CN112771079A - 用于治疗tag72阳性肿瘤的tag72靶向性嵌合抗原受体修饰的t细胞 - Google Patents
用于治疗tag72阳性肿瘤的tag72靶向性嵌合抗原受体修饰的t细胞 Download PDFInfo
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Abstract
描述了靶向TAG72的嵌合抗原受体及其在治疗卵巢癌和其他癌症中的用途。
Description
技术领域
本公开涉及肿瘤相关糖蛋白72(TAG72)-特异性嵌合抗原受体(CAR)-工程化改造的T细胞,配制方法以及用作选择性针对TAG72-阳性细胞的抗癌剂的方法。
发明背景
CD19+B细胞恶性肿瘤患者的嵌合抗原受体(CAR)工程化改造的T细胞疗法已显示出令人印象深刻的临床响应,其最近已导致用于白血病和淋巴瘤患者的两项标志性FDA批准(Maude SL,Teachey DT,Porter DL,Grupp SA.CD19-targeted chimeric antigenreceptor T-cell therapy for acute lymphoblastic leukemia.Blood.2015;125(26):4017-23;Jain MD,Davila ML.Concise Review:Emerging Principles from theClinical Application of Chimeric Antigen Receptor T Cell Therapies for B CellMalignancies.Stem cells.2018;36(1):36-44)。这些研究表明,即使在高度难治性疾病的条件下,也可优化CAR T细胞以诱导癌症患者的持久且完全的响应。开发用于实体癌的有效CAR T细胞疗法的主要障碍是避免由于缺乏真正限制性肿瘤抗原而导致的脱瘤中靶毒性,以及获得受T细胞持久性和肿瘤运输限制的持久响应(Priceman SJ,Forman SJ,BrownCE.Smart CARs engineered for cancer immunotherapy.Current opinion inoncology.2015;27(6):466-74;Chen N,Li X,Chintala NK,Tano ZE,AdusumilliPS.Driving CARs on the uneven road of antigen heterogeneity in solidtumors.Current opinion in immunology.2018;51:103-10)。迄今为止,大多数用于引导CAR T细胞特异性的肿瘤抗原已靶向过表达的蛋白,包括但不限于间皮素、PSMA、PSCA、HER2/neu、EGFR和IL13Rα2(Priceman SJ,Forman SJ,Brown CE.Smart CARs engineeredfor cancer immunotherapy.Current opinion in oncology.2015;27(6):466-74;YongCSM,Dardalhon V,Devaud C,Taylor N,Darcy PK,Kershaw MH.CAR T-cell therapy ofsolid tumors.Immunology and cell biology.2017;9 5(4):356-63)。尽管该领域仍在不断发展,但是靶向实体瘤中这些蛋白的CAR T细胞的临床疗效受到一定限制(CastellarinM,Watanabe K,June CH,Kloss CC,Posey AD,Jr.Driving cars to the clinic forsolid tumors.Gene therapy.2018.Epub 2018/06/09.),以及识别额外的靶标还有解决有限的T细胞持久性仍然是限制CAR T细胞疗法成功的突出问题。
肿瘤上细胞表面蛋白的异常糖基化长期以来都与肿瘤发展有关,并且具有独特的糖蛋白特征,这些特征是用于免疫疗法的有吸引力的靶标,包括CAR T细胞(Steentoft C,Migliorini D,King TR,Mandel U,June CH,Posey AD,Jr.Glycan-Directed Car-TCells.Glycobiology.2018.Epub 2018/01/26;RodrIguez E,Schetters STT,van KooykY.The tumour glyco-code as a novel immune checkpoint for immunotherapy.Naturereviews Immunology.2018;18(3):204-11.Epub 2018/02/06)。已知包括结肠癌、乳腺癌、胰腺癌和卵巢癌在内的多种癌症类型过表达糖蛋白,包括粘蛋白MUC16和MUC1,以及与肿瘤相关的糖蛋白72(TAG72)(Hollingsworth MA,Swanson BJ.Mucins in cancer:protectionand control of the cell surface.Nature reviews Cancer.2004;4(1):45-60.Epub2003/12/19),这将它们与正常上皮区分开来。TAG72是高分子量粘蛋白,与丝氨酸和苏氨酸残基有大量的邻糖苷连接(Julien S,Videira PA,Delannoy P.Sialyl-tn in cancer:(how)did we miss the target?Biomolecules.2012;2(4):435-66.Epub 2012/01/01)。已在卵巢癌患者组织样本中显示出TAG72、MUC1和MUC16的高表达,几乎100%的卵巢癌利用这三种抗原同时染色鉴定(Chauhan SC,Vinayek N,Maher DM,Bell MC,Dunham KA,Koch MD,Lio Y,Jaggi M.Combined staining of TAG72,MUC1,and CA125 improves labelingsensitivity in ovarian cancer:antigens for multi-targeted antibody-guidedtherapy.The journal of histochemistry and cytochemistry.2007;55(8):867-75)。重要的是,大约90%的上皮性卵巢癌是TAG72阳性,表明其在卵巢癌的多种组织学亚型中的丰度。
已开发了对TAG72的肿瘤相关唾液酰基Tn抗原(STn抗原)特异的几种单克隆抗体,包括经过充分研究的克隆CC49(Muraro R,Kuroki M,Wunderlich D,Poole DJ,Colcher D,Thor A,Greiner JW,Simpson JF,Molinolo A,Noguchi P,et al.Generation andcharacterization of B72.3 second generation monoclonal antibodies reactivewith the tumor-associated glycoprotein 72antigen.Cancer research.1988;48(16):4588-96)。CC49随后在使用诊断成像和放射疗法的多种临床前和临床研究中应用,并且还涉及抗体人源化的多次尝试(Cheng KT.Radioiodinated anti-TAG72 CC49 Fab'antibodyfragment.Molecular Imaging and Contrast Agent Database(MICAD).BethesdaMD2004;Pavlinkova G,Booth BJ,Batra SK,Colcher D.Radioimmunotherapy of humancolon cancer xenografts using a dimeric single-chain Fv antibodyconstruct.Clinical cancer research:an official journal of the AmericanAssociation for Cancer Research.1999;5(9):2613-9;Kashmiri SV,Shu L,Padlan EA,Milenic DE,Schlom J,Hand PH.Generation,characterization,and in vivo studiesof humanized anticarcinoma antibody CC49.Hybridoma.1995;14(5):461-73;DePascalis R,Gonzales NR,Padlan EA,Schuck P,Batra SK,Schlom J,Kashmiri SV.Invitro affinity maturation of a specificity-determining region-graftedhumanized anticarcinoma antibody:isolation and characterization of minimallyimmunogenic high-affinity variants.Clinical cancer research:an officialjournal of the American Association for Cancer Research.2003;9(15):5521-31;Gonzales NR,Padlan EA,De Pascalis R,Schuck P,Schlom J,Kashmiri SV.Minimizingimmunogenicity of the SDR-grafted humanized antibody CC49 by geneticmanipulation of the framework residues.Molecular immunology.2003;40(6):337-49;Pavlinkova G,Colcher D,Booth BJ,Goel A,Wittel UA,Batra SK.Effects ofhumanization and gene shuffling on immunogenicity and antigen binding ofanti-TAG72single-chain Fvs.International journal of cancer.2001;94(5):717-26;Hege KM,Bergsland EK,Fisher GA,Nemunaitis JJ,Warren RS,McArthur JG,Lin AA,Schlom J,June CH,Sherwin SA.Safety,tumor trafficking and immunogenicity ofchimeric antigen receptor(CAR)-T cells specific for TAG72 in colorectalcancer.Journal for immunotherapy of cancer.2017;5:22)。
发明概述
本文描述了使用TAG72靶向性CAR T细胞治疗多种癌症(如,卵巢癌)的方法。
本文描述了包含编码嵌合抗原受体(CAR)的核苷酸序列的核酸分子,其中所述嵌合抗原受体包含:靶向Tag-72的scFv,间隔子,跨膜域,41-BB共刺激域或CD28共刺激域,以及CD3ζ信号传导域。
在各个实施方案中,跨膜域选自:CD4跨膜域或其具有1-5个氨基酸修饰的变体,CD8跨膜域或其具有1-5个氨基酸修饰的变体,CD28跨膜域或其具有1-5个氨基酸修饰的变体;间隔子包含20-150个氨基酸,并且位于scFv和跨膜域之间;跨膜域是CD4跨膜域或其具有1-5个氨基酸修饰的变体;跨膜域是CD4跨膜域;嵌合抗原受体包含选自以下的跨膜域:CD4跨膜域或其具有1-2个氨基酸修饰的变体,CD8跨膜域或其具有1-2个氨基酸修饰的变体,CD28跨膜域或其具有1-2个氨基酸修饰的变体;间隔子区包含选自SEQ ID NO:2-12的氨基酸序列或其具有1-5个氨基酸修饰的变体;间隔子包含IgG铰链区;间隔子包含10-50个氨基酸;4-1BB共刺激域包含SEQ ID NO:24的氨基酸序列或其具有1-5个氨基酸修饰的变体;CD3ζ信号传导域包含SEQ ID NO:21的氨基酸序列;3至15个氨基酸的接头位于4-1BB共刺激域和CD3ζ信号传导域或其变体之间;CAR包含SEQ ID NO:29的氨基酸序列或其具有1-5个氨基酸修饰的变体;scFv包含SEQ ID NO:1,SEQ ID NO:33或SEQ ID NO:34的氨基酸序列。
本文还公开了:包含本文所述核酸分子的病毒载体;由包含本文所述核酸分子的载体转导的人T细胞群(例如,包含中央记忆T细胞的群)。
本文还描述了治疗患者中的实体瘤的方法,其包括施用由包含本文所述核酸分子的载体转导的自体或同种异体人T细胞群,其中所述实体瘤包含表达Tag-72的细胞。在各个实施方案中,局部或全身施用嵌合抗原受体;表达TAG72的细胞是卵巢癌细胞;以及通过单次或重复给药施用嵌合抗原受体。
在各个实施方案中,嵌合抗原受体包含:TAG72 scFv(例如,scFv,其包含氨基酸序列:
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYNERFKGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASARESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:1),具有至多5个或至多10个单氨基酸取代)。
在各个实施方案中,嵌合抗原受体包含:TAG72 V15 scFv(例如,scFv,其包含氨基酸序列:
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYSQKFQGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDIVMSQSPDSLAVSLGERVTLNCKSSQSVLYSSNSKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLSFGAGTKLELK(SEQ ID NO:33),具有至多5个或至多10个单氨基酸取代)。
在各个实施方案中,嵌合抗原受体包含:TAG72 V59_V15 scFv(例如,scFv,其包含氨基酸序列:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQRLEWMGYFSPGNDDFKYSQKFQGRVTITADTSASTAYMELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNSKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQPYSYPLSFGAGTKLELK(SEQ ID NO:34),具有至多5个或至多10个单氨基酸取代)。
还描述了含有表达CAR的载体的T细胞。在各个实施方案中,至少20%、30%或40%的转导的人T细胞是中央记忆T细胞;至少30%的转导的人T细胞是CD4+和CD62L+或CD8+和CD62L+;人T细胞群对于患者是自体的;以及人T细胞群对于患者是同种异体的。
TAG72靶向性CAR
本文所述的TAG72靶向性CAR包括靶向TAG72的scFv(例如(如,scFv,其包含氨基酸序列:
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYNERFKGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASARESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:1)或包含由柔性接头连接的序列
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIG YFSPGNDDFKYNERFKGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSL NMAYWGQGTLVTVSSGSTS(SEQ ID NO:__)和序列
SSDIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQ SPKLLIYWASARESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYS YPLTFGAGTKLELK(SEQ ID NO:__);
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYSQKFQGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDIVMSQSPDSLAVSLGERVTLNCKSSQSVLYSSNSKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLSFGAGTKLELK(SEQ ID NO:33);或
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQRLEWMGYFSPGNDDFKYSQKFQGRVTITADTSASTAYMELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNSKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQPYSYPLSFGAGTKLELK(SEQ ID NO:34)。
有用的TAG72 CAR可由以下组成或包含以下:SEQ ID NO:__(缺少信号序列的成熟CAR)的氨基酸序列,或者TAG72 CAR可由以下组成或包含以下:SEQ ID NO:29、31或31的氨基酸序列(具有GMCSFRa信号序列的未成熟CAR)。CAR可以以包括信号序列如人GM-CSF受体α信号序列(MLLLVTS LLLCELPHPAFLLIP;SEQ ID NO:__)的形式表达。CAR可用其他可用于监测表达的序列来表达,例如T2A跳跃序列和截短的EGFRt。因此,CAR可包含以下或由以下组成:SEQ ID NO:29、31或32的氨基酸序列,或者可包含以下或由以下组成:与SEQ ID NO:29、31或32至少95%、96%、97%、98%、或99%相同的氨基酸序列。CAR可包含以下或由以下组成:SEQ ID NO:29、31或32中任何一个的具有至多1、2、3、4或5个氨基酸变化(优选保守氨基酸变化)的氨基酸序列。
间隔子区
本文所述的CAR可包括位于TAG72靶向域(即,TAG72靶向性ScFv或其变体)和跨膜域之间的间隔子。可使用多种不同的间隔子。它们中的一些包括人Fc区的至少一部分,例如人Fc区的铰链部分或CH3域或其变体。下表1提供了可用于本文所述CAR中的各种间隔子。
表1间隔子的示例
一些间隔子区包含全部或部分的免疫球蛋白(例如,IgG1、IgG2、IgG3、IgG4)铰链区,即落在免疫球蛋白的CH1和CH2域之间的序列,例如IgG4 Fc铰链或CD8铰链。一些间隔子区包含免疫球蛋白CH3域或者CH3域和CH2域两者。免疫球蛋白衍生的序列可包含一个或多个氨基酸修饰,例如1、2、3、4或5个取代,例如减少脱靶结合的取代。
铰链/接头区也可包含具有序列ESKYGPPCPSCP(SEQ ID NO:4)或
ESKYGPPCPPCP(SEQ ID NO:3)的IgG4铰链区。铰链/接头区还可包含序列ESKYGPPCPPCP(SEQ ID NO:3),其后是接头序列GGGSSGGGSG(SEQ ID NO:2),其后是IgG4CH3序列GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:12)。因此,整个接头/间隔子区可包含序列:ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:11)。在一些情况下,与SEQ ID NO:11相比,间隔子具有1、2、3、4或5个单氨基酸变化(例如,保守变化)。在某些情况下,IgG4 Fc铰链/接头区以减少Fc受体(FcR)结合的方式在两个位点(L235E;N297Q)突变。
跨膜域
多种跨膜域可用于其中。表2包括合适的跨膜域的示例。当存在间隔子区时,跨膜域位于该间隔子区的羧基端。
表2跨膜域的示例
共刺激域
共刺激域可以是适合与CD3ζ信号传导域一起使用的任何域。在某些情况下,共信号传导域是4-1BB共信号传导域,其包含与
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:24)至少90%、至少95%、至少98%相同或者与其相同的序列。在某些情况下,与SEQ ID NO:24相比,4-1BB共信号传导域具有5个氨基酸变化(优选保守的)中的1、2、3、4个。
共刺激域位于跨膜域和CD3ζ信号传导域之间。表3包括合适的共刺激域以及CD3ζ信号传导域序列的示例。
表3CD3ζ域和共刺激域的示例
在各个实施方案中,共刺激域选自:表3中描述的共刺激域或其具有1-5个(例如,1个或2个)氨基酸修饰的变体,CD28共刺激域或其具有1-5个(例如,1个或2个)氨基酸修饰的变体,4-1BB共刺激域或其具有1-5个(例如,1个或2个)氨基酸修饰的变体,以及OX40共刺激域或其具有1-5个(例如,1个或2个)氨基酸修饰的变体。在某些实施方案中,存在4-1BB共刺激域或其具有1-5个(例如,1个或2个)氨基酸修饰的变体。在一些实施方案中,存在两个共刺激域,例如CD28共刺激域或其具有1-5个(例如,1个或2个)氨基酸修饰(例如,取代)的变体和4-1BB共刺激域或其具有1-5个(例如,1个或2个)氨基酸修饰(例如,取代)的变体。在各个实施方案中,1-5个(例如,1个或2个)氨基酸修饰是取代。共刺激域在CD3ζ信号域的氨基端,并且由2-10个(例如,3个氨基酸,如GGG)组成的短接头可位于共刺激域和CD3ζ信号传导域之间。
CD3ζ信号传导域
CD3ζ信号传导域可以是适合与CD3ζ信号传导域一起使用的任何域。在某些情况下,CD3ζ信号域包含与以下至少90%、至少95%、至少98%相同或与以下相同的序列:RVKFSRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:21)。在一些情况下,与SEQ ID NO:21相比,CD3ζ信号传导具有5个氨基酸变化(优选保守的)中的1、2、3、4个。
截短的EGFR
CD3ζ信号传导域后面可以是核糖体跳跃序列(例如,LEGGGEGRGSLLTCGDVEENPGPR;SEQ ID NO:27)和具有与以下至少90%、至少95%、至少98%相同或与以下相同的序列的截短的EGFR:LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM(SEQ ID NO:28)。在一些情况下,与SEQ ID NO:28相比,截短的EGFR具有5个氨基酸变化(优选保守的)中的1、2、3、4个。
氨基酸修饰是指蛋白或肽序列中的氨基酸取代、插入和/或缺失。“氨基酸取代”或“取代”是指用另一种氨基酸替换亲本肽或蛋白序列中特定位置的氨基酸。取代可以非保守方式(即,通过将属于具有特定大小或特征的一组氨基酸的氨基酸密码子改变为属于另一组的氨基酸)或以保守方式(即,通过将属于具有特定大小或特征的一组氨基酸的氨基酸密码子改变为属于同一组的氨基酸)改变所得蛋白的氨基酸。这种保守变化通常导致所得蛋白的结构和功能的较少改变。以下是氨基酸不同分组的示例:1)具有非极性R基团的氨基酸:丙氨酸,缬氨酸,亮氨酸,异亮氨酸,脯氨酸,苯丙氨酸,色氨酸,甲硫氨酸;2)具有不带电荷的极性R基团的氨基酸:甘氨酸,丝氨酸,苏氨酸,半胱氨酸,酪氨酸,天冬酰胺,谷氨酰胺;3)具有带电荷的极性R基团的氨基酸(pH 6.0时带负电荷):天冬氨酸,谷氨酸;4)碱性氨基酸(pH 6.0时带正电荷):赖氨酸,精氨酸,组氨酸(pH 6.0时)。另一组可以是具有苯基的那些氨基酸:苯丙氨酸,色氨酸和酪氨酸。
在某些情况下,TAG72 CAR可使用载体产生,在该载体中,CAR开放阅读框后面是T2A核糖体跳跃序列和无细胞质信号传导尾巴的截短的EGFR(EGFRt)。在这种排列下,EGFRt的共表达提供了惰性、非免疫原性表面标志物,其允许精确测量基因修饰的细胞,并实现正选择基因修饰的细胞,以及有效细胞追踪过继转移后体内的治疗性T细胞。有效控制增殖以避免细胞因子风暴和脱靶毒性是T细胞免疫疗法成功的重要障碍。TAG72 CAR慢病毒载体中掺入的EGFRt可用作自杀基因,以在治疗相关毒性的情况下消融CAR+T细胞。
本文所述CAR可通过本领域已知的任何方法来产生,尽管优选使用重组DNA技术来产生。可通过本领域已知的分子克隆标准技术(基因组文库筛选,重叠PCR,引物辅助的连接,定点诱变等),方便地制备编码嵌合受体的几个区的核酸并将其组装成完整的编码序列。优选地,将所得编码区插入表达载体并用于转化合适的表达宿主细胞系,优选T淋巴细胞细胞系,最优选自体T淋巴细胞细胞系。
附图简述
图1A-1D显示TAG72-BBζCAR T细胞的图画表示,并显示用纯化TAG72培养的TAG72-BBζCAR T细胞的结果。(A)具有TAG72-CAR的慢病毒表达盒的图,该TAG72-CAR包含靶向TAG72的人源化scFv(CC49克隆),以及129个氨基酸修饰的人IgG4 Fc接头(无CH2域,ΔCH2),CD4跨膜域,胞质4-1BB共刺激域和溶细胞CD3ζ域。表达了截短的非信号传导CD19(CD19t)(通过T2A核糖体跳跃序列与CAR序列分开),用于鉴定慢病毒转导的T细胞。(B)模拟(未转导)和TAG72-BBζCAR T细胞通过流式细胞术评估CD19t表达以检测CAR的慢病毒转导(左图)或评估蛋白L以检测scFv(右图)。(C)模拟(上图)和TAG72-BBζCAR T细胞(下图)中的CD4和CD8表达。(D)利用针对可溶性或板结合的纯化TAG72抗原以所示蛋白量(单位)体外刺激24小时的CAR T细胞,通过流式细胞术评估激活(CD137的表达)。
图2A-2B显示针对纯化TAG72抗原的TAG72-BBζCAR T细胞激活的结果。(A)利用针对可溶性或板结合的纯化TAG72抗原以所示蛋白量(单位)体外刺激24小时的CAR T细胞,通过流式细胞术评估激活(CD69的表达)。(B)通过ELISA来自针对板结合的纯化TAG72抗原的TAG72-BBζCAR T细胞的IFNγ产生。
图3A-3H显示用TAG72阳性和TAG72阴性癌细胞培养的TAG72-BBζCAR T细胞的实验结果。(a)对多种卵巢和结肠直肠(LS174T)癌细胞系的TAG72表面表达的流式细胞术分析。(b)如材料和方法中所述与抗原阳性和阴性肿瘤靶标共培养24小时和72小时后,定量TAG72-BBζCAR T细胞相对于模拟的肿瘤杀伤。(c)与所示肿瘤靶标共培养后24小时和72小时的TAG72-BBζCAR T细胞扩增。(d,e)与所示肿瘤靶标共培养24小时和72小时后,通过ELISA定量来自模拟或TAG72-BBζCAR T细胞的上清液中的IFNγ和IL-2水平。(F)培养72小时后,从患者腹水(OAS)收获的原代人卵巢癌细胞上的TAG72表面表达的流式细胞术分析。与新鲜解冻的OAS细胞共培养72小时后,TAG72-BBζCAR T细胞相对于模拟的(G)肿瘤杀伤和(H)IFNγ产生的定量。
图4显示来自OVCAR3或OV90荷瘤小鼠的腹水上的TAG72表达的流式细胞术分析结果。
图5显示在24小时共培养测定中存在或不存在10个单位的可溶TAG7下TAG72-BBζCAR T细胞介导的OVCAR3细胞肿瘤杀伤的结果。
图6A-6F显示来自OVCAR3荷瘤小鼠的局部腹膜内递送TAG72-BBζCAR T细胞实验的结果。(A)示出在NSG小鼠中i.p.植入5.0x106个OVCAR3(eGFP/ffluc)肿瘤细胞,接着在肿瘤注射后第14天i.v.或i.p.递送5.0x106个模拟或TAG72-BBζCAR T细胞的示意图。(B)用模拟或TAG72-BBζCAR T细胞i.v.或i.p.治疗的小鼠的代表性生物发光通量成像。(C)来自用模拟或TAG72-BBζCAR T细胞i.v.或i.p.治疗的OVCAR3(eGFP/ffluc)荷瘤小鼠的通量(每只小鼠)定量。每组N=3。(D)模拟和TAG72-BBζCAR T细胞治疗的小鼠的Kaplan–Meier存活曲线。每组N≥4只小鼠。数据代表两个独立实验或是来自两个独立实验的组合。(E)治疗后6天、13天和29天定量每μL血液中的TAG72-BBζCAR T细胞。每组N=4。(F)治疗后第6天和第13天的荷瘤小鼠i.p.腔中人CD45+(hCD45)和小鼠CD45+(mCD45)细胞频率的代表性流式细胞术分析。来自两个独立实验的代表性图像。
图7显示OVCAR3模型中人CD45+细胞的定量;治疗后6天,13天和29天定量每μL血液中的人CD45+细胞。每组N=4。
图8A-8B显示OV90荷瘤小鼠中通过i.p.或通过i.v.体内递送抗肿瘤活性TAG72-BBζCAR T细胞的结果。(A)来自用模拟或TAG72-BBζCAR T细胞i.v.或i.p.治疗的OV90(eGFP/ffluc)荷瘤小鼠的通量(每只小鼠)定量。(B)模拟和TAG72-BBζCAR T细胞治疗的小鼠的Kaplan-Meier存活曲线。每组N≥4只小鼠。
图9A-9F显示OV90荷瘤小鼠中单次或重复局部施用TAG72-BBζCAR T细胞的实验结果。(A)示出NSG小鼠中i.p.植入5.0x106个OV90(eGFP/ffluc)肿瘤细胞,在肿瘤感染后第8天接着用5.0x106个模拟或TAG72-BBζCAR T细胞单次或重复i.p.治疗的示意图。(B)用模拟或TAG72-BBζCAR T细胞单次或重复处理i.p.治疗的小鼠的代表性生物发光通量图像。(C)来自用模拟或TAG72-BBζCAR T细胞单次或重复i.p.治疗的OV90(eGFP/ffluc)荷瘤小鼠的通量(每只小鼠)定量。(D)所有小鼠在治疗开始(上图)和峰值治疗(下图)时间点的相对肿瘤生长动力学的分析。进行Mann-Whitney检验以计算p值。(E)模拟和TAG72-BBζCAR T细胞治疗的小鼠的Kaplan-Meier存活。每组N≥5只小鼠。(F)肿瘤注射后42天和70天从单次和重复治疗的小鼠收获的肿瘤中人CD3细胞的组织学(上图:10倍放大,下图:40倍放大)。数据代表两个独立实验。
图10A-10B显示OV90模型中人CD45+细胞的定量;治疗后7天、14天和34天每μL血液中人CD45+细胞的定量。每组N=4。
图11A-11E显示卵巢癌中肿瘤相关糖蛋白抗原异质性以及定量CAR T细胞介导的抗原逃逸的实验的结果。(A)OVCAR8、OVCAR3和OV90人卵巢癌细胞系上的TAG72、MUC16和MUC1表面表达的流式细胞术分析。(B)治疗后99天从模拟和TAG72-BBζCAR T细胞治疗的OVCAR3荷瘤小鼠收获的i.p.实体瘤中TAG72、MUC16和MUC1表达的组织学。10倍放大。(C)肿瘤注射后42天、70天和105天从单次和重复治疗的OV90荷瘤小鼠收获的实体瘤中TAG72表达的组织学。10倍放大。(D)在所示时间点从接受单次或重复i.p.治疗的小鼠腹水收获的OV90肿瘤细胞中TAG72表达的流式细胞术分析。(E)用模拟或TAG72-BBζCAR T细胞(1:10E:T比)共培养后4天的OVCAR3细胞以及28天长出的肿瘤细胞上的TAG72表达。
图12显示hTag72scFv-IgG4(HL-CH3)-CD4tm-41BB-Zeta-T2A-CD19t的带注释的多肽序列(含有T2A和CD19t的SEQ ID NO:26;不含T2A和CD19t的SEQ ID NO:29)。不含GMCSFRa信号肽,T2A和CD19t的SEQ ID NO:35。
图13A-13C显示人源化TAG72 CAR T细胞的肿瘤杀伤、激活和T细胞增殖。(A)将OV90和OVCAR3细胞与模拟、IDEC、V15或V59/15变体TAG72 CAR T细胞以1:2的E:T共培养72小时。肿瘤杀伤表示为相对于模拟治疗条件的杀伤%。(B)通过流式细胞术染色表面CD137表达,从72小时共培养测定中分析T细胞激活。(C)相对于第0天接种的T细胞计数,确定72小时的T细胞增殖(倍数扩增)。
图14A-14B显示OV90荷瘤小鼠中人源化TAG72 CAR T细胞的单次或重复局部施用的实验结果。(A)通过流式细胞术确定OV90肿瘤细胞系上的TAG72抗原的内源性表达。将OV90-ffluc细胞注射至NSG小鼠的腹膜内(i.p.)腔中,并且通过生物发光成像追踪,报告为通量(光子/秒)。肿瘤注射后8天,将单次或重复剂量的5.0x106个模拟、TAG72 CAR T细胞的IDEC或V15变体局部施用至荷瘤小鼠的i.p.腔中。(B)通过生物发光成像定量单次或重复T细胞治疗小鼠的肿瘤负荷。垂直虚线表示T细胞初始和重复治疗的时间点。
图15A-15C显示OVCAR3荷瘤小鼠中i.v.施用人源化TAG72 CAR T细胞的结果。(A)通过流式细胞术分析OVCAR3肿瘤细胞的内源性表面TAG72表达。然后将OVCAR3-ffluc肿瘤注射至NSG小鼠的i.p.腔中,并用单剂量5.0x106个模拟、IDEC或V15变体TAG72 CAR T细胞i.v.治疗。(B)通过生物发光成像定量单剂量治疗的小鼠的肿瘤负荷,并报告为通量(光子/秒)。垂直虚线表示用T细胞治疗的时间点。(C)T细胞治疗后6天、13天和29天小鼠中通过流式细胞术定量血液中的模拟或IDEC和V15 TAG72 CAR T细胞的持久性和增殖(每μL血液的CAR+细胞),并且突出显示了V15变体TAG72 CAR T细胞相比IDEC的持久性和增殖增强。
图16A-16E显示不同人源化V15-CAR设计对体外抗肿瘤T细胞功能活性的影响。(A)七种TAG72-CAR T细胞变体(具有V15 scFv克隆)的CAR表达稳定性。(B-E)CAR T细胞针对TAG72阴性(DU145、OVCAR8)和TAG72阳性(OVCAR3、OV90和OVCAR8-sTn)表达的肿瘤细胞的体外肿瘤杀伤活性、T细胞增殖、CD137+激活指示物和PD-1+耗竭指示物(72小时)。
图17显示不同人源化V15-CAR设计对TAG72-CAR T细胞的体外细胞因子产生的影响。CAR T细胞针对TAG72-阴性(DU145、OVCAR8)和TAG72阳性(OVCAR3、OV90、OVCAR8-sTn)表达的肿瘤细胞的体外IFNγ产生(24小时)。
图18A-18B显示人源化TAG72 CAR T细胞的实时长期杀伤和增殖。(A)使用xCelligence技术用OV90细胞进行实时细胞毒性测定。将四个T细胞群以1比20的效应物与靶标比铺板并观察10天。细胞指数指示活肿瘤计数。(B)终点时,收集剩余的细胞并通过流式细胞术分析。
图19显示不含T2A和CD19t的Tag72scFv(IDEC)-IgG4(HL-CH3)-CD4tm-41BB-Zeta的带注释的多肽序列(SEQ ID NO:30)。不含GMCSFRa信号肽的SEQ ID NO:35。
图20显示不含T2A和CD19t的Tag72scFv(v15)-IgG4(HL-CH3)-CD4tm-41BB-Zeta的带注释的多肽序列(SEQ ID NO:31)。不含GMCSFRa信号肽的SEQ ID NO:3。
图21显示不含T2A和CD19t的Tag72scFv(v59_v15)-IgG4(HL-CH3)-CD4tm-41BB-Zeta的带注释的多肽序列(SEQ ID NO:32)。不含GMCSFRa信号肽的SEQ ID NO:37。
发明详述
在本公开中,描述了具有人源化抗人TAG72 scFv抗原结合域和4-1BB细胞内共刺激信号传导域(TAG72-BBζ)的第二代CAR T细胞的产生和抗肿瘤功效。TAG72-BBζCAR T细胞对多种表达TAG72的人卵巢癌细胞系和源自细胞培养中培养的患者卵巢癌腹水的上皮细胞表现出有效的抗原依赖性细胞毒性。TAG72-BBζCAR T细胞在腹膜卵巢肿瘤模型中的局部腹膜内体内递送可消除抗原阳性疾病并延长小鼠的总生存期。相反,静脉内CAR T细胞递送在控制疾病方面无效。此外,与单一治疗相比,重复局部输注TAG72-BBζCAR T细胞促进更持久控制疾病。这些临床前发现支持TAG72-BBζCAR T细胞作为卵巢癌可行的治疗选择,也突出显示其在表达TAG72的多种实体癌中的更广泛应用。
实施例
本发明还描述了以下实施例,其并不限制权利要求书中描述的发明范围。
材料和方法
以下材料和方法在本文所述的实施例中使用。
细胞系
上皮卵巢癌系OVCAR3(ATCC HTB-161)在含有20%胎牛血清(FBS,Hyclone)和1X抗生素-抗真菌药(1XAA,Gibco)的RPMI-1640(Lonza)(完全RPMI)中培养。将源自转移性腹水OV90的上皮卵巢癌系(CRL-11732)在MCDB 105培养基(Sigma)和Medium 199(Thermo)的1:1混合物(用氢氧化钠(Sigma)调节pH值为7.0,且最终20%FBS和1X AA)中培养。上皮-子宫内膜样卵巢癌系COV362.4(Sigma)在含有10%FBS、1XAA、25mM HEPES(Irvine Scientific)和2mM L-谷氨酰胺(Fisher)的Dulbecco改良Eagles培养基(DMEM,Life Technologies)(完全DMEM)中培养。上皮卵巢癌系OVCAR8由希望之城的Carlotta Glackin博士馈赠,并在完全RPMI-1640中培养。上皮卵巢癌系SKOV3(ATCC HTB-77)和结肠上皮癌系LS174T(ATCC CL-188)在完全DMEM中培养。所有细胞均培养于37℃,5%CO2。
DNA构建体和慢病毒产生
如前所述(22),通过用在EF1α启动子的控制下携带eGFP/ffluc融合物的epHIV7慢病毒转导,将肿瘤细胞工程化改造为表达增强绿色荧光蛋白和萤火虫萤光素酶(eGFP/ffluc)。CAR构建体中使用的人源化scFv序列是从靶向TAG72的单克隆抗体克隆huCC49获得的(17)。细胞外间隔子域包括129个氨基酸的中等长度CH2缺失形式(ΔCH2)的IgG4 Fc间隔子(23)。包含的细胞内共刺激信号传导域是具有CD4跨膜域的4-1BB。CD3ζ细胞溶解域先前已有描述(22)。通过T2A核糖体跳跃序列将CAR序列与截短的CD19基因(CD19t)分离,并在EF1α启动子控制下克隆到epHIV7慢病毒主链中。
如前所述(22,24)产生慢病毒。简而言之,使用修改的磷酸钙方法,用包装质粒和CAR慢病毒骨架质粒转染293T细胞。3至4天后收集病毒上清液,并用2mM镁和25U/mL内切核酸酶(EMD Millipore)处理。上清液通过高速离心浓缩,将慢病毒沉淀重悬于磷酸盐缓冲盐水(PBS)-乳糖溶液(每100mL PBS中4g乳糖),等分取样并保存于-80℃。慢病毒滴度使用HT1080细胞基于CD19t表达进行定量。
T细胞分离、慢病毒转导和离体扩增
根据希望之城内部审查委员会(IRB)批准的协议,从同意的研究参与者(健康捐赠者)获得白细胞单采术产物。在白细胞单采术当天,外周血单个核细胞(PBMC)通过在Ficoll-Paque(GE Healthcare)上进行密度梯度离心,然后在PBS/EDTA(Miltenyi Biotec)中多次洗涤进行分离。将细胞于室温下放置在旋转器上过夜,然后洗涤并重悬于含有10%FBS的X-VIVO T细胞培养基(Lonza)(完全X-VIVO)。根据制造商的方案,使用系统(Miltenyi Biotec),将多达5.0x109个PBMC与抗CD14和抗CD25微珠(MiltenyiBiotec)于室温孵育30分钟并磁性消除,这些被称为经消除的PBMC(dPBMC)。在进一步处理前,将dPBMC冷冻于(StemCell Technologies)中。
如先前所述(22)进行T细胞激活和转导。简而言之,将新鲜解冻的dPBMC洗涤一次,然后在含有100U/mL重组人IL-2(rhIL-2,Novartis Oncology)和0.5ng/mL重组人IL-15(rhIL-15,CellGenix)的完全X-VIVO中培养。对于CAR慢病毒转导,珠刺激后1天,将T细胞与CD3/CD28(Life Technologies)、硫酸鱼精蛋白(APP Pharmaceuticals)、细胞因子混合物(如上所述)和期望的慢病毒(以1的感染复数(MOI))一起培养。然后,将细胞在含有细胞因子的完全X-VIVO中培养并且每隔2-3天补充新鲜的含有细胞因子的完全X-VIVO。7天后,将珠磁性除去,并将细胞在含有细胞因子的完全X-VIVO中进一步扩增,以达到所期望的细胞产量。根据制造商的方案,CAR T细胞使用EasySepTMCD19阳性富集试剂盒I或II(StemCell Technologies)对CD19t进行正选择。进一步扩增后,在进行体外功能测定和体内肿瘤模型之前,将细胞冷冻于中。通过流式细胞术验证CAR T细胞的纯度和表型。
流式细胞术
为了进行流式细胞术分析,将细胞重悬于FACS缓冲液(Hank’s平衡盐溶液(HBSS-/-,Life Technologies),不含Ca2+、Mg2+或酚红,含有2%FBS和1xAA)。将细胞与一抗在黑暗中于4℃孵育30分钟。对于二次染色,细胞先洗涤两次,然后用Brilliant Violet510(BV510)、异硫氰酸荧光素(FITC)、藻红蛋白(PE)、多甲藻素叶绿素蛋白复合物(PerCP)、PerCP-Cy5.5、PE-Cy7、别藻蓝蛋白(APC)或APC-Cy7(或APC-eFluor780)缀合抗体在黑暗中于4℃孵育30分钟。使用针对CD3(BD Biosciences,克隆:SK7),CD4(BD Biosciences,克隆:SK3),CD8(BD Biosciences,克隆:SK1),CD14(BD Biosciences,克隆:MΦP9),CD19(BDBiosciences,克隆:SJ25C1)),CD25(BD Biosciences,克隆:2A3),小鼠CD45(BioLegend,克隆:30-F11),CD45(BD Biosciences,克隆:2D1),CD69(BD Biosciences,克隆:L78),CD137(BD Biosciences,克隆):4B4-1),MUC1(BioLegend,克隆16A),MUC16(Abcam,克隆X75或EPSISR23),生物素化的L蛋白(GenScript USA)(25),TAG72(克隆,muCC49),驴抗兔Ig(Invitrogen),山羊抗小鼠Ig(BD Biosciences)以及链霉亲合素(BD Biosciences)的抗体。细胞活力使用4',6-二脒基-2-苯基吲哚(DAPI,Sigma)测定。流式细胞术在MACSQuant分析仪10(Miltenyi Biotec)上进行,并且数据使用FlowJo软件(v10,TreeStar)分析。
体外肿瘤杀伤和T细胞功能测定
对于肿瘤杀伤分析,在无外源细胞因子的情况下,在完全X-VIVO中将CAR T细胞和肿瘤靶标以所示效应物:肿瘤(E:T)比于96孔板中共培养24到72小时,并通过如上所述的流式细胞术分析。CAR T细胞的肿瘤杀伤通过相对于靶标与模拟(未转导)T细胞共培养时观察到的CD45阴性细胞计数比较CD45阴性细胞计数来计算。对于T细胞激活测定,在无外源细胞因子的情况下,在完全X-VIVO中将CAR T细胞和肿瘤靶标以所示E:T比于96孔板中共培养所示的时间点,并通过流式细胞术分析T细胞激活的特异性标志物。将冷冻未培养的患者原发性卵巢癌腹水(OAS3、OAS4和OAS7)解冻,直接分析TAG72表达,并通过T细胞功能测定评估。简而言之,卵巢癌患者的腹水由希望之城国家医疗中心(COH)手术人员在无菌真空容器中获得,并经COH机构审查委员会(IRB)和人类受试者保护办公室批准。COH IRB放弃了书面知情同意的需要,因为所有样品均被脱识别,而腹水为丢弃物,如前所述(26)。
为了对板结合抗原进行T细胞激活测定,将纯化的可溶TAG72抗原(BioRad)以所示TAG72单位于4℃下接种在96孔平底高亲和力板(Corning)的1XPBS中过夜,一式两份。然后将总共104份TAG72-BBζCAR T细胞以固定体积100μL加入到每个孔中,并孵育所示时间,然后收集细胞,通过流式细胞术分析激活标志物(CD69,CD137)。还收集上清液以分析细胞因子产生。
ELISA细胞因子测定
在所示时间收集来自肿瘤杀伤测定或对板结合TAG72抗原进行CAR T细胞激活测定的上清液,并于-20℃冷冻以进一步使用。然后分别根据人IFNγ和IL-2ELISAReady-SET-GO!试剂盒制造商的方案,分析上清液中的分泌性人IFNγ和IL-2。使用WallacVictor3 1420计数器(Perkin-Elmer)和Wallac 1420工作站软件在450nm处读取板。
体内肿瘤研究
所有动物实验均在希望之城机构动物护理和使用委员会批准的方案下进行。为了进行体内肿瘤研究,OVCAR3和OV90细胞(5.0x106)以最终体积500μl HBSS-/-制备,并通过腹膜内(i.p.)注射将其植入6至8周龄的雌性NSG小鼠。通过生物光子成像(Xenogen,LagoX)至少每周监测一次肿瘤生长,并使用Living Image软件(Xenogen)分析通量信号。为了成像,以4.29mg/小鼠用悬浮于PBS的150μL D-萤光素钾盐(Perkin Elmer)进行i.p.注射小鼠。一旦通量信号达到所期望的水平,对于OV90是第8天和对于OVCAR3是第14天,在1X PBS中制备T细胞,并用500μL i.p.或200μL静脉内(i.v.)注射5.0x106个模拟或TAG72-BBζCART细胞治疗小鼠。在OV90肿瘤模型中,我们测试了肿瘤植入后从第8天开始用i.p.TAG72-BBζCAR T细胞重复治疗,接着在其他所示天数时治疗的影响。使用人道终点确定存活。出现痛苦症状如由腹水所致的腹胀、呼吸费力或呼吸困难、明显体重减轻、受损活动或垂死迹象时,对小鼠实施安乐死。在预定时间点或垂死状态下,对小鼠实施安乐死,并收集并处理组织和/或腹水进行如下所述的流式细胞术和免疫组织化学。
通过肝素化毛细管(Chase Scientific)从异氟烷麻醉的小鼠眶后(RO)放血收集外周血至含有肝素/PBS溶液(1000单位/mL,Sagent Pharmaceuticals)的聚苯乙烯管。记录每次RO抽血的体积(约120μL/小鼠),用于每微升血液的细胞定量。按照制造商的方案,红细胞(RBC)用1X红细胞裂解缓冲液(Sigma)裂解,然后如上所述进行洗涤、染色和流式细胞术分析。来自i.p.腹水流体的细胞通过i.p.腔内注射5mL冷的1X PBS从安乐死的小鼠收集,通过注射器抽取并在进一步处理前保存在冰上。将消除RBC的腹水洗涤、染色并通过流式细胞术使用上述抗体和方法分析肿瘤相关糖蛋白表达和CAR T细胞。
免疫组织化学
将肿瘤组织固定于4%多聚甲醛(4%PFA,Boston BioProducts)至多3天,并在进一步处理前于70%乙醇保存。免疫组织化学由希望之城病理学中心进行。简而言之,将石蜡包埋的切片(10μm)用苏木精&伊红(H&E,Sigma-Aldrich)、小鼠抗人CD3(DAKO)、小鼠抗人TAG72(AB16838,Abcam)、兔抗人MUC1(AB45167,Abcam)、MUC16(AB1107,Abcam)染色。使用Nanozoomer2.0HT数字玻片扫描仪和相关NDP.view2软件(Hamamatzu)获得图像。
统计分析
除非另有说明,数据以平均值±SEM表示。除非另有说明,使用非配对双尾学生t检验进行组之间的统计比较,以计算p值。*p<0.05,**p<0.01,***p<0.001;NS,不显著。
实施例1:构建含有4-1BB细胞内共刺激域的TAG72-CAR T细胞并验证TAG72-BBζCAR T细胞显示针对TAG72的活性
为了确定含有4-1BB细胞内共刺激域的TAG72-CAR T细胞是否对纯化TAG72有效显示激活,使上述细胞在存在增加量的可溶性TAG72或结合板的TAG72下生长,并测量激活指标物CD137表达。
结果
如先前所述(Priceman SJ,Gerdts EA,Tilakawardane D,Kennewick KT,MuradJP,Park AK,Jeang B,Yamaguchi Y,Yang X,Urak R,Weng L,Chang WC,Wright S,Pal S,Reiter RE,Wu AM,Brown CE,Forman SJ.Co-stimulatory signaling determines tumorantigen sensitivity and persistence of CAR T cells targeting PSCA+metastaticprostate cancer.Oncoimmunology.2018;7(2):e1380764),将TAG72-BBζCAR慢病毒用于转导消除CD14+和CD25+细胞的人健康供体来源的外周血单个核细胞(dPBMC)。TAG72-BBζCART细胞在制造过程中富集(基于CD19t+选择),并在T细胞表面稳定表达(图1B)。CAR T细胞以与模拟(未转导的)T细胞类似的动力学和相当的CD4:CD8比离体扩增(图1C)。重要的是,作为CAR T细胞针对TAG72激活的第一度量,当与板结合而非可溶的纯化TAG72一起培养时,TAG72-BBζCAR T细胞在表面上显示剂量依赖性CD137表达(图1D)。此外,当与板结合而非可溶的纯化TAG72一起培养时,TAG72-BBζCAR T细胞显示剂量依赖性诱导其他激活剂指示物,特别是细胞表面CD69表达和IFNγ释放(图2)。
实施例2:验证在体外TAG72-BBζCAR T细胞选择性靶向并显示激活TAG72阳性卵巢癌细胞
为了确定TAG72-BBζCAR T细胞是否对TAG72阳性癌细胞显示选择性活性,使TAG72-BBζCAR T细胞在TAG72阳性或TAG72阴性卵巢癌细胞存在下生长,并定量杀伤卵巢癌细胞的百分比。
结果
作为评估TAG72-BBζCAR T细胞针对TAG72阳性癌细胞的选择性活性(包括靶向和赋予靶细胞死亡)的第一步,评估人卵巢癌细胞系(包括SKOV3、OVCAR8、COV362.4、OVCAR3、OV90以及TAG72+结肠癌细胞系LS174T)的TAG72表达,以鉴定TAG72阳性癌细胞系。先前的研究已通过卵巢肿瘤患者样品的免疫组织化学和人卵巢癌细胞系的Western免疫印迹证明了TAG72的表达(Chauhan SC,Vinayek N,Maher DM,Bell MC,Dunham KA,Koch MD,Lio Y,Jaggi M.Combined staining of TAG-72,MUC1,and CA125 improves labelingsensitivity in ovarian cancer:antigens for multi-targeted antibody-guidedtherapy.The journal of histochemistry and cytochemistry:official journal ofthe Histochemistry Society.2007;55(8):867-75;Ponnusamy MP,Venkatraman G,SinghAP,Chauhan SC,Johansson SL,Jain M,Smith L,Davis JS,Remmenga SW,BatraSK.Expression of TAG-72in ovarian cancer and its correlation with tumor stageand patient prognosis.Cancer letters.2007;251(2):247-57)。通过流式细胞术,TAG72在OVCAR3细胞上表达(约42%),并在OV90细胞上表达更高(约90%),而在COV362.4细胞上检测到极低的水平(图3A)。SKOV3和OVCAR8细胞上没有TAG72。肿瘤细胞的免疫荧光染色在细胞表面以及细胞内证实了TAG72表达和细胞定位。值得注意的是,与体外培养的细胞相比,观察到TAG72在从荷瘤动物腹水收集的OVCAR3和OV90细胞上的更高表达(图4)。
为了评估我们的TAG72-BBζCAR T细胞的抗原依赖性活性,将其以1:1至1:2的E:T比与TAG72阳性和阴性卵巢肿瘤靶标进行共培养测定,以确定它们的杀伤潜力。24小时后,TAG72-BBζCAR T细胞相对于模拟T细胞具有明显的抗原特异性T细胞介导的杀伤活性(图3B)。在表达TAG72的靶标中,平均59%的LS174T、79%的OVCAR3和67%的OV90细胞被杀死。72小时后,同一肿瘤系的杀死分别增加至77%、90%和97%。TAG72-BBζCAR T细胞显示最小杀伤TAG72阴性或低表达的SKOV3、OVCAR8和COV362.4细胞。72小时时,针对TAG72阳性肿瘤细胞的TAG72-BBζCAR T细胞扩增2到3倍(图3C)。在1:10的较低E:T比时观察到类似的肿瘤杀伤,表明TAG72-BBζCAR T细胞的强大杀伤能力。TAG72-BBζCAR T细胞的肿瘤杀伤能力受从肿瘤细胞脱落的可溶性TAG72的影响最小(图5)。测量了来自CAR T细胞的细胞因子产生,作为T细胞活性的另一度量。仅当TAG72-BBζCAR T细胞与抗原阳性肿瘤靶标OVCAR3、LS174T和OV90共培养时,才观察到IFNγ和IL-2细胞因子产生(图3D和图3E)。尽管IL-2产生在早期时间点(T=24小时)达到峰值,并且仅在较晚的时间点(T=72小时)针对OVCAR3才检测到,但相比之下,IFNγ水平在整个72小时均显示升高的水平。
实施例3:在体外确认TAG72-BBζCAR T细胞选择性靶向来自卵巢癌腹水的TAG72阳性细胞
为了进一步确认TAG72是卵巢癌CAR靶标以及我们的TAG72-BBζCAR T细胞的抗肿瘤活性,使TAG72-BBζCAR T细胞在来自三名患者(OAS3、OAS4、OAS7)的人卵巢癌腹水存在下生长。
结果
通过流式细胞术,来自OAS3、OAS4和OAS7的新鲜解冻的腹水分别表达62%、80%和67%的TAG72,但在培养72小时后分别减少至2%、53%和19%(图3F)。不希望受特定理论的束缚,TAG72表达的减少可能反映了离体培养条件对TAG72表达维持的影响(Horan Hand P,Colcher D,Salomon D,Ridge J,Noguchi P,Schlom J.Influence of spatialconfiguration of carcinoma cell populations on the expression of a tumor-associated glycoprotein.Cancer research.1985;45(2):833-40)。TAG72-BBζCAR T细胞与腹水共培养72小时后显示细胞溶解活性,并显示有效且选择性CAR介导的杀伤TAG72阳性OAS4和OAS7细胞,针对TAG72阴性OAS3细胞未检测到抗肿瘤活性(图3G)。TAG72-BBζCAR T细胞针对OAS4产生IFNγ和IL-2,但针对OAS3和OAS7细胞未产生(图3H)。这些数据表明,TAG72-CAR T细胞在体外选择性靶向来自卵巢癌腹水的TAG72阳性细胞。
实施例4:验证在小鼠模型中体内局部递送至卵巢腹水的TAG72-BBζCAR T细胞显示有效的抗肿瘤活性并使小鼠延长寿命
为了评估TAG72-BBζCAR T细胞在体内的治疗潜力,测试了TAG72-BBζCAR T细胞在免疫受损的NSG小鼠中选择性靶向TAG72阳性OVCAR3肿瘤的能力;该小鼠模型模拟在晚期人疾病中观察到的模仿腹膜卵巢肿瘤。TAG72-BBζCAR T细胞通过腹膜内(i.p.)注射递送。
结果
慢病毒转导OVCAR3细胞来表达eGFP/ffluc,以通过无创光学成像追踪肿瘤的生长。肿瘤i.p.注射后14天,小鼠通过全身静脉内(i.v.)或局部i.p.递送模拟或TAG72-BBζCAR T细胞(5.0x106个)进行治疗(图6A)。在通过局部i.p.递送TAG72-BBζCAR T细胞治疗的小鼠中观察到快速抗肿瘤作用,在治疗后1-2周达到最大的抗肿瘤响应(图6B和图6C)。与区局部递送相比,i.v递送TAG72-BBζCAR T细胞显示有限的抗肿瘤响应。小鼠中抗肿瘤响应持续3-4周,但最终在小鼠中观察到肿瘤复发。局部i.p.递送TAG72-BBζCAR T细胞显著延长小鼠的存活,通过i.v.递送观察到有限的益处(图6D)。
为了解决i.p.和i.v.疗法之间观察到的潜在差异,对小鼠血液和腹水中的CAR T细胞进行定量。引人注目的是,i.p.治疗后6天,在小鼠血液中发现相当数量的CAR T细胞(huCD45+CD19t+),在同一时间点i.v.治疗小鼠的血液中CAR T细胞减少了5倍以上(图6E和图7)。然而,在随后时间点i.p.和i.v.治疗小鼠的血液中观察到了相等数量的CAR T细胞,从1-2周扩增,在治疗后4周明显减少。i.p.治疗后第6天,治疗小鼠腹水中的CAR T细胞继续存在于肿瘤部位,在同一时间点i.v.治疗小鼠中未检测到CAR T细胞。然而,在治疗后第13天,在i.v.和i.p.治疗小鼠中观察到相似水平的CAR T细胞(图6F)。不希望受特定理论的束缚,这些数据共同表明,CAR T细胞在i.v.递送后最终到达肿瘤,但与i.p.递送相比动力学延迟,这可能部分归因于缺乏通过这种施用途径的观察疗法。CD45阴性细胞(可能多数是OVCAR3肿瘤细胞)在i.p.TAG72-BBζCAR T细胞治疗的小鼠中显著消除,但在i.p.或i.v.模拟T细胞或i.v.TAG72-BBζCAR T细胞治疗的小鼠中未消除。这些数据支持TAG72-CAR T细胞的局部腹膜内递送作为靶向小鼠腹膜卵巢肿瘤的有效方法。
实施例5:验证TAG72-BBζCAR T细胞选择性靶向OV90 i.p.模型中的TAG72阳性细胞,并比较单次相比多次给药方案施用TAG72-BBζCAR T细胞的功效
为了评估TAG72-BBζCAR T细胞在OV90 i.p.模型中选择性靶向TAG72阳性细胞的功效,将TAG72-BBζCAR T细胞以单次或多次重复剂量递送,并随时间评估肿瘤的大小。
结果
值得注意的是,与OVCAR3相比,OV90 i.p.模型在体外显示更均匀的TAG72表达(图3A)。与OVCAR3模型i.p.相比,OV90 i.p.模型中的局部CAR T细胞递送显示选择性靶向TAG72细胞。相反,i.v.TAG72-BBζCAR T细胞治疗在OV90模型中未显示抗肿瘤功效(图8)。在用i.p.递送TAG72-BBζCAR T细胞的模型中,总生存期仅延迟了约25天(图8),可能是由于该模型的侵略性。鉴于此观察结果,评估了重复TAG72-BBζCAR T细胞给药相比单次剂量的功效,并发现其改善治疗响应(图9A)。与单次剂量的TAG72-BBζCAR T细胞相比,在OV90模型中一个月的过程内的重复给药显示更持久的抗肿瘤响应(图9B和图9C)。当绘制为相对肿瘤生长动力学时,与单次给药相比,重复给药促进了更广泛的肿瘤消退以及更持久的肿瘤控制(图9D)。
在该研究中,与单次剂量(30天受益)相比,接受重复剂量TAG72-BBζCAR T细胞的小鼠(55天获益)的总生存期显著延长(图9E)。重复治疗的小鼠腹膜肿瘤中观察到更多的T细胞数量(图9F)。然而,重要的是,与OVCAR3模型相比,观察到负荷OV90的小鼠血液中CAR T细胞减少的数量、扩增和持久性(图10)。不希望受特定理论的束缚,这些结果表明,这种更具侵略性的肿瘤模型也可能具有妨碍T细胞功能和总体CAR T细胞功效的抑制机制。总的来说,这些数据表明TAG72-BBζCAR T细胞在卵巢癌异种移植模型中的有效抗肿瘤活性,并且还表明与单次剂量相比,重复给药局部递送的CAR T细胞可提供对肿瘤的更好控制。
实施例6:确定TAG72-CAR T细胞疗法后肿瘤复发显示抗原逃逸
鉴于在前面实施例中观察到TAG72-BBζCAR T细胞在肿瘤复发之前减少,定量肿瘤中TAG72随时间的表达,以确定TAG72表达的丧失是否与减少的TAG72-BBζCAR T细胞数量相关。
结果
对CAR T细胞疗法的主要抗性机制之一是实体肿瘤中存在的肿瘤抗原异质性,其促进最终的抗原损失或逃逸(Chen N,Li X,Chintala NK,Tano ZE,AdusumilliPS.Driving CARs on the uneven road of antigen heterogeneity in solidtumors.Current opinion in immunology.2018;5 1:103-10)。鉴于(前面实施例的)两个体内模型中CAR T细胞的损失在肿瘤复发之前,因此肿瘤中TAG72表达的潜在损失发生与CAR T细胞的损失有关。为了评估前者,在治疗前和后随时间测量了来自模拟和TAG72-BBζCAR T细胞治疗的小鼠肿瘤的TAG72的表达。由于TAG72、MUC1和MUC16均已被鉴定为卵巢癌的潜在靶标,因此定量了这些细胞表面抗原在TAG72阴性OVCAR8和TAG72阳性OVCAR3和OV90细胞上的表达。OVCAR8似乎只表达低水平MUC1且不存在TAG72和MUC16,而OVCAR3以不同水平表达所有三种抗原,OV90显示MUC1的低表达且不存在MUC16(图11A)。因此,定量了这些抗原在用模拟或TAG72-BBζCAR T细胞治疗的小鼠的OVCAR3肿瘤中的表达。在T细胞输注后12周,来自模拟治疗的小鼠的肿瘤显示TAG72(类似于细胞系的流式细胞术分析),MUC16和MUC1的异质表达(图11B)。然而,用TAG72-BBζCAR T细胞治疗的小鼠在早期时间点的肿瘤复发显示TAG72表达急剧降低,而保持MUC16和MUC1的表达。类似地,TAG72-BBζCAR T细胞在OV90肿瘤模型中的重复治疗也显示在治疗后早期复发肿瘤中TAG72表达降低(图11E)。值得注意的是,在实体瘤以及腹水中肿瘤复发的较后时间点,检测到TAG72以高水平表达(图11C和图11D)。为了进一步在体外证实这一发现,定量CAR T细胞共培养后的肿瘤细胞中TAG72的表达,并且发现与未与CAR T细胞共培养时长出的肿瘤细胞相比降低(图11E)。总之,这些数据表明,抗原逃逸可能在TAG72-BBζCAR T细胞疗法后的肿瘤复发中起关键作用。
实施例7:在体外验证TAG72-BBζCAR T细胞选择性靶向TAG72阳性卵巢癌细胞并显示针对TAG72阳性卵巢癌细胞的激活
为了确定人源化TAG72-BBζCAR T细胞是否也有效杀伤TAG72阳性癌细胞,在TAG72阳性卵巢癌细胞存在下培养人源化TAG72-BBζCAR T细胞,并定量杀伤的卵巢癌细胞百分比。
结果
创建了一系列表达来自抗TAG-72抗体克隆CC49(IDEC-TAG72-BBz、V15-TAG72-BBz或V59/15-TAG72-BBz)的人源化变体的IDEC、V15或组合的V59/V15抗原结合域(scFv)的代表性4-1BB共刺激的CAR T细胞;图19-21(显示为羧基端不含T2A和CD1t序列)。这些CAR都利用相同的胞外域(由IgG4铰链组成,其中铰链第10个氨基酸突变为P;具有序列GGGSSGGGSG的接头和人IgG CH3域),CD4跨膜域和4-1BB细胞内共刺激信号域。使这些人源化TAG72-BBζCAR T细胞在OV90或OVCAR3卵巢癌细胞存在下生长,并定量杀死的卵巢癌细胞百分比。在体外,暴露于表达TAG72的卵巢癌细胞系(图13A-13C)后,IDEC和V15 TAG72-BBz CAR T细胞均显示相当的有效T细胞介导的抗原依赖性细胞毒性、激活和T细胞增殖。在该分析中,V59/15TAG72-BBz CAR显示很低的活性,已从进一步的实验中去除。
实施例8:验证在OV90 i.p.模型中人源化TAG72-BBζCAR T细胞选择性靶向TAG72阳性细胞并比较TAG72-BBζCAR T细胞单次相比多次给药方案施用的功效
为了评估在OV90 i.p.模型中人源化TAG72-BBζCAR T细胞选择性靶向TAG72阳性细胞的功效,将人源化TAG72-BBζCAR T细胞以单次或多次重复剂量递送,并随时间评估肿瘤的大小。
结果
通过流式细胞术确定在OV90肿瘤细胞系上TAG72抗原的内源性表达。将OV90-ffluc细胞注射至NSG小鼠的腹膜内(i.p.)腔中,并通过生物发光成像跟踪且报告为通量(光子/秒)。肿瘤注射后第8天,将5.0x106个模拟、IDEC或V15变体TAG72 CAR T细胞的单次或重复剂量局部施用至荷瘤小鼠的i.p.腔中(图14A)。通过生物发光成像定量单次或重复T细胞治疗小鼠的肿瘤负荷。垂直虚线表示用T细胞初始和重复治疗的时间点。有趣的是,我们显示,使用体内卵巢肿瘤模型,用V15-TAG72-BBz进行局部腹膜内治疗比IDEC-TAG72-BBzCAR更大程度地降低抗原阳性靶标(OV90植入肿瘤)的肿瘤负荷(图14A-14B)。鉴于此观察结果,评估了与单次剂量相比重复TAG72-BBζCAR T细胞给药的功效,并发现其改善治疗响应(图14B)。在OV90模型中,与单剂量TAG72-BBζCAR T细胞相比,在超过50天的过程中重复给药表明更持久的抗肿瘤响应(图14B)。
实施例9:验证人源化TAG72-BBζCAR T细胞选择性靶向OVCAR3 i.p.模型中的TAG72阳性细胞并比较单次给药方案施用TAG72-BBζCAR T细胞的持久性
为了评估人源化TAG72-BBζCAR T细胞选择性靶向OVCAR3 i.p.模型中TAG72阳性细胞的功效,将人源化TAG72-BBζCAR T细胞以单剂量递送,并随时间评估肿瘤的大小。
结果
通过流式细胞术分析OVCAR3肿瘤细胞上的内源性表面TAG72表达。然后,将OVCAR3-ffluc肿瘤注射至NSG小鼠i.p.腔中,所述小鼠用单次剂量的5.0x106个模拟、IDEC或V15变体TAG72 CAR T细胞进行i.v.治疗(图15A)。通过生物发光成像定量单剂量治疗的小鼠的肿瘤负荷,并报告为通量(光子/秒)。垂直虚线表示用T细胞治疗的时间点。有趣的是,静脉内(i.v.)施用的V15-TAG72-BBz CAR T细胞而非IDEC-TAG72-BBz CAR T细胞能够介导针对负荷OVCAR3肿瘤的小鼠的强大抗肿瘤响应(图15B)。与IDEC-TAG72-BBz相比,V15-TAG72-BBz CAR的这种体内抗肿瘤响应部分是由增加的增殖介导,从而增加了其响应的持续时间(图15C)。
实施例10:确定人源化TAG72-BBζCAR T细胞设计影响肿瘤杀伤、T细胞增殖、激活、耗竭和细胞因子产生
为了评估人源化TAG72 CAR T细胞的设计,创建了一系列代表性TAG72CAR T细胞,以V15 scFv为特征并改变了接头、跨膜和共刺激域。
结果
具有V15 scFv克隆的所有七个代表性人源化TAG72-CAR T细胞变体均显示CAR表达稳定性(图16A)。在体外肿瘤杀伤活性中,使人源化TAG72-CAR T细胞在TAG72阳性(OVCAR3、OV90和OVCAR8-sTn)或TAG72阴性(DU145、OVCAR8)卵巢癌细胞存在下生长,并定量杀伤卵巢癌细胞的百分比。所有七个代表性人源化TAG72-CAR T细胞变体均显示对TAG72阳性OVCAR3、OV90和OVCAR8-sTn细胞的有效且选择性CAR介导的杀伤,但对TAG72阴性DU145和OVCAR8细胞未检测到抗肿瘤活性(图16B)。T细胞增殖改变,并且在TAG72阳性OVCAR3、OV90和OVCAR8-sTn细胞中比在TAG72阴性DU145和OVCAR8细胞中更高(图16C)。CD137+激活指示物表明,代表性人源化TAG72-CAR T细胞变体改变,并且在TAG72阳性OVCAR3、OV90和OVCAR8-sTn细胞中比在TAG72阴性DU145和OVCAR8细胞中更高(图16D)。CAR T细胞针对TAG72阴性(DU145、OVCAR8)和TAG72阳性(OVCAR3、OV90和OVCAR8-sTn)表达的肿瘤细胞的PD-1+耗竭指示物(72小时)(图16E)。
不同V15-CAR设计还影响TAG72-CAR T细胞的体外细胞因子产生。CAR T细胞针对TAG72阴性(DU145、OVCAR8)和TAG72阳性(OVCAR3、OV90、OVCAR8-sTn)表达的肿瘤细胞的体外IFNγ产生(24小时)。尽管具有CD28tm-BBz构建体的CAR显示与CD4tm-BBz构建体相比相似的抗肿瘤活性,但CD28tm使某些TAG72阳性肿瘤细胞产生更多的细胞因子(图17)。
使用xCelligence技术对OV90细胞和一些代表性人源化TAG72-CAR T细胞变体进行实时细胞毒性测定。将四个T细胞群以1比20的效应物与靶标比铺板,并观察10天。细胞指数指示活肿瘤计数。在该长期杀伤测定中,所有三种代表性人源化TAG72-CAR T细胞变体均显示有效抗肿瘤活性(图18A)。在长期杀伤测定终点,收集剩余细胞并通过流式细胞术分析。所有三种代表性人源化TAG72-CAR T细胞变体都证明了T细胞扩增(图18B)。
Claims (22)
1.核酸分子,其包含编码嵌合抗原受体(CAR)的核苷酸序列,其中所述嵌合抗原受体包含:靶向Tag-72的scFv,间隔子,跨膜域,CD28或41-BB共刺激域,以及CD3ζ信号传导域。
2.权利要求1的核酸分子,其中所述跨膜域选自:CD4跨膜域或其具有1-5个氨基酸修饰的变体,CD8跨膜域或其具有1-5个氨基酸修饰的变体,CD28跨膜域或其具有1-5个氨基酸修饰的变体。
3.权利要求1的核酸分子,其中所述TAG72 scFV选自IDEC、V15和V59_V15。
4.权利要求1的核酸分子,其中所述跨膜域是CD4跨膜域或其具有1-5个氨基酸修饰的变体。
5.权利要求1的核酸分子,其中所述跨膜域是CD4跨膜域。
6.权利要求1的核酸分子,其中所述嵌合抗原受体包含选自以下的跨膜域:CD4跨膜域或其具有1-2个氨基酸修饰的变体,CD8跨膜域或其具有1-2个氨基酸修饰的变体,CD28跨膜域或其具有1-2个氨基酸修饰的变体。
7.权利要求1的核酸分子,其中所述间隔子区包含选自SEQ ID NO:2-12的氨基酸序列或其具有1-5个氨基酸修饰的变体。
8.权利要求1的核酸分子,其中所述间隔子包含IgG铰链区。
9.权利要求1的核酸分子,其中所述间隔子包含10-50个氨基酸。
10.权利要求1的核酸分子,其中所述4-1BB共刺激域包含SEQ ID NO:24的氨基酸序列或其具有1-5个氨基酸修饰的变体。
11.权利要求1的核酸分子,其中所述CD3ζ信号传导域包含SEQ ID NO:21的氨基酸序列。
12.权利要求1的核酸分子,其中3至15个氨基酸的接头位于所述4-1BB共刺激域和所述CD3ζ信号传导域或其变体之间。
13.权利要求1的核酸分子,其中所述CAR包含SEQ ID NO:29的氨基酸序列或其具有1-5个氨基酸修饰的变体。
14.权利要求7的核酸分子,其中所述scFv包含SEQ ID NO:1、31或32的氨基酸序列。
15.表达载体,其包含权利要求1的核酸分子。
16.病毒载体,其包含权利要求1的核酸分子。
17.人T细胞群,其由包含权利要求1的核酸分子的载体转导。
18.权利要求17的人T细胞群,其中所述人T细胞群包含中央记忆T细胞。
19.治疗患者中的实体瘤的方法,其包括施用由包含权利要求1的核酸分子的载体转导的自体或同种异体人T细胞群,其中所述实体瘤包含表达Tag-72的细胞。
20.权利要求19的方法,其中局部或全身施用所述嵌合抗原受体。
21.权利要求1的方法,其中所述表达TAG72的细胞是卵巢癌细胞。
22.权利要求19的方法,其中通过单次或重复给药施用所述嵌合抗原受体。
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WO2017088012A1 (en) * | 2015-11-27 | 2017-06-01 | Cartherics Pty. Ltd. | Genetically modified cells and uses thereof |
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WO2005000899A2 (en) * | 2003-06-27 | 2005-01-06 | Biogen Idec Ma Inc. | Modified binding molecules comprising connecting peptides |
WO2017040529A1 (en) * | 2015-08-31 | 2017-03-09 | Bluebird Bio, Inc. | Anti-sialyl tn chimeric antigen receptors |
CN108136014A (zh) * | 2015-08-31 | 2018-06-08 | 蓝鸟生物公司 | 抗唾液酸tn嵌合抗原受体 |
WO2017088012A1 (en) * | 2015-11-27 | 2017-06-01 | Cartherics Pty. Ltd. | Genetically modified cells and uses thereof |
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US20210308184A1 (en) | 2021-10-07 |
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WO2020028721A1 (en) | 2020-02-06 |
JP7440489B2 (ja) | 2024-02-28 |
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AU2019312673A1 (en) | 2021-02-25 |
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