域以及长度优选地在1、2、3、4、5、6、7、8、9或10个氨基酸之间的短寡肽或多肽接头,所述接头连接CAR的TM结构域和细胞内信号传导结构域。甘氨酸-丝氨酸接头提供特别适合的接头。
在优选实施方案中,抗CD33 VHH CAR包括细胞内信号传导结构域,所述细胞内信号传导结构域包括一个或多个“共刺激信号传导结构域”和“初级信号传导结构域”。
以刺激性方式起作用的初级信号传导结构域可含有信号传导基元,所述信号传导基元称为免疫受体酪氨酸活化基元或ITAM。
适于在特定实施方案中所涵盖的抗CD33 VHH CAR中使用的含ITAM的初级信号传导结构域的说明性实例包括源自FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d的那些。在特定的优选实施方案中,CAR包含CD3ζ初级信号传导结构域和一个或多个共刺激信号传导结构域。细胞内初级信号传导结构域和共刺激信号传导结构域可以按任何次序串联连接至跨膜结构域的羧基末端。
在特定实施方案中,抗CD33 VHH CAR包含一个或多个共刺激信号传导结构域,用以增进表达CAR受体的T细胞的功效和扩增。
适于在特定实施方案中所涵盖的抗CD33 VHH CAR中使用的此类共刺激分子的说明性实例包括但不限于TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DAP10、LAT、SLP76、TRAT1、TNFR2和ZAP70。在一个实施方案中,CAR包含一个或多个选自由CD28、CD137和CD134组成的组的共刺激信号传导结构域,以及一个CD3ζ初级信号传导结构域。
在各种实施方案中,抗CD33 VHH CAR包含:结合CD33的VHH;从选自由以下组成的组的多肽分离的跨膜结构域:CD4、CD8α、CD154和PD-1;从选自由以下组成的组的多肽分离的一个或多个细胞内共刺激信号传导结构域:CD28、CD134和CD137;和从选自由以下组成的组的多肽分离的信号传导结构域:FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。
在各种实施方案中,抗CD33 VHH CAR包含:结合CD33的VHH;从选自由以下组成的组的多肽分离的跨膜结构域:CD4、CD8α、CD154和PD-1;从选自由以下组成的组的多肽分离的一个或多个细胞内共刺激信号传导结构域:CD28、CD134和CD137;和从选自由以下组成的组的多肽分离的信号传导结构域:FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。
在优选的实施方案中,抗CD33 VHH CAR包括包含SEQ ID NO:2-21中任一者所示的氨基酸序列的VHH、CD8α铰链结构域、CD8α跨膜结构域、4-1BB共刺激结构域和CD3ζ初级信号传导结构域。
在优选的实施方案中,抗CD33 VHH CAR包括包含SEQ ID NO:10中所示的氨基酸序列的VHH、CD8a铰链结构域、CD8a跨膜结构域、4-1BB共刺激结构域和CD3ζ初级信号传导结构域。
在特定实施方案中,抗CD33 VHH CAR包含SEQ ID NO:62-81中任一者所示的序列。
在特定实施方案中,抗CD33 VHH CAR包含SEQ ID NO:70或SEQ ID NO:80中所示的序列。
E.多肽
本文涵盖各种多肽,包括但不限于CD33 VHH DARIC、CD33 VHH DARIC结合组分、CD33 DARIC信号传导组分、抗CD33 VHH CAR以及它们的片段。在优选实施方案中,多肽包含SEQ ID NO:2-82中任一者所示的氨基酸序列。除非作相反说明,否则“多肽”、“肽”和“蛋白质”可互换使用,并且根据常规含义,即,定义为氨基酸序列。在一个实施方案中,“多肽”包括融合多肽和其它变体。多肽可使用多种众所周知的重组和/或合成技术中的任一种制备。多肽不限于特定长度,例如其可以包含全长蛋白质序列、全长蛋白质的片段、或融合蛋白,并且可以包括多肽的翻译后修饰,例如糖基化、乙酰化、磷酸化等,以及本领域中已知的天然存在的和非天然存在的其它修饰。在特定的优选实施方案中,从一种或多种人多肽制备、获得或分离融合多肽、多肽、其片段和其它变体。
如本文所使用,“分离的肽”或“分离的多肽”等是指从细胞环境中以及从与细胞的其它组分的缔合中体外分离和/或纯化的肽或多肽分子,即所述肽或多肽分子未与体内物质显著缔合。在特定实施方案中,分离的多肽是合成多肽、半合成多肽或从重组来源获得或衍生的多肽。
多肽包括“多肽变体”。多肽变体与天然存在的多肽的不同之处可以在于一个或多个取代、缺失、添加和/或插入。此类变体可以是天然存在的,例如剪接变体,或者可以通过合成生成,例如通过修饰上述多肽序列中的一个或多个。例如,在特定实施方案中,可以期望通过将一个或多个取代、缺失、添加和/或插入引入到多肽中来改进多肽的结合亲和力和/或其它生物性质。在特定实施方案中,多肽包含与本文所涵盖的参考序列中的任何一个有至少约65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、86%、97%、98%或99%氨基酸一致性的多肽,通常其中变体维持参考序列的至少一种生物活性。在特定实施方案中,生物活性是结合亲和力。在特定实施方案中,生物活性是细胞溶解活性。
多肽变体包括生物活性“多肽片段”。生物活性多肽片段的说明性实例包括抗CD33VHH结构域、细胞内信号传导结构域等。如本文所使用,术语“生物活性片段”或“最小生物活性片段”是指保留至少100%、至少90%、至少80%、至少70%、至少60%、至少50%、至少40%、至少30%、至少20%、至少10%、或至少5%的天然存在的多肽活性的多肽片段。在某些实施方案中,多肽片段可包含至少5个至约1700个氨基酸长的氨基酸链。应了解,在某些实施方案中,片段是至少5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、55、60、65、70、75、80、85、90、95、100、110、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1100、1200、1300、1400、1500、1600、1700个或更多个氨基酸长。
在特定实施方案中,本文列出的多肽可包含一个或多个表示为可互换使用的“X”或“Xaa”的氨基酸。“X”如果存在于氨基酸SEQ ID NO中,则是指任何一个或多个氨基酸。在特定实施方案中,表示融合蛋白的SEQ ID NO包括累积地代表任何氨基酸序列的连续X残基的序列。在特定实施方案中,“XX”表示任何两个氨基酸的组合。在某些实施方案中,“XX”表示两个丝氨酸,即SS。在某些实施方案中,“XX”表示降低免疫原性的任何两个氨基酸的组合。
在优选的实施方案中,“XX”表示氨基酸KP。
如上文所述,多肽可以通过不同方式改变,包括氨基酸取代、缺失、截短和插入。用于此类操作的方法是本领域中一般已知的。例如,参考多肽的氨基酸序列变体可以通过DNA中的突变制备。用于诱变和核苷酸序列改变的方法是本领域中众所周知的。参见,例如Kunkel(1985,Proc.Natl.Acad.Sci.USA.82:488-492),Kunkel等人,(1987,Methods inEnzymol,154:367-382),美国专利第4,873,192号,Watson,J.D.等人(Molecular Biologyof the Gene,Fourth Edition,Benjamin/Cummings,Menlo Park,Calif.,1987)和其中引用的参考文献。关于不影响目的蛋白质的生物活性的适当氨基酸取代的指导可见于Dayhoff等人,(1978)Atlas of Protein Sequence and Structure(Natl.Biomed.Res.Found.,Washington,D.C.)的模型中。
在某些实施方案中,多肽变体包括一个或多个保守取代。“保守性取代”是氨基酸被具有类似特性的另一个氨基酸取代,使得肽化学领域的技术人员能预期多肽的二级结构和亲水性质基本上不变的取代。修饰可以针对特定实施方案中所涵盖的多核苷酸和多肽的结构进行并且仍然获得了对具有期望特性的变体或衍生多肽进行编码的功能分子。当希望改变多肽的氨基酸序列以产生等效或甚至改良的变体多肽时,本领域技术人员例如可以改变编码DNA序列的一个或多个密码子,例如根据表1。
表1-氨基酸密码子
可以使用本领域中公知的计算机程序如DNASTAR、DNA Strider、Geneious、MacVector或Vector NTI软件找到关于确定哪些氨基酸残基可以被取代、插入或缺失而不消除生物活性的指南。优选地,本文所公开的蛋白质变体的氨基酸改变是保守氨基酸改变,即带类似电荷或不带电荷的氨基酸的取代。保守性氨基酸变化涉及侧链相关的一组氨基酸中的一个的取代。天然存在的氨基酸一般被分成四组:酸性氨基酸(天冬氨酸、谷氨酸)、碱性氨基酸(赖氨酸、精氨酸、组氨酸)、非极性氨基酸(丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)和不带电极性氨基酸(甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸)。苯丙氨酸、色氨酸和酪氨酸有时共同归类为芳香族氨基酸。在肽或蛋白质中,适合的氨基酸保守性取代是本领域技术人员已知的且一般可以在不改变所得分子的生物活性的情况下进行。本领域技术人员应认识到,一般说来,多肽非必需区中的单一氨基酸取代基本上不会改变生物活性(参见例如Watson等人Molecular Biologyofthe Gene,第4版,1987,The Benjamin/Cummings Pub.Co.,p.224)。
在一个实施方案中,在期望表达两个或更多个多肽的情况下,如本文中其它地方所公开的,对所述两个或多个多肽进行编码的多核苷酸序列可通过IRES序列或编码核糖体跳跃序列的多核苷酸序列分开。
特定实施方案中所涵盖的多肽包括融合多肽。在特定实施方案中,提供了融合多肽和编码融合多肽的多核苷酸。融合多肽和融合蛋白是指具有至少二个、三个、四个、五个、六个、七个、八个、九个或十个多肽区段的多肽。在优选实施方案中,融合多肽包含一种或多种CD33 VHH DARIC组分。在其他优选的实施方案中,融合多肽包含一种或多种CD33 VHHDARIC。
在另一个实施方案中,两种或更多种CD33 VHH DARIC组分和/或其他多肽可以表达为融合蛋白,该融合蛋白包含本文其它地方公开的多肽之间的一条或多条自裂解肽序列。
在特定实施方案中,融合多肽包含CD33 DARIC信号传导组分、自裂解多肽序列或核糖体跳跃序列和CD33 VHH DARIC结合组分。
在特定实施方案中,融合多肽包含CD33 DARIC信号传导组分、自裂解多肽序列或核糖体跳跃序列、CD33 VHH DARIC结合组分、另一种自裂解多肽序列或核糖体跳跃序列和针对另一种靶抗原的另一种DARIC结合组分。
融合多肽可以包括:一个或多个多肽结构域或片段,包括但不限于信号肽、细胞渗透性肽结构域(CPP)、结合结构域、信号传导结构域、表位标签(例如,麦芽糖结合蛋白(“MBP”)、谷胱甘肽S转移酶(GST)、HIS6、MYC、FLAG、V5、VSV-G和HA);多肽接头;以及多肽裂解信号。融合多肽典型地是C末端连接至N末端,不过它们还可以是C末端连接至C末端、N末端连接至N末端、或N末端连接至C末端。在特定实施方案中,融合蛋白的多肽可以遵循任何次序。融合多肽或融合蛋白还可以包括保守性修饰的变体、多态性变体、等位基因、突变体、子序列和种间同系物,只要保存融合多肽所希望的活性即可。融合多肽可以通过化学合成方法或通过二个部分之间的化学键联制造,或一般可以使用其它标准技术制备。构成融合多肽的连接的DNA序列可操作地连接至如本文别处所公开的适合转录或翻译控制元件。
融合多肽可以任选地包括可以用于连接多肽内的一个或多个多肽或结构域的一个或多个接头。肽接头序列可以用于将任何两种或更多种多肽组分隔开一定距离,该距离足以确保每一多肽折叠成其适当二级结构和三级结构,从而允许多肽结构域发挥其所希望的功能。此类肽接头序列是使用本领域中的标准技术并入融合多肽中。适合肽接头序列可以基于以下因素选择:(1)其能够呈现柔性延长构象;(2)其无法呈现能与第一多肽和第二多肽上的功能性表位相互作用的二级结构;以及(3)缺乏可能与多肽功能性表位反应的疏水性或带电残基。在特定实施方案中,优选的肽接头序列含有Gly、Asn和Ser残基。其它接近中性的氨基酸,如Thr和Ala,也可以用于接头序列中。可以有用地用作接头的氨基酸序列包括Maratea等人,Gene 40:39-46,1985;Murphy等人,Proc.Natl.Acad.Sci.美国83:8258-8262,1986;美国专利第4,935,233号和美国专利第4,751,180号。当特定融合多肽区段含有可用于隔开功能性结构域并防止空间干扰的非必需N末端氨基酸区域时,不需要接头序列。在特定实施方案中,优选的接头通常是被合成为重组融合蛋白的一部分的柔性氨基酸子序列。接头多肽可以是在1个与200个氨基酸之间的长度、在1个与100个氨基酸之间的长度或在1个与50个氨基酸之间的长度,包括其间的所有整数值。
示例性多肽裂解信号包括多肽裂解识别位点,如蛋白酶裂解位点、核酸酶裂解位点(例如罕见限制酶识别位点、自裂解核酶识别位点)和自裂解病毒寡肽(参见deFelipeand Ryan,2004.Traffic,5(8);616-26)。
合适的蛋白酶切割位点和自切割肽是技术人员已知的(参见,例如Ryan等人,1997.J.Gener.Virol.78,699-722;Scymczak等人.(2004)Nature Biotech.5,589-594)。示例性蛋白酶裂解位点包括但不限于马铃薯Y病毒(potyvirus)NIa蛋白酶(例如烟草蚀刻病毒蛋白酶)、马铃薯Y病毒HC蛋白酶、马铃薯Y病毒P1(P35)蛋白酶、大麦花叶病毒(byovirus)NIa蛋白酶、大麦花叶病毒RNA-2编码的蛋白酶、口疮病毒(aphthovirus)L蛋白酶、肠病毒2A蛋白酶、鼻病毒2A蛋白酶、细小核糖核酸3C蛋白酶、豇豆花叶病毒(comovirus)24K蛋白酶、线虫传多面体病毒(nepovirus)24K蛋白酶、水稻东格鲁球状病毒(rice tungro sphericalvirus,RTSV)3C样蛋白酶、欧防风黄点病毒(parsnip yellow fleck virus,PYVF)3C样蛋白酶、肝素、凝血酶、因子Xa以及肠激酶的裂解位点。在一个实施方案中,TEV(烟草蚀刻病毒)蛋白酶裂解位点因其较高的裂解严格度而成为优选的,例如EXXYXQ(G/S)(SEQ ID NO:95),例如ENLYFQG(SEQ ID NO:96)和ENLYFQS(SEQ ID NO:97),其中X表示任何氨基酸(TEV裂解发生于Q与G或Q与S之间)。
在特定实施方案中,多肽裂解信号是病毒自裂解肽或核糖体跳跃序列。
核糖体跳跃序列的说明性实例包括但不限于:2A或2A样位点、序列或结构域(Donnelly等人,2001.J.Gen.Virol.82:1027-1041)。在特定实施方案中,病毒2A肽是口疮病毒2A肽、马铃薯Y病毒2A肽或心病毒2A肽。
在一个实施方案中,病毒2A肽选自由以下组成的组:口蹄疫病毒(FMDV)2A肽、马鼻炎A病毒(ERAV)2A肽、明脉扁刺蛾β四体(TaV)2A肽、猪捷申病毒-1(PTV-1)2A肽、泰勒病毒(Theilovirus)2A肽以及脑心肌炎病毒2A肽。
2A位点的示例性实例提供于表2中。
表2:
在优选实施方案中,多肽或融合多肽包含一种或多种CD33VHH DARIC组分、CD33VHH DARIC或抗CD33 VHH CAR。
在优选的实施方案中,融合多肽包含由自裂解多肽序列分开的CD33DARIC信号传导组分和CD33 VHH DARIC结合组分。
在特定实施方案中,融合多肽包含SEQ ID NO:32-61中任一者所示的序列。在特定实施方案中,融合多肽包含SEQ ID NO:40、50或60中任一者所示的序列。
在特定实施方案中,融合多肽包含:CD33 DARIC信号传导组分,其包括FRB T2098L多聚化结构域、CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域;病毒自裂解2A多肽;和CD33 VHH DARIC结合组分,其包括抗CD33 VHH、FKBP12多聚化结构域多肽和CD4跨膜结构域。
在特定实施方案中,融合多肽包含SEQ ID NO:32-41中任一者所示的序列。在具体实施方案中,融合多肽包含SEQ ID NO:40中所示的序列。
在特定实施方案中,融合多肽包含:CD33 DARIC信号传导组分,其包括FRB T2098L多聚化结构域、CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域;病毒自裂解2A多肽;和抗CD33 VHH、CD4跨膜结构域;以及任选的CD27、CD28、TNFRS14、TNFRS18、TNFRS25、OX40或TNFR2共刺激结构域。
在特定实施方案中,融合多肽包含SEQ ID NO:42-51中任一者所示的序列。在具体实施方案中,融合多肽包含SEQ ID NO:50中所示的序列。
在特定实施方案中,融合多肽包含SEQ ID NO:52-61中任一者所示的序列。在具体实施方案中,融合多肽包含SEQ ID NO:60中所示的序列。
F.多核苷酸
在特定实施方案中,提供了编码CD33 VHH DARIC、CD33 VHH DARIC结合组分、CD33DARIC信号传导组分、抗CD33 VHH CAR以及它们的片段的多核苷酸。如本文所使用,术语“多核苷酸”或“核酸”是指脱氧核糖核酸(DNA)、核糖核酸(RNA)和DNA/RNA杂交体。多核苷酸可以是单链或双链的并且是重组、合成或分离的。聚核苷酸包括但不限于:前信使RNA(pre-mRNA)、信使RNA(mRNA)、RNA、合成RNA、合成mRNA、基因组DNA(gDNA)、PCR扩增DNA、互补DNA(cDNA)、合成DNA或重组DNA。多核苷酸是指至少5个、至少10个、至少15个、至少20个、至少25个、至少30个、至少40个、至少50个、至少100个、至少200个、至少300个、至少400个、至少500个、至少1000个、至少5000个、至少10000个或至少15000个或更多个核苷酸长度,以及所有中间长度的核苷酸,即核糖核苷酸或脱氧核糖核苷酸或任一类型核苷酸的修饰形式的聚合形式。容易理解的是,在此情形中,“中间长度”意思指在引述值之间的任何长度,如6、7、8、9等;101、102、103等;151、152、153等;201、202、203等。在特定实施方案中,多核苷酸或变体与参考序列具有至少或约50%、55%、60%、65%、70%、71%、72%、73%、74%、75%,76%、77%、78%、79%、80%、81%、82%、83%、84%,85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%序列同一性。
如本文所使用,“分离的多核苷酸”是指已经从其两侧的处于天然存在状态的序列纯化的多核苷酸,例如,已经从通常与其相邻的序列移除的DNA片段。在特定实施方案中,“分离的多核苷酸”还指互补DNA(cDNA)、重组DNA或自然中不存在且已经通过人手制造的其它多核苷酸。在特定实施方案中,分离的多核苷酸是合成多核苷酸、半合成多核苷酸或从重组来源获得或衍生的多核苷酸。
在各种实施方案中,多核苷酸包括编码本文所涵盖的多肽的mRNA。在某些实施方案中,所述mRNA包含帽、一个或多个核苷酸和聚腺苷酸尾。
在特定实施方案中,对一种或多种CD33 VHH DARIC组分进行编码的多核苷酸可被密码子优化。如本文所使用,术语“密码子优化的”是指取代编码多肽的多核苷酸中的密码子以便增加所述多肽的表达、稳定性和/或活性。影响密码子优化的因素包括但不限于以下一个或多个:(i)两种或更多种生物体或基因之间密码子偏好的变化或合成地构造的偏好表;(ii)生物体、基因或基因集内密码子偏好程度的变化;(iii)包括环境在内的密码子的系统性变化;(iv)根据解码tRNA得到的密码子变化;(v)根据总体或三联体一个位置中的GC%得到的密码子变化;(vi)与参考序列,例如天然存在序列相似程度的变化;(vii)密码子频率截止值的变化;(viii)由DNA序列转录的mRNA的结构特性;(ix)有关作为设计密码子取代集合的基础的DNA序列的功能的先验知识;(x)每个氨基酸的密码子集的系统性变化;和/或(xi)伪翻译起始位点的分离移除。
如本文所使用,术语“核苷酸”是指与磷酸化糖的N-糖苷键联中的杂环含氮碱基。核苷酸应理解为包括天然碱基,以及多种本领域认可的经过修饰的碱基。此类碱基一般位于核苷酸糖部分的1′位处。核苷酸一般包含碱基、糖和磷酸酯基。在核糖核酸(RNA)中,糖是核糖,并且在脱氧核糖核酸(DNA)中,糖是脱氧核糖,即,不含核糖中存在的羟基的糖。
多核苷酸的说明性实例包括但不限于编码SEQ ID NO:2-82中任一者所示的多肽的多核苷酸。
在各种例示性实施方案中,本文所涵盖的多核苷酸包括但不限于编码一种或多种CD33 VHH DARIC组分、CD33 VHH DARIC受体、抗CD33 VHH CAR、融合多肽的多核苷酸,以及包含本文所涵盖的多核苷酸的表达载体、病毒载体和转移质粒。
如本文所使用,术语“多核苷酸变体”和“变体”等是指与参考多核苷酸序列展示相当大的序列同一性的多核苷酸或在下文所定义的严格条件下与参考序列杂交的多核苷酸。这些术语还涵盖因至少一个核苷酸的添加、缺失、取代或修饰而有别于参考多核苷酸的多核苷酸。因此,术语“多核苷酸变体”和“变体”包括一个或多个核苷酸被添加或缺失或修饰,或被置换成不同核苷酸的多核苷酸。就这一点而言,本领域中应充分理解,可以对参考多核苷酸进行包括突变、添加、缺失和取代在内的某些改变,由此使改变的多核苷酸保持参考多核苷酸的生物功能或活性。
如本文所使用,叙述“序列同一性”或例如包含“与……50%一致的序列”是指在核苷酸与核苷酸基础上或在氨基酸与氨基酸基础上,序列在比较窗内一致的程度。因此,“序列同一性百分比”可以通过以下方式计算:在比较窗内比较二个最佳地比对的序列,测定的一致核酸碱基(例如A、T、C、G、I)或一致氨基酸残基(例如Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys以及Met)在两个序列中出现的位置的数目以得到相配位置的数目,用相配位置的数目除以比较窗中的位置总数(即,窗大小)并将结果乘以100,得到序列同一性百分比。包含与本文所描述的参考序列中的任何一个具有至少约50%、55%、60%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、86%、97%、98%或99%序列同一性的核苷酸和多肽。
如本文其它地方所公开的或如本领域已知的,无论编码序列本身的长度如何,本文所涵盖的多核苷酸可以与其它DNA序列组合,如启动子和/或增强子、非转译区(UTR)、信号序列、Kozak序列、多腺苷酸化信号、另外的限制酶位点、多克隆位点、内部核糖体进入位点(IRES)、重组酶识别位点(例如,LoxP位点、FRT位点和Att位点)、终止密码子、转录终止信号以及对自裂解多肽、表位标记进行编码的多核苷酸,使得所述多核苷酸的总长度可以显著变化。因此,涵盖可以采用几乎任何长度的多核苷酸片段,其总长度优选地受制备的容易性和在预期的重组DNA方案中的使用的限制。
多核苷酸可以使用本领域中已知并且可用的多种公认技术中的任一种制备、操作、表达和/或递送。为了表达所希望的多肽,可将编码所述多肽的核苷酸序列插入适当载体中。
载体的示例性实例包括但不限于质粒、自主复制序列和转座元件,例如SleepingBeauty、PiggyBac。
载体的另外的说明性实例包括但不限于:质粒、噬菌粒、粘粒、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1源性人工染色体(PAC)、如λ噬菌体或M13噬菌体等噬菌体以及动物病毒。
可用作载体的病毒的示例性实例包括但不限于逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(例如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒以及乳多泡病毒(例如SV40)。
表达载体的示例性实例包括但不限于用于在哺乳动物细胞中表达的pClneo载体(Promega);用于慢病毒介导的基因转移和在哺乳动物细胞中表达的pLenti4/V5-DESTTM、pLenti6/V5-DESTTM和pLenti6.2/V5-GW/lacZ(Invitrogen)。在特定实施方案中,本文所公开的多肽的编码序列可连接至此类表达载体中以在哺乳动物细胞中表达所述多肽。
在特定实施方案中,载体是游离型载体或维持在染色体外的载体。如本文所使用,术语“附加型”是指能够复制而不整合到宿主的染色体DNA中且不会从分裂的宿主细胞中逐渐丧失的载体,这还意味着所述载体在染色体外或附加地复制。
表达载体中存在的“表达控制序列”、“控制元件”或“调节序列”是载体的那些非转译区,包括复制起点、选择盒、启动子、增强子、翻译起始信号(Shine Dalgamo序列或Kozak序列)内含子、多腺苷酸化序列、5′和3′非转译区,其与宿主细胞蛋白相互作用以进行转录和转译。这些元件在其强度和特异性方面可能有所变化。取决于所利用的载体系统和宿主,可使用多种适合的转录与翻译元件,包括遍在型启动子和诱导型启动子。
在特定实施方案中,多核苷酸包括载体,所述载体包括但不限于表达载体和病毒载体。载体可以包括一种或多种外源、内源或异源控制序列,如启动子和/或增强子。“内源控制序列”是与基因组中的给定基因天然连接的序列。“外源控制序列”是通过基因操纵(即分子生物学技术)与被放置成与基因并置使得此基因的转录由所连接的增强子/启动子引导的序列。“异源控制序列”是与被基因操纵的细胞来自不同物种的外源序列。“合成”控制序列可以包括一种或多种内源和/或外源序列和/或在体外或计算机中所确定的为特定疗法提供最佳启动子和/或增强子活性的序列的元件。
如本文所使用,术语“启动子”是指RNA聚合酶结合的多核苷酸(DNA或RNA)的识别位点。RNA聚合酶启动并转录与启动子可操作地连接的多核苷酸。在特定实施方案中,在哺乳动物细胞中起作用的启动子包括位于起始转录位点上游大约25个到30个碱基的AT富含区和/或发现距离转录起始上游70个到80个碱基的另一个序列,即N可以是任何核苷酸的CNCAAT区。
术语“增强子”是指含有能够提供增强的转录的序列并且在一些情况下可以不依赖于其相对于另一个控制序列的朝向而起作用的DNA的区段。增强子可以与启动子和/或其他增强子元件协同地或相加性地起作用。术语“启动子/增强子”是指含有能够提供启动子和增强子两者的功能的序列的DNA的区段。
术语“可操作地连接”是指所描述的组分的关系允许其以其预定方式起作用的并接。在一个实施方案中,术语是指核酸表达控制序列(如启动子和/或增强子)与第二多核苷酸序列,例如,所关注的多核苷酸之间的功能性连接,其中表达控制序列引导对应于第二序列的核酸的转录。
如本文所使用,术语“组成型表达控制序列”是指连续地或连续不断地允许可操作地连接的序列的转录的启动子、增强子或启动子/增强子。组成型表达控制序列可以是允许在各种各样的细胞和组织类型中表达的“普遍存在的”启动子、增强子或启动子/增强子或分别允许在受限种类的细胞和组织类型中表达的“细胞特异性”、“细胞类型特异性”、“细胞谱系特异性”或“组织特异性”启动子、增强子或启动子/增强子。
适合于在特定实施方案中使用的示例性普遍存在的表达控制序列包括但不限于:巨细胞病毒(CMV)立即早期启动子、病毒猿猴病毒40(SV40)(例如,早期或晚期)、莫洛尼鼠白血病病毒(MoMLV)LTR启动子、Rous肉瘤病毒(RSV)LTR、单纯疱疹病毒(HSV)(胸苷激酶)启动子、H5、来自牛痘病毒的P7.5启动子和P11启动子、延伸因子1-α(EF1a)启动子、早期生长应答1(EGR1)、铁蛋白H(FerH)、铁蛋白L(FerL)、甘油醛3-磷酸脱氢酶(GAPDH)、真核翻译起始因子4A1(EIF4A1)、热休克70kDa蛋白5(HSPA5)、热休克蛋白90kDaβ成员1(HSP90B1)、热休克蛋白70kDa(HSP70)、β-驱动蛋白(β-KIN)、人ROSA 26基因座(Irions等人,,NatureBiotechnology 25,1477-1482(2007))、泛素C启动子(UBC)、磷酸甘油酸激酶-1(PGK)启动子、巨细胞病毒增强子/鸡β-肌动蛋白(CAG)启动子、β-肌动蛋白启动子和骨髓增殖性肉瘤病毒增强子阴性对照区缺失并且d1587rev引物结合位点取代的(MND)U3启动子(Haas等人,Journal of Virology.2003;77(17):9439-9450)。
在一个实施方案中,载体包括MNDU3启动子。
在一个实施方案中,载体包括包含人EF1a基因的第一内含子的EF1a启动子。
在一个实施方案中,载体包括缺少人EF1a基因的第一内含子的EF1a启动子。
在特定实施方案中,可能期望使用细胞、细胞类型、细胞谱系或组织特异性表达控制序列来实现期望的多核苷酸序列的细胞类型特异性、谱系特异性或组织特异性表达(例如,仅在细胞类型、细胞谱系或组织的亚群中或在发育的特定阶段期间表达编码多肽的特定核酸)。
在特定实施方案中,可能需要将多核苷酸表达为T细胞特异性启动子。
如本文所使用,“条件表达”可以是指任何类型的条件表达,包括但不限于:诱导型表达;可阻遏型表达;在具有特定生理、生物或疾病状态等的细胞或组织中的表达。此定义不旨在排除细胞类型或组织特异性表达。某些实施方案提供了所关注多核苷酸的条件表达,例如通过使细胞、组织、生物体等经受导致多核苷酸表达或导致由所关注多核苷酸编码的多核苷酸的表达增加或减少的治疗或病状来控制表达。
诱导型启动子/系统的说明性实例包括但不限于:类固醇诱导型启动子如编码糖皮质激素或雌激素受体的基因的启动子(通过用对应的激素处理可诱导)、金属硫蛋白启动子(通过各种重金属处理可诱导)、MX-1启动子(通过干扰素可诱导)、“基因开关(GeneSwitch)”米非司酮可调节系统(Sirin等人,2003,Gene,323:67)、cumate诱导型基因开关(WO 2002/088346)、四环素依赖性调节系统等。诱导剂包括但不限于糖皮质激素、雌激素、米非司酮(RU486)、金属、干扰素、小分子、cumate、四环素、多西环素及其变体。
如本文所使用,“内部核糖体进入位点”或“IRES”是指促进内部核糖体直接进入顺反子(蛋白编码区)的如ATG等起始密码子由此导致基因的非帽依赖性转译的元件。参见,例如,Jackson等人,1990.Trends Biochem Sci 15(12):477-83)以及Jackson andKaminski.1995.RNA l(10):985-1000。本领域的技术人员通常采用的IRES的实例包括美国专利第6,692,736号中所描述的那些。本领域已知的“IRES”的其它实例包括但不限于可从微小RNA病毒获得的IRES(Jackson等人,1990)和可从病毒或细胞mRNA来源获得的IRES,例如免疫球蛋白重链结合蛋白(BiP)、血管内皮生长因子(VEGF)(Huez等人,1998.Mol.Cell.Biol.18(11):6178-6190)、成纤维细胞生长因子2(FGF-2)和胰岛素样生长因子(IGFID、翻译起始因子eIF4G和酵母转录因子TFIID和HAP4、可从Novagen商购的Ephelomencycarditis病毒(EMCV)(Duke等人,1992.J.Virol 66(3):1602-9)和VEGF IRES(Huez等人,1998.Mol Cell Biol 18(11):6178-90)。已经报道了小核糖核酸病毒科(Picomaviridae)、二顺反子病毒科(Dicistroviridae)和黄病毒科(Flaviviridae)物种的病毒基因组中以及HCV、弗里德鼠白血病病毒(Friendmurine leukemia virus,FrMLV)和莫洛尼氏鼠白血病病毒(Moloney murine leukemia virus,MoMLV)中的IRES。
在一个实施方案中,本文所涵盖的多核苷酸中使用的IRES是EMCVIRES。
在特定实施方案中,多核苷酸是共有Kozak序列。如本文所使用,术语“Kozak序列”是指大大促进mRNA与核糖体的小亚基的初始结合并增加转译的短核苷酸序列。共有Kozak序列是(GCC)RCCATGG(SEQ ID NO:83),其中R是嘌呤(A或G)(Kozak,1986.Cell.44(2):283-92,和Kozak,1987.NucleicAcids Res.15(20):8125-48)。
引导异源核酸转录物的高效终止和聚腺苷酸化的元件将增加异源基因的表达。转录终止信号一般见于聚腺苷酸化信号的下游。在特定实施方案中,载体在编码待表达多肽的多核苷酸的3′端包含聚腺苷酸化序列。如本文所使用,术语“聚腺苷酸位点(polyAsite)”或“聚腺苷酸序列(polyA sequence)”表示引导由RNA聚合酶II引起的初生RNA转录物终止和聚腺苷酸化的DNA序列。聚腺苷酸化序列可以通过在编码序列的3′端添加聚腺苷酸尾来促进mRNA稳定性,并因此促使翻译效率增加。裂解和聚腺苷酸化是由RNA中的聚腺苷酸序列引导。哺乳动物mRNA前体(pre-mRNA)的核心聚腺苷酸序列有两个识别元件侧接裂解-聚腺苷酸化位点上。典型地,几乎不变的AAUAAA六聚物位于富含U或GU残基的可变性较高的元件上游20-50个核苷酸处。初生转录物的裂解发生于这两个元件之间并偶合以将多达250个腺苷添加至5′裂解产物中。在特定实施方案中,核心聚腺苷酸序列是理想的聚腺苷酸序列(例如AATAAA、ATTAAA、AGTAAA)。在特定实施方案中,聚腺苷酸序列是SV40聚腺苷酸序列、牛生长激素聚腺苷酸序列(BGHpA)、兔β-珠蛋白聚腺苷酸序列(rβgpA)、其变体、或本领域中已知的另一种适合的异源或内源性聚腺苷酸序列。在特定实施方案中,聚腺苷酸序列是合成的。
在特定实施方案中,对一种或多种多肽或融合多肽进行编码的多核苷酸可以通过非病毒和病毒方法引入到免疫效应细胞中,例如T细胞。在特定实施方案中,一种或多种多核苷酸的递送可以通过相同的方法或通过不同的方法和/或通过相同的载体或通过不同的载体来提供。
术语“载体”在本文中用以指能够转移或输送另一个核酸分子的核酸分子。被转移的核酸一般被连接至载体核酸分子,例如插入载体核酸分子中。载体可以包括引导在细胞中自主复制的序列,或可以包括足以允许整合至宿主细胞DNA中的序列。在特定实施方案中,使用了非病毒载体将本文所涵盖的一个或多个多核苷酸递送至T细胞中。
非病毒载体的示例性实例包括但不限于质粒(例如DNA质粒或RNA质粒)、转座子、粘粒和细菌人工染色体。
非病毒递送特定实施方案中所涵盖的多核苷酸的示例性方法包括但不限于:电穿孔、声致穿孔、脂质体转染、显微注射、基因枪法、病毒粒子、脂质体、免疫脂质体、纳米粒子、聚阳离子或脂质:核酸偶联物、裸DNA、人工病毒粒子、DEAE-葡聚糖介导的转移、基因枪以及热休克。
适用于特定实施方案中所涵盖的特定实施方案中使用的多核苷酸递送系统的说明性实例包括但不限于由Amaxa Biosystems、Maxcyte,Inc.、BTX Molecular DeliverySystems和Copemicus Therapeutics Inc.提供的那些。脂质转染试剂是商业销售的(例如,TransfectamTM和LipofectinTM)。已经在文献中描述了适合于多核苷酸的高效受体识别脂质转染的阳离子脂质和中性脂质。参见例如Liu等人(2003)Gene Therapy.10:180-187;和Balazs等人(2011)Journal ofDrug Delivery.2011:1-12。在特定实施方案中还涵盖了抗体靶向的、源自细菌的、基于无生命纳米细胞的递送。
在特定实施方案中所涵盖的包含多核苷酸的病毒载体可以通过施用至个体患者体内递送,通常通过全身施用(例如,静脉内、腹膜内、肌内、皮下或颅内输注)或局部施用,如下所述。可替代地,载体可以离体递送到细胞,如从个体患者移植的细胞(例如,动员的外周血、淋巴细胞、骨髓抽吸物、组织活检等)或通用供体造血干细胞,然后将细胞再植入患者体内。
在一个实施方案中,将包含本文所涵盖的多核苷酸的病毒载体直接施用至生物体以在体内转导细胞。可替代地,可以施用裸DNA。通过通常用于引入分子与血液或组织细胞最终接触的任何途径施用,包括但不限于注射、输注、局部施用和电穿孔。施用这种核酸的合适的方法是本领域技术人员可获得的并且是公知的,并且尽管可以使用多于一种途径来施用特定组合物,但是特定途径通常可以提供比另一条途径更直接且更有效的反应。
适合于在特定实施方案中所涵盖的特定实施方案中使用的病毒载体系统的说明性实例包括但不限于腺相关病毒(AAV)、逆转录病毒、单纯疱疹病毒、腺病毒以及痘苗病毒载体。
在各种实施方案中,通过用包含一种或多种多核苷酸的重组腺相关病毒(rAAV)转导细胞,将编码本文所涵盖的一种或多种CD33 VHH DARIC组分和/或其他多肽的一种或多种多核苷酸引入免疫效应细胞,例如T细胞。
AAV是一种小型(约26nm)复制缺陷型、主要是附加型无包膜病毒。AAV可以感染分裂和非分裂细胞,并且可以将其基因组并入到宿主细胞的基因组中。重组AAV(rAAV)通常至少由转基因及其调节序列和5′和3′AAV反向末端重复序列(ITR)构成。ITR序列的长度为约145bp。在特定实施方案中,rAAV包括从AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9或AAV10分离的ITR和衣壳序列。
在一些实施方案中,使用嵌合rAAV,从一种AAV血清型分离ITR序列,并从不同的AAV血清型分离衣壳序列。例如,具有源自AAV2的ITR序列和源自AAV6的衣壳序列的rAAV被称为AAV2/AAV6。在特定实施方案中,rAAV载体可以包括来自AAV2的ITR以及来自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9或AAV10中任一种的衣壳蛋白。在优选实施方案中,rAAV包括源自AAV2的ITR序列和源自AAV6的衣壳序列。在优选实施方案中,rAAV包括源自AAV2的ITR序列和源自AAV2的衣壳序列。
在一些实施方案中,工程和选择方法可以应用于AAV衣壳,以使其更有可能转导所关注细胞。
已经公开了rAAV载体的构建、制备和其纯化,例如在美国专利第9,169,494号;第9,169,492号;第9,012,224号;第8,889,641号;第8,809,058号;以及第8,784,799号,上述美国专利中的每一个以全文引用的方式并入本文中。
在各种实施方案中,通过用包含一种或多种多核苷酸的逆转录病毒,例如慢病毒转导细胞,将编码本文所涵盖的一种或多种CD33 VHH DARIC组分和/或其他多肽的一种或多种多核苷酸引入免疫效应细胞,例如T细胞。
如本文所使用,术语“逆转录病毒”是指RNA病毒,所述RNA病毒将其基因组RNA逆转录为线性双链DNA拷贝并且随后将其基因组DNA共价整合到宿主基因组中。适用于特定实施方案的示例性逆转录病毒包括但不限于:莫洛尼氏鼠白血病病毒(M-MuLV)、莫洛尼氏鼠肉瘤病毒(MoMSV)、哈维鼠肉瘤病毒(HaMuSV)、鼠乳腺肿瘤病毒(MuMTV)、长臂猿白血病病毒(GaLV)、猫白血病病毒(FLV)、泡沫病毒、弗里德鼠白血病病毒、鼠干细胞病毒(MSCV)和劳斯肉瘤病毒(RSV)以及慢病毒。
如本文所使用,术语“幔病毒”是指复杂逆转录病毒的组(或属)。说明性慢病毒包括但不限于:HIV(人免疫缺陷病毒;包含HIV 1型和HIV 2型);维斯纳-梅迪病毒(visna-maedi virus,VMV)病毒;山羊关节炎-脑炎病毒(CAEV);马传染性贫血病毒(EIAV);猫免疫缺陷病毒(FIV);牛免疫缺陷病毒(BIV);以及猿猴免疫缺陷病毒(SIV)。在一个实施方案中,优选基于HIV的载体主链(即,HIV顺式作用序列元件)。
在各种实施方案中,本文所涵盖的慢病毒载体包括一个或多个LTR,以及以下辅助元件中的一个或多个或全部:cPPT/FLAP、Psi(Ψ)包装信号、输出元件、poly(A)序列,并且可以任选地包括WPRE或HPRE、绝缘子元件、可选择标志物和细胞自杀基因,如本文其它地方所讨论的。
在特定实施方案中,本文所涵盖的慢病毒载体可以是整合的或非整合的或整合缺陷型慢病毒。如本文所使用,术语“整合缺陷型慢病毒”或“IDLV”是指具有缺乏将病毒基因组整合到宿主细胞的基因组的能力的整合酶的慢病毒。专利申请WO 2006/010834中已经描述了无整合能力的病毒载体,所述专利申请以全文引用的方式并入本文中。
适于降低整合酶活性的HIV-1 pol基因中的示例性突变包括但不限于:H12N、H12C、H16C、H16V、S81 R、D41A、K42A、H51A、Q53C、D55V、D64E、D64V、E69A、K71A、E85A、E87A、D116N、D1161、D116A、N120G、N1201、N120E、E152G、E152A、D35E、K156E、K156A、E157A、K159E、K159A、K160A、R166A、D167A、E170A、H171A、K173A、K186Q、K186T、K188T、E198A、R199c、R199T、R199A、D202A、K211A、Q214L、Q216L、Q221 L、W235F、W235E、K236S、K236A、K246A、G247W、D253A、R262A、R263A和K264H。
术语“长末端重复序列(LTR)”是指位于逆转录病毒DNA的末端处的碱基对的结构域,其在其天然序列环境中是直接重复序列并含有U3、R和U5区。
如本文所使用,术语“FLAP元件”或“cPPT/FLAP”指代序列包含逆转录病毒(例如,HIV-1和HIV-2)的中心多嘌呤段和中心终止序列(cPPT和CTS)的核酸。合适的FLAP元件描述于美国专利第6,682,907号和Zennou等人,2000,Cell.101:173。
如本文所用,术语“包装信号”或“包装序列”是指位于逆转录病毒基因组内的psi[Ψ]序列,其是病毒RNA插入病毒衣壳或颗粒所需的,参见例如Clever等人,1995.J.ofVirology,第69卷,第4期;第2101-2109页。
术语“输出元件”是指调节RNA转录物从细胞的细胞核向细胞质转运的顺式作用转录后调节元件。RNA输出元件的实例包括但不限于人免疫缺陷病毒(HIV)rev应答元件(RRE)(参见例如Cullen等人,1991.J.Virol.65:1053;以及Cullen等人1991.Cell 58:423),和乙型肝炎病毒转录后调节元件(HPRE)。
在特定实施方案中,通过将转录后调节元件、高效的多腺苷酸化位点和任选地转录终止信号结合到载体中来增加异源序列在病毒载体中的表达。多种转录后调节元件可以增加异源核酸在蛋白质上的表达,例如土拨鼠肝炎病毒转录后调节元件(WPRE;Zufferey等人,1999,J.Virol.,73:2886);存在于乙型肝炎病毒(HPRE)中的转录后调节元件(Huang等人,Mol.Cell.Biol.,5:3864);等(Liu等人,1995,Genes Dev.,9:1766)。
由于修饰LTR,慢病毒载体优选地含有几种安全性增强。“自失活”(SIN)载体是指复制缺陷型载体,例如逆转录病毒载体或慢病毒载体,其中被称为U3区的右(3′)LTR增强子-启动子区已经被修饰(例如,通过缺失或取代)以防止病毒转录超过第一轮病毒复制。优选地通过在载体DNA的3′LTR的U3区引入缺失(即,用于产生载体RNA的DNA)来实现自失活。因此,在逆转录期间,此缺失被转移到前病毒DNA的5′LTR。在特定实施方案中,期望消除足够的U3序列以大大减少或完全消除LTR的转录活性,从而大大减少或消除转导细胞中全长载体RNA的产生。在基于HIV的慢病毒载体的情况下,已经发现这种载体耐受显著的U3缺失,包含去除LTR TATA盒(例如,从-418至-18的缺失),而没有显著降低载体滴度。
通过用异源启动子替代5′LTR的U3区来提供另外的安全性增强以驱动病毒颗粒产生期间病毒基因组的转录。可以使用的异源启动子的实例包含例如病毒性猿猴病毒40(SV40)(例如,早期或晚期)、巨细胞病毒(CMV)(例如,立即早期)、莫洛尼氏鼠白血病病毒(MoMLV)、劳斯肉瘤病毒(RSV)和单纯性疱疹病毒(HSV)(胸苷激酶)启动子。
如本文所使用,术语“假型”或“假型包装”是指其病毒包膜蛋白已经被具有优选特性的另一病毒的病毒包膜蛋白取代的病毒。例如,HIV可以用水疱性口炎病毒G蛋白(VSV-G)包膜蛋白进行假型包装,这允许HIV感染更广泛的细胞,因为HIV包膜蛋白(由env基因编码)通常将病毒靶向CD4+呈递细胞。
在某些实施方案中,根据已知方法产生慢病毒载体。参见例如Kutner等人,BMCBiotechnol.2009;9:10.doi:10.1186/1472-6750-9-10;Kutner等人Nat.Protoc.2009;4(4):495-505.doi:10.1038/nprot.2009.22。
根据本文所涵盖的某些特定实施方案,大多数或所有病毒载体骨架序列衍生自慢病毒,例如HIV-1。然而,应理解,可以使用或组合许多不同来源的逆转录病毒和/或慢病毒序列,或者可以容置慢病毒序列中的某些慢病毒序列的多种取代和改变而不损害转移载体执行本文所描述的功能的能力。此外,本领域已知多种慢病毒载体,参见Naldini等人,(1996a、1996b和1998);Zufferey等人,(1997);Dull等人,1998,美国专利第6,013,516号、第5,994,136号,其中许多可以适于产生本文所涵盖的病毒载体或转移质粒。
在各种实施方案中,通过用包含一种或多种多核苷酸的腺病毒转导细胞,将编码本文所涵盖的一种或多种CD33 VHH DARIC组分和/或其他多肽的一种或多种多核苷酸引入免疫效应细胞。
基于腺病毒的载体能够在许多细胞类型中具有极高转导效率并且并不需要细胞分裂。使用这种载体,已经获得了高滴度和高表达水平。此载体可以在相对简单的系统中大量制备。将大多数腺病毒载体工程改造,使得转基因取代Ad E1a、E1b和/或E3基因;随后,在以反式提供缺失的基因功能的人293细胞中繁殖复制缺陷型载体。Ad载体可以在体内转导多种类型的组织,包含如在肝、肾和肌肉中发现的非分裂的分化细胞。常规Ad载体具有很大的承载能力。
复制缺陷的当前腺病毒载体的产生和繁殖可以利用命名为293的独特辅助细胞系,所述辅助细胞系通过Ad5 DNA片段从人胚胎肾细胞转化并组成性地表达E1蛋白(Graham等人,1977)。由于E3区可从腺病毒基因组中分配出来(Jones和Shenk,1978),所以目前的腺病毒载体在293细胞的帮助下在E1、D3区或两个区中携带外源DNA(Graham和Prevec,1991)。腺病毒载体已经用于真核基因表达(Levrero等人,1991;Gomez-Foix等人,1992)和疫苗开发(Grunhaus&Horwitz,1992;Graham&Prevec,1992)。向不同的组织施用重组腺病毒方面的研究包含气管滴注(Rosenfeld等人,1991;Rosenfeld等人,1992)、肌肉注射(Ragot等人,1993)、外周静脉注射(Herz&Gerard,1993)以及立体定向脑内接种(Le Gal La Salle等人,1993)。在临床试验中使用Ad载体的实例涉及用于伴随肌内注射的抗肿瘤免疫的多核苷酸疗法(Sterman等人,Hum.Gene Ther.7:1083-9(1998))中所述。
在各种实施方案中,通过用包含一种或多种多核苷酸的单纯疱疹病毒,例如HSV-1、HSV-2转导细胞,将编码本文所涵盖的一种或多种CD33 VHH DARIC组分和/或其他多肽的一种或多种多核苷酸引入免疫效应细胞。
成熟HSV病毒粒子由包膜的二十面体衣壳组成,其中病毒基因组由152kb的线性双链DNA分子组成。在一个实施方案中,基于HSV的病毒载体缺乏一个或多个必需或非必需的HSV基因。在一个实施方案中,基于HSV的病毒载体为复制缺陷型。大多数复制缺陷型HSV载体含有缺失以去除一个或多个中早期、早期或晚期HSV基因从而防止复制。例如,HSV载体可能缺乏选自由以下组成的组的立即早期基因:ICP4、ICP22、ICP27、ICP47及其组合。HSV载体的优点是其进入可以导致长期DNA表达的潜伏期的能力,以及其可以容纳高达25kb的外源DNA插入物的大型病毒DNA基因组。基于HSV的载体描述于例如美国专利第5,837,532号、第5,846,782号和第5,804,413号以及国际专利申请WO 9I/02788、WO 96/04394、WO 98/15637和WO 99/06583中,所述专利中的每一个以全文引用的方式并入本文中。
G.经过基因修饰的细胞
在各种实施方案中,对细胞进行修饰以表达用于治疗癌症的本文所涵盖的CD33VHH DARIC、一种或多种CD33 VHH DARIC组分、抗CD33 VHH CAR和/或融合蛋白。可以对细胞进行非基因修饰以表达本文所涵盖的多肽中的一种或多种多肽,或者在特定的优选实施方案中,可以对细胞进行基因修饰以表达本文所涵盖的多肽中的一种或多种多肽。如本文所使用,术语“基因工程改造”或“基因修饰”是指将额外的遗传物质以DNA或RNA的形式添加到细胞中的总遗传物质中。在特定实施方案中,术语“基因修饰细胞”、“修饰细胞”和“重新定向的细胞”可互换使用。
在特定实施方案中,本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHHCAR被引入免疫效应细胞中并在其中表达,以改进免疫效应细胞的功效。
“免疫效应细胞”是免疫系统的任何细胞,其具有一种或多种效应功能(例如,细胞毒性细胞杀伤活性、细胞因子分泌、ADCC和/或CDC的诱导)。本文所涵盖的说明性免疫效应细胞是T淋巴细胞,包括但不限于细胞毒性T细胞(CTL;CD8+T细胞)、TIL和辅助T细胞(HTL;CD4+细胞)。在特定实施方案中,所述细胞包括αβ T细胞。在特定实施方案中,所述细胞包括γδ T细胞。在一个实施方案中,免疫效应细胞包括自然杀伤(NK)细胞。在一个实施方案中,免疫效应细胞包括自然杀伤T(NKT)细胞。免疫效应细胞可以是自体的/自身的(“自我”)或非自体的(“非自我”,例如同种异体的、同基因的或异种的)。
如本文所使用,“自体”是指来自同一受试者的细胞。如本文所使用,“同种异体”是指来自同一物种的在基因上与比较细胞不同的细胞。如本文所使用,“同基因”是指来自不同受试者的在基因上与比较细胞相同的细胞。如本文所使用,“异种”是指来自与比较细胞不同的物种的细胞。在优选实施方案中,细胞是人自体免疫效应细胞。
适用于引入本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR的说明性免疫效应细胞包括T淋巴细胞。术语“T细胞”或“T淋巴细胞”是本领域公认的并且旨在包含胸腺细胞、未成熟T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或活化T淋巴细胞。T细胞可以是T辅助(Th)细胞,例如T辅助1(Th1)或T辅助2(Th2)细胞。T细胞可以是辅助T细胞(HTL;CD4+T细胞)、细胞毒性T细胞(CTL;CD8+T细胞)、CD4+CD8+T细胞、CD4-CD8-T细胞或任何其他的T细胞亚群。在特定实施方案中,T细胞表达T细胞受体。T细胞受体包含两个亚基,即α链和β链亚基(αβTCR),或γ链和δ链亚基(γδTCR),其中每一个是通过每个T细胞基因组中的重组事件产生的独特蛋白质。在特定实施方案中,T细胞是αβTCR T细胞(αβ T细胞)。在特定实施方案中,T细胞是γδTCR T细胞(γδ T细胞)。适用于特定实施方案中的其它示例性T细胞群包括原生T细胞和记忆T细胞。
如本领域技术人员所理解的,其他细胞还可用作包含本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR的免疫效应细胞。在特定实施方案中,免疫效应细胞还包含NK细胞、NKT细胞、嗜中性粒细胞和巨噬细胞。免疫效应细胞还包含效应细胞的祖细胞,其中可以在体内或体外诱导这种祖细胞分化成免疫效应细胞。因此,在特定实施方案中,免疫效应细胞包含免疫效应细胞的祖细胞,如源自脐带血、骨髓或动员的外周血的CD34+细胞群内含有的造血干细胞(HSC),所述HSC在受试者中施用后分化为成熟免疫效应细胞,或可以在体外诱导所述HSC分化为成熟免疫效应细胞。
如本文所使用,术语“CD34+细胞”是指在其细胞表面上表达CD34蛋白的细胞。如本文所使用,“CD34”是指通常充当细胞-细胞粘附因子并且参与T细胞进入淋巴结的细胞表面糖蛋白(例如,唾液黏蛋白)。CD34+细胞群含有造血干细胞(HSC),其在施用给患者时分化并促成所有造血谱系,包含T细胞、NK细胞、NKT细胞、嗜中性粒细胞和单核细胞/巨噬细胞谱系的细胞。
在特定实施方案中,提供了用于制备表达本文所涵盖的一种或多种CD33 VHHDARIC组分或抗CD33 VHH CAR的免疫效应细胞的方法。在一个实施方案中,该方法包括转染或转导从个体分离的免疫效应细胞,使得免疫效应细胞具有一种或多种核酸和/或载体,例如包含编码本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR的核酸的慢病毒载体。在一个实施方案中,该方法包括转染或转导从个体分离的免疫效应细胞,使得免疫效应细胞表达本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR。在某些实施方案中,免疫效应细胞从个体分离并经基因修饰而无需在体外进一步操纵。然后,可以将这种细胞直接再次施用到个体。在另外的实施方案中,免疫效应细胞在基因修饰之前首先在体外被活化和刺激以增殖。在这方面,可以在基因修饰之前和/或之后培养免疫效应细胞。
在特定实施方案中,在本文所述的免疫效应细胞的体外操纵或基因修饰之前,从受试者获得细胞来源。在特定实施方案中,经过修饰的免疫效应细胞包括T细胞。
T细胞可以从多种来源获得,包括但不限于周边血液单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、肋膜积液、脾组织以及肿瘤。在某些实施方案中,可以使用技术人员已知的任何数目的技术如沉降(例如,FICOLLTM分离)从采集自受试者的单位血液中获得T细胞。
在其它实施方案中,使用分离的或纯化的T细胞群。在一些实施方案中,在PBMC的分离之后,可以在活化、扩增和/或基因修饰之前或之后将细胞毒性和辅助T淋巴细胞分类为原初、记忆和效应T细胞亚群。
在一个实施方案中,分离的或纯化的T细胞群表达包括但不限于以下的标志物中的一个或多个:CD3+、CD4+、CD8+或其组合。
在某些实施方案中,T细胞从个体分离,并且在进行修饰以表达一种或多种CD33VHH DARIC组分或抗CD33VHH CAR之前首先进行体外活化和刺激以增殖。
为了实现足够治疗剂量的T细胞组合物,通常对T细胞进行一轮或多轮刺激、活化和/或扩增。在特定实施方案中,通常可以使用如例如以下美国专利中所述的方法使T细胞活化和扩增:第6,352,694号;第6,534,055号;第6,905,680号;第6,692,964号;第5,858,358号;第6,887,466号;第6,905,681号;第7,144,575号;第7,067,318号;第7,172,869号;第7,232,566号;第7,175,843号;第5,883,223号;第6,905,874号;第6,797,514号;和第6,867,041号,上述美国专利中的每一个以全文引用的方式并入本文中。在特定实施方案中,在引入编码本文所涵盖的一种或多种CD33 VHHDARIC组分或抗CD33VHH CAR的载体或多核苷酸,使T细胞活化和扩增约6小时、约12小时、约18小时或约24小时。
H.组合物和配制物
本文所涵盖的组合物可包含一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR、编码一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR的多核苷酸、包含它们的载体、经过基因修饰的免疫效应细胞、桥接因子等。组合物包括但不限于药物组合物。“药物组合物”是指单独地或与一种或多种其它治疗方式组合地施用于细胞或动物的在药学上可接受的或生理学上可接受的溶液中配制的组合物。还应理解,如果需要,组合物也可以与其他药剂组合施用,例如细胞因子、生长因子、激素、小分子、化学治疗剂、前药、药物、抗体或其他各种药学活性剂。对组合物中还可以包含的其它组分实际上不存在限制,条件是另外的药剂不会不利地影响组合物递送预期疗法的能力。
短语“药学上可接受的”在本文中用于指在正确医学判断的范围内适合于与人和动物的组织接触使用而不会产生过多毒性、刺激、过敏反应或其它问题或并发症的、与合理的益处/风险比相称的那些化合物、材料、组合物和/或剂型。
术语“药学上可接受的载剂”是指对桥接因子、多肽、多核苷酸、包括其的载体或经过基因修饰的免疫效应细胞施用的稀释剂、佐剂、赋形剂或媒介物。药物载剂的说明性实例可以是无菌液体,如细胞培养基、水和油,包含石油、动物、植物或合成来源的油,如花生油、大豆油、矿物油、芝麻油等。还可以使用生理盐水溶液和水性右旋糖以及甘油溶液作为液体载体,特别是用于可注射溶液。在特定实施方案中,适合的药物赋形剂包含淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、水稻、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。除了任何常规培养基或药剂与活性成分不相容的情况之外,涵盖了其在药物组合物中的用途。补充性活性成分也可以并入到组合物中。
在一个实施方案中,包括药学上可接受的载剂的组合物适于施用于受试者。在特定实施方案中,包括载剂的组合物适于肠胃外施用,例如血管内(静脉内或动脉内)、腹膜内或肌内施用。在特定实施方案中,包括药学上可接受的载剂的组合物适合于心室内、脊柱内或鞘内施用。药学上可接受的载剂包含无菌水溶液、细胞培养基或分散体。这些培养基和药剂用于药物活性物质的用途在本领域中是熟知的。除了任何常规培养基或药剂与桥接因子、多肽、多核苷酸、包括其的载体或经过基因修饰的免疫效应细胞不相容的情况之外,涵盖其在药物组合物中的用途。
在特定实施方案中,本文所涵盖的组合物包括经过基因修饰的T细胞和药学上可接受的载剂。本文所涵盖的包括基于细胞的组合物的组合物可以通过肠内或肠胃外施用方法单独地或与其它适合的化合物组合地施用以实现期望的治疗目的。
在特定实施方案中,本文所涵盖的组合物包括桥接因子和药学上可接受的载剂。
药学上可接受的载剂必须具有足够高的纯度和足够低的毒性以使其适于施用于正在治疗的人类受试者。药学上可接受的载剂应当保持或增加组合物的稳定性。药学上可接受的载剂可以是液体的或固体的并且在涵盖计划的施用方式的情况下被选择以在与组合物的其它组分组合时提供期望的体积、同一性等。例如,药学上可接受的载剂可以是但不限于结合剂(例如,预胶化玉米淀粉、聚乙烯吡咯烷酮或羟丙基甲基纤维素等)、填充剂(例如,乳糖和其它糖、微晶纤维素、果胶、明胶、硫酸钙、乙基纤维素、聚丙烯酸酯、磷酸氢钙等)、润滑剂(例如,硬脂酸镁、滑石、硅石、胶体二氧化硅、硬脂酸、金属硬脂酸盐、氢化植物油、玉米淀粉、聚乙二醇、苯甲酸钠、乙酸钠等)、崩解剂(例如,淀粉、羧基乙酸淀粉钠等)或润湿剂(例如,月桂基硫酸钠等)。用于本文所涵盖的组合物的其它适合的药学上可接受的载剂包括但不限于水、盐溶液、醇、聚乙二醇、明胶、直链淀粉、硬脂酸镁、滑石、硅酸、粘性石蜡、羟甲基纤维素、聚乙烯吡咯烷酮等等。
此类载剂溶液还可以含有缓冲剂、稀释剂和其它适合的添加剂。如本文所使用,术语“缓冲剂”是指化学成分中和酸或碱而不显著改变pH的溶液或液体。本文所涵盖的缓冲剂的实例包括但不限于杜尔贝克氏(Dulbecco′s)磷酸盐缓冲盐水(PBS)、林格氏(Ringer′s)溶液、5%葡萄糖水溶液(D5W)、正常(normal)/生理(physiologic)盐水(0.9%NaCl)。
药学上可接受的载剂可以存在的量足以使组合物的pH保持处于约7。可替代地,组合物的pH的范围为约6.8至约7.4,例如6.8、6.9、7.0、7.1、7.2、7.3和7.4。在仍另一个实施方案中,所述组合物的pH为约7.4。
本文所涵盖的组合物可以包括无毒的药学上可接受的培养基。组合物可以是悬浮液。如本文所使用,术语“悬浮液”是指细胞未连接到固相支持物的非粘附性条件。例如,可以搅拌或搅动保持为悬浮液的细胞并且所述细胞未粘附到支持物,如培养皿。
在特定实施方案中,本文所涵盖的组合物在悬浮液中配制,其中经过修饰的T细胞分散在静脉注射(IV)袋等中的可接受的液体培养基或溶液例如盐水或无血清培养基内。可接受的稀释剂包括但不限于水、PlasmaLyte、林格氏溶液、等渗氯化钠(盐水)溶液、无血清细胞培养基和适合于低温储存的培养基,例如培养基。
在某些实施方案中,药学上可接受的载剂基本上不含人或动物来源的天然蛋白质,并且适合于储存包括经过修饰的T细胞群的组合物。药物组合物旨在施用到人类患者中并且因此基本上不含细胞培养组分,如牛血清白蛋白、马血清和胎牛血清。
在一些实施方案中,所述组合物在药学上可接受的细胞培养基中配制。此类组合物适于施用于人类受试者。在特定实施方案中,药学上可接受的细胞培养基是无血清培养基。
无血清培养基相比于含有血清的培养基具有若干优点,包含组合物得到简化且得到更好定义、污染物程度得到减小、可能的传染剂来源得到消除以及成本降低。在各种实施方案中,无血清培养基是无动物的并且可以任选地是无蛋白质的。任选地,培养基可以含有生物药学上可接受的重组蛋白。“无动物”培养基是指组合物源自非动物来源的培养基。重组蛋白替代无动物培养基中的天然动物蛋白质,并且营养物从合成的、植物或微生物来源获得。相比之下,“无蛋白质”培养基被定义为基本上不含蛋白质。
特定实施方案中使用的无血清培养基的说明性实例包括但不限于QBSF-60(Quality Biological,Inc.)、StemPro-34(Life Technologies)和X-VIVO 10。
在一个实施方案中,包括经过修饰的T细胞的组合物在PlasmaLyte中配制。
在各种实施方案中,包括经过修饰的T细胞的组合物在冷冻保存培养基中配制。例如,可以使用具有低温保存药剂的低温保存培养基在解冻后保持高细胞存活率结果。特定实施方案中使用的低温保存培养基的说明性实例包括但不限于CryoStor CS10、CryoStorCS5和CryoStor CS2。
在一个实施方案中,所述组合物在包括50:50PlasmaLyte A:CryoStor CS10的溶液中配制。
在特定实施方案中,所述组合物基本上不含支原体、内毒素和微生物污染。关于内毒素,“基本上不含”是指每剂细胞的内毒素含量低于FDA针对生物制剂允许的含量,其为每天5EU/kg体重的总内毒素,针对平均70kg的人而言为每总剂量细胞350EU。在特定实施方案中,本文所涵盖的组合物含有约0.5EU/mL至约5.0EU/mL、或约0.5EU/mL、1.0EU/mL、1.5EU/mL、2.0EU/mL、2.5EU/mL、3.0EU/mL、3.5EU/mL、4.0EU/mL、4.5EU/mL或5.0EU/mL。
在特定实施方案中,对药学上可接受的载剂溶液的配制对本领域的技术人员来说是熟知的,这和开发用于将本文所描述的特定组合物用于各种治疗方案中的适合的给药和治疗方案一样,包含例如肠内和肠胃外,例如血管内、静脉内、动脉内、骨内、心室内、脑内、颅内、脊柱内、鞘内和髓内施用和配制。本领域技术人员应当理解,本文所涵盖的特定实施方案可以包括其它制剂,例如在药物领域中众所周知的那些,并且描述于例如Remington:The Science and Practice ofPharmacy,第I卷和第II卷,第22版编著:Loyd V.AllenJr.Philadelphia,PA:Pharmaceutical Press;2012中,上述美国专利中的每一个以全文引用的方式并入本文中。
在特定实施方案中,组合物包含一定量的免疫效应细胞,该免疫效应细胞包含编码本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR的多核苷酸。在特定实施方案中,组合物包含一定量的免疫效应细胞,该免疫效应细胞表达本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR。如本文所使用,术语“量”是指包括本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR等以在存在桥接因子的情况下实现有益或期望的预防或治疗结果(包括临床结果)的“有效的量”或“有效量”的细胞。
“预防有效量”是指有效实现期望的预防结果的包括本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR等的细胞的量。通常,但不一定,因为预防剂量是在疾病之前或疾病早期用于受试者的,所以预防有效量小于治疗有效量。
“治疗有效量”是指在存在桥接因子的情况下有效“治疗”受试者(例如,患者)的包括本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33VHH CAR的细胞的量。当指示治疗量时,待施用的组合物、细胞、桥接因子等的精确量可以由内科医生涵盖年龄、体重、肿瘤大小、感染程度或转移程度以及患者(受试者)的病状的个体差异来确定。
通常可以说,包括本文所述的免疫效应细胞的药物组合物可以以102至1010个细胞/kg体重,优选地105至106个细胞/kg体重(包含那些范围内的所有整数值)的剂量施用。细胞的数目将取决于组合物的期望最终用途,其中包含的细胞的类型也是如此。对于本文所提供的用途,细胞的体积通常为一升或更少,可以为500ml或更少,甚至250ml或100ml或更少。因此,所期望细胞的密度通常大于106个细胞/mL,并且通常大于107个细胞/mL,通常108个细胞/mL或更大。临床相关数目的免疫细胞可以分配到累积等于或超过105、106、107、108、109、1010、1011或1012个细胞的多次输注中。在一些实施方案中,特别是由于所有输注的细胞将被重新定向至特定靶抗原,所以可以施用106/千克(106-1011/患者)范围内的较低数量的细胞。
如果需要,治疗还可以包含施用如本文所描述的有丝分裂原(例如,PHA)或淋巴因子、细胞因子和/或趋化因子(例如,IFN-γ、IL-2、IL-12、TNF-α、IL-18和TNF-β、GM-CSF、IL-4、IL-13、F1t3-L、RANTES、MIP1α等)以增强免疫应答的诱导。
通常,包括如本文所描述的活化和扩增的细胞的组合物可以用于治疗和预防免疫受损个体中出现的疾病。特别地,本文所涵盖的组合物用于治疗癌症。在特定实施方案中,免疫效应细胞可以单独施用,或作为药物组合物与载剂、稀释剂、赋形剂和/或与如IL-2等其它组分或其它细胞因子或细胞群组合施用。
在特定实施方案中,药物组合物包括与一种或多种药学上或生理学上可接受的载剂、稀释剂或赋形剂组合的一定量的经过基因修饰的T细胞。
在特定实施方案中,药物组合物包括一定量的与一种或多种药学上或生理学上可接受的载剂、稀释剂或赋形剂组合的桥接因子。
在特定实施方案中,组合物包括有效量的免疫效应细胞,该免疫效应细胞包括本文所涵盖的单独或与桥接因子和/或一种或多种治疗剂组合的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR,诸如放射疗法、化学疗法、移植、免疫疗法、激素疗法、光动力疗法等。所述组合物还可以与抗生素组合施用。本领域可接受这种治疗剂作为如本文所描述的如特定癌症等特定疾病状态的标准治疗。所涵盖的示例性治疗剂包含细胞因子、生长因子、类固醇、NSAID、DMARD、抗炎剂、化学治疗剂、放射治疗剂、治疗性抗体或其它活性剂和辅助剂。
在特定实施方案中,将包含有效量的免疫效应细胞的组合物施用于受试者,该免疫效应细胞包含编码本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR的多核苷酸,并将包含有效量的桥连因子的组合物在细胞组合物之前、期间、与细胞组合物组合或之后施用于受试者,并且任选地重复施用于受试者。
在某些实施方案中,包括免疫效应细胞的组合物可以与任何数目的抗炎剂、化学治疗剂或治疗性抗体等结合施用,该免疫效应细胞包括编码本文所涵盖的一种或多种CD33VHH DARIC组分或抗CD33 VHH CAR的多核苷酸。
I.治疗方法
经修饰以表达编码本文所涵盖的一种或多种CD33 VHH DARIC组分或抗CD33 VHHCAR的多核苷酸的免疫效应细胞提供了改进的过继性免疫疗法的方法,以用于预防、治疗和改善免疫病症例如癌症或预防、治疗或改善与其相关的至少一种症状。
包含CD33 DARIC信号传导组分、CD33 VHH DARIC结合组分或抗CD33 VHH CAR的免疫效应细胞提供了改进的过继性免疫疗法的方法,以用于预防、治疗和改善免疫病症例如癌症或预防、治疗或改善与其相关的至少一种症状。
在特定实施方案中,经修饰以表达CD33 VHH DARIC的免疫效应细胞提供了改进的过继性免疫疗法的方法以在降低中靶抗原、脱靶细胞毒性(识别正常非靶细胞上的靶抗原)的风险的同时微调针对表达靶抗原的靶细胞(例如,肿瘤细胞)的细胞毒性应答的安全性和功效。
在特定实施方案中,预防、治疗或改善癌症的至少一种症状的方法包括向受试者施用有效量的包含CD33 VHH DARIC或抗CD33 VHH CAR的一种或多种组分的经过修饰的免疫效应细胞或T细胞,以将细胞重定向到靶细胞。通过转导可化学调节的免疫刺激信号,经过基因修饰的细胞是更有效且安全的细胞免疫疗法。
在特定实施方案中,对一种或多种免疫效应细胞(例如,T细胞)进行修饰以表达CD33 VHH DARIC结合组分和CD33 DARIC信号传导组分两者。在这种情况下,向有需要的受试者施用经过修饰的细胞,并且通过在免疫效应细胞上表达的CD33 VHH结合组分与在靶细胞上表达的CD33的相互作用使其归巢到靶细胞。在已经向受试者施用经过修饰的细胞之前、与向受试者施用经过修饰的细胞几乎相同的时间或已经向受试者施用经过修饰的细胞之后,向受试者施用桥接因子。在存在桥接因子的情况下,在CD33 VHH DARIC结合组分、桥接因子和CD33 DARIC信号传导组分之间形成三元复合物。在形成三元复合物之后,CD33VHH DARIC将免疫刺激信号转导至免疫效应细胞,该免疫效应细胞进而引发来自免疫效应细胞的针对靶细胞的细胞毒性应答。
在特定实施方案中,对一种或多种免疫效应细胞(例如,T细胞)进行修饰以表达CD33 DARIC信号传导组分。在这种情况下,向有需要的受试者施用经过修饰的细胞。在已经向受试者施用经过修饰的细胞之前、与向受试者施用经过修饰的细胞几乎相同的时间或已经向受试者施用经过修饰的细胞之后,向受试者施用CD33 VHH DARIC结合组分。另外,可以通过以下方式向受试者施用CD33 VHH DARIC结合组分:在与桥接因子的预成型复合物中;与桥接因子相同的时间,但是在单独的组合物中;或在与桥接因子不同的时间。在存在或不存在桥接因子的情况下,CD33 VHH结合组分结合在靶细胞上表达的CD33。在存在桥接因子的情况下,在CD33 VHHDARIC结合组分、桥接因子和CD33 DARIC信号传导组分之间形成三元复合物。在形成三元复合物之后,CD33 VHH DARIC将免疫刺激信号转导至免疫效应细胞,该免疫效应细胞进而引发来自免疫效应细胞的针对靶细胞的细胞毒性应答。
在特定实施方案中,对一种或多种免疫效应细胞(例如,T细胞)进行修饰以表达CD33 DARIC信号传导组分。在这种情况下,向有需要的受试者施用经过修饰的细胞。在已经向受试者施用经过修饰的细胞之前、与向受试者施用经过修饰的细胞几乎相同的时间或已经向受试者施用经过修饰的细胞之后,向受试者施用CD33 VHH DARIC结合组分。另外,可以通过以下方式向受试者施用CD33 VHH DARIC结合组分:在与桥接因子的预成型复合物中;与桥接因子相同的时间,但是在单独的组合物中;或在与桥接因子不同的时间。在存在或不存在桥接因子的情况下,CD33结合组分结合在靶细胞上表达的靶抗原。在存在桥接因子的情况下,在CD33 VHH DARIC结合组分、桥接因子和CD33 DARIC信号传导组分之间形成三元复合物。在形成三元复合物之后,CD33 VHH DARIC将免疫刺激信号转导至免疫效应细胞,该免疫效应细胞进而引发来自免疫效应细胞的针对靶细胞的细胞毒性应答。在特定实施方案中,可以在缓解或消退不完全并且病状复发或变得难治的情况下诱导CD33 VHH DARIC活化。
在特别优选的实施方案中,通过用一种或多种CD33 VHH DARIC组分对T细胞(例如,原代T细胞)进行基因修饰,将原代T细胞的特异性重定向到表达CD33的肿瘤细胞或癌细胞。
在特别优选的实施方案中,通过用针对靶抗原的工程改造的抗原受体和一种或多种CD33 VHH DARIC组分对T细胞(例如,原代T细胞)进行基因修饰,将原代T细胞的特异性重新定向到表达CD33的肿瘤细胞或癌细胞。
在特定实施方案中,本文所涵盖的经过修饰的免疫效应细胞用于治疗实体瘤或癌症。
在特定实施方案中,本文所涵盖的经过修饰的免疫效应细胞用于治疗实体瘤或癌症,包括但不限于:肾上腺癌、肾上腺皮质癌、肛门癌、阑尾癌、星形细胞瘤、非典型畸胎样/横纹肌样瘤、基底细胞癌、胆管癌、膀胱癌、骨癌、脑/CNS癌、乳腺癌、支气管肿瘤、心脏肿瘤、子宫颈癌、胆管癌、软骨肉瘤、脊索瘤、结肠癌、结直肠癌、颅咽管瘤、原位导管癌(DCIS)子宫内膜癌、室管膜瘤、食管癌、鼻腔神经胶质瘤、尤文氏肉瘤、颅外生殖细胞肿瘤、性腺外生殖细胞肿瘤、眼癌、输卵管癌、纤维组织肉瘤、纤维肉瘤、胆囊癌、胃癌、胃肠道类癌、胃肠道间质瘤(GIST)、生殖细胞肿瘤、胶质瘤、成胶质细胞瘤、头颈癌、血管母细胞瘤、肝细胞癌、下咽癌、眼内黑色素瘤、卡波西肉瘤、肾癌、喉癌、平滑肌肉瘤、唇癌、脂肪肉瘤、肝癌、肺癌、非小细胞肺癌、肺类癌、恶性间皮瘤、髓样癌、成神经管细胞瘤、脑膜瘤、黑色素瘤、梅克尔细胞癌、中线癌、口腔癌、粘膜肉瘤、骨髓增生异常综合征、骨髓增生性肿瘤、鼻腔和鼻窦癌、鼻咽癌、神经母细胞瘤、少突神经胶质瘤、口腔癌、口腔癌、口咽癌、骨肉瘤、卵巢癌、胰腺癌、胰岛细胞肿瘤、乳头状癌、副神经节瘤、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、松果体瘤、垂体瘤、胸膜肺母细胞瘤、原发性腹膜癌、前列腺癌、直肠癌、视网膜母细胞瘤、肾细胞癌、肾盂和输尿管癌、横纹肌肉瘤、唾液腺癌、皮脂腺癌、皮肤癌、软组织肉瘤、鳞状细胞癌、小细胞肺癌、小肠癌、胃癌、汗腺癌、滑膜瘤、睾丸癌、咽喉癌、胸腺癌、甲状腺癌,尿道癌症、子宫癌、子宫肉瘤、阴道癌、血管癌、外阴癌和Wilms肿瘤。
在特定实施方案中,本文所涵盖的经过修饰的免疫效应细胞用于治疗实体瘤或癌症,包括但不限于非小细胞肺癌、头部和颈部鳞状细胞癌、结直肠癌、胰腺癌、乳腺癌、甲状腺癌、膀胱癌、子宫颈癌、食管癌、卵巢癌、胃癌、子宫内膜癌、神经胶质瘤、成胶质细胞瘤和少突神经胶质瘤。
在特定实施方案中,本文所涵盖的经过修饰的免疫效应细胞用于治疗实体瘤或癌症,包括但不限于非小细胞肺癌、转移性结直肠癌、成胶质细胞瘤、头颈癌、胰腺癌和乳腺癌。
在特定实施方案中,本文所涵盖的经过修饰的免疫效应细胞用于治疗成胶质细胞瘤。
在特定实施方案中,本文所涵盖的经过修饰的免疫效应细胞用于治疗液体癌或血液癌。
在特定实施方案中,本文所涵盖的经过修饰的免疫效应细胞用于治疗B细胞恶性肿瘤,包括但不限于:白血病、淋巴瘤和多发性骨髓瘤。
在特定实施方案中,本文所涵盖的经过修饰的免疫效应细胞用于治疗液体癌,包括但不限于白血病、淋巴瘤和多发性骨髓瘤:急性淋巴细胞白血病(ALL)、急性髓性白血病(AML)、成髓细胞白血病、早幼粒细胞白血病、髓单核细胞白血病、单核细胞白血病、红白血病、毛细胞白血病(HCL)、慢性淋巴细胞白血病(CLL)和慢性粒细胞白血病(CML)、慢性髓单核细胞白血病(CMML)和真性红细胞增多症、霍奇金淋巴瘤(Hodgkin lymphoma)、结节性淋巴细胞为主的霍奇金淋巴瘤、伯基特淋巴瘤(Burkitt lymphoma)、小淋巴细胞淋巴瘤(SLL)、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、免疫母细胞性大细胞淋巴瘤、前体B淋巴母细胞淋巴瘤、套细胞淋巴瘤、边缘区淋巴瘤、蕈样真菌病、间变性大细胞淋巴瘤、塞扎里氏综合征(Sézary syndrome)、前体T淋巴母细胞淋巴瘤、多发性骨髓瘤、明显多发性骨髓瘤、冒烟型多发性骨髓瘤、浆细胞白血病、非分泌性骨髓瘤、IgD骨髓瘤、骨硬化性骨髓瘤、骨的孤立性浆细胞瘤以及髓外浆细胞瘤。
在特定实施方案中,本文所涵盖的经过修饰的免疫效应细胞用于治疗急性髓性白血病(AML)。
在本文所涵盖的方法中使用的优选细胞包含自体/自身(“自我”)细胞、优选地造血细胞、更优选地T细胞以及更优选地免疫效应细胞。
在特定实施方案中,方法包括向有需要的患者施用治疗有效量的表达一种或多种CD33 VHH DARIC组分的经过修饰的免疫效应细胞,并且还向受试者施用桥接因子。在某些实施方案中,细胞用于治疗处于发展免疫病症的风险中的患者。因此,特定实施方案包括治疗或预防或改善免疫病症例如癌症的至少一种症状,包括向有需要的受试者施用治疗有效量的本文所涵盖的经过修饰的免疫效应细胞和桥接因子。
在特定实施方案中,方法包括向有需要的患者施用治疗有效量的表达抗CD33 VHHCAR的经过修饰的免疫效应细胞。在某些实施方案中,细胞用于治疗处于发展免疫病症的风险中的患者。因此,特定实施方案包括治疗或预防或改善免疫病症例如癌症的至少一种症状,包括向有需要的受试者施用治疗有效量的本文所涵盖的经过修饰的免疫效应细胞和桥接因子。
在特定实施方案中,方法包括向有需要的患者施用治疗有效量的表达CD33 DARIC信号传导组分的经过修饰的免疫效应细胞或包含其的组合物,并且还向受试者施用CD33VHH DARIC结合组分和桥接因子,任选地其中CD33VHH DARIC结合组分在施用之前与桥接因子结合。在某些实施方案中,细胞用于治疗处于发展免疫病症的风险中的患者。因此,特定实施方案包括治疗或预防或改善免疫病症例如癌症的至少一种症状,包括向有需要的受试者施用治疗有效量的经过修饰的免疫效应细胞,该经过修饰的免疫效应细胞细胞表达表达CD33 DARIC信号传导组分和任选的工程改造的抗原受体或另一种DARIC结合组分、CD33VHH DARIC结合组分和桥连因子。
施用经过修饰的免疫效应细胞、CD33 DARIC VHH结合组分和/或桥接因子的数量和频率将由如患者的病状以及患者的疾病的类型和严重性等因素确定,即使适当的剂量和剂量时间表可以通过临床试验确定。
在一个说明性实施方案中,提供给受试者的有效量的经过修饰的免疫效应细胞为至少2×106个细胞/kg、至少3×106个细胞/kg、至少4×106个细胞/kg、至少5×106个细胞/kg、至少6×106个细胞/kg、至少7×106个细胞/kg、至少8×106个细胞/kg、至少9×106个细胞/kg、或至少10×106个细胞/kg或更多个细胞/kg,包含所有中间剂量的细胞。
在另一个说明性实施方案中,提供给受试者的有效量的经过修饰的免疫效应细胞为约2×106个细胞/kg、约3×106个细胞/kg、约4×106个细胞/kg、约5×106个细胞/kg、约6×106个细胞/kg、约7×106个细胞/kg、约8×106个细胞/kg、约9×106个细胞/kg、或约10×106个细胞/kg或更多个细胞/kg,包含所有中间剂量的细胞。
在另一个说明性实施方案中,提供给受试者的有效量的经过修饰的免疫效应细胞为约2×106个细胞/kg至约10×106个细胞/kg、约3×106个细胞/kg至约10×106个细胞/kg、约4×106个细胞/kg至约10×106个细胞/kg、约5×106个细胞/kg至约10×106个细胞/kg、2×106个细胞/kg至约6×106个细胞/kg、2×106个细胞/kg至约7×106个细胞/kg、2×106个细胞/kg至约8×106个细胞/kg、3×106个细胞/kg至约6×106个细胞/kg、3×106个细胞/kg至约7×106个细胞/kg、3×106个细胞/kg至约8×106个细胞/kg、4×106个细胞/kg至约6×106个细胞/kg、4×106个细胞/kg至约7×106个细胞/kg、4×106个细胞/kg至约8×106个细胞/kg、5×106个细胞/kg至约6×106个细胞/kg、5×106个细胞/kg至约7×106个细胞/kg、5×106个细胞/kg至约8×106个细胞/kg或6×106个细胞/kg至约8×106个细胞/kg,包含所有中间剂量的细胞。
本领域普通技术人员将认识到,可能需要多次施用特定实施方案中所涵盖的组合物以实现期望的疗法。例如,组合物可以在1周、2周、3周、1个月、2个月、3个月、4个月、5个月、6个月、1年、2年、5年、10年或更长时间的跨度内施用1次、2次、3次、4次、5次、6次、7次、8次、9次或10次或更多次。经过修饰的免疫效应细胞、CD33 VHH DARIC组分和桥接因子可以通过以下方式施用:在同一或不同组合物中;同时在一种或多种组合物中;或在不同的时间在多于一种组合物中。经过修饰的免疫效应细胞、CD33VHH DARIC组分和桥接因子可以通过相同的施用途径或不同途径施用。
在某些实施方案中,可能希望向受试者施用活化的T细胞,并且随后重新抽血(或进行单采血液成分术)、从中活化T细胞并且向患者重新输注这些活化的和扩增的T细胞。可以每隔几周进行多次此过程。在某些实施方案中,可以抽取10cc到400cc的血来活化T细胞。在某些实施方案中,抽取20cc、30cc、40cc、50cc、60cc、70cc、80cc、90cc、100cc、150cc、200cc、250cc、300cc、350cc或400cc或更多量的血来活化T细胞。不受理论束缚,使用此多次抽血/多次重新输注方案可以用于选出某些T细胞群。
在一个实施方案中,治疗被诊断患有癌症的受试者的方法包括:从受试者中除去免疫效应细胞;通过将编码一种或多种CD33 VHH DARIC组分的一种或多种载体引入到细胞中来修饰免疫效应细胞并且产生经过修饰的免疫效应细胞群;以及向同一受试者施用经过修饰的免疫效应细胞群。在优选实施方案中,免疫效应细胞包括T细胞。
在一个实施方案中,治疗被诊断患有癌症的受试者的方法包括:从受试者中除去免疫效应细胞;通过将编码抗CD33 VHH CAR的一种或多种载体引入到细胞中来修饰免疫效应细胞并且产生经过修饰的免疫效应细胞群;以及向同一受试者施用经过修饰的免疫效应细胞群。在优选实施方案中,免疫效应细胞包括T细胞。
用于施用特定实施方案中所涵盖的细胞组合物的方法包含任何有效导致重新引入经过离体修饰的免疫效应细胞或重新引入免疫效应细胞的经过修饰的祖细胞的方法,所述免疫效应细胞在引入受试者时分化为成熟免疫效应细胞。一种方法包括通过将编码一种或多种CD33 VHH DARIC组分或抗CD33 VHH CAR的一种或多种载体引入到细胞中并将转导的细胞返回到受试者体内来离体修饰外周血T细胞。
本说明书中引用的所有出版物、专利申请和授权专利都以引用的方式并入本文中,其引用的程度如同特定且个别地指示每一个别出版物、专利申请或授权专利以引用的方式并入一般。
尽管已经出于清楚理解的目的,通过说明和实例相当详细地描述了前述实施方案,但根据本文所涵盖的传授内容,本领域的普通技术人员将易于了解,可以在不脱离所附权利要求书的精神或范围的情况下对其作出某些改变和修改。提供以下实例仅作为说明并且不具有限制性。本领域技术人员将容易认识到可以在特定实施方案中被改变或修改以产生本质上类似的结果的各种非关键参数。
实施例
实施例1
CD33 VHH DARIC T细胞表现出抗肿瘤应答
设计、构建并验证抗CD33 VHH DARIC结合组分和信号传导组分。构建CD33特异性VHHDARIC慢病毒载体,其包含与编码以下的多核苷酸可操作地连接的MNDU3启动子:DARIC信号传导组分(CD8α-信号肽、FRB变体(T82L)、CD8α跨膜结构域、细胞内4-1BB共刺激结构域和CD3ζ信号传导结构域);P2A序列;以及DARIC结合组分(Igκ-信号肽、CD33特异性VHH结合结构域(骆驼科或人源化)、G4S接头、FKBP12结构域和具有截短的胞内结构域的CD4衍生跨膜结构域)(图1B)。参见例如,SEQ ID NO:32-41。用抗CD33 DARIC慢病毒载体转导的T细胞表达图1A中所示的膜结合多肽。抗CD33 scFv CAR或DARIC设计用作对照。
将来自三个供体的T细胞各自用编码不同CD33特异性VHH DARIC、抗CD33scFvDARIC或抗CD33 scFv CAR的LVV转导,并扩增10天。将未转导的T细胞、用抗CD33 scFv对照构建体转导的T细胞或抗CD33 VHH DARIC T细胞用重组CD33-Fc试剂染色。仅将对照CAR和DARIC T细胞用CD33-Fc染色阳性染色(图2A,下图,图2B)。然而,当用VHH结构域特异性的单克隆抗体分析时,大多数抗CD33 VHH DARIC T细胞,而非对照CAR或DARIC T细胞染色呈阳性(图2A,上图)。如部分地通过CD62L和CD45RA染色确定的,对照CAR和DARIC T细胞和抗CD33 VHH DARIC T细胞均具有类似的T细胞表型(图3A和图3B)。
将未转导的T细胞、用抗CD33 scFv对照构建体转导的T细胞或抗CD33VHH DARIC T细胞与CD33+THP-1细胞以1∶1的E∶T比率在存在或不存在AP21967的情况下共培养24小时。抗CD33 scFv CAR对照细胞在存在或不存在雷帕霉素类似物的情况下均具有强的细胞因子产生。抗CD33 scFv DARICT细胞和抗CD33 VHH DARIC T细胞仅当在存在AP21967的情况下与THP-1细胞一起培养时才表现出稳健的细胞因子应答(图4A和图4B)。在未转导的对照中检测到最小的细胞因子产生。
另外,针对全长CD33(CD33M)以及表达较短的截短CD33(CD33m)的剪接变体评估VHH9和VHH10 DARIC的特异性。用编码全长CD33M或剪接变体CD33m的mRNA电穿孔人293T细胞(图4C)。将DARIC T细胞与经过修饰的293T细胞以1∶1的E∶T比率在存在或不存在AP21967的情况下共培养24小时,并评估活化,如通过细胞因子分泌测量的(图4C)。VHH9DARIC T细胞对CD33M或CD33m 293T细胞表现出稳健的细胞因子应答,而VHH10 DARIC T细胞仅在存在CD33M的情况下被活化。
实施例2
CD33 VHH DARIC T细胞对CD33抗原作出特异性应答
如实施例1所述生成抗CD33 VHH DARIC T细胞。将来自三个供体的T细胞各自用编码不同抗CD33特异性VHH DARIC的LVV转导,并扩增10天。对照包括未转导的(UTD)T细胞和用CD33 CAR转导的T细胞。AML细胞系MV4-11正常表达CD33。将MV4-11细胞工程改造以敲除CD33基因(CD33-KO细胞)。所得CD33-KO细胞系在细胞表面上缺乏CD33表达。图5A。将抗CD33VHH DARIC T细胞与MV4-11细胞或CD33-KO细胞以1∶1的E∶T比率在存在或不存在二聚化药物的情况下共培养24小时。抗CD33VHH DARIC T细胞在存在MV4-11靶细胞的情况下产生细胞因子,但在存在CD33-KO细胞的情况下不产生细胞因子。图5B。
对CD33-KO细胞系进行修饰以表达CD33m剪接变体(CD33-KO-C2)。将MV4-11细胞和CD33-KO-C2细胞与UTD细胞、抗CD33 CAR T细胞或抗CD33 VHH DARIC T细胞在存在或不存在二聚化药物的情况下共培养,并在24h后分析细胞因子的产生。抗CD33 VHH9 DARIC同时识别正常CD33和CD33m剪接变异体,并在与MV4-11细胞或CD33-KO-C2细胞共培养时产生细胞因子(图5C)。抗CD33 CAR T细胞或抗CD33VHH2 DARIC对照T细胞仅对MV4-11靶细胞具有活性。
实施例3
CD33VHH DARIC T细胞不受可溶性CD33蛋白的抑制
如实施例1所述生成抗CD33VHH DARIC T细胞。将来自三个供体的T细胞各自用编码不同抗CD33特异性VHH DARIC的LVV转导,并扩增10天。将抗CD33 VHH DARIC T细胞与CD33+THP-1细胞以1∶1的E∶T比率在存在或不存在雷帕霉素的情况下共培养24小时。在共培养期间添加各种量的重组CD33-Fc蛋白。在存在和不存在重组可溶性CD33蛋白的情况下,抗CD33 VHH DARIC T细胞在存在雷帕霉素的情况下表现出稳健的细胞因子应答。图6.
实施例4
CD33VHH DARIC T细胞对低水平的CD33抗原作出应答
如实施例1所述生成抗CD33VHH DARIC T细胞。将来自三个供体的T细胞各自用编码不同抗CD33特异性VHH DARIC的LVV转导,并扩增10天。将抗CD33 VHH DARIC T细胞与AP21967二聚化剂和用不同量的编码CD33的mRNA转染的CD33neg293T细胞共培养。24h后收集上清液并分析细胞因子产生。抗CD33VHH DARIC T细胞在与CD33转染的靶细胞共培养后,即使在极低的mRNA浓度下,也显示出IFNγ产生的剂量依赖性增加(图7)。
实施例5
CD33 VHH DARIC T细胞控制体内肿瘤生长
如实施例1所述生成抗CD33 VHH DARIC T细胞。通过用表达荧光素酶报告基因的HL60 AML肿瘤细胞接种小鼠,在免疫缺陷型NSG小鼠中建立表达CD33的肿瘤。通过发光来监测肿瘤生长。10天后,向小鼠施用与媒介物或雷帕霉素共同施用的10×106个抗CD33 VHHDARIC T细胞。对照包括仅接受雷帕霉素或未转导的(UTD)T细胞的小鼠。肿瘤生长在治疗组和对照组两者之间相当。图8A。与用UTD T细胞和雷帕霉素治疗的小鼠相比,用抗CD33 VHHDARIC T细胞和雷帕霉素治疗的小鼠显示出增强的肿瘤控制。图8B。
通常,在以下权利要求书中,所使用的术语不应被解释为将权利要求限制于说明书和权利要求中所公开的具体实施方案,而是应被解释为包含所有可能的实施方案连同这些权利要求所要求的等效物的全部范围。因此,权利要求书不受本公开限制。
序列表
<110> 蓝鸟生物公司(blubird bio, Inc.)
印希彼有限公司(Inhibrx, Inc.)
乔丹·贾儒尔(Jarjour, Jordan)
马克·波格森(Pogson, Mark)
梁炜亨(Leung, Wai-Hang)
凯尔·琼斯(Jones, Kyle)
威廉·克拉戈(Crago, William)
安吉莉卡·萨纳布里亚(Sanabria, Angelica)
安德鲁·霍兰兹(Hollands, Andrew)
雅各布·加诺(Gano, Jacob)
米尔顿·马(Ma, Milton)
约翰·C·蒂默(Timmer, John C.)
布伦丹·P·埃克尔曼(P.(Eckelman, Brendan P.)
<120> CD33靶向免疫疗法
<130> BLBD-119/02WO 315698-3173
<150> US 62/898,392
<151> 2019-09-10
<150> US 62/845,304
<151> 2019-05-08
<160> 119
<170> PatentIn version 3.5
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20 25 30
Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val
35 40 45
Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr Val Glu Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His
85 90 95
Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Lys Pro
115
<210> 5
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val
35 40 45
Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr Tyr Ala Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Lys Pro
115
<210> 6
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<400> 6
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Ser
20 25 30
Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr Val Glu Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His
85 90 95
Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr Arg Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Lys Pro
115 120
<210> 7
<211> 124
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<400> 7
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Ser Tyr Asn
20 25 30
Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn Glu Arg Gly Gly Arg
35 40 45
Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp Thr Asn Tyr Ala Asn
50 55 60
Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr
65 70 75 80
Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr Thr Tyr Arg Asp Gln
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro
115 120
<210> 8
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<400> 8
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val
20 25 30
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala
35 40 45
Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp
50 55 60
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
65 70 75 80
Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala
85 90 95
His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Lys Pro
115
<210> 9
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<400> 9
Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val Gln Val Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp
20 25 30
His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Asn Cys Asn
85 90 95
Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Lys Pro
115 120
<210> 10
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<400> 10
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val
20 25 30
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala
35 40 45
Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp
50 55 60
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
65 70 75 80
Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala
85 90 95
His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Lys Pro
115
<210> 11
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<400> 11
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp
20 25 30
His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Glu Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Lys Pro
115 120
<210> 12
<211> 125
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (124)..(125)
<223> Xaa是任何氨基酸或不存在
<400> 12
Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg Ala Ile Asn Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg Tyr Ala Asp Ser Val
50 55 60
Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asp Asn Thr Met Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr His Cys
85 90 95
Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr Met Glu Ala Asp Ala
100 105 110
Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Xaa Xaa
115 120 125
<210> 13
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (119)..(120)
<223> Xaa是任何氨基酸或不存在
<400> 13
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Gly Tyr
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu Tyr Ala Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe Val Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Xaa Xaa
115 120
<210> 14
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (117)..(118)
<223> Xaa是任何氨基酸或不存在
<400> 14
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Ser Ala Ser
20 25 30
Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val
35 40 45
Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr Val Glu Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His
85 90 95
Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Xaa Xaa
115
<210> 15
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (117)..(118)
<223> Xaa是任何氨基酸或不存在
<400> 15
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val
35 40 45
Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr Tyr Ala Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Xaa Xaa
115
<210> 16
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (120)..(121)
<223> Xaa是任何氨基酸或不存在
<400> 16
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Ser
20 25 30
Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr Val Glu Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys His
85 90 95
Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr Arg Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Xaa Xaa
115 120
<210> 17
<211> 124
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (123)..(124)
<223> Xaa是任何氨基酸或不存在
<400> 17
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Ser Tyr Asn
20 25 30
Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn Glu Arg Gly Gly Arg
35 40 45
Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp Thr Asn Tyr Ala Asn
50 55 60
Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr
65 70 75 80
Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr Thr Tyr Arg Asp Gln
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Xaa Xaa
115 120
<210> 18
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (115)..(116)
<223> Xaa是任何氨基酸或不存在
<400> 18
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val
20 25 30
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala
35 40 45
Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp
50 55 60
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
65 70 75 80
Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala
85 90 95
His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Xaa Xaa
115
<210> 19
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (119)..(120)
<223> Xaa是任何氨基酸或不存在
<400> 19
Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val Gln Val Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp
20 25 30
His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Asn Cys Asn
85 90 95
Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Xaa Xaa
115 120
<210> 20
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (115)..(116)
<223> Xaa是任何氨基酸或不存在
<400> 20
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ser Gly Ile Asp Val
20 25 30
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala
35 40 45
Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala Ser Leu Ala Asp
50 55 60
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
65 70 75 80
Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala
85 90 95
His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Xaa Xaa
115
<210> 21
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH序列
<220>
<221> MOD_RES
<222> (119)..(120)
<223> Xaa是任何氨基酸或不存在
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Asn Ile Asp
20 25 30
His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala Glu Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Xaa Xaa
115 120
<210> 22
<211> 292
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 22
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val
20 25 30
Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg Ala
35 40 45
Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg Tyr
65 70 75 80
Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asp
85 90 95
Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
100 105 110
Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr Met
115 120 125
Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
145 150 155 160
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
165 170 175
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
180 185 190
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
195 200 205
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
210 215 220
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
225 230 235 240
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
245 250 255
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
260 265 270
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His
275 280 285
Arg Arg Arg Gln
290
<210> 23
<211> 287
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 23
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val
20 25 30
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr
35 40 45
Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu Tyr
65 70 75 80
Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe Val
115 120 125
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly
130 135 140
Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe
145 150 155 160
Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu
165 170 175
Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys
180 185 190
Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val
195 200 205
Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp
210 215 220
Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala
225 230 235 240
Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met
245 250 255
Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly
260 265 270
Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
275 280 285
<210> 24
<211> 285
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 24
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val
20 25 30
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly
35 40 45
Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys Gln
50 55 60
Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr Val
65 70 75 80
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly
130 135 140
Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys
145 150 155 160
Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly
165 170 175
Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met
180 185 190
Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln
195 200 205
Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala
210 215 220
Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu
225 230 235 240
Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu
245 250 255
Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly
260 265 270
Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
275 280 285
<210> 25
<211> 285
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 25
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val
20 25 30
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
35 40 45
Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly
50 55 60
Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr Tyr
65 70 75 80
Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly
130 135 140
Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys
145 150 155 160
Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly
165 170 175
Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met
180 185 190
Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln
195 200 205
Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala
210 215 220
Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu
225 230 235 240
Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu
245 250 255
Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly
260 265 270
Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
275 280 285
<210> 26
<211> 288
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 26
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val
20 25 30
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile
35 40 45
Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr Val
65 70 75 80
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr Arg
115 120 125
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly
130 135 140
Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
145 150 155 160
Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu
165 170 175
Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
180 185 190
Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly
195 200 205
Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro
210 215 220
Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His
225 230 235 240
Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg
245 250 255
Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile
260 265 270
Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
275 280 285
<210> 27
<211> 291
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 27
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val
20 25 30
Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile
35 40 45
Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn Glu
50 55 60
Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp Thr
65 70 75 80
Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp Asp
85 90 95
Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu Asp
100 105 110
Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr Thr
115 120 125
Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro
130 135 140
Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp
145 150 155 160
Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr
165 170 175
Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn
180 185 190
Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp
195 200 205
Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr
210 215 220
Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile
225 230 235 240
Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu
245 250 255
Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu
260 265 270
Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg
275 280 285
Arg Arg Gln
290
<210> 28
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 28
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val
20 25 30
Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser Ser
35 40 45
Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg
50 55 60
Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala
65 70 75 80
Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr
85 90 95
Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln
115 120 125
Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln
130 135 140
Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly
145 150 155 160
Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys
165 170 175
Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly
180 185 190
Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser
195 200 205
Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly
210 215 220
Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe
225 230 235 240
Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val
245 250 255
Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe
260 265 270
Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
275 280
<210> 29
<211> 287
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 29
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val
20 25 30
Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr
35 40 45
Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
100 105 110
Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn
115 120 125
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly Gly
130 135 140
Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe
145 150 155 160
Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu
165 170 175
Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys
180 185 190
Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val
195 200 205
Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp
210 215 220
Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala
225 230 235 240
Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met
245 250 255
Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly
260 265 270
Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
275 280 285
<210> 30
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 30
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val
20 25 30
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ser
35 40 45
Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg
50 55 60
Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala Ala
65 70 75 80
Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr
85 90 95
Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly Gln
115 120 125
Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln
130 135 140
Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly
145 150 155 160
Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys
165 170 175
Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly
180 185 190
Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser
195 200 205
Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly
210 215 220
Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe
225 230 235 240
Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val
245 250 255
Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe
260 265 270
Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
275 280
<210> 31
<211> 287
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC结合组分
<400> 31
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val
20 25 30
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr
35 40 45
Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr Ala
65 70 75 80
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln Asn
115 120 125
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly Gly
130 135 140
Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe
145 150 155 160
Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu
165 170 175
Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys
180 185 190
Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val
195 200 205
Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp
210 215 220
Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala
225 230 235 240
Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg Met
245 250 255
Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly
260 265 270
Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
275 280 285
<210> 32
<211> 617
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 32
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Thr Leu Arg Glu Ser
340 345 350
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys
355 360 365
Gly Ser Gly Arg Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly
385 390 395 400
Gly His Thr Arg Tyr Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser
405 410 415
Arg Asp Asn Ala Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys
420 425 430
Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His
435 440 445
Arg Ile Ala Thr Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr
450 455 460
Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu
465 470 475 480
Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr
485 490 495
Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp
500 505 510
Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln
515 520 525
Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly
530 535 540
Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr
545 550 555 560
Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val
565 570 575
Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly
580 585 590
Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys
595 600 605
Val Arg Cys Arg His Arg Arg Arg Gln
610 615
<210> 33
<211> 612
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 33
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Arg Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn
385 390 395 400
Leu Ser Thr Glu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
405 410 415
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg
420 425 430
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser
435 440 445
Ser Gly Asp Phe Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys
450 455 460
Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
465 470 475 480
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
485 490 495
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
500 505 510
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
515 520 525
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
530 535 540
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
545 550 555 560
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
565 570 575
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
580 585 590
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His
595 600 605
Arg Arg Arg Gln
610
<210> 34
<211> 610
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 34
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln
370 375 380
Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly
385 390 395 400
Arg Ala Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
420 425 430
Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro
435 440 445
Ile Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly
450 455 460
Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly
465 470 475 480
Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly
485 490 495
Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys
500 505 510
Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu
515 520 525
Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile
530 535 540
Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro
545 550 555 560
Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly
565 570 575
Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu
580 585 590
Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg
595 600 605
Arg Gln
610
<210> 35
<211> 610
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 35
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln
370 375 380
Ala Pro Gly Lys Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly
385 390 395 400
Asp Thr Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
405 410 415
Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg
420 425 430
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly
435 440 445
Val Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly
450 455 460
Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly
465 470 475 480
Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly
485 490 495
Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys
500 505 510
Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu
515 520 525
Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile
530 535 540
Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro
545 550 555 560
Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly
565 570 575
Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu
580 585 590
Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg
595 600 605
Arg Gln
610
<210> 36
<211> 613
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 36
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly
385 390 395 400
Thr Thr Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
420 425 430
Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr
435 440 445
Asp Arg Gly Tyr Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
450 455 460
Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro
465 470 475 480
Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His
485 490 495
Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp
500 505 510
Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg
515 520 525
Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys
530 535 540
Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly
545 550 555 560
Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys
565 570 575
Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly
580 585 590
Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg
595 600 605
His Arg Arg Arg Gln
610
<210> 37
<211> 616
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 37
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser
340 345 350
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln
370 375 380
Leu Ser Gly Asn Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn
385 390 395 400
Ala Tyr Gly Asp Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr
405 410 415
Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn
420 425 430
Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met
435 440 445
Leu Glu Asn Tyr Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln
450 455 460
Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr
465 470 475 480
Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys
485 490 495
Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser
500 505 510
Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu
515 520 525
Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln
530 535 540
Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly
545 550 555 560
His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu
565 570 575
Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly
580 585 590
Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val
595 600 605
Arg Cys Arg His Arg Arg Arg Gln
610 615
<210> 38
<211> 608
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 38
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser
340 345 350
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala
355 360 365
Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala
370 375 380
Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp
385 390 395 400
Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp
405 410 415
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu
420 425 430
Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val
435 440 445
Gly Ala Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly
450 455 460
Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
465 470 475 480
Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu
485 490 495
Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
500 505 510
Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly
515 520 525
Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro
530 535 540
Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His
545 550 555 560
Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg
565 570 575
Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile
580 585 590
Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
595 600 605
<210> 39
<211> 612
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 39
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser
340 345 350
Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala
385 390 395 400
Gly Pro Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
420 425 430
Glu Asp Thr Ala Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser
435 440 445
Gly Leu Pro Gln Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys
450 455 460
Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
465 470 475 480
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
485 490 495
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
500 505 510
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
515 520 525
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
530 535 540
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
545 550 555 560
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
565 570 575
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
580 585 590
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His
595 600 605
Arg Arg Arg Gln
610
<210> 40
<211> 608
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 40
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala
370 375 380
Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp
385 390 395 400
Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp
405 410 415
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu
420 425 430
Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val
435 440 445
Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly
450 455 460
Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
465 470 475 480
Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu
485 490 495
Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
500 505 510
Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly
515 520 525
Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro
530 535 540
Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His
545 550 555 560
Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg
565 570 575
Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile
580 585 590
Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln
595 600 605
<210> 41
<211> 612
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC融合蛋白
<400> 41
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala
385 390 395 400
Gly Pro Asn Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala
420 425 430
Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser
435 440 445
Gly Leu Pro Gln Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys
450 455 460
Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
465 470 475 480
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
485 490 495
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
500 505 510
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
515 520 525
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
530 535 540
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
545 550 555 560
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
565 570 575
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
580 585 590
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His
595 600 605
Arg Arg Arg Gln
610
<210> 42
<211> 649
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 42
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Thr Leu Arg Glu Ser
340 345 350
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys
355 360 365
Gly Ser Gly Arg Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly
385 390 395 400
Gly His Thr Arg Tyr Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser
405 410 415
Arg Asp Asn Ala Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys
420 425 430
Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His
435 440 445
Arg Ile Ala Thr Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr
450 455 460
Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu
465 470 475 480
Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr
485 490 495
Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp
500 505 510
Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln
515 520 525
Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly
530 535 540
Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr
545 550 555 560
Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val
565 570 575
Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly
580 585 590
Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala
595 600 605
Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys
610 615 620
Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala
625 630 635 640
Asp Ala His Ser Thr Leu Ala Lys Ile
645
<210> 43
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 43
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Arg Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn
385 390 395 400
Leu Ser Thr Glu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
405 410 415
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg
420 425 430
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser
435 440 445
Ser Gly Asp Phe Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys
450 455 460
Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
465 470 475 480
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
485 490 495
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
500 505 510
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
515 520 525
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
530 535 540
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
545 550 555 560
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
565 570 575
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
580 585 590
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg
595 600 605
Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly
610 615 620
Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr
625 630 635 640
Leu Ala Lys Ile
<210> 44
<211> 642
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 44
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln
370 375 380
Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly
385 390 395 400
Arg Ala Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
420 425 430
Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro
435 440 445
Ile Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly
450 455 460
Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly
465 470 475 480
Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly
485 490 495
Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys
500 505 510
Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu
515 520 525
Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile
530 535 540
Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro
545 550 555 560
Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly
565 570 575
Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu
580 585 590
Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp
595 600 605
Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe
610 615 620
Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala
625 630 635 640
Lys Ile
<210> 45
<211> 642
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 45
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln
370 375 380
Ala Pro Gly Lys Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly
385 390 395 400
Asp Thr Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
405 410 415
Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg
420 425 430
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly
435 440 445
Val Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly
450 455 460
Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly
465 470 475 480
Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly
485 490 495
Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys
500 505 510
Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu
515 520 525
Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile
530 535 540
Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro
545 550 555 560
Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly
565 570 575
Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu
580 585 590
Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp
595 600 605
Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe
610 615 620
Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala
625 630 635 640
Lys Ile
<210> 46
<211> 645
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 46
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly
385 390 395 400
Thr Thr Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
420 425 430
Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr
435 440 445
Asp Arg Gly Tyr Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
450 455 460
Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro
465 470 475 480
Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His
485 490 495
Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp
500 505 510
Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg
515 520 525
Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys
530 535 540
Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly
545 550 555 560
Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys
565 570 575
Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly
580 585 590
Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu
595 600 605
Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly
610 615 620
Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser
625 630 635 640
Thr Leu Ala Lys Ile
645
<210> 47
<211> 648
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 47
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser
340 345 350
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln
370 375 380
Leu Ser Gly Asn Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn
385 390 395 400
Ala Tyr Gly Asp Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr
405 410 415
Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn
420 425 430
Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met
435 440 445
Leu Glu Asn Tyr Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln
450 455 460
Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr
465 470 475 480
Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys
485 490 495
Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser
500 505 510
Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu
515 520 525
Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln
530 535 540
Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly
545 550 555 560
His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu
565 570 575
Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly
580 585 590
Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu
595 600 605
Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro
610 615 620
Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp
625 630 635 640
Ala His Ser Thr Leu Ala Lys Ile
645
<210> 48
<211> 640
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 48
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser
340 345 350
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala
355 360 365
Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala
370 375 380
Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp
385 390 395 400
Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp
405 410 415
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu
420 425 430
Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val
435 440 445
Gly Ala Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly
450 455 460
Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
465 470 475 480
Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu
485 490 495
Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
500 505 510
Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly
515 520 525
Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro
530 535 540
Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His
545 550 555 560
Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg
565 570 575
Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile
580 585 590
Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg
595 600 605
Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr
610 615 620
Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile
625 630 635 640
<210> 49
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 49
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser
340 345 350
Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala
385 390 395 400
Gly Pro Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
420 425 430
Glu Asp Thr Ala Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser
435 440 445
Gly Leu Pro Gln Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys
450 455 460
Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
465 470 475 480
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
485 490 495
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
500 505 510
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
515 520 525
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
530 535 540
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
545 550 555 560
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
565 570 575
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
580 585 590
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg
595 600 605
Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly
610 615 620
Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr
625 630 635 640
Leu Ala Lys Ile
<210> 50
<211> 640
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 50
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala
370 375 380
Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp
385 390 395 400
Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp
405 410 415
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu
420 425 430
Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val
435 440 445
Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly
450 455 460
Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
465 470 475 480
Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu
485 490 495
Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
500 505 510
Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly
515 520 525
Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro
530 535 540
Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His
545 550 555 560
Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg
565 570 575
Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile
580 585 590
Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg
595 600 605
Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr
610 615 620
Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile
625 630 635 640
<210> 51
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.OX40融合蛋白
<400> 51
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala
385 390 395 400
Gly Pro Asn Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala
420 425 430
Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser
435 440 445
Gly Leu Pro Gln Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys
450 455 460
Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
465 470 475 480
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
485 490 495
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
500 505 510
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
515 520 525
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
530 535 540
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
545 550 555 560
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
565 570 575
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
580 585 590
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Ala Leu Tyr Leu Leu Arg
595 600 605
Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly
610 615 620
Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr
625 630 635 640
Leu Ala Lys Ile
<210> 52
<211> 781
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 52
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Thr Leu Arg Glu Ser
340 345 350
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys
355 360 365
Gly Ser Gly Arg Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly
385 390 395 400
Gly His Thr Arg Tyr Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser
405 410 415
Arg Asp Asn Ala Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys
420 425 430
Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His
435 440 445
Arg Ile Ala Thr Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr
450 455 460
Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Val Gln Val Glu
465 470 475 480
Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr
485 490 495
Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp
500 505 510
Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln
515 520 525
Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly
530 535 540
Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr
545 550 555 560
Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val
565 570 575
Glu Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly
580 585 590
Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys
595 600 605
Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro
610 615 620
Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu
625 630 635 640
Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser
645 650 655
Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly
660 665 670
Val Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser
675 680 685
Asp Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile
690 695 700
Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln
705 710 715 720
Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro
725 730 735
Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser
740 745 750
Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro
755 760 765
Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser
770 775 780
<210> 53
<211> 776
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 53
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Arg Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn
385 390 395 400
Leu Ser Thr Glu Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
405 410 415
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg
420 425 430
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser
435 440 445
Ser Gly Asp Phe Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys
450 455 460
Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
465 470 475 480
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
485 490 495
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
500 505 510
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
515 520 525
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
530 535 540
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
545 550 555 560
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
565 570 575
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
580 585 590
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys
595 600 605
Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg
610 615 620
Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser
625 630 635 640
Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg
645 650 655
Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly
660 665 670
Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly
675 680 685
Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser
690 695 700
Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met
705 710 715 720
Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val
725 730 735
Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro
740 745 750
Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val
755 760 765
Pro Asp Ala Gly Met Lys Pro Ser
770 775
<210> 54
<211> 774
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 54
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln
370 375 380
Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly
385 390 395 400
Arg Ala Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
420 425 430
Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro
435 440 445
Ile Arg Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly
450 455 460
Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly
465 470 475 480
Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly
485 490 495
Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys
500 505 510
Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu
515 520 525
Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile
530 535 540
Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro
545 550 555 560
Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly
565 570 575
Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu
580 585 590
Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln
595 600 605
Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr
610 615 620
Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser
625 630 635 640
Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro
645 650 655
Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly
660 665 670
Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His
675 680 685
Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser
690 695 700
Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp
705 710 715 720
Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe
725 730 735
Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr
740 745 750
Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp
755 760 765
Ala Gly Met Lys Pro Ser
770
<210> 55
<211> 774
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 55
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln
370 375 380
Ala Pro Gly Lys Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly
385 390 395 400
Asp Thr Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser
405 410 415
Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg
420 425 430
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly
435 440 445
Val Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly
450 455 460
Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly
465 470 475 480
Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly
485 490 495
Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys
500 505 510
Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu
515 520 525
Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile
530 535 540
Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro
545 550 555 560
Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly
565 570 575
Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu
580 585 590
Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln
595 600 605
Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr
610 615 620
Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser
625 630 635 640
Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro
645 650 655
Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly
660 665 670
Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His
675 680 685
Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser
690 695 700
Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp
705 710 715 720
Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe
725 730 735
Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr
740 745 750
Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp
755 760 765
Ala Gly Met Lys Pro Ser
770
<210> 56
<211> 777
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 56
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly
385 390 395 400
Thr Thr Asn Tyr Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala
420 425 430
Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr
435 440 445
Asp Arg Gly Tyr Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
450 455 460
Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro
465 470 475 480
Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His
485 490 495
Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp
500 505 510
Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg
515 520 525
Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys
530 535 540
Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly
545 550 555 560
Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys
565 570 575
Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly
580 585 590
Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu
595 600 605
Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala
610 615 620
Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro
625 630 635 640
Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg
645 650 655
Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser
660 665 670
Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro
675 680 685
Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys
690 695 700
Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr
705 710 715 720
Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln
725 730 735
Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr
740 745 750
Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly
755 760 765
Val Pro Asp Ala Gly Met Lys Pro Ser
770 775
<210> 57
<211> 780
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 57
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser
340 345 350
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln
370 375 380
Leu Ser Gly Asn Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn
385 390 395 400
Ala Tyr Gly Asp Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr
405 410 415
Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn
420 425 430
Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met
435 440 445
Leu Glu Asn Tyr Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln
450 455 460
Val Thr Val Lys Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr
465 470 475 480
Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys
485 490 495
Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser
500 505 510
Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu
515 520 525
Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln
530 535 540
Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly
545 550 555 560
His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu
565 570 575
Leu Leu Lys Leu Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly
580 585 590
Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys
595 600 605
Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala
610 615 620
Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile
625 630 635 640
Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala
645 650 655
Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val
660 665 670
Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp
675 680 685
Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val
690 695 700
Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala
705 710 715 720
Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys
725 730 735
Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln
740 745 750
Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu
755 760 765
Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser
770 775 780
<210> 58
<211> 772
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 58
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser
340 345 350
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala
355 360 365
Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala
370 375 380
Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp
385 390 395 400
Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp
405 410 415
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu
420 425 430
Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val
435 440 445
Gly Ala Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Gly Gly Gly
450 455 460
Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
465 470 475 480
Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu
485 490 495
Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
500 505 510
Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly
515 520 525
Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro
530 535 540
Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His
545 550 555 560
Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg
565 570 575
Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile
580 585 590
Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln Arg Glu
595 600 605
Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gln Gly
610 615 620
Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser Ser
625 630 635 640
Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro Thr Arg
645 650 655
Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly Glu Ala
660 665 670
Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His Gly Thr
675 680 685
Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser Asp His
690 695 700
Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp Thr Asp
705 710 715 720
Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Lys
725 730 735
Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu Leu
740 745 750
Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly
755 760 765
Met Lys Pro Ser
770
<210> 59
<211> 776
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 59
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Gln Ser
340 345 350
Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala
385 390 395 400
Gly Pro Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
420 425 430
Glu Asp Thr Ala Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser
435 440 445
Gly Leu Pro Gln Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys
450 455 460
Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
465 470 475 480
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
485 490 495
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
500 505 510
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
515 520 525
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
530 535 540
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
545 550 555 560
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
565 570 575
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
580 585 590
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys
595 600 605
Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg
610 615 620
Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser
625 630 635 640
Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg
645 650 655
Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly
660 665 670
Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly
675 680 685
Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser
690 695 700
Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met
705 710 715 720
Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val
725 730 735
Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro
740 745 750
Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val
755 760 765
Pro Asp Ala Gly Met Lys Pro Ser
770 775
<210> 60
<211> 772
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 60
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Arg Ser Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala
370 375 380
Pro Gly Lys Glu Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp
385 390 395 400
Thr Asn Tyr Ala Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp
405 410 415
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu
420 425 430
Asp Thr Ala Val Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val
435 440 445
Gly Ala Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Gly Gly Gly
450 455 460
Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr
465 470 475 480
Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu
485 490 495
Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe
500 505 510
Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly
515 520 525
Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro
530 535 540
Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His
545 550 555 560
Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Arg
565 570 575
Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile
580 585 590
Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys Leu Gln Arg Glu
595 600 605
Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gln Gly
610 615 620
Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser Ser
625 630 635 640
Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro Thr Arg
645 650 655
Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly Glu Ala
660 665 670
Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly Gly His Gly Thr
675 680 685
Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser Ser Ser Asp His
690 695 700
Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met Gly Asp Thr Asp
705 710 715 720
Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Lys
725 730 735
Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu Leu
740 745 750
Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly
755 760 765
Met Lys Pro Ser
770
<210> 61
<211> 776
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC.TNFR2融合蛋白
<400> 61
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser
290 295 300
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
305 310 315 320
Asn Pro Gly Pro Ser Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
325 330 335
Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser
340 345 350
Gly Gly Gly Glu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
355 360 365
Ala Ser Gly Ser Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln
370 375 380
Ala Pro Gly Lys Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala
385 390 395 400
Gly Pro Asn Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
405 410 415
Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala
420 425 430
Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser
435 440 445
Gly Leu Pro Gln Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys
450 455 460
Pro Gly Gly Gly Gly Ser Gly Val Gln Val Glu Thr Ile Ser Pro Gly
465 470 475 480
Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr
485 490 495
Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg
500 505 510
Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly
515 520 525
Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu
530 535 540
Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile
545 550 555 560
Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu
565 570 575
Glu Gly Gly Arg Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu
580 585 590
Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Lys Lys Lys Pro Leu Cys
595 600 605
Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro Ala Asp Lys Ala Arg
610 615 620
Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu Ile Thr Ala Pro Ser
625 630 635 640
Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg
645 650 655
Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly
660 665 670
Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly
675 680 685
Gly His Gly Thr Gln Val Asn Val Thr Cys Ile Val Asn Val Cys Ser
690 695 700
Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln Ala Ser Ser Thr Met
705 710 715 720
Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro Lys Asp Glu Gln Val
725 730 735
Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser Gln Leu Glu Thr Pro
740 745 750
Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val
755 760 765
Pro Asp Ala Gly Met Lys Pro Ser
770 775
<210> 62
<211> 372
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 62
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg
35 40 45
Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg
65 70 75 80
Tyr Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala
85 90 95
Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr
115 120 125
Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val
130 135 140
Ser Ser Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
145 150 155 160
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
165 170 175
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
180 185 190
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
195 200 205
Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys
210 215 220
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
225 230 235 240
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
245 250 255
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
260 265 270
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
275 280 285
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
290 295 300
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
305 310 315 320
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
325 330 335
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
340 345 350
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
355 360 365
Leu Pro Pro Arg
370
<210> 63
<211> 367
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 63
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg
35 40 45
Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu
65 70 75 80
Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe
115 120 125
Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala
130 135 140
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
145 150 155 160
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
165 170 175
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
180 185 190
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
195 200 205
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
210 215 220
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
225 230 235 240
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
245 250 255
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
260 265 270
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
275 280 285
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
290 295 300
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
305 310 315 320
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
325 330 335
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
340 345 350
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 64
<211> 365
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 64
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys
50 55 60
Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr
65 70 75 80
Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala Thr Thr
130 135 140
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
145 150 155 160
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
165 170 175
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
180 185 190
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
195 200 205
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
210 215 220
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
225 230 235 240
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
245 250 255
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
260 265 270
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
275 280 285
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
290 295 300
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
305 310 315 320
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
325 330 335
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
340 345 350
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 65
<211> 365
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 65
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr
65 70 75 80
Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala Thr Thr
130 135 140
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
145 150 155 160
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
165 170 175
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
180 185 190
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
195 200 205
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
210 215 220
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
225 230 235 240
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
245 250 255
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
260 265 270
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
275 280 285
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
290 295 300
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
305 310 315 320
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
325 330 335
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
340 345 350
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 66
<211> 368
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 66
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr
65 70 75 80
Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr
115 120 125
Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala
130 135 140
Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
145 150 155 160
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
165 170 175
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
180 185 190
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
195 200 205
Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
210 215 220
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
225 230 235 240
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
245 250 255
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
260 265 270
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
275 280 285
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
290 295 300
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
305 310 315 320
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
325 330 335
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
340 345 350
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 67
<211> 371
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 67
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn
50 55 60
Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp
65 70 75 80
Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu
100 105 110
Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr
115 120 125
Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Lys
130 135 140
Pro Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
145 150 155 160
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
165 170 175
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
180 185 190
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
195 200 205
Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu
210 215 220
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
225 230 235 240
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
245 250 255
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
260 265 270
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
275 280 285
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
290 295 300
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
305 310 315 320
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
325 330 335
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
340 345 350
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
355 360 365
Pro Pro Arg
370
<210> 68
<211> 363
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 68
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser
35 40 45
Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala
65 70 75 80
Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly
115 120 125
Gln Gly Thr Gln Val Thr Val Lys Pro Ala Ala Ala Thr Thr Thr Pro
130 135 140
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
145 150 155 160
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
165 170 175
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
180 185 190
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
195 200 205
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
210 215 220
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
225 230 235 240
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
245 250 255
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
260 265 270
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
275 280 285
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
290 295 300
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
305 310 315 320
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
325 330 335
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
340 345 350
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 69
<211> 367
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 69
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser
20 25 30
Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr
65 70 75 80
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
100 105 110
Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln
115 120 125
Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Ala Ala Ala
130 135 140
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
145 150 155 160
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
165 170 175
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
180 185 190
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
195 200 205
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
210 215 220
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
225 230 235 240
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
245 250 255
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
260 265 270
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
275 280 285
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
290 295 300
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
305 310 315 320
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
325 330 335
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
340 345 350
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 70
<211> 363
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 70
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser
35 40 45
Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala
65 70 75 80
Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala Thr Thr Thr Pro
130 135 140
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
145 150 155 160
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
165 170 175
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
180 185 190
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
195 200 205
Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
210 215 220
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
225 230 235 240
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
245 250 255
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
260 265 270
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
275 280 285
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
290 295 300
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
305 310 315 320
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
325 330 335
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
340 345 350
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 71
<211> 367
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 71
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr
65 70 75 80
Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln
115 120 125
Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Ala Ala Ala
130 135 140
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
145 150 155 160
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
165 170 175
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
180 185 190
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
195 200 205
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
210 215 220
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
225 230 235 240
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
245 250 255
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
260 265 270
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
275 280 285
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
290 295 300
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
305 310 315 320
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
325 330 335
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
340 345 350
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 72
<211> 369
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 72
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg
35 40 45
Ala Ile Asn Thr Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg
65 70 75 80
Tyr Ala Asp Ser Val Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala
85 90 95
Asp Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Val Tyr His Cys Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr
115 120 125
Met Glu Ala Asp Ala Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val
130 135 140
Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
145 150 155 160
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
165 170 175
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
180 185 190
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
195 200 205
Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr
210 215 220
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
225 230 235 240
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
245 250 255
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
260 265 270
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
275 280 285
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
290 295 300
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
305 310 315 320
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
325 330 335
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
340 345 350
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
355 360 365
Arg
<210> 73
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 73
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg
35 40 45
Thr Phe Ser Gly Tyr Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Leu Val Ala Arg Ile Ser Gly Asn Asn Leu Ser Thr Glu
65 70 75 80
Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Ala Glu Tyr Asp Tyr Ser Ser Gly Asp Phe
115 120 125
Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr
130 135 140
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
145 150 155 160
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
165 170 175
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
180 185 190
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
195 200 205
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
210 215 220
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
225 230 235 240
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
245 250 255
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
260 265 270
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
275 280 285
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
290 295 300
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
305 310 315 320
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
325 330 335
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
340 345 350
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 74
<211> 362
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 74
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Gly Phe Ser Ala Ser Leu Met Ser Trp His Arg Gln Ala Pro Gly Lys
50 55 60
Gln Arg Asp Leu Val Ala Ser Ile Thr Arg Asp Gly Arg Ala Asn Tyr
65 70 75 80
Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys His Ala Tyr Ser Phe Asp Tyr Pro Ile Arg Ser Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr Pro Ala
130 135 140
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
145 150 155 160
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
165 170 175
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
180 185 190
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
195 200 205
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
210 215 220
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
225 230 235 240
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
245 250 255
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
260 265 270
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
275 280 285
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
290 295 300
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
305 310 315 320
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
325 330 335
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
340 345 350
Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 75
<211> 362
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 75
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Gly Arg Glu Trp Val Ala Ala Ile Thr Thr Ser Gly Asp Thr Thr Tyr
65 70 75 80
Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Ala His Arg Gly Gly Gly Val Ile Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr Pro Ala
130 135 140
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
145 150 155 160
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
165 170 175
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
180 185 190
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
195 200 205
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
210 215 220
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
225 230 235 240
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
245 250 255
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
260 265 270
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
275 280 285
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
290 295 300
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
305 310 315 320
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
325 330 335
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
340 345 350
Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 76
<211> 365
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 76
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Ile Phe Ser Ile Ser Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Leu Val Ala Ser Thr Thr Ser Ser Gly Thr Thr Asn Tyr
65 70 75 80
Val Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys His Ala Tyr Ile Ala Thr Thr Thr Asp Arg Gly Tyr
115 120 125
Arg Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr
130 135 140
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
145 150 155 160
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
165 170 175
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
180 185 190
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
195 200 205
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
210 215 220
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
225 230 235 240
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
245 250 255
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
260 265 270
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
275 280 285
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
290 295 300
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
305 310 315 320
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
325 330 335
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
340 345 350
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 77
<211> 368
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 77
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Ile Ser Ser Tyr Asn Val Val Gly Trp Tyr Arg Gln Leu Ser Gly Asn
50 55 60
Glu Arg Gly Gly Arg Thr Met Val Ala Gln Ile Asn Ala Tyr Gly Asp
65 70 75 80
Thr Asn Tyr Ala Asn Ala Val Val Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asp Ala Lys Asn Thr Val Tyr Leu His Met Ser Asn Leu Lys Pro Glu
100 105 110
Asp Thr Gly Val Tyr Tyr Cys Asn Gly Gln Arg Met Leu Glu Asn Tyr
115 120 125
Thr Tyr Arg Asp Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Lys
130 135 140
Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
145 150 155 160
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
165 170 175
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
180 185 190
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
195 200 205
Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
210 215 220
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
225 230 235 240
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
245 250 255
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
260 265 270
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
275 280 285
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
290 295 300
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
305 310 315 320
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
325 330 335
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
340 345 350
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360 365
<210> 78
<211> 360
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 78
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu
20 25 30
Val Gln Ala Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Arg Ser
35 40 45
Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala
65 70 75 80
Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Val Tyr Leu Gln Met Lys Asn Leu Lys Pro Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly
115 120 125
Gln Gly Thr Gln Val Thr Val Lys Pro Thr Thr Thr Pro Ala Pro Arg
130 135 140
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
145 150 155 160
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
165 170 175
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
180 185 190
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
195 200 205
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
210 215 220
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
225 230 235 240
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
245 250 255
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
260 265 270
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
275 280 285
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
290 295 300
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
305 310 315 320
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
325 330 335
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
340 345 350
His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 79
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 79
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Gly Gly Ser
20 25 30
Val Gln Val Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr
65 70 75 80
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
100 105 110
Val Tyr Asn Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln
115 120 125
Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Lys Pro Thr Thr Thr
130 135 140
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
145 150 155 160
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
165 170 175
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
180 185 190
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
195 200 205
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
210 215 220
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
225 230 235 240
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
245 250 255
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
260 265 270
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
275 280 285
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
290 295 300
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
305 310 315 320
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
325 330 335
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
340 345 350
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 80
<211> 360
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 80
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser
35 40 45
Ser Gly Ile Asp Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Leu Val Ala Glu Ile Ser Gly Val Gly Asp Thr Asn Tyr Ala
65 70 75 80
Ala Ser Leu Ala Asp Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Asn Ala His Ser Phe Leu Asp Leu Val Gly Ala Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr Pro Ala Pro Arg
130 135 140
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
145 150 155 160
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
165 170 175
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
180 185 190
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
195 200 205
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
210 215 220
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
225 230 235 240
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
245 250 255
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
260 265 270
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
275 280 285
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
290 295 300
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
305 310 315 320
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
325 330 335
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
340 345 350
His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 81
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH嵌合抗原受体
<400> 81
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Thr Leu Asn Ile Asp His Ile Gly Trp Tyr Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Leu Val Gly Val Ile Ser Ser Gly Ala Gly Pro Asn Tyr
65 70 75 80
Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys Asn Ala Trp Ile Asp Tyr Gly Ser Gly Leu Pro Gln
115 120 125
Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro Thr Thr Thr
130 135 140
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
145 150 155 160
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
165 170 175
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
180 185 190
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
195 200 205
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
210 215 220
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
225 230 235 240
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
245 250 255
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
260 265 270
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
275 280 285
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
290 295 300
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
305 310 315 320
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
325 330 335
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
340 345 350
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
355 360
<210> 82
<211> 301
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 实验室制备-抗CD33 VHH DARIC信号传导组分
<400> 82
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Ile Leu Trp His Glu Met Trp His Glu
20 25 30
Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys
35 40 45
Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly
50 55 60
Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp
65 70 75 80
Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn
85 90 95
Val Lys Asp Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg
100 105 110
Arg Ile Ser Lys Ala Ser Ala Gly Thr Gly Ser Asp Ile Tyr Ile Trp
115 120 125
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
130 135 140
Thr Met His Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
145 150 155 160
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
165 170 175
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
180 185 190
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
195 200 205
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
210 215 220
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
225 230 235 240
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
245 250 255
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
260 265 270
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
275 280 285
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
290 295 300
<210> 83
<211> 10
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 共有Kozak序列
<400> 83
gccrccatgg 10
<210> 84
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 84
Asp Gly Gly Gly Ser
1 5
<210> 85
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 85
Thr Gly Glu Lys Pro
1 5
<210> 86
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 86
Gly Gly Arg Arg
1
<210> 87
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 87
Gly Gly Gly Gly Ser
1 5
<210> 88
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 88
Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp
1 5 10
<210> 89
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 89
Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser
1 5 10 15
Leu Asp
<210> 90
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 90
Gly Gly Arg Arg Gly Gly Gly Ser
1 5
<210> 91
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 91
Leu Arg Gln Arg Asp Gly Glu Arg Pro
1 5
<210> 92
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 92
Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro
1 5 10
<210> 93
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 93
Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg Pro
1 5 10 15
<210> 94
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 示例性接头序列
<400> 94
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 95
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TEV蛋白酶裂解序列
<220>
<221> misc_feature
<222> (2)..(3)
<223> Xaa是任何氨基酸
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa是任何氨基酸
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> Xaa=Gly或Ser
<400> 95
Glu Xaa Xaa Tyr Xaa Gln Xaa
1 5
<210> 96
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TEV蛋白酶裂解序列
<400> 96
Glu Asn Leu Tyr Phe Gln Gly
1 5
<210> 97
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TEV蛋白酶裂解序列
<400> 97
Glu Asn Leu Tyr Phe Gln Ser
1 5
<210> 98
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 98
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
1 5 10 15
Glu Glu Asn Pro Gly Pro
20
<210> 99
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 99
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 100
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 100
Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
1 5 10
<210> 101
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 101
Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
1 5 10 15
Glu Asn Pro Gly Pro
20
<210> 102
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 102
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 103
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 103
Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro
1 5 10
<210> 104
<211> 23
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 104
Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp
1 5 10 15
Val Glu Ser Asn Pro Gly Pro
20
<210> 105
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 105
Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 106
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 106
Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro
1 5 10
<210> 107
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 107
Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala
1 5 10 15
Gly Asp Val Glu Ser Asn Pro Gly Pro
20 25
<210> 108
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 108
Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val
1 5 10 15
Glu Ser Asn Pro Gly Pro
20
<210> 109
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 109
Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro
1 5 10
<210> 110
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 110
Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn
1 5 10 15
Pro Gly Pro
<210> 111
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 111
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn
1 5 10 15
Pro Gly Pro
<210> 112
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 112
Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro
1 5 10
<210> 113
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 113
Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly
1 5 10 15
Pro
<210> 114
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 114
Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 115
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 115
Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 116
<211> 40
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 116
Val Thr Glu Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro
1 5 10 15
Arg Pro Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys
20 25 30
Ile Val Ala Pro Val Lys Gln Thr
35 40
<210> 117
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 117
Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro
1 5 10 15
Gly Pro
<210> 118
<211> 40
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 118
Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys Ile Val
1 5 10 15
Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
20 25 30
Asp Val Glu Ser Asn Pro Gly Pro
35 40
<210> 119
<211> 33
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 包含2A位点的自裂解多肽
<400> 119
Glu Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Thr Leu
1 5 10 15
Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly
20 25 30
Pro
Claims (94)
1.一种非天然细胞,其包含:
(a)第一多肽,所述第一多肽包含:FRB多聚化结构域多肽或其变体;CD8α跨膜结构域或CD4跨膜结构域;CD137共刺激结构域;和/或CD3ζ初级信号传导结构域;以及
(b)第二多肽,所述第二多肽包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体;FKBP多聚化结构域多肽或其变体;和CD4跨膜结构域或CD8α跨膜结构域;
其中桥接因子促进在所述非天然细胞的表面上形成多肽复合物,其中所述桥接因子与所述第一多肽和所述第二多肽的多聚化结构域缔合并且置于所述多聚化结构域之间。
2.根据权利要求1所述的非天然细胞,其中所述FKBP多聚化结构域是FKBP12。
3.根据权利要求1或权利要求2所述的非天然细胞,其中所述FRB多肽是FRB T2098L。
4.根据权利要求1至3中任一项所述的非天然细胞,其中所述桥接因子选自由以下组成的组:AP21967、西罗莫司、依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。
5.根据权利要求1至4中任一项所述的非天然细胞,其中所述第一多肽包含信号肽、CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域。
6.根据权利要求1至5中任一项所述的非天然细胞,其中所述第二多肽包含信号肽和CD4跨膜结构域。
7.根据权利要求1至6中任一项所述的非天然细胞,其中所述第二多肽包含共刺激结构域。
8.根据权利要求7所述的非天然细胞,其中所述第二多肽的所述共刺激结构域选自由以下组成的组的共刺激分子:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNFRS14、TNFRS18、TNRFS25和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
9.根据权利要求7或权利要求8所述的非天然细胞,其中所述第二多肽的所述共刺激结构域是从OX40或TNFR2中分离的共刺激结构域。
10.根据权利要求1至9中任一项所述的非天然细胞,其中所述第二多肽包含SEQ IDNO:22-31中任一者所示的序列。
11.一种非天然细胞,其包含多肽复合物,所述多肽复合物包含:
(a)第一多肽,所述第一多肽包含:FRB多聚化结构域多肽或其变体;CD8α跨膜结构域或CD4跨膜结构域;CD137共刺激结构域;和/或CD3ζ初级信号传导结构域;
(b)第二多肽,所述第二多肽包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体;FKBP多聚化结构域多肽或其变体;和CD4跨膜结构域或CD8α跨膜结构域;以及
(c)桥接因子,所述桥接因子与所述第一多肽和所述第二多肽的多聚化结构域缔合并且置于所述多聚化结构域之间。
12.根据权利要求11所述的非天然细胞,其中所述FKBP多聚化结构域是FKBP12。
13.根据权利要求11或权利要求12所述的非天然细胞,其中所述FRB多肽是FRBT2098L。
14.根据权利要求11至13中任一项所述的非天然细胞,其中所述桥接因子选自由以下组成的组:AP21967、西罗莫司、依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。
15.根据权利要求11至14中任一项所述的非天然细胞,其中所述第一多肽包含信号肽、CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域。
16.根据权利要求11至15中任一项所述的非天然细胞,其中所述第二多肽包含信号肽和CD4跨膜结构域。
17.根据权利要求11至16中任一项所述的非天然细胞,其中所述第二多肽包含共刺激结构域。
18.根据权利要求17所述的非天然细胞,其中所述第二多肽的所述共刺激结构域选自由以下组成的组的共刺激分子:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNFRS14、TNFRS18、TNRFS25和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
19.根据权利要求17或权利要求18所述的非天然细胞,其中所述第二多肽的所述共刺激结构域是从OX40或TNFR2中分离的共刺激结构域。
20.根据权利要求11至19中任一项所述的非天然细胞,其中所述第二多肽包含SEQ IDNO:22-31中任一者所示的序列。
21.根据权利要求1至20中任一项所述的非天然细胞,其中所述细胞是造血细胞。
22.根据权利要求1至21中任一项所述的非天然细胞,其中所述细胞是T细胞、αβT细胞或γδT细胞。
23.根据权利要求1至22中任一项所述的非天然细胞,其中所述细胞是CD3+、CD4+和/或CD8+细胞。
24.根据权利要求1至23中任一项所述的非天然细胞,其中所述细胞是免疫效应细胞。
25.根据权利要求1至24中任一项所述的非天然细胞,其中所述细胞是细胞毒性T淋巴细胞(CTL)、肿瘤浸润淋巴细胞(TIL)或辅助T细胞。
26.根据权利要求1至25中任一项所述的非天然细胞,其中所述细胞是自然杀伤(NK)细胞或自然杀伤T(NKT)细胞。
27.根据权利要求1至26中任一项所述的非天然细胞,其中所述细胞的来源是外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织或肿瘤。
28.根据权利要求1至27中任一项所述的非天然细胞,其中当所述第一多肽和所述第二多肽被表达时,所述FRB多聚化结构域和所述FKBP多聚化结构域在细胞外定位。
29.一种融合多肽,其包括:
(a)第一多肽,所述第一多肽包含:FRB多聚化结构域多肽或其变体;CD8α跨膜结构域或CD4跨膜结构域;CD137共刺激结构域;和/或CD3ζ初级信号传导结构域;
(b)多肽裂解信号;以及
(c)第二多肽,所述第二多肽包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体;FKBP多聚化结构域多肽或其变体;和CD4跨膜结构域或CD8α跨膜结构域。
30.根据权利要求29所述的融合多肽,其中所述FKBP多聚化结构域是FKBP12。
31.根据权利要求29或权利要求30所述的融合多肽,其中所述FRB多肽是FRB T2098L。
32.根据权利要求29至31中任一项所述的融合多肽,其中所述桥接因子选自由以下组成的组:AP21967、西罗莫司、依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。
33.根据权利要求29至32中任一项所述的融合多肽,其中所述第一多肽包含信号肽、CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域。
34.根据权利要求29至33中任一项所述的融合多肽,其中所述第二多肽包含信号肽和CD4跨膜结构域。
35.根据权利要求29至34中任一项所述的非天然细胞,其中所述融合多肽包含SEQ IDNO:32-41中任一者所示的序列。
36.根据权利要求29至35中任一项所述的融合多肽,其中所述第二多肽包含共刺激结构域。
37.根据权利要求36所述的融合多肽,其中所述第二多肽的所述共刺激结构域选自由以下组成的组的共刺激分子:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNFRS14、TNFRS18、TNRFS25和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
38.根据权利要求36或权利要求37所述的融合多肽,其中所述第二多肽的所述共刺激结构域是从OX40或TNFR2中分离的共刺激结构域。
39.根据权利要求29至38中任一项所述的融合多肽,其中所述多肽裂解信号是病毒自裂解多肽。
40.根据权利要求29至39中任一项所述的融合多肽,其中所述多肽裂解信号是病毒自裂解2A多肽。
41.根据权利要求29至40中任一项所述的融合多肽,其中所述多肽裂解信号是选自由以下组成的组的病毒自裂解多肽:口蹄疫病毒(FMDV)(F2A)肽、马A型鼻炎病毒(ERAV)(E2A)肽、明脉扁刺蛾β四体病毒(TaV)(T2A)肽、猪捷申病毒-1(PTV-1)(P2A)肽、泰勒病毒2A肽和脑心肌炎病毒2A肽。
42.根据权利要求29至41中任一项所述的非天然细胞,其中所述融合多肽包含SEQ IDNO:42-61中任一者所示的序列。
43.根据权利要求29至42中任一项所述的融合多肽,其中当所述第一多肽和所述第二多肽被表达时,所述FRB多聚化结构域和所述FKBP多聚化结构域在细胞外定位。
44.一种多肽复合物,其包括:
(a)第一多肽,所述第一多肽包含:FRB多聚化结构域多肽或其变体;CD8α跨膜结构域或CD4跨膜结构域;CD137共刺激结构域;和/或CD3ζ初级信号传导结构域;
(b)第二多肽,所述第二多肽包含:具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体;FKBP多聚化结构域多肽或其变体;和CD4跨膜结构域或CD8α跨膜结构域;以及
(c)桥接因子,所述桥接因子与所述第一多肽和所述第二多肽的多聚化结构域缔合并且置于所述多聚化结构域之间。
45.根据权利要求44所述的多肽复合物,其中所述FKBP多聚化结构域是FKBP12。
46.根据权利要求44或权利要求45所述的多肽复合物,其中所述FRB多肽是FRBT2098L。
47.根据权利要求44至46中任一项所述的多肽复合物,其中所述桥接因子选自由以下组成的组:AP21967、西罗莫司、依维莫司、诺氟莫司、吡美莫司、地磷莫司、他克莫司、替西罗莫司、乌米莫司和佐他莫司。
48.根据权利要求44至47中任一项所述的多肽复合物,其中所述第一多肽包含CD8α跨膜结构域、CD137共刺激结构域和CD3ζ初级信号传导结构域。
49.根据权利要求44至48中任一项所述的多肽复合物,其中所述第二多肽包含CD4跨膜结构域。
50.根据权利要求44至49中任一项所述的多肽复合物,其中所述第二多肽包含共刺激结构域。
51.根据权利要求50所述的多肽复合物,其中所述第二多肽的所述共刺激结构域选自由以下组成的组的共刺激分子:Toll样受体1(TLR1)、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、半胱天冬酶募集结构域家族成员11(CARD11)、CD2、CD7、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD94、CD134(OX40)、CD137(4-1BB)、CD278(ICOS)、DNAX-活化蛋白10(DAP10)、用于活化T细胞家族成员1的接头(LAT)、含SH2结构域的76kD白细胞蛋白(SLP76)、T细胞受体相关的跨膜衔接子1(TRAT1)、TNFR2、TNFRS14、TNFRS18、TNRFS25和T细胞受体相关的蛋白激酶70的ζ链(ZAP70)。
52.根据权利要求50或权利要求51所述的多肽复合物,其中所述第二多肽的所述共刺激结构域是从OX40或TNFR2中分离的共刺激结构域。
53.根据权利要求44至52中任一项所述的多肽复合物,其中所述细胞是造血细胞。
54.根据权利要求44至53中任一项所述的多肽复合物,其中所述细胞是T细胞、αβT细胞或γδT细胞。
55.根据权利要求44至54中任一项所述的多肽复合物,其中所述细胞是CD3+、CD4+和/或CD8+细胞。
56.根据权利要求44至55中任一项所述的多肽复合物,其中所述细胞是免疫效应细胞。
57.根据权利要求44至56中任一项所述的多肽复合物,其中所述细胞是细胞毒性T淋巴细胞(CTL)、肿瘤浸润淋巴细胞(TIL)或辅助T细胞。
58.根据权利要求44至57中任一项所述的多肽复合物,其中所述细胞是自然杀伤(NK)细胞或自然杀伤T(NKT)细胞。
59.根据权利要求44至58中任一项所述的多肽复合物,其中所述细胞的来源是外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织或肿瘤。
60.根据权利要求44至59中任一项所述的多肽复合物,其中当所述第一多肽和所述第二多肽被表达时,所述FRB多聚化结构域和所述FKBP多聚化结构域在细胞外定位。
61.一种嵌合抗原受体(CAR),其包含:
a)具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体;
b)铰链结构域;
c)跨膜结构域;
d)一个或多个细胞内共刺激信号传导结构域;和/或
e)初级信号传导结构域。
62.根据权利要求61所述的CAR,其中所述CAR从5’至3’包含:
a)具有SEQ ID NO:2-21中任一者所示的氨基酸序列的抗CD33 VHH抗体;
b)铰链结构域;
c)跨膜结构域;
d)一个或多个细胞内共刺激信号传导结构域;和/或
e)初级信号传导结构域。
63.根据权利要求61或权利要求62所述的CAR,其中所述铰链结构域和跨膜结构域从CD8α、CD27、CD28、CD33、CD37、CD45、CD64、CD71、CD80、CD86、CD 134、CD137、CD152、CD154、AMN和PD1中分离。
64.根据权利要求61至63中任一项所述的CAR,其中所述一个或多个共刺激信号传导结构域从选自由以下组成的组的共刺激分子分离:CD28、CD134、CD137和CD278。
65.根据权利要求61至64中任一项所述的CAR,其中所述CAR包含CD8α信号肽、CD8α铰链结构域和跨膜结构域、CD134共刺激结构域和CD3ζ初级信号传导结构域。
66.一种CAR,其包含SEQ ID NO:62-81中任一者所示的序列。
67.一种多核苷酸,其编码根据权利要求1至28中任一项所述的第一多肽或第二多肽、根据权利要求29至43中任一项所述的融合多肽、或根据权利要求61-66中任一项所述的CAR。
68.一种cDNA,其编码根据权利要求1至28中任一项所述的第一多肽或第二多肽、根据权利要求29至43中任一项所述的融合多肽、或根据权利要求61-66中任一项所述的CAR。
69.一种RNA,其编码根据权利要求1至28中任一项所述的第一多肽或第二多肽、根据权利要求29至43中任一项所述的融合多肽、或根据权利要求61-66中任一项所述的CAR。
70.一种载体,其包含根据权利要求67至69中任一项所述的多核苷酸。
71.根据权利要求70所述的载体,其中所述载体是表达载体。
72.根据权利要求70所述的载体,其中所述载体是转座子。
73.根据权利要求72所述的载体,其中所述载体是piggyBAC转座子或Sleeping Beauty转座子。
74.根据权利要求70所述的载体,其中所述载体是病毒载体。
75.根据权利要求74所述的载体,其中所述载体是腺病毒载体、腺相关病毒(AAV)载体、疱疹病毒载体、痘苗病毒载体或逆转录病毒载体。
76.根据权利要求75所述的载体,其中所述逆转录病毒载体是慢病毒载体。
77.根据权利要求76所述的载体,其中所述慢病毒载体选自由以下组成的组:人免疫缺陷病毒1(HIV-1);人免疫缺陷病毒2(HIV-2)、维斯纳-梅迪病毒(VMV)病毒;山羊关节炎-脑炎病毒(CAEV);马传染性贫血病毒(EIAV);猫免疫缺陷病毒(FIV);牛免疫缺陷病毒(BIV);以及猿猴免疫缺陷病毒(SIV)。
78.一种细胞,其包含根据权利要求1至28中任一项所述的第一多肽和第二多肽、根据权利要求29至43中任一项所述的融合多肽、或根据权利要求61-66中任一项所述的CAR。
79.根据权利要求78所述的细胞,其中所述细胞是造血细胞。
80.根据权利要求78或79所述的细胞,其中所述细胞是免疫效应细胞。
81.根据权利要求78至80中任一项所述的细胞,其中所述细胞是T细胞、αβT细胞或γδT细胞。
82.根据权利要求78至81中任一项所述的细胞,其中所述细胞表达CD3+、CD4+、CD8+或它们的组合。
83.根据权利要求78至82中任一项所述的细胞,其中所述细胞是细胞毒性T淋巴细胞(CTL)、肿瘤浸润淋巴细胞(TIL)或辅助T细胞。
84.根据权利要求78至83中任一项所述的细胞,其中所述细胞是自然杀伤(NK)细胞或自然杀伤T(NKT)细胞。
85.一种组合物,其包含根据权利要求1-28和78至84中任一项所述的细胞。
86.一种组合物,其包含生理上可接受的载体和根据权利要求1-28和78至84中任一项所述的细胞。
87.一种治疗有需要的受试者的方法,所述方法包括向所述受试者施用有效量的根据权利要求85或权利要求86所述的组合物。
88.一种治疗、预防或改善癌症、传染病、自身免疫性疾病、炎性疾病和免疫缺陷的至少一种症状或与其相关的病状的方法,所述方法包括向受试者施用有效量的根据权利要求85或权利要求86所述的组合物。
89.一种治疗实体癌的方法,所述方法包括向受试者施用有效量的根据权利要求85或权利要求86所述的组合物。
90.根据权利要求89所述的方法,其中所述实体癌选自由以下组成的组:肺癌、鳞状细胞癌、结直肠癌、胰腺癌、乳腺癌、甲状腺癌、膀胱癌、子宫颈癌、食道癌、卵巢癌、胃癌、子宫内膜癌或脑癌。
91.根据权利要求89或权利要求90所述的方法,其中所述实体癌是非小细胞肺癌、头部和颈部鳞状细胞癌、结直肠癌、胰腺癌、乳腺癌、甲状腺癌、膀胱癌、子宫颈癌、食管癌、卵巢癌、胃癌、子宫内膜癌、神经胶质瘤、成胶质细胞瘤或少突神经胶质瘤。
92.一种治疗血液恶性肿瘤的方法,所述方法包括向受试者施用有效量的根据权利要求85或权利要求86所述的组合物。
93.根据权利要求92所述的方法,其中所述血液恶性肿瘤是白血病、淋巴瘤或多发性骨髓瘤。
94.根据权利要求92所述的方法,其中所述恶性血液病是急性骨髓性白血病(AML)。
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