CN107735407B - 治疗剂 - Google Patents
治疗剂 Download PDFInfo
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- CN107735407B CN107735407B CN201680039617.1A CN201680039617A CN107735407B CN 107735407 B CN107735407 B CN 107735407B CN 201680039617 A CN201680039617 A CN 201680039617A CN 107735407 B CN107735407 B CN 107735407B
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Abstract
一种免疫应答细胞,例如T细胞,该免疫应答细胞表达(i)第二代嵌合抗原受体,其包含:(a)信号传导区域;(b)共刺激信号传导区域;(c)跨膜结构域;和(d)与靶抗原上的第一表位特异性地相互作用的结合元件;以及(ii)嵌合共刺激受体,其包含(e)与(b)不同的共刺激信号传导区域;(f)跨膜结构域;和(g)与靶抗原上的第二表位特异性地相互作用的结合元件。这种排列被称为平行嵌合活化受体(pCAR)。这种类型的细胞在治疗中是有用的,并且描述和要求保护使用它们的试剂盒和方法以及用于制备它们的方法。
Description
本发明涉及编码新颖的嵌合抗原受体(CAR)的核酸,并且涉及CAR本身、掺入这些核酸的细胞及其在治疗中的用途,尤其涉及其中这些核酸用于促进对所选择的靶标的T细胞应答的方法。
背景技术
嵌合抗原受体(CAR)也可以被称为人工T细胞受体、嵌合T细胞受体(TCR)或嵌合免疫受体,是工程化受体,是本领域所熟知的。它们主要用于转化免疫效应细胞(特别是T细胞),以便为那些细胞提供特定的特异性。它们在癌症免疫疗法领域中尤其受到研究,在该领域中它们可以用于诸如过继性细胞转移等技术。在这些疗法中,将T细胞从患者中去除并进行修饰,以便它们表达对在特定癌症形式中发现的抗原具有特异性的受体。将T细胞重新引入患者中,这些T细胞然后可以识别并杀死癌细胞。
第一代CAR最通常使用CD3ζ(z)来提供TCR样信号,并因此引起杀肿瘤的功能。然而,在伴随的共刺激信号不存在的情况下,CD3z链融合受体的参与可能不足以引起实质的IL-2分泌和/或增殖。在生理学T细胞应答中,最佳的淋巴细胞激活需要一种或多种共刺激受体(信号2)如CD28或4-1BB的参与。因此,T细胞也已经被工程化,以致于它们以肿瘤抗原依赖性方式接受共刺激信号。
就这一点而言,一个重要的发展是“第二代CAR”的成功设计,该第二代CAR在人类原代T细胞中转导功能性抗原依赖性共刺激信号,从而除了杀肿瘤活性之外允许T细胞增殖。第二代CAR最通常使用来源于CD28或4-1BB的模块来提供共刺激。与其第一代对应物(单独的CD3z信号)相比,共刺激加CD3ζ信号的联合递送使得第二代CAR在功能方面明显优越。在美国专利号7,446,190中找到了第二代CAR的实例。
最近,已经准备了所谓的“第三代CAR”。这些组合了多个信号传导结构域(如CD28+4-1BB+CD3z或CD28+OX40+CD3z)以进一步增大效力。在第3代CAR中,所述信号传导结构域串联排列在CAR胞内结构域中并置于CD3z的上游。
然而通常,这些第三代CAR所取得的成果令人失望地表现为仅仅比第二代配置略有改善。
也已经建议使用用多种构建体转化的细胞。例如,Kloss等人,NatureBiotechnology[自然生物技术]2012,doi:10.1038/nbt.2459描述了用靶向第一抗原的CAR和靶向第二抗原的嵌合共刺激受体(CCR)转导T细胞,该CAR包含信号激活区域(CD3ζ链),该CCR包含CD28和4-1BB共刺激区域。这两种构建体以不同的结合亲和力与它们各自的抗原结合,并且这导致‘肿瘤感知’效应,该效应可以增强治疗的特异性以便减少副作用。
开发这样的***是令人希望的,通过这些***,T细胞可以通过经由表达抗原的肿瘤靶细胞进行的几个重复回合的刺激来维持在它们可以生长、产生细胞因子并递送杀死信号的状态。提供次优的共刺激导致T细胞在再刺激时迅速失去这些效应子功能,进入被称为“无反应性”的状态。当CAR T细胞在体外顺序地被再刺激时,它们逐渐失去效应子特性(例如IL-2产生、增殖能力)并分化以变得更像效应子-换句话说,不太可能表现出共刺激的效应。这对于癌症免疫疗法是不令人希望的,因为更多分化的细胞当在肿瘤微环境中反复被刺激时倾向于具有较短的寿命和降低的进行进一步生长/激活的能力。
发明内容
诸位申请人已经发现可以使用构建体的组合来产生有效的T细胞应答,在这些构建体的组合中多个共刺激区域排列在不同构建体中。
根据本发明的第一方面,提供了免疫应答细胞,该免疫应答细胞表达
(i)第二代嵌合抗原受体,其包含:
(a)信号传导区域;
(b)共刺激信号传导区域;
(c)跨膜结构域;和
(d)与靶抗原上的第一表位特异性地相互作用的结合元件;以及
(ii)嵌合共刺激受体,其包含
(e)与(b)不同的共刺激信号传导区域;
(f)跨膜结构域;和
(g)与靶抗原上的第二表位特异性地相互作用的结合元件。
诸位申请人已经发现该***的功效良好,并且特别地可能比使用具有类似元件的传统第三代CAR所达到的功效更好。本发明的该类型的构建体可以被称为“平行嵌合活化受体”或“pCAR”。
另外,细胞的增殖,其维持其细胞毒性效力和释放IL-2的能力,被维持了用表达抗原的肿瘤细胞进行的许多个重复回合的刺激。
不受理论束缚,pCAR中元件的排列可能正在促进活性。例如,根据定义,第三代CAR中的一个共刺激模块必须置于远离其靠近质膜的内叶的自然位置。这可能会导致它不能正常发信号,由于对专性膜相关伴侣分子的访问受损。可替代地,在第三代CAR中2个共刺激信号传导模块的紧密接近可能导致空间问题,阻止了一个或多个下游信号传导途径的完全参与。这两个问题在本发明的排列中都被避免了。信号传导部分(b)和(e)可以与跨膜结构域直接融合,确保它们都与细胞内的质膜相邻。此外,它们可以在细胞内的不同位点间隔开,以致于不会彼此在空间上相互作用。
在本发明的第一方面中使用的合适的免疫应答细胞包括T细胞,如细胞毒性T细胞、辅助T细胞或调节T细胞以及自然杀伤(NK)细胞。特别地,该免疫应答细胞是T细胞。
以上合适的元件(a)可以包括任何合适的信号传导区域,包括包含基于免疫受体酪氨酸的活化基序(ITAM)的任何区域,如例如由Love等人,Cold Spring HarborPerspect.Biol[冷泉港实验室生物学展望]2010 2(6)la002485所综述的。在具体的实施例中,该信号传导区域包含如例如在美国专利号7,446,190中所描述的人CD3[ζ]链的胞内结构域或其变体。
特别地,这包含跨越全长人CD3ζ链的氨基酸残基52-163的结构域。它具有许多多态性形式(例如,序列ID:gb|AAF34793.1和gb|AAA60394.1),其分别如SEQ ID NO 1和2所示:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO 1)
RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(SEQ ID NO 2)
如本文所使用的,术语“变体”是指多肽序列,该多肽序列是基本序列的天然存在的多态性形式以及合成的变体,其中链内的一个或多个氨基酸被***、去除或替换。然而,该变体产生与基本序列类似的生物学效应。例如,上述变体将以与人CD3[ζ]链的胞内结构域类似的方式起作用。氨基酸取代可以被认为是“保守的”,其中氨基酸被具有广泛相似性质的同一类别中的不同氨基酸替换。非保守取代是其中氨基酸被不同类型或类别的氨基酸替换。
将氨基酸类别定义如下:
如本领域技术人员所熟知的,通过保守取代改变肽的一级结构可能不会显著改变该肽的活性,因为***序列中的氨基酸的侧链可以能够形成与已经被取代掉的氨基酸的侧链类似的键和接触。即使当取代处于决定该肽的构象的关键的区域时,也是如此。
非保守取代也可以是可能的,条件是它们不妨碍如上所描述的多肽的功能。广义而言,在不改变多肽的生物学活性的情况下,可能将存在更少的非保守取代。
一般而言,变体将具有与基本序列(例如SEQ ID NO 1或SEQ ID NO 2)是至少70%,例如至少71%、75%、79%、81%、84%、87%、90%、93%、95%、96%或98%同一的氨基酸序列。在该上下文中的同一性可以用SEQ ID NO 2或片段(特别是如下所描述的片段)作为基本序列使用BLASTP计算机程序来确定。在以下网站可公开获得BLAST软件:http://blast.ncbi.nlm.nih.gov/Blast.cgi(于2009年3月12日访问)。
共刺激信号序列(b)合适地位于跨膜结构域(c)和信号传导区域(a)之间并且远离结合元件(d)。类似地,共刺激信号序列(e)合适地位于跨膜结构域(f)附近并且远离结合元件(g)。
用作以上元件(b)和(e)的合适的共刺激信号传导区域在本领域中也是熟知的,并且包括B7/CD28家族的成员,例如B7-1、B7-2、B7-H1、B7-H2、B7-H3、B7-H4、B7-H6、B7-H7、BTLA、CD28、CTLA-4、Gi24、ICOS、PD-1、PD-L2或PDCD6;或ILT/CD85家族蛋白,例如LILRA3、LILRA4、LILRB1、LILRB2、LILRB3或LILRB4;或肿瘤坏死因子(TNF)超家族成员,例如4-1BB、BAFF、BAFF R、CD27、CD30、CD40、DR3、GITR、HVEM、LIGHT、淋巴毒素-α、OX40、RELT、TACI、TL1A、TNF-α或TNF RII;或SLAM家族的成员,例如2B4、BLAME、CD2、CD2F-10、CD48、CD58、CD84、CD229、CRACC、NTB-A或SLAM;或TIM家族的成员,例如TIM-1、TIM-3或TIM-4;或其他共刺激分子,例如CD7、CD96、CD160、CD200、CD300a、CRTAM、DAP12、Dectin-1、DPPIV、EphB6、整合素α4β1、整合素α4β7/LPAM-1、LAG-3或TSLP R。
共刺激信号传导区域的选择可以取决于转化细胞的预期的特定用途来选择。特别地,针对以上(b)和(e)所选择的共刺激信号传导区域是可以共同合作或一起协同起作用的那些。例如,(b)和(e)的共刺激信号传导区域可以选自CD28、CD27、ICOS、4-1BB、OX40、CD30、GITR、HVEM、DR3或CD40。
在具体的实施例中,(b)或(e)中的一个是CD28并且另一个是4-1BB或OX40。
在具体的实施例中,(b)是CD28。
在另一个具体的实施例中,(e)是4-1BB或OX40并且特别地是4-1BB。在另一个实施例中,(e)是CD27。
以上(c)和(f)的跨膜结构域可以是相同或不同的,但特别地是不同的以确保构建体在细胞表面上分离。选择不同的跨膜结构域还可以增强载体的稳定性,因为在病毒载体中包含直接重复核酸序列使得其倾向于重排,伴随在正向重复之间的序列缺失。然而,在(c)和(f)的跨膜结构域是相同的情况下,可以通过修饰或“摆动(wobbling)”所选择的编码相同蛋白质序列的密码子来降低这种风险。
合适的跨膜结构域是本领域已知的,并且包括例如CD8α、CD28、CD4或CD3z跨膜结构域。
在共刺激信号传导区域包含如上所描述的CD28的情况下,该CD28跨膜结构域代表合适的选择。全长CD28蛋白质是SEQ ID NO 3的220个氨基酸的蛋白质
MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO 3)
其中跨膜结构域以粗体显示。
特别地,共刺激信号传导区域之一是基于铰链区,并且合适地还基于CD28的跨膜结构域和胞内结构域。特别地,其包含SEQ ID NO 3的氨基酸114-220,如下面SEQ ID NO 4所示。
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO 4)
在具体的实施例中,以上共刺激信号传导区域(b)或(e)中的一个是SEQ ID NO 4的修饰形式,其包含SEQ ID No 5的c-myc标签。
该c-myc标签是熟知的并且属于SEQ ID NO 5
EQKLISEEDL(SEQ ID NO 5)。
可以将该c-myc标签通过***胞外结构域或通过替换胞外结构域中的区域来添加到共刺激信号传导区域(b)或(e)中,该c-myc标签因此位于SEQ ID NO 3的氨基酸1-152的区域内。
在具体的优选实施例中,该c-myc标签替换CD28序列中的MYPPPY基序。这个基序代表潜在危险的序列。它负责CD28及其天然配体CD80和CD86之间的相互作用,以致于当CAR T细胞遇到表达这些配体之一的靶细胞时其提供脱靶毒性的可能性。通过用如上所描述的标签序列替换该基序,降低了不需要的副作用的可能性。
因此在具体的实施例中,该构建体的共刺激信号传导区域(b)是SEQ ID NO 6
IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO 6)
此外,包含c-myc表位意味着促进了使用单克隆抗体检测CAR T细胞。这是非常有用的,因为当使用一些可用的抗体时已经证明流式细胞术检测不可靠。
另外,提供c-myc表位标签可以促进靶向CAR T细胞的抗原独立扩增,例如通过使用适当的单克隆抗体在溶液中或固定在固相(例如,一个袋子)上对CAR进行交联来促进。
此外,在TCR的可变区内抗人c-myc抗体(9e10)的表位的表达先前已显示足以使抗体介导的和补体介导的细胞毒性在体外和体内都成为可能(Kieback等人(2008)Proc.Natl.Acad.Sci.USA[美国科学院院刊],105(2)623-8)。因此,提供这样的表位标签还可以用作“******(suicide system)”,借此在毒性事件下可以使用抗体来耗尽CAR T细胞。
结合元件(d)和(g)将是不同的并且将结合相同的、重叠的或不同的表位。在具体的实施例中,第一表位和第二表位与相同的受体或抗原缔合。因此,如上所描述的第一表位和第二表位在一些情况下可以是相同的或重叠的,以致于结合元件(d)和(g)将在它们的结合中竞争。可替代地,取决于所设想的特定疗法,第一表位和第二表位可以是不同的并且与相同或不同的抗原缔合。在一个实施例中,抗原是不同的,但可以与相同的疾病例如相同的特定癌症相关。
如本文所使用的,术语“抗原”是指将与结合元件结合的特异性结合对的任何成员。因此该术语包括靶细胞上的受体。
因此,合适的结合元件(d)和(g)可以是为pCAR提供识别感兴趣的靶标的能力的任何元件。本发明的pCAR针对的靶标可以是期望诱导T细胞应答的临床感兴趣的任何靶标。这将包括与各种类型的癌症相关的标记,包括例如一种或多种ErbB受体或αvβ6整合素,与***癌(例如,使用结合***特异性膜抗原(PSMA)的结合元件)、乳腺癌(例如,使用靶向Her-2(也称为ErbB2)的结合元件)和成神经细胞瘤(例如,使用靶向GD2的结合元件)、黑色素瘤、小细胞或非小细胞肺癌、肉瘤以及脑肿瘤相关的标记。在具体的实施例中,靶标是如上所描述的一种或多种ErbB二聚体或者集落刺激因子-1的受体(CSF-1R)或αvβ6整合素,所有这些都涉及几种实体瘤。
本发明的pCAR中使用的结合元件可以包含识别所选靶标的抗体。为了方便,用作结合元件的抗体优选是来自骆驼科动物、人类或其他物种的单链抗体(scFv)或单结构域抗体。单链抗体可以克隆自对所希望的靶标特异的杂交瘤的V区基因。这样的杂交瘤的产生已经变得常规,并且在此将不重复该程序。在Orlandi等人,Proc.Natl Acad.Sci.(USA)[美国科学院院刊]86:3833-3837(1989)中已经描述了可以用于克隆可变区重链(VH)和可变区轻链(VL)的技术。简言之,从杂交瘤细胞系中分离mRNA,并且例如使用逆转录酶聚合酶链式反应(RT-PCR)试剂盒将其逆转录成互补DNA(cDNA)。使用与VH和VL基因序列对应的序列特异性引物。克隆产物的序列分析以及与VH和VL基因的已知序列的比较可以用于显示克隆的VH基因符合预期。然后将VH和VL基因连接在一起,例如使用编码(gly4-ser)3接头的寡核苷酸。
可替代地,pCAR的结合元件可以包含配体,例如T1E肽(结合ErbB同源二聚体和异源二聚体)、集落刺激因子-1(CSF-1)或IL-34(均结合CSF-1受体)。T1E肽是由整个的成熟的人EGF蛋白质构成的嵌合融合蛋白,不包括五个最N末端氨基酸(促表皮生长因子前体(NP_001954.2)的氨基酸971-975),这五个最N末端氨基酸被成熟的人TGF-α蛋白的七个最N末端氨基酸(促转化生长因子α亚型1(NP_003227.1)的氨基酸40-46)替换。
在另一个实施例中,pCAR的结合元件包含αvβ6整合素特异性结合剂。现在整合素αVβ6被认为是癌症的靶标,因为已经发现其在许多类型的癌症中被强烈地上调。通过经由病毒衣壳蛋白VP1中的RGD基序的结合,αvβ6已经被鉴定为体外***病毒(FMDV)的受体。因此,如例如在美国专利号8,383,593中所描述的,源自FMDV的一系列肽,并且特别是起源于FMDV的VP1蛋白并包含RGD基序的肽显示增加的结合效力和结合特异性。特别地,这些肽包含序列基序
RGDLX5X6L(SEQ ID NO 7)或
RGDLX5X6I(SEQ ID NO 8),
其中LX5X6L或LX5X6I包含在α螺旋结构内,其中X5和X6是促螺旋残基,它们在[α]螺旋的中间被发现具有大于1.0的构象偏好(来自Creighton,1993以及Pace C.N.和ScholtzJ.M.(1998),Biophysical Journal[生物物理学杂志],第75卷,第422-427页)。特别地,此类残基独立地选自下组,该组由以下各项组成:Glu、Ala、Leu、Met、Gln、Lys、Arg、Val、Ile、Trp、Phe和Asp。
此类序列的具体实例包括SEQ ID No 9-11或其变体:
YTASARGDLAHLTTTHARHL(SEQ ID NO 9)
GFTTGRRGDLATIHGMNRPF(SEQ ID NO 10)
或
NAVPNLRGDLQVLAQKVART(SEQ ID NO 11)
这些肽可以形成用于本申请的CAR的结合元件的特定的组。
对于所选的恶性肿瘤例如霍奇金淋巴瘤和一些乳腺癌,两种天然配体是CSF-1和IL-34,并且这些形成特别适合于(d)和(g)的结合元件。然而,它们的确以不同的亲和力结合。结合的亲和力可以影响观察到的活性。在这种情况下确保将具有较低结合亲和力的结合元件用作结合元件(d)并将具有较高结合亲和力的结合元件用作结合元件(g)可能是有益的。特别地,在一个实施例中,第二代CAR(i)对其同源靶标的相对亲和力低于配对的基于TNFR的嵌合共刺激受体(ii)的相对亲和力。这并不妨碍在每个位置使用高或低亲和力靶向部分,其条件是维持这种相对亲和关系。因此在本发明的情况下,在具体的实施例中,结合元件(d)是具有相对低的结合亲和力的CSF-1,而结合元件(g)包含具有较高的结合亲和力的IL-34。
所述结合元件适合与促进在细胞表面上进行表达的前导序列缔合。许多前导序列是本领域已知的,并且这些包括巨噬细胞集落刺激因子受体(FMS)前导序列或CD124前导序列。
在另外的实施例中,表达pCAR的细胞被工程化以共表达嵌合细胞因子受体,特别是4αβ嵌合细胞因子受体。在4αβ中,IL-4受体-α链的胞外结构域与IL-2/15受体-β的跨膜结构域和胞内结构域连接。这允许通过在合适的支持培养基中培养这些细胞来离体选择性扩增和富集基因工程化的T细胞,该支持培养基在4αβ的情况下将包含IL-4作为唯一的细胞因子支持物。类似地,该***可以与嵌合细胞因子受体一起使用,在该嵌合细胞因子受体中该IL-4受体-α链的胞外结构域与另一受体的跨膜结构域和胞内结构域连接,所述另一受体天然地被还结合共同的γ链的细胞因子结合。
如所讨论的,这些细胞可用于治疗以刺激对靶细胞群体的T细胞介导的免疫应答。因此,本发明的第二方面提供了一种用于刺激在对其有需要的患者中的靶细胞群体的T细胞介导的免疫应答的方法,所述方法包括向该患者给予如上所描述的免疫应答细胞群体,其中所述结合元件(d)和(g)对该靶细胞具有特异性。
在本发明的第三方面,提供了一种用于制备根据前述权利要求中任一项所述的免疫应答细胞的方法,所述方法包括用编码如上所定义的结构(i)的CAR的第一核酸转导细胞;并且用编码如上所定义的结构(ii)的CAR的第二核酸转导细胞。
特别地,在该方法中,将来自患者的淋巴细胞用编码(i)和(ii)的CAR的核酸进行转导。特别地,例如通过逆转录病毒介导的转导将T细胞进行遗传修饰以将CAR编码核酸引入宿主T细胞基因组中,由此允许稳定的CAR表达。然后可以任选地在扩增之后将它们重新引入患者,以提供有益的治疗效果。在细胞(例如T细胞)被工程化以共表达嵌合细胞因子受体例如4αβ的情况下,扩增步骤可以包括在包含细胞因子的培养基(例如在4αβ的情况下包含IL-4作为唯一细胞因子支持物的培养基)中的离体培养步骤。可替代地,该嵌合细胞因子受体可以包含IL-4受体-α链的胞外结构域,该胞外结构域与具有不同性质的常见γ细胞因子例如IL-7所使用的胞内结构域连接。在这种设置下,细胞在IL-4中的扩增可能导致较少的细胞分化,这利用IL-7的天然能力来实现这种效果。以这种方式,可以确保具有所希望的分化状态的基因工程化的T细胞的选择性扩增和富集。
在本发明的第四方面,提供了编码上述(i)的CAR的第一核酸与编码上述(ii)的CCR的第二核酸的组合。如先前所指出的,这一组合被称为pCAR。所述核酸的合适序列对于本领域技术人员来说将是显而易见的。所述序列可以被优化用于所需的免疫应答细胞中。然而,在一些情况下,如上所讨论的,为了避免重复序列,密码子可以从最佳发生变化或是“摆动的”。此类核酸的具体实例将编码以上所描述的优选实施例。
为了实现转导,将本发明的第四方面的核酸适当地引入载体例如质粒或逆转录病毒载体中。此类载体(包括质粒载体)或含有它们的细胞系构成本发明的另外的方面。
第一和第二核酸或含有它们的载体可以组合在试剂盒中,提供该试剂盒的目的在于原位产生本发明的第一方面的免疫应答细胞。
由以上所描述的核酸编码的平行嵌合活化受体(pCAR)构成本发明的另外的方面。
具体实施方式
现在将通过举例的方式并参考以下附图具体描述本发明,其中:
图1是显示体现本发明的一组CAR和pCAR(命名为C34B和34CB)的示意图。所有CAR和pCAR在具有4αβ(一种嵌合细胞因子受体)的SFG逆转录病毒载体中共表达,在所述嵌合细胞因子受体中该IL-4受体-α胞外结构域已经融合至IL-2受体-β的跨膜结构域和胞内结构域。4αβ的使用允许通过在IL-4(因为它募集伽马c(γc)链)中培养来选择性富集并扩增基因修饰的T细胞。
图2显示使用图1中所示的CAR的实验的结果。将表达这些CAR和pCAR的T细胞(1×106个细胞)(或未转导的(UT)作为对照)与T47D肿瘤细胞在体外共同培养24小时,所述T47D肿瘤细胞表达(T47D-FMS)或缺乏(T47D)同源靶抗原(由c-fms编码的集落刺激因子-1受体(CSF-1R))。然后通过MTT测定来定量剩余的活肿瘤细胞。
图3显示将表达图1的CAR和pCAR的T细胞(或未转导(untrans或untransduced)的T细胞作为对照)在不存在外源性细胞因子的情况下经历连续回合的Ag刺激的代表性实验。通过每周在T47D FMS单层上进行培养来提供刺激,并按指定的时间间隔计算T细胞数目。
图4显示来自与图3中所示相似的7个重复实验的合并数据,这表明在每个刺激周期后一周中发生的CAR T细胞的扩增倍增。
图5显示在图3所示的实验中的刺激周期2、6、9和12的时候进行的说明性细胞毒性测定。这是在每个再刺激周期的时间之后24小时测试T细胞杀死T47D FMS和未修饰的T47D单层(MTT测定)的能力而得出的。
图6显示通过ELISA针对IL-2和IFN-γ含量而测试在每个刺激周期之后的一天从培养物中去除的上清液的结果。
图7表明表达CSF-1R的间变性大细胞淋巴瘤的体内异种移植模型的建立,其允许随后测试CAR和pCAR工程化的T细胞的抗肿瘤活性。使用被工程化以表达萤火虫萤光素酶(luc)和红色荧光蛋白(RFP)的K299细胞来建立该模型。图7A显示静脉注射所示剂量的K299luc细胞后、通过生物发光成像(BLI)进行定量的肿瘤形成。图7B中显示在接受200万个肿瘤细胞的小鼠中的代表性BLI图像。显示了RFP+肿瘤细胞在所示组织中的表达(图7C),这表明在该模型中仅在***中形成肿瘤。图7D中显示CSF-1R在五个代表性***肿瘤上的表达。
图8显示治疗性研究的结果,在所述治疗性研究中将K299 luc细胞静脉注射到SCID Beige小鼠中(每组n=9,分成2个单独的实验)。5天后,用CAR T细胞处理小鼠。图8A中显示来自肿瘤的合并的生物发光发射。图8B中显示来自个体小鼠的生物发光发射并且图8C中显示小鼠的存活。
图9显示随着时间的推移在治疗研究中使用的动物的重量。
图10-13显示对来自本发明的表达双重CAR(C34B)的T细胞的“耗尽标志物”的表达的分析结果,其中图10显示PD1分析的结果,图11显示TIM3分析的结果,图12显示LAG3分析的结果并且图13显示2B4分析的结果。
图14是靶向整合素αvβ6的一组CAR和构建体的示意图,这些CAR和构建体已经被制备以包括体现本发明的pCAR(命名为SFG T1E-41BB/A20-28z)。A20-28z是使用源自***病毒的A20肽进行靶向的第二代CAR。A20以高亲和力与αvβ6结合,并且以85-1000倍较低的亲和力与其他结合RGD的整合素结合。C20-28z是一种匹配的对照,在所述C20-28z中A20的关键元件已被突变以废除整合素结合活性。如图1所描述的,将所有CAR都与4αβ,进行共表达。
图15是通过流式细胞术获得的一系列直方图,说明整合素在A375puro和Panc1细胞中表达。将细胞用抗β6(百健艾迪公司(Biogen Idec))染色,然后用第二抗小鼠PE、抗αvβ3或抗αvβ5(两种都是APC轭合的,生物技术公司(Bio-Techne))染色。根据单独的第二抗体或同种型对照来设定门控。
图16是说明靶向αvβ6的CAR(包括本发明的pCAR)的细胞毒性的一系列图。将表达所示CAR和pCAR的T细胞与αvβ6阴性肿瘤细胞(Panc1和A375 puro)或αvβ6阳性肿瘤细胞(Bxpc3和A375 puroβ6)进行共培养。数据显示2-7个独立实验的平均值±_SEM,每个实验一式三份地进行。*p<0.05;**p<0.01;***p<0.001。
图17是显示通过靶向αvβ6的CAR(包括本发明的pCAR)产生IFN-γ的一系列图。将表达所示CAR和pCAR的T细胞与αvβ6阴性肿瘤细胞(Panc1和A375 puro)或αvβ6阳性肿瘤细胞(Bxpc3和A375 puroβ6)进行共培养。数据显示5-6个独立实验的平均值±_SEM,每个实验一式两份地进行。*p<0.05;**p<0.01;***p<0.001;ns–不显著。
图18显示再刺激实验的结果,所述再刺激实验使用以上所描述的CAR和pCAR工程化的T细胞并指示A20-28z/T1E-41BB pCAR T细胞经受反复抗原刺激的能力,伴随着T细胞的扩增以及表达αvβ6整合素的靶细胞(Bxpc3)或不表达αvβ6整合素的靶细胞(Panc1)的破坏。
图19显示使用pCAR工程化的T细胞的再刺激实验的结果,在所述再刺激实验中将A20-28z与T1E-41BB、T1E-CD27或T1E-CD40进行共表达,允许通过TNF受体家族的另外的成员对共刺激进行比较评价。对照T细胞是非转导的(NT),而CAR包含截短的(tr)胞内结构域。在表达αvβ6整合素的靶细胞(Bxpc3)或不表达αvβ6整合素的靶细胞(Panc1)上再刺激T细胞,与未刺激的T细胞进行比较。在Bxpc3细胞的情况下,观察到A20-28z/T1E-CD27 T细胞的优良扩增(图19A),伴随着持续的细胞毒性活性(图19B)。相比之下,在Panc1细胞的情况下,观察到A20-28z/T1E-CD27 T细胞的优良扩增(图19A),伴随着持续的细胞毒性活性(图19B)。这些数据表明,TNF受体家族的另外的成员也可以使用pCAR形式递送共刺激。
实例1
制备靶向在霍奇金淋巴瘤、间变性大细胞淋巴瘤和一些实体瘤例如三阴性乳腺癌中过度表达的CSF-1受体(由c-FMS编码)的一组CAR,并在图1中进行示意性地说明。该组CAR包括具有两种天然配体CSF-1或IL-34中的任一种作为靶向部分的第二代CAR和第三代CAR。尽管CSF-1和IL-34都与CSF-1受体结合,但IL-34以高得多的亲和力结合(比CSF-1高34倍)。
将构建体SFG C28ζ和SFG CTr作为NcoI/XhoI片段克隆到SFG逆转录病毒载体中,确保它们的起始密码子在先前被缺失的env基因占据的天然存在的NcoI位点处。基因表达由莫洛尼鼠白血病病毒(MoMLV)长末端重复(LTR)(其具有启动子活性)实现,并且通过MoMLVψ包装信号确保RNA的病毒包装,所述MoMLVψ包装信号侧翼是剪接供体和受***点。
使用聚合酶不完全引物延伸(PIPE)克隆方法设计和克隆所有其他构建体。PIPE克隆方法是常规限制性内切酶和连接依赖性克隆方法的基于PCR的替代方法。它消除了对并入限制性位点的需求,所述限制性位点可能将另外的不需要的残基编码到表达的蛋白质中。PIPE方法依赖于PCR反应的最后循环中扩增过程的低效率(可能是由于dNTP的可用性降低),这导致产生具有突出的5’端的部分单链的(PIPE)PCR产物。将一组载体特异性引物用于PCR载体线性化,并且然后将具有5’-载体-端重叠序列的另一组引物用于***物扩增,通过PIPE产生不完全延伸产物。在接下来的步骤中,将PIPE产物混合,并且将单链重叠序列进行退火并组装成完整的SFG CAR构建体。通过诊断的限制性消化确认成功的克隆。对所有构建体进行DNA测序以确认存在预测的编码序列而没有任何PCR诱导的突变(英国的源生物科学公司(Source Bioscience,UK))。
该组包括两个“双靶向的”嵌合活化受体(pCAR),其中CSF-1或IL-34与28z和4-1BB偶联,或反之亦然。然后使用含有Thosea Asigna(T)2A的逆转录病毒载体,将双靶向的pCAR组合在相同T细胞群体上进行化学计量共表达。这些CAR中的一种被命名为“C34B”(CSF1-28z加IL34-41BB),并且另一种被命名为“34CB”(IL34-28z加CSF1-41BB)。
在这些双靶向的CAR T细胞中,将两种共刺激基序(CD28/4-1BB)置于其靠近膜的自然位置,彼此物理地分离,并在相同的T细胞中共表达。
使用载体中的另外的T2A元件,将所有CAR都与IL-4响应性4αβ受体进行共表达。这使得能够使用IL-4富集/扩增T细胞,使选择后比较这些不同细胞群体的功能更容易。
实验的主要焦点是测试T细胞对于用表达或缺乏FMS/CSF-1受体靶标的肿瘤靶细胞进行重复再刺激的行为。在每个循环中,将100万个所示的IL-4扩增的CAR T细胞悬浮于RPMI+人AB血清中并用表达抗原的靶标(T47D FMS)或无抗原的靶标(T47D)的成片单层(24孔盘)进行培养。
此后,如果CAR T细胞持续并破坏该单层,则每周将100万个T细胞除去并以相同的方式再刺激。推测在每个时间点的总细胞数,这取决于在每个每周一次的周期中发生的T细胞的扩增。
贯穿所有这些实验,将T细胞在不存在任何外源性细胞因子例如IL-2或IL-4的情况下进行培养,所以它们必须制造它们自己的细胞因子以持续并扩增。通过ELISA在从T细胞/肿瘤细胞共培养物收获的上清液中测量细胞因子(IFN-γ和IL-2)产生,这提供了有效共刺激的第二标记。
据发现(图2),当它们首次暴露于表达靶标(FMS编码的CSF-1受体)的肿瘤单层时,所有被预测杀死的CAR都是这样(来自12个实验的合并数据)。对照是UT(未转导的)、P4(靶向无关抗原PSMA)和其中胞内结构域被截短的CT4。正如预期的,没有一个CAR T细胞杀死缺乏CSF-1受体的肿瘤细胞(T47D)。
图3中显示代表性再刺激实验。图4中显示来自7个实验的合并的再刺激数据。在这种情况下,尽管IL-34靶向的第二代和第三代构建体较差,但第一个周期的增殖对于大多数构建体是相似的。这可能是因为IL-34靶向部分的亲和力太高。
然而,在后来的周期中,C34B双pCAR组合(与IL-34靶向的4-1BB共刺激基序共表达的CSF-1靶向的28z第二代CAR)一致地显现出明显优越性。
在图3所示的实验中,在每个再刺激周期的时间之后24小时收集上清液,并通过ELISA对其分析细胞因子含量(IFN-γ和IL-2)。通过MTT测定来测量剩余肿瘤细胞活力的百分比(图5中所示的代表性实例)。细胞因子产生结果示于图6中。据发现,只有C34B CAR T细胞在贯穿每个刺激周期中都保留了制造IL-2的能力。在第一周期之后,所有其他CAR组合都失去了该能力。通过递归再刺激而持续保留制造IL-2的能力通常不可见于CAR T细胞,并且这表明这种双重共刺激的递送从根本上改变了这些细胞在体外的分化,延缓了无反应性的发作。
还监测在连续Ag刺激周期上单层破坏后活T细胞的数量,并且结果示于图5中。再刺激的第二个周期后,除C34B外的所有CAR都开始丧失实现CSF-1R依赖性肿瘤细胞杀死的能力。相比之下,表达C34B的T细胞在这种细胞毒性测定中保持抗原依赖性效力长达13个再刺激的迭代周期,但从未引起针对未修饰的T47D细胞的细胞毒性。
此外,还通过流式细胞术测量了这些T细胞上的所谓“耗尽标志物”(PD1、TIM3、2B4和LAG3)。结果示于图10-13中。正如预期的,表达各种耗尽标志物的T细胞的百分比在再刺激的T细胞上逐渐增加,但是这不阻碍C34B细胞在抗原刺激时的增殖、肿瘤细胞破坏或细胞因子释放。这表明C34B的优越功能不是耗尽标志物的延迟上调的结果。
总之,本发明的pCAR方法似乎将细胞维持在这样一种状态:借此状态这些细胞通过更多个再刺激周期而保持对抗原的应答性。有迹象表明,它可以阻止超出控制记忆状态的分化,并且似乎延迟了无反应性的发作,同时保持细胞在活化时制造IL-2的能力。
实例2
体内效果分析
使用其中CSF-1受体靶标以低水平表达并且其中疾病在整个***中传播的高侵袭性体内异种移植物模型,对以上实例1中使用的一组CAR进行抗肿瘤活性测试(图7)。用萤火虫萤光素酶标记肿瘤细胞,这允许非侵入性监测疾病负担。
将SCID/Beige小鼠随机分为6组(每组9只动物,通过两个独立实验组合),并静脉内(IV)接种重悬于200μL PBS中的2x106个K299肿瘤细胞。在第5天,将各组用以下所示的治疗方案之一进行处理:
·C4B组:20x106个C4B T细胞IV
·C34B组:20x106个C34B T细胞IV
·43428Bz:20x106个43428Bz T细胞IV
·34CB组:20x106个34CB T细胞IV
·UT(未转导)组:20x106个未转导的T细胞IV
·NT(未处理)组:200μL PBS IV
在研究期间的适当时间点使用生物发光成像(BLI)监测肿瘤生长。
结果示于图8中。再次,通过较低的平均BLI发射(图8A-B)、延迟的肿瘤进展或肿瘤回归导致小鼠存活延长所示(图8C),表现最好的***是pCAR、C34B的***。
在整个实验中称重动物,并且没有观察到显著的毒性(图9)。
实例3
选择靶向部分来工程化以αvβ6-依赖性方式引发T细胞活化的pCAR。
制备单独地或与扩展的ErbB家族一起靶向αvβ6整合素的一组CAR,并且示意性地示于图14中。在这种情况下使用的结合元件是源自***病毒(血清型01BFS)(US 8,927,501)的衣壳蛋白VP1的GH环的A20肽(SEQ ID NO 11)。将其置于CD124信号肽的下游并融合到CD28和CD3ζ胞内结构域以形成第二代CAR,即A20-28ζ。制备对照(C20-28ζ),其包含类似的构建体,但是具有乱序的靶向肽(命名为C20),在该靶向肽中关键的RGDL基序被AAAA替换。第二个对照包含与CD28截短的胞内结构域(A20-Tr)融合的A20。
为了产生本发明的pCAR(命名为T1E-41BB/A20-28z),将A20-28z与嵌合共刺激受体进行共表达,所述嵌合共刺激受体包含与CD8α跨膜结构域和41BB胞内结构域融合的pan-ErbB靶向肽(T1E)。
在所示的情况下,将CAR与4αβ嵌合细胞因子受体进行共表达,以允许体外IL-4介导的富集。使用Thosea Asigna(T)2A核糖体跳跃肽(skip peptide)实现IL-4响应性4αβ嵌合细胞因子受体的等摩尔共表达,其中将IL-4受体α胞外结构域融合到共享的IL-2/15受体β的跨膜结构域和胞内结构域。这些嵌合分子通过逆转录病毒基因转移而在人T细胞中表达。
使用流式细胞术评估癌细胞系A375的整合素表达模式(图15),并将它们分成αvβ6阴性肿瘤细胞(Panc1和A375 puro)或αvβ6阳性肿瘤细胞(Bxpc3和A375 puroβ6)。将这些细胞与CAR T细胞以1:1的效应物:靶标比例进行共培养24、28或72小时,在这之后,将细胞毒性通过MTT测定来评估并相对于未处理的肿瘤细胞来表达。结果示出于图16中。
这些数据显示,A20-28z CAR T细胞杀死表达αvβ6整合素的所有靶细胞(Bxpc3和A375β6puro),但饶恕了缺乏这种整合素的靶标(Panc1和A375 puro)。其次,对照CAR C20-28z和A20-Tr在这些测定中是无活性的。第三,表达T1E-41BB/A20-28z pCAR的T细胞引起表达αvβ6整合素的靶细胞(Bxpc3和A375β6puro)的有效杀死。所有这些结果都如预期的那样。然而,值得注意的是,表达T1E-41BB/A20-28z pCAR的T细胞也引起了缺乏αvβ6的靶细胞(Panc1和A375 puro)的杀死。这表明,在pCAR构型内,A20肽以低亲和力结合非αvβ6整合素的能力足以触发这些工程化T细胞的活化。
然后评估通过pCAR和对照工程化的T细胞产生IFN-γ。将缺乏αvβ6的肿瘤细胞(Panc1和A375 puro)或表达αvβ6的肿瘤细胞(Bxpc3和A375 puroβ6)与基因工程化的T细胞以1:1的效应物:靶标比例进行共培养,并在24、48或72小时后收集上清液。通过ELISA(eBioscience公司)定量IFN-γ的水平。结果示于图17中。正如预期的,当与αvβ6阳性肿瘤细胞(Bxpc3和A375 puroβ6)一起培养时,对照不产生显著量的IFN-γ,而A20-28z CAR T细胞释放IFN-γ。值得注意的是,当与αvβ6阳性肿瘤细胞(Bxpc3)一起培养时,表达本发明的pCAR(T1E-41BB/A20z)的T细胞比A20-28z T细胞产生更多的IFN-γ。另外,当与αvβ6阴性肿瘤细胞(Panc1和A375 puro)一起培养时,T1E-41BB/A20z+T细胞产生IFN-γ。再一次,这表明,在pCAR构型中,A20肽与非αvβ6整合素的低亲和力结合足以触发这些工程化的T细胞的活化。
接下来,在Panc1肿瘤细胞(αvβ6阴性)或Bxpc3肿瘤细胞(αvβ6阳性)上不存在IL-2支持物的情况下,对CAR T细胞群体每两周进行一次再刺激。将肿瘤细胞与源自患有胰腺导管腺癌(PDAC)的患者的CAR T细胞以1:1的效应物:靶标比例进行共培养(图18)。首先以2x105个细胞/孔添加T细胞,并在共培养72小时后计数以评估扩增(顶图)。通过MTT测定在添加T细胞后72小时评估细胞毒性(底图)。如果有足够数量的T细胞(2x105),则在新鲜的肿瘤单层上对T细胞进行再刺激,并且在另外的72小时后重复该过程。
结果示于图18中。这些说明A20-28z/T1E-41BB+T细胞经历了伴随着IL-2释放(数据未显示)和αvβ6+Bxpc3细胞破坏的许多轮扩增。再一次,它们还经历了伴随着IL-2释放和Panc1肿瘤细胞破坏的许多轮扩增。
总体而言,结果清楚地表明,与靶向αvβ6的第二代CAR相比,包含A20-28z/T1E-41BB的pCAR表现出增强的体外功能性。此外,A20-28z/T1E-41BB+T细胞还经历了通过Panc1或A375 puro细胞的激活,所述Panc1或A375 puro细胞表达最低至不可检测水平的这种整合素。考虑到使用C34B pCAR获得的发现(实例1和2),这表明pCAR构型允许当与41BB CCR发生高亲和力结合相互作用而与28z第二代CAR发生较低亲和力相互作用时在一系列再刺激后发生T细胞活化。
实例4
使用替代性TNF受体家族成员CD27来工程化功能性pCAR。
使用A20-28z/T1E-41BB pCAR作为起始材料,对另外的pCAR进行工程化,其中该41BB模块被TNF受体家族的替代性成员即CD27或CD40替换。对对照pCAR进行工程化,其中胞内结构域被截短(tr)。将表达αvβ6的靶细胞(Bxpc3)或缺乏αvβ6的靶细胞(Panc1)以24孔板的每孔5x104个细胞的密度进行铺板。24小时后,将5x104个pCAR T细胞添加到靶细胞或空孔(“未刺激的”)中,而不添加外源细胞因子支持物。另外72小时后,从各孔中收获T细胞并对其进行计数(图19A)。进行MTT测定以确定剩余的靶细胞的活力百分比,从而与在未添加T细胞的情况下进行铺板的对照靶细胞进行比较(图19B)。如果T细胞在每个刺激周期后增殖,则在新鲜的靶细胞上对这些T细胞进行再刺激,完全如上所描述的。像之前那样在72小时后进行pCAR T细胞的增殖(图19A)和MTT测定(图19B)。以这种方式继续对pCAR T细胞进行迭代再刺激和对靶细胞杀死进行评估,直到T细胞在每个72小时周期的过程中不再增殖。
这些数据再一次证实,当T细胞在Panc1靶细胞上被刺激时,通过持续的T细胞增殖和肿瘤细胞杀死所示,A20-28z/T1E-41BB pCAR的优越功能性。这进一步证实,A20肽与非αvβ6整合素的低亲和力结合足以触发这些工程化的T细胞的活化。然而,值得注意的是,当在表达αvβ6的Bxpc3细胞上再刺激时,A20-28z/T1E-CD27 pCAR达到最高水平的增殖(图19A)和持续的肿瘤细胞杀死(图19B)。相比之下,基于CD40的pCAR在这些测定中表现出适度的功能。总的来说,这些数据表明可以使用许多TNF受体家族成员(通过CD27或41BB示例)来工程化表现出优越功能性的pCAR。
序列表
<110> 伦敦国王学院(King's College London)
<120> 治疗剂
<130> P3052/WO
<150> GB1513540.3
<151> 2015-07-31
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<170> BiSSAP 1.3.6
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Claims (14)
1.一种免疫应答细胞,该免疫应答细胞表达
(i)第二代嵌合抗原受体,其包含:
(a)一个信号传导区域;
(b)一个共刺激信号传导区域;
(c)一个跨膜结构域;和
(d)与靶抗原上的第一表位特异性地相互作用的结合元件;以及
(ii)嵌合共刺激受体,其包含
(e)与(b)不同的一个共刺激信号传导区域;
(f)一个跨膜结构域;和
(g)与靶抗原上的第二表位特异性地相互作用的结合元件;
其中
该免疫应答细胞是T细胞或自然杀伤细胞,
信号传导区域(a)是人CD3ζ链的细胞内结构域;共刺激信号传导区域(b)或(e)中的一个是CD28并且另一个是4-1BB、CD27或OX40,并且(i)结合元件(d)的结合亲和力低于结合元件(g)的结合亲和力。
2.根据权利要求1所述的免疫应答细胞,其中该信号传导区域(a)选自SEQ ID NO:1或SEQ ID NO:2。
3.根据前述权利要求中任一项所述的免疫应答细胞,其中(c)和(f)的跨膜结构域选自CD8α和CD28跨膜结构域。
4.根据前述权利要求中任一项所述的免疫应答细胞,其中所述第一表位和第二表位与相同的受体或抗原缔合。
5.根据前述权利要求中任一项所述的免疫应答细胞,该免疫应答细胞共表达嵌合细胞因子受体。
6.根据权利要求5所述的免疫应答细胞,其中该嵌合细胞因子受体是4αβ。
7.根据前述权利要求中任一项所述的免疫应答细胞,其中结合元件(d)或结合元件(g)中的至少一个是ErbB二聚体的配体、集落刺激因子-1的受体(CSF-1R)或αvβ6整合素特异性结合剂。
8.根据前述权利要求中任一项所述的免疫应答细胞,其中结合元件(d)是CSF-1并且结合元件(g)是IL-34。
9.根据权利要求1至7中任一项所述的免疫应答细胞,其中结合元件(d)是αvβ6整合素特异性结合剂,该结合剂是包含SEQ ID NO:9-11的任一项的肽;并且结合元件(g)是T1E肽。
10.根据权利要求1-6中任一项所述的免疫应答细胞,其中所述结合元件(d)和(g)之一是T1E肽。
11.一种用于制备根据前述权利要求中任一项所述的免疫应答细胞的方法,所述方法包括用编码如权利要求1中所定义的结构(i)的嵌合抗原受体的第一核酸转导细胞;并且用编码如权利要求1中所定义的结构(ii)的嵌合共刺激受体的第二核酸转导细胞。
12.根据权利要求11所述的方法,其中该免疫应答细胞包含嵌合细胞因子受体,并且其中扩增步骤在所述细胞因子的存在下进行。
13.一种第一核酸和第二核酸的组合,该第一核酸编码如权利要求1中所定义的(i)的嵌合抗原受体,该第二核酸编码如权利要求1中所定义的(ii)的嵌合共刺激受体。
14.一种载体或载体组合,其包含根据权利要求13所述的组合。
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KR102411571B1 (ko) | 2022-06-21 |
US20200331981A1 (en) | 2020-10-22 |
GB201513540D0 (en) | 2015-09-16 |
RU2747733C1 (ru) | 2021-05-13 |
US10899818B2 (en) | 2021-01-26 |
WO2017021701A1 (en) | 2017-02-09 |
MX2018001009A (es) | 2018-06-07 |
EP3328880B1 (en) | 2021-07-07 |
US10703794B2 (en) | 2020-07-07 |
CA2993746A1 (en) | 2017-02-09 |
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