CN115279400A - 用于跨血脑屏障递送免疫治疗剂以治疗脑癌的方法和组合物 - Google Patents
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Abstract
本申请涉及增强跨血脑屏障(BBB)的免疫治疗剂的穿透的序列、包含所述序列的组合物、及其用于治疗例如胶质母细胞瘤(GBM)等脑癌的方法。进一步公开了许多鉴定为当插入至腺相关病毒(AAV)的衣壳时增强跨BBB的潜在靶向肽序列。
Description
优先权声明
本申请要求2020年1月10日提交的美国临时申请序列号62/959,625的权益,前述的全部内容通过引用并入本文。
技术领域
本文描述了增强免疫治疗剂跨血脑屏障的穿透的序列、包含所述序列的组合物、及其用于治疗例如胶质母细胞瘤(GBM)等脑癌的方法。
背景技术
多形性胶质母细胞瘤(GBM)是成年人中最常见和致命的脑肿瘤,中位总生存期仅为15个月1。在美国,每年约有12,000例新发GBM病例确诊,发病率为每100,000人中3.2例2。尽管在了解GBM的组织学、分子景观(molecular landscape)和肿瘤微环境方面取得了重大进展3-6,但自2005年以来几乎没有治疗进展。将我们对GBM的丰富知识转化为有效疗法的一个关键障碍是对GBM肿瘤部位的药物递送效率低。静脉内施用是一种方便且广泛适用的给药途径,因为GBM肿瘤具有良好的结构上的血管化(vascularized structurally)7,理论上可以实现良好的肿瘤覆盖。然而,设计跨血脑屏障(BBB)和/或血液-肿瘤屏障的药物仍然具有挑战性。
发明内容
胶质母细胞瘤是一种极其致命的脑癌,很难使用常规方法治疗。全身给药的癌症基因疗法是用于治疗胶质母细胞瘤的一种新的治疗范式。本文描述了脑穿透性AAV病毒载体,将其工程化用于建立用于胶质母细胞瘤基因疗法的血管内基因递送平台,例如,以全身递送PD-L1抗体用于胶质母细胞瘤的治疗。
因此,本文提供用于将免疫治疗剂递送至受试者中的癌症的方法。所述方法包括向受试者施用腺相关病毒(AAV),所述腺相关病毒(AAV)包含(i)包含氨基酸序列的衣壳蛋白,所述氨基酸序列包含来自序列TVSALFK(SEQ ID NO:8);TVSALK(SEQ ID NO:4);KLASVT(SEQ ID NO:83);或KFLASVT(SEQ ID NO:84)的至少四个连续氨基酸,和(ii)编码免疫治疗剂的转基因,任选地,其中癌症细胞在人类受试者的脑中。
在一些实施方案中,氨基酸序列包含来自序列TVSALK(SEQ ID NO:4);TVSALFK(SEQ ID NO:8);KLASVT(SEQ ID NO:83);或KFLASVT(SEQ ID NO:84)的至少五个连续氨基酸。
在一些实施方案中,氨基酸序列包含来自序列TVSALK(SEQ ID NO:4);TVSALFK(SEQ ID NO:8);KLASVT(SEQ ID NO:83);或KFLASVT(SEQ ID NO:84)的至少六个连续氨基酸。
本文还提供用于将免疫治疗剂递送至受试者中的癌症的方法。所述方法包括向受试者施用腺相关病毒(AAV),所述腺相关病毒(AAV)包含(i)包含氨基酸序列的衣壳蛋白,所述氨基酸序列包含来自序列V[S/p][A/m/t/]L(SEQ ID NO:79)、TV[S/p][A/m/t/]L(SEQ IDNO:80)、TV[S/p][A/m/t/]LK(SEQ ID NO:81)或TV[S/p][A/m/t/]LFK(SEQ ID NO:82)的至少四个连续氨基酸,和(ii)编码免疫治疗剂的转基因,任选地,其中癌症细胞在人类受试者的脑中。
在一些实施方案中,靶向序列包含VPALR(SEQ ID NO:1);VSALK(SEQ ID NO:2);TVPALR(SEQ ID NO:3);TVSALK(SEQ ID NO:4);TVPMLK(SEQ ID NO:12);TVPTLK(SEQ IDNO:13);FTVSALK(SEQ ID NO:5);LTVSALK(SEQ ID NO:6);TVSALFK(SEQ ID NO:8);TVPALFR(SEQ ID NO:9);TVPMLFK(SEQ ID NO:10)或TVPTLFK(SEQ ID NO:11)。
在一些实施方案中,所述编码免疫治疗剂的转基因编码靶向PD-1或PD-L1的抗体。
在一些实施方案中,所述受试者是哺乳动物受试者。
在一些实施方案中,所述AAV是AAV9。
在一些实施方案中,所述AAV9包括AAV9 VP1。
在一些实施方案中,所述靶向序列插入在对应于包含SEQ ID NO:85的AAV9 VP1的氨基酸588和589的位置。
在一些实施方案中,所述细胞在受试者的脑中,并且所述AAV通过肠胃外;脑内;或鞘内递送来施用。
在一些实施方案中,所述肠胃外递送是经由静脉内、动脉内、皮下、腹膜内、或肌内递送。
在一些实施方案中,所述鞘内递送是经由腰椎注射、小脑延髓池注射、或脑实质内注射。
在一些实施方案中,所述方法进一步包括向受试者施用化学疗法、放射、和/或手术切除。
在一些实施方案中,所述化学疗法包含替莫唑胺(temozolamide)、洛莫司汀(lomustine)、或其组合。
除非另有定义,否则本文中使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常所理解的相同的含义。本文描述了用于本发明的方法和材料;也可以使用本领域已知的其它的合适的方法和材料。材料、方法和实例仅是说明性的,而不旨在进行限制。本文中提及的所有出版物、专利申请、专利、序列、数据库条目、和其它参考文献均通过引用以其整体并入本文。在有冲突的情况下,本说明书包括定义将占主导。
本发明的其它特征和优点将从以下详细描述和附图以及权利要求中显而易见。
附图说明
图1A-1C描述通过将细胞穿透肽(CPP)插入AAV9的衣壳来工程化AAV9的示例性策略。图1A是AAV9病毒的3D模型。插入在衣壳中氨基酸588和589(VP1编号)之间的个体CPP显示在3倍轴上,在该轴上可能发生受体结合。图1B说明个体AAV的生产方法。将包括pRC(工程化或非工程化的)、pHelper和pAAV的三种质粒共转染至HEK 293T细胞中,并使用碘克沙醇梯度收获和纯化AAV。图1C是包含编码抗PDL1抗体的序列的示例性载体的质粒图。
图2A-2B描述在静脉内施用低剂量候选AAV后小鼠脑切片的代表性图像(图2A)及其定量分析(图2B)。使用具有混合遗传背景的小鼠。候选AAV的不同之处在于它们的插入的CPP(参见表3),但都表达核红色荧光蛋白(RFP)作为报告蛋白(reporter)。具有低产量的候选AAV被排除在进一步筛选之外。AAV的剂量为每个动物1×1010vg(病毒基因组)。图2A中的各个白点代表RFP标记的细胞。在图2B中,*P<0.05,相对于AAV9,ANOVA。
图2C-2D描述在重复实验中在静脉内施用AAV.CPP.11和AAV.CPP.12后小鼠脑切片的代表性图像(图2C)及其定量分析(图2D)。AAV.CPP.11和AAV.CPP.12分别包含CPP BIP1和BIP2(参见表3)。AAV的剂量增加至每个动物1×1011vg。候选AAV表达核红色荧光蛋白(RFP)作为报告蛋白。图2C中的各个白点代表RFP标记的细胞。在图2D中,*P<0.05,**P<0.01,相对于AAV9,ANOVA。
图3A描述BIP靶向序列的优化以便进一步朝向更好的脑转导地工程化AAV9。能够使AAV9更有效地转导脑(如在AAV.CPP.11中)的BIP1(VPALR,SEQ ID NO:1)源自大鼠的蛋白Ku70。人、小鼠和大鼠Ku70蛋白的不同之处在于它们的确切氨基酸序列。BIP2(VSALK,SEQID NO:2)如在AAV.CPP.12中是与BIP1相关的“合成”肽。进一步的工程化聚焦于VSALK序列,以期最小化最终工程化的AAV的物种特异性(species specificity)。为了产生新的靶向序列,将感兴趣的氨基酸添加至VSALK序列中,并且在其它情况下,调换(switched)个别氨基酸的位置。将所有新的源自BIP2的序列再次插入AAV9衣壳以产生用于筛选的新的候选AAV。依次出现的序列是SEQ ID NOs:69、70、71、1-6、72、7和8。
图3B-3C描述在静脉内施用更多候选AAV后小鼠脑切片的代表性图像(图3B)及其定量分析(图3C)。所有候选AAV都表达核红色荧光蛋白(RFP)作为报告蛋白。AAV的剂量为每个动物1×1011vg。图3B中的各个白点代表RFP标记的细胞。AAV.CPP.16和AAV.CPP.21因其强大而广泛的脑转导被识别为热门(top hit)。在图3C中,*P<0.05,**P<0.01,***P<0.001,相对于AAV9,ANOVA。
图3D描述在静脉内施用候选AAV后肝脏中的转导效率的定量分析。显示转导的肝脏细胞的百分比。AAV的剂量为每个动物1×1011vg。***P<0.001,相对于AAV9,ANOVA。
图4A-4E描述在人血脑屏障的体外球状体模型中筛选选定的候选AAV。图4A说明包含在表面形成屏障的人微血管内皮细胞的球状体,以及球状体内部的人周细胞和星形胶质细胞。评价候选AAV从周围介质穿透至球状体内部并转导内部细胞的能力。图4B-4D显示AAV9(图4B)、AAV.CPP.16(图4C)和AAV.CPP.21(图4D)处理的球状体的图。图4E显示不同AAV处理的球状体的相对RFP强度。***P<0.001,相对于AAV9,ANOVA。
图5A-5B描述在C57BL/6J近交系小鼠中在静脉内施用AAV9、AAV.CPP.16和AAV.CPP.21后脑切片的代表性图像(图5A)及其定量分析(图5B)。所有候选AAV都表达核红色荧光蛋白(RFP)作为报告蛋白。AAV的剂量为每个动物1×1012vg。图5A中的各个白点代表RFP标记的细胞。在图5B中,*P<0.05,***P<0.001,ANOVA。
图6A-6B描述在BALB/cJ近交系小鼠中在静脉内施用AAV9、AAV.CPP.16和AAV.CPP.21后脑切片的代表性图像(图6A)及其定量分析(图6B)。所有候选AAV都表达核红色荧光蛋白(RFP)作为报告蛋白。AAV的剂量为每个动物1×1012vg。图6A中的各个白点代表RFP标记的细胞。在图6B中,***P<0.001,ANOVA。
图7A-7B描述在C57BL/6J近交系小鼠中在静脉内施用高剂量的AAV.CPP.16和AAV.CPP.21后脑切片的代表性图像(图7A)及其定量分析(图7B)。两种候选AAV都表达核红色荧光蛋白(RFP)作为报告蛋白。AAV的剂量为每个动物4×1012vg。图7A中的各个白点代表RFP标记的细胞。在图7B中,*P<0.05,学生检验。
图8A显示AAV.CPP.16和AAV.CPP.21在包括皮层、中脑和海马体的小鼠中的多个脑区域中转导成体神经元(由NeuN抗体标记)。转导的神经元由NeuN抗体和RFP共同标记。在成年C57BL/6J小鼠(6周龄)中静脉内施用4×1012vg的AAV。
图8B描述AAV.CPP.16和AAV.CPP.21相对于AAV9显示增强的靶向小鼠的脊髓和运动神经元的能力。在新生小鼠(出生后1天)中静脉内施用4×1010vg的AAV。使用CHAT抗体染色可视化脊髓的腹角中的运动神经元。RFP和CHAT信号的共定位表明了运动神经元的特异性转导。
图9A描述AAV.CPP.16相对于AAV9显示增强的靶向成年小鼠的心脏的能力。在成年C57BL/6J小鼠(6周龄)中静脉内施用1×1011vg的AAV。显示RFP标记的细胞相对于所有DAPI染色的细胞的百分比。*P<0.05,学生检验。
图9B描述AAV.CPP.16相对于AAV9显示增强的靶向成年小鼠的骨骼肌的能力。在成年C57BL/6J小鼠(6周龄)中静脉内施用1×1011vg的AAV。显示RFP标记的细胞相对于所有DAPI染色的细胞的百分比。*P<0.05,学生检验。
图9C描述AAV.CPP.16相对于AAV9显示增强的靶向成年小鼠的背根神经节(DRG)的能力。在成年C57BL/6J小鼠(6周龄)中静脉内施用1×1011vg的AAV。显示RFP标记的细胞相对于所有DAPI染色的细胞的百分比。*P<0.05,学生检验。
图10A描述在非人灵长类动物中静脉内施用后,AAV.CPP.16和AAV.CPP.21相对于AAV9显示增强的转导初级视觉皮层中的脑细胞的能力。将2×1013vg/kg AAVs-CAG-AADC(作为报告基因)静脉内注射至3月龄的具有低预先存在的中和抗体的食蟹猴中。使用针对AADC的抗体染色可视化AAV转导的细胞(以黑色显示)。放大左图中的正方形区域,如右图所示。AAV.CPP.16相对于AAV9转导显著更多的细胞。AAV.CPP.21相对于AAV9也转导了更多的细胞,尽管其效果与AAV.CPP.16相比不太明显。
图10B描述在非人灵长类动物中静脉内施用后,AAV.CPP.16和AAV.CPP.21相对于AAV9显示增强的转导顶叶皮层中的脑细胞的能力。将2×1013vg/kg AAVs-CAG-AADC(作为报告基因)静脉内注射至3月龄的具有低预先存在的中和抗体的食蟹猴中。使用针对AADC的抗体染色可视化AAV转导的细胞(以黑色显示)。放大左图中的正方形区域,如右图所示。AAV.CPP.16相对于AAV9转导显著更多的细胞。AAV.CPP.21相对于AAV9也转导了更多的细胞,尽管其效果与AAV.CPP.16相比不太明显。
图10C描绘了在非人灵长类动物中静脉内施用后,AAV.CPP.16和AAV.CPP.21相对于AAV9显示增强的转导丘脑中的脑细胞的能力。将2×1013vg/kg AAVs-CAG-AADC(作为报告基因)静脉内注射至3月龄的具有低预先存在的中和抗体的食蟹猴中。使用针对AADC的抗体染色可视化AAV转导的细胞(以黑色显示)。放大左图中的方形区域,如右图所示。AAV.CPP.16相对于AAV9转导显著更多的细胞。AAV.CPP.21相对于AAV9也转导了更多的细胞,尽管其效果与AAV.CPP.16相比不太明显。
图10D描述在非人灵长类动物中静脉内施用后,AAV.CPP.16和AAV.CPP.21相对于AAV9显示增强的转导小脑中的脑细胞的能力。将2×1013vg/kg AAVs-CAG-AADC(作为报告基因)静脉内注射至3月龄的具有低预先存在的中和抗体的食蟹猴中。使用针对AADC的抗体染色可视化AAV转导的细胞(以黑色显示)。放大左图中的正方形区域,如右图所示。AAV.CPP.16和AAV.CPP.21二者相对于AAV9都转导了显著更多的细胞。
图11A-11B描述AAV.CPP.16和AAV.CPP.21未结合至LY6A。LY6A用作AAV.PHP.B及其变体包括AAV.PHP.eB的受体(如在US9102949、US20170166926中所描述的),并介导AAV.PHP.eB在某些小鼠品系中跨BBB的强大作用(Hordeaux等,Mol Ther 2019 27(5):912-921;Huang等,2019,dx.doi.org/10.1101/538421)。在培养的293细胞中过表达小鼠LY6A显著增加AAV.PHP.eB与细胞表面的结合(图11A)。相反,过表达LY6A不增加对AAV9、AAV.CPP.16或AAV.CPP.21的病毒结合(图11B)。这表明AAV.CPP.16或AAV.CPP.21不与AAV.PHP.eB共享LY6A作为受体。
图12A-12C描述AAV.CPP.21可用于在胶质母细胞瘤(GBM)的小鼠模型中将治疗性基因全身递送至脑肿瘤。如图11A中所示,静脉内施用的AAV.CPP.21-H2BmCherry显示出靶向肿瘤块(tumor mass),特别是肿瘤扩张边界(tumor expanding frontier)(图12A)。在图11B(图像)和图11C(定量分析)中,当与前药更昔洛韦(ganciclovir)组合时,使用AAV.CPP.21以全身递送“自杀基因”HSV.TK1,引起脑肿瘤块的收缩。HSV.TK1将原本“休眠的”更昔洛韦转变为杀死肿瘤的药物。*P<0.05,学生检验。
图13描述与AAV9相比,当局部注射于成年小鼠脑时,AAV.CPP.21引起更广泛和更有力的脑组织的转导。在成年小鼠(>6周龄)中进行AAV的脑内注射(1×1011vg),并在AAV注射后3周收获并检查脑组织。**P<0.01,学生检验。
图14是比较使用AAV9(上)和AAV.CPP16(下)在小鼠模型中对GBM肿瘤微环境的递送效率的一组图像。如插图(右)所示,AAV.CPP16提供了更大的递送功效。
图15A-C显示AAV.CPP.16-抗PD-L1介导的免疫疗法在鼠GBM模型中延长存活。图15A,实验方案的示意图。图15B,如指示处理的动物的存活率。图15C,使用AAV.CPP16-抗-PDL1治疗的动物的长期存活率。LTS:长期存活率。
图16A-C显示在所有长期存活的小鼠中根除了GBM肿瘤。图16A,肿瘤注射部位后部和前部的脑切片的H&E染色。在任何切片中均无残留的GBM。图16B,肿瘤植入后7天的生物发光成像表明了初始肿瘤植入成功。图16C,具有瘢痕样组织的GBM肿瘤植入部位。
图17A-17B显示通过免疫印迹法测量的HA标记的抗PD-L1抗体在GBM肿瘤中的表达。在小鼠中肿瘤植入后5天,静脉内注射1e12 vg的AAV或PBS。IV注射后14天收获肿瘤组织。HA标签染色的强度(图17A)被定量为抗PD-L1抗体表达的测量值(图17B)。
具体实施方式
有关跨BBB递送相关的困难阻碍了包括癌症等脑病症的治疗剂的开发。腺相关病毒(AAV)已成为一种用于将治疗性基因递送至大脑、脊髓和眼睛的重要的研究和临床工具;参见,例如,US9102949;US9585971;和US20170166926。随着最近Luxturna和Zolgensma的批准,由AAV介导的基因疗法取得了重大进展。批准Zolgensma用于两岁以下的脊髓性肌萎缩症患者的血管内治疗尤为令人鼓舞,因为它证明了使用跨BBB的AAV载体用于中枢神经系统(CNS)的全身性基因疗法的可行性。尽管在年轻患者中取得了成功,但Zolgensma中使用的AAV血清型AAV9,其跨BBB的效率低,特别是在成年人中,这限制了其用于其它CNS疾病的应用8,9。本文描述了在啮齿动物和非人灵长类动物二者中实现比当前行业标准(即AAV9)至少5-10倍增强的下一代的、脑穿透性AAV载体(即AAV.CPP16),其可用于新的跨BBB的AAV平台用于GBM癌症基因疗法。
通过基于已知的细胞穿透肽(CPP)的合理设计和靶向筛选(参见,例如,Gomez等,Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides.Biochem.Soc.Trans.2007;35(Pt 4):797-801),已经发现靶向序列在被工程化至AAV的衣壳中时,将基因至大脑的递送效率提高了多达三个数量级。这些方法用于工程化AAV载体,所述载体在胶质母细胞瘤的动物模型中显著减少肿瘤大小。
此外,大脑是“免疫豁免的(immune privileged)”,这使得GBM的免疫疗法具有挑战性。“引发(priming)”免疫应答是希望将免疫学上“冷(cold)”GBM肿瘤转变为免疫原性的“热(hot)”肿瘤。本发明的方法利用本文所述的载体来递送可以实现该目的的免疫治疗剂,例如,抗PD-L1抗体。不希望受理论束缚,据信AAV载体本身通过增加细胞毒性T细胞的肿瘤浸润来“引发”免疫系统,而在肿瘤部位和在整个CNS中表达的抗PD-L1抗体激活原本“耗竭(exhausted)”的T细胞。
靶向序列
本方法识别了许多潜在的靶向肽,这些肽例如在插入至例如AAV1、AAV2、AAV8、或AAV9等AAV的衣壳时、或当化学地或通过表达为融合蛋白与例如抗体或其它大生物分子等生物剂缀合时,增强通过BBB的穿透。
在一些实施方案中,靶向肽包含至少5个氨基酸的序列。在一些实施方案中,氨基酸序列包含序列VPALR(SEQ ID NO:1)和VSALK(SEQ ID NO:2)的至少4个、例如5个连续的氨基酸。
在一些实施方案中,靶向肽包含X1 X2 X3 X4 X5的序列,其中:
(i)X1、X2、X3、X4是V、A、L、I、G、P、S、T或M的任何四个不相同的氨基酸;和
(ii)X5是K、R、H、D或E(SEQ ID NO:73)。
在一些实施方案中,靶向肽包含至少6个氨基酸的序列。在一些实施方案中,氨基酸序列包含序列TVPALR(SEQ ID NO:3)、TVSALK(SEQ ID NO:4)、TVPMLK(SEQ ID NO:12)和TVPTLK(SEQ ID NO:13)的至少4个、例如5个或6个连续的氨基酸。
在一些实施方案中,靶向肽包含X1 X2 X3 X4 X5 X6的序列,其中:
(i)X1是T;
(ii)X2、X3、X4、X5是V、A、L、I、G、P、S、T或M的任何四个不相同的氨基酸;和
(iii)X6是K、R、H、D或E(SEQ ID NO:74)。
在一些实施方案中,靶向肽包含X1 X2 X3 X4 X5 X6的序列,其中:
(i)X1、X2、X3、X4是来自V、A、L、I、G、P、S、T或M的任何四个不相同的氨基酸;
(ii)X5是K、R、H、D或E;和
(iii)X6是E或D(SEQ ID NO:75)。
在一些实施方案中,靶向肽包含至少7个氨基酸的序列。在一些实施方案中,氨基酸序列包含序列FTVSALK(SEQ ID NO:5)、LTVSALK(SEQ ID NO:6)、TVSALFK(SEQ ID NO:8)、TVPALFR(SEQ ID NO:9)、TVPMLFK(SEQ ID NO:10)和TVPTLFK(SEQ ID NO:11)的至少4个、例如5个、6个或7个连续的氨基酸。在一些其它实施方案中,靶向肽包含X1 X2 X3 X4 X5 X6 X7的序列,其中:
(i)X1是F、L、W或Y;
(ii)X2是T;
(iii)X3、X4、X5、X6是V、A、L、I、G、P、S、T或M的任何四个不相同的氨基酸;和
(iv)X7是K、R、H、D或E(SEQ ID NO:76)。
在一些实施方案中,靶向肽包含X1 X2 X3 X4 X5 X6 X7的序列,其中:
(i)X1是T;
(ii)X2、X3、X4、X5是V、A、L、I、G、P、S、T或M的任何四个不相同的氨基酸;
(iii)X6是K、R、H、D或E;和
(iv)X7是E或D(SEQ ID NO:77)。
在一些实施方案中,靶向肽包含X1 X2 X3 X4 X5 X6 X7的序列,其中:
(i)X1、X2、X3、X4是V、A、L、I、G、P、S、T或M的任何四个不相同的氨基酸;
(ii)X5是K、R、H、D或E;
(iii)X6是E或D;和
(iv)X7是A或I(SEQ ID NO:78)。
在一些实施方案中,靶向肽包含V[S/p][A/m/t/]L的序列(SEQ ID NO:79),其中大写字母在该位置是优选的。在一些实施方案中,靶向肽包含TV[S/p][A/m/t/]L的序列(SEQID NO:80)。在一些实施方案中,靶向肽包含TV[S/p][A/m/t/]LK的序列(SEQ ID NO:81)。在一些实施方案中,靶向肽包含TV[S/p][A/m/t/]LFK的序列(SEQ ID NO:82)。
在一些实施方案中,靶向肽不是由VPALR(SEQ ID NO:1)或VSALK(SEQ ID NO:2)组成的。
包括上述5、6或7-氨基酸序列的特定示例性氨基酸序列列于表1中。
表1–靶向序列
也可以使用包括反向序列的靶向肽,例如,KLASVT(SEQ ID NO:83)和KFLASVT(SEQID NO:84)。
本文公开的靶向肽可以根据本领域已知的用于产生拟肽模物(peptidomimetics)的方法进行修饰。参见,例如,Qvit等,Drug Discov Today.2017年2月;22(2):454-462;Farhadi和Hashemian,Drug Des Devel Ther.2018;12:1239–1254;Avan等,Chem.Soc.Rev.,2014,43,3575-3594;Pathak等,Indo American Journal ofPharmaceutical Research,2015.8;Kazmierski,W.M.,编辑,PeptidomimeticsProtocols,Human Press(Totowa NJ 1998);Goodman等,编辑,Houben-Weyl Methods ofOrganic Chemistry:Synthesis of Peptides and Peptidomimetics,Thiele Verlag(NewYork 2003);以及Mayo等,J.Biol.Chem.,278:45746(2003)。在一些情况下,相对于非拟肽的肽,本文公开的肽和片段的这些修饰的拟肽形式表现出增强的体内稳定性。
产生拟肽的方法包括用D-氨基酸对映异构体替代肽序列中的一个或更多个、例如全部氨基酸。这样的序列在本文中称为“逆向(retro)”序列。在另一种方法中,将氨基酸残基的N-末端至C-末端的顺序反转,使得原始肽的N-末端至C-末端的氨基酸残基的顺序变成修饰的拟肽中C-末端至N-末端的氨基酸残基的顺序。这样的序列可称为“反转(inverso)”序列。
拟肽可以是逆向和反向形式,即,本文公开的肽的“逆向-反转”形式。新的拟肽可以由排列成以使拟肽中N-末端至C-末端的氨基酸残基的顺序对应于原始肽中C-末端至N-末端的氨基酸残基的顺序的D-氨基酸组成。
制备拟肽的其它方法包括用化学上不同但公认的氨基酸功能类似物、即人工氨基酸类似物替换肽中的一个或更多个氨基酸残基。人工氨基酸类似物包括β-氨基酸、β-取代的β-氨基酸(“β3-氨基酸”),氨基酸的含磷类似物诸如α-氨基膦酸和α-氨基次膦酸,以及具有非肽键的氨基酸。可以使用人工氨基酸产生拟肽,诸如类肽低聚物(例如,类肽酰胺或酯类似物)、β-肽、环肽、寡聚脲或寡聚氨基甲酸酯肽;或杂环分子。示例性逆向-反转靶向拟肽包括KLASVT和KFLASVT,其中序列包括所有的D-氨基酸。这些序列可以例如通过氨基末端的生物素化和羧基末端的酰胺化来修饰。
AAVs
用于本方法和组合物中的病毒载体包括重组逆转录病毒、腺病毒、腺相关病毒、α病毒、和慢病毒,其包含本文所述的靶向肽和任选地用于在靶组织中表达的转基因。
在本方法中用于递送核酸的优选的病毒载体系统是腺相关病毒(AAV)。AAV是一种具有25nm衣壳的微小的无包膜病毒。没有疾病已知或已显示与野生型病毒相关。AAV具有单链DNA(ssDNA)基因组。AAV已显示表现出长期的游离型转基因表达,并且AAV已证明在大脑中、特别是在神经元中具有优异的转基因表达。包含少至300个碱基对的AAV载体可被包装并且可整合。外源DNA的空间限制为约4.7kb。诸如在Tratschin等,Mol.Cell.Biol.5:3251-3260(1985)中描述的AAV载体等AAV载体可用于将DNA引入细胞。已经使用AAV载体将多种核酸引入至不同的细胞类型(参见例如Hermonat等,Proc.Natl.Acad.Sci.USA 81:6466-6470(1984);Tratschin等,Mol.Cell.Biol.4:2072-2081(1985);Wondisford等,Mol.Endocrinol.2:32-39(1988);Tratschin等,J.Virol.51:611-619(1984);和Flotte等,J.Biol.Chem.268:3781-3790(1993)。存在许多可供选择的AAV变体(已克隆超过100种),并且已根据所需特性识别AAV变体。在一些实施方案中,AAV是AAV1、AAV2、AAV4、AAV5、AAV6、AV6.2、AAV7、AAV8、AAV9、rh.10、rh.39、rh.43或CSp3;对于CNS使用,在一些实施方案中,AAV是AAV1、AAV2、AAV4、AAV5、AAV6、AAV8或AAV9。作为一个实例,已显示AAV9比较有效地跨血脑屏障。使用本方法,可以通过将如本文所述的靶向序列插入至衣壳蛋白中、例如插入至AAV9衣壳蛋白VP1中氨基酸588和589之间,来基因工程化AAV衣壳以增加跨BBB的穿透、或增加向特定组织的穿透。
示例性野生型AAV9衣壳蛋白VP1(Q6JC40-1)序列如下所示:
因此本文提供了包括一种或更多种本文所述的靶向肽序列的AAV,例如,包括含有本文所述的靶向序列的衣壳蛋白的AAV,例如,包含SEQ ID NO:1的衣壳蛋白,其中靶向肽序列已插入至该序列中,例如,在氨基酸588和589之间。
免疫治疗性转基因
在一些实施方案中,AAV还包括编码例如本文所述或本领域已知的免疫治疗剂的转基因序列(即异源序列)。转基因优选连接至促进/驱动转基因在靶组织中的表达的序列。
用作免疫治疗剂的示例性转基因包括编码免疫检查点抑制性抗体或其抗原结合片段、例如充当检查点抑制剂的单链可变区片段(scFv)抗体的转基因。
免疫疗法的实例包括但不限于旨在诱导T淋巴细胞识别癌细胞的过继性T细胞疗法或癌症疫苗制剂,以及检查点抑制剂,诸如抗CD137抗体(例如,BMS-663513)、抗PD1抗体(例如,纳武单抗(Nivolumab)、派姆单抗(pembrolizumab)/MK-3475、匹地利珠单抗(Pidilizumab)(CT-011))、抗PDL1抗体(例如,BMS-936559、MPDL3280A)、或抗CTLA-4抗体(例如,伊匹单抗(ipilumimab);参见,例如,Krüger等,(2007)Histol Histopathol.22(6):687-96;Eggermont等,(2010)Semin Oncol.37(5):455-9;Klinke(2010)Mol.Cancer.9:242;Alexandrescu等,(2010)J.Immunother.33(6):570-90;Moschella等,(2010)Ann N YAcad Sci.1194:169-78;Ganesan和Bakhshi,(2010)Natl.Med.J.India23(1):21-7;以及Golovina和Vonderheide,(2010)Cancer J.16(4):342-7。
可用于本文所述方法的示例性抗PD-1抗体包括与人PD-1结合的那些;示例性PD-1蛋白序列提供于NCBI登录号NP_005009.2。示例性抗体描述于美国专利号8,008,449;9,073,994;和美国公开号2011/0271358,包括例如PF-06801591、AMP-224、BGB-A317、BI754091、JS001、MEDI0680、PDR001、REGN2810、SHR-1210、TSR-042、派姆单抗、纳武单抗、阿维鲁单抗(avelumab)、西米普利单抗(Cemiplimab)、斯巴达珠单抗(Spartalizumab)、卡瑞利珠单抗(Camrelizumab)、信迪利单抗(Sintilimab)、匹地利珠单抗、替雷利珠单抗(Tislelizumab)、特瑞普利单抗(Toripalimab)、AMP-224、AMP-514和阿特珠单抗(atezolizumab)。
可用于本文所述方法的示例性抗-CD40抗体包括与人CD40结合的那些;示例性CD40蛋白前体序列提供于NCBI登录号NP_001241.1、NP_690593.1、NP_001309351.1、NP_001309350.1和NP_001289682.1。示例性抗体包括描述于国际公开号WO2002/088186;WO2007/124299;WO2011/123489;WO2012/149356;WO2012/111762;WO2014/070934;美国公开号2013/0011405;2007/0148163;2004/0120948;2003/0165499;和美国专利号8,591,900中的那些;包括例如达西组单抗(dacetuzumab)、卢卡木单抗(lucatumumab)、布来鲁单抗(bleselumab)、替奈昔单抗(teneliximab)、ADC-1013、CP-870,893、Chi Lob 7/4、HCD122、SGN-4、SEA-CD40、BMS-986004和APX005M。在一些实施方案中,抗CD40抗体是CD40激动剂,而不是CD40拮抗剂。
可用于本文所述方法的示例性抗PD-L1抗体包括与人PD-L1结合的那些;示例性PD-L1蛋白序列提供于NCBI登录号NP_001254635.1、NP_001300958.1和NP_054862.1。示例性抗体描述于美国公开号2017/0058033;国际公开号WO2017/118321A1;WO2016/061142A1;WO2016/007235A1;WO2014/195852A1和WO2013/079174A1,包括例如BMS-936559(MDX-1105)、FAZ053、KN035、阿特珠单抗(Tecentriq,MPDL3280A)、阿维鲁单抗(Bavencio)、德瓦鲁单抗(Durvalumab)(Imfinzi,MEDI-4736)、恩弗利单抗(Envafolimab)(KN035)、CK-301、CS-1001、SHR-1316(HTI-1088)、CBT-502(TQB-2450)、BGB-A333和BMS-986189。也可以使用非抗体肽抑制剂,例如AUNP12、CA-170。也参见Akinleye&Rasool,Journal of Hematology&Oncology,12:92(2019)doi:10.1186/s13045-019-0779-5。
在一些实施方案中,免疫治疗剂是抗PD-L1抗体的抗原结合部分或包含抗PD-L1抗体的抗原结合部分,例如针对人PD-L1蛋白(PD-L1.Hu)的单链可变区片段(scFv)抗体;编码抗PDL1抗体scFv的示例性序列示于SEQ ID NO:105或其一部分,例如缺失信号肽、HA-标签和Myc-标签的一个、两个或更多个,例如包含SEQ ID NO:105的氨基酸(aa)31-513:
示例性抗PDL1 scFv序列(信号肽(aa 1-21);HA-标签,aa 21-30;Myc-标签,aa514-523)
以下是示例性抗PD-L1核酸序列(信号肽(nt 1-63);HA-标签,nt 64-90;Myc-标 签,nt1540-1569)
其它抗体、以及产生编码这些抗体的核酸的方法是本领域已知的;参见,例如,Li等,Int J Mol Sci.2016Jul;17(7):1151;Engeland等,Mol Ther.2014Nov;22(11):1949–1959和上文中的参考文献。
病毒还可以包括一种或更多种促进转基因表达的序列,例如一种或更多种启动子序列;增强子序列,例如5’非翻译区(UTR)或3’UTR;多聚腺苷酸化位点;和/或隔离子序列。在一些实施方案中,启动子是脑组织特异性启动子,例如神经元特异性或神经胶质特异性启动子。在某些实施方案中,启动子是选自以下的基因的启动子:神经元核(NeuN)、神经胶质原纤维酸性蛋白(GFAP)、MeCP2、腺瘤性息肉病(APC)、离子化钙结合适配分子1(Iba-1)、突触蛋白I(SYN)、钙/钙调蛋白依赖性蛋白激酶II、微管蛋白αI、神经元特异性烯醇酶和血小板源性生长因子β链。在一些实施方案中,启动子是泛细胞型启动子,例如,巨细胞病毒(CMV)、β葡糖醛酸酶(GUSB)、泛素C(UBC)、或劳斯肉瘤病毒(RSV)启动子。也可以使用土拨鼠肝炎病毒转录后反应元件(WPRE)。在一些实施方案中,使用人信号或前导序列,例如,IgK前导序列。在一些实施方案中,替代使用人信号序列,如下表所示(表改编自novoprolabs.com/support/articles/commonly-used-leader-peptide-sequences-for-efficient-secretion-of-a-recombinant-protein-expressed-in-mammalian-cells-201804211337.html):
*,Barash等,Biochem Biophys Res Commun.2002Jun 21;294(4):835-42。在一些实施方案中,使用促进抗体的分泌的分泌序列,例如,如von Heijne,J Mol Biol.1985年7月5日;184(1):99-105;Kober等,Biotechnol.Bioeng.2013;110:1164–1173;Tsuchiya等,Nucleic Acids Research Supplenzent No.3 261-262(2003)中所描述的。
在一些实施方案中,AAV还具有一种或更多种另外的突变,所述突变增加向例如CNS等靶组织的递送、或减少组织外靶向,例如在预期CNS、心脏或肌肉递送时减少肝脏递送的突变(例如,如Pulicherla等,(2011)Mol Ther19:1070-1078中所描述的);或添加其它靶向肽,例如,如Chen等,(2008)Nat Med 15:1215-1218或Xu等,(2005)Virology 341:203-214或US9102949;US9585971;和US20170166926中所描述的。也参见Gray和Samulski(2011)“Vector design and Considerations for CNS applications”,Gene Vector Designand Application to Treat Nervous System Disorders ed.Glorioso J.,编辑,(Washington,DC:Society for Neuroscience;)1-9,在可获取自sfn.org/~/media/SfN/Documents/Short%20Courses/2011%20Short%20Course%20I/2011_SC1_Gray.ashx。
使用方法
本文所述的方法和组合物可用于将免疫治疗组合物递送至组织,例如递送至中枢神经系统(脑)、心脏、肌肉或背根神经节或脊髓(周围神经系统)。在一些实施方案中,所述方法包括递送至特定的脑区域,例如,皮层、小脑、海马体、黑质、或杏仁核。在一些实施方案中,所述方法包括递送至神经元、星形胶质细胞、和/或神经胶质细胞。
在一些实施方案中,所述方法和例如AAV等组合物用于将编码免疫治疗剂的核酸序列递送至患有脑癌的受试者。脑癌包括神经胶质瘤(例如,多形性胶质母细胞瘤(GBM))、转移癌(例如,来自肺癌、乳腺癌、黑色素瘤、或结肠癌)、脑膜瘤、垂体腺瘤、和听神经瘤。因此,所述方法可以包括向已诊断患有脑癌的受试者例如静脉内等全身施用含有如本文所述的靶向肽并编码免疫治疗剂的AAV(例如AAV9)(例如,其中插入有CPP 16的AAV9,在本文中也称为AAV.CPP16)。
在一些实施方案中,所述方法还包括共同施用化学治疗剂。在一些实施方案中,化学治疗剂是毒素或细胞毒性药物,包括但不限于替莫唑胺、洛莫司汀、或其组合。参见,例如Herrlinger等,Lancet.2019年2月16日;393(10172):678-688。所述方法还可包括施用放射、手术切除或两者。
药物组合物和施用方法
本文所述的方法包括使用含有作为活性成分的AAV的药物组合物,所述AAV包含(i)靶向肽和(ii)编码免疫治疗剂的序列。
药物组合物通常包含药学上可接受的载体。如本文中所用的,术语“药学上可接受的载体”包括与药物施用相容的盐水、溶剂、分散介质、包衣、抗菌剂和抗真菌剂、等渗剂和吸收延迟剂等。
药物组合物通常配制成与其预期的给药途径相容。给药途径的实例包括肠胃外,例如静脉内、动脉内、皮下、腹膜内、肌内或注射或输注施用。因此,递送可以是全身的或局部的。
配制合适的药物组合物的方法是本领域已知的,参见,例如,Remington:TheScience and Practice of Pharmacy,21st ed.,2005;以及Drugs and thePharmaceutical Sciences:a Series of Textbooks and Monographs(Dekker,NY)系列书籍。例如,用于肠胃外应用的溶液或悬浮液可包括以下组分:无菌稀释剂,诸如注射用水、盐水溶液、固定油、聚乙二醇、甘油、丙二醇、或其它合成溶剂;抗菌剂,诸如苯甲醇或对羟基苯甲酸甲酯;抗氧化剂,诸如抗坏血酸或亚硫酸氢钠;螯合剂,诸如乙二胺四乙酸;缓冲剂,诸如乙酸盐、柠檬酸盐或磷酸盐,以及用于调节张力(tonicity)的试剂,例如氯化钠或葡萄糖。可以用诸如盐酸或氢氧化钠等酸或碱调节pH。肠胃外制剂可以封装在安瓿、一次性注射器或者玻璃或塑料制成的多剂量小瓶中。
适用于可注射用途的药物组合物可包括无菌水溶液(水溶性)或分散液,和用于临时制备无菌可注射溶液或分散液的无菌粉末。对于静脉内施用,合适的载体包括生理盐水、抑菌水、Cremophor ELTM(BASF,Parsippany,NJ)或磷酸盐缓冲液(PBS)。在所有情况下,所述组合物必须是无菌的,并且应该以易于注射的程度流动。它在制造和储存条件下应该是稳定的,并且必须防止例如细菌和真菌等微生物的污染作用。载体可以是溶剂或分散介质,其含有例如水、乙醇、多元醇(例如甘油、丙二醇和液体聚乙二醇等)、以及它们的适当混合物。例如,可以通过使用诸如卵磷脂等包衣、在分散液的情况下通过保持所需的粒度和通过使用表面活性剂来保持适当的流动性。可以通过各种抗菌剂和抗真菌剂来实现防止微生物的作用,例如对羟基苯甲酸酯、三氯叔丁醇、苯酚、抗坏血酸、硫柳汞等。在许多情况下,优选在所述组合物中包括等渗剂,例如糖、多元醇如甘露醇、山梨糖醇、氯化钠。可通过在所述组合物中包含延迟吸收的试剂,例如单硬脂酸铝和明胶,可以实现可注射组合物的延长吸收。
可以通过将所需量的活性化合物与所需的上述列举的成分的一种或其组合掺入适当的溶剂中,然后过滤灭菌来制备无菌可注射溶液。通常,通过将活性化合物掺入无菌溶媒(vehicle)中来制备分散液,所述溶媒含有基本分散介质和来自上述列举的那些的所需的其它成分。在用于制备无菌注射液的无菌粉末的情况下,优选的制备方法是真空干燥和冷冻干燥,这样可以从先前无菌过滤的溶液中产生活性成分和任何其它所需成分的粉末。
在一个实施方案中,将治疗性化合物与将保护治疗性化合物免于从体内快速消除的载体一起制备,诸如作为控释制剂,包括植入物和微囊化的给药系统。可以使用可生物降解的、生物相容性的聚合物,例如乙烯乙酸乙烯酯、聚酸酐、聚乙醇酸、胶原蛋白、聚原酸酯、和聚乳酸。此类制剂可以使用标准技术制备,或例如从Alza Corporation和NovaPharmaceuticals,Inc.等处商购获得。脂质体悬浮液(包括靶向具有针对细胞抗原的单克隆抗体的选定细胞的脂质体)也可以用作药学上可接受的载体。这些可以根据本领域技术人员已知的方法来制备,例如,如美国专利号4,522,811中所描述的。
药物组合物可以与施用说明一起包含在试剂盒、容器、包装或分配器(dispenser)中。
实施例
在以下实施例中进一步描述本发明,这些实施例不限制权利要求中描述的本发明的范围。
材料和方法
在以下实施例中使用了以下材料和方法。
1.衣壳变体的产生
为了生成衣壳变体质粒,使用CloneEZ无缝克隆技术(GenScript)合成了编码细胞穿透肽(表3)的DNA片段(GenScript),并在氨基酸位置588和589(VP1氨基酸编号)之间插入至AAV9 Rep-cap质粒(pRC9)的骨架中。CPPs BIP1(VPALR,SEQ ID NO:1)和BIP2(VSALK,SEQID NO:2)以及它们的诸如AAV.CPP.16中的TVSALK(SEQ ID NO:4)和AAV.CPP.21中的TVSALFK(SEQ ID NO:8)等衍生物,源自Ku70蛋白,其序列如下所示:
此外,AAV9、AAV.CPP.16和AAV.CPP.21的VP1蛋白序列如下所示:
2.重组AAV的生产
使用标准的三质粒共转染方案(pRC质粒、pHelper质粒和pAAV质粒)包装重组AAV。使用聚乙烯亚胺(PEI,Polysciences)将pRC9(或其变体)、pHelper和携带转基因(例如由普遍存在的EF1a启动子驱动的核定向RFPH2B-mCherry)的pAAV共转染至HEK 293T细胞中。在转染后72小时和120小时从无血清培养基中收集rAAV载体,并在转染后120小时从细胞中收集rAAV载体。使用由8%PEG-8000(wt/vol)的PEG沉淀法浓缩培养基中的AAV颗粒。重悬浮含有病毒颗粒的细胞沉淀并且通过超声处理裂解。在37℃下用DNase和RNase处理来自PEG沉淀和细胞裂解物的组合的病毒载体30分钟,然后使用超速离心法(VTi 50转子,40,000r.p.m,18℃,1小时)通过碘克沙醇梯度(15%、25%、40%和60%)进行纯化。然后使用Millipore Amicon过滤器单元(UFC910008,100K MWCO)浓缩rAAV,并在含有0.001%Pluronic F68(Gibco)的杜氏磷酸盐缓冲液(PBS)中配制。
3.AAV滴定法
通过使用定量PCR测定DNase抗性基因组拷贝来确定病毒滴度。用PVUII(NEB)消化pAAV-CAG-GFP以产生质粒ITR的自由末端,并用于产生标准曲线。用DNase I孵育病毒样品以消除污染的DNA,然后用氢氧化钠处理以溶解病毒衣壳并释放病毒基因组。使用ITR正向引物5’-GGAACCCCTAGTGATGGAGTT(SEQ ID NO:91)和ITR反向引物5’-CGGCCTCAGTGAGCGA(SEQ ID NO:92)进行定量PCR。将载体滴度相对于rAAV-2参考标准材料(RSMs,ATCC,目录号:VR-1616,Manassas,VA)归一化。
4.小鼠中AAV的施用
对于静脉内施用,将稀释于无菌盐水(0.2ml)中的AAV通过尾静脉注射施用于成年小鼠(6周龄以上)。使动物存活三周,然后安乐死以收获组织。对于脑内注射,使用汉密尔顿(Hamilton)注射器注射稀释于PBS(10ul)中的AAV,来自前囟的坐标:右1.0mm,后0.3mm,深2.6mm。所有动物研究均在IACUC批准的AAALAC认可的设施中进行。
5.小鼠组织处理
麻醉后的动物用冷磷酸盐缓冲液(PBS)、接着用4%多聚甲醛(PFA)进行心脏灌注。将组织在4%PFA中后固定(post-fix)过夜,然后在30%蔗糖溶液中浸泡两天,随后将其在OCT中包埋并速冻。通常,切出80um厚脑切片用于自然荧光成像,40um厚脑切片用于IHC。
6.体外人BBB球状体模型
将热的1%琼脂糖(w/v,50ul)添加至96孔板中以冷却/固化。然后将原代人星形胶质细胞(Lonza Bioscience)、人脑微血管周细胞(HBVP,ScienCell ResearchLaboratories)和人脑微血管内皮细胞(hCMEC/D3;Cedarlane)以1:1:1的比例(每种类型1500个细胞)接种至琼脂糖凝胶上。在5%CO2培养箱中在37℃下培养细胞48~72小时以自发组装多细胞BBB球状体。据报道,在球状体的外围形成了多细胞屏障,模仿了BBB。将AAVs-H2B-mCherry添加至培养基中,并在4天后使用4%PFA固定所有球状体,将球状体转移至Nunc Lab-Tek II薄玻璃8孔室盖玻片(Thermo Scientific)中,并使用Zeiss LSM710共聚焦显微镜成像。检查球状体内的RFP信号强度并将其用作“读数”。
7.非人灵长类(NHP)中的AAV的施用
所有NHP研究均由CRO在IACUC批准的AAALAC认可的设施中进行。对食蟹猴进行很少或没有的预先存在的针对AAV9的中和抗体的预筛(<1:5的滴度)。使用蠕动泵静脉内(通过头静脉或股静脉)注射稀释于PBS/0.001%F68中的AAV。3周后,用PBS、接着用4%PFA对动物进行心脏灌注。然后收集组织并处理以用于石蜡包埋和切片。
8.免疫组化
使用稀释于含有10%驴血清和2%Triton X-100的PBS的一抗对小鼠组织切片进行漂浮染色。使用的一抗包括:鸡抗GFP(1:1000);兔抗RFP(1:1000);小鼠抗NeuN(1:500);大鼠抗GFAP(1:500);山羊抗GFAP(1:500);小鼠抗CD31(1:500)。针对一抗的宿主物种,以1:200的稀释应用与Alexa Fluor 488、Alexa Fluor 555或Alexa Fluor 647荧光团缀合的二抗。
对于NHP组织的石蜡切片,进行DAB染色以可视化由AAV-AADC转导的细胞。兔抗AADC抗体(1:500,Millipore)用作一抗。
9.AAV结合试验
在5%CO2培养箱中在37℃下培养HEK 293T细胞。在将HEK 293T细胞以每孔250,000个细胞的密度接种至24孔板中一天后,使用200ul DMEM(31053028;Gibco)、1ug DNA质粒和3ug的PEI的转染混合物将LY6A的cDNA质粒瞬时转染至细胞中。转染后48小时,将细胞置于冰上冷却10分钟。然后将培养基更换为500ul的含有MOI为10000的rAAVs-mCherry的冰冷无血清DMEM培养基。在冰上孵育1小时后,用冷PBS洗涤推测表面结合有AAV的细胞3次,然后进行基因组DNA提取。通过使用对mCherry特异性的引物的qPCR定量结合细胞的病毒颗粒,并使用人GCG作为参照将其归一化至HEK293T基因组。
10.胶质母细胞瘤小鼠模型
所有实验均按照布里格姆妇女医院(Brigham and Women’s Hospital)和哈佛医学院(Harvard Medical School)动物护理和使用委员会(IACUC)批准的方案进行。使用重量20+/-1g(Envigo)的同系免疫活性C57BL/6雌性小鼠。使用具有26号针头的10μl注射器(80075;Hamilton)颅内注射重悬浮于2μl磷酸盐缓冲液(PBS)中的GL261-Luc(100,000个小鼠胶质母细胞瘤细胞)。使用立体定向框架(stereotactic frame)定位植入部位(前囟坐标,以mm为单位:右2,前0.5,皮层深度3.5)。7天后,一次施用200ul AAV-HSV-TK1(1E+12病毒基因组,IV),并且每天施用更昔洛韦(50mg/kg),持续10天。
实施例1.AAV9衣壳的修饰
为了鉴定可增强生物分子或病毒跨血脑屏障的穿透的肽序列,使用AAV肽展示技术,将表3中列出的个体细胞穿透肽插入至AAV9衣壳中在氨基酸588和589(VP1编号)之间,如图1A中所示。通过修饰RC质粒进行插入,RC质粒是用于AAV包装的三种共转染质粒之一;图1B显示了实验的示例性示意图。分别产生和筛选个体AAV变体。有关更多详细信息,参见材料和方法#1-3。
表3
#,SEQ ID NO:
Syn,合成的
实施例2.第一轮体内筛选
将表达核RFP(H2B-RFP)的AAV静脉内注射至具有混合的C57BL/6和BALB/c遗传背景的成年小鼠。3周后,收获脑组织并切片以显示RFP标记的细胞(图2A和2C中的白点,分别在图2B和2D中定量)。将CPP BIP1和BIP2分别插入至AAV.CPP.11和AAV.CPP.12的衣壳中。有关更多详细信息,参见材料和方法#4-5。
实施例3.修饰的AAV9衣壳的优化
通过优化BIP靶向序列进一步工程化AAV.CPP.11和AAV.CPP.12。BIP插入物来源于蛋白Ku70(完整序列参见图3A和材料/方法#1)。选择作为“合成”来源的BIP序列VSALK作为研究重点,以最小化工程化的AAV载体的潜在物种特异性。产生AAV,并与AAV9相比,分别测试了AAV的脑转导效率(参见图3B-C)。在图3D中显示了IV注射一些递送报告基因RFP的AAV变体后3周的小鼠肝脏中的细胞转导的百分比。有关更多详细信息,参见材料和方法#1-5。
实施例4.体外模型-BBB穿透筛选
使用体外球状体BBB模型筛选了一些AAV变体跨人BBB的能力。所述球状体包含在表面形成屏障的人微血管内皮细胞、以及人周细胞和星形胶质细胞。评价了携带核RFP作为报告蛋白的AAV从周围介质穿透至球状体内并转导内部细胞的能力。图4A显示了实验示意图。图4B-D分别显示了wtAAV9、AAV.CPP.16和AAV.CPP.21的结果,这些和其它肽在图4E中定量。在该模型中,肽11、15、16和21产生了进入球状体的最大穿透作用。有关更多详细信息,参见材料和方法#6。
实施例5.体内BBB穿透筛选
以如上文实施例2中所述进行的实验,选择了AAV.CPP.16和AAV.CPP.21用于在体内模型中进一步评价。所有AAV都携带核RFP作为报告蛋白。在静脉内施用于C57BL/6J成年小鼠(图5A中的脑切片中的白点,在图5B中定量)和BALB/c成年小鼠(图6A中的脑切片中的白点,在图6B中定量)后,两者都显示出了相对于AAV9增强的转导脑细胞的能力。
高剂量的AAV.CPP.16和AAV.CPP.21(每只小鼠4×1012vg,IV施用)引起了小鼠中广泛的脑转导。两种AAV都携带核RFP作为报告蛋白(图7A中的脑切片中的白点,在图7B中定量)。
实施例6.修饰的AAV的体内分布
如图8A中所示,AAV.CPP.16和AAV.CPP.21在包括皮层、中脑和海马体的小鼠中的多个脑区域中优先靶向神经元(由NeuN抗体标记)。两种AAV都携带核RFP作为报告蛋白。
AAV.CPP.16和AAV.CPP.21相对于AAV9也显示增强的靶向小鼠中的脊髓和运动神经元的能力。所有的AAV都携带核RFP作为报告蛋白,并静脉内施用于新生小鼠(4×1010vg)。使用CHAT抗体染色可视化运动神经元。图8B中,RFP和CHAT信号的共定位表明了运动神经元的特异性转导。
还评价了AAV-CAG-H2B-RFP和AAV.CPP.16-CAG-H2B-RFP转导小鼠中各种组织的相对能力。静脉内注射1×1011vg。将转导的细胞数归一化为DAPI核染色标记的总细胞数。结果表明,AAV.CPP.16在靶向小鼠的心脏(图9A);骨骼肌(图9B)和背根神经节(图9C)组织方面比AAV9更有效。
实施例7.非人灵长类模型中的BBB穿透
将2×1013vg/kg AAVs-CAG-AADC(作为报告基因)静脉内注射至3月龄的食蟹猴。使用针对AADC的抗体染色可视化AAV转导的细胞(以黑色显示)。如图10A-D中所示,在静脉内施用于非人类灵长类动物后,AAV.CPP.16和AAV.CPP.21相对于AAV9显示增强的转导脑细胞的能力。AAV.CPP.16在初级视觉皮层(图10A)、顶叶皮层(图10B)、丘脑(图10C)、和小脑(图10D)中比wtAAV9转导显著更多的细胞。有关更多详细信息,参见材料和方法#7-8。
实施例8.AAV.CPP.16和AAV.CPP.21不结合LY6A
LY6A作为AAV.PHP.eB的受体并介导AAV.PHP.eB在某些小鼠品系中跨BBB的强大作用。在培养的293细胞中过表达小鼠LY6A显著增加了AAV.PHP.eB与细胞表面的结合(参见图11A)。相反,过表达LY6A不增加与AAV9、AAV.CPP.16或AAV.CPP.21的病毒结合(参见图11B)。这表明了AAV.CPP.16或AAV.CPP.21不与AAV.PHP.eB共享LY6A作为受体。有关更多细节,参见材料和方法#9。
实施例9.使用AAV.CPP.21将治疗性蛋白递送至脑
AAV.CPP.21用于在脑肿瘤小鼠模型中全身递送“自杀基因”HSV.TK1(材料和方法#10)。HSV.TK1将原本“休眠”的更昔洛韦转变为杀死肿瘤的药物。静脉内施用AAV.CPP.21-H2BmCherry(图12A,左下图和右中图)显示出靶向肿瘤块,尤其是肿瘤扩张边界。如图12B-C中所示,当与前药更昔洛韦结合时,使用AAV.CPP.21全身递送“自杀基因”HSV.TK1引起脑肿瘤肿块的收缩。这些结果表明AAV.CPP.21可用于将治疗性基因全身递送至脑肿瘤中。有关更多详细信息,参见材料和方法#10。
实施例10.AAV.CPP.21的脑内施用
除了全身施用(例如在实施例2中),将如本文所述的AAV局部施用于小鼠的脑。AAV9-H2B-RFP和AAV.CPP.21-H2B-RFP(图13)的脑内注射导致了AAV.CPP.21处理的脑切片中相对于AAV9处理的脑切片更广泛和更高强度的RFP信号。有关更多详细信息,参见材料和方法#4。
实施例11.AAV.CPP.16向胶质母细胞瘤肿瘤微环境的全身递送
使用全身施用(例如在实施例2中),将如本文所述的AAV递送至原位免疫活性小鼠胶质母细胞瘤模型(GL261模型)的脑中。(如材料和方法#10中所述)。如图14中所示,AAV.CPP16远远优于AAV9,大量递送至肿瘤和周围微环境。
为了确定这种提高的递送效率是否会转化为改善的治疗功效,对小鼠GBM模型施用各种治疗;图15A提供了实验方案的示意图。结果,如图15B-C中所示,证明了AAV.CPP.16-抗PD-L1介导的免疫疗法在鼠GBM模型中显著延长存活。如图15B中所示,用AAV9-抗PD-L1治疗的8只小鼠中的1只长期存活,而用AAV.CPP.16-抗PD-L1治疗的8只小鼠中的6只长期存活(超过100天)。图15C显示了所有6只长期存活者(5只用AAV.CPP.16-抗PD-L1治疗的加上1只用AAV9-抗PD-L1治疗的;1只用AAV.CPP.16-抗PD-L1治疗的长期存活者由于技术原因在再攻击(re-challenge)手术期间死亡)在肿瘤植入后200天仍然存活。因此,静脉内注射表达靶向小鼠PD-L1的抗体的AAV.CPP.16可在75%的小鼠中根除GBM肿瘤,而未经治疗的小鼠在肿瘤植入后一个月内死亡。
在200天时处死长期存活的小鼠,并检查它们的大脑。如图16A中所示,没有证据表明有肿瘤残余。图16B显示了对具有延长生存期的小鼠之一拍摄的生物发光图像,显示在植入后7天存在肿瘤细胞。图16C显示了初始的肿瘤植入物没有残留肿瘤,并且仅残余胶质瘢痕组织,表明了完全的肿瘤根除。
此外,免疫组织化学显示CB8+细胞毒性T细胞也存在于GBM肿瘤部位,进一步证明了免疫反应。
实施例12.HA标记的抗PD-L1抗体在GBM肿瘤中的表达
图17A-17B中显示了通过免疫印迹法测量的HA标记的抗PD-L1抗体在GBM肿瘤中的表达。在小鼠中肿瘤植入后5天静脉内注射1e12 vg的AAV或PBS。IV注射后14天收获肿瘤组织。HA标签染色的强度(图17A)被定量为抗PD-L1抗体表达的测量值(图17B)。
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其它实施方式
应当理解,虽然已经结合本发明的详细描述说明了本发明,但是前述描述旨在进行说明而非限制本发明的范围,本发明的范围由所附权利要求的范围限定。其它方面、优点和修改也在所附权利要求的范围之内。
Claims (15)
1.一种将免疫治疗剂递送至受试者中的癌症的方法,所述方法包括向所述受试者施用腺相关病毒(AAV),所述腺相关病毒(AAV)包含(i)包含氨基酸序列的衣壳蛋白,所述氨基酸序列包含来自序列TVSALFK(SEQ ID NO:8);TVSALK(SEQ ID NO:4);KLASVT(SEQ ID NO:83);或KFLASVT(SEQ ID NO:84)的至少四个连续氨基酸,和(ii)编码免疫治疗剂的转基因,任选地,其中癌症细胞在人类受试者的脑中。
2.根据权利要求1所述的方法,其中所述氨基酸序列包含来自序列TVSALK(SEQ ID NO:4);TVSALFK(SEQ ID NO:8);KLASVT(SEQ ID NO:83);或KFLASVT(SEQ ID NO:84)的至少五个连续氨基酸。
3.根据权利要求1所述的方法,其中所述氨基酸序列包含来自序列TVSALK(SEQ ID NO:4);TVSALFK(SEQ ID NO:8);KLASVT(SEQ ID NO:83);或KFLASVT(SEQ ID NO:84)的至少六个连续氨基酸。
4.一种将免疫治疗剂递送至受试者中的癌症的方法,所述方法包括向所述受试者施用腺相关病毒(AAV),所述腺相关病毒(AAV)包含(i)包含氨基酸序列的衣壳蛋白,所述氨基酸序列包含来自序列V[S/p][A/m/t/]L(SEQ ID NO:79)、TV[S/p][A/m/t/]L(SEQ ID NO:80)、TV[S/p][A/m/t/]LK(SEQ ID NO:81)或TV[S/p][A/m/t/]LFK(SEQ ID NO:82)的至少四个连续氨基酸,和(ii)编码免疫治疗剂的转基因,任选地,其中癌症细胞在人类受试者的脑中。
5.根据权利要求4所述的方法,其中靶向序列包括VPALR(SEQ ID NO:1);VSALK(SEQ IDNO:2);TVPALR(SEQ ID NO:3);TVSALK(SEQ ID NO:4);TVPMLK(SEQ ID NO:12);TVPTLK(SEQID NO:13);FTVSALK(SEQ ID NO:5);LTVSALK(SEQ ID NO:6);TVSALFK(SEQ ID NO:8);TVPALFR(SEQ ID NO:9);TVPMLFK(SEQ ID NO:10)或TVPTLFK(SEQ ID NO:11)。
6.根据权利要求1-5所述的方法,其中所述编码免疫治疗剂的转基因编码靶向PD-1或PD-L1的抗体。
7.根据权利要求6所述的方法,其中所述受试者是哺乳动物受试者。
8.根据权利要求7所述的方法,其中所述AAV是AAV9。
9.根据权利要求8所述的方法,其中所述AAV9包括AAV9 VP1。
10.根据权利要求9所述的方法,其中所述靶向序列插入在对应于包含SEQ ID NO:85的AAV9 VP1的氨基酸588和589的位置。
11.根据权利要求7所述的方法,其中所述细胞在所述受试者的脑中,并且所述AAV通过肠胃外递送;脑内;或鞘内递送来施用。
12.根据权利要求11所述的方法,其中所述肠胃外递送是经由静脉内、动脉内、皮下、腹膜内或肌内递送。
13.根据权利要求12所述的方法,其中所述鞘内递送是经由腰椎注射、小脑延髓池注射或脑实质内注射。
14.根据权利要求1-13中任一项所述的方法,其还包括向所述受试者施用化学疗法、放射和/或手术切除术。
15.根据权利要求14所述的方法,其中所述化学疗法包括替莫唑胺、洛莫司汀或其组合。
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JP2023510784A (ja) | 2023-03-15 |
EP4087602A4 (en) | 2023-11-29 |
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