CN105431456A - 用修饰的嵌合抗原受体靶向肿瘤新生血管 - Google Patents
用修饰的嵌合抗原受体靶向肿瘤新生血管 Download PDFInfo
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Abstract
可以向宿主施用转导有嵌合抗原受体的T细胞以杀伤癌细胞。所述嵌合抗原受体可包括对ανβ3整合素具有强结合亲和力的靶向部分,其包括但不限于锯鳞血抑肽多肽。所述靶向部分还可以被修饰以具有减小的对α5β1整合素的结合亲和力。
Description
相关申请的交叉引用
本申请要求享有于2013年6月14日提交的美国临时申请第61/835147号的优先权,通过引用将其整体并入本文。
政府资助:
NIH参考编号R01CA132792。
发明背景
目前正在开发数个有希望的免疫疗法以对抗癌症。例如,一种类型的免疫疗法是利用抗原特异性T细胞在细胞介导的免疫中的优势。T细胞通过T细胞受体(TCR)来识别其目标,TCR与由癌细胞表面上的主要组织相容性复合物(MHC)所呈现的抗原肽相结合。在存在共刺激分子的情况下,这一结合导致T细胞活化,并随后裂解所结合的靶细胞(VanderMerwePA,etal.,MolecularinteractionsmediatingTcellantigenrecognition,Annu.Rev.Immunol.21:659-84(2003))。然而,大多数的肿瘤相关抗原是自身蛋白,且免疫系统已发展出对它们的耐受。因此,癌症免疫疗法所面临的主要挑战之一是,难以产生足够数量的高结合亲和力的肿瘤特异性T细胞(KammertoensT,etal.,Makingandcircumventingtolerancetocancer,Eur.J.Immunol.39:2345-53(2009))。
研究人员最近试图通过移植用嵌合抗原受体(CAR)来遗传修饰T细胞(被称为T-CAR)来应对免疫系统对癌细胞抗原的耐受(JenaB,etal.,RedirectingT-cellspecificitybyintroducingatumorspecificchimericantigenreceptor,Blood116:1035-44(2010))。通常通过将单链抗体(scFv)加到细胞内信号结构域(通常是TCR/CD3复合体的ζ链)以生成CAR。最近构建的CAR还包含共刺激分子如CD28或41BB,其能够改善效应细胞的存活和增殖(CarpenitoC,etal.,Controloflarge,establishedtumorxenograftswithgeneticallyretargetedhumanTcellscontainingCD28andCD137domains,Proc.Natl.Acad.Sci.USA106:3360-5(2009))。对于癌症疗法,T-CAR比天然T细胞受体具有至少三重优势。首先,通常scFv的抗原结合亲和力比大多数TCR的结合部分更高。高亲和力结合是有效T细胞活化所需要的。其次,由于scFv介导的抗原结合的性质,T-CAR识别是非-MHC限制性的且独立于抗原加工。这扩大了T-CAR对具有不同MHC单倍型的患者的用途。再次,由于T-CAR识别是非-MHC限制性的,它们靶向癌细胞的能力不受癌细胞下调MHC的能力(肿瘤细胞逃避癌症免疫疗法的重要机制)的限制。
之前已经用结合多种肿瘤相关抗原的scFv构建了CAR(DaviesDM,etal.,AdoptiveT-cellimmunotherapyofcancerusingchimericantigenreceptor-graftedTcells,Arch.Immunol.Ther.Exp.(Warsz)58:165-78(2010))。令人鼓舞的前临床数据,已促进了一系列的使用移植有这些CAR的T细胞的过继性转移来治疗不同组织来源的肿瘤(包括黑素瘤、淋巴瘤、神经母细胞瘤和结肠直肠癌)的临床试验(DaviesDM,etal.,AdoptiveT-cellimmunotherapyofcancerusingchimericantigenreceptor-graftedTcells,Arch.Immunol.Ther.Exp.(Warsz)58:165-78(2010);RobbinsPF,etal.,TumorregressioninpatientswithmetastaticsynovialcellsarcomaandmelanomausinggeneticallyengineeredlymphocytesreactivewithNY-ESO-1,J.Clin.Oncol.29:917-24(2011);KochenderferJN,etal.,EradicationofB-lineagecellsandregressionoflymphomainapatienttreatedwithautologousTcellsgeneticallyengineeredtorecognizeCD19,Blood116:4099-102(2010);PorterDL,etal.,ChimericantigenreceptormodifiedTcellsinchroniclymphoidleukemia.N.Engl.J.Med.365:725-33(2011);PuleMA,etal.,Virus-specificTcellsengineeredtocoexpresstumor-specificreceptors:persistenceandantitumoractivityinindividualswithneuroblastoma,Nat.Med.14:1264-70(2008);ParkhurstMR,etal.,Tcellstargetingcarcinoembryonicantigencanmediateregressionofmetastaticcolorectalcancerbutinduceseveretransientcolitis.Mol.Ther.19:620-6(2011))。这些试验中的许多试验都显示出有希望的结果,在一些情况下甚至使已确认的肿瘤完全好转。
尽管T-CAR相比于天然T效应细胞有着令人印象深刻的改善,但也有显著的缺点。例如,T-CAR不主动迁移至肿瘤位点,且其缺乏活性机制以渗透至肿瘤组织。开发的规避细胞迁移问题的一种策略包括将T细胞工程化以表达趋化因子受体,其可以响应肿瘤相关的趋化因子环境(JenaB,etal.,RedirectingT-cellspecificitybyintroducingatumorspecificchimericantigenreceptor,Blood116:1035-44(2010);DiStasiA,etal.,TlymphocytescoexpressingCCR4andachimericantigenreceptortargetingCD30haveimprovedhomingandantitumoractivityinaHodgkintumormodel,Blood113:6392-402(2009))。然而,尽管这一策略改善了效应细胞迁移至肿瘤的能力,但其几乎不能促进其渗透至肿瘤组织的能力。
另一种需要改善的治疗形式是纳米颗粒介导的药物递送。在最近几年,纳米颗粒作为有希望的化疗的递送载体已被广泛研究。纳米颗粒具有超越许多小分子药物的弱生物制药性质并改变其药代动力学的潜力。然而,尽管它们具备巨大的前景,纳米颗粒介导的抗肿瘤药物递送还是比预期更不乐观。纳米颗粒对恶性组织的优先生物分布和滞留,依赖于弱的组织性、经常渗漏的血管和实体瘤内缺乏淋巴管,这一特征被称为增强的渗透和滞留(EPR)效应(GreishK,Enhancedpermeabilityandretention(EPR)effectforanticancernanomedicinedrugtargeting,MethodsMol.Biol.624:25-37(2010))。然而,EPR促进纳米颗粒递送至实体肿瘤的能力仍存在争议。可以促进纳米颗粒分布于肿瘤组织,这一明确限定的机制将大大提高其在临床应用中的用途。
因此,本领域需要可以克服血管壁屏障的修饰的T细胞,以使修饰的T细胞可以在全身施用后进入肿瘤细胞。本领域还需要增加肿瘤血管渗透性的方法和组合物,以允许纳米颗粒优先沉积至肿瘤组织。
发明概述
在一个实施方案中,T细胞可被移植有嵌合抗原受体,该受体包括对ανβ3整合素具有强结合亲和力的靶向部分。在另一个实施方案中,所述靶向部分可以是锯鳞血抑肽(echistatin)多肽。在另一个实施方案中,所述靶向部分可以被修饰以具有减小的对α5β1整合素的结合亲和力。
在一个实施方案中,可将转导有嵌合抗原受体的T细胞施用于宿主以杀伤癌细胞。嵌合抗原受体可包括对ανβ3整合素具有强结合亲和力的靶向部分,其包括但不限于锯鳞血抑肽多肽。在一个实施方案中,所述靶向部分可以被修饰以具有减小的对α5β1整合素的结合亲和力。
附图简述
结合附图将更好地理解前述概述以及下述详细说明。仅出于说明的目的,在此示出了附图中的某些实施方案。然而,应当理解的是,本文所公开的发明概念并不限于图中所示的精确安排和手段。
图1A根据一个实施方案示出了在逆转录病毒载体构建体中的eCAR的示意图。图1B根据一个实施方案示出了eCAR的转导效率。
图2A根据一个实施方案示出了HUVEC上αvβ3表达的流式细胞术分析。图2B根据一个实施方案示出了通过T-eCAR的HUVEC的细胞溶解。图2C根据一个实施方案示出了在T-eCAR和T-Her2CAR介导的靶细胞杀伤期间IL-2释放的定量。图2D根据一个实施方案示出了在T-eCAR和T-Her2CAR介导的靶细胞杀伤期间IFN-γ释放的定量。
图3A根据一个实施方案示出了在B16-F0细胞上αvβ3表达的流式细胞术分析。图3B-3C根据一个实施方案示出了T-eCAR针对B-16-GFPluc的细胞溶解效应。
图4A-4B根据一个实施方案示出了T-eCAR全身施用后肿瘤血管的选择性破坏。
图5A-5B根据一个实施方案示出了T-eCAR针对已确认的黑素瘤(5A)或前列腺癌(5B)的实体瘤的治疗效果。
图6A-6B根据一个实施方案示出了全身递送后,T-eCAR增强罗丹明标记的纳米颗粒的肿瘤分布。
图7示出了T-eCAR预施用加强了抗血管生成药物(AAD)的治疗效果。
发明详述
在详细解释至少一个实施方案之前,应当理解的是,本文所述的发明概念并不将其应用局限于下列描述或附图所示的构建细节或组分安排。还应当理解的是,本文所采用的措辞和术语仅仅是出于描述的目的而并不应当被认为是限制性的。
还应当理解的是,所述特征中的任一特征可以单独使用或与其它特征组合使用。查阅本文的附图和详细说明后,其它本发明的系统、方法、特征和优势,对于本领域技术人员而言是或将是显而易见的。本文意图在于所有的这些其他的系统、方法、特征和优势都受所附权利要求的保护。
本申请所引用的所有参考文献以其整体并入本文。
几乎所有的嵌合抗原受体(CAR)都被构建,以结合表达于肿瘤细胞表面上的肿瘤相关抗原。在一个实施方案中,一种可替代的方法是制备靶向肿瘤相关的新生血管的CAR。这一方法克服了与其它T-CAR相关的低效肿瘤软组织渗透问题。例如,在一个实施方案中,CAR包括作为靶向部分的锯鳞血抑肽(下称“eCAR”)。在另一个实施方案中,通过将来自锯鳞血抑肽的肽序列连接到T细胞的ζ链上来构建CAR。锯鳞血抑肽是49个氨基酸的去整合素,其可存在于锯鳞蝰(Echiscarinatus)毒液中(SEQID:001)。其对αvβ3整合素具有很强的结合亲和力,αvβ3整合素大量表达于肿瘤新生血管的内皮细胞表面(KumarCC,et.al.,Biochemicalcharacterizationofthebindingofechistatintointegrinalphavbeta3receptor,J.Pharmacol.Exp.Ther.283:843-53(1997);CaiW,et.al.,ChenX.Anti-angiogeniccancertherapybasedonintegrinalphavbeta3antagonism,AnticancerAgentsMed.Chem.6:407-28(2006))。
在另一个实施方案中,所选择的锯鳞血抑肽包括修饰的DNA序列,其中第28位氨基酸甲硫氨酸被替换为亮氨酸,以减少其对α5β1(SEQID:002)的结合(Wierzbicka-PatynowskiI,etal.,Structuralrequirementsofechistatinfortherecognitionofalpha(v)beta(3)andalpha(5)beta(1)integrins,J.Biol.Chem.274:37809-14(1999))。避免锯鳞血抑肽结合α5β1是重要的,这是因为与αvβ3不同的是α5β1通常表达于许多健康组织中。
在另一个实施方案中,T细胞被移植有eCAR(T-eCAR)。在体外,T-eCAR可以有效裂解表达αvβ3整合素的人脐静脉内皮细胞和肿瘤细胞。在另一个实施方案中,全身性的T-eCAR施用可导致肿瘤血管的广泛破坏,其是由肿瘤组织中明显的出血而没有证据表明破坏正常组织的血管来判断的。在另一个实施方案中,T-eCAR对肿瘤血管的破坏可显著抑制所确定的整块肿瘤的生长。
在一个实施方案中,以策略性设计的时间顺序与纳米颗粒共同递送的T-eCAR可以显著提高纳米颗粒在肿瘤细胞中的沉积。在另一个实施方案中,T-eCAR可以与纳米载体共同递送,以增加其选择性递送抗肿瘤药物至肿瘤组织的能力。
靶向肿瘤新生血管的CAR
在一个实施方案中,CAR被构建成靶向肿瘤新生血管。例如,在一个实施方案中,锯鳞血抑肽序列可被连接到T细胞(T-eCAR)的ζ链。在另一个实施方案中,锯鳞血抑肽可以通过将第28位氨基酸甲硫氨酸替换成亮氨酸来进行修饰。这一修饰可以基本上防止T-eCAR破坏健康组织。例如,野生型锯鳞血抑肽对整合素家族的三个成员αvβ3、α5β1和αIIbβ3具有强的结合亲和力。αvβ3和αIIbβ3具有窄的分布。例如,αvβ3主要表达于活化的内皮细胞表面上,而αIIbβ3由血小板表达。但α5β1更广泛地分布(CoxD,etal.,Integrinsastherapeutictargets:lessonsandopportunities,Nat.Rev.DrugDiscov.9:804-20(2010))。在修饰的锯鳞血抑肽中以亮氨酸取代甲硫氨酸,降低了锯鳞血抑肽对α5β1的结合亲和力(Wierzbicka-PatynowskiI,etal.,Structuralrequirementsofechistatinfortherecognitionofalpha(v)beta(3)andalpha(5)beta(1)integrins,J.Biol.Chem.274:37809-14(1999))。此外,这一修饰不会显著影响T-eCAR对αvβ3或αIIbβ3的结合亲和力。
图1A仅以示例的方式示出了eCAR(SEQID:003)的构建的一个实施方案。标记了逆转录病毒载体的5'和3'长末端重复序列。还标记了锯鳞血抑肽的编码序列、CD28(包含跨膜结构域)和ζ链。锯鳞血抑肽的DNA序列(Echi)可以编码锯鳞血抑肽的修饰形式。例如,第28位氨基酸甲硫氨酸可以被亮氨酸取代,以降低其对非αvβ3整合素的结合亲和力(Wierzbicka-PatynowskiI,etal.,Structuralrequirementsofechistatinfortherecognitionofalpha(v)beta(3)andalpha(5)beta(1)integrins,J.Biol.Chem.274:37809-14(1999))。还可以合成所述序列并将该序列插入到用于T细胞稳定转染的逆转录病毒载体中。可以将信号肽(SP)添加到融合基因的5'端。出于联合仿真(co-simulation)功能的目的,可以将CD28结构域插入到锯鳞血抑肽和ζ链之间。可以将c-Myc标签插入到锯鳞血抑肽和CD28之间,以促进eCAR表达的检测。在一个实施方案中,在载体构建的过程中插入c-Myc标签。
在一个实施方案中,可以测量eCAR的转导效率。可以用eCAR或含有绿色荧光蛋白(GFP)的逆转录病毒(SFG-GFP)转导脾细胞。在一个实施方案中,可以通过加入GFP标记物基因来构建脾细胞。空转导的细胞可以作为阴性对照。在用双色流式细胞术分析之前,可以用缀合有PE的抗-c-Myc抗体将细胞染色,以检测GFP和eCAR。仅以示例的方式参考图1B,几乎一半的eCAR-转导的脾细胞是PE阳性的,而没有SFG-GFP转导的细胞或对照细胞显示出任何明显的PE染色。另一方面,高百分比的SFG-GFP-转导的细胞是GFP阳性的,而eCAR转导的细胞或对照细胞都没有检测出。其结果是,在一个实施方案中,可以有效地通过逆转录病毒构建体将eCAR移植到脾细胞中。
移植有eCAR的T细胞可以有选择性地并有效地杀伤人脐静脉内皮细胞
在一个实施方案中,为了确定eCAR的效力,可以将其与表达αvβ3整合素的人脐静脉内皮细胞(HUVEC)共孵育。如图2A所示,可以用缀合有FITC的RGD肽(其还对αvβ3整合素具有高结合亲和力)将HUVEC染色。流式细胞术分析示出,αvβ3整合素在大多数的HUVEC上高表达。在另一个实施方案中,如图2B所示,活化的T-eCAR和对照SFG-GFP构建体可以在24小时期间内以不同速率与HUVEC混合,以测试T-eCAR杀伤表达αvβ3整合素的靶细胞的能力。脾细胞可以获得自C57BL/6供体,并用包括T-eCAR或对照SFG-GFP构建体的逆转录病毒转导。然后可以通过洗涤去除T细胞,可以用结晶紫将剩余的单层染色,以确定HUVEC的细胞活力。对照孔可以仅代表HUVAC。在一个实施方案中,移植有SFG-GFP的脾细胞在HUVEC上没有表现出任何显著的毒性。在另一个实施方案中,T-eCAR可以完全裂解所有的细胞,甚至是在具有最低的效应细胞与靶细胞的比率的孔中。
在另一个实施方案中,如图2C-2D所示,可以测量在T-eCAR介导的HUVEC杀伤过程中所释放的细胞因子。此结果也可以与Her2-CAR介导的表达Her2的肿瘤细胞杀伤相比较。在一个实施方案中,IL-2和干扰素γ(IFN-γ)这两种免疫活性信号分子,在由这两种CAR介导的细胞溶解过程中以几乎相等的水平进行释放。这表明,两种CAR共享相同的杀伤机制。此外,先前的研究已表明,移植有抗原特异性TCR的CD4和CD8T细胞可以作为效应细胞来有效地杀伤肿瘤细胞(FrankelTL,etal.,BothCD4andCD8TcellsmediateequallyeffectiveinvivotumortreatmentwhenengineeredwithahighlyavidTCRtargetingtyrosinase,J.Immunol.184:5988-98(2010);KerkarSP,etal.,GeneticengineeringofmurineCD8+andCD4+Tcellsforpreclinicaladoptiveimmunotherapystudies,J.Immunother.34:343-52(2011))。因此,在另一个实施方案中,移植有eCAR的CD4和CD8T细胞可以用作效应细胞来有效地杀伤靶细胞。
除了活化的内皮细胞,也有报道称已经发现一些与肿瘤转移相关的肿瘤细胞表达水平升高的αvβ3整合素(HiekenTJ,etal.,Beta3integrinexpressioninmelanomapredictssubsequentmetastasis.J.Surg.Res.63:169-73(1996);DuanX,etal.,Associationofalphavbeta3integrinexpressionwiththemetastaticpotentialandmigratoryandchemotacticabilityofhumanosteosarcomacells,Clin.Exp.Metastasis21:747-53(2004))。一种具有较高水平的αvβ3整合素表达的肿瘤细胞系是B16鼠类黑素瘤细胞系(GongW,etal.,IFN-gammawithdrawalafterimmunotherapypotentiatesB16melanomainvasionandmetastasisbyintensifyingtumorintegrinalphavbeta3signaling,Int.J.Cancer123:702-8(2008))。在一个实施方案中,如图3A所示,用缀合有FITC的RGD肽将细胞染色后,可以通过流式细胞术测定B16-F0(B16-GFpluc的亲本系)中的αvβ3整合素的表达。在一个实施方案中,高百分比的B16-F0表达了αvβ3整合素。
在另一个实施方案中,如图3B-3C所示,可以分析T-eCAR杀伤已稳定转导有含有GFP和荧光素酶(B16GFPluc)的融合基因的B16细胞系的能力。例如,可以通过GFP的直接可视化来方便地评估细胞溶解的杀伤效应(图3B)。转导有包含eCAR或对照SFG-GFP构建体的逆转录病毒的脾细胞可以按10:1、5:1和2.5:1的比例与B16-GFpluc混合。分析前可以将细胞培养48小时。在一个实施方案中,经荧光显微镜的可视化示出了,B16-GFPluc细胞与T-eCAR的孵育(E(效应细胞):T(靶细胞)比例=5:1)显著降低了孔中GFP阳性细胞的数量。在另一个实施方案中,经荧光显微镜的可视化示出了,转导有对照SFG-GFP构建体的脾细胞对细胞单层的完整性几乎没有或没有作用。
在另一个实施方案中,可以通过测量荧光素酶活性进行细胞溶解的非放射性定量分析来方便地评估细胞溶解的杀伤效应(图3C)(FuX,etal.,Asimpleandsensitivemethodformeasuringtumor-specificTcellcytotoxicity.PLoSOne2010;5:e11867(2010))。转导有包含eCAR或对照SFG-GFP构建体的逆转录病毒的脾细胞,可以与B16-GFpluc按10:1、5:1和2.5:1的比例混合。对照孔可以仅包含肿瘤细胞。细胞杀伤的百分比可以由以下公式进行计算:细胞杀伤(%)=[1-(有效应细胞的孔的读数)/(没有效应细胞的孔的读数)]×100。与SFG-GFP转导的效应细胞相比,*p<0.05。分析前可以将细胞培养48小时。在一个实施方案中,萤光素酶活性的定量分析表明,T-eCAR甚至可以在最低的E:T比例(2.5:1)时杀伤至少60%的B16-GFPluc细胞。在另一个实施方案中,荧光素酶活性的定量分析表明,移植有SFG-GFP构建体的T细胞仅在最高的E:T比例(10:1)时导致B16细胞的温和裂解。
在一个实施方案中,如果肿瘤细胞表达升高水平的αvβ3整合素,T-eCAR具有同时破坏肿瘤新生血管以及肿瘤软组织的能力。本文所述的方法适用于表达αvβ3的任何实体肿瘤。
体内施用T-eCAR诱导肿瘤而不是正常组织中的广泛出血。
图4A仅以示例的方式示出了,T-eCAR可以对体内肿瘤血管起作用。在一个实施方案中,可以通过皮下植入2×105个新鲜收获的B16细胞来在同系的C57BL/6小鼠的右侧腹建立肿瘤块。一旦肿瘤直径达到约8mm,小鼠可以通过尾静脉接受5×106个转导有eCAR或SFG-GFP的脾细胞的注射(全身输注)。另一组小鼠可以仅接受PBS作为阴性对照。然后,可以在过继性细胞转移后第3天,对小鼠实施安乐死。在一个实施方案中,可以收集肿瘤和正常器官组织,以制备用于组织学检查和H&E染色的石蜡包埋后的组织切片。在一个实施方案中,在用T-eCAR处理的肿瘤中存在广泛的出血(图4A)。肿瘤软组织可填充有红血细胞和其它血细胞成分。在另一个实施方案中,用SFG-GFP-转导的脾细胞处理的肿瘤表现出几乎没有至没有出血。eCAR和SFG-GFP构建体之间的唯一区别在于,后者的锯鳞血抑肽编码序列被GFP基因取代。因此,在一个实施方案中,T-eCAR施用后的肿瘤血管破坏主要是由于在eCAR中并入了锯鳞血抑肽序列。在另一个实施方案中,T-eCAR施用后的肿瘤血管破坏主要是由于T-eCAR结合新生血管相关的αvβ3整合素以触发T细胞介导的杀伤效应的能力。
在一个实施方案中,包括来自肺、肝和肾的正常器官组织,在T-eCAR施用后没有显示出任何显著的出血(图4B)。在另一个实施方案中,T-eCAR处理的肿瘤中的出血源自引入的T细胞对肿瘤血管的选择性破坏。
T-eCAR的过继性转移显著抑制已确定的实体肿瘤的生长。
在一个实施方案中,为了确定T-eCAR破坏肿瘤血管的结果,可以通过初始皮下移植1×105的B16肿瘤细胞(同系鼠类黑素瘤)或PC-3人前列腺癌细胞(异种移植肿瘤)至C57BL/6小鼠(用于B16细胞)和SCID小鼠(用于PC-3细胞)的右侧腹来进行体内实验。五天后,当肿瘤变得易察觉时,小鼠可接受PBS或4×106个转导有eCAR或SFG-GFP的脾细胞的静脉内全身输注。第三组中的小鼠仅给予PBS。可以每周测量肿瘤以确定肿瘤的体积。与SFG-GFP和PBS相比,*p<0.05,+p<0.01。
在一个实施方案中,如图5A所示,在用PBS或转导有SGF-GFP的脾细胞处理的小鼠中,肿瘤基本上可以不间断地生长。在开始处理B16黑素瘤后的第28天,两个组中的肿瘤都可以达到大尺寸且动物可能需要被安乐死,这是由于到达了预设的终点。相比之下,在另一个实施方案中,施用T-eCAR可以有效减缓肿瘤的生长。处理开始后的第42天,T-eCAR处理组中的肿瘤可以相对较小,且大多数动物可能还活着。在图5B中的另一个实施方案中,T-eCAR处理导致了在处理后的第11天和第14天时显著更小尺寸的肿瘤。因此,在两个实施方案中,T-eCAR介导的肿瘤血管的破坏,可导致对不同组织来源的已确定的实体肿瘤的显著治疗益处。
在图5A中的实施方案中,过继性转移的T-eCAR可以攻击肿瘤血管和肿瘤细胞。如此,初始肿瘤血管破坏可以允许将T-eCAR有效渗透至肿瘤软组织,以尽可能近地接触肿瘤细胞。这可以避免积极外渗的需要,而这是一种T-eCAR可能不具备的特性。因此,在另一个实施方案中,血管破坏和随后的T-eCAR渗透以直接杀伤肿瘤细胞的组合,可以协同作用而比任意一种单独作用能获得更好的治疗效果。
在一个实施方案中,通过将其与抗血管生成剂组合来使得T-eCAR的作用最大化,所述抗血管生成剂是例如血管生成素2、血管抑素、内皮抑素、血小板因子4、阿瓦斯丁(avastin)、阿柏西普(aflibercept)、索拉非尼(sorafenib)、舒尼替尼(sunitinib)、帕唑帕尼(pazopanib)、凡德他尼(vandetanib)、瓦他拉尼(vatalanib)、西地尼布(cediranib)、阿西替尼(axitinib),其可以在施用T-eCAR后防止新的肿瘤血管形成。在另一个实施方案中,此类组合可以产生协同效应,如T-eCAR介导的肿瘤血管破坏可以将肿瘤血管生成的相对缓慢的过程转换成可以将针对抗血管生成化合物的治疗响应最大化的急性事件。
经T-eCAR的肿瘤血管破坏可以增加纳米颗粒的肿瘤渗透。
在另一个实施方案中,如图4A所示,在输注T-eCAR后可以在恶性组织中观察到显著的出血。这表明T-eCAR可以用作在全身施用后增加递送到肿瘤的纳米颗粒的手段。因此,在一个实施方案中,T-eCAR可以随纳米颗粒施用,以增加肿瘤的纳米颗粒渗透性。这类方法适用于所有的肿瘤血管,这是因为所有的肿瘤血管基本上都包含高水平的αvβ3整合素,且从而对T-eCAR的效果敏感。
在另一个实施方案中,可以向小鼠施用转导有SFG-GFP对照构建体的T-eCAR或T细胞(图4A-4B)。例如,在一个实施方案中,可以在C57BL/6小鼠的右侧腹建立鼠类黑素瘤。一旦肿瘤达到约8mm直径,小鼠可以接受静脉输注的4×106个T-eCAR或SFG-GFP转导的脾细胞。48小时后,向小鼠静脉注射10mg/kg的罗丹明标记的脂质体纳米颗粒。然后可以在施用脂质体纳米颗粒后的一至两天时,对动物进行安乐死。在一个实施方案中,如图6A所示,可以收集肿瘤和主要的器官以制备可用于低温荧光显微镜检查的冷冻切片。在一个实施方案中,在接受SFG-GFP转导的T细胞的小鼠所制备的组织切片中,仅可稀疏地看到罗丹明染色。相比之下,在另一个实施方案中,在来自接受有T-eCAR的小鼠的整个肿瘤软组织中,可以看到普遍的罗丹明染色。可见的血管似乎有破损区域(表示为白箭头)。在另一个实施方案中,如图6B所示,在肿瘤组织中进行罗丹明的定量证实了,SFG-GFP和T-eCAR处理之间可以存在显著的差异。可以使用MicroSuiteTMFIVE软件定量肿瘤组织。由软件给出作为值强度(valueintensity)的结果,代表了在3个切片(各个肿瘤样品之一)中的15个随机选择的区域的均值。与SFG-GFP相比,*p<0.01。
正常器官组织切片的检查表明,除了肺几乎没有罗丹明沉积的证据,其中血管可以看到轻微的罗丹明染色。这一观察结果与早期报道的在全身递送后脂质体纳米颗粒倾向于被困于肺中这一结果相一致(LiuY,etal.,Factorsinfluencingtheefficiencyofcationicliposome-mediatedintravenousgenedelivery,Nat.Biotechnol.15:167-73(1997))。尽管在肺中有这一机械陷阱,但几乎没有纳米颗粒在肺的软组织中分布的证据。这一事实,再结合不能在肺(和其它正常器官组织)中检测出任何显著的出血(图4B),一起支持了这一假设。总结这些结果表明:1)尽管报道在肿瘤中存在增强的渗透和滞留(EPR)效应,但其不允许在全身递送后,纳米颗粒有效沉积以进入肿瘤间质,2)T-eCAR在正常器官组织中并不导致显著的血管损伤,尽管其对肿瘤新生血管有强破坏作用,和3)由T-eCAR介导的肿瘤血管的破坏,允许全身性递送纳米颗粒以有效地深入肿瘤软组织。因此,在一个实施方案中,T-eCAR可以与许多纳米颗粒介导的药物递送的抗肿瘤小分子组合,这些小分子是例如5-氟尿嘧啶(5-FU)、6-巯基嘌呤(6-MP)、卡培他滨(Capecitabine)克拉屈滨(Cladribine)、氯法拉滨(Clofarabine)、阿糖胞苷氟尿苷、氟达拉滨(Fludarabine)、吉西他滨(Gemcitabine)羟基脲、甲氨蝶呤、培美曲塞(Pemetrexed)喷司他丁(Pentostatin)、硫鸟嘌呤、二氯甲基二乙胺(氮芥)、苯丁酸氮芥、环磷酰胺异环磷酰胺、美法仑(melphalan)、链佐星(streptozocin)、卡莫司汀(carmustine)(BCNU)、洛莫司汀(lomustine)、白消安(busulfan)、达卡巴嗪(dacarbazine)(DTIC)、替莫唑胺(temozolomide)塞替派(thiotepa)、六甲蜜胺(altretamine)、顺铂(cisplatin)、卡铂(carboplatin)、奥沙利铂(oxalaplatin)、柔红霉素(Daunorubicin)、多柔比星(Doxorubicin)表阿霉素(Epirubicin)、伊达比星(Idarubicin)、紫杉醇(paclitaxel)多西他赛(docetaxel)伊沙匹隆(ixabepilone)长春花碱(vinblastine)长春新碱(vincristine)长春瑞滨(vinorelbine)雌莫司汀(Estramustine)
此外,在一个实施方案中,递送罗丹明标记的纳米颗粒后,检查主要的器官没有发现任何显著的血管渗漏。并可以在T-eCAR施用示出了显著的肿瘤血管破损的相同动物中获得这一观察结果。此结果再结合在肺和其它正常器官组织中不能检查出任何显著的出血,一起表明了T-eCAR对正常组织没有显著的毒性。在另一个实施方案中,虽然正常组织的一些内皮细胞与癌症血细胞一样表达了αvβ3整合素,但表达的水平低于容易被T-eCAR检测到的阈值。
在进一步的实施方案中,T-eCAR可以与任何抗血管生成药物(AAD)共同施用,以提高后者的治疗效果。抗血管生成药物可以包括但不限于:血管生成素2、血管抑素、内皮抑素、血小板因子4、阿瓦斯丁、阿柏西普、索拉非尼、舒尼替尼、帕唑帕尼、凡德他尼、瓦他拉尼、西地尼布、阿西替尼等。抗血管生成治疗是基于实体的命题(solidproposition),即血管生成是实体肿瘤的重要体现。近年来已经开发了数种选择性抗血管生成药物(AAD)。然而,迄今这些化合物的骨干仅产生了很大程度上为中度的影响,且没有一个显示出对总生存率有任何改善。AAD较少有最佳治疗结果的主要原因之一是,由于肿瘤血管形成是相对缓慢的过程。这需要长时间的处理,在此期间,肿瘤常常发展出对治疗的耐受。T-eCAR与AAD的组合可以解决这一问题。例如,在一个实施方案中,由T-eCAR对肿瘤血管的初始破坏可以将肿瘤血管生成相对缓慢的过程转换成为急性事件,这将增加肿瘤对抗血管生成治疗的响应。作为另一个示例,在一个实施方案中并如图7所示,向荷瘤动物预施用T-eCAR显著增强了AAD(如帕唑帕尼)的治疗效果。在一个实施方案中,可以通过将CT26肿瘤细胞移植至Balb/c小鼠的右侧腹来建立大肠癌。当肿瘤达到约5mm直径时,可以用下述方式处理肿瘤:1)PBS,2)仅T-eCAR,3)仅AAD,和4)T-eCAR加AAD。在另一个实施方案中,可以从T-eCAR加AAD组合治疗肿瘤中获得最佳的治疗结果,这表明T-eCAR介导的肿瘤血管破坏加强了AAD的治疗效果。可以通过T-eCAR加强任意AAD的治疗效果以增强治疗效果,这是因为所有的AAD都抑制血管生成。
应当理解的是,上述描述旨在进行说明而非限制。所呈现的材料使任何的本领域技术人员能够制造并使用本文所述的发明概念,且提供于具体实施方案的上下文中,其变体对于本领域技术人员而言是显而易见的(例如,一些本公开的实施方案可以相互组合使用)。阅读上述描述后,许多其它的实施方案对于本领域技术人员而言将是显而易见的。因此,本发明的范围应当参照所附的权利要求来确定,且还应包括这些权利要求的等效物的全部范围。在所附的权利要求中,所使用的术语“包括(including)”和“其中(inwhich)”分别是术语“包括(comprising)”和“其中(wherein)”的通俗英语等价物。
实施例
细胞系。人脐静脉内皮细胞(HUVEC)和鼠类黑素瘤细胞系B16-F0获得自ATCC(Manassas,VA)。HUVEC培养于ATCC配制的含有20%胎牛血清(FBS)的杜尔伯克氏改良的伊格尔氏培养基(DMEM;目录号30-2002)中,B16-F0细胞生长于含有100μg/ml链霉素和100U/ml青霉素的10%FBSDMEM中。通过将pIR-eGFP-luc和pCMV-piggyBac质粒共转染进B16-F0,然后通过如前所述的流式细胞术分选及单细胞克隆,在本发明人的实验室建立B16-GFPluc细胞(FuX,etal.,Asimpleandsensitivemethodformeasuringtumor-specificTcellcytotoxicity.PLoSOne2010;5:e11867(2010))。
逆转录病毒载体构建和制备。逆转录病毒载体的构建示意性示于图1A中。通过含有限制性位点XhoI和NcoI的IDT(IntegratedDNATechnologies,Coralville,Iowa)合成Leu-28-锯鳞血抑肽的编码序列(MECESGPCCRCKFLKEGTICKRARGDDLDDYCNGKTCDCPRNPHKGPAT;GenBank:M27213.1)和Myc-标签(EQKLISEEDL)。然后,通过取代HER2ScFv编码序列,将此构建体克隆进载体SFG6FRG5-CD28-ζ中(AhmedN,etal.,RegressionofexperimentalmedulloblastomafollowingtransferofHER2-specificTcells,CancerRes.67:5957-64(2007))。将信号肽(SP)添加至融合基因的5'端。此外,c-Myc标签(c-Myc)已插入到锯鳞血抑肽和CD28之间,以促进eCAR表达的检测。该结构被命名为eCAR。为了构建SFG-GFP,将减去终止密码子的GFP基因类似地插入到SFG-FRG5-CD28-ζ。为了制备病毒母液,使用6转染试剂(RocheAppliedScienceIndianapolis,IN),将逆转录病毒载体构建体转染到逆转录病毒包装细胞系铂-E(Platinum-E)中(MoritaS,etal.,Plat-E:anefficientandstablesystemfortransientpackagingofretroviruses.GeneTher7:1063-6(2000))。48和72小时后,收获上清液,并通过0.45μm的过滤器进行过滤。将纯化的上清液合并,并滴定至293细胞以确定病毒产率。
用逆转录病毒载体转导鼠类脾细胞。脾细胞收集自C57BL/6小鼠,并培养于补充有25mmHEPES、200nML-谷氨酰胺、10%FBS、1%MEM非必需氨基酸、1mM丙酮酸钠、50μΜβ-巯基乙醇、100μg/ml链霉素和100U/ml青霉素的RPMI1640培养基中。用伴刀豆球蛋白A(2μg/ml;Sigma,St.Louis,MO)和鼠IL-2(1ng/ml;ProSpec,EastBrunswick,NJ)刺激悬浮液中的细胞(2×106/ml)24小时,之后将其转移到RetroNectin(TakaraBio.Inc.,Shiga,Japan)包被的非组织培养的24孔板上,用于eCAR或SFG-GFP逆转录病毒的转导。然后,将转导的脾细胞在新鲜的补充有10ng/ml鼠类IL-2的培养基中培养48小时。
流式细胞术分析eCAR和GFP的表达。用含有2%胎牛血清的PBS洗涤转导有eCAR逆转录病毒的脾细胞一次,之后用含有大鼠抗-小鼠CD16/CD32抗体的小鼠BDFc块(BDBiosciences,SanJose,CA)于4℃孵育30分钟。用PBS洗涤两次后,在黑暗中于4℃用缀合有PE的Myc-标签小鼠抗体(Cellsignaling,Danvers,MA)或同种型抗体染色细胞30分钟。用于分析前,将细胞洗涤两次。SFG-GFP转导的细胞直接用于分析而不进行任何染色。然后在BDFACSAriaTMII(BDBiosciences,SanJose,California)上分析两种细胞制备物,数据分析>10,000个事件。为了确定αvβ3整合素的表达,4℃用10μg缀合有异硫氰酸荧光素(FITC)的精氨酸-甘氨酸-天冬氨酸(RGD)肽(AnaSpec,Fremont,CA)于100μl的1%FBS-PBS中,将HUVEC或B16-F0细胞染色30分钟。用PBS洗涤3次后,用相同的BDFACSAriaTMII分析细胞。
逆转录病毒转导的脾细胞的细胞毒性分析。通过可视化或通过最近报道的非放射性定量测量,来分析逆转录病毒转导的脾细胞对靶细胞的细胞毒性(FuX,etal.,Asimpleandsensitivemethodformeasuringtumor-specificTcellcytotoxicity.PLoSOne2010;5:e11867(2010))。对于可视化检测,将5×104的靶细胞孔初始接种到48孔板上。加入逆转录病毒转导的脾细胞(效应细胞)24小时后,效应细胞与靶细胞(E:T)的比例范围为20:1至2.5:1。细胞固定24或48小时后,并用1%的结晶紫在20%的乙醇中染色,以在光学显微镜下可视化并成像。为了定量测量逆转录病毒转导的脾细胞的细胞毒性,首先将1×104靶细胞接种于96孔板上。加入效应细胞24小时后,E:T比例范围为20:1至2.5:1。48小时后,除去培养基,并用PBS漂洗细胞。然后,每孔加入50μl的Bright-GloTM(萤光素酶分析系统,Promega,Madison,WI)。将板轻轻振摇2分钟,使细胞完全裂解。然后将细胞裂解物转移进96孔不透明板,用多模式酶标仪(MolecularDevices,Sunnyvale,CA)进行发光测量。通过下式计算细胞毒性:细胞杀伤(%)=[1-(有效应细胞的孔的读数)/(没有效应细胞的孔的读数)]×100。
细胞因子释放的测量。脾细胞获得自C57BL/6供体。它们是未转染的(UT)或转导有SFG-GFP、eCAR(T-eCAR)或Her2CAR(T-Her2CAR)。Her2CAR构建的细节已报道于本发明人以前的出版物(FuX,etal.,Asimpleandsensitivemethodformeasuringtumor-specificTcellcytotoxicity.PLoSOne2010;5:el1867(2010))中。为了测量CAR介导的细胞溶解期间的细胞因子释放,将HUVEC或表达Her2的4T1-Her2与相应的T-CAR,以1:5的比例在48孔板中混合。由于所有的T-CAR都是由获取自C57BL/6小鼠的脾细胞制备的,它们对于4T1-Her2靶标呈现为同种异体效应T细胞。24小时培养后收集培养物上清液。按照各个制造商的说明,通过ELISA(R&DSystems,Minneapolis,MN)测定IL-2和IFN-γ的量。
动物实验。为了确定肿瘤,将1×105的B16-F0鼠类黑素瘤细胞移植进6至8周大的雄性有免疫活性的C57BL/6小鼠的右侧腹(TaconicFarms,Hudson,NY)。当肿瘤可触及(约5天)时,向小鼠静脉注射eCAR或SFG-GFP逆转录病毒转导的脾细胞(在100μl的RPMI1640中的4×106个)或PBS(每组n=10只小鼠)。每周测量两次肿瘤尺寸,直至实验结束。通过下式计算肿瘤体积:肿瘤体积(mm3)=[长度(mm)]×[宽度(mm)]2×0.52。
为了确定逆转录病毒转染的脾细胞对肿瘤血管的效果,将带有大小相当的B16-F0肿瘤(约8mm直径)的小鼠静脉内注射eCAR或SFG-GFP逆转录病毒转导的脾细胞(在100μlRPMI1640中的5×106个)或PBS(每组n=3只小鼠)。3天后将小鼠人道处死,并切除它们的肿瘤。肿瘤被固定于10%的福尔马林中24小时,然后,在70%乙醇中再放置24小时。然后在ShandonExcelsiorES组织处理仪TM(ThermoScientific,Waltham,MA)中脱水过夜。切割连续的5μm厚的切片,在二甲苯中脱水并逐渐降低乙醇浓度(从100%至50%)。然后用苏木精和曙红将切片染色,以用于在显微镜下观察和拍照。
为了研究肿瘤血管破裂后的纳米颗粒递送,将eCAR或SFG-GFP逆转录病毒转导的脾细胞静脉注射到如上所述的荷瘤小鼠中。48小时后,给小鼠静脉注射标记有罗丹明DHPE(FormuMaxScientific,Inc.PaloAlto,CA)(按10mg/kg稀释于100μl的PBS的剂量)的DSPC/CHOL/mPEG2000-DSPE脂质体纳米颗粒(100μm大小)。脂质体注射后24小时,处死小鼠,收集肿瘤以及包括肺、肾和肝的主要器官。将所收集的肿瘤和器官于4℃固定于4%的多聚甲醛中24小时,然后用25%的蔗糖于4℃再处理24小时,之后将其包埋于OCT中。制备连续的5μm厚的冷冻切片,以用于在荧光显微镜(OlympusBX51)下观察并拍照。用MicroSuiteTMFIVE软件定量罗丹明图像的强度。简言之,在每个切片中随机点击5个区域以获得强度值的读数。使用总共3个切片(每只动物一个)用于定量,以获得各个处理组的平均值。
统计分析。所有定量数据报告为平均值+/-SD。使用方差分析和学生t-测试进行统计学分析以用于多重比较。P值<0.05被认为是统计学上显著的。
Claims (20)
1.用于杀伤癌细胞的化合物,其包括:
移植有嵌合抗原受体(CAR)的T细胞,其中所述CAR包含对αvβ3整合素具有强结合亲和力的靶向部分。
2.根据权利要求1所述的化合物,其中所述靶向部分是锯鳞血抑肽多肽。
3.根据权利要求2所述的化合物,其中所述锯鳞血抑肽多肽中的肽序列连接到T细胞的ζ链。
4.根据权利要求1所述的化合物,其中所述靶向部分是对α5β1整合素具有降低的结合亲和力的突变的锯鳞血抑肽多肽。
5.根据权利要求4所述的化合物,其中所述突变的锯鳞血抑肽多肽在内源性锯鳞血抑肽多肽的氨基酸28上有亮氨酸取代。
6.将嵌合抗原受体(CAR)移植到T细胞的方法,其包括:
用逆转录病毒载体或慢病毒载体转导T细胞,所述逆转录病毒载体或慢病毒载体包含T细胞ζ链以及对αvβ3整合素具有强亲和力的靶向部分的编码序列。
7.根据权利要求6所述的方法,其中所述靶向部分是锯鳞血抑肽多肽。
8.根据权利要求6所述的方法,其中所述靶向部分是对α5β1整合素具有降低的结合亲和力的突变的锯鳞血抑肽多肽。
9.根据权利要求8所述的方法,其中所述突变的锯鳞血抑肽多肽在内源性锯鳞血抑肽多肽的氨基酸28上有亮氨酸取代。
10.根据权利要求6所述的方法,其中所述逆转录病毒载体或慢病毒载体还包含信号肽。
11.根据权利要求7所述的方法,其中所述逆转录病毒载体或慢病毒载体还包含在所述T细胞ζ链和所述锯鳞血抑肽多肽的编码序列之间的CD28结构域。
12.根据权利要求11所述的方法,其中所述逆转录病毒载体或慢病毒载体还包含在所述CD28结构域和所述锯鳞血抑肽多肽的编码序列之间的c-Myc标签。
13.杀伤宿主中的癌细胞的方法,其包括:
向所述宿主施用转导有嵌合抗原受体(CAR)的T细胞,所述CAR包含对αvβ3整合素具有强结合亲和力的靶向部分。
14.根据权利要求13所述的方法,其中所述靶向部分是锯鳞血抑肽多肽。
15.根据权利要求14所述的方法,其中所述锯鳞血抑肽多肽中的肽序列连接到T细胞的ζ链。
16.根据权利要求13所述的方法,其中所述靶向部分是对α5β1整合素具有降低的结合亲和力的突变的锯鳞血抑肽多肽。
17.根据权利要求16所述的方法,其中所述突变的锯鳞血抑肽多肽在内源性锯鳞血抑肽多肽的氨基酸28上有亮氨酸取代。
18.根据权利要求13所述的方法,其中转导的T细胞与一种或多种抗肿瘤小分子共同施用。
19.根据权利要求13所述的方法,其中转导的T细胞与一种或多种抗血管生成剂共同施用。
20.根据权利要求13所述的方法,其中所述抗血管生成剂包括以下的至少一种:血管生成素2、血管抑素、内皮抑素、血小板因子4、阿瓦斯丁、阿柏西普、索拉非尼、舒尼替尼、帕唑帕尼、凡德他尼、瓦他拉尼、西地尼布和阿西替尼。
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US (1) | US20140369977A1 (zh) |
EP (1) | EP3008092A4 (zh) |
JP (1) | JP2016526536A (zh) |
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US10189908B2 (en) | 2014-02-05 | 2019-01-29 | The University Of Chicago | Chimeric antigen receptors recognizing cancer-specific TN glycopeptide variants |
WO2016123143A1 (en) | 2015-01-26 | 2016-08-04 | The University Of Chicago | CAR T-CELLS RECOGNIZING CANCER-SPECIFIC IL 13Rα2 |
EP3250609A4 (en) | 2015-01-26 | 2018-07-11 | The University of Chicago | Il13ra alpha 2 binding agents and use thereof in cancer treatment |
JP2018510160A (ja) * | 2015-03-20 | 2018-04-12 | ブルーバード バイオ, インコーポレイテッド | ベクター製剤 |
KR102623244B1 (ko) | 2016-12-21 | 2024-01-09 | 주식회사 엔케이맥스 | 면역 세포 및 포나티닙을 포함하는 약학 조성물 및 방법 |
RU2020113680A (ru) | 2017-09-19 | 2021-10-20 | Массачусетс Инститьют Оф Текнолоджи | Композиции для т-клеточной терапии с химерным антигенным рецептором и их применения |
EP3856763A1 (en) | 2018-09-28 | 2021-08-04 | Massachusetts Institute of Technology | Collagen-localized immunomodulatory molecules and methods thereof |
US11183799B2 (en) * | 2019-04-05 | 2021-11-23 | Stephen G. Kimmet | Electrical power inlet connection device and method |
US11642409B2 (en) | 2019-06-26 | 2023-05-09 | Massachusetts Insttute of Technology | Immunomodulatory fusion protein-metal hydroxide complexes and methods thereof |
WO2021061648A1 (en) | 2019-09-23 | 2021-04-01 | Massachusetts Institute Of Technology | Methods and compositions for stimulation of endogenous t cell responses |
US20230092787A1 (en) * | 2020-02-17 | 2023-03-23 | University Of Virginia Patent Foundation | Car t cells targeting the integrin alphav beta3 exhibit robust anti-tumor responses against gliomas and other solid tumor malignancies |
EP4118112A1 (en) | 2020-03-10 | 2023-01-18 | Massachusetts Institute of Technology | Compositions and methods for immunotherapy of npm1c-positive cancer |
WO2021183675A2 (en) | 2020-03-10 | 2021-09-16 | Massachusetts Institute Of Technology | Methods for generating engineered memory-like nk cells and compositions thereof |
US20210340524A1 (en) | 2020-05-01 | 2021-11-04 | Massachusetts Institute Of Technology | Methods for identifying chimeric antigen receptor-targeting ligands and uses thereof |
US20210338833A1 (en) | 2020-05-01 | 2021-11-04 | Massachusetts Institute Of Technology | Chimeric antigen receptor-targeting ligands and uses thereof |
WO2023081715A1 (en) | 2021-11-03 | 2023-05-11 | Viracta Therapeutics, Inc. | Combination of car t-cell therapy with btk inhibitors and methods of use thereof |
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US20090257952A1 (en) * | 2006-10-04 | 2009-10-15 | The Board Of Trustees Of The Leland Stanford Junior University | Engineered Integrin Binding Peptides |
WO2012138858A1 (en) * | 2011-04-08 | 2012-10-11 | Baylor College Of Medicine | Reversing the effects of the tumor microenvironment using chimeric cytokine receptors |
US20130040836A1 (en) * | 2006-07-05 | 2013-02-14 | F-Star Biotechnologische Forschungs-Und Entwicklungsges.M.B.H. | Methods for engineering t-cell receptors |
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- 2014-06-13 JP JP2016519666A patent/JP2016526536A/ja active Pending
- 2014-06-13 US US14/303,769 patent/US20140369977A1/en not_active Abandoned
- 2014-06-13 WO PCT/US2014/042239 patent/WO2014201319A1/en active Application Filing
- 2014-06-13 EP EP14811656.9A patent/EP3008092A4/en not_active Withdrawn
- 2014-06-13 CN CN201480036188.3A patent/CN105431456A/zh active Pending
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US20130040836A1 (en) * | 2006-07-05 | 2013-02-14 | F-Star Biotechnologische Forschungs-Und Entwicklungsges.M.B.H. | Methods for engineering t-cell receptors |
US20090257952A1 (en) * | 2006-10-04 | 2009-10-15 | The Board Of Trustees Of The Leland Stanford Junior University | Engineered Integrin Binding Peptides |
WO2012138858A1 (en) * | 2011-04-08 | 2012-10-11 | Baylor College Of Medicine | Reversing the effects of the tumor microenvironment using chimeric cytokine receptors |
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XINPING FU ET AL.,: "Genetically modified T cells targeting neovasculature efficiently destroy tumor blood vessels, shrink established solid tumors and increase nanoparticle delivery", 《INT J CANCER》 * |
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US20140369977A1 (en) | 2014-12-18 |
EP3008092A4 (en) | 2017-01-11 |
EP3008092A1 (en) | 2016-04-20 |
JP2016526536A (ja) | 2016-09-05 |
WO2014201319A1 (en) | 2014-12-18 |
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