CN104395462A - 增强亲和力的t细胞受体及其制备方法 - Google Patents
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Abstract
本公开提供通过用表达Delta-like-1或Delta-like-4的基质细胞培养的表达抗原特异的TCRα的造血祖细胞的激动剂选择产生增强亲和力的T细胞受体的方法,由此种方法制备的组合物,和其用途。
Description
对相关申请的交叉引用
在35U.S.C.§119(e)下,本申请要求2012年5月3日提交的美国临时申请61/642,358的权益,所述申请以其整体通过引用并入本文。
关于序列表的声明
以文本形式替代纸件拷贝提供与本申请相关的序列表,并且通过引用在此并入本说明书。含有序列表的文本文件的名称为360056_412WO_SEQUENCE_LISTING.TXT。该文本文件为129KB,于2013年5月2日建立并且通过EFS-Web电子提交。
政府利益的声明
以在由美国国立卫生研究院(National Institute of Health)/美国国立癌症研究所(Naional Cancer Institute)授予的合同No.P01 CA 18029下的政府支持完成本发明。在本发明中,政府具有某些权利。
背景
技术领域
本公开涉及增强亲和力的T细胞受体(TCRs)和,更特别地,涉及使用表达抗原特异的TCRα的造血祖细胞的激动剂选择产生增强亲和力的TCRs,和其用途。
相关技术描述
TCR基因治疗是新兴的治疗方法,其可能克服很多与常规T细胞过继免疫治疗相关的困难,比如分离,表征,和扩展肿瘤抗原特异的T细胞克隆所需的大量的时间和劳动(Schmitt,Ragnarsson,&Greenberg,2009,Hum.Gene Ther.20:1240-1248)。基因治疗的进一步好处包括利用能够长期体内留存的明确的T细胞群体的能力(Berger等人,2008,J.Clin.Invest.118:294-305;Hinrichs等人,2009,Proc.Natl.Acad.Sci.USA 106:17469-17474)。此种T细胞可以用编码良好表征的对于肿瘤抗原具有高亲和力的TCRs的基因转导,从而增加介导抗肿瘤效果的可能性。实际上,近期的以遗传修饰的表达靶向自身/肿瘤-抗原的高亲和力嵌合受体的T细胞靶向晚期B细胞白血病的治疗的报道强调使用设计的高亲合力T细胞用于治疗白血病的可能(Kalos等人,2011,Sci.Transl.Med.3:95ra73)。然而,因为由T细胞免疫治疗靶向的大多数肿瘤抗原是过表达的自身蛋白,因此特异于这些抗原的高亲和力T细胞一般在胸腺中接受负选择。因此,基于T细胞的免疫治疗的一个重要限制通常是对非突变肿瘤抗原具有足够高亲和力的表达内源性TCR的T细胞的有限的利用度。
已经开发了一些策略以增强意在用于TCR基因治疗的TCRs的亲和力(Richman&Kranz,2007,Biomol.Eng.24:361-373;Udyavar等人,2009,J.Immunol.182:4439-4447;Zhao等人,2007,J.Immunol.179:5845-5854)。这些方法通常需要产生TCR突变体文库,其经过数轮诱变和随后筛选赋予对靶肽/MHC配体的更高亲和力的突变。突变通常在已知与肽/MHC相互作用的CDR区域进行。CDR1和CDR2区域显著地与MHC分子接触,而高变的CDR3区域主要接触肽(Wucherpfennig等人,2010,Cold SpringHarbor Perspectives in Biology 2:a005140-a005140)。位点定向的诱变策略通常靶向所有这三个区域的选择的部分,但仍然不总是成功产生更高亲和力变体,并且改善被限制于仅在具体靶向的区域的改变。此外,引入MHC接触残基的突变具有可能增加TCR对MHC的亲和力同时减少受体对其同源肽的总体特异性的风险。由于此原因,理想的是,引入以增强TCR的亲和力的大多数突变将被限制于CDR3区域。然而,目前的方法学限制于产生CDR3多样性的能力,因为位点定向的诱变受限于CDR3区域的原长度。
鉴于分离识别与抗原相关的相关肿瘤的高亲和力T细胞的困难,存在对用于产生增强亲和力的TCRs的替代方法的持续需要。
简短概述
在一个方面,本公开提供产生增强亲和力的TCR的方法,所述方法包括:a)在足以诱导造血祖细胞分化为DN TCRαβ+胸腺细胞条件下将造血祖细胞与基质细胞和肽抗原接触达足以诱导造血祖细胞分化为DNTCRαβ+胸腺细胞的时间,其中所述造血祖细胞包含编码来自特异于肽抗原的亲本TCR的TCRα链的非内源性核酸序列,并且其中所述基质细胞包含编码Delta-like-1或Delta-like-4的非内源性核酸序列和编码MHC分子的核酸序列;b)从DN TCRαβ+胸腺细胞分离编码不同TCRβ链的核酸序列并且将编码TCRβ链的核酸序列引入能够在细胞表面上表达TCR和包含编码来自步骤a)的TCRα链的核酸序列的细胞;并且鉴定增强亲和力的TCR(例如,通过MHC四聚体测定检测或选择高亲和力TCRαβ候选物,并随后测量与亲本TCRαβ相比的结合亲和力)。
在进一步的方面,提供由本文公开的方法产生的增强亲和力的TCRs,所述增强亲和力的TCRs可以是结合细胞的或可溶形式,并且可以进一步被密码子优化以增强在T细胞中的表达。
在更进一步的方面,本公开的增强亲和力的TCRs可以通过施用包含增强亲和力的TCRs的组合物用于在受试者中治疗疾病(比如癌症,感染性疾病,或自身免疫病)。在进一步实施方案中,本公开的增强亲和力的TCRs可以用于诊断方法或成像方法,包括用于本文中所鉴定的适应证或病症相关的这些方法。
当参考以下详述和附图时,本发明的这些或其它方面将变得显而易见。本文公开的所有参考文献由此像各自分别并入一样,以其整体通过引用并入。
附图简述
图1A-D:如表明的,分选来自OT-1转基因小鼠的胸腺细胞的TCRβ-TCRγδ-CD4-CD8-CD117+CD44+DN1和DN2祖细胞并在不同浓度的卵清蛋白SIINFEKL肽(SEQ ID NO:1)的存在下,培养在表达I类MHCH-2Kb分子的OP9-DL1细胞上达20天。(A,B,C)通过在指定时间点的流式细胞术分析培养物。(D)在培养的第20天确定各个培养物的总细胞性。
图2:将尚未通过从B6或OT-1转基因小鼠分选的阳性选择的CD69-DP胸腺细胞在卵清蛋白SIINFEKL肽(SEQ ID NO:1)的存在下培养在表达I类MHC H-2Kb分子OP9-DL1细胞上。
图3A-C:分选B6胸腺细胞的CD4-CD8-CD117+CD44+DN1和DN2祖细胞并用亲和力增强的WT1特异的TCR 3D克隆的TCRα链转导,并存在或缺少1μM的WT1肽RMFPNAPYL(SEQ ID NO:2)的情况下培养在表达I类MHC H-2Db分子的OP9-DL1细胞上。(A)在培养的第16天,通过流式细胞术分析转导的(hCD2+)和未转导的(hCD2-)细胞。(B)在存在1uM WT1肽RMFPNAPYL(SEQ ID NO:2)的情况下OP9-DL1培养的第21天,根据指定方案分选DN TCRαβ+细胞。(C)溶解分选的细胞,分离DNA,并且使用Vb10-特异的正向引物和Cb2-特异的反向引物进行PCR。随后将Vb10 PCR产物定向TOPO-克隆在载体pENTR/D-TOPO中,使用技术转入逆转录病毒载体MigR1-attR,并且产生逆转录病毒上清并用于转导鼠58-/-细胞以用于如描述的文库筛选。
图4A-C:所述逆转录病毒TCRβ文库用于转导CD8+3Dα+58-/-细胞。(A)如表明的,初始将转导的细胞仅对GFP表达分选(结果未显示),接着两次另外的对GFP和高MHC-WT1肽四聚体表达的分选。还对分选的58-/-细胞分析对于作为非特异性四聚体结合的对照的GP33非特异的,但MHCH-2Db-肽四聚体特异的染色。(B)分离的TCRβ链的序列分析。(C)通过序列分析鉴定了四条候选TCRβ链,并转入回MigR1-attR逆转录病毒载体。产生逆转录病毒上清,并用于转导CD8+3Dα+58-/-细胞。
图5A-C:(A)将各个与3Dα成对的候选TCRβ链转导的58-/-细胞用MHC-WT1肽特异四聚体,以及几个非特异的MHC H-2Db-肽四聚体染色,以评估针对MHC的可能的非依赖于肽的反应。(B)通过用MHC-肽四聚体染色各个转导的细胞系,然后后流式细胞术确定三种最高亲和力TCRs的相对亲和力。使用PE-缀合的四聚体的六个2-倍稀释度进行KD测量,并且由获得半数最大结合的配体的浓度通过非线性回归从结合曲线确定了表观KD值。(C)将最高亲和力TCRβ链(克隆#1)密码子优化,并且将四聚体结合与原增强亲和力的3Dαβ构建体相比较。
图6A-B:分析来自3D-PYYα-IRES-hCD2和7431α-IRES-hCD2逆基因(retrogenic)小鼠的TCRβ+胸腺细胞(A)和脾细胞(B)的CD4和CD8表达。来自3D-PYYα-IRES-hCD2和7431α-IRES-hCD2逆基因小鼠的TCRβ+胸腺细胞(A)的Vβ10和Vβ9表达。
图7:分析来自6天的体外间皮素(mesothelin)肽刺激+IL2的WT1之后的逆基因小鼠的脾细胞。
详细描述
本公开提供产生增强的或高亲和力TCRs的方法和组合物,其中来自抗原-特异的TCR的TCRα链用于从头(de novo)选择在体外T细胞发育过程中产生的与抗原-特异的TCRα链配对的TCRβ链,以形成新的、增强亲和力的受体,所述增强亲和力的受体可以有利地驱使T细胞独立于负选择通过新的选择过程成熟从而靶向研究的抗原。
在一个方面,本公开提供产生增强亲和力的T细胞受体(TCR)的方法,所述方法通过在存在肽抗原的情况下将造血祖细胞(含有编码抗原特异的TCRα链的非内源性核酸序列)与基质细胞(含有编码Delta-like-1或Delta-like-4的非内源性核酸序列和编码MHC分子的核酸序列)培养,其将诱导造血祖细胞分化为DN TCRαβ+胸腺细胞。随后,将编码来自DNTCRαβ+胸腺细胞的不同TCRβ链的核酸序列分离并引入能够在细胞表面上表达TCR并且还表达上文指出的TCRα链的细胞。最后,通过比较候选TCRαβ与亲本TCRαβ的结合亲和力鉴定增强亲和力的TCR。
另外,本公开提供使用此方法产生的增强亲和力的TCRs,以及用于在不同治疗应用(包括治疗受试者中的疾病(例如,癌症,感染性疾病,自身免疫病))中使用本公开的增强亲和力的TCRs的组合物和方法。
在更详细陈述本公开之前,对要在本文中使用的某些数据提供定义对于其理解可以有帮助。贯穿本公开陈述其它定义。
在本说明书中,术语“约”和“基本上由...组成”意为指出的范围、值、或结构的±20%,除非另有指示。应该理解,本文所使用的术语“一个(a)”和“一个(an)”是指“一个以上”列举的组分。备选方案(例如,“或”)的使用应该被理解为意为备选方案中的一个、二者或其任意组合。如本文使用的,术语“包括,”“具有”和“包含”同义使用,所述术语和其变体意在被理解为不受限制的。
“T细胞受体”(TCR)是指在T细胞(或T淋巴细胞)表面发现的分子,与CD3联合,通常负责识别结合于主要组织相容性复合体(MHC)分子的抗原。在大多数T细胞中,所述TCR具有二硫键连接的高度变化的α和β链(也分别称为TCRα和TCRβ)的异源二聚体。在小的子集的T细胞中,所述TCR由可变的γ和δ链(也分别称为TCRγ和TCRδ)的异源二聚体构成。TCR的各个链是免疫球蛋白超家族的成员并且具有一个N-末端免疫球蛋白可变结构域,一个免疫球蛋白恒定结构域,跨膜区域,和在C-末端的短胞质尾区(参见Janeway等人,Immunobiology:The Immune System inHealth and Disease,第3版,Current Biology Publications,p.4:33,1997)。在本公开中所使用的TCR可以来自不同动物物种,包括人、小鼠、大鼠、或其它哺乳动物。TCR可以是结合细胞的或可溶的形式。
如果它们以例如,大于或等于约105M-1,106M-1,107M-1,108M-1,109M-1,1010M-1,1011M-1,1012M-1,或1013M-1的亲和力或Ka(即,单位为1/M的特定结合相互作用的平衡缔合常数)结合靶分子,本公开的TCRs和其结合结构域可以是″免疫特异的″或能够结合到所需程度,包括″特异地或选择性地结合″靶而不显著结合存在于测试样品中的其它组分。″高亲和力″结合结构域是指具有至少107M-1,至少108M-1,至少109M-1,至少1010M-1,至少1011M-1,至少1012M-1,至少1013M-1,或获更大的Ka的那些结合结构域。备选地,亲和力可以被定义为单位为M的特定结合相互作用的平衡解离常数(Kd)(例如,10-5M至10-13M)。根据本公开的TCRs和结合结构域多肽的亲和力可容易使用常规技术确定(参见,例如,Scatchard等人(1949)Ann.N.Y.Acad.Sci.51:660;和美国专利号5,283,173;5,468,614,分析,或相当物)。因此,“增强亲和力的T细胞受体”(增强亲和力的TCR)是指选择的或改造的TCR,所述TCR具有比野生型(或亲本)TCR更强的对靶抗原的结合。增强的亲和力可以通过对靶抗原的Ka(平衡缔合常数)高于野生型(也成为亲本或原)TCR对靶抗原的Ka的TCR,对靶抗原的Kd(解离常数)低于野生型(也成为亲本或原)TCR对靶抗原的Kd的TCR,或对靶抗原的解离速率(Koff)小于野生型(或亲本)TCR对靶抗原的解离速率的TCR表征。
“主要组织相容性复合体分子”(MHC分子)是指递送肽抗原至细胞表面的糖蛋白。I类MHC分子是异源二聚体,其由跨膜α链(具有三个α结构域)和非共价关联的β2微球蛋白组成。II类MHC分子由两个跨膜糖蛋白,α和β构成,二者都跨膜。各个链具有两个结构域。I类MHC分子递送源自胞质中的肽至细胞表面,在那里,肽:MHC复合体被CD8+T细胞识别。II类MHC分子递送源自囊泡系统中的肽至细胞表面,在那里它们被CD4+T细胞识别。MHC分子可以来自不同动物物种,包括人、小鼠、大鼠或其它哺乳动物。
“造血祖细胞”是源自造血干细胞或胎儿组织的细胞,其能够进一步分化为成熟细胞类型(例如,T细胞系的细胞)。在一个特定实施方案中,使用CD24lo Lin-CD117+造血祖细胞。如本文限定的,造血祖细胞可以包括胚胎干细胞,所述胚胎干细胞能够进一步分化为T细胞系的细胞。造血祖细胞可以来自不同动物物种,包括人、小鼠、大鼠或其它哺乳动物。
“胸腺细胞祖细胞”或“胸腺细胞”是存在于胸腺中的造血祖细胞。
“造血干细胞”是指未分化的造血细胞,其能够基本上在体内或离体无限繁殖并且能够分化为其它细胞类型,包括T细胞系的细胞。造血干细胞可以分离自,例如,胎儿肝,骨髓,和脐带血。
“T细胞系的细胞”是指显示至少一个区别于来自其它淋巴细胞,和红细胞或骨髓系细胞的细胞的T细胞或其前体或祖先表型特征的细胞。此种表型特征可以包括表达一个以上T细胞特异的(例如,CD8+)的蛋白,或T细胞特异的生理、形态、功能、或免疫学特征。例如,T细胞系的细胞可以是明确是T细胞系的祖先或前体细胞;CD25+未成熟和灭活的T细胞;经历CD4或CD8系定型(定型)的细胞;CD4+CD8+双阳性的胸腺细胞祖细胞;单阳性的CD4+或CD8+;TCRαβ或TCRγδ;或成熟和功能性或激活的T细胞。
“基质细胞”是任何器官的结缔组织细胞。在一个特定实施方案中,所述基质细胞是骨髓基质细胞。可以被改造以表达DLL1或DLL4的基质细胞系的实例包括小鼠基质细胞系MS5(Itoh,等人,Exp.Hematol.1989,17:145-153)和S17,和人基质细胞系HGS2.11,HGS2.52,HGS.18,HGS3.30,HGS3.65,HGS.3.66,HGS3.103,和HGS3.114(可从Human Genome SciencesInc.,MD获得,参见美国公开申请20020001826)。在一个特定实施方案中,使用OP9细胞(Kodama等人,1994,Exp.Hematol.22:979-984;可从RIKEN细胞库获得)。之前已经描述了表达DLL1和DLL4的OP9细胞(参见,例如,Schmitt等人,2002,Immunity:17:749-756;美国专利号7,575,925)
“双阴性TCRαβ胸腺细胞”(DN TCRαβ胸腺细胞)是指不表达CD4和CD8共受体,但表达TCRα和β链的胸腺细胞群体。
“肽抗原”是指长度在约7个氨基酸至约25个氨基酸范围内,作为抗原被TCR,或其结合结构域特异识别的氨基酸序列,并且其可以源自或基于更长的靶生物分子(例如,多肽,蛋白)或其衍生物的片段。抗原可以表达在细胞表面,细胞内,或作为完整的膜蛋白。抗原可以是源自宿主的(例如,肿瘤抗原,自身免疫抗原)或具有外部来源(例如,细菌的,病毒的)。
“核酸序列”,或多核苷酸,可以是RNA或DNA的形式,其包括cDNA,基因组DNA,和合成的DNA。核酸序列可以双链或单链的,并且如果是单链的,可以是编码链或非编码(反义链)。编码序列可以与本领域已知的编码序列相同或可以是不同的编码序列,其作为遗传密码冗余或简并的结果,或通过剪接,编码相同多肽。
“非内源性”是指不存在于向其中引入(例如,重组引入)分子的宿主细胞/样品中的分子(例如,核酸序列)。非内源性分子可以来自相同物种或不同物种。
Notch配体“Delta-like-1”(DL1或DLL1)和“Delta-like-4”(DL4或DLL4)是Notch Delta配体的同系物并且是δ/serrate/jagged蛋白家族的成员。它们在造血作用过程中介导细胞命运抉择方面发挥作用并且可以在细胞细胞间通讯中发挥作用。示例性Delta-like-1序列包括Genbank登录号.NM_005618.3(SEQ ID NO:3)和NP_005609.3(SEQ ID NO:4)(分别是智人转录本和蛋白序列),和Genbank登录号.NM_007865.3(SEQ ID NO:5)和NP_031891.2(SEQ ID NO:6)(分别是小家鼠转录本和蛋白序列)。示例性Delta-like-4序列包括Genbank登录号.NM_019074.3(SEQ ID NO:7)和NP_061947.1(SEQ ID NO:8)(分别是智人转录本和蛋白序列)和Genbank登录号.NM_019454.3(SEQ ID NO:9)和NP_062327.2(SEQ IDNO:10)(分别是小家鼠转录本和蛋白序列)。Notch配体可市购或可以通过标准重组DNA技术生产并纯化至不同程度。
“胚胎干细胞”或“ES细胞”或“ESCs”是指未分化的胚胎干细胞,其具有整合入并变成发育中的胚胎的生殖系的部分的能力。胚胎干细胞能够分化为造血祖细胞。适于本文中用途的胚胎干细胞包括来自J1ES细胞系,129JES细胞系,鼠干细胞系D3(American Type Culture Collection目录#CRL1934),源自129/Sv小鼠的R1或E14K细胞系,源自Balb/c和C57Bl/6小鼠的细胞系,和人胚胎干细胞(例如来自WiCell Research Institute,WI;或EScell International,Melbourne,Australia)的细胞.
“WT1”是指肾母细胞瘤1(Wilm′s tumor1),一种转录因子,其含有C末端四个锌指基序和N末端的富脯氨酸/谷氨酰胺的DNA结合结构域。WT1对泌尿生殖系统的正常发育具有重要作用并且在一小群患有肾母细胞瘤(Wilm′s tumor)的患者中是突变的。已经在不同癌症中观察到WT1的高表达包括,乳腺癌,卵巢癌,急性白血病,血管肿瘤,黑素瘤,结肠癌,肺癌,甲状腺癌,骨和软组织肉瘤,和食管癌。对于WT1已经指出了可变剪接。示例性WT1序列包括Genbank登录号:NM_000378.4(SEQ ID NO:11)(人转录本),NP_000369.3(SEQ ID NO:12)(人蛋白);NM_024424.3(SEQ ID NO:13)(人转录本),NP_077742.2(SEQ ID NO:14)(人蛋白);NM_024426.4(SEQ ID NO:15)(人转录本),NP_077744.3(SEQ ID NO:16);NM_001198552.1(SEQ ID NO:17),NP_001185481.1(SEQ ID NO:18)(人蛋白);NM_001198551.1(SEQ ID NO:19)(人转录本),NP_001185480.1(SEQ ID NO:20)(人蛋白);NM_144783.2(SEQ ID NO:21)(小鼠转录本),和NP_659032.3(SEQ ID NO:22)(小鼠蛋白)。
“间皮素(mesothelin)”(MSLN)是指编码前体蛋白的基因,所述前体蛋白被切割为两个产物,巨核细胞强化因子和间皮素(mesothelin).巨核细胞强化因子作为可以刺激骨髓巨核细胞中集落形成的细胞因子行使功能。间皮素是糖基磷脂酰肌醇锚定的细胞-表面蛋白,其可以作为细胞粘附蛋白行使功能。该蛋白在上皮性间皮瘤,卵巢癌中和在特定鳞状细胞癌中过表达。选择性剪切导致产生多个转录本变体。示例性间皮素序列包括Genbank登录号:NM_001177355.1(SEQ ID NO:23),NP_001170826.1(SEQ ID NO:24)(分别是人转录本和前蛋白序列);NM_005823.5(SEQ ID NO:25),NP_005814.2(SEQ ID NO:26)(分别是人转录本和前蛋白序列);NM_013404.4(SEQ ID NO:27),NP_037536.2(SEQ ID NO:28)(分别是人转录本和前蛋白序列);NM_018857.1(SEQ ID NO:29),NP_061345.1(SEQID NO:30)(分别是小鼠转录本和前体蛋白序列)。
“MHC-肽四聚体染色”是指用于检测抗原-特异性T细胞的测定,其特征是MHC分子的四聚体,各包含与至少一个抗原同源(例如,相同或相关)的氨基酸序列的相同肽,其中所述复合体能够结合对同源抗原特异的T细胞。每个MHC分子可以用生物素分子标签标记。通过加入链亲和素将生物素化的MHC/肽四聚化,所述链亲和素典型地是荧光标记的。可以由流式细胞术通过荧光标记检测四聚体。在某些实施方案中,MHC-肽四聚体测定用于检测或选择本公开的高亲和力TCRs。
产生增强亲和力的TCRs的方法
通过背景,在胸腺中T细胞发育过程中,祖先胸腺细胞接受许多TCR-介导的检查点。这些中第一个被称为β-选择,并且发生在鼠T细胞发育的双阴性3(DN3)阶段。在Tcrb基因座产生成功重排的DN3细胞可以在细胞表面表达与不变的前体Tα蛋白配对的TCRβ蛋白。该受体被称为前体TCR,并且其以不依赖于配体的方式发信号以促进增殖,αβ系细胞分化至CD4/CD8双阳性(DP)阶段,和Tcrα基因座的重排(Boehmer等人,1999,Curr.Opin.Immunol.11:135-142)。在β-选择之前,在TCRα座位钝化并接近TCR基因重排的同时,TCRγ和-δ座位二者在发育的DN3阶段都经历重排,并且在这两个位置的成功重排导致成熟γδ-TCR的表达,其可提供驱动向γδT细胞系分化的信号-在发育过程中γδT细胞不通过DP阶段分化,并且通常保持DN或CD8αα+。αβ/γδ细胞命运抉择由在该发育阶段TCR信号的强度确定,因为发育的T细胞通过与成熟γδTCR相关的更强信号区分前体TCR信号和γδTCR信号(Pennington,Silva-Santos,&Hayday,2005,Curr.Opin.Immunol.17:108-115)。有趣的是,很多αβTCR转基因小鼠在胸腺中具有大量的成熟CD24-TCRαβ阳性CD4/CD8双阴性(DN)细胞,已经显示其代表在β-选择检查点作为来自成熟αβ转基因TCR的更强信号的结果发育的“γδ效仿者(wanna-be)”细胞(Egawa等人,2000,PLOS One 3:1512)。
本文公开的是产生增强亲和力的TCRs的方法,其中在β-选择前抗原-特异的TCRα链的异位表达使得在体外T细胞分化过程中在存在同源抗原的情况下分化时表达对于相同抗原高亲和力TCR的T细胞发育。使用该方法,表达高亲和力受体的T细胞通过采用响应T细胞发育的DN3阶段的激动剂信号的DN TCRαβ+系命运绕过负选择。
在某些实施方案中,本公开提供产生增强亲和力TCR的方法,所述方法包括:a)在足以诱导造血祖细胞分化为DN TCRαβ+胸腺细胞的条件下,将造血祖细胞与基质细胞和肽抗原接触达足以诱导造血祖细胞分化为DNTCRαβ+胸腺细胞的时间,其中所述造血祖细胞包含编码来自特异于所述肽抗原的亲本TCR的TCRα链的非内源性核酸序列,并且其中所述基质细胞包含编码Delta-like-1或Delta-like-4的非内源性核酸序列和编码MHC分子的核酸序列;b)从DN TCRαβ+胸腺细胞分离编码不同TCRβ链的核酸序列并将编码所述TCRβ链的核酸序列引入能够在细胞表面上表达TCR的和包含编码来自步骤a)的TCRα链的核酸序列的细胞;并且鉴定增强亲和力的TCR(例如,通过MHC四聚体测定检测或选择高亲和力TCRαβ候选物,并随后测量与亲本TCRαβ相比的结合亲和力。
在某些实施方案中,造血祖细胞包括胸腺细胞祖细胞或胚胎干细胞。在其它实施方案中,造血祖细胞源自胎儿肝组织。在其它实施方案中,造血祖细胞包含源自或来源于骨髓,脐带血,或外周血的造血干细胞。在进一步的其它实施方案中,造血祖细胞源自人、小鼠、大鼠、或其它哺乳动物。在一个特定实施方案中,使用CD24lo Lin-CD117+胸腺细胞祖细胞。
已经将造血祖细胞改进为包含编码来自特异于肽抗原的亲本TCR的TCRα链的非内源性核酸序列。在一个特定实施方案中,所述TCRβ链也分离自所述亲本TCR。可以使用本领域已知的标准分子生物学技术进行TCRα和β链的克隆。克隆TCR链的方法是本领域已知的(参见,例如,Walchli等人,2011,PLoS ONE 6:e27930;Birkholz等人,2009,J.Immunol.Methods 346:45-54;Kurokawa等人,2001,Clin.Exp.Immunol.123:340-345)。
“基质细胞”是任何器官的结缔组织细胞。可以根据本发明使用的基质细胞包括人和小鼠基质细胞。可以被改造以表达DL1或DL4的基质细胞系的实例包括小鼠基质细胞系MS5(Itoh,等人,Exp.Hematol.1989,17:145-153)和S17,和人基质细胞系HGS2.11,HGS2.52,HGS.18,HGS3.30,HGS3.65,HGS.3.66,HGS3.103,和HGS3.114(可从Human GenomeSciences Inc.,MD获得,参见美国公开申请20020001826)。在某些实施方案中,基质细胞是骨髓基质细胞。在进一步的实施方案中,使用OP9细胞。
在某些实施方案中,基质细胞包括编码DL1,比如人DL1的非内源性核酸序列。示例性Delta-like-1序列包括Genbank登录号.NM_005618.3(SEQ ID NO:3)和NP_005609.3(SEQ ID NO:4)(分别是智人转录本和蛋白序列)和Genbank登录号.NM_007865.3(SEQ ID NO:5)和NP_031891.2(SEQ ID NO:6)(分别是小家鼠转录本和蛋白序列)。在某些实施方案中,基质细胞包含编码DL4,比如人DL4的非内源性核酸序列。示例性Delta-like-4序列包括Genbank登录号.NM_019074.3(SEQ ID NO:7)和NP_061947.1(SEQ ID NO:8)(分别是智人转录本和蛋白序列)和Genbank登录号.NM_019454.3(SEQ ID NO:9)和NP_062327.2(SEQ ID NO:10)(分别是小家鼠转录本和蛋白序列)。Notch配体是可市购的或可以通过标准重组DNA技术生产并纯化到不同程度。
在更进一步的实施方案中,基质细胞是表达DL1,比如人DL1的OP9细胞或其衍生物。之前已经描述了表达DL1和DL4的OP9细胞(Schmitt等人,2002,Immunity 17:749-756;美国专利No.7,575,925)。
在某些实施方案中,基质细胞也包含编码MHC分子的核酸序列。在特定实施方案中,基质细胞包含编码I类MHC分子的核酸序列,并且可以任选地还包含编码β2微球蛋白的核酸序列。所述I类MHC和β2微球蛋白分子可以源自人,小鼠,大鼠,或其它哺乳动物物种I类MHC分子,其基因和蛋白序列为本领域已知的。在其它实施方案中,所述基质细胞包含编码II类MHC分子的核酸序列。所述II类MHC分子可以源自人,小鼠,大鼠,或其它哺乳动物物种MHC分子,其基因和蛋白序列为本领域已知的。
给定的T细胞仅在其结合宿主细胞的MHC分子时将识别肽抗原(MHC-限制的抗原识别)。选择对于已知肽抗原具有特异性的亲本TCR用于使用所公开的方法增强TCR亲和力。因此,也选择结合特定肽抗原的MHC分子并在基质细胞中表达以允许所公开的体外系统中的MHC-限制的抗原识别。鉴定结合肽抗原MHC分子的方法是本领域已知的(参见,例如,Akatsuka等人,2002,Tissue Antigens 59:502-511)。在某些实施方案中,MHC分子包含HLA-A2和β-2微球蛋白,优选人来源的,其可以结合于,例如,WT1肽RMFPNAPYL(SEQ ID NO:2)。在其它实施方案中,MHC分子包含小鼠H-2Db,其可以结合,例如,WT1肽RMFPNAPYL或Hung等人,2007,Gene Therapy 14:921-929的图3A中所公开的不同间皮素肽,或H-2Kb,其可以结合,例如,Hung等人的图3A中所公开的不同间皮素肽。在Hung等人中公开的可能的H-2Db限制性间皮素表位包括:ISKANVDVL(SEQ ID NO:42),GQKMNAQAI(SEQ ID NO:43),SAFQNVSGL(SEQ ID NO:44),和LLGPNIVDL(SEQ ID NO:45)。在Hung等人中公开的可能的H-2Kb限制的间皮素表位包括:EIPFTYEQL(SEQID NO:46)和GIPNGYLVL(SEQ ID NO:47)。
公开的方法中使用的肽抗原是指亲本TCR特异结合的抗原的肽序列,或靶生物分子(例如,多肽,蛋白)。肽序列可以源自在细胞表面,细胞内表达的,或为完整膜蛋白的抗原。所述抗原可以是源自宿主的抗原(例如,肿瘤/癌抗原,和自身免疫抗原),或外源性抗原(例如,病毒,细菌,原生动物抗原)。肿瘤或癌抗原可以源自不同癌症,比如本文中指出的那些。在一些实施方案中,癌抗原包括白血病抗原。在某些实施方案中,肽抗原源自肾母细胞瘤1(WT1),比如包含氨基酸序列RMFPNAPYL(SEQ ID NO:2)的WT1肽。在其它实施方案中,肽抗原源自间皮素,比如在Hung等人,2007,Gene Therapy 14:921-929的图3A中公开的间皮素肽。在一些实施方案中,所述间皮素肽包含氨基酸序列GQKMNAQAI(SEQ ID NO:31)。在其它实施方案中,所述间皮素肽包含含有ISKANVDVL(SEQ ID NO:42),GQKMNAQAI(SEQ ID NO:43),SAFQNVSGL(SEQ ID NO:44),和LLGPNIVDL(SEQ ID NO:45),EIPFTYEQL(SEQ ID NO:46),或GIPNGYLVL(SEQ ID NO:47)的氨基酸序列。自身免疫抗原是由特异于自身抗原的自体反应性TCRs识别的抗原,其具有引起自身免疫病,使自身免疫病恶化,促进自身免疫病进展,引起或使与自身免疫病相关的症状恶化的随之而来的免疫效应功能。例如,特异于胶原蛋白肽的自体反应的TCRs可以用于类风湿性关节炎(rheumatoid arthritis)中Tregs的抑制性基因治疗。自身免疫抗原还可以是位于其它引起自身免疫病或介导自身免疫病症状的免疫细胞(例如,产生自身抗体的B细胞)上的抗原。例如,CD20肽抗原可以用于产生靶向参与或与类风湿性关节炎相关的B细胞的增强亲和力的TCRs。可以将肽抗原加入针对如本文描述的造血祖细胞和基质细胞的培养系统。备选地,包含编码研究的肽抗原的核酸序列的基质细胞可以用于在细胞培养中表达此种抗原。不希望受理论限制,不论是否作为外源肽抗原加入培养系统或通过基质细胞表达,肽抗原都与由基质细胞表达的MHC分子形成复合体从而形成MHC-肽抗原复合体。MHC-肽抗原复合体允许在培养系统中通过TCRs的MHC-限制性肽抗原识别。在某些实施方案中,将OP9细胞用核酸序列转导从而表达WT1抗原肽RMFPNAPYL(SEQ ID NO:2)。在其它实施方案中,将OP9细胞用核酸序列转导从而表达间皮素抗原肽GQKMNAQAI(SEQ ID NO:31)。
结合I类MHC分子的肽通常形成约7至约10个氨基酸的长度。结合II类MHC分子的肽长度上可变,通常长约10-25个氨基酸。在某些实施方案中,亲本TCR的肽抗原特异性是已知的。在其它实施方案中,亲本TCR的肽抗原特异性需要使用本领域已知的方法来确定。(Borras等人,2002,J.Immunol.Methods 267:79-97;Hiemstra等人,2000,Cur.Opin.Immunol.12:80-4)。例如,如果亲本TCR的靶抗原已知,尽管不是特定肽序列,源自所述靶抗原多肽序列的肽文库可以用于筛选和鉴定对于亲本TCR的特定肽抗原。
“载体”是能够转运其它核酸的核酸分子。载体可以是,例如,质粒,粘粒,病毒,或噬菌体。“表达载体”是当其存在于适当的环境中时,能够引导由载体携带的一个以上基因编码的蛋白的表达的载体。
“逆转录病毒”是具有RNA基因组的病毒。“γ逆转录病毒”是指逆转录病毒科的一个属。示例性γ逆转录病毒包括但不限于,小鼠干细胞病毒,鼠白血病病毒,猫白血病病毒,猫肉瘤病毒,禽网状内皮组织增殖病毒。
“慢病毒”是指逆转录病毒的一个属,其能够感染分裂细胞和非分裂细胞。慢病毒的一些实例包括HIV(人免疫缺陷病毒:包括1型HIV,和2型HIV);马感染性贫血病毒;猫免疫缺陷病毒(FIV);牛免疫缺陷病毒(BIV);和猴免疫缺陷病毒(SIV)。
编码核心病毒的载体也称为“病毒载体”。存在大量的适于用于本发明的可用病毒载,包括鉴定用于人基因治疗应用的那些,比如由Pfeifer和Verma描述的那些(Pfeifer,A.和I.M.Verma.2001.Ann.Rev.GenomicsHum.Genet.2:177-211)。合适的病毒载体包括基于RNA病毒的载体,比如源自逆转录病毒的载体,例如,源自莫洛尼鼠白血病病毒(MLV)的载体,并且包括更复杂的源自逆转录病毒的载体,例如,源自慢病毒的载体。源自HIV-1的载体属于该类型。其它实例包括源自HIV-2,FIV,马感染性贫血病毒,SIV,和梅迪/维斯那病毒的慢病毒载体。使用逆转录病毒和慢病毒病毒载体和用含有TCRs转基因的病毒粒子包装细胞用于转导哺乳动物靶细胞的方法是本领域公知的并且之前例如,在美国专利8,119,772;Walchli等人,2011,PLoS One 6:327930;Zhao等人,J.Immunol.,2005,174:4415-4423;Engels等人,2003,Hum.Gene Ther.14:1155-68;Frecha等人,2010,Mol.Ther.18:1748-57;Verhoeyen等人,2009,Methods Mol.Biol.506:97-114中描述。逆转录病毒和慢病毒载体构建体和表达系统也可以市购。
在一个特定实施方案中,使用病毒载体将编码特异于肽抗原的TCRα链的非内源性核酸序列引入造血祖细胞中。在另一个实施方案中,病毒载体用于将编码DL1或DL4的非内源性核酸序列和编码MHC分子的核酸序列引入基质细胞中。所述病毒载体可以是逆转录病毒载体或慢病毒载体。所述病毒载体还可以包括编码用于转导的标记的核酸序列。用于病毒载体的转导标记是本领域已知的并且包括选择标记,所述选择标记可以赋予药物抗性,或可检测标记,比如荧光标记或细胞表面蛋白,所述可检测标记可以通过比如流式细胞术的方法被检测。在一个特定实施方案中,所述病毒载体进一步包含用于转导的基因标记,所述基因标记包括绿色荧光蛋白或人CD2的细胞外结构域。然而病毒载体基因组包含多于一条要在宿主细胞中表达的核酸序列作为分离的转录本,所述病毒载体还可以在两个(或更多)转录本之间包含另外的序列以允许双顺反子或多顺反子的表达。用于病毒载体的此种序列的实例包括内部核糖体进入位点(IRES),弗林蛋白酶切割位点,病毒2A肽。
其它载体也可以用于多核苷酸递送,所述载体包括DNA病毒载体,所述DNA病毒载体包括,例如基于腺病毒的载体和基于腺病毒伴随病毒(AAV)的载体;源自单纯疱疹病毒的载体(HSVs),所述源自单纯疱疹病毒的载体包括扩增子载体,复制缺陷HSV和减毒HSV(Krisky等人,1998,Gene Ther.5:1517-30)。
近期开发用于基因治疗用途的其它载体也可以用于本公开的方法。此种载体包括源自杆状病毒和α-病毒的那些。(Jolly D J.1999.Emerging viralvectors.pp 209-40在Friedmann T.编辑1999.The development of humangene therapy.New York:Cold Spring Harbor Lab中)。
在足以诱导造血祖细胞分化为DN TCRαβ+胸腺细胞条件下,将造血祖细胞与包含编码非内源性DL1或DL4的核酸序列和编码MHC分子的核酸序列的基质细胞培养足以诱导造血祖细胞分化为DN TCRαβ+胸腺细胞的时间。在某些实施方案中,将造血祖细胞培养在6cm或10cm组织培养-处理的盘中。培养物中的造血祖细胞浓度可以在1-109,或1x102至1x106,或1x103至1x104之间。在一些实施方案中,将造血祖细胞(约1-5x104细胞)培养在表达DL1的单层OP9细胞上。
可以向培养中添加一种以上促进造血祖细胞定型和分化的细胞因子。所述细胞因子可以源自人或其它物种。培养中的细胞因子浓度可以从约1ng/ml至约50ng/ml。可以使用的细胞因子的代表性实例包括:FGF家族的所有成员,包括FGF-4和FGF-2;Flt-3-配体,干细胞因子(SCF),血小板生成素(TPO),和IL-7。细胞因子可以与黏多糖,比如硫酸肝素联合使用。细胞因子可市购或可以通过重组DNA技术生产并纯化至不同程度。一些细胞因子可以通过标准生化技术从细胞培养基纯化。
可以将造血祖细胞培养在培养基中,所述培养基包括条件培养基,非条件培养基,或胚胎干细胞培养基。合适的条件培养基的实例包括IMDM,DMEM,或αMEM,用胚胎成纤维细胞(例如,人胚胎成纤维细胞)为条件的,或相当培养基。合适的非条件培养基的实例包括Iscove’s改良的Delbucco’s培养基(IDMD),DMEM,或αMEM,或相当的培养基。培养基可以包含血清(例如,牛血清,胎牛血清,小牛血清,马血清,人血清,或人工血清替代物)或其可以无血清。
培养条件需要将造血祖细胞培养足够时间以诱导造血祖细胞分化为DN TCRαβ+胸腺细胞。通常将细胞维持培养达约4-5天,优选约5至20天。将理解细胞可以被维持达获得所要结果(即,所要的细胞组合物)所需要的适当的时间量。例如,为了产生主要包含不成熟的钝化的T细胞的细胞组合物,可以将所述细胞维持培养达约5至20天。可以将细胞维持培养达20至30天以产生主要包含成熟T细胞的细胞组合物。还可以在不同时间点,比如从约几天至约25天从培养物收集非黏附细胞。之前已经描述了用于造血干细胞在基质细胞系上的培养方法(美国专利#7,575,925;Schmitt等人,2004,Nat.Immunol.5:410-417;Schmitt等人,2002,Immunity 17:749-756)。
造血祖细胞向DN TCRαβ+胸腺细胞的分化可以被检测并且使用标准流式细胞术方法分离这些细胞。可以使用一种以上细胞分选以分离DNTCRαβ+胸腺细胞。例如,第一细胞分选可以鉴定表达转导标记(即,用于TCRα表达的标记)的造血祖细胞。在某些实施方案中,转导标记是人CD2的细胞外结构域。在进一步的实施方案中,转导标记阳性细胞可以接受第二细胞分选以筛选CD4-和CD8-的细胞。对DN细胞的第三细胞分选可以筛选表达TCRβ的细胞。可以使用细胞表面或转导标记的不同组合设计这些分选的部分,或单种或多种细胞分选,从而鉴定所需的DN TCRαβ+胸腺细胞亚群,这对于本领域技术人员而言将是显而易见的。分选DN TCRαβ+细胞的方法是本领域已知的(U.S.7,575,925和Schmitt等人,2002,Immunity:17:749-756)。
分离编码来自DN TCRαβ+胸腺细胞的不同TCRβ链的核酸序列并引入包含编码来自亲本TCR的TCRα链的核酸序列的T细胞。如本文讨论的,从细胞克隆TCRβ链的方法是本领域公知的并已经在之前描述。在某些实施方案中,一旦将编码候选TCRβ链的核酸序列从DN TCRαβ+胸腺细胞分离,则所述核酸序列可以接受进一步选择过程,借此选择具有由亲本TCRβ链使用的相同Vβ基因的TCRβ链用于引入T细胞。可以使用用于PCR的Vβ基因特异引物在分选的细胞群体内鉴定含有TCRβ链的亲本Vβ基因。与增强抗原-特异的TCRs体外亲和力相关的一个问题是,一些改进可能仅增加受体对MHC的亲和力,而不是肽/MHC,从而增加TCR将自体反应的可能性。将候选TCRβ链限制于含有亲本Vβ基因的那些增加保留接触MHC的TCR CDR1和CDR2结构域,和限制CDR3可变性的可能性。如之前讨论的,病毒载体,比如逆转录病毒载体和慢病毒载体,适于将编码不同TCRβ链和/或亲本TCRα的核酸序列引入T细胞。在一些实施方案中,所述病毒载体进一步包含用于转导的基因标记(例如绿色荧光蛋白)。
能够在细胞表面表达TCR的细胞用于用编码来自DN TCRαβ+胸腺细胞的不同TCRβ链的核酸序列转化或转导。能够在细胞表面表达TCR的细胞表达CD3分子。“CD3”六条链的多蛋白复合体,其稳定地与细胞表面上的TCR相关。在哺乳动物中,所述复合体包含CD3γ链,CDδ链,两条CD3ε,CD3ζ链的同源二聚体。所述CD3γ,CD3δ,和CD3ε是含有单个免疫球蛋白结构域的免疫球蛋白超家族的高度相关的细胞表面蛋白。CD3γ,CD3δ,和CD3ε的跨膜区域带负电,其是允许该链与带正电的TCR链关联的特征。CD3γ,CD3δ,和CD3ε链的胞质结构域含有基于免疫受体酪氨酸的活化基序(ITAMs),其使得它们在受体刺激之后与胞质蛋白酪氨酸激酶关联并且从而发信号至细胞内部。TCR的细胞-表面表达需要CD3蛋白(参见Janeway等人,Immunobiology:The Immune System in Health andDisease,第3版,Current Biology Publications,p.4:39,1997)。
在一些实施方案中,能够在细胞表面上表达TCR的细胞是T细胞,包括源自人,小鼠,大鼠,或其它哺乳动物的原代细胞或细胞系。如果获自哺乳动物,T细胞可以从很多来源获得,包括血液,骨髓,淋巴结,胸腺,或其它组织或液体。T细胞可以富集的或纯化的。T细胞系是本领域公知的,其中一些描述于Sandberg等人,2000,Leukemia 21:230-237。在某些实施方案中,使用缺少TCRα和β链的内源性表达的T细胞。此种T细胞可以天然缺少TCRα和β链的内源性表达或可以被修饰以阻断表达(例如,来自不表达TCRα和β链的转基因小鼠或操作以抑制TCRα和β链的表达的细胞系的T细胞)。在某些实施方案中,使用58α-β-细胞,一种缺少内源性TCRα和TCRβ链的鼠T细胞系(Letourneur和Malissen,1989,Eur.J.Immunol.19:2269-74)。在其它实施方案中,使用H9 T细胞系(目录#HTB-176,ATCC,Manassas,VA)。在某些实施方案中,能够在细胞表面表达TCR的细胞不是T细胞或T细胞系的细胞,除了已被修饰以表达CD3,使TCR能够在细胞表面表达的细胞(例如,293细胞或3T3细胞)。之前已经描述了不是T细胞系的TCRs在细胞上的细胞表面表达(Szymczak等人,2004,Nat.Biotechnol.22:589-594).
为了鉴定可能增强亲和力的TCR,一旦将能够在还表达亲本TCRα链的细胞表面表达TCR的细胞用候选TCRβ链文库转化或转导,使用MHC-肽四聚体染色分选或鉴定抗原-特异的细胞。MHC-肽四聚体染色特征是MHC分子的四聚体,各自包含具有与至少一种抗原同源(例如,相同或相关)的氨基酸序列的相同肽,其中所述复合体能够结合对同源抗原特异的T细胞。各个MHC分子可以用生物素分子标签标记。通过加入链亲和素将生物素化的MHC/肽四聚体化,所述链亲和素典型地是荧光标记的。可以由流式细胞术通过荧光标记检测四聚体。检测抗原特异的T细胞的MHC-肽四聚体染色方法是本领域公知的(例如,Altman等人,1996,Science 274:94-96;Kalergis等人,2000,J.Immunol.Methods 234:61-70;Xu和Screaton,2002,J.Immunol.Methods 268:21-8;James等人,J.Vis.Exp.25:1167)。在某些实施方案中,所述MHC-肽四聚体包含I类MHC分子。在其它实施方案中,所述MHC-肽四聚体包含II类MHC分子。在进一步实施方案中,用于公开的方法中培养步骤的相同肽抗原与整合入MHC-肽四聚体的肽相同。在其它实施方案中,公开的方法的培养步骤中由基质细胞表达的MHC分子与MHC-肽四聚体中的MHC分子相同。可以通过流式细胞术分选MHC-肽四聚体染色的细胞一次以上。第一分选可以选择表达可检测转导标记(例如,绿色荧光蛋白)的转导的细胞。还可以分选转导阳性细胞一次以上,以获得表达与亲本TCR相同的Vβ链的细胞。可以使用细胞表面或转导标记的不同组合设计这些分选的部分,或单种或多种细胞分选,从而鉴定所需的细胞亚群,这对于本领域技术人员而言将是显而易见的。
通过比较候选TCRαβ与亲本TCRαβ的结合亲和力鉴定增强亲和力的TCR。随后可以使用标准分子生物学技术将抗原-特异的T细胞克隆和测序。候选TCRβ克隆随后可以用于转导包含亲本TCRα链的T细胞并且MHC-肽四聚体染色可以用于比较与亲本TCRαβ的染色水平,如之前描述的。以候选TCRβ观察到的增加的染色可以表征与亲本TCRαβ相比增强亲和力。然而,如果亲本TCRαβ被密码子优化以增加在T细胞中的表达,直接比较以候选TCRβ的四聚体染色水平可能是不可能的。候选TCRβ链也可以被密码子优化,以直接与亲本TCRβ比较。
如果其具有比亲本TCRαβ对肽抗原更强的结合,则候选TCRαβ与亲本TCRαβ相比具有增强的亲和力。增强的亲和力可以由具有对于靶抗原的Ka(平衡缔合常数)比亲本TCR对于靶抗原的Ka高的TCR,对于靶抗原的KD(解离常数)比亲本TCR对于靶抗原的KD小的TCR,或对于靶抗原的解离速率(Koff)比野生型(或亲本)TCR对于靶抗原的解离速率小的TCR表示。测量TCR结合亲和力的方法已经在之前描述(例如,Laugel等人,2007,J.Biol.Chem.282:23799-23810;Garcia等人,2001,Proc.Natl.Acad.Sci.USA 98:6818-6823)。
增强亲和力TCRs和组合物
在另一个方面,提供由本文公开的方法产生的增强亲和力的TCRs。增强亲和力的TCR可以是结合细胞的(例如,表达在成熟T细胞的表面)或可溶的形式。在某些实施方案中,增强亲和力的TCRs可以被密码子优化以增强在T细胞中的表达(Scholten等人,2006,Clin.Immunol.119:135-145)。
在其它实施方案中,增强亲和力的TCRs还可以是融合蛋白的组分,所述融合蛋白可以进一步包含细胞毒性组分(例如,化疗药物比如长春地辛(vindesine),抗叶酸剂(antifolates);细菌毒素,蓖麻毒素,抗病毒剂),所述细胞毒性组分用于特异的杀死癌症细胞或感染的细胞或使癌症细胞或感染的细胞无效,或可检测组分(例如,生物素,荧光部分,放射性核素),所述可检测部分用于成像癌症细胞,感染的细胞,或自身免疫攻击下的组织。
本公开还提供药物组合物,所述药物组合物包含由本文公开的方法产生的增强亲和力的TCR和药学可接受的载体,稀释剂,或赋形剂。合适的赋形剂包括水、盐水、葡萄糖、甘油、乙醇等和其组合。
应用
由本公开的方法产生的增强亲和力的TCRs可以用于通过施用包含增强亲和力的TCRs组合物治疗受试者中疾病(比如癌症,感染疾病,或自身免疫病)。
可以用增强亲和力TCR治疗治疗的疾病包括癌症,感染性疾病(病毒,细菌,原生动物感染),和自身免疫病。TCR基因治疗是对于不同类型癌症(Morgan等人,2006,Science 314:126-129;在Schmitt等人,2009,Human Gene Therapy中综述的;在2007年6月,J.Clin.Invest.117:1466-1476中综述的)和感染疾病(Kitchen等人,2009,PLoS One 4:38208;Rossi等人,2007,Nat.Biotechnol.25:1444-54;Zhang等人,PLoS Pathog.6:e1001018;Luo等人,2011,J.Mol.Med.89:903-913)的有希望的治疗手段。使用包含自体反应性TCRs的调节性T细胞用于自身免疫病的免疫抑制基因治疗也是新兴的治疗手段(Fujio等人,2006,J.Immunol.177:8140-8147;Brusko等人,2008,Immunol.Rev.223:371-390)。
多种癌症,包括实体肿瘤和白血病可用本文公开的组合物和方法处理。可以被治疗的癌症类型包括:乳腺癌、前列腺癌、和结肠腺癌;所有形式的肺支气管癌;髓细胞瘤;黑素瘤;肝细胞瘤(hepatoma);成神经细胞瘤(neuroblastoma);乳头状瘤(papilloma);apud瘤(apudoma);迷芽瘤(choristoma);鳃原瘤(branchioma);恶性类癌综合征(malignant carcinoidsyndrome);类癌心脏病(carcinoid heart disease);和癌(例如,沃克癌(Walker),基底细胞癌(basal cell),基底鳞状癌(basosquamous),布-皮癌(Brown-Pearce),导管癌(ductal),埃利希癌(Ehrlich tumor),Krebs 2,merkel细胞癌,粘蛋白癌(mucinous),非小细胞肺癌,燕麦细胞癌(oat cell),乳头状癌(papillary),硬癌(scirrhous),细支气管癌(bronchiolar),支气管原癌(bronchogenic),鳞状细胞癌(squamous cell),和移行细胞癌(transitional cell)。可以被治疗的另外的癌症类型包括:组织细胞疾病(histiocytic disorders);白血病;恶性组织细胞增多病(histiocytosis malignant);霍奇金病(Hodgkin′sdisease);免疫增生小(immunoproliferative small);非霍奇金淋巴瘤(non-Hodgkin′s lymphoma);浆细胞瘤(plasmacytoma);网状内皮组织增殖(reticuloendotheliosis);黑素瘤;软骨母细胞瘤(chondroblastoma);软骨瘤(chondroma);软骨肉瘤(chondrosarcoma);纤维瘤(fibroma);纤维肉瘤(fibrosarcoma);巨细胞瘤(giant cell tumors);组织细胞瘤(histiocytoma);脂肪瘤(lipoma);脂肪肉瘤(liposarcoma);间皮瘤(mesothelioma);粘液瘤(myxoma);粘液肉瘤(myxosarcoma);骨瘤(osteoma);骨肉瘤(osteosarcoma);脊索瘤(chordoma);颅咽管瘤(craniopharyngioma);无性细胞瘤(dysgerminoma);错构瘤(hamartoma);间质瘤(mesenchymoma);中肾瘤(mesonephroma);肌肉瘤(myosarcoma);成釉细胞瘤(ameloblastoma);牙骨质瘤(cementoma);牙瘤(odontoma);畸胎瘤(teratoma);胸腺瘤(thymoma);滋养细胞瘤(trophoblastic tumor)。进一步地,以下类型的癌症也被认为是可由治疗处理的:腺瘤(adenoma);胆管瘤(cholangioma);胆脂瘤(cholesteatoma);圆柱瘤(cyclindroma);囊腺癌(cystadenocarcinoma);囊腺瘤(cystadenoma);颗粒细胞瘤(granulosa cell tumor);两性胚细胞瘤(gynandroblastoma);肝细胞瘤(hepatoma);汗腺腺瘤(hidradenoma);胰岛细胞瘤(islet cell tumor);睾丸间质细胞瘤(Leydig cell tumor);乳头状瘤(papilloma);支持细胞瘤(sertoli cell tumor);卵泡膜细胞瘤(theca cell tumor);平滑肌瘤(leimyoma);平滑肌肉瘤(leiomyosarcoma);成肌细胞瘤(myoblastoma);肌瘤(myomma);肌肉瘤(myosarcoma);横纹肌瘤(rhabdomyoma);横纹肌肉瘤(rhabdomyosarcoma);室管膜细胞瘤(ependymoma);神经节细胞瘤(ganglioneuroma);神经胶质瘤(glioma);成神经管细胞瘤(medulloblastoma);脑脊膜瘤(meningioma);神经鞘瘤(neurilemmoma);成神经细胞瘤(neuroblastoma);神经上皮瘤(neuroepithelioma);纤维神经瘤(neurofibroma);神经瘤(neuroma);副神经节瘤(paraganglioma);非嗜铬副神经节瘤(paraganglioma nonchromaffin)。可以被治疗的癌症类型还包括:血管角质瘤(angiokeratoma);血管淋巴样增生伴嗜酸细胞增多(angiolymphoid hyperplasia with eosinophilia);血管瘤硬化(angioma sclerosing);多发性血管瘤(angiomatosis);血管球瘤(glomangioma);血管内皮瘤(hemangioendothelioma);血管瘤(hemangioma);血管外皮细胞瘤(hemangiopericytoma);血管肉瘤(hemangiosarcoma);淋巴管瘤(lymphangioma);淋巴管肌瘤(lymphangiomyoma);淋巴管肉瘤(lymphangiosarcoma);松果体瘤(pinealoma);癌肉瘤(carcinosarcoma);软骨肉瘤(chondrosarcoma);叶状囊肉瘤(cystosarcoma phyllodes);纤维肉瘤(fibrosarcoma);血管肉瘤(hemangiosarcoma);平滑肌肉瘤(leiomyosarcoma);白色肉瘤(leukosarcoma);脂肪肉瘤(liposarcoma);淋巴管肉瘤(lymphangiosarcoma);肌肉瘤(myosarcoma);粘液肉瘤(myxosarcoma);卵巢癌;横纹肌肉瘤(rhabdomyosarcoma);肉瘤(sarcoma);肿瘤(neoplasms);多发性神经纤维瘤(nerofibromatosis);和宫颈非典型增生(cervicaldysplasia)。
需要增强的TCR治疗的过度增殖性疾病的多样性的示例是B-细胞癌症,包括B-细胞淋巴瘤(比如不同形式的霍奇金病,非霍奇金淋巴瘤(NHL)或中枢神经系统淋巴瘤),白血病(比如急性淋巴细胞白血病(acutelymphoblastic leukemia)(ALL),慢性淋巴细胞白血病(chronic lymphocyticleukemia)(CLL),多毛细胞白血病(Hairy cell leukemia)和慢性肌细胞白血病(chronic myoblastic leukemia))和骨髓瘤(myelomas)(比如多发性骨髓瘤(multiple myeloma))。其它B细胞癌症包括小淋巴细胞性淋巴瘤(smalllymphocytic lymphoma),B细胞幼淋巴细胞白血病(B-cell prolymphocyticleukemia),淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma),splenic脾边缘区淋巴瘤(marginal zone lymphoma),浆细胞骨髓瘤(plasma cellmyeloma),孤立性骨浆细胞瘤(solitary plasmacytoma of bone),骨外浆细胞瘤(extraosseous plasmacytoma),黏膜相关淋巴组织结外边缘区B细胞淋巴瘤(extra-nodal marginal zone B-cell lymphoma of mucosa-associated)(MALT)淋巴组织),结边缘区B细胞淋巴瘤(nodal marginal zone B-cell lymphoma),滤泡性淋巴瘤(follicular lymphoma),套细胞淋巴瘤(mantle cell lymphoma),弥漫性大B细胞淋巴瘤(diffuse large B-cell lymphoma),纵隔(胸腺)大B细胞淋巴瘤(mediastinal(thymic)large B-cell lymphoma),血管内大B细胞淋巴瘤(intravascular large B-cell lymphoma),原发性渗出性淋巴瘤(primaryeffusion lymphoma),伯基特氏淋巴瘤/白血病(Burkitt′s lymphoma/leukemia),不确定恶性潜能的B细胞增殖(B-cell proliferations of uncertain malignantpotential),淋巴瘤样肉芽肿病(lymphomatoid granulomatosis),和移植后淋巴组织增生性疾病(post-transplant lymphoproliferative disorder)。
自身免疫病包括:关节炎,类风湿性关节炎(rheumatoid arthritis),幼年型类风湿性关节炎(juvenile rheumatoid arthritis),骨关节炎(osteoarthritis),多软骨炎(polychondritis),银屑病关节炎(psoriatic arthritis),银屑病(psoriasis),皮炎(dermatitis),多肌炎(polymyositis)/皮肌炎(dermatomyositis),包涵体肌炎(inclusion body myositis),炎症性肌炎(inflammatory myositis),毒性表皮坏死(toxic epidermal necrolysis),系统性硬皮病和硬化(systemic scleroderma and sclerosis),CREST综合征,炎性肠病相关的反应(responses associated with inflammatory bowel disease),克罗恩病(Crohn′s disease),溃疡性结肠炎(ulcerative colitis),呼吸窘迫综合征(respiratory distress syndrome),急性呼吸窘迫综合征(adult respiratorydistress syndrome)(ARDS),脑膜炎(meningitis),脑炎(encephalitis),葡萄膜炎(uveitis),结肠炎(colitis),血管球性肾炎(glomerulonephritis),过敏性疾病,湿疹(eczema),哮喘(asthma),包括T细胞浸润和慢性炎症反应的病症,动脉粥样硬化(atherosclerosis),自身免疫性心肌炎(autoimmunemyocarditis),白细胞粘附缺陷(leukocyte adhesion deficiency),系统性红斑狼疮(systemic lupus erythematosus)(SLE),亚急性皮肤红斑狼疮(subacutecutaneous lupus erythematosus),盘状狼疮(discoid lupus),狼疮性脊髓炎(lupus myelitis),狼疮性脑炎(lupus cerebritis),青少年型糖尿病(juvenileonset diabetes),多发性硬化(multiple sclerosis),过敏性脑脊髓炎(allergicencephalomyelitis),视神经脊髓炎(neuromyelitis optica),风湿热(rheumaticfever),西登哈姆氏舞蹈病(Sydenham′s chorea),细胞因子和T-淋巴细胞介导的急性和迟发型超敏反应性相关免疫反应,结核病(tuberculosis),结节病(sarcoidosis),肉芽肿病(granulomatosis)(包括韦格纳肉芽肿(Wegener′sgranulomatosis)和丘-施综合征(Churg-Strauss disease)),粒细胞缺乏症(agranulocytosis),脉管炎(vasculitis)(包括超敏性血管炎/脉管炎(hypersensitivity vasculitis/angiitis),ANCA和类风湿性脉管炎(rheumatoidvasculitis)),再生障碍性贫血(aplastic anemia),戴-布贫血(DiamondBlackfan anemia),免疫性溶血性贫血(immune hemolytic anemia)(包括自身免疫溶血性贫血(autoimmune hemolytic anemia)(AIHA),恶性贫血(pernicious anemia),纯红细胞再生障碍(pure red cell aplasia)(PRCA),因子VIII缺乏(Factor VIII deficiency),血友病A(hemophilia A),自身免疫性嗜中性白血球减少症(autoimmune neutropenia),全血细胞减少症(pancytopenia),白细胞减少症(leukopenia),包括血细胞渗出(leukocyte diapedesis)的疾病,中枢神经系统(CNS)炎性疾病,多器官损伤综合征(multiple organ injurysyndrome),重症肌无力(myasthenia gravis),抗原-抗体复合体介导的疾病,抗肾小球基底膜疾病(anti-glomerular basement membrane disease),抗磷脂抗体综合征(anti-phospholipid antibody syndrome),过敏性神经炎(allergicneuritis),眼-口-生殖器三联综合征(Behcet disease),卡斯尔曼病(Castleman′s syndrome),古德帕斯丘综合征(Goodpasture′s syndrome),兰伯特-伊顿综合征(Lambert-Eaton Myasthenic Syndrome),雷诺综合征(Reynaud′s syndrome),舍格仑综合征(Sjorgen′s syndrome),斯蒂文森-约翰逊综合征(Stevens-Johnson syndrome),实体器官移植排斥,移植物抗宿主病(graft versus host disease)(GVHD),大疱性类天疱疮(bullous pemphigoid),天疱疮(pemphigus),自身免疫性多内分泌腺病(autoimmunepolyendocrinopathies),血清阴性脊柱关节病(seronegativespondyloarthropathies),莱特尔病(Reiter′s disease),僵人综合征(stiff-mansyndrome),巨细胞性动脉炎(giant cell arteritis),免疫复合物性肾炎(immune complex nephritis),IgA肾病,IgM多发性神经病或IgM介导的神经病,特发性血小板减少性紫癜(idiopathic thrombocytopenic purpura)(ITP),血栓性血小板减少性紫癜(thrombotic throbocytopenic purpura)(TTP),过敏性紫癜(Henoch-Schonlein purpura),自身免疫性血小板减少症(autoimmunethrombocytopenia),睾丸和卵巢的自身免疫病(包括自身免疫性睾丸炎和卵巢炎(autoimmune orchitis and oophoritis),原发性甲状腺机能减退(primaryhypothyroidism);自身免疫内分泌疾病,包括自身免疫甲状腺炎(autoimmune thyroiditis),慢性甲状腺炎(chronic thyroiditis)(桥本甲状腺炎(Hashimoto′s Thyroiditis)),亚急性甲状腺炎(subacute thyroiditis),特发性甲状腺机能减退(idiopathic hypothyroidism),艾迪生病(Addison′s disease),格雷夫斯病(Grave′s disease),自身免疫多腺体综合征(autoimmunepolyglandular syndromes)(或多腺体内分泌综合征(polyglandularendocrinopathy syndromes)),I型糖尿病,也称为胰岛素依赖型糖尿病(insulin-dependent diabetes mellitus)(IDDM)和席汉综合征(Sheehan′ssyndrome);自身免疫肝炎(autoimmune hepatitis),淋巴样间质性肺炎(lymphoid interstitial pneumonitis)(HIV),闭塞性细支气管炎(bronchiolitisobliterans)(非移植性)相对NSIP,吉兰-巴雷综合征(Guillain-BarréSyndrome),大血管血管炎(large vessel vasculitis)(包括风湿性多肌痛(polymyalgia rheumatica)和巨细胞性(高安)动脉炎(giant cell(Takayasu′s)arteritis),中等血管的血管炎(medium vessel vasculitis)(包括川崎病(Kawasaki′s disease)和结节性多动脉炎(polyarteritis nodosa)),结节性多动脉炎(polyarteritis nodosa)(PAN)强直性脊柱炎(ankylosing spondylitis),贝惹病(Berger′s disease)(IgA肾病(IgA nephropathy)),急进性肾小球肾炎(rapidly progressive glomerulonephritis),原发性胆汁性肝硬化(primarybiliary cirrhosis),乳糜泻(Celiac sprue)(麸质肠病(gluten enteropathy)),冷球蛋白血症(cryoglobulinemia),肝炎相关冷球蛋白血症(cryoglobulinemiaassociated with hepatitis),肌萎缩侧索硬化(amyotrophic lateral sclerosis)(ALS),冠心病(coronary artery disease),家族性地中海热(familialMediterranean fever),显微镜下多血管炎(microscopic polyangiitis),科根综合征(Cogan′s syndrome),维-奥二氏综合征(Whiskott-Aldrich syndrome)和血栓闭塞性脉管炎(thromboangiitis obliterans)。
在一个特定实施方案中,用通过本文公开的方法产生的增强亲和力的TCRs治疗受试者的方法包括患有急性髓细胞白血病(acute myelocyticleukemia),急性淋巴细胞性白血病(acute lymphocytic leukemia),或慢性粒细胞白血病(chronic myelocytic leukemia)的受试者。
感染性疾病包括与感染剂相关的那些,并且包括多种细菌中的任一种(例如,致病性大肠杆菌(E.coli),鼠伤寒沙门氏菌(S.typhimurium),铜绿假单胞菌(P.aeruginosa),炭疽芽胞杆菌(B.anthracis),肉毒杆菌(C.botulinum),艰难梭菌(C.difficile),产气荚膜梭菌(C.perfringens),H.pylori,霍乱弧菌(V.cholerae),李斯特菌属(Listeria spp.),立克次氏体属(Rickettsiaspp.),衣原体属(Chlamydia spp.),等等),分枝杆菌(mycobacteria),和寄生虫(包括任何已知的原生动物寄生成员)。感染性病毒包括真核病毒(例如,腺病毒,本雅病毒,疱疹病毒,乳头瘤病毒,副粘病毒,小核糖核酸病毒(picornavirus),棒状病毒(例如,狂犬病(Rabies)),正粘病毒(orthomyxovirus)(例如,流感),痘病毒(例如,牛痘(Vaccinia)),呼肠孤病毒(reovirus),逆转录病毒,慢病毒(例如,HIV),虫媒病毒(例如,HCV)等)。在某些实施方案中,用本发明的增强亲和力的TCRs治疗以胞质病原(其抗原被I类MHC分子加工和展示)的感染。
可以以结合细胞的形式向受试者施用增强亲和力的TCRs(即,靶细胞群体(成熟T细胞(例如,CD8+T细胞)或T细胞系的其它细胞)的基因治疗)。在一个特定实施方案中,向受试者施用的包含增强亲和力的TCRs的T细胞系细胞是自体细胞。在另一个实施方案中,增强亲和力的TCRs可以以可溶的形式施用于受试者。可溶的TCRs是本领域已知的(参见,例如,Molloy等人,2005,Curr.Opin.Pharmacol.5:438-443;美国专利#6,759,243)。
“治疗(treat)”和“治疗(treatment)”是指受试者(即,可以是人或非人哺乳动物(例如,灵长类,小鼠,大鼠)的个体)的疾病,病症,或病状的医疗管理。通常,合适的剂量和治疗方案以足以提供治疗或预防利益的量提供本文描述的增强亲和力的TCRs,和任选地佐剂。治疗盒预防利益包括改善的临床结果;与疾病相关症状的减少或缓解;减少的症状出现;改善的生活质量;更长的无病状态;疾病程度的减少,疾病状态的稳定;疾病进程的延迟;缓和;存活;或延长的存活。
可以以由医学领域的技术人员所确定的适于要治疗的(或预防的)疾病或病状的方式施用包括增强亲和力的受体的药物组合物。施用组合物的适当的剂量,合适的持续时间,和频率将由以下因素确定,如患者状况,疾病的尺寸、类型和严重度,活性成分的特定形式,和施用方法。
在进一步的实施方案中,本公开的增强亲和力的TCRs可以用于诊断方法或成像方法,包括于本文鉴定的适应症或病症施用的这些方法。
实施例
以下实施例表明,在OP9-DL1培养中,当暴露于其同源抗原时,由本公开所提供的,例如,TCR转基因胸腺细胞有效分化为“γδ样”CD4-CD8-CD24-TCRβ+系。此外,仅表达来自特异于肿瘤抗原WT1的T细胞克隆的TCRα链的祖先胸腺细胞在OP9-DL1培养中也可以分化为该成熟TCRαβ+系。从分选自这些培养物中的DN TCRαβ+细胞群体产生TCRβ链文库,并当与抗原-特异的TCRα链配对时筛选WT1 MHC四聚体反应。使用该方法,鉴定了一些TCRβ链,所述TCRβ链能够与抗原-特异的TCRα链配对以产生具有对WT1肽比原TCR至多高10-倍的亲和力的TCRs。
实施例1:在OP9-DL1细胞上的分化过程中肽激动剂的参与可以驱使成
熟TCRαβ+DN细胞从纯化自TCR转基因小鼠的T细胞祖先的分化。
在β-选择之前通过αβTCR的激动剂信号导致体内T细胞发育过程中“γδ样”双阴性(DN)TCRαβ+细胞的分化,并且DN3阶段的TCR交联导致在OP9-DL1细胞上的体外T细胞分化过程中相似系的分化。为了确定来自TCR转基因小鼠的祖先T细胞也能够在DN3阶段响应同源肽抗原分化为DN TCRαβ+,从转基因OT-1小鼠(表达对呈递到I类MHC H-2Kb上的卵清蛋白肽序列SIINFEKL(SEQ ID NO:1)特异的TCR;Stock#003831,Jackson Laboratory,ME;还参见Hogquist等人,1994,细胞76:17-27)分选TCRαβ-CD4-CD8-CD117+CD44+DN1和DN2祖先胸腺细胞并在缺少肽的情况下,或以增加的卵清蛋白-特异肽(SEQ ID NO:1)浓度,与转导以表达小鼠I类MHC分子H-2Kb的OP9-DL1细胞(Schmitt等人,2002,Immunity 17:749-756;美国专利号7,575,925)培养20天并在不同时间点通过流式细胞术分析。在缺少肽的情况下,双阳性(DP)T细胞可以在第16天被检测到,并且在第20天构成培养物的主要部分(图1A)。然而,由甚至非常低的肽浓度(0.0001μM)减少了DP T细胞的发育或存活,并且来自含有0.01μM或更多肽的培养物中DP是完全缺失的(图1A),表明DP细胞由OP9-DL1培养中的强烈的激动剂信号转导负性选择。
为了确定是否增加的强烈激动剂信号驱使TCRαβ+DN细胞发育,分析DN群体CD24和TCRβ的表达,所述CD24是在所有未成熟祖先T细胞群体上以高水平表达的成熟标记。发现大多数细胞在第5天表达高水平的CD24并且缺少TCRβ表达(图1B),但在第16天,来自所有培养条件的大多数DN细胞表达TCRβ,尽管从含有0.01μM或更多肽的培养物中观察到基本上更大量的CD24-细胞(与无肽培养物中6.9%TCR+CD24-相比,在含有0.01和1.0μM的肽的培养物中分别有38.2%和31.4%TCR+CD24-细胞)(图1B)。第20天,在含有0.01μM或1.0μM肽的培养物中,所有DN细胞中~60%为TCRβ+CD24-形式,而在未接受或接受低浓度(0.0001μM)肽的培养中,DNs中仅~20%为TCRβ+CD24-,并且接近50%为TCRβ-(图1B,1C)。此外,当在不同培养条件之间比较TCR表面表达水平时,响应高水平肽发育的TCRβ+细胞在细胞表面表达更高水平的TCRβ(图1C)。不希望受理论限制,可能一些TCRαβ+DN细胞在无添加肽情况下在培养中的发育是由于与OP9-DL1培养系统中的其它肽-MHC配体交叉反应。为了证实在这些培养物中观察到的TCRαβ+DN细胞不通过DP阶段发育,从B6或OT-1胸腺分选尚未阳性选择的CD69-DP细胞并且在存在或缺少卵清蛋白SIINFEKL肽(SEQ ID NO:1)的情况下培养。B6 DP细胞不受SIINFEKL肽(SEQ ID NO:1)的存在影响,但当在存在SIINFEKL(SEQID NO:1)的情况下,将OT-1 DP胸腺细胞培养在OP9-DL1细胞上时,观察到所有负选择的特征,包括细胞性的大量损失和共受体下调(图2)。重要的是,在这些培养物中观察到的DN细胞相同地都是TCR阴性的(图2)。
这些数据表明,在OP9-DL1细胞上分化的过程中肽激动剂的参与可以驱动成熟TCRαβ+DN细胞从纯化自TCR转基因小鼠的T细胞祖先的分化。
实施例2:转基因TCRα链与内源性TCRβ链配对以驱动OP9-DL1培养
系统中DN CD24
-
TCRαβ
+
“γδ效仿者”细胞的发育
为了确定在β-选择前仅TCRα链表达是否也应该导致表达与引入能够以超过某亲和力阈值参与OP9-DL1培养系统中的肽-MHC配体的TCRα链配对的内源性TCRβ链的DN3 T细胞祖先系转向,从B6小鼠分选CD4-CD8-CD117+CD44+DN1和DN2祖先胸腺细胞并用来自肾母细胞瘤抗原(WT1)特异的T细胞克隆3D的TCRα链转导,所述T细胞克隆3D之前被鉴定为亲和力增强的变体,分离自3Dα的CDR3区域的饱和诱变文库。所述3Dα表达构建体含有内部核糖体进入序列基序,其后是人CD2的细胞外结构域(Genbank登录号.NM_001767.3(SEQ ID NO:48)和NP_001758.2(SEQ ID NO:49)(分别是全长CD2的转录本和蛋白序列))(IRES-hCD2)作为标记转导。在存在或缺少1.0μM的I类MHC H-2Db限制的WT1肽RMFPNAPYL(SEQ ID NO:2)的情况下,将转导的祖先胸腺细胞培养达14天,并随后通过流式细胞术分析。不论在培养条件是否存在肽,hCD2阴性级分内的DN细胞几乎不含的TCRαβ+细胞。相反,来自未接受肽的培养物的hCD2阳性级分(其表达3Dα基因)含有6.8%TCRβ+细胞,并且当添加1.0μM WT1肽时,TCRαβ+细胞的数量增加至16.6%(图3A)。这些数据表明在β-选择之前,显著的TCRαβ+DN细胞群体可以从异位表达TCRα链的早期祖先胸腺细胞发育。此外,当同源肽(对于引入的TCRα链)存在时该TCRαβ+DN细胞群体增加的事实提示这些细胞的大量级分响应WT1抗原-特异的信号发育。
一并考虑,这些数据表明,TCRαβ+DN群体可能含有表达可能与引入的3Dα配对以形成对MHC-WT1肽四聚体的亲和力比原增强亲和力的受体更高,并且比可以从普通T细胞组库(repertoire)分离显著更高的TCR的TCRβ链的细胞。
因此,3Dα-转导的CD4-CD8-CD117+CD44+DN1和DN2祖先胸腺细胞在表达小鼠1类MHC H-2Db的OP9-DL1细胞上分化并且还被转导以表达WT1。在几天直到第21天收集非黏附细胞并分选hCD2+CD4-CD8-TCRβ+细胞于TRIzol试剂中(Invitrogen)(图3B)。汇集来自个别天的细胞分选物;纯化RNA,并产生cDNA。亲本3D TCR使用Vb10可变区。为了保留接触MHC的TCR CDR1和CDR2结构域,将候选TCRβ链限制于含有该可变区的那些。因此,通过使用Vβ10特异的正向引物,和Cβ2特异的反向引物的PCR分离分选的细胞群体内含有Vβ10的TCRβ链(图3C)。设计Vb10-特异的正向引物含有CACC序列,以允许定向TOPO-克隆入pENTRTM/D-载体(Invitrogen),接着使用技术转移以重组(Invitrogen)入逆转录病毒载体MigR1-attR(MigR1载体的版本(Pear等人,1998,Blood 92:3780-3792),其被修饰含有attR位点和ccdB基因以用于克隆)。MigR1-TCRβ文库用于转导PlatE逆转录病毒包装细胞(Morita等人,2000,Gene Therapy 7:1063-1066;Cell Biolabs,Inc.),从而产生逆转录病毒上清,所述逆转录病毒上清随后用于通过逆转录病毒转导58α-β-细胞,所述58α-β-细胞为缺少内源性TCRα和TCRβ链,(58-/-)的鼠T细胞系(Letourneur和Malissen,1989,Eur.J.Immunol.19:2269-74)。
滴定逆转录病毒TCRβ文库上清,并且使用转导后导致少于20%转导的细胞的稀释度以保证大多数细胞仅含有一个逆转录病毒整合。首先分选转导的细胞的GFP阳性细胞,并且随后再在也具有高MHC-WT1肽四聚体染色水平的Vβ10+细胞上分选两次(图4A)。第二次分选之后,分析细胞的具有不相关的,但对GP33是MHC H-2Db-肽四聚体特异的染色,从而评估MHC-WT1肽四聚体阳性细胞是否以不依赖于肽的方式结合MHC残基(图4A)。
在对MHC-WT1肽四聚体高的,文库-转导的58-/-细胞的第三分选之后,将分选的细胞扩增,溶解,并分离DNA。通过使用MigR1-attR载体特异引物的PCR回收逆转录病毒插入物,所述引物被设计包括来自载体的AttB克隆位点。使用两步法,首先使用重组克隆技术将插入物克隆入pDONRTM载体(Invitrogen)并随后回到MigR1-attR。从重组克隆反应中挑取个体细菌克隆并测序。序列分析>30个克隆之后,鉴定了四个最普遍的TCRβ链用于进一步分析。有趣的是,几个克隆具有与原3Dβ链共有多个保守残基的CDR3β序列(图4B)。发现所述克隆中的一个(克隆#1)与原3Dβ几乎相同,除了P108Q置换和G112S置换(图4B)。将四个候选TCRβ链通过逆转录病毒转导入3Dα+58-/-细胞并通过流式细胞术分析(图4C)。当转导入3Dα+58-/-细胞时,所有四个候选克隆结合MHC-WT1肽四聚体,尽管克隆#4以比其它的显著低的水平结合MHC-WT1肽四聚体并不再进一步分析。亲本3Dβ链之前被密码子优化,并且因此在细胞表面表达更高水平的TCR,妨碍3Dβ和分离的克隆之间四聚体染色水平的直接比较。
为了更直接地评估各个TCRβ链对MHC-WT1肽四聚体的相对亲和力,3Dα+58-/-细胞转导以3Dα,并且将各个候选TCRβ链用MHC-WT1肽四聚体的六个2倍系列稀释度染色并由产生半数最大结合的配体浓度通过非线性回归将MFI值与饱和结合曲线匹配(图5A)。发现全部三个候选TCRβ链当与3Dα配对时的表观亲和力高于亲本3Dβ,并且克隆#1具有高~10倍的亲和力(图5A)。因此,为了直接比较与克隆#1配对的3Dα相对3Dβ的四聚体染色,将克隆#1密码子优化以致原3Dβ和克隆#1之间仅有的序列差异在CDR3区域中。将两种构建体转导入58-/-细胞并且通过流式细胞术评估MHC-WT1肽四聚体染色。当克隆#1被密码子优化时,发现其如预期以比原3Dβ更高的水平结合四聚体(图5B)。
与增强抗原-特异的TCRs体外亲和力相关的一个问题是一些修饰可能仅增加受体对MHC的亲和力,而不是肽/MHC,从而增加TCR将自体反应的可能性。通过将TCRβ文库限制于共有相同的可变结构域(Vb10)的TCRβ链从而限制CDR3的可变性来降低该风险。为了确定所述候选TCRβ链中任一个赋予增加的以不依赖于肽的方式结合MHC H-2Db分子倾向,将转导的58-/-细胞用一系列MHC H-2Db四聚体(肽:WT1,GP33,E4,MESN,SQV)染色。与3Dα相比,所有三种候选TCRβ链都被MHC-WT1肽四聚体以高水平染色,类似于原3Dβ(图5C)。当用其它MHC H-2Db-肽四聚体染色时,全部三种TCRβ链对于四聚体染色均为阴性,提示对于这些受体所观察到的亲和力的增加不仅是增加的对MHC的亲和力的结果(图5C)。
实施例3:通过在早期体外人T细胞发育过程中抗原-特异的TCRα链的 异位表达产生高亲和力WT1-特异的T细胞。
在白血病细胞的表面肾母细胞瘤(WT1)抗原以异常高水平表达。筛选HLAA2/WT1-特异的T细胞克隆,获得具有高特异活性的克隆。从C4克隆分离TCRα和TCRβ链,所述C4克隆被确定具有对WT1的最高亲和力。将包含C4 TCR并赋予高水平表达的慢病毒载体进行2012年计划的TCR基因治疗临床试验。为了进一步增强C4 TCR对WT1抗原的亲和力,与具有表达C4 TCRα链的人脐带血祖细胞一起使用在之前的实施例中描述的体外分化系统。
WT1-特异的T细胞的产生:
产生在实施例1中描述的OP9-DL1细胞系的变体,所述变体表达人I类MHC分子HLA-A2(Genbank登录号.U18930.1(SEQ ID NO:50)和AAA87076.1(SEQ ID NO:51),分别为转录本和蛋白序列,)和人I类MHCβ2微球蛋白(β2M)分子(Genbank登录号.NM_004048.2(SEQ ID NO:52)和NP_004039.1(SEQ ID NO:53),分别为转录本和蛋白序列)。使用还编码作为转导标记的绿色荧光蛋白(GFP)的逆转录病毒载体,通过逆转录病毒转导将C4 TCR克隆的TCRα链稳定转导入源自脐带血的造血祖细胞。通过流式细胞术分选表达GFP的祖细胞并且在存在或缺少WT1肽RMFPNAPYL(SEQ ID NO:2)的情况下培养在OP9-DL1-A2/β2M基质细胞上。在OP9-DL1培养中人造血祖细胞容易增殖和分化至由表型CD34+CD1a+CD4+表征的人T细胞发育阶段(La Motte-Mohs等人,2005,Blood 105:1431-1439),此时它们经历在β,γ,和δ位置处的TCR基因重排(Spits,2002,Nat.Rev.Immunol.2:760-772)。假设,与其它鼠对应物一样,在TCRβ位置产生框内重排的表达TCRα的人T细胞祖先将改变两个细胞中一个的命运:表达不与转基因TCRα良好配对的,或与转基因TCRα配对,但不通过该αβTCR接受强信号的TCRβ链的那些,将响应通过前体TCR的信号转导分化至DP阶段;另一方面,产生能够与转基因TCRα配对并通过该成熟αβTCR接受足够强信号的TCRβ链的那些将接收信号以向DN TCRαβ+γδ-样系分化。因为DP细胞在无阳性选择信号的情况下仅存活~3-4天,并且因为有效阳性选择不存在于OP9-DL1培养物中,绝大多数不通过αβTCR接受激动剂信号的细胞将从培养物中排除,使由于早期αβTCR信号转导而发育的γδ-样细胞积累。
分离候选TCRβ链:
在培养的不同点,通过细胞分选收集具有DN TCRαβ+γδ-样表型的并且是WT1肽/A2 MHC-四聚体阳性的非黏附细胞。因为培养物中抗原的持续存在可以导致TCR下调,其可能降低四聚体染色至低于检测值,因此检测WT1四聚体阳性细胞可是不可能的。此外,因为这些细胞可能不表达CD8αβ,不是非CD8依赖性的高亲和力受体不可通过四聚体染色检测。因此,可能需要从培养物中出现的所有DN TCRαβ+细胞筛选TCRβ链(参见下文)。可能还需要限制候选T细胞至与由原C4 TCRβ链使用的相同Vβ片段(Vβ17)的那些,从而保留亲本C4 TCR的CDR1和CDR2 MHC接触。
细胞分选之后,通过纯化总RNA,用C-β1或C-β2引物进行全长RACE RT-PCR,和将PCR产物克隆入pENTRTM/载体(Invitrogen)克隆内源性TCRβ链,其允许定向TOPO-克隆并整合允许快速和有效地使用Invitrogen’s技术重组系统转移至逆转录病毒载体Mig-attR(MigR1的变体(Pear等人,1998,Blood 92:3780-3792),其含有用于插入目的基因的attR位点)的attL位点。将重组反应的产物电穿孔入高效大肠杆菌,并将克隆刮在一起并大量提取(maxiprepped)以产生可能WT1-反应性TCRβ链的逆转录病毒文库。
筛选高亲和力WT1-特异的TCRs:
通过将TCRβ文库转导入已转导表达C4 TCRα链的人T细胞系H9(目录#HTB-176,ATCC,Manassas,VA)(H9-C4α)鉴定能够与C4 TCRα链配对以形成高亲和力WT1-特异的TCR的TCRβ链。将转导的细胞通过流式细胞术分选高水平的MHC-WT1肽四聚体染色并且将通过PCR从分选的群体扩增逆转录病毒插入物。通过TOPO-克隆PCR产物接着序列分析鉴定候选TCRβ链。将选择的TCRβ链和亲本C4α转导入H9-C4α细胞并且将通过将转导的细胞用2倍系列稀释的PE-缀合的四聚体染色计算对MHC-WT1肽四聚体的相对亲和力(如在实施例2中描述的)。通过将各个稀释度的MFI与通过非线性回归的结合曲线和限定为获得半数最大结合的四聚体浓度的KD匹配确定亲和力值。进一步表征可以与C4 TCRα配对以产生通过MHC-肽四聚体染色比野生型C4受体更高亲和力的TCR的TCRβ链的安全性和有效性。
实施例4:使用靶向WT1的TCR基因治疗的体内小鼠模型表征候选高亲 和力TCRs的疗效和安全性。
在靶向WT1的基因治疗的HLA-A2转基因小鼠模型中测试在实施例3中所鉴定的增强亲和力的人WT1-特异的TCRs的安全性和疗效。
评估增强的TCRs的脱靶(off-target)活性:
通过测量由在WT1肽存在或缺少的情况下TCR-转导的T细胞响应一系列表达A2的靶细胞导致的细胞因子产生评估高亲和力TCRs的混杂的活化。与亲本C4 TCR相比,展现出对WT1阴性靶细胞的脱靶识别的TCRs不进入进一步研究。
体内对正常组织的增强亲和力的TCRs活性:
正常组织中的WT1表达在小鼠和人二者中类似,并且由C4 TCR识别的WT1肽在小鼠中相同并且已知被小鼠细胞加工和呈递(Gaiger等人,2000,Blood 96:1480-9)。已经使用HLA-A2转基因小鼠测试正常组织被表达人高亲和力WT1-特异的TCRs的T细胞的识别(Kuball等人,2009,J.Exp.Med.206:463-475)。
为了评价在之前实施例中公开的体外产生的增强亲和力的TCRs的安全性,将来自B6.A2/Db小鼠的CD8+T细胞(表达编码融合于Db的α3(用于结合小鼠CD8)的A2的α1和α2结构域的转基因)(Newberg等人,1996,J.Immunol.156:2473-2480),转导以表达候选增强亲和力的TCRs。在转导至含有小鼠而不是人Cα和Cβ结构域之前修饰TCRs,其增加小鼠T细胞中的表达(Pouw等人,2007,J.Gene Med.9:561-570)。将TCR-转导的T细胞转移至小鼠之后约4-6周,通过组织学分析已知天然表达WT1的组织(例如,肺和肾)中T细胞浸润和组织损伤的证据,并且通过流式细胞术评估骨髓中表达WT1的造血祖细胞的大量减少。
增强的亲和力与改善的靶点识别和功能的关联:
存在对于TCRs的亲和力阈值可以存在的证据,超过所述阈值进一步增强将不增加T细胞功能并且可能实际上降低抗原灵敏性(Schmid等人,2010,J.Immunol.184:4936-46)。因此,将高亲和力TCR-转导的CD8+T细胞对用有限肽浓度脉冲的靶细胞的反应与表达亲本C4 TCR的T细胞相比较。分析细胞因子产生(IFNγ/IL-2)和增殖,以及细胞溶解活性。呈现增加的亲和力和增强的功能的TCRs进入进一步研究和可能在TCR基因治疗试验中使用。
实施例5:体内产生高亲和力WT1-特异的T细胞。
使用体内小鼠模型(TCRα逆基因小鼠)以确定TCRβ+双阴性(DN)细胞是否可以在胸腺中发育。与转基因方法相比,逆基因(通过逆病毒转导的)小鼠允许快速产生表达特异的TCR转基因的小鼠。制备逆基因小鼠的方法是本领域已知的(参见,例如,Holst等人,2006,Nat.Protoc.1:406-417;Holst等人,2006,Nat.Methods 3:191-197;Bettini等人,2012,Immunology 136:265-272)。简而言之,从B6小鼠骨髓纯化造血祖先/干细胞并转导以表达来自高亲和力WT1特异的3D-PYY TCR或低亲和力间皮素特异的TCR 7431之一的TCRα链。3D-PYY TCR是从3D TCR改造的更高亲和力TCR,其使用T细胞展示系统和用WT1/Db Ig DimerX(BDBiosciences)的选择来鉴定(Stone等人,2011,J.Immunol.186:5193-5200;Chervin等人,2008,J.Immunol.Methods 339:175-184)。包含3D-PYYTCRα或7431α转基因的逆转录病毒构建体还包括作为转导标记的人CD2细胞外结构域,并在两个转基因之间具有IRES。将转导的源自骨髓的祖先转移入致死剂量照射的B6宿主小鼠以产生表达引入的TCRα链的骨髓嵌合体。体内转移TCRα-转导的骨髓细胞之后六周,牺牲小鼠。通过流式细胞术分析来自胸腺和脾的细胞的CD4和CD8表达(图6A,6B)。通过胸腺中的TCRβ+细胞分析CD4和CD8表达(图6A)显示可以在发育早期异位表达TCRα链的转导的胸腺细胞中体内检测到大量的双阴性TCRβ+细胞,并且该群体在表达来自高亲和力TCR的TCRα(例如,3D-PYYα)的小鼠中更明显。还分别分析来自3D-PYYα和7431α逆基因小鼠的DN TCRβ+胸腺细胞中Vβ10和Vβ9的表达(图6A)。这些数据显示对于使用相同Vβ基因片段作为原抗原特异的TCR的细胞,富集DN TCRβ+群体。结合在一起,这些数据支持这样的假想:DN TCRβ+细胞响应由与在胸腺中表达的靶抗原(即,WT1或间皮素)的同源相互作用导致的相对强的TCR信号而发育。TCRβ+逆基因脾细胞的CD4和CD8表达分析显示,这些DNTCRβ+细胞也存在于逆基因小鼠的外周(图6B)。
将来自3D-PYYα和7431α逆基因小鼠的脾细胞分别用WT1肽和间皮素肽刺激,并在IL-2的存在下体外培养6天。将IL-2添加至培养中以可能扩增抗原特异的细胞,从而它们可以通过四聚体染色被检测。通过流式细胞术,在TCRβ+门内分析培养物中CD4和CD8表达,以及亲本TCRVβ基因的表达(图7)。同样,观察亲本Vβ基因家族的富集,尤其是对于高亲和力3D-PYY。还通过用WT1或间皮素肽/MHC四聚体染色分析培养的T细胞中抗原-特异的T细胞的存在(图7)。这些数据显示,尤其对于高亲和力3D-PYYα逆基因小鼠,大量的抗原特异的T细胞存在于这些培养物中。在TCRα-转导的(hCD2+)群体内发现了四聚体阳性细胞的事实表明,这些细胞作为早期表达TCRα链的结果而发育。这表明在这些小鼠中发育的DN TCRβ+细胞实际上含有高亲和力抗原特异的T细胞。因为这些是DN细胞,它们不具有CD8的贡献以帮助四聚体结合-则这些TCRs是“CD8-不依赖的”-CD8-不依赖的四聚体结合需要高亲和力TCR。
可以将上文描述的不同实施方案组合以提供进一步实施方案。将本说明书中提及的和/或申请材料表中列出的所有美国专利,美国专利申请公开,美国专利申请,外国专利,外国专利申请和非专利出版物以其整体通过引用并入本文。如果需要,使用不同专利、申请和公开的观点,实施方案的方面可以被改进以提供更进一步实施方案。
根据上文详述的说明,可以对所述实施方案进行这些和其它改变。通常,在以下权利要求中,使用的术语应该不被认为将权利限制于说明书和权利要求中公开的特定实施方案,而应该被认为包括和此权利要求被赋予的相应物的全部范围一起的所有可能的实施方案。因此,权利要求不受公开所限制。
Claims (41)
1.一种产生增强亲和力的T细胞受体(TCR)的方法,所述方法包括:
a.在足以诱导造血祖细胞分化为DN TCRαβ+胸腺细胞的条件和时间下将造血祖细胞与基质细胞和肽抗原接触,
b.从所述DN TCRαβ+胸腺细胞分离编码不同TCRβ链的核酸序列并且将编码所述TCRβ链的核酸序列引入能够在细胞表面上表达TCR和包含编码来自步骤(a)的TCRα链的核酸序列的细胞;和
c.鉴定增强亲和力的TCR,
其中所述造血祖细胞包含编码来自特异于所述肽抗原的亲本TCR的TCRα链的非内源性核酸序列,并且
其中所述基质细胞包含编码Delta-like-1或Delta-like-4的非内源性核酸序列和编码MHC分子的核酸序列。
2.权利要求1所述的方法,其中所述TCRβ链分离自所述亲本TCR。
3.权利要求1所述的方法,其中所述造血祖细胞包括胸腺细胞祖细胞或胚胎干细胞。
4.权利要求1所述的方法,其中所述造血祖细胞包括源自骨髓或脐带血的造血干细胞。
5.权利要求1所述的方法,其中使用病毒载体将编码特异于所述肽抗原的TCRα链的非内源性核酸序列引入所述造血祖细胞。
6.权利要求5所述的方法,其中所述病毒载体是逆转录病毒载体。
7.权利要求5所述的方法,其中所述病毒载体是慢病毒载体。
8.权利要求5所述的方法,其中所述病毒载体进一步包含用于转导的基因标记。
9.权利要求8所述的方法,其中所述用于转导的基因标记包括绿色荧光蛋白或人CD2的细胞外结构域。
10.权利要求1所述的方法,其中所述基质细胞表达Delta-like-1。
11.权利要求1所述的方法,其中所述基质细胞源自OP9。
12.权利要求1所述的方法,其中所述方法进一步包括通过MHC-肽四聚体染色来选择由步骤(b)产生的能够在细胞表面上表达TCR的细胞。
13.权利要求12所述的方法,其中通过多次MHC-肽四聚体染色来选择由步骤(b)产生的能够在细胞表面上表达TCR的细胞。
14.权利要求1所述的方法,其中使用病毒载体将编码来自步骤(b)的不同TCRβ链的核酸序列引入能够在细胞表面上表达TCR的细胞。
15.权利要求14所述的方法,其中所述病毒载体是逆转录病毒载体。
16.权利要求14所述的方法,其中所述病毒载体是慢病毒载体。
17.权利要求14所述的方法,其中所述病毒载体进一步包含用于转导的基因标记。
18.权利要求17所述的方法,其中所述用于转导的基因标记包含绿色荧光蛋白。
19.权利要求1所述的方法,其中能够在细胞表面上表达TCR的细胞源自TCRα-/β-58T细胞杂交瘤。
20.权利要求1所述的方法,其中所述增强亲和力的TCR是人TCR。
21.权利要求1所述的方法,其中所述MHC分子包括I类MHC分子或II类MHC分子。
22.权利要求21所述的方法,其中所述MHC分子包括HLA-A2和人β-2-微球蛋白(β2M)。
23.权利要求1所述的方法,其中所述肽抗原选自由以下各项组成的组:病毒抗原,细菌抗原,癌抗原,和自身免疫抗原。
24.权利要求23所述的方法,其中所述肽抗原是WT1肽抗原或间皮素肽抗原。
25.权利要求24所述的方法,其中所述WT1肽抗原包含氨基酸序列RMFPNAPYL(SEQ ID NO:2)。
26.权利要求24所述的方法,其中所述间皮素肽抗原包含氨基酸序列GQKMNAQAI(SEQ ID NO:31)。
27.权利要求1所述的方法,其中将所述肽抗原添加到培养中的造血祖细胞和基质细胞。
28.权利要求1所述的方法,其中所述基质细胞包含编码所述肽抗原的核酸序列。
29.权利要求1所述的方法,其中在将选择的TCRβ链引入能够在细胞表面上表达TCR的细胞之前,从DN TCRαβ+胸腺细胞分离编码不同TCRβ链的核酸序列进一步包括选择具有与亲本TCRβ链相同的Vβ基因的TCRβ链。
30.一种增强亲和力的TCR,所述增强亲和力的TCR由权利要求1所述的方法产生。
31.一种融合蛋白,所述融合蛋白包含由权利要求1所述的方法产生的增强亲和力的TCR和细胞毒性或可检测组分。
32.一种增强亲和力的TCR,所述TCR由权利要求23或24所述的方法产生。
33.一种药物组合物,所述药物组合物包含由权利要求1所述的方法产生的增强亲和力的TCR,和药学可接受的载体,稀释剂,或赋形剂。
34.一种治疗受试者中疾病的方法,所述方法包括施用由权利要求1所述的方法产生的增强亲和力的TCR。
35.权利要求34所述的方法,其中所述疾病选自由以下各项组成的组:病毒感染,细菌感染,癌症,和自身免疫病。
36.权利要求34所述的方法,其中所述受试者是人。
37.权利要求34所述的方法,其中将所述增强的TCR以可溶的TCR施用于受试者。
38.权利要求34所述的方法,其中向所述受试者施用包含所述增强亲和力的TCR的T细胞。
39.权利要求38所述的方法,其中所述T细胞包括调节性T细胞。
40.权利要求38所述的方法,其中所述T细胞包括CD8+T细胞或CD4+T细胞。
41.权利要求38所述的方法,其中所述T细胞是自体T细胞。
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