CN105682679B - 用于表达前列腺相关抗原的载体 - Google Patents
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- CN105682679B CN105682679B CN201480059609.4A CN201480059609A CN105682679B CN 105682679 B CN105682679 B CN 105682679B CN 201480059609 A CN201480059609 A CN 201480059609A CN 105682679 B CN105682679 B CN 105682679B
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Abstract
本公开提供(a)包含编码两个、三个或更多个免疫原性PAA多肽的多抗原构建体的载体;(b)包含所述载体的组合物,(c)涉及所述载体和组合物在引发免疫应答或治疗前列腺癌中的用途的方法。
Description
相关申请的引用
本申请要求2013年11月1日提交的美国临时专利申请第61/898,966号的优先权,其整体援引加入本文。
序列表的引用
本申请与电子格式的序列表一起提交。所述序列表提供为.txt格式的题为“PC72055A_FF_SEQ_LISTING_ST25.txt”的文件,其创建于2014年10月6日且大小为429KB。所述.txt文件所含的序列表是本说明书的部分,并且整体援引加入本文。
发明领域
本发明大体上涉及免疫疗法,并且特别地涉及治疗或预防肿瘤性病症的疫苗以及方法。
背景技术
前列腺癌是全世界发达国家的男性中第二最常诊断的癌症以及癌症相关死亡的第四主要原因。已显示与正常对应物相比,各种前列腺相关抗原(PAA)如前列腺特异性抗原(PSA)、前列腺特异性膜抗原(PSMA)和前列腺干细胞抗原(PSCA)被前列腺癌细胞过量表达。因此,这些抗原代表用于通过使用基于疫苗的免疫疗法诱导针对表达所述抗原的癌症的特异性免疫应答的可能靶标。(参见例如Marrari,A.,M.lero,et al.(2007)."Vaccinationtherapy in prostate cancer."Cancer Immunol lmmunother 56(4):429-45)
PSCA为123-氨基酸膜蛋白。天然全长人PSCA由SEQ ID NO:21的氨基酸1和4-125组成(在第二和第三位置分别没有丙氨酸和丝氨酸残基)。PSCA具有高组织特异性,并且在超过85%的前列腺癌样本上表达,表达水平随着较高的Gleason评分而升高且具有雄激素独立性。其在80-100%的前列腺癌的骨转移中表达。
PSA是专门由前列腺的腺泡和导管内壁的柱状上皮细胞产生的激肽释放酶样丝氨酸蛋白酶。PSA mRNA翻译为失活的261-氨基酸preproPSA前体。PreproPSA具有构成前区(信号多肽)的24个额外的残基和多肽原。多肽原的释放导致237-氨基酸的成熟胞外形式,其是酶促活性的。天然人PSA的全长序列由SEQ ID NO:15的氨基酸4-263组成。PSA是器官特异性的,结果其由良性前列腺增生(BPH)组织、原发性前列腺癌组织和转移性前列腺癌组织的上皮细胞产生。
也称作叶酸水解酶1(FOLH1)的PSMA由750个氨基酸组成。全长人PSMA的氨基酸序列在SEQ ID NO:1中提供。PSMA包括胞质结构域(氨基酸1-19)、跨膜结构域(氨基酸20-43)和胞外结构域(氨基酸44-750)。据发现PSMA在前列腺癌细胞中以比正常组织高1000倍的水平表达。其在各种其他实体瘤如结肠癌、乳腺癌、肝癌、膀胱癌、胰腺癌、肺癌、肾癌以及黑素瘤和肉瘤的新生血管上大量表达。因此,认为PSMA不仅是前列腺癌细胞特异性的靶标,而且还是其他癌症的pan-癌靶标。
虽然已鉴定大量肿瘤相关抗原,并且已探索许多这些抗原作为基于蛋白或基于DNA的疫苗用于治疗或预防癌症,但是到目前为止大部分临床试验未能产生治疗产品。开发癌症疫苗的挑战之一在于癌症抗原通常是自我衍生的,因此是弱免疫原性的,因为免疫系统自我调节为不识别自身蛋白。因此,需要增强癌症疫苗的免疫原性或疗效的方法。
已探索许多方法用于增强免疫原性或增强癌症疫苗的抗肿瘤效力。这样的方法之一包括使用各种免疫调节剂如TLR激动剂、TNFR激动剂、CTLA-4抑制剂和蛋白激酶抑制剂。
Toll样受体(TLR)是在造血和非造血细胞上表达的1型膜受体。在TLR家族中已鉴定至少11个成员。这些受体的特征在于它们能够识别病原生物体表达的病原相关分子模式(PAMP)。先天免疫系统中的这些受体对随后获得的免疫应答的极性施加控制。在TLR中,因为其在免疫应答中的功能,已广泛研究TLR9。通过增加促炎细胞因子的产生和共刺激分子呈递至T细胞,TLR9受体的刺激指导抗原呈递细胞(APC)启动有效的TH1为主的T-细胞应答。据发现TLR9的配体CpG寡核苷酸是一类有效的免疫刺激因子。已测试CpG疗法抗小鼠中的各种肿瘤模型,并且一致地显示促进肿瘤抑制或退化。
细胞毒性的T-淋巴细胞抗原4(CTLA-4)是免疫球蛋白超家族的成员,并且在辅助性T细胞的表面上表达。CTLA-4是CD28依赖性T细胞激活的负调节物,并且充当适应性免疫应答的抑制检验点。与T-细胞共刺激蛋白CD28相似,CTLA-4结合至抗原呈递细胞上的CD80和CD86。CTLA-4将抑制信号传递至T细胞,而CD28传递刺激信号。针对人CTLA-4的人抗体已描述为许多疾病状况中的免疫刺激调节剂,如治疗或预防病毒和细菌感染以及用于治疗癌症(WO 01/14424和WO 00/37504)。各种临床前研究已显示通过单克隆抗体阻断CTLA-4会增强针对免疫原性肿瘤的宿主免疫应答,并且甚至可以排斥建立的肿瘤。已在临床试验中于各种类型实体瘤的治疗中研究两种全人抗人CTLA-4单克隆抗体(mAb)依匹木单抗(MDX-010)和曲美木单抗(Tremelimumab)(也称作CP-675206)。
肿瘤坏死因子(TNF)超家族是吸引特定同源细胞表面受体TNF受体(TNFR)超家族的一组细胞因子。肿瘤坏死因子超家族的成员通过配体介导的三聚化来发挥作用,引起几种胞内接头的募集已激活多个信号转导途径如凋亡、NF-kB途径、JNK途径以及免疫和炎症应答。TNF超家族的实例包括CD40配体、OX40配体、4-1BB配体、CD27、CD30配体(CD153)、TNF-α、TNF-β、RANK配体、LT-α、LT-β、GITR配体和LIGHT。TNFR超家族包括例如CD40、OX40、4-1BB、CD70(CD27配体)、CD30、TNFR2、RANK、LT-βR、HVEM、GITR、TROY和RELT。在TNF成员中,已广泛探索CD40激动剂在治疗中的使用,包括各种CD40激动性抗体,如全人激动剂CD40单克隆抗体CP870893。
蛋白激酶是催化蛋白中特定残基的磷酸化的酶家族。在临床研究中已研究许多激酶抑制剂用于抗癌疗法,其包括例如MK0457、VX-680、ZD6474、MLN8054、AZD2171、SNS-032、PTK787/ZK222584、索拉菲尼(BAY43-9006)、SU5416、SU6668AMG706、Zactima(ZD6474)、MP-412、达沙替尼、CEP-701、(来妥替尼)、XL647、XL999、Tykerb、(拉帕替尼)、MLN518、(以前称作CT53518)、PKC412、ST1571、AMN107、AEE 788、OSI-930、OSI-817、苹果酸舒尼替尼(Sutent;SU11248)、伐他拉尼(PTK787/ZK222584)、SNS-032、SNS-314以及阿昔替尼(AG-013736)。吉非替尼和厄洛替尼是两种可口服的EGFR-TKI。
发明概述
本公开涉及用于表达两个或更多个免疫原性PAA多肽的构建自黑猩猩腺病毒ChAd68基因组序列的载体。所述载体包含(1)C68DNA序列,(2)用于表达两个或更多个免疫原性PAA多肽的多抗原构建体,以及(3)控制抗原产物(即,所述免疫原性PAA多肽)的转录和翻译的调节序列。所述载体中包括的C68DNA序列是通过一个或多个病毒基因的功能缺失源自C68基因组序列,但是足以产生感染性病毒颗粒。在一特定实施方案中,所述载体中使用的C68DNA序列是整个C68基因组,仅在E1和E3区中具有功能缺失。
所述载体携带的多抗原构建体包含编码选自免疫原性PSMA多肽、免疫原性PSA多肽和免疫原性PSCA多肽的两个或更多个免疫原性PAA多肽的核苷酸序列。在一些实施方案中,所述载体携带的多抗原构建体包含(1)编码至少一个免疫原性PSMA多肽的核苷酸序列,(2)编码至少一个免疫原性PSA多肽的核苷酸序列,以及(3)编码至少一个免疫原性PSCA多肽的核苷酸序列。所述多抗原构建体还可以包括使得能够表达所述构建体编码的不同PAA多肽的分隔物序列。分隔物序列的实例包括2A肽序列和IRES。在一些实施方案中,所述载体包含具有以下结构之一的多抗原构建体:
(1)PSA-F2A-PSMA-mIRES-PSCA;
(2)PSA-F2A-PSMA-T2A-PSCA;
(3)PSA-T2A-PSCA-F2A-PSMA;和
(4)PSCA-F2A-PSMA-mIRES-PSA.
在一些实施方案中,编码免疫原性PSA多肽的核苷酸序列包含SEQ ID NO:58的核苷酸1115-1825或包含SEQ ID NO:58的核苷酸1106-1825,编码免疫原性PSCA多肽的核苷酸序列包含SEQ ID NO:58的核苷酸1892-2257或包含SEQ ID NO:58的1886-2257,并且编码免疫原性PSMA多肽的核苷酸序列包含SEQ ID NO:58的核苷酸2333-4543或包含SEQ ID NO:58的核苷酸2324-4543。在一些具体实施方案中,所述多抗原构建体包含选自SEQ ID NO:33、34、35和36的核苷酸序列。在一特定实施方案中,所述多抗原构建体包含编码SEQ ID NO:60的多肽序列的核苷酸序列。在另一特定实施方案中,所述多抗原构建体包含SEQ ID NO:61的核苷酸序列。
本公开还提供包含所述载体的组合物。在一些实施方案中,所述组合物为免疫原性组合物,其可用于在哺乳动物如小鼠、狗、猴或人中引发针对PAA的免疫应答。在一些实施方案中,所述组合物为疫苗组合物,其可用于免疫哺乳动物如人,用于抑制异常细胞增殖,用于提供针对癌症发展的保护(用作预防剂),或者用于治疗与PAA过量表达相关的病症(用作治疗剂),如癌症,特别是前列腺癌。
本公开还涉及利用所述载体或组合物引发针对PAA的免疫应答或者治疗哺乳动物(特别是人)中的癌症(如前列腺癌)的方法。在一些实施方案中,将所述载体或组合物(包括疫苗组合物)与增强所述载体或组合物的免疫原性或效果的一种或多种免疫调节剂联合向哺乳动物(特别是人)给药。在一些特定实施方案中,所述方法包括共给药本发明提供的疫苗联合至少一种免疫抑制细胞抑制剂和至少一种免疫效应细胞增强剂。
附图说明
图1.PJV7563载体的示意图。
图2.5种病毒2A盒的氨基酸比对。跳过的甘氨酸-脯氨酸键用星号表示。
图3.优选的EMCV IRES的序列。翻译起始位点用星号表示。最小IRES元件不包括下划线的EMCV L蛋白的前5个密码子。
图4.示出来自代表性研究的小鼠组的Kaplan-Meier存活曲线的图,所述代表性研究评价在包含皮下TUBO肿瘤的BALB/neuT小鼠中苹果酸舒尼替尼(Sutent)和抗小鼠CTLA-4单克隆抗体(克隆9D9)对癌症疫苗(疫苗)的抗肿瘤效力的影响。
图5.示出来自代表性研究的IFNγELISPOT结果的图,所述代表性研究评价CpG7909和抗CD40抗体(Bioxcell#BE0016-2)对癌症疫苗(rHER2)诱导的抗原特异性T细胞应答的影响。
图6.示出代表性研究结果的图,所述代表性研究利用胞内细胞因子染色测定评价CpG7909对癌症疫苗(PMED)诱导的免疫应答性能的免疫调节活性,其中测量细胞因子阳性CD8T细胞。(*表示通过学生T-检验P<0.05)。
图7.示出代表性研究结果的图,所述代表性研究利用胞内细胞因子染色测定评价CpG7909对癌症疫苗(PMED)诱导的免疫应答性能的免疫调节活性,其中测量细胞因子阳性CD4T细胞(图7)。(*表示通过学生T-检验P<0.05)。
图8.示出代表性研究结果的图,所述代表性研究利用胞内细胞因子染色测定评价激动性抗小鼠CD40单克隆抗体对癌症疫苗(PMED)诱导的免疫应答性能的免疫调节活性,其中测量细胞因子阳性CD8T细胞。(*表示通过学生T-检验P<0.05)
图9.示出代表性研究结果的图,所述代表性研究利用胞内细胞因子染色测定评价激动性抗小鼠CD40单克隆抗体对癌症疫苗(PMED)诱导的免疫应答性能的免疫调节活性,其中测量细胞因子阳性CD4T细胞。(*表示通过学生T-检验P<0.05)
图10.示出来自代表性研究的小鼠组的Kaplan-Meier存活曲线的图,所述代表性研究评价在包含自发性乳腺肿瘤的BALB/neuT小鼠中低剂量苹果酸舒尼替尼(Sutent)对癌症疫苗的抗肿瘤效力的影响。
图11.示出AdC68-734的基因组结构的图。
CMV Enh/pro=人巨细胞病毒即时早期增强子和启动子;
tet op=四环素操纵子;T2A=Thosea asigna病毒2A;F2A=口蹄疫病毒2A;SV40pA=猿猴病毒40多腺苷酸化信号;LITR=左侧反向末端重复;RITR=右侧反向末端重复。
图12.点图,其示出通过流式细胞术,在用表达三抗原的AdC68载体转导的A549细胞表面上PSMA和PSCA的表达。
图13.来自AdC68载体感染的A549的裂解物的蛋白印迹。
发明详述
A.定义
术语“佐剂”指能够增强、加速或延长疫苗免疫原引发的免疫应答的物质。
术语“激动剂”指促进(诱导、引起、增强或增加)另一分子或受体的活性的物质。术语激动剂涵盖结合受体的物质(例如,抗体、来自另一物种的天然配体的同系物)以及促进受体功能但不与其结合(例如,通过激活相关蛋白)的物质。
术语“拮抗剂”或“抑制剂”指部分或完全阻断、抑制或中和另一分子或受体的生物活性的物质。
术语“共给药”指在治疗期间向相同个体给药两种或更多种药剂。所述两种或更多种药剂可以包含在单一制剂中,因此同时给药。或者,所述两种或更多种药剂可以在不同物理制剂中,并且顺序或同时向个体分别给药。术语“同时给药(administeredsimultaneously)”或“同时给药(simultaneous administration)”表示第一药剂的给药和第二药剂的给药在时间上互相重叠,而术语“顺序给药(administered sequentially)”或“顺序给药(sequential administration)”表示第一药剂的给药和第二药剂的给药在时间上互相不重叠。
术语“胞质”表示通过宿主细胞表达编码特定多肽的核苷酸序列之后,所表达的多肽保留在宿主细胞内。
术语“简并变体”指这样的核苷酸序列,与参考核苷酸序列相比其具有碱基取代,但是由于遗传密码的简并性,其编码与参考核苷酸序列相同的氨基酸序列。
术语“有效量”指向哺乳动物给药的足以在所述哺乳动物中引起期望的效应的量。
术语给定多肽的“片段”指比给定多肽短且与给定多肽的序列享有100%相同性的多肽。
在两个或更多个核酸或多肽序列的上下文中,术语“相同”或百分比“相同性”指在比较和比对最大对应性时,相同或者具有特定百分比的相同氨基酸残基或核苷酸的两个或更多个序列或自序列。
术语“免疫-效应子-细胞增强子”或“IEC增强子”指能够增加或增强哺乳动物的一种或多种类型的免疫效应细胞的数量、性能或功能的物质。免疫效应细胞的实例包括胞质CD8T细胞、CD40T细胞、NK细胞和B细胞。
术语“免疫调节剂”指能够改变(例如,抑制、减少、增加、增强或刺激)哺乳动物的先天、体液或细胞免疫系统的任何组分的工作的物质。因此,术语“免疫调节剂”涵盖如本文定义的“免疫-效应子-细胞”和如本文定义的“免疫-抑制-细胞”,以及影响哺乳动物免疫系统的其他组分的物质。
术语“免疫应答”指宿主脊椎动物的免疫系统对特定物质(如抗原或免疫原)的任何可检测的应答,包括但不限于先天免疫应答(例如,Toll受体信号级联的激活)、细胞介导的免疫应答(例如,T细胞如抗原特异性T细胞和免疫系统的非特异性细胞介导的应答)和体液免疫应答(例如,B细胞介导的应答,抗体的产生和分泌入血浆、淋巴和/或组织液)。免疫应答的实例包括Toll样受体激活的改变(例如,增加)、淋巴因子(例如,细胞因子(例如,Th1、Th2或Th17型细胞因子)或趋化因子)表达或分泌、巨噬细胞激活、树突细胞激活、T细胞(例如,CD4+或CD8+T细胞)激活、NK细胞激活、B细胞激活(例如,抗体产生和/或分泌)、免疫原(例如,抗原(例如,免疫原性多肽))结合至MHC分子、细胞毒性T淋巴细胞("CTL")应答的诱导、B细胞应答(例如,抗体产生)的诱导、免疫系统细胞(例如,T细胞和B细胞)的扩大(例如,细胞群体的生长)以及抗原呈递细胞的抗原加工和呈递增加。术语“免疫应答”还涵盖在体外脊椎动物免疫系统的一种或多种组分对特定物质(如抗原或免疫原)的任何可检测的应答。
术语“免疫原性”指针对特定抗原,物质引起、引发、刺激或诱导免疫应答的能力,或者提高、增强、增加或延长预先存在的免疫应答的能力,无论单独或当连接至载体时,存在或不存在佐剂。
术语“免疫原性PSA多肽”指多肽,其针对人PSA蛋白或针对表达人PSA蛋白的细胞是免疫原性的。
术语“免疫原性PSCA多肽”指多肽,其针对人PSCA蛋白或针对表达人PSCA蛋白的细胞是免疫原性的。
术语“免疫原性PSMA多肽”指多肽,其针对人PSMA蛋白或针对表达人PSMA蛋白的细胞是免疫原性的。
术语“免疫原性PAA多肽”指如上文定义的“免疫原性PSA多肽”、“免疫原性PSCA多肽”或“免疫原性PSMA多肽”。
术语“免疫-抑制-细胞抑制剂”或“ISC抑制剂”指能够诱导或抑制哺乳动物免疫抑制细胞的数量或功能的物质。免疫抑制细胞的实例包括调节T细胞(“T reg”)、髓源性抑制细胞和肿瘤相关的巨噬细胞。
在向包括人在内的哺乳动物给药物质如治疗剂或免疫调节剂的上下文中,术语“皮内给药(intradermal administration)”或“皮内给药(administeredintradermally)”指将所述物质递送入哺乳动物皮肤的真皮层。哺乳动物的皮肤由3层组成-表皮层、真皮层和皮下层。表皮是比较薄、坚韧、外层的皮肤。表皮中的大多数细胞为角质形成细胞。皮肤的下一层真皮是纤维和弹性组织的厚层(大部分由胶原、弹性蛋白和肌原纤蛋白组成),其给予皮肤灵活性和强度。真皮包含神经末梢、汗腺和油腺(皮脂腺)、毛囊和血管。根据皮肤的位置,真皮的厚度不同。在人中,在眼睑上其为约0.3mm,而在背上其为约3.0mm。皮下层由容纳较大血管和神经的脂肪和结缔组织组成。这层的厚度在整个身体不同,并且人与人不同。术语“皮内给药”指将物质递送至真皮层内。相比之下,“皮下给药”指将物质给药入皮下层,而“体表给药”指将物质给药至皮肤表面上。
术语“局部给药(local administration)”或“(administered locally)”涵盖“体表给药”、“皮内给药”和“皮下给药”,各自如上文所定义。这个术语还涵盖“肿瘤内给药”,肿瘤内给药指将物质给药至肿瘤内。局部给药意图允许在给药部位周围高局部浓度一段时间,直至达到所给药的物质的全身生物分布,而“全身给药”意图将所给药的物质吸收入血液并通过循环系统分布至整个身体的器官或组织来达到快速全身暴露。
术语“哺乳动物”指哺乳动物纲的任何动物物种。哺乳动物的实例包括:人;非人灵长类如猴;实验室动物如大鼠、小鼠、豚鼠;家养动物如猫、狗、兔、牛、绵羊、山羊、马和猪;以及圈养野生动物如狮子、老虎、大象等。
术语“膜结合”表示通过宿主细胞表达编码特定多肽的核苷酸序列之后,所表达的多肽结合至、连接至细胞的膜或以其他方式与细胞的膜关联。
术语“肿瘤性病症”指其中细胞以异常高和不受控制的速度增殖的疾病状况,所述速度超过周围正常组织的速度且与周围正常组织的速度不协调。其通常导致称作“肿瘤”的实体病变或肿块。这个术语涵盖良性和恶性肿瘤性病症。在本公开中可与术语“癌症”交换使用的术语“恶性肿瘤性病症”指这样的肿瘤性病症,其特征在于肿瘤细胞扩散至体内的其他位置的能力(称作“转移”)。术语“良性肿瘤性病症”指其中肿瘤细胞缺少转移能力的肿瘤性病症。
术语“可操作地连接”指并列,其中这样描述的组分的关系允许它们以它们预期的方式发挥功能。“可操作地连接”至转基因的控制序列以这样的方式连接,从而在与控制序列相容的条件下实现转基因的表达。
术语“药学可接受的赋形剂”指免疫原性或疫苗组合物中除活性成分(例如,抗原、编码抗原的核酸、免疫调节剂或佐剂)以外的物质,其与活性成分相容,并且不会在给药的个体中引起显著的不良反应。
术语“肽”、“多肽”和“蛋白”在本文中可交换使用,并且指任何长度的聚合形式的氨基酸,其可以包括编码和非编码的氨基酸、化学或生物化学修饰或衍生的氨基酸、以及具有修饰的多肽骨架的多肽。
术语“预防(preventing)”或“预防(prevent)”指(a)阻止病症出现或(b)延迟病症的发生或病症症状的发生。
术语“前列腺相关抗原”(或PAA)指TAA(如本文定义),其特异性地在前列腺肿瘤细胞上表达,或者以通过肿瘤细胞比通过相同组织类型的非肿瘤细胞高的频率或密度表达。PAA的实例包括PSA、PSCA和PSMA。
在多肽的上下文中,术语“分泌”表示通过宿主细胞表达编码多肽的核苷酸序列之后,所表达的多肽分泌至宿主细胞外。
当用来描述免疫调节剂如蛋白激酶抑制剂的量时,术语“亚适量”指免疫调节剂的剂量,其低于在向患者单独给药免疫调节剂时对治疗的疾病产生期望的疗效所需要的最小量。
术语“治疗(treating)”、“治疗(treatment)”或“治疗(treat)”指消除病症、减少病症的严重程度、或者减少病症症状的严重程度或发生频率。
术语“肿瘤相关抗原”或“TAA”指抗原,其由肿瘤细胞特异性地表达,或者以通过肿瘤细胞比通过相同组织类型的非肿瘤细胞高的频率或密度表达。肿瘤相关抗原可以是宿主不正常表达的抗原;它们可以是宿主正常表达的突变、截短、错误折叠或其他异常表现的分子;它们可以与正常表达的分子相同但以异常高的水平表达;或者它们可以在异常的背景或环境中表达。肿瘤相关抗原可以是例如蛋白或蛋白片段、复合碳水化合物、神经节苷脂、半抗原、核酸或者这些或其他生物分子的任何组合。
术语“疫苗”指用于向哺乳动物给药以引发针对特定抗原的免疫应答的免疫原性组合物。
术语“载体”指能够转运或转移外源核酸分子的核酸分子。外源核酸分子称作“插入物”或“转基因”。载体一般由插入物和充当载体骨架的较大序列组成。术语“载体”涵盖表达载体和转录载体。术语“表达载体”指能够在靶细胞中表达插入物的载体。其一般包含驱动插入物表达的控制序列,如增强子、启动子和终止子序列。术语“转录载体”指能够转录但不翻译的载体。转录载体用来扩增它们的插入物。基于载体的结构或来源,载体的主要类型包括质粒载体、粘粒载体、噬菌体载体如λ噬菌体、病毒载体如腺病毒(Ad)载体以及人工染色体。
B.包含多抗原构建体的载体
在一方面,本公开提供用于表达两个或更多个免疫原性PAA多肽的构建自黑猩猩腺病毒ChAd68基因组的病毒载体。黑猩猩腺病毒ChAd68在文献中还称作猿猴腺病毒25、C68、Chad68、SAdV25、PanAd9或Pan9。为了方便,黑猩猩腺病毒ChAd68在本说明书中可以称作“C68”,而构建自黑猩猩腺病毒ChAd68基因组的病毒载体称作“C68载体”。C68的全长基因组序列可获得自Genbank(登录号AC_000011.1),并且在SEQ ID NO:57中提供。此外,C68的全长基因组序列以及腺病毒基因E1a、E1b、E2a、E2b、E3、E4、l1、l2、L3、L4和L5位置还在美国专利6,083,716中提供。
本公开提供的C68载体包含(1)C68DNA序列,和(2)用于表达两个或更多个免疫原性PAA多肽的多抗原构建体。所述载体还可以包含控制抗原产物的转录和翻译的非天然调节序列。所述非天然调节序列指不是C68基因组部分的序列。C68DNA序列、多抗原构建体和调节序列互相可操作地连接。
C68载体可以是可复制的,条件可复制的或复制缺陷的。可复制的C68载体可以在典型的宿主细胞(即,通常能够被腺病毒感染的细胞)中复制。可复制的病毒载体与野生型腺病毒相比可以在腺病毒基因组中具有一个或多个突变(例如,一个或多个缺失、插入和/或取代),所述突变不抑制宿主细胞中的病毒复制。条件复制的C68载体是已工程化以在预定条件下复制的病毒载体。例如,可以将复制必不可少的基因功能如腺病毒早期区域编码的基因功能可操作地连接至可诱导、可抑制或组织特异性的转录控制序列如启动子。复制缺陷的C68载体是需要补充复制所需的病毒基因组的一个或多个基因功能或区域的病毒载体,作为例如一个或多个复制必不可少的基因功能或区域缺陷的结果,所述载体不在典型的宿主细胞中复制,特别是待所述载体感染的人中的宿主细胞。
所述载体可用于克隆或表达免疫原性PAA多肽,或者用于将组合物中的多抗原构建体如疫苗递送至宿主细胞或宿主动物如人。在一些特定实施方案中,本公开提供选自以下的载体:(i)包含SEQ ID NO:58的核苷酸序列或由SEQ ID NO:58的核苷酸序列组成的载体;(ii)包含SEQ ID NO:58的核苷酸9–34811或由SEQ ID NO:58的核苷酸9–34811组成的载体;以及(iii)包含SEQ ID NO:63的核苷酸序列或由SEQ ID NO:63的核苷酸序列组成的载体。
本公开提供的C68载体还涵盖本公开中具体描述或示例的载体的功能变体。“功能变体”指相对于本公开中具体描述或示例的载体(“亲本载体”)的序列包含突变(例如,添加、缺失或取代)但保留亲本载体的功能或特性的载体。例如,功能变体可以包含对应于本公开中示例的亲本载体的密码子优化的序列。
B1.C68DNA序列
术语“C68DNA序列”指是C68基因组序列部分的DNA序列。所述载体中包括的C68DNA序列通过一个或多个病毒基因或基因组区的功能缺失衍生自C68基因组序列。术语“功能缺失”表示例如通过突变或修饰来去除或改变足量的病毒基因区,从而基因区不再能够产生基因表达的功能产物或进行其正常功能。通常破坏复制必不可少的基因功能不需要缺失整个基因区。但是,为了在C68基因组中为一个或多个转基因提供足够空间,去除一个或多个基因区的大部分可以是可取的。虽然优选缺失遗传物质,但是通过添加或取代来突变遗传物质对于破坏基因功能也是适当的。
在一些实施方案中,所述载体的C68DNA序列通过功能缺失整个或足够部分的腺病毒即时早期基因E1a和延迟早期基因E1b衍生自C68基因组序列。在其他实施方案中,除了E1a和E1b的功能缺失,还可以对一个或多个其他基因如延迟早期基因E2a,延迟早期基因E3、E4,晚期基因L1至L5中的任一种,即时基因IX和IVa2进行功能缺失。因此,用于构建本发明的载体的C68DNA可以仅在E1中包含缺失。或者,有效破坏它们的生物活性的整个基因或其部分的缺失可以以任何组合使用。例如,在一示例性载体中,C68DNA序列通过E1基因和E4基因的功能缺失,或者E1、E2a和E3基因的缺失,或者E1和E3基因的功能缺失,或者有或无E3缺失的E1、E2a和E4的功能缺失等衍生自C68基因组序列。此外,这类缺失可以与其他突变如温度敏感突变组合使用以获得期望的结果。在一特定实施方案中,C68DNA序列是整个C68基因组,仅在E1和E3区中具有功能缺失。
在一些特定实施方案中,E1基因的功能缺失通过缺失SEQ ID NO:57的核苷酸577-3403或通过缺失SEQ ID NO:57的核苷酸456-3012来完成,而E3基因的功能缺失通过缺失SEQ ID NO:57的核苷酸27125–31831或通过缺失SEQ ID NO:57的核苷酸27812-31330来完成。在其他特定实施方案中,所述载体中包括的C68DNA序列包含SEQ ID NO:57的核苷酸3013–27811。在其他特定实施方案中,所述载体中包括的C68DNA序列包含SEQ ID NO:57的核苷酸3013–27811和31331–36519。
可以将多抗原构建体插入腺病毒基因组的任何缺失区域。还可以将多抗原构建体插入现有基因区以破坏该区的功能。在一些实施方案中,将多抗原构建体插入缺失的E1基因的位置。
B2.多抗原构建体
术语“多抗原构建体”指编码两个或更多个PAA多肽的核酸分子或序列。这类分子或序列在本公开中还可以称作“多抗原疫苗”或“多抗原质粒”。多抗原构建体可以携带两个编码核苷酸序列,其中所述编码核苷酸序列各自表达单独的免疫原性PAA多肽。这样的构建体在本公开中还称作“双抗原构建体”、“双抗原疫苗”或“双抗原质粒”。多抗原构建体还可以携带三个编码核苷酸序列,其中所述编码核苷酸序列各自表达单独的免疫原性PAA多肽。这样的构建体在本公开中还称作“三抗原构建体”、“三抗原疫苗”或“三抗原质粒”。多抗原构建体编码的单独的PAA多肽可以是对相同抗原如PSMA、PSA或PSCA免疫原性的。例如,双抗原构建体可以表达对PSMA均为免疫原性的两种不同的PAA抗原。多抗原构建体编码的单独的PAA多肽可以是对不同抗原免疫原性的,例如,双抗原构建体可以表达分别对PSMA和PSA是免疫原性的两个PAA多肽。优选多抗原构建体编码对不同抗原是免疫原性的两个或更多个单独的PAA多肽。
在一些实施方案中,多抗原构建体编码以下组合中任一种的至少两个免疫原性PAA多肽:
1)免疫原性PSMA多肽和免疫原性PSA多肽;
2)免疫原性PSMA多肽和免疫原性PSCA多肽;以及
3)免疫原性PSA多肽和免疫原性PSCA多肽。
在一些特定实施方案中,多抗原构建体编码至少一个免疫原性PSMA多肽、至少一个免疫原性PSA多肽和至少一个免疫原性PSCA多肽。
多抗原构建体表达的免疫原性PSMA多肽可以是胞质、分泌或膜结合的,但是优选膜结合。在一些实施方案中,免疫原性PSMA多肽选自以下的氨基酸序列:
1)SEQ ID NO:1的氨基酸序列,
2)SEQ ID NO:1的氨基酸15-750;
3)SEQ ID NO:3的氨基酸序列;
4)SEQ ID NO:5的氨基酸序列;
5)SEQ ID NO:7的氨基酸序列;
6)SEQ ID NO:3的氨基酸4–739;
7)SEQ ID NO:5的氨基酸4-739;
8)SEQ ID NO:7的氨基酸4-739;
9)SEQ ID NO:9的氨基酸序列;以及
10)SEQ ID NO:9的氨基酸4–739。
多抗原构建体表达的免疫原性PSA多肽可以是胞质、分泌或膜结合的,但是优选胞质。在一些实施方案中,免疫原性PSA多肽选自以下的氨基酸序列:
1)SEQ ID NO:15的氨基酸27-263;
2)SEQ ID NO:17的氨基酸序列;以及
3)SEQ ID NO:17的氨基酸4-240。
多抗原构建体表达的免疫原性PSCA多肽可以是全长人PSCA蛋白。在一些实施方案中,免疫原PSCA多肽选自以下的氨基酸序列:
1)SEQ ID NO:21的氨基酸序列;
2)SEQ ID NO;21的氨基酸2-125;以及
3)SEQ ID NO:21的氨基酸4-125。
在一些其他实施方案中,多抗原构建体编码至少一个免疫原性PSA多肽、至少一个免疫原性PSCA多肽和至少一个免疫原性PSMA多肽,其中所述免疫原性PSA多肽包含SEQ IDNO:17的氨基酸序列或SEQ ID NO:17的氨基酸4–240,其中所述免疫原性PSCA多肽包含SEQID NO:21的氨基酸序列或SEQ ID NO:21的氨基酸2-125,并且其中所述免疫原性PSMA多肽包含选自以下的氨基酸序列:
1)SEQ ID NO:1的氨基酸15-750;
2)SEQ ID NO:9的氨基酸4–739;以及
3)SEQ ID NO:9的氨基酸序列。
在一些特定实施方案中,多抗原构建体包含编码SEQ ID NO:60或64的氨基酸序列的核苷酸序列。
在一些特定实施方案中,多抗原构建体包含:(i)编码免疫原性PSA多肽的核苷酸序列,(ii)编码免疫原性PSCA多肽的核苷酸序列,以及(iii)编码免疫原性PSMA多肽的核苷酸序列,其中:
(1)编码免疫原性PSA多肽的核苷酸序列选自:(i)SEQ ID NO:18的核苷酸序列;(ii)SEQ ID NO:20的序列;(iii)包含SEQ ID NO:18的核苷酸10-720的核苷酸序列;(iv)包含SEQ ID NO:58或SEQ ID NO:63的核苷酸1115-1825的核苷酸序列;(v)包含SEQ ID NO:58或SEQ ID NO:63的核苷酸1106-1825的核苷酸序列;以及(vi)上文(i)–(v)中提供的任何核苷酸序列的简并变体。
(2)编码免疫原性PSCA多肽的核苷酸序列选自:(i)SEQ ID NO:22的核苷酸序列;(ii)包含SEQ ID NO:22的核苷酸10-375的核苷酸序列;(iii)包含SEQ ID NO:58或SEQ IDNO:63的核苷酸1892-2257的核苷酸序列;(iv)包含SEQ ID NO:58或SEQ ID NO:63的核苷酸1886-2257的核苷酸序列;以及(v)上文(i)–(iv)中提供的任何核苷酸序列的简并变体;以及
(3)编码免疫原性PSMA多肽的核苷酸序列选自:(i)包含SEQ ID NO:2的核苷酸43-2250的核苷酸序列;(ii)SEQ ID NO:4的核苷酸序列;(iii)SEQ ID NO:6的核苷酸序列;(iv)SEQ ID NO:8的核苷酸序列;(v)SEQ ID NO:10的核苷酸序列;(vi)包含SEQ ID NO:4的核苷酸10–2217的核苷酸序列;(vii)包含SEQ ID NO:6的核苷酸10–2217的核苷酸序列;(viii)包含SEQ ID NO:8的核苷酸10–2217的核苷酸序列;(ix)包含SEQ ID NO:10的核苷酸10–2217的核苷酸序列;(x)包含SEQ ID NO:58或SEQ ID NO:63的核苷酸2333-4543的核苷酸序列;(xi)包含SEQ ID NO:58或SEQ ID NO:63的核苷酸2324-4543的核苷酸序列;以及(xii)上文(i)–(xi)中提供的任何核苷酸序列的简并变体。
在另一具体实施方案中,多抗原构建体包含编码免疫原性PSA多肽的核苷酸序列、编码免疫原性PSCA多肽的核苷酸序列和编码免疫原性PSMA多肽的核苷酸序列,其中:编码免疫原性PSA多肽的核苷酸序列包含SEQ ID NO:58或SEQ ID NO:63的核苷酸1106-1825;编码免疫原性PSCA多肽的核苷酸序列包含SEQ ID NO:58或SEQ ID NO:62的核苷酸1886-2257;并且编码免疫原性PSMA多肽的核苷酸序列包含SEQ ID NO:58或SEQ ID NO:63的核苷酸2324-4543。
为了使得能够从载体携带的单一多抗原构建体表达不同免疫原性PAA多肽,在编码单独的免疫原性PAA多肽(即,PSA、PSCA和PSMA多肽)的序列之间包含间隔序列。这些间隔序列使得能够单独翻译下游免疫原性PAA多肽。这样的间隔序列在本说明书中称作“分隔物序列”。可以用于从单一载体共表达多个多肽的任何序列均可以在本公开提供的载体中用作分隔物序列。可用的分隔物序列的实例包括内部核糖体进入位点(IRES)和2A肽序列。
也称作切割盒或CHYSEL(顺式作用水解酶元件)的2A肽和2A肽样序列约20个氨基酸长,具有高度保守的羧基端D-V/I-EXNPGP基序(图2)。该序列在自然中罕见,最常在病毒如口蹄疫病毒(FMDV)、马鼻炎A病毒(ERAV)、脑心肌炎病毒(EMCV)、猪捷申病毒(teschovirus)(PTV)和Thosea asigna病毒(TAV)中找到(Luke,G.A.,P.de Felipe,et al.(2008)."Occurrence,function and evolutionary origins of'2A-like'sequences invirus genomes."J Gen Virol 89(Pt 4):1036-1042)。用基于2A的多抗原表达策略,将编码多个靶抗原的基因在单一开放阅读框中连接在一起,通过2A序列分开。将整个开放阅读课克隆入具有单一启动子和终止子的载体。当将构建体递送至宿主细胞时,编码多个抗原的mRNA作为单一多聚蛋白转录和翻译。在2A序列的翻译期间,核糖体跳过C-末端甘氨酸和脯氨酸之间的键。核糖体跳过作用像从下游释放上游蛋白的共翻译自催化“切割”。关于在共表达多个多肽的载体中使用各种2A肽序列的一般信息可以在Andrea L.Szymczak&Darrio AA Vignali:Development of 2A peptide-based strategies in the design ofmulticistronic vectors.Expert Opinion Biol.Ther.(2005)5(5)627-638中找到,其公开援引加入本文。两个蛋白抗原之间2A序列的并入导致将~20个氨基酸添加至上游多肽的C-末端,并且将1个氨基酸(脯氨酸)添加至下游蛋白的N-末端。在这种方法的改编中,可以将蛋白酶切割位点并入在2A盒的N末端,从而普遍存在的蛋白酶会从上游蛋白切割盒(Fang,J.,S.Yi,et al.(2007)."An antibody delivery system for regulatedexpression of therapeutic levels of monoclonal antibodies in vivo."Mol Ther15(6):1153-1159)。
可以用于本发明的具体2A-肽序列的实例在Andrea L.Szymczak&Darrio AAVignali:Development of 2A peptide-based strategies in the design ofmulticistronic vectors.Expert Opinion Biol.Ther.(2005)5(5)627-638中公开,并且在表1中提供。
表1. 2A-肽序列
内部核糖体进入位点(IRES)是在某些RNA分子的5’非翻译区中找到的RNA元件(图3)(Bonnal,S.,C.Boutonnet,et al.(2003)."IRESdb:the Internal Ribosome EntrySite database."Nucleic Acids Res 31(1):427-428)。它们将真核核糖体吸引至RNA以促进下游开放阅读课的翻译。不像正常的细胞7-甲基鸟苷帽依赖性翻译,IRES介导的翻译可以在RNA分子内远处的AUG密码子处起始。可以开发该高效过程用于多顺反子表达载体(Bochkov,Y.A.and A.C.Palmenberg(2006)."Translational efficiency of EMCV IRESin bicistronic vectors is dependent upon IRES sequence and gene location."Biotechniques 41(3):283-284,286,288)。优选的EMCV IRES(pIRES)的序列在图3和SEQID NO:59中提供。最小EMCV IRES(mIRES)不包括如图3所示的EMCV L蛋白的下划线的前5个密码子。通常,将两个转基因插入载体的启动子和转录终止子之间,作为通过IRES分开的两个独立的开放阅读框。当将构建体递送至宿主细胞时,会转录编码两个转基因的单一长转录物。第一ORF会以传统的帽依赖性方式翻译,在IRES上游的终止密码子处终止。第二ORF会利用IRES以帽不依赖性方式翻译。以这种方式,可以从转录自载体的具有单一表达盒的单一mRNA产生两个独立的蛋白。在一些实施方案中,多抗原构建体包含EMCV IRES,其包含SEQID NO:59的核苷酸1-553。
通常,在多抗原构建体上的两个编码免疫原性PAA多肽的序列之间仅需要一个分隔物序列。在多抗原构建体上分隔物序列和编码PAA多肽的核苷酸序列的顺序在式(I)中示出:
PAA1-SS1-PAA2-SS2-PAA3 (I)
其中:(i)PAA1、PAA2和PAA3各自为编码免疫原性PSA多肽的核苷酸序列、编码免疫原性PSCA多肽的核苷酸序列或编码免疫原性PSMA多肽的核苷酸序列,只要PAA1、PAA2和PAA3编码不同的PAA多肽,以及(ii)SS1和SS2为分隔物序列,并且可以相同或不同。
在一些实施方案中,所述载体包含式(I)的多抗原构建体,其中:
(i)PAA1为编码免疫原性PSA多肽的核苷酸序列;
(ii)PAA2为编码免疫原性PSCA或PSMA多肽的核苷酸序列。(其中PAA2为编码免疫原性PSCA的核苷酸序列,则PAA3为编码免疫原性PSMA的核苷酸序列,或者反之亦然);
(iii)SS1为2A-肽序列;以及
(iv)SS2为2A-肽序列或IRES。
在一些特定实施方案中,多抗原构建体具有选自以下的结构:
(1)PSA-F2A-PSMA-mIRES-PSCA;
(2)PSA-F2A-PSMA-T2A-PSCA;
(3)PSA-T2A-PSCA-F2A-PSMA;以及
(4)PSCA-F2A-PSMA-mIRES-PSA
在一具体实施方案中,所述载体包含具有式(I)的结构的多抗原构建体:
PAA1-SS1-PAA2-SS2-PAA3 (I)
其中:
(i)PAA1为编码免疫原性PSA多肽的核苷酸序列,并且包含SEQ ID NO:58的核苷酸1115-1825或者包含SEQ ID NO:58或63的1106-1114;
(ii)PAA2为编码免疫原性PSCA多肽的核苷酸序列,并且包含SEQ ID NO:58的核苷酸1892-2257或者包含SEQ ID NO:58或63的1886-2257;
(iii)PAA3是编码免疫原性PSMA多肽的核苷酸序列且包含SEQ ID NO:58的核苷酸2333-4543或包含SEQ ID NO:58或63的2324-4543;
(iv)SS1为编码T2A的核苷酸序列;以及
(v)SS2为编码F2A的核苷酸序列。
多抗原构建体还可以包括位于编码免疫原性PAA多肽(即,免疫原性PSA、PSCA或PSMA多肽)的核苷酸序列和下游分隔物序列之间的接头序列。这样的接头序列的一个实例为编码甘氨酸-丝氨酸的核苷酸序列。
在一些特定实施方案中,多抗原构建体包含编码SEQ ID NO:60的氨基酸序列或编码SEQ ID NO:61的氨基酸序列的核苷酸序列。在一特定实施方案中,多抗原构建体包含选自以下的核苷酸序列:SEQ ID NO:61的核苷酸序列、SEQ ID NO:65的核苷酸序列、SEQ IDNO:66的核苷酸序列以及所述核苷酸序列中任一种的简并变体。
B3.调节序列
除了上文描述的分隔物序列和接头序列,所述载体可以包含其他非天然的调节序列以驱动所编码的PAA多肽的高效表达。调节序列的实例包括:(1)转录起始、终止、启动子和增强子序列;(2)高效的RNA加工信号如剪接和多腺苷酸化信号;(3)稳定胞质mRNA的序列;(4)增强翻译效率的序列(即,Kozak共有序列);(5)增强蛋白稳定性的序列;以及(6)增强蛋白分泌的序列。可以用于本公开提供的载体以驱动在哺乳动物细胞中高效表达的启动子系统的实例包括SV40启动子、鸡B肌动蛋白启动子、人延伸因子启动子、人巨细胞病毒(CMV)启动子、猿猴CMV启动子、小鼠CMV启动子、伪狂犬病启动子、劳氏肉瘤病毒启动子、磷酸甘油酸激酶启动子、小鼠白血病病毒LTR启动子、禽类白血病病毒LTR启动子、小鼠乳腺瘤病毒LTR启动子、moloney小鼠白血病病毒LTR启动子、纤溶酶原激活物抑制剂启动子、CYR61、腺病毒主要晚期启动子、小鼠金属硫蛋白启动子、小鼠磷酸烯醇丙酮酸羧激酶启动子、牛B-乳球蛋白启动子、牛催乳素启动子、泛素C启动子以及单纯疱疹病毒胸苷激酶启动子。转录终止信号的实例包括SV40多腺苷酸化(polyA);牛生长激素polyA;兔B珠蛋白polyA;HSV胸苷激酶、糖蛋白B和糖蛋白D;HPV E和L,以及合成的终止子。
在一些实施方案中,C68载体包含人巨细胞病毒(CMV)启动子,任选具有CMV增强子,以及SV40polyA。
C.包含携带多抗原构建体的载体的组合物(载体组合物)
本公开还提供一种包含本公开提供的载体的组合物(在本文中“载体组合物”)。所述载体组合物可用于在包括人在内的哺乳动物中于体外或体内引发针对PAA蛋白的免疫应答。所述载体组合物可以包含单独的载体,或者还可以包含赋形剂。
在一些实施方案中,所述载体组合物为药物组合物,其包含本公开提供的载体和药学可接受的赋形。用于药物组合物的合适的赋形剂是本领域已知的。赋形剂可以包括水性溶液、非水性溶液、悬浮液和乳液。非水性赋形剂的实例包括丙二醇、聚乙二醇、植物油如橄榄油和可注射的有机酯如油酸乙酯。水性赋形剂的实例包括水、醇/水性溶液、乳液或悬浮液,包括盐水和缓冲介质。合适的赋形剂还包括辅助载体的细胞摄取的物质。
在一些实施方案中,所述药物组合物是向人给药的疫苗组合物,用于抑制异常细胞增殖、提供针对癌症发展的保护(用作预防剂),或者用于治疗与PAA过量表达相关的癌症(用作治疗剂),或者用于引发对特定人PAA如PSMA、PSA和PSCA的免疫应答。所述疫苗组合物还可以包含一种或多种佐剂。可以包括在所述疫苗组合物中的佐剂的实例在下文中提供。
D.所述载体和载体组合物的用途
在其他方面,本公开提供使用所述载体或包含上述的载体的组合物的方法。
在一方面,本公开提供一种在哺乳动物,特别是人中引发针对PAA的免疫应答的方法,所述方法包括向所述哺乳动物给药有效量的(1)包含多抗原构建体的载体,或者(2)包含这类载体的组合物。
在另一方面,本公开提供一种抑制人中的异常细胞增殖的方法,其中所述异常细胞增殖与PAA的过量表达有关。所述方法包括向哺乳动物给药有效量的(1)包含编码两个或更多个免疫原性PAA多肽的多抗原构建体的载体,或者(2)包含这类载体的组合物。在一些实施方案中,所述方法用于抑制人的前列腺中的异常细胞增殖。在一特定实施方案中,本公开提供一种抑制过量表达PSMA的前列腺中的异常细胞增殖的方法。在一些实施方案中,本公开提供一种治疗人中的前列腺癌的方法,所述方法包括向所述人给药有效量的(1)包含多抗原构建体的载体,或者(2)包含这类载体的组合物。在一优选实施方案中,多抗原构建体是编码免疫原性PSMA多肽、免疫原性PSA多肽和免疫原性PSCA多肽的三抗原构建体。
所述载体或载体组合物可以通过本领域已知的许多方法向包括人在内的动物给药。合适的方法的实例包括:(1)肌肉内、皮内、表皮内、静脉内、动脉内、皮下或腹腔内给药,(2)口服给药,以及(3)局部施用(如眼部、鼻内和阴道内施用)。皮内或表皮内给药核酸疫苗组合物的一种特定方法包括使用基因枪递送技术,如PowderMed销售的Particle MediatedEpidermal Delivery(PMEDTM)疫苗递送装置。肌肉内给药核酸疫苗的另一特定方法是注射然后电穿孔。
在给定方法中待给药的所述载体或载体组合物的有效量可以由本领域技术人员容易地确定,并且会取决于许多因素。在治疗癌症如前列腺癌的方法中,在确定有效量中可以考虑的因素包括但不限于:(1)待治疗的个体,包括个体的免疫状态和健康;(2)待治疗的癌症的严重程度或阶段,(3)表达的具体免疫原性PAA多肽,(4)期望的保护或治疗程度,(5)给药方法和时间表,(6)使用的制剂,以及(7)其他治疗剂(如佐剂或免疫调节剂)的共给药。例如,当核酸疫苗组合物配制为水性溶液并通过皮下注射针头注射或气动注射给药时,所述载体的有效量可以在2μg/剂量–10mg/剂量的范围中,而当核酸制备为包衣金珠并利用基因枪技术递送时,可以仅需要16ng/剂量–16μg/剂量。
本公开提供的包括疫苗组合物在内的载体或载体组合物可以与一种或多种佐剂一起使用。合适的佐剂的实例包括:(1)水包油乳液制剂(有或无其他特定免疫刺激剂如胞壁酰多肽或细菌细胞壁组分),如MF59TM(包含5%角鲨烯、0.5%吐温80和0.5%失水山梨醇三油酸酯)和SAF(包含10%角鲨烯、0.4%吐温80、5%pluronic-嵌段聚合物L121和thr-MDP);(2)皂草苷佐剂,如QS21、STIMULONTM(Cambridge Bioscience,Worcester,MA)、(Isconova,Sweden)或(Commonwealth Serum Laboratories,Australia);(3)完全弗氏佐剂(CFA)和不完全弗氏佐剂(IFA);(4)包含CpG基序的寡核苷酸,即包含至少一个CG二核苷酸,其中胞嘧啶是未甲基化的(例如,Krieg,Vaccine(2000)19:618-622;Krieg,Curr Opin Mol Ther(2001)3:15-24;WO 98/40100、WO 98/55495、WO98/37919和WO 98/52581);以及(5)包括铝盐在内的金属盐(如明矾、磷酸铝、氢氧化铝);(12)皂草苷和水包油乳液(例如WO 99/11241)。
本公开提供的载体或载体组合物可以与一种或多种免疫调节剂一起使用。在另一方面,本公开提供一种治疗哺乳动物,特别是人中的前列腺癌的方法,所述方法包括向所述哺乳动物给药:(1)有效量的本发明提供的载体、载体组合物或疫苗;(2)有效量的至少一种免疫抑制细胞抑制剂(ISC抑制剂);以及(3)有效量的至少一种免疫效应细胞增强剂(IEC增强剂)。这种方法在本公开中也称作“基于疫苗的免疫治疗方案”(或“VBIR”)。
IEC增强剂和ISC抑制剂可以通过任何合适的方法和途径给药,包括(1)全身给药如静脉内、肌肉内或口服给药,以及(2)局部给药如皮内和皮下给药。当适当或合适时,局部给药一般优选于全身给药。任何IEC增强剂和ISC抑制剂的局部给药可以在适合药物的局部给药的哺乳动物身体的任何位置进行;但是,更优选靠近疫苗引流淋巴结局部给药这些免疫调节剂。
来自单一类别的IEC增强剂的两种或更多种特定IEC增强剂(例如,两种CTLA-拮抗剂)可以与ISC抑制剂组合给药。此外,来自两种或更多种不同类别的IEC增强剂的两种或更多种特定IEC增强剂(例如,一种CTLA-4拮抗剂和一种TLR激动剂,或者一种CTLA-4拮抗剂和一种PD-1拮抗剂)可以一起给药。相似地,来自单一类别的ISC抑制剂的两种或更多种特定ISC抑制剂(例如,两种或更多种蛋白激酶抑制剂)可以与IEC增强剂组合给药。此外,来自两种或更多种不同类别的ISC抑制剂的两种或更多种特定ISC抑制剂(例如,一种蛋白激酶抑制剂和一种COX-2抑制剂)可以一起给药。
所述载体或载体组合物可以与所用的任何或全部免疫调节剂(即,ISC抑制剂和IEC增强剂)同时或顺序给药。相似地,当使用两种或更多种免疫调节剂时,它们互相可以同时或顺序给药。在一些实施方案中,将载体或载体组合物与一种免疫调节剂同时给药(例如,在混合物中),但是与一种或多种额外的免疫调节剂顺序给药。所述载体或载体组合物以及免疫调节剂的共给药可以包括这样的情况,其中给药疫苗和至少一种免疫调节剂,从而每种同时存在于给药部位,如疫苗引流淋巴结,即使抗原和免疫调节剂未同时给药。疫苗和免疫调节剂的共给药还可以包括这样的情况,其中从给药部位清除疫苗或免疫调节剂,但是所清除的疫苗或免疫调节剂的至少一种细胞效应在给药部位如疫苗引流淋巴结持续存在,至少直至向给药部位给药一种或多种额外的免疫调节剂。在其中核酸疫苗与CpG组合给药的情况下,疫苗和CpG可以包含在单一制剂中,并且通过任何合适的方法一起给药。在一些实施方案中,将共制剂(混合物)中的核酸疫苗和CpG通过肌肉内注射联合电穿孔给药。
任何ISC抑制剂均可以与本发明提供的载体或载体组合物组合使用。ISC抑制剂的类别的实例包括PD-1/PD-L1拮抗剂、蛋白激酶抑制剂、环加氧酶-2(COX-2)抑制剂、磷酸二酯酶5型(PDE5)抑制剂和DNA交联剂。PD-1/PD-L1拮抗剂的实例包括抗PD-1和PD-L1单克隆抗体。COX-2抑制剂的实例包括赛来考昔和罗非考昔。PDE5抑制剂的实例包括阿伐那非、洛地那非、米罗那非、西地那非、他达拉非、伐地那非、乌地那非和扎普司特。DNA交联剂的实例为环磷酰胺。具体蛋白激酶抑制剂的实例在下文中详细描述。
术语“蛋白激酶抑制剂”指充当蛋白激酶的选择性或非选择性抑制剂的任何物质。术语“蛋白激酶”指催化腺苷三磷酸的末端磷酸转移至蛋白底物中的酪氨酸、丝氨酸或苏氨酸残基的酶。蛋白激酶包括受体酪氨酸激酶和非受体酪氨酸激酶。受体酪氨酸激酶的实例包括EGFR(例如,EGFR/HER1/ErbB1、HER2/Neu/ErbB2、HER3/ErbB3、HER4/ErbB4)、INSR(胰岛素受体)、IGF-IR、IGF-II1R、IRR(胰岛素受体相关受体)、PDGFR(例如,PDGFRA、PDGFRB)、c-KIT/SCFR、VEGFR-1/FLT-1、VEGFR-2/FLK-1/KDR、VEGFR-3/FLT-4、FLT-3/FLK-2、CSF-1R、FGFR 1-4、CCK4、TRK A-C、MET、RON、EPHA 1-8、EPHB 1-6、AXL、MER、TYRO3、TIE、TEK、RYK、DDR1-2、RET、c-ROS、LTK(白细胞酪氨酸激酶)、ALK(间变性淋巴瘤激酶)、ROR 1-2、MUSK、AATYK1-3和RTK 106。非受体酪氨酸激酶的实例包括BCR-ABL、Src、Frk、Btk、Csk、Abl、Zap70、Fes/Fps、Fak、Jak、Ack和LIMK。在本公开提供的基于疫苗的免疫治疗方案中,以亚适量向哺乳动物给药蛋白激酶抑制剂。术语“亚适量”指低于在单一治疗中给药酪氨酸激酶抑制剂(即,单独给药蛋白激酶抑制剂,没有任何其他治疗剂)用于靶标肿瘤性病症时的最小有效剂量的剂量。
适合用于基于疫苗的免疫治疗方案的具体蛋白激酶抑制剂包括拉帕替尼、AZD2171、ET18OCH 3、靛玉红-3'-肟、NSC-154020、PD 169316、槲皮素、roscovitine、曲西立滨、ZD1839、5-碘代杀结核菌素、adaphostin、aloisine、alsterpaullone、aminogenistein、API-2、芹菜素、牛蒡苷元、ARRY-334543、阿昔替尼、AY-22989、AZD 2171、双吲哚基马来酰亚胺IX、CCl-779、白屈菜赤碱、DMPQ、DRB、依地福新、ENMD-981693、erbstatin类似物、厄洛替尼、法舒地尔、吉非替尼(ZD1839)、H-7、H-8、H-89、HA-100、HA-1004、HA-1077、HA-1100、羟基法舒地尔、kenpaullone、KN-62、KY12420、LFM-A13、木犀草素、LY294002、LY-294002、mallotoxin、ML-9、MLN608、NSC-226080、NSC-231634、NSC-664704、NSC-680410、NU6102、奥罗莫星、羟吲哚I、PD 153035、PD 98059、根皮苷、四羟反式芪、鬼臼苦素、PKI、PP1、PP2、PTK787/ZK222584、PTK787/ZK-222584、purvalanol A、雷帕鸣、雷帕霉素、Ro 31-8220、楸毒素、SB202190、SB203580、西罗莫司、SL327、SP600125、星形孢菌素、STI-571、SU1498、SU4312、SU5416、司马沙尼、SU6656、SU6668、syk抑制剂、TBB、TCN、酪氨酸磷酸化抑制剂AG1024、酪氨酸磷酸化抑制剂AG 490、酪氨酸磷酸化抑制剂AG 825、酪氨酸磷酸化抑制剂AG957、U0126、W-7、渥曼青霉素、Y-27632、zactima、ZM 252868、吉非替尼、苹果酸舒尼替尼、厄洛替尼、拉帕替尼、卡纳替尼、semaxinib、伐他拉尼、索拉菲尼、伊马替尼、达沙替尼、来氟米特、凡德他尼以及尼洛替尼。
在一些实施方案中,蛋白激酶抑制剂为多激酶抑制剂,其为作用于一种以上特异性激酶的抑制剂。多激酶抑制剂的实例包括伊马替尼、索拉菲尼、拉帕替尼、BIRB-796、和AZD-1152、AMG706、zactima、MP-412、索拉菲尼、达沙替尼、来妥替尼、XL647、XL999、拉帕替尼、MLN518、(也称作CT53518)、PKC412、ST1571、AEE 788、OSI-930、OSI-817、苹果酸舒尼替尼、厄洛替尼、吉非替尼、阿昔替尼、波舒替尼、坦罗莫司以及尼洛替尼。在一些特定实施方案中,酪氨酸激酶抑制剂为舒尼替尼、索拉菲尼,或者舒尼替尼或索拉菲尼的药学可接受的盐或衍生物(如苹果酸盐或甲苯磺酸盐)。
由Pfizer Inc.以商品名SUTENT销售的苹果酸舒尼替尼在化学上描述为具有N-[2-(二乙基氨基)乙基]-5-[(Z)-(5-氟-1,2-二氢-2-氧代-3H-亚吲哚-3-基)甲基]-2,4-二甲基-1H-吡咯-3-羧酰胺的丁二酸、羟基-、(2S)-化合物(1:1)。所述化合物、其合成和特定多晶型物描述于美国专利第6,573,293号,美国专利公开号2003-0229229、2003-0069298和2005-0059824,以及J.M.Manley,M.J.Kalman,B.G.Conway,C.C.Ball,J.L Havens andR.Vaidyanathan,"Early Amidation Approach to 3-[(4-amido)pyrrol-2-yl]-2-indolinones,"J.Org.Chew.68,6447-6450(2003)。舒尼替尼及其L-苹果酸盐的制剂描述于PCT公开号WO 2004/024127。苹果酸舒尼替尼在美国已批准用于治疗具有不可切除的局部晚期或转移性疾病的患者中的胃肠道间质瘤,晚期肾细胞癌,以及进展性、分化良好的胰腺神经内分泌肿瘤。用于人的胃肠道间质瘤(GIST)和晚期肾细胞癌(RCC)的苹果酸舒尼替尼的推荐剂量为每天口服一次50mg,时间表为4周治疗然后2周间歇(时间表4/2)。用于胰腺神经内分泌肿瘤(pNET)的苹果酸舒尼替尼的推荐剂量为每天口服一次37.5mg。
在基于疫苗的免疫治疗方案中,苹果酸舒尼替尼可以以单一剂量或多剂量口服给药。通常,将苹果酸舒尼替尼递送2周、3周、4周或连续更多周剂量,然后约1或2周或更多的“间歇”期,其中不递送苹果酸舒尼替尼。在一实施方案中,将所述剂量递送约4周,具有2周间歇。在另一实施方案中,将苹果酸舒尼替尼递送2周,具有1周间歇。但是,其还可以在整个治疗期间递送,没有“间歇”期。在基于疫苗的免疫治疗方案中向人口服给药的苹果酸舒尼替尼的有效量通常低于40mg/人/剂量。例如,其可以以37.5、31.25、25、18.75、12.5、6.25mg/人/天口服给药。在一些实施方案中,将苹果酸舒尼替尼在1–25mg/人/剂量的范围中口服给药。在一些其他实施方案中,将苹果酸舒尼替尼在6.25、12.5或18.75mg/人/剂量的范围中口服给药。其他剂量方案和变化是可预见的,并且会通过医生指导来确定。
以商品名NEXAVAR销售的甲苯磺酸索拉菲尼也是多激酶抑制剂。其化学名为4-(4-{3-[4-氯-3-(三氟甲基)苯基]脲基}苯氧基)-N-甲基吡-啶-2-羧酰胺。其在美国批准用于治疗原发性肾癌(晚期肾细胞癌)和晚期原发性肝癌(肝细胞癌)。推荐每日剂量为400mg,每天口服两次。在本公开提供的基于疫苗的免疫治疗方案中,口服给药的甲苯磺酸索拉菲尼的有效量通常低于400mg/人/天。在一些实施方案中,口服给药的甲苯磺酸索拉菲尼的有效量在10-300mg/人/天的范围中。在一些其他实施方案中,口服给药的甲苯磺酸索拉菲尼的有效量为10-200mg/人/天,如10、20、60、80、100、120、140、160、180或200mg/人/天。
以商品名INLYTA销售的阿昔替尼是VEGF受体1、2和3的选择性抑制剂。其化学名为(N-甲基-2-[3-((E)-2-吡啶-2-基-乙烯基)-1H-吲唑-6-基硫烷基]-苯甲酰胺。其批准用于在一次以前的全身治疗失败之后治疗晚期肾细胞癌。起始剂量为5mg,每天口服两次。可以基于个体安全和耐受性进行剂量调整。在本公开提供的基于疫苗的免疫治疗方案中,口服给药的阿昔替尼的有效量通常低于5mg,每天两次。在一些其他实施方案中,口服给药的阿昔替尼的有效量为1-5mg,每天两次。在一些其他实施方案中,口服给药的阿昔替尼的有效量为每天两次1、2、3、4或5mg。
在基于疫苗的免疫治疗方案中可以使用任何IEC增强剂。它们可以是小分子或大分子(如蛋白、多肽、DNA、RNA和抗体)。可以使用的IEC增强剂的实例包括TNFR激动剂、CTLA-4拮抗剂、TLR激动剂、程序性细胞死亡蛋白1(PD-1)拮抗剂(如抗PD-1抗体CT-011)、和程序性细胞死亡蛋白1配体1(PD-L1)拮抗剂(如BMS-936559)、淋巴细胞-激活基因3(LAG3)拮抗剂、以及包含T细胞免疫球蛋白和粘蛋白结构域的分子-3(TIM-3)拮抗剂。具体TNFR激动剂、CTLA-4拮抗剂和TLR激动剂的实例在下文中详细提供。
TNFR激动剂.
TNFR激动剂的实例包括OX40、4-1BB(如BMS-663513)、GITR(如TRX518)和CD40的激动剂。具体CD40激动剂的实例在下文中详细描述。
CD40激动剂是结合至细胞上的CD40受体且能够增加一种或多种CD40或CD40L相关活性的物质。因此,CD40“激动剂”涵盖CD40“配体”。
CD40激动剂的实例包括CD40激动抗体,CD40激动抗体的片段,CD40配体(CD40L),以及CD40L的片段和衍生物如寡聚(例如,二价、三聚CD40L)、融合蛋白以及它们的变体。
用于本发明的CD40配体包括任何肽、多肽或蛋白,或者编码可以结合并激活细胞上的一种或多种CD40受体的肽、多肽或蛋白的核酸。合适的CD40配体描述于例如美国专利第6,482,411号;美国专利第6,410,711号;美国专利第6,391,637号;和美国专利第5,981,724号,所有专利和申请以及其中公开的CD40L序列均整体援引加入本文。虽然会优选人CD40配体用于人治疗,但是来自任何物种的CD40配体均可以用于本发明。为了用于其他动物物种,如在兽医实施方案中,优选匹配所治疗的动物的物种的CD40配体。在某些实施方案中,CD40配体为gp39肽或蛋白寡聚体,包括天然形成的gp39肽、多肽或蛋白寡聚体,以及包含寡聚序列的gp39肽、多肽、蛋白(和编码核酸)。虽然在本发明的某些方面优选寡聚体如二聚体、三聚体和四聚体,但是在本发明的其他方面,考虑使用较大的寡聚结构,只要所述寡聚结构保留结合并激活一种或多种CD40受体的能力。
在某些其他实施方案中,CD40激动剂为抗CD40抗体或其抗原结合片段。所述抗体可以是人、人源化或部分人嵌合抗CD40抗体。具体抗CD40单克隆抗体的实例包括G28-5、mAb89、EA-5或S2C6单克隆抗体以及CP870893。在一特定实施方案中,抗CD40激动剂抗体为CP870893或达西珠单抗(dacetuzumab)(SGN-40)。
CP-870,893为全人激动性CD40单克隆抗体(mAb),其已在临床上研究作为抗肿瘤疗法。CP870,893的结构和制备公开于WO2003041070(其中该抗体通过内部鉴定的“21.4.1”来鉴定)。CP-870,893的重链和轻链的氨基酸序列分别在SEQ ID NO:40和SEQ ID NO:41中示出。在临床试验中,CP870,893以一般在0.05-0.25mg/kg每输液的范围中的剂量通过静脉内输液给药。在I期临床研究中,估计CP-870893的最大耐受剂量(MTD)为0.2mg/kg,并且剂量限制性毒性包括3级CRS和3级荨麻疹。[Jens Ruter et al.:Immune modulation withweekly dosing of an agonist CD40antibody in a phase I study of patients withadvanced solid tumors.[Cancer Biology&Therapy 10:10,983-993;November 15,2010.]。在本公开提供的基于疫苗的免疫治疗方案中,CP-870,893可以皮内、皮下或体表给药。优选其皮内给药。方案中待给药的CP870893的有效量一般低于0.2mg/kg,通常在0.01mg–0.15mg/kg或0.05–0.1mg/kg的范围中。
达西珠单抗(也称作SGN-40或huS2C6;CAS编号88-486-59-9)是另一抗CD40激动剂抗体,其已在临床试验中研究用于惰性淋巴瘤、弥漫性大B细胞淋巴瘤和多发性骨髓瘤。在临床试验中,达西珠单抗以2mg/kg-16mg/kg的每周剂量静脉内给药。在本公开提供的基于疫苗的免疫治疗方案中,达西珠单抗可以皮内、皮下或体表给药。优选其皮内给药。在基于疫苗的免疫治疗方案中待给药的达西珠单抗的有效量一般低于16mg/kg,通常在0.2mg–14mg/kg或0.5–8mg/kg或1-5mg/kg的范围中。
CTLA-4抑制剂.
用于本公开提供的基于疫苗的免疫治疗方案的合适的抗CTLA-4拮抗剂包括但不限于抗CTLA-4抗体(如人抗CTLA-4抗体、小鼠抗CTLA-4抗体、哺乳动物抗CTLA-4、人源化抗CTLA-4抗体、单克隆抗CTLA-4抗体、多克隆抗CTLA-4抗体、嵌合抗CTLA-4抗体、抗CTLA-4结构域抗体)、抗CTLA-4抗体的片段(如(单链抗CTLA-4片段、重链抗CTLA-4片段和轻链抗CTLA-4片段)以及对抗共刺激途径的CTLA-4抑制剂。在一些实施方案中,CTLA-4抑制剂为依匹木单抗或曲美木单抗。
作为YERVOY销售的依匹木单抗(也称作MEX-010或MDX-101)为人抗人CTLA-4抗体。依匹木单抗还可以通过其CAS注册号477202-00-9来提到,并且在整体援引加入本文的PCT公开第WO01/14424号中公开为抗体10DI。包含依匹木单抗的药物组合物的实例在PCT公开第WO2007/67959中提供。依匹木单抗在美国批准用于治疗不可切除或转移性的黑素瘤。作为单一治疗的依匹木单抗的推荐剂量为每3周通过静脉内给药3mg/kg,总计4个剂量。在本发明提供的方法中,将依匹木单抗局部给药,特别是皮内或皮下给药。局部给药的依匹木单抗的有效量通常在5–200mg/剂量/人的范围中。在一些实施方案中,依匹木单抗的有效量在10–150mg/剂量/人/剂量的范围中。在一些特定实施方案中,依匹木单抗的有效量为约10、25、50、75、100、125、150、175或200mg/剂量/人。
曲美木单抗(也称作CP-675,206)为全人IgG2单克隆抗体,并且具有CAS编号745013-59-6。曲美木单抗在美国专利第6,682,736号中公开为抗体11.2.1,该专利整体且为了所有目的援引加入本文。曲美木单抗的重链和轻链的氨基酸序列分别在SEQ ID NO:42和43中示出。曲美木单抗已在临床试验中研究用于治疗各种肿瘤,包括黑素瘤和乳腺癌;其中曲美木单抗以0.01-15mg/kg的剂量范围每4或12周作为单一剂量或多剂量静脉内给药。在本发明提供的方案中,将曲美木单抗局部给药,特别是皮内或皮下给药。皮内或皮下给药的曲美木单抗的有效量通常在5–200mg/剂量/人的范围中。在一些实施方案中,曲美木单抗的有效量在10–-150mg/剂量/人/剂量的范围中。在一些特定实施方案中,曲美木单抗的有效量为约10、25、37.5、40、50、75、100、125、150、175或200mg/剂量/人。
Toll样受体(TLR)激动剂.
术语“toll样受体激动剂”或“TLR激动剂”指充当toll样受体(TLR)的激动剂的化合物。这包括TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10和TLR11或者它们的组合的激动剂。除非另有说明,提到TLR激动剂化合物可以包括任何药学可接受形式的化合物,包括任何异构体(例如,非对映体或对映体)、盐、溶剂合物、多晶型物等。特别地,如果化合物是旋光的,提到化合物可以包括每种化合物的对映体以及对映体的外消旋混合物。而且,化合物可以鉴定为一种或多种特定TLR的激动剂(例如,TLR7激动剂、TLR8激动剂或TLR7/8激动剂)。
在一些实施方案中,所述TLR激动剂为TLR9激动剂,特别是CpG寡核苷酸(或CpG.ODN)。CpG寡核苷酸为短核酸分子,其包含胞嘧啶然后通过磷酸键连接的鸟嘌呤,其中胞嘧啶的嘧啶环是未甲基化的。CpG基序是一种碱基模式,其包括未甲基化的中心CpG,被至少一个碱基包围在中心CpG的侧翼(在3'和5'侧)。CpG寡核苷酸包括D和K寡核苷酸。整个CpG寡核苷酸可以是未甲基化的,或者部分可以是未甲基化的。可用于本公开提供的方法的CpG寡核苷酸的实例包括美国专利号6194388、6207646、6214806、628371、6239116和6339068中公开的那些CpG寡核苷酸。
可用于本公开提供的方法的特定CpG寡核苷酸的实例包括:
5'TCGTCGTTTTGTCGTTTTGTCGTT3'(CpG 7909);
5'TCGTCGTTTTTCGGTGCTTTT3'(CpG 24555);和
5'TCGTCGTTTTTCGGTCGTTTT3'(CpG 10103).
合成的24聚体单链寡核苷酸CpG7909已广泛研究用于治疗癌症,作为单一治疗和与化疗剂组合,以及作为佐剂用于针对癌症和传染性疾病的疫苗。据报道多达1.05mg/kg的单一静脉内剂量的CpG 7909耐受良好,没有临床效果和显著毒性,而单一剂量皮下的CpG7909具有0.45mg/kg的最大耐受剂量(MTD),具有肌痛和体质效应的剂量限制性毒性。[参见Zent,Clive S,et al:Phase I clinical trial of CpG 7909(PF-03512676)in patientswith previously treated chronic lymphocytic leukemia.Leukemia and Lymphoma,53(2):211-217(7)(2012)]。在本公开提供的方案中,CpG7909可以通过注射入肌肉或通过任何其他合适的方法给药。优选其在接近疫苗引流淋巴结处局部给药,特别是通过皮内或皮下给药。为了与核酸疫苗如DNA疫苗一起使用,CpG可以优选与疫苗共配制在单一制剂中,并且通过肌肉内注射加上电穿孔来给药。通过肌肉内、皮内或皮下给药的CpG7909的有效量通常在10μg/剂量-10mg/剂量的范围中。在一些实施方案中,CpG7909的有效量在0.05mg–14mg/剂量的范围中。在一些特定实施方案中,CpG7909的有效量为约0.05、0.1、0.2、0.3、0.4、0.5、05 1mg/剂量。包括CpG 24555和CpG 10103在内的其他CpG寡核苷酸可以以相似方式和剂量水平给药。
在一些特定实施方案中,本公开提供一种增强疫苗的免疫原性或疗效用于治疗人中的肿瘤性病症的方法,所述方法包括向人给药(1)有效量的至少一种ISC抑制剂和(2)有效量的至少一种IEC增强剂,其中所述至少一种ISC抑制剂是选自甲苯磺酸索拉菲尼、苹果酸舒尼替尼、阿昔替尼、厄洛替尼、吉非替尼、阿昔替尼、波舒替尼、坦罗莫司或尼洛替尼的蛋白激酶抑制剂,并且其中所述至少一种IEC增强剂选自CTLA-4抑制剂、TLR激动剂或CD40激动剂。在一些优选实施方案中,方案包括向人给药(1)有效量的至少一种ISC抑制剂和(2)有效量的至少一种IEC增强剂,其中所述至少一种ISC抑制剂是选自阿昔替尼、甲苯磺酸索拉菲尼或苹果酸舒尼替尼的蛋白激酶抑制剂,并且其中所述至少一种IEC增强剂是选自依匹木单抗或曲美木单抗的CTLA-4抑制剂。在一些进一步优选实施方案中,方案包括向人给药(1)有效量的至少一种ISC抑制剂和(2)有效量的至少两种IEC增强剂,其中所述至少一种ISC抑制剂是选自舒尼替尼或阿昔替尼的蛋白激酶抑制剂,并且其中所述至少两种IEC增强剂是曲美木单抗以及选自CpG7909、CpG2455或CpG10103的TLR激动剂。
在一些其他实施方案中,本公开提供一种治疗人中的前列腺癌的方法,所述方法包括向人给药(1)有效量的能够引发针对人PAA的免疫应答的疫苗,(2)有效量的至少一种ISC抑制剂,和(3)有效量的至少一种IEC增强剂,其中所述至少一种ISC抑制剂是选自甲苯磺酸索拉菲尼、苹果酸舒尼替尼、阿昔替尼、厄洛替尼、吉非替尼、阿昔替尼、波舒替尼、坦罗莫司或尼洛替尼的蛋白激酶抑制剂,并且其中所述至少一种IEC增强剂选自CTLA-4抑制剂、TLR激动剂或CD40激动剂。在一些优选实施方案中,所述方法包括向人给药(1)有效量的能够引发针对人PAA的免疫应答的疫苗,(2)有效量的至少一种ISC抑制剂,和(3)有效量的至少一种IEC增强剂,其中所述至少一种ISC抑制剂是选自甲苯磺酸索拉菲尼、苹果酸舒尼替尼或阿昔替尼的蛋白激酶抑制剂,并且其中所述至少一种IEC增强剂是选自依匹木单抗或曲美木单抗的CTLA-4抑制剂。
在一些其他具体实施方案中,所述方法包括向人给药(1)有效量的至少一种ISC抑制剂和(2)有效量的至少两种IEC增强剂,其中所述至少一种ISC抑制剂是选自舒尼替尼或阿昔替尼的蛋白激酶抑制剂,并且其中所述至少两种IEC增强剂是曲美木单抗以及选自CpG7909、CpG2455或CpG10103的TLR激动剂。
额外的治疗剂.
本公开提供的基于疫苗的免疫治疗方案还可以包含额外的治疗剂。可以使用各种癌症治疗剂,包括化疗剂和激素治疗剂。术语“化疗剂”指化学或生物物质,其可以引起癌细胞死亡,或者干扰癌细胞的生长、分裂、修复和/或功能。特定化疗剂的实例包括:醋酸阿比特龙、卡巴他赛(cabazitaxel)、地加瑞克、地舒单抗、多西他赛、enzalutamide、醋酸亮丙立德、泼尼松、西普鲁塞-T和二氯化镭223。术语“激素治疗剂”指化学或生物物质,其抑制或消除激素的产生,或者抑制或抵消激素对癌细胞的生长和/或存活的影响。特定激素治疗剂的实例包括亮丙立德、戈舍瑞林、曲普瑞林、组氨瑞林、比卡鲁胺、氟他胺和尼鲁米特。本公开提供的VBIR还可以与其他治疗联合使用,包括(1)手术方法,其取出全部或部分参与激素产生的器官或腺体,如卵巢、睾丸、肾上腺和垂体腺,以及(2)辐射治疗,其中使患者的器官或腺体接受一定量的辐射,所述量足以抑制或消除靶激素的产生。
E.实施例
提供以下实施例以说明本发明的某些实施方案。它们不应当理解为以任何方式限制本发明的范围。从以上讨论和这些实施例,本领域技术人员可以确定本发明的基本特征,并且不背离其精神和范围,可以进行本发明的各种改变和修改以使其适应各种使用和条件。
实施例1.衍生自人PSMA蛋白的胞质、分泌和膜结合形式的抗原
1A.免疫原性PSMA多肽的设计
基于天然人PSMA蛋白序列构建编码胞质、分泌和修改形式的免疫原性PSMA多肽的DNA构建体,并且测试它们诱导抗肿瘤效应子免疫应答的能力。每种人PSMA抗原形式的结构和制备提供如下。
1A1.人PSMA胞质抗原.设计胞质形式的免疫原性PSMA多肽,以便一旦表达,将免疫原性多肽保留在细胞内。去除人PSMA的细胞质结构域(氨基酸1-19)和跨膜结构域(氨基酸20-43),得到由SEQ ID NO:1的人PSMA的氨基酸44-750(胞外结构域或ECD)组成的胞质PSMA多肽。可以将最佳的Kozak序列“MAS”添加至多肽的N-末端以增强表达或促进克隆。
1A2.人PSMA分泌抗原.设计分泌形式的免疫原性PSMA多肽,以便一旦表达,将多肽分泌至细胞外。分泌多肽用SEQ ID NO:1的人PSMA的氨基酸44-750(ECD)和具有氨基酸序列ETDTLLLWVLLLWVPGSTGD的Igκ分泌元件和N-末端的两个氨基酸接头(AA)制成,以便一旦表达,最大化PSMA抗原的分泌。
1A3.人PSMA膜结合抗原.设计免疫原性PSMA膜结合多肽以将多肽稳定在细胞表面上。去除人PSMA蛋白的前14个氨基酸,并且所得的免疫原性多肽由SEQ ID NO:1的人PSMA蛋白的氨基酸15–750组成。由SES ID NO:1的天然人PSMA蛋白的氨基酸15–750组成且与天然人PSMA蛋白享有100%序列相同性的免疫原性多肽在本公开中也称作“人PSMA修改”、“hPSMA修改”或“hPSMAmod”抗原。还产生以下3种免疫原性PSMA多肽(称作“改组的PSMA修改抗原”),它们是人PSMA修改抗原(SEQ ID NO:9)的变体。
(1)具有SEQ ID NO:3的氨基酸序列的改组的PSMA修改抗原1;
(2)具有SEQ ID NO:5的氨基酸序列的改组的PSMA修改抗原2;以及
(3)具有SEQ ID NO:7的氨基酸序列的改组的PSMA修改抗原3。
编码改组的PSMA修改抗原1、2和3的核苷酸序列分别在SEQ ID NO:4、6和8中示出。
1B.制备DNA质粒用于表达PSMA抗原
将编码PSMA胞质、PSMA分泌和PSMA修改抗原的DNA构建体分别克隆入适合动物中的体内测试的PJV7563载体(图1)。将PJV7563载体中的DNA的两条链测序以证实设计完整性。
大规模的质粒DNA制品(Qiagen/CsCl)制备自序列证实的克隆。通过高260/280比例、高超螺旋/切口DNA比例、低内毒素水平(<10U/mg DNA)和阴性生物负荷证实质粒DNA的性能。
1C.PSMA构建体在哺乳动物细胞中的表达
通过FACS确定PSMA胞质、分泌和修改抗原的表达。将哺乳动物293细胞用编码各种免疫原性PSMA多肽的PJV7563PMED载体转染。3天之后,将293细胞用小鼠抗PSMA抗体染色,然后用荧光缀合的(FITC)大鼠抗小鼠二抗染色。结果在表2中示出。数据报道为相对于阴性对照的平均荧光强度(MFI),证实人PSMA修改抗原在细胞表面上表达。
表2.人PSMA修改抗原在细胞表面上的表达
样品 | 平均荧光强度 |
未转染的293细胞 | 231 |
用全长人PSMA(SEQ ID NO:1)转染的293细胞 | 6425 |
用人PSMA修改抗原(SEQ ID NO:9)转染的293细胞 | 12270 |
实施例2.各种免疫原性PSA多肽的设计
3A.免疫原性PSA多肽的构建
与实施例1中对3种不同免疫原性PSMA多肽形式(例如,胞质、膜结合和分泌形式)所描述的相似,基于人PSA序列也设计了3种形式的免疫原性PSA多肽。通过缺失分泌信号和pro结构域(氨基酸1-24)来构建由天然人PSA的氨基酸25-261组成的胞质形式的免疫原性PSA多肽。这种胞质免疫原性PSA多肽的氨基酸序列在SEQ ID NO:17中提供。PSA多肽的分泌形式为天然全长人PSA(氨基酸1-261)。通过将免疫原性PSA多肽胞质形式(天然人PSA的氨基酸25-261)连接至人PSMA跨膜结构域(人PSMA的氨基酸15-54)来构建膜结合形式的免疫原性PSA多肽。
3B.Pasteur和HLA A24小鼠中的免疫应答
研究设计.如实施例1所述,将8-10周龄的HLA A2Pasteur小鼠或HLAA24小鼠在引发/加强/加强(prime/boost/boost)方案中利用实施例3A提供的PMED用表达各种PSA抗原的DNA免疫,每次免疫接种之间有2周间隔。在最后一次免疫接种之后7天于干扰素-γ(IFNγ)ELISPOT测定和夹心ELISA中测量抗原特异性的T和B细胞应答。
表3中示出的ELISpot数据表明胞质和膜结合形式的免疫原性PSA多肽均能诱导T细胞识别用腺病毒转导以表达胞质PSA抗原的人肿瘤细胞(SKmel5-Ad-PSA),但不能识别用腺病毒转导以表达eGFP的细胞(SKmel5-Ad-eGFP)。这两种抗原还引发对PSA蛋白的应答。PSA分泌抗原不能诱导对SKmel5-Ad-PSA或PSA蛋白的T细胞。SFC>50考虑为阳性。
表3.通过PSA抗原诱导T细胞应答
在Pasteur小鼠至PSA+HLA A2.1+SKmel5人癌细胞中
表4.抗PSA抗体应答的诱导
如通过夹心ELISA测定测量的
表4中的数据证实分泌和膜结合形式的免疫原性PSA多肽均能诱导抗PSA抗体应答。
实施例3.多抗原疫苗构建体的构建
在这个实施例中,描述了利用不同策略构建包含多抗原构建体的质粒。这些质粒享有如pPJV7563的相同的一般质粒骨架。除非另有说明,多抗原构建体中包括的基因编码(1)SEQ ID NO:9的免疫原性PSMA多肽,(2)包含SEQ ID NO:21的氨基酸2-125的免疫原性PSCA多肽,以及(3)SEQ ID NO:17的免疫原性PSA多肽。
实施例3A.包含双抗原构建体的质粒
3A1.利用多启动子构建质粒
质粒460(PSMA/PSCA双启动子).利用位点定向诱变、PCR和限制性片段插入技术构建质粒460。首先,利用位点定向诱变用MD5和MD6引物根据制造商的方案(Quickchange试剂盒,Agilent Technologies,Santa Clara,CA)将Kpn I限制位点引入质粒5259中CMV启动子的上游。其次,利用携带Kpn I限制位点的引物(MD7和MD8)通过PCR从质粒5166扩增由最小CMV启动子、人PSMA和兔B球蛋白转录终止子组成的表达盒。将PCR扩增子用Kpn I消化并插入牛小肠碱性磷酸酶(CIP)处理的质粒5259的新引入的Kpn I位点。
3A2.利用2A肽构建双抗原构建体
质粒451(PSMA-T2A-PSCA).利用重叠PCR和限制片段交换技术构建质粒451。首先,利用质粒5166作为模板用引物119和117通过PCR扩增编码人PSMA氨基酸15-750的基因。利用质粒5259作为模板用引物118和120通过PCR扩增编码全长人PSCA的基因。PCR导致在PSMA的3’端和PSCA的5’端添加重叠的TAV 2A(T2A)序列。将扩增子混合在一起并用引物119和120通过PCR扩增。将PSMA-T2A-PSCA扩增子用Nhe I和Bgl II消化并插入相似消化的质粒5166。在PSMA和T2A盒之间包括甘氨酸-丝氨酸接头以促进高切割效率。
质粒454(PSCA-F2A-PSMA).利用PCR和限制片段交换技术产生质粒454。首先,利用质粒5259作为模板用引物42和132通过PCR扩增编码全长人PSCA的基因。将扩增子用BamH I消化并插入相似消化的、CIP处理的质粒5300。在PSCA和FMDV 2A(F2A)盒之间包括甘氨酸-丝氨酸接头以促进高切割效率。
质粒5300(PSA-F2A-PSMA).利用重叠PCR和限制片段交换技术构建质粒5300。首先,通过PCR用引物MD1和MD2从质粒5297扩增编码PSA氨基酸25-261的基因。通过PCR用引物MD3和MD4从质粒5166扩增编码人PSMA氨基酸15-750的基因。PCR导致在PSA的3’端和PSMA的5’端添加重叠的F2A序列。将扩增子混合在一起并通过PCR延伸。将PSA-F2A-PSMA扩增子用Nhe I和Bgl II消化并插入相似消化的质粒pPJV7563。
3A3.利用内部核糖体进入位点的双抗原构建体
质粒449(PSMA-mIRES-PSCA).利用重叠PCR和限制片段交换技术构建质粒449。首先,通过PCR用引物124和123从质粒5259扩增编码全长人PSCA的基因。通过PCR用引物101和125从pShuttle-IRES扩增最小EMCV IRES。将重叠的扩增子混合在一起并用引物101和123通过PCR扩增。将IRES-PSCA扩增子用Bgl II和BamH I消化并插入Bgl II消化的、CIP处理的质粒5166。为了修正IRES内的自发突变,将包含Avr II至Kpn I序列的IRES用来自pShuttle-IRES的等同片段代替。
质粒603(PSCA-pIRES-PSMA).利用PCR和无缝克隆技术构建质粒603。通过PCR用引物SD546和SD547从质粒455扩增在其3’端连接至优选EMCV IRES的编码全长人PSCA的基因。通过PCR利用引物SD548和SD550从质粒5166扩增编码人PSMA氨基酸15-750的基因。根据制造商的说明书通过无缝克隆(Invitrogen,Carlsbad,CA)将两个重叠的PCR扩增子插入NheI和Bgl II消化的pPJV7563。
质粒455(PSCA-mIRES-PSA).利用重叠PCR和限制片段交换技术构建质粒455。首先,通过PCR用引物115和114从质粒5297扩增编码人PSA氨基酸25-261的基因。通过PCR用引物101和116从pShuttle-IRES扩增最小EMCV IRES。将重叠的扩增子混合在一起并用引物101和114通过PCR扩增。将IRES-PSA扩增子用Bgl II和BamH I消化并插入Bgl II消化的、CIP处理的质粒5259。为了修正这个克隆内的自发突变,用来自新鲜的重叠PCR反应的等同片段代替Bgl II至BstE II序列。
实施例3B.包含三抗原构建体的质粒
一般策略.选择包括PSA-F2A-PSMA、PSMA-mIRES-PSCA、PSMA-T2A-PSCA、PSA-T2A-PSCA、PSCA-F2A-PSMA、PSCA-pIRES-PSMA和PSMA-mIRES-PSA在内的许多双抗原质粒用于以各种组合并入三抗原质粒载体。在所有情况下,质粒载体是基于亲本pPJV7563质粒骨架。4种质粒载体(质粒456、457、458和459)利用单一完全CMV启动子与兔B球蛋白转录终止子来驱动全部3个抗原的表达。2种其他质粒载体(质粒846和850)并入双启动子策略联合IRES或2A以驱动3个抗原的表达。将具有多个2A盒的质粒工程化以携带不同的盒,以便最小化质粒/载体扩增期间第一个和第二个盒之间重组的可能性。通过流式细胞术(图7A和7B)和蛋白印迹(图8A和8B)证实抗原表达。
质粒456(PSA-F2A-PSMA-mIRES-PSCA).通过限制片段交换构建质粒456。将质粒5300用Nhe I和Hpa I消化,并且将~1.8kb插入物连接入相似消化的质粒449。
质粒457(PSA-F2A-PSMA-T2A-PSCA).通过限制片段交换构建质粒457。将质粒5300用Nhe I和Hpa I消化,并且将~1.8kb插入物连接入相似消化的质粒451。
质粒458(PSA-T2A-PSCA-F2A-PSMA).利用PCR和限制片段交换技术构建质粒458。通过PCR用引物119和139从质粒5297扩增编码人PSA氨基酸25-261的基因,导致在3’端添加T2A序列和Nhe I限制位点。将扩增子用Nhe I消化并插入相似消化的质粒454。
质粒459(PSCA-F2A-PSMA-mIRES-PSA).通过限制片段交换构建质粒459。将质粒454用Nhe I和Bgl II消化,并且将包含PSCA-F2A-PSMA的插入物连接入相似消化的质粒455。
质粒846(CBA-PSA,CMV-PSCA-pIRES-PSMA).利用PCR和无缝克隆技术构建质粒846。首先,合成表达盒,其由以下组成:1)来自鸡β肌动蛋白(CBA)基因的启动子和5’非翻译区,2)杂合鸡β肌动蛋白/兔β珠蛋白内含子,3)编码人PSA氨基酸25-261的基因,以及4)牛生长激素终止子。通过PCR用引物3SalICBA和5SalIBGH从质粒796扩增这个PSA表达盒。利用GeneArt无缝克隆和组装试剂盒(Invitrogen,Carlsbad,CA)将扩增子克隆入质粒603的SalI位点。当将这种质粒递送入细胞时,PSA表达会从CBA启动子驱动,而PSCA和PSMA表达会从CMV启动子驱动。
质粒850(CBA-PSA,CMV-PSCA-F2A-PSMA).利用PCR和无缝克隆技术构建质粒850。首先,通过PCR用引物3SalICBA和5SalIBGH从质粒796扩增CBA启动子驱动的PSA表达盒。利用GeneArt无缝克隆将扩增子克隆入质粒454的SalI位点。当将这种质粒递送入细胞时,PSA表达会从CBA启动子驱动,而PSCA和PSMA表达会从CMV启动子驱动。
质粒916((PSA-T2A-PSCA-F2A-PSMA).利用PCR和Gibson克隆技术构建质粒916。通过PCR扩增编码3种PAA多肽的基因并通过Gibson克隆技术将其连接入pPJV7563的Nhe I/Bgl II位点。质粒916的完整核苷酸序列如SEQ ID NO:62所示。质粒458和质粒916编码包含3种免疫原性PAA多肽的相同氨基酸序列,所述氨基酸序列如SEQ ID NO:60所示。编码包含3种PAA多肽的氨基酸序列的质粒916中的核苷酸序列是密码子优化的,并且还如SEQ ID NO:61所示。
表21.在构建多抗原质粒中使用的引物列表
实施例3C.三抗原腺病毒载体
一般策略.当用DNA质粒,可以将病毒载体工程化以递送多个前列腺癌抗原。将上文所述用于多抗原构建体的3种多抗原表达策略-双启动子、2A肽和内部核糖体进入位点–以各种组合合并以产生三抗原腺病毒载体。简单地说,将多抗原表达盒克隆入pShuttle-CMV质粒,将所述pShuttle-CMV载体修改以携带2个拷贝的四环素操纵子序列(TetO2)。利用AdEasy载体系统根据制造商的说明书(Agilent Technologies,Inc.,Santa Clara,CA)产生重组腺病毒血清型5载体。将病毒在HEK293细胞中扩增,并且根据标准方案通过双氯化铯条带进行纯化。在体内研究之前,将病毒储备完全表征病毒颗粒浓度、感染性滴度、无菌性、内毒素、基因组和转基因完整性、转基因性质和表达。
腺病毒-733(PSA-F2A-PSMA-T2A-PSCA).Ad-733是质粒457的病毒等同物。3种抗原的表达从单一CMV启动子驱动,四环素操纵子用于在表达Tet阻遏物的HEK293系中大规模制备期间抑制转基因表达。多抗原表达策略包括两种不同的2A序列。
腺病毒-734(PSA-T2A-PSCA-F2A-PSMA).Ad-734是质粒458的病毒等同物。3种抗原的表达从单一CMV启动子驱动,四环素操纵子用于在表达Tet阻遏物的HEK293系中大规模制备期间抑制转基因表达。多抗原表达策略包括两种不同的2A序列。
腺病毒-735(PSCA-F2A-PSMA-mIRES-PSA).Ad-735是质粒459的病毒等同物。3种抗原的表达从单一CMV启动子驱动,四环素操纵子用于在表达Tet阻遏物的HEK293系中大规模制备期间抑制转基因表达。多抗原表达策略包括2A序列和IRES。
腺病毒-796(CBA-PSA,CMV-PSCA-pIRES-PSMA).Ad-796是质粒846的病毒等同物。PSA的表达从鸡β肌动蛋白启动子驱动,而PSCA和PSMA的表达从CMV-TetO2启动子驱动。多抗原表达策略包括两个启动子和一个IRES。
腺病毒-809(CBA-PSA,CMV-PSCA-F2A-PSMA).Ad-809是质粒850的病毒等同物。PSA的表达从鸡β肌动蛋白启动子驱动,而PSCA和PSMA表达从CMV-TetO2启动子驱动。多抗原表达策略包括两个启动子和一个2A序列。
实施例4.疫苗与舒尼替尼和抗CTLA-4抗体组合的抗癌效力
在包含皮下TUBO肿瘤的BALB/neuT小鼠中研究癌症疫苗与舒尼替尼和抗CTLA-4单克隆抗体(克隆9D9)组合的抗肿瘤效力。
研究方法.简单地说,在肿瘤植入后第10天开始直至第64天,用20mg/kg的媒介物或苹果酸舒尼替尼向10只小鼠/组每天口服剂量给药。在第13天开始用不编码抗原(对照疫苗)或编码SEQ Id NO:54的大鼠Her-2抗原(癌症疫苗)的DNA构建体作为腺病毒载体免疫接种,随后通过DNA用各抗原(HBV抗原或rHer-2)进行2次每周免疫接种、2次每两周免疫接种和7次每周免疫接种。在PMED注射之后第20、27、41、55、62、69、76、83、90和97天将用抗小鼠CTLA-4单克隆抗体处理的小鼠组(实心圆形和空心三角形)用250μg抗体腹腔内注射。
结果.图4示出来自代表性研究的小鼠组的Kaplan-Meier存活曲线,其评价舒尼替尼和抗小鼠CTLA-4单克隆抗体(克隆9D9)与癌症疫苗组合的抗肿瘤效力。在用Sutent与对照疫苗(空心圆形)、抗小鼠CTLA-4单克隆抗体(空心三角形)或癌症疫苗(实心三角形)处理的小鼠中观察到存活时间增加。在用Sutent和癌症疫苗与抗小鼠CTLA-4组合(实心圆形)处理的小鼠中观察到存活的进一步增加。通过log-rank检验计算P值。
实施例5.CPG或CD40激动剂对癌症疫苗诱导的免疫应答的影响
BALB/c小鼠中的免疫原性研究
在BALB/c小鼠中研究局部给药免疫调节剂对癌症诱导的抗原特异性免疫应答的数量和性能的影响,其中通过利用IFNγELISPOT测定或胞内细胞因子染色测定测量rHER2特异性T细胞应答来评价免疫应答。简单地说,通过PMED递送系统将所示的4-6只雌性BALB/c小鼠/组用编码rHER2抗原序列(SEQ ID NO:54)的DNA质粒表达构建体免疫。PMED驱动之后通过在接近疫苗引流腹股沟淋巴结处皮内注射来局部给药免疫调节剂CpG7909(PF-03512676)和抗CD40单克隆激动抗体。根据下文所述方法通过IFNγELISPOT或胞内细胞因子染色测定测量抗原特异性T细胞应答。
胞内细胞因子染色(ICS)测定
通过ICS测定从分离自个体动物的脾细胞或PBMC测量rHer-2特异性多功能(多细胞因子阳性)T细胞免疫应答。通常,将1e6脾细胞与1μg/ml的布雷菲德菌素A和10μg/ml的肽刺激剂(rHer-2特异性CD8p66、rHer-2特异性CD4p169或无关的HBV p87)在5%CO2培养箱中于37℃下温育5hr。刺激之后,将脾细胞洗涤并用Fc封闭剂(抗小鼠CD16/CD32)在4℃下封闭10min,然后用活/死aqua染色、抗小鼠CD3ePE-Cy7、抗小鼠CD8a Pacific蓝和抗小鼠CD45R/B220PerCP-Cy5.5染色20min。将细胞洗涤,用4%多聚甲醛在4℃下固定过夜,用BD fix/perm溶液在RT下透化30min并与抗小鼠IFNγAPC、抗小鼠TNF Alexa488和抗小鼠IL-2PE在RT下温育30min。将细胞洗涤,并且通过流式细胞术获得20,000个CD4或CD8T细胞用于分析。通过疫苗特异性应答减去对无关肽HBV的rHer-2特异性应答并归一化至从脾分离的脾细胞总数量来计算抗原特异性单一、二或三细胞因子阳性T的总数量/每只动物的总脾。
IFNγELISPOT测定结果
图5示出来自代表性研究的小鼠组的IFNγELISPOT结果,所述代表性研究评价当给予所示的免疫调节剂时通过rHER2疫苗诱导的抗原特异性T细胞应答的数量级。简单地说,将每个治疗组的每只小鼠(n=4)通过PMED用编码rHER2抗原序列(SEQ ID NO:54)的DNA质粒表达构建体,然后立即用所示的100ug对照大鼠IgG单克隆抗体(Bioxcell#BE0089:对照mAb)或50g CpG7909或100ug抗CD40单克隆抗体(Bioxcell#BE0016-2:a-CD40mAb)。在PMED驱动之后7天,通过IFNγELISPOT测定从与10μg/ml浓度的对照或rHer-2特异性p66肽混合的5e5脾细胞测量抗原特异性免疫应答。来自包含1e5CD8T细胞的脾细胞的总IFNγ分泌细胞数量计算自来自个体动物的ELISPOT结果和脾细胞中CD8T细胞的%,并且将每组的平均值和平均值的标准误差作图。如所示,与接收对照抗体的小鼠相比,CpG7909和抗CD40单克隆抗体显著增加rHer-2DNA诱导的抗原特异性免疫应答的数量级。
胞内细胞因子染色(ICS)测定结果.图6和7示出代表性研究的结果,所述代表性研究通过胞内细胞因子染色测定评价CpG 7909对疫苗诱导的免疫应答的性能的免疫调节活性。简单地说,将每只动物以4-周间隔用通过PMED递送的编码rHER2抗原序列(SEQ ID NO:54)的DNA质粒表达构建体免疫两次。在通过PMED进行DNA免疫之后立即在接近右侧疫苗引流腹股沟淋巴结处向每组中的小鼠(n=5)皮内注射PBS(PMED组)或50g的CpG 7909(PMED+CpG组)。通过PMED最后一次免疫之后7天,对分离自每只个体小鼠的脾细胞进行ICS测定以检测分泌IFNγ、TNF和/或IL-2的抗原特异性多功能CD8或CD4T细胞。与PBS相比,从用局部递送的CpG 7909处理的小鼠检测到rHer-2特异性多细胞因子阳性CD8和CD4T细胞应答的显著增加。与PBS相比,在接受局部递送的CpG7909给药的动物中观察到单一细胞因子阳性CD8群体的增加。
图8和9示出代表性研究的结果,所述代表性研究通过胞内细胞因子染色测定评价激动性抗CD40单克隆抗体对疫苗诱导的免疫应答的性能的免疫调节活性。简单地说,将每只动物以4周间隔用通过PMED递送的编码rHER2抗原序列(SEQ ID NO:54)的DNA质粒表达构建体免疫两次。在通过PMED给药第一次免疫之后一天,在接近右侧疫苗引流腹股沟淋巴结处向每组中的小鼠(n=6)给予100g皮内注射的同种型IgG对照(用IgG进行PMED)或抗CD40单克隆抗体(用CD40进行PMED)。在最后一次PMED之后7天,对分离自每只个体小鼠的脾细胞进行ICS测定以检测分泌IFNγ、TNF和/或IL-2的rHer-2特异性多功能CD8或CD4T细胞。与同种型IgG对照相比,从用局部递送的抗CD40单克隆抗体处理的小鼠检测到rHer-2特异性三细胞因子阳性CD8和CD4T细胞应答的显著增加。通过局部给予抗CD40单克隆抗体,rHer-2特异性单一和双细胞因子阳性CD4T细胞也有显著增加。
实施例6.癌症疫苗与低剂量舒尼替尼组合的抗癌效力
在具有自发性乳腺垫肿瘤的BALB/neuT小鼠中研究抗癌疫苗与低剂量舒尼替尼组合的抗肿瘤效力。
动物处理.简单地说,一天两次向13-14周龄的雌性小鼠口服给予5mg/kg的苹果酸舒尼替尼(Sutent),持续112天。在第3天通过腺病毒注射给予不递送抗原的对照疫苗和递送SEQ ID NO:54的大鼠Her-2抗原(rHer-2)的癌症疫苗作为引发,然后通过PMED两周一次给药分别递送HBV抗原(对照疫苗)或rHer-2(癌症疫苗)的DNA 7次。当全部10个乳腺垫变为肿瘤阳性时或者当任何乳腺肿瘤的体积达到2000mm3时确定存活终点。
结果.结果在图10中示出。与以前发表的Sutent的药物动力学谱(Mendel,D.,Laird,D.,et al.:“In vivo antitumor activity of SU11248,a novel tyrosinekinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors:determination of a pharmacokinetic/pharmacodynamic relationship”.Clinical Cancer Research,203,9:327-337)相比,在一天两次以5mg/kg剂量给药的小鼠中Sutent的CMax预期显著低于在小鼠和人中达到有效抗肿瘤效力所必需的最小血液水平。数据显示用低剂量Sutent单一治疗处理的小鼠的存活快速且暂时提高。但是当给予癌症疫苗时,观察到存活更持久且显著提高(通过Log rank检验P<0.0001)。
实施例7.通过局部给药CpG增强疫苗诱导的免疫应答
在猴研究中研究局部给药CpG(PF-03512676)对本发明提供的人PSMA核酸诱导的免疫应答的免疫增强,其中通过利用IFNγELISPOT测定测量PSMA特异性T细胞应答来评价免疫应答。
动物处理和样品采集.将6组食蟹猴(每个测试组6只(#1-6))用通过电穿孔递送的编码人PSMA修改抗原(SEQ ID NO:9的多肽)的质粒DNA免疫。简单地说,在第0天所有动物接受两侧肌肉内注射的5mg质粒DNA然后电穿孔(DNA EP)。在电穿孔之后,第2组在接近DNA注射部位处接受两侧肌肉内注射的与1mg明矾混合的2mg的CpG。第3和4组分别在第0天或第3天在接近DNA注射部位处接受两侧肌肉内注射的没有明矾递送的2mg的CpG。第5组在第3天接受在接近疫苗引流腹股沟淋巴结处递送的2mg两侧皮内注射的CpG。第6组在第0天接受两侧注射的与DNA溶液混合的200g的CpG,将其共电穿孔入肌肉。
IFNγELISPOT测定方法.DNA免疫之后15天从每只动物采集外周血样品。从血液样品分离外周血单核细胞(PBMC)并使其进行IFNγELISPOT测定以测量PSMA特异性T细胞应答。简单地说,将来自个体动物的4e5PBMC平板接种在IFNγELISPOT平板中的每个孔中,所述孔具有各自2ug/ml的PSMA特异性肽或非特异性对照肽(人HER2肽池)的池。每个PSMA特异性肽池的组成在表24A中提供。将平板在37℃和5%CO2下温育16hr,洗涤并在温育之后按照制造商的说明书显影。通过CTL读数器计数IFNγ斑点形成细胞(SFC)的数量。每个条件一式两份进行。
结果.表6示出代表性IFNγELISPOT测定的结果,所述代表性IFNγELISPOT测定评价并比较没有(第1组)或者有通过肌肉内(第2、3、4和5组)或皮内注射(第6组)局部给予的CpG(PF-03512676)的疫苗诱导的IFNγT细胞应答。通过PSMA肽池的SFC平均数减去非特异性对照肽(人HER2肽池)的SFC平均数并归一化至用1e6PBMC观察的SFC来计算报道的PSMA特异性应答。^表示计数不准,因为斑点的数量太多不能计数。ND表示未确定。
在全部6只动物(第1组)中检测在不存在CpG(PF-03512676)下对多PSMA特异性肽池的PSMA特异性IFNγT细胞应答。在DNA电穿孔之后3天通过肌肉内(第4组:3/6)或皮内(第5组:2/6)注射额外接受CpG(PF-03512676)的一些动物中测量的对PSMA肽的总应答适度升高。但是,当在电穿孔之后同一天递送CpG时(第2和3组),有几只动物不能产生高应答(第2组:4/6和第3组:3/6),无论与明矾混合或不与明矾混合。但是,与未接受CpG的动物(第1组)相比,当将低10倍剂量的CpG与DNA溶液共电穿孔入肌肉时(第6组),在4/6动物中检测到较高的净应答,具有统计上较高的对肽池H1和R1的应答(P<0.05)。数据表明当共电穿孔入肌肉时,低剂量的CpG可以有效地增强DNA疫苗诱导的IFNγT细胞应答。
表6.DNA电穿孔之后15天,通过IFNγELISPOT测定从PBMC测量没有(第1组)或有CpG(第2、3、4、5和6组)的DNA疫苗诱导的PSMA特异性IFNγT细胞应答
实施例8.通过局部给药抗CTLA-4抗体增强疫苗诱导的免疫应答
在猴研究中研究低剂量皮下给药的抗CTLA-4单克隆抗体(CP-675,206)对恒河猴PSMA核酸诱导的免疫应答的影响,其中通过利用IFNγELISPOT测定测量PSMA特异性T细胞应答来评价免疫应答。研究中使用的恒河猴PSMA核酸具有如SEQ ID NO:56所示的序列,并且编码SEQ ID NO:55的免疫原性PSMA多肽。
动物处理和样品采集.将5组雄性印度恒河猴(每个测试组7只(#1-7))用通过两侧肌肉内注射(2x 5e10V.P.)递送的编码恒河猴PSMA修改多肽的腺病毒免疫。腺病毒注射之后,第1组立即接受媒介物(vehicle),并且第2-4组在接近疫苗引流淋巴结处分别立即接受剂量2x 25mg、2x 16.7mg和2x 8.4mg的两侧皮下注射的抗CTLA-4抗体(CP-675,206)。
免疫之后9天,从每只动物分离外周血单核细胞(PBMC)并使其进行IFNγELISPOT测定以测量恒河猴PSMA特异性T细胞应答。简单地说,将来自个体动物的4e5PBMC平板接种在IFNγELISPOT平板中的每个孔中,所述孔具有各自2ug/ml的恒河猴PSMA特异性肽(表24A中定义的P1、P2、P3或R1+R2)或非特异性对照肽(人HER2肽池)的池。将平板在37℃和5%CO2下温育16hr,洗涤并在温育之后按照制造商的说明书显影。通过CTL读数器计数IFNγ斑点形成细胞(SFC)的数量。每个条件一式两份进行。将来自恒河猴PSMA特异性肽池的背景调整的SFC的一式两份的平均值归一化至1e6PBMC中的应答。来自每个个体动物的对肽池的个体和总应答在表29中示出。
IFNγELISPOT测定方法.在4℃下将IFNγ特异性的捕获抗体.(BD Bioscience,#51-2525kc)包被在微孔板中的聚偏氟乙烯(PVDF)膜上过夜。将平板用血清/蛋白封闭以防止非特异性结合至抗体。封闭之后,将效应细胞(如分离自免疫小鼠的脾细胞或分离自恒河猴的PBMC)和靶标(如来自肽文库的PSMA肽、用抗原特异性肽脉冲的靶细胞或表达相关抗原的肿瘤细胞)添加至孔中,并且在5%CO2培养箱中于37℃下温育过夜。通过PVDF膜表面上的包被抗体捕获效应细胞分泌的细胞因子。去除细胞和培养基之后,将100μl生物素化的多克隆抗人IFNγ抗体添加至每个孔中用于检测。按照制造商(Mabtech)的方案,通过添加链霉抗生物素蛋白-辣根过氧化物酶和沉淀底物3-氨基-9-乙基咔唑(AEC)以产生红色斑点来使斑点可见。每个斑点代表单一细胞因子产生T细胞。
结果.表7示出代表性IFNγELISPOT测定的结果,所述代表性IFNγELISPOT测定比较没有(第1组)或有(第2-4组)在接近疫苗引流淋巴结处通过皮下注射局部给予的抗CTLA-4单克隆抗体(CP-675,206)的疫苗诱导的T细胞应答。疫苗产生免疫应答(第1组),通过局部给药剂量50mg的抗CTLA-4抗体(CP-675,206)显著增强所述免疫应答(第2组,利用低估值通过学生T-检验P=0.001)。分别为33.4mg(第3组:利用低估值通过学生T-检验P=0.004)和16.7mg(第4组:通过学生T-检验P=0.05)的低剂量抗CTLA-4抗体也显著增强所述应答。数据表明通过皮下注射递送的低剂量抗CTLA-4可以显著增强疫苗诱导的免疫应答。
表7.没有(第1组)或有皮下注射的抗CTLA-4抗体(CP-675,206)的疫苗诱导的IFNγT细胞应答。^表示由于高斑点数量,计数是低估的。TNTC表示太多而不能计数。
实施例9.通过低剂量舒尼替尼免疫调节髓源性抑制细胞
提供以下实施例以说明在非肿瘤小鼠模型中,在体内低剂量舒尼替尼对髓源性抑制细胞(MDSC)的免疫调节效应。
研究方法.
为了产生MDSC富集的脾细胞,将TUBO细胞(1x106)植入5只BALB/neuT小鼠的胁腹,并且保留约20-30天直至肿瘤体积达到1000-1500mm3。然后将小鼠处死,取出脾并回收MDSC富集的脾细胞。将脾细胞用5μM CFSE标记10分钟,用PBS洗涤并计数。随后将标记的细胞以5x107个脾细胞/ml重悬于PBS中,并且通过i.v.尾静脉注射过继转移入幼稚BALB/c受体小鼠。继承性转移之前3天,受体小鼠开始用5mg/kg、10mg/kg和20mg/kg的媒介物或苹果酸舒尼替尼(Sutent)每天两次剂量给药。过继转移之后,受体小鼠继续接受每天两次剂量给药的媒介物或舒尼替尼另外两天,该点之后将小鼠处死,取出脾,回收脾细胞并加工用于表型分析。
将脾细胞计数并以5x106个细胞/ml重悬于FACS染色缓冲液(PBS、0.2%(w/v)牛血清白蛋白和0.02%(w/v)叠氮钠)中。为了脾细胞的流式细胞术染色,首先将2.5x106个细胞与CD16/CD32的抗体在4℃下温育10分钟以封闭Fc受体并最小化非特异性结合。然后将脾细胞用小鼠细胞表面标记的适当荧光团缀合的抗体(Biolegend)在4℃下染色20分钟。对于T细胞(抗CD3(Pacific Blue),克隆17A2),而对于MDSC(抗GR-1(APC),克隆RB6-8C5和抗CD11b(PerCp Cy5.5),克隆M1/70)。还包括活/死染色。抗体温育之后,将染色的脾细胞用2ml的FACS缓冲液洗涤,在BD CANTOII流式细胞仪上进行数据采集之前通过离心沉淀并重悬于0.2ml的FACS缓冲液中。为了监测舒尼替尼或媒介物对过继转移的MDSC存活的影响,我们计算了活的、单重门中的CFSE+、CD3-、GR1+、CD11b+百分比。然后我们通过计算总脾细胞计数代表的实际细胞数量百分比来确定每个脾过继转移的MDSC的数量。通过FloJo和Graphpad软件分析数据。
结果.表27中示出的数据代表过继转移之后2天,来自用媒介物或者5mg/kg、10mg/kg和20mg/kg舒尼替尼每天两次剂量给药的小鼠的每个脾回收的过继转移的CSFE+、CD3-、GR1+、CD11b+细胞的平均数量。利用Dunnett’s多重比较检验通过单向ANOVA确定统计显著性,比较舒尼替尼剂量给药组与0mg/kg(媒介物)组。数据证实每天两次剂量给药的舒尼替尼在体内对MDSC具有免疫调节效应,甚至当低至5mg/kg剂量给药时,仍导致与媒介物处理的对照组相比时回收的数量在统计上显著减少。
表8.从脾回收的CFSE+、CD3-、GR1+、CD11b+MDSC的平均数量
实施例10.三抗原腺病毒和DNA构建体的免疫原性
提供以下实施例以说明本发明提供的表达3种抗原PSMA、PSCA和PSA的三抗原疫苗构建体(腺病毒载体或DNA质粒形式)在非人灵长类中引发对全部3种编码的抗原的特异性T细胞应答的能力。
体内研究方法.引发之后9天,将本发明提供的各自表达3种抗原的5种腺病毒载体(PSMA、PSCA和PSA;Ad-733、Ad-734、Ad-735、Ad-796和Ad-809)的T细胞免疫原性与各自仅表达单一抗原(PSMA、PSA或PSCA)的3种腺病毒载体的混合物进行比较。评价对表达单一抗原的单一腺病毒(第1-3组)的应答以证实特异性。简单地说,将印度恒河猴(对于第1和3组,n=6;对于第2组,n=7;而对于第4-9组,n=8)用总计1e11V.P.肌肉内注射,然后在同一天皮内注射10mg/kg的抗CTLA-4。注射之后9天,从每只动物分离外周血单核细胞(PBMC)并使其进行IFNΓELISPOT测定以测量PSMA、PSA和PSCA特异性T细胞应答。
腺病毒和抗CTLA-4注射之后13周,当T细胞应答已收缩时,猴接受通过电穿孔递送的DNA(第1组:PSMA,质粒5166;第2组:PSA,质粒5297;第3组:PSCA,质粒5259;第4组:PSMA、PSA和PSCA的混合物,质粒5166、5259和5297;第4组:质粒457;第6组:质粒458;第7组:质粒459;第8组:质粒796以及第9组:质粒809)增强免疫接种。总的来说,每只动物接受总计5mg的本发明提供的质粒DNA,其递送引发中使用的腺病毒中编码的相同表达盒。增强免疫接种之后,从每只动物分离外周血单核细胞(PBMC)并使其进行IFNγELISPOT测定。
IFNγELISPOT测定.简单地说,将来自个体动物的4e5PBMC平板接种在IFNγELISPOT平板中的每个孔中,所述孔具有各自2ug/ml的PSMA特异性肽池P1、P2、P3或H1和H2(表9A),PSA特异性池1或2(表9B)、PSCA特异性池(表10)或者非特异性对照肽(人HER2肽池)。将平板在37℃和5%CO2下温育16hr,洗涤并在温育之后按照制造商的说明书显影。通过CTL读数器计数IFNγ斑点形成细胞(SFC)的数量。每个条件一式两份进行。将来自抗原特异性肽池的背景调整的SFC的一式两份的平均值归一化至1e6PBMC中的应答。表中的抗原特异性应答表示对相应抗原特异性肽或肽池的应答的总和。
结果:表11代表研究,其在印度恒河猴中通过IFNγELISPOT评价与各自表达单一抗原的3种腺病毒的混合物相比,各自表达全部3种抗原的5种不同腺病毒的T细胞免疫原性。大多数仅接受Ad-PSMA(第1组)注射的动物诱导对PSMA的特异性应答,但不诱导对PSA或PSCA的特异性应答(学生T-检验,P<0.03。将诱导对PSCA的应答的一只动物(#4)优先从统计分析去除)。仅接受Ad-PSA注射的动物(第2组)诱导对PSA的特异性应答,但不诱导对PSMA或PSCA的特异性应答(学生T-检验,P<0.02)。仅接受Ad-PSCA注射的动物(第3组)诱导对PSCA的特异性应答,但不诱导对PSMA或PSA的特异性应答(学生T-检验,P<0.03)。全部5种三抗原表达腺病毒载体(第5-9组)均诱导对全部3种抗原的IFNγT细胞应答,数量级随动物而变化。三抗原表达腺病毒诱导的对PSCA的应答的数量级与单独抗体的混合物相似(第4组)。但是,Ad-809(第9组)诱导的对PSMA的应答和Ad-796(第8组)诱导的对PSA的数量级分别各自显著优于混合物(学生T-检验,P=0.04和P=0.02)。这些结果表明在非人灵长类中用表达三抗原的腺病毒免疫接种可以引发与用单独腺病毒的混合物免疫接种等同或更优的T细胞免疫应答。
表12示出代表研究的IFNγELISPOT结果,所述研究评价通过腺病毒引发联合抗CTLA-4然后相应质粒DNA的电穿孔增强来递送的本发明提供的5种不同三抗原表达盒的免疫原性。将免疫应答与通过腺病毒引发与抗CTLA-4以及DNA电穿孔增强免疫接种相似递送的表达单一抗原的3种构建体的混合物进行比较。
仅接受Ad-PSMA与抗CTLA-4然后质粒-PSMA(第1组)免疫接种的所有动物诱导对PSMA的特异性应答,但不诱导对PSA或PSCA的特异性应答。相似地,仅接受Ad-PSA与抗CTLA-4然后质粒-PSA免疫接种的所有动物(第2组)诱导对PSA的特异性应答,但不诱导对PSMA或PSCA的特异性应答,最后,仅接受Ad-PSCA与抗CTLA-4然后质粒-PSCA(第3组)免疫接种的所有动物诱导对PSCA的特异性应答,但不诱导对PSMA或PSA的特异性应答(学生T-检验,P<0.01)。
已用三抗原表达载体(第5-9组)或混合物(第4组)免疫的所有动物均诱导对全部3种抗原的IFNγT细胞应答。检测的PSCA或PSA特异性IFγT细胞的频率分别在所有这些组(第4-9组)相似。但是接受三抗原表达载体免疫接种的构建体组7和9产生比3种单一抗原表达构建体的混合物(第4组)显著高的对PSMA的应答频率。这些结果表明在一个盒中表达三抗原的腺病毒和DNA疫苗可以在非人灵长类引发与表达单一抗原的腺病毒和DNA的混合物等同或更优的IFNγT细胞应答。
表9A.PSMA肽池*
表9B.PSA肽池:示出与来自PSA肽文库的13个氨基酸重叠的15个氨基酸肽的氨基酸位置和序列。
表10.PSCA肽池:示出与来自PSCA肽文库的13个氨基酸重叠的15个氨基酸肽的氨基酸位置和序列。
表11.通过ELISPOT分析的腺病毒引发与抗CTLA-4之后单一抗原(第1组:Ad-PSMA;第2组:Ad-PSA;第3组:Ad-PSCA;第4组:Ad-PSMA、Ad-PSA和Ad-PSCA的混合物)或三抗原表达腺病毒载体(第4组:Ad-733;第6组:Ad-734;第7组:Ad-735;第8组:Ad-796和第9组:Ad-809)诱导的IFNγT细胞应答。
表12.通过ELISPOT测定分析的腺病毒引发与抗CTLA-4以及DNA电穿孔增强免疫接种之后单一抗原(第1组:PSMA;第2组:PSA;第3组:PSCA;第4组:PSMA、PSA和PSCA的混合物)或三抗原表达载体(第5–9组)诱导的IFNγT细胞应答。
实施例11.C68载体的构建
11A.载体AdC68-734构建
AdC68-734是基于黑猩猩腺病毒C68的复制缺陷型腺病毒,其编码3种免疫原性PAA多肽–免疫原性PSA多肽、免疫原性PSCA多肽和免疫原性PSMA多肽。在生物信息学中设计载体序列。首先,基线全长C68序列获得自Genbank(定义:猿猴腺病毒25,完整基因组;登录号AC_000011.1)。将文献中描述的5个点突变引入该序列。(Roshorm,Y.,M.G.Cottingham,etal.(2012)."T cells induced by recombinant chimpanzee adenovirus alone and inprime-boost regimens decrease chimeric EcoHIV/NDK challenge virus load."Eur JImmunol 42(12):3243-3255)然后将2.6千碱基的病毒早期转录区1(E1)缺失以使得病毒不可复制,并且将3.5千碱基的早期转录区3(E3)去除以在载体中为转基因表达盒产生空间。(Tatsis,N.,L.Tesema,et al.(2006).Chimpanzee-origin adenovirus vectors asvaccine carriers.Gene Ther.13:421-429)然后将高效真核表达盒引入E1区。表达盒包括以下组分:(A)巨细胞病毒(CMV)即时早期增强子/启动子;(B)Tet操纵子(四环素阻遏物的结合位点);(C)多抗原构建体,其包含(1)编码人PSA的氨基酸25-261的核苷酸序列,(2)编码甘氨酸-丝氨酸接头和Thosea asigna病毒2A肽(T2A)的顺式作用水解酶元件,(3)编码人PSCA的氨基酸2-123的核苷酸序列,(4)编码甘氨酸-丝氨酸接头和口蹄疫病毒2A肽(F2A)的顺式作用水解酶元件,以及(5)编码人PSMA的氨基酸15-750的核苷酸序列;以及(D)SV40polyA转录终止信号。最后,在病毒基因组的每个末端插入PacI限制位点以促进从亲本杆粒释放基因组。在病毒扩增期间去除来自PacI限制位点的核苷酸,因此不掺入载体产物的基因组本身。包括PacI限制位点在内的整个载体AdC68-734的核苷酸序列在SEQ ID NO:58中示出。并入载体AdC68-734(以及质粒458)的多抗原构建体(PSA-T2A-PSCA-F2A-PSMA)还在SEQ ID NO:61中示出。SEQ ID NO:61的多抗原构建体编码的氨基酸序列在SEq ID NO:60中示出。载体AdC68-734的组分在表13中提供。
表13.载体AdC68-734的组分
在生物信息学中设计之后,在多阶段方法中利用体外寡核苷酸合成以及随后在大肠杆菌和酵母中重组介导的中间组装来生物化学合成34,819碱基对序列。最终将病毒基因组插入细菌人工染色体(pCC1BAC-LCyeast-TRP Trunc)用于扩增。在产生过程中的多个步骤进行下一代测序(MiSeq技术),包括用来产生病毒种子原液的最终杆粒17.3.3.22批次。通过用PacI消化杆粒17.3.3.22以从BAC骨架释放AdC68-734基因组来产生病毒种子原液。将线性化的核酸转染入E1补足贴壁HEK293细胞系中,并且当可见细胞病理效应和腺病毒集落形成时,通过多轮冻/融收获培养物以从细胞释放病毒。通过标准技术扩增并纯化病毒。杆粒17.3.3.22的基因结构在图11中提供。
11B.额外的C68载体的构建
以与AdC68-734相似的方式构建额外的三抗原C68载体。一些额外的载体包括与载体AdC68-734中略微不同的C68基因组中的功能缺失,而其他并入不同的多抗原构建体。基于这些实施例和本公开的其他描述,本领域技术人员能够从C68构建额外的载体用于表达各种多抗原构建体,其全部在本发明的范围内。
(1)AdC68X-734和AdC68W-734
载体AdC68X-734构建自C68,其通过缺失SEQ ID NO:57的C68基因组的核苷酸577-3403(E1区)和27125–31831(E2区)来功能缺失C68基因组的E1和E3区以及在缺失的E1区中插入SEQ ID NO:61的三抗原构建体(PSA-T2A-PSCA-F2A-PSMA)来构建。载体AdC68W-734与载体AdC68-734相同,除了AdC68W-734在C68NDA序列中包含一个或多个突变。
(2)AdC68X-733和AdC68X-735
载体AdC68X-733和AdC68X-735分别是通过用SEQ ID NO:65和66的三抗原构建体代替AdC68X-734载体中并入的三抗原构建体来产生的。并入载体AdC68X-733的多抗原构建体(即,PSA-F2A-PSMA-T2A-PSCA)与并入质粒457的相同,并且并入载体AdC68X-735的多抗原构建体(即,PSCA-F2A-PSMA-mIRES-PSA)与质粒459相同。
11C.研究生产力表征
产生各种研究等级批次的AdC68-734并测试生产力。将杆粒用PacI消化以从BAC骨架释放载体基因组,并且将线性化的核酸转染入E1补足贴壁HEK293细胞系中。当大量细胞病理效应和腺病毒集落可见时,通过多轮冻/融收获培养物以从细胞释放病毒。将来自这些第0代(P0)培养物的病毒在组织培养瓶中扩增至少一个额外的世代,然后用作种子原液用于研究规模的生产运行(~0.5-3e13个总病毒颗粒/批)。总计实施11个生产运行(5个在HEK293悬浮细胞中,6个在HEK293贴壁细胞中)。平均特异性生产力为15,000+/-6,000个纯化的病毒颗粒/初始感染的细胞,具有55的病毒颗粒:感染单位比例。研究规模的生产力总结于表14中。
表14.研究规模的生产批次的特异性生产力和感染性
*在生产中使用的晚代HEK293悬浮细胞
11D.抗原表达
通过流式细胞术测量PSMA和PSCA的表面表达(图12),并且通过来自MOI=10,000的AdC68-载体感染的A549细胞的蛋白印迹分析测量PSMA、PSCA和PSA的总细胞表达(图13)。感染之后2天,将模拟和AdC68感染的细胞用抗PSCA(异硫氰酸荧光素缀合的单克隆抗体1G8[1:200])和PSMA抗体(别藻蓝蛋白缀合的单克隆抗体J591[1:200])染色用于流式细胞术分析。从大多数用不同三抗原表达AdC68载体感染的细胞检测到PSCA和PSMA的表面表达,具有不同水平。从AdC68X-809感染的细胞检测到相对较高水平的PSCA和PSMA表达,并且从AdC68X-733感染的细胞检测到较低水平。感染之后2天,将来自约1x105个感染细胞的总细胞裂解物上样至十二烷基硫酸钠聚丙烯酰胺凝胶的每条泳道。随后将凝胶转移至膜用于检测PSA、PSMA和PSCA蛋白,利用PSA、PSMA和PSCA特异性的一抗通过蛋白印迹分析进行检测。在感染细胞中检测到全部3种抗原的不同程度的表达。虽然从AdC68-734和AdC68X-735感染裂解物检测到相对相似水平的PSMA和PSCA,但是与AdC68X-735相比,从AdC68-734检测到较高水平的PSA。
11E.免疫原性
进行各种三抗原AdC68载体诱导的CD8IFNγ应答的直接比较。将每组小鼠(每组n=5)在四头肌中用1e9或1e10VP的AdC68-734、AdC68X-735、AdC68X-809或Ad5-734免疫。通过在免疫之后第13天从每只动物收集脾来测量小鼠中的IFNγCD8+T细胞应答。分离脾细胞并使其进行IFNγELISPOT测定以测量PSMA、PSCA和PSA特异性的T细胞应答。简单地说,将来自免疫动物的2.5-5x105个脾细胞在10μg/ml的单独人PSMA、PSCA或PSA特异性肽的存在下进行培养。以前确定15-聚体肽包含每个前列腺抗原的CD8+T细胞表位。单独用培养基培养的脾细胞用作对照。每个条件一式三份进行。将平板在37℃和5%CO2下温育20h,洗涤,并且在温育之后按照制造商的说明书显影。通过CTL读数器计数IFNγSFC的数量。结果示出PSMA、PSCA和PSA特异性SFC的平均数量,将其减去单独培养基背景值并归一化至1x106个脾细胞。
总的来说,所有三抗原表达AdC68载体均诱导对全部3种抗原的免疫应答,但是至不同数量级。在1e9VP下,通过AdC68载体的对PSMA的应答与Ad5相似。通过3种AdC68载体的对PSCA的应答与Ad5诱导的应答相似或低于Ad5诱导的应答,但是与所有测试的载体相比,用Ad68-735对PSA的应答较低。但是在1e10VP下,与AdC68-734、AdC68-735或Ad5相比,AdC68-809诱导相似或更好的对全部3种抗原的应答。结果在表15中示出。
表15.通过IFNγELISPOT测定的在C57BL6小鼠中通过共表达PSMA、PSA和PSCA的AdC68载体的IFNγT细胞免疫原性
选择原始序列
SEQ ID NO:1.全长人PSMA的氨基酸序列
MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA
SEQ ID NO:2.编码SEQ ID NO:1的全长人PSMA的核苷酸序列
atgtggaatctccttcacgaaaccgactcggctgtggccaccgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttcgggtggtttataaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctagcacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctagccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcc
SEQ ID NO:3.PSMA改组抗原1的氨基酸序列
SEQ ID NO:4.编码SEQ ID NO:3的PSMA改组抗原1的氨基酸序列的核苷酸序列
SEQ ID NO:5.PSMA改组抗原2的氨基酸序列
SEQ ID NO:6.编码SEQ ID NO:5的PSMA改组抗原2的氨基酸序列的核苷酸序列
SEQ ID NO:7.PSMA改组抗原3的氨基酸序列
SEQ ID NO:8.编码SEQ ID NO:7的PSMA改组抗原3的氨基酸序列的核苷酸序列
SEQ ID NO:9.膜结合PSMA抗原的氨基酸序列
MASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA
SEQ ID NO:10.编码SEQ ID NO:9的膜结合PSMA抗原的氨基酸序列的核苷酸序列
atggctagcgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttcgggtggtttataaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctggcacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctggccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcc
SEQ ID NO:11.胞质PSMA抗原的氨基酸序列
SEQ ID NO:12.编码SEQ ID NO:11的胞质PSMA抗原的氨基酸序列的核苷酸序列
SEQ ID NO:13.分泌PSMA抗原的氨基酸序列
SEQ ID NO:14.编码SEQ ID NO:13的分泌PSMA抗原的氨基酸序列的核苷酸序列
SEQ ID NO:15.全长人PSA的氨基酸序列
MASWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP
SEQ ID NO:16.编码SEQ ID NO:15的全长人PSA的氨基酸序列的核苷酸序列
atggctagctgggtcccggttgtcttcctcaccctgtccgtgacgtggattggcgctgcgcccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagcttgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaacccc
SEQ ID NO:17.胞质PSA抗原的氨基酸序列
MASIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP
SEQ ID NO:18.编码SEQ ID NO:17的胞质PSA抗原的氨基酸序列的核苷酸序列
atggctagcattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagcttgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaacccc
SEQ ID NO:19.膜结合PSA抗原的氨基酸序列
MASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPGIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP
SEQ ID NO:20.编码SEQ ID NO:19的膜结合PSA抗原的氨基酸序列的核苷酸序列
atggctagcgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttcgggtggtttataaaatcctccaatgaagctactaacattactccaggaattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagcttgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaacccctga
SEQ ID NO:21.全长人PSCA的氨基酸序列
MASKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQL
SEQ ID NO:22.编码SEQ ID NO:21的全长人PSCA的氨基酸序列的核苷酸序列
atggctagcaaggctgtgctgcttgccctgttgatggcaggcttggccctgcagccaggcactgccctgctgtgctactcctgcaaagcccaggtgagcaacgaggactgcctgcaggtggagaactgcacccagctgggggagcagtgctggaccgcgcgcatccgcgcagttggcctcctgaccgtcatcagcaaaggctgcagcttgaactgcgtggatgactcacaggactactacgtgggcaagaagaacatcacgtgctgtgacaccgacttgtgcaacgccagcggggcccatgccctgcagccggctgccgccatccttgcgctgctccctgcactcggcctgctgctctggggacccggccagcta
SEQ ID NO:23.质粒5166的核苷酸序列
SEQ ID NO:24.质粒5259的核苷酸序列
SEQ ID NO:25.质粒5297的核苷酸序列
SEQ ID NO:26.质粒460的核苷酸序列
SEQ ID NO:27.质粒451的核苷酸序列
SEQ ID NO:28.质粒454的核苷酸序列
SEQ ID NO:29.质粒5300的核苷酸序列
SEQ ID NO:30.质粒449的核苷酸序列
SEQ ID NO:31.质粒603的核苷酸序列
SEQ ID NO:32.质粒455的核苷酸序列
SEQ ID NO:33.质粒456的核苷酸序列
SEQ ID NO:34.质粒457的核苷酸序列
SEQ ID NO:35.质粒458的核苷酸序列
ggcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcaaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacaggtcgacaatattggctattggccattgcatacgttgtatctatatcataatatgtacatttatattggctcatgtccaatatgaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttacgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacaccaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggattccccgtgccaagagtgactcaccgtccggatctcagcaagcaggtatgtactctccagggtgggcctggcttccccagtcaagactccagggatttgagggacgctgtgggctcttctcttacatgtaccttttgcttgcctcaaccctgactatcttccaggtcaggatcccagagtcaggggtctgtattttcctgctggtggctccagttcaggaacagtaaaccctgctccgaatattgcctctcacatctcgtcaatctccgcgaggactggggaccctgtgacgaacatggctagcattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagcttgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaaccccGGATCCgaaggtaggggttcattattgacctgtggagatgtcgaagaaaacccaggacccgctagcaaggctgtgctgcttgccctgttgatggcaggcttggccctgcagccaggcactgccctgctgtgctactcctgcaaagcccaggtgagcaacgaggactgcctgcaggtggagaactgcacccagctgggggagcagtgctggaccgcgcgcatccgcgcagttggcctcctgaccgtcatcagcaaaggctgcagcttgaactgcgtggatgactcacaggactactacgtgggcaagaagaacatcacgtgctgtgacaccgacttgtgcaacgccagcggggcccatgccctgcagccggctgccgccatccttgcgctgctccctgcactcggcctgctgctctggggacccggccagctaGgatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaatggcaagcgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttcgggtggtttataaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctggcacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctggccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcctaaagatctgggccctaacaaaacaaaaagatggggttattccctaaacttcatgggttacgtaattggaagttgggggacattgccacaagatcatattgtacaaaagatcaaacactgttttagaaaacttcctgtaaacaggcctattgattggaaagtatgtcaaaggattgtgggtcttttgggctttgctgctccatttacacaatgtggatatcctgccttaatgcctttgtatgcatgtatacaagctaaacaggctttcactttctcgccaacttacaaggcctttctaagtaaacagtacatgaacctttaccccgttgctcggcaacggcctggtctgtgccaagtgtttgctgacgcaacccccactggctggggcttggccataggccatcagcgcatgcgtggaacctttgtggctcctctgccgatccatactgcggaactcctagccgcttgttttgctcgcagccggtctggagcaaagctcataggaactgacaattctgtcgtcctctcgcggaaatatacatcgtttcgatctacgtatgatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggaattctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactc
SEQ ID NO:36.质粒459的核苷酸序列
SEQ ID NO:37.PSHUTTLE IRES的核苷酸序列
SEQ ID NO:38.Her-2抗原的氨基酸序列:
SEQ ID NO:39.编码SEQ ID NO:38的Her-2抗原氨基酸序列的核酸序列
SEQ ID NO:40.抗CD40抗体CP870,893的重链的氨基酸序列:
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
SEQ ID NO:41.抗CD40抗体CP870,893的轻链的酸序列:
MRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
SEQ ID NO:42.抗CTLA-4抗体曲美木单抗的重链的酸序列
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:43.抗CTLA-4抗体曲美木单抗的轻链的酸序列
DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:44.CpG 7909的核苷酸序列
5'TCGTCGTTTTGTCGTTTTGTCGTT3'
SEQ ID NO:45.CpG 24555的核苷酸序列
5'TCGTCGTTTTTCGGTGCTTTT3'
SEQ ID NO:46.CpG 10103的核苷酸序列
5'TCGTCGTTTTTCGGTCGTTTT3'
SEQ ID NO:47.eGFP的氨基酸序列
SEQ ID NO:48.HBV核心抗原的氨基酸序列
SEQ ID NO:49.HBV表面抗原的氨基酸序列
SEQ ID NO:50.恒河猴PSMA ECD蛋白的氨基酸序列:
SEQ ID NO:51.大鼠Her-2p66肽(H-2d T细胞表位)的氨基酸序列
SEQ ID NO:52.大鼠Her-2p169肽(H-2d T细胞表位)的氨基酸序列
SEQ ID NO:53.HBV核心抗原p87肽的氨基酸序列
SEQ ID NO:54.大鼠Her-2抗原(rHer-2)的氨基酸序列:
SEQ ID NO:55.恒河猴PSMA抗原的氨基酸序列:
SEQ ID NO:56.编码SEQ ID NO:55”的恒河猴PSMA抗原的核苷酸序列
SEQ ID NO:57.猿猴腺病毒25(C68)的完整基因组
ccatcttcaataatatacctcaaactttttgtgcgcgttaatatgcaaatgaggcgtttgaatttggggaggaagggcggtgattggtcgagggatgagcgaccgttaggggcggggcgagtgacgttttgatgacgtggttgcgaggaggagccagtttgcaagttctcgtgggaaaagtgacgtcaaacgaggtgtggtttgaacacggaaatactcaattttcccgcgctctctgacaggaaatgaggtgtttctgggcggatgcaagtgaaaacgggccattttcgcgcgaaaactgaatgaggaagtgaaaatctgagtaatttcgcgtttatggcagggaggagtatttgccgagggccgagtagactttgaccgattacgtgggggtttcgattaccgtgtttttcacctaaatttccgcgtacggtgtcaaagtccggtgtttttacgtaggtgtcagctgatcgccagggtatttaaacctgcgctctccagtcaagaggccactcttgagtgccagcgagaagagttttctcctccgcgccgcgagtcagatctacactttgaaagatgaggcacctgagagacctgcccgatgagaaaatcatcatcgcttccgggaacgagattctggaactggtggtaaatgccatgatgggcgacgaccctccggagccccccaccccatttgagacaccttcgctgcacgatttgtatgatctggaggtggatgtgcccgaggacgatcccaatgaggaggcggtaaatgatttttttagcgatgccgcgctgctagctgccgaggaggcttcgagctctagctcagacagcgactcttcactgcatacccctagacccggcagaggtgagaaaaagatccccgagcttaaaggggaagagatggacttgcgctgctatgaggaatgcttgcccccgagcgatgatgaggacgagcaggcgatccagaacgcagcgagccagggagtgcaagccgccagcgagagctttgcgctggactgcccgcctctgcccggacacggctgtaagtcttgtgaatttcatcgcatgaatactggagataaagctgtgttgtgtgcactttgctatatgagagcttacaaccattgtgtttacagtaagtgtgattaagttgaactttagagggaggcagagagcagggtgactgggcgatgactggtttatttatgtatatatgttctttatataggtcccgtctctgacgcagatgatgagacccccactacaaagtccacttcgtcacccccagaaattggcacatctccacctgagaatattgttagaccagttcctgttagagccactgggaggagagcagctgtggaatgtttggatgacttgctacagggtggggttgaacctttggacttgtgtacccggaaacgccccaggcactaagtgccacacatgtgtgtttacttgaggtgatgtcagtatttatagggtgtggagtgcaataaaaaatgtgttgactttaagtgcgtggtttatgactcaggggtggggactgtgagtatataagcaggtgcagacctgtgtggttagctcagagcggcatggagatttggacggtcttggaagactttcacaagactagacagctgctagagaacgcctcgaacggagtctcttacctgtggagattctgcttcggtggcgacctagctaggctagtctacagggccaaacaggattatagtgaacaatttgaggttattttgagagagtgttctggtctttttgacgctcttaacttgggccatcagtctcactttaaccagaggatttcgagagcccttgattttactactcctggcagaaccactgcagcagtagccttttttgcttttattcttgacaaatggagtcaagaaacccatttcagcagggattaccagctggatttcttagcagtagctttgtggagaacatggaagtgccagcgcctgaatgcaatctccggctacttgccggtacagccgctagacactctgaggatcctgaatctccaggagagtcccagggcacgccaacgtcgccagcagcagcagcaggaggaggatcaagaagagaacccgagagccggcctggaccctccggcggaggaggaggagtagctgacctgtttcctgaactgcgccgggtgctgactaggtcttcgagtggtcgggagagggggattaagcgggagaggcatgatgagactaatcacagaactgaactgactgtgggtctgatgagtcgcaagcgcccagaaacagtgtggtggcatgaggtgcagtcgactggcacagatgaggtgtcggtgatgcatgagaggttttctctagaacaagtcaagacttgttggttagagcctgaggatgattgggaggtagccatcaggaattatgccaagctggctctgaggccagacaagaagtacaagattactaagctgataaatatcagaaatgcctgctacatctcagggaatggggctgaagtggagatctgtctccaggaaagggtggctttcagatgctgcatgatgaatatgtacccgggagtggtgggcatggatggggttacctttatgaacatgaggttcaggggagatgggtataatggcacggtctttatggccaataccaagctgacagtccatggctgctccttctttgggtttaataacacctgcatcgaggcctggggtcaggtcggtgtgaggggctgcagtttttcagccaactggatgggggtcgtgggcaggaccaagagtatgctgtccgtgaagaaatgcttgtttgagaggtgccacctgggggtgatgagcgagggcgaagccagaatccgccactgcgcctctaccgagacgggctgctttgtgctgtgcaagggcaatgctaagatcaagcataatatgatctgtggagcctcggacgagcgcggctaccagatgctgacctgcgccggcgggaacagccatatgctggccaccgtacatgtggcttcccatgctcgcaagccctggcccgagttcgagcacaatgtcatgaccaggtgcaatatgcatctggggtcccgccgaggcatgttcatgccctaccagtgcaacctgaattatgtgaaggtgctgctggagcccgatgccatgtccagagtgagcctgacgggggtgtttgacatgaatgtggaggtgtggaagattctgagatatgatgaatccaagaccaggtgccgagcctgcgagtgcggagggaagcatgccaggttccagcccgtgtgtgtggatgtgacggaggacctgcgacccgatcatttggtgttgccctgcaccgggacggagttcggttccagcggggaagaatctgactagagtgagtagtgttctggggcgggggaggacctgcatgagggccagaataactgaaatctgtgcttttctgtgtgttgcagcagcatgagcggaagcggctcctttgagggaggggtattcagcccttatctgacggggcgtctcccctcctgggcgggagtgcgtcagaatgtgatgggatccacggtggacggccggcccgtgcagcccgcgaactcttcaaccctgacctatgcaaccctgagctcttcgtcgttggacgcagctgccgccgcagctgctgcatctgccgccagcgccgtgcgcggaatggccatgggcgccggctactacggcactctggtggccaactcgagttccaccaataatcccgccagcctgaacgaggagaagctgttgctgctgatggcccagctcgaggccttgacccagcgcctgggcgagctgacccagcaggtggctcagctgcaggagcagacgcgggccgcggttgccacggtgaaatccaaataaaaaatgaatcaataaataaacggagacggttgttgattttaacacagagtctgaatctttatttgatttttcgcgcgcggtaggccctggaccaccggtctcgatcattgagcacccggtggatcttttccaggacccggtagaggtgggcttggatgttgaggtacatgggcatgagcccgtcccgggggtggaggtagctccattgcagggcctcgtgctcgggggtggtgttgtaaatcacccagtcatagcaggggcgcagggcatggtgttgcacaatatctttgaggaggagactgatggccacgggcagccctttggtgtaggtgtttacaaatctgttgagctgggagggatgcatgcggggggagatgaggtgcatcttggcctggatcttgagattggcgatgttaccgcccagatcccgcctggggttcatgttgtgcaggaccaccagcacggtgtatccggtgcacttggggaatttatcatgcaacttggaagggaaggcgtgaaagaatttggcgacgcctttgtgcccgcccaggttttccatgcactcatccatgatgatggcgatgggcccgtgggcggcggcctgggcaaagacgtttcgggggtcggacacatcatagttgtggtcctgggtgaggtcatcataggccattttaatgaatttggggcggagggtgccggactgggggacaaaggtaccctcgatcccgggggcgtagttcccctcacagatctgcatctcccaggctttgagctcggagggggggatcatgtccacctgcggggcgataaagaacacggtttccggggcgggggagatgagctgggccgaaagcaagttccggagcagctgggacttgccgcagccggtggggccgtagatgaccccgatgaccggctgcaggtggtagttgagggagagacagctgccgtcctcccggaggaggggggccacctcgttcatcatctcgcgcacgtgcatgttctcgcgcaccagttccgccaggaggcgctctccccccagggataggagctcctggagcgaggcgaagtttttcagcggctt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aaagatatttgcaattggagtgtattagtttatacatgtgagggagttaatcttaccattgtcaatgccacctcagctcaaaatggtagaattcaaggacaaagtgtcagtgtatctaatgggtattttacccaacatacttttatctatgacgttaaagtcataccactgcctacgcctagcccacctagcactaccacacagacaacccacactacacagacaaccacatacagtacattaaatcagcctaccaccactacagcagcagaggttgccagctcgtctggggtccgagtggcatttttgatgtgggccccatctagcagtcccactgctagtaccaatgagcagactactgaatttttgtccactgtcgagagccacaccacagctacctccagtgccttctctagcaccgccaatctctcctcgctttcctctacaccaatcagtcccgctactactcctagccccgctcctcttcccactcccctgaagcaaacagacggcggcatgcaatggcagatcaccctgctcattgtgatcgggttggtcatcctggccgtgttgctctactacatcttctgccgccgcattcccaacgcgcaccgcaagccggtctacaagcccatcattgtcgggcagccggagccgcttcaggtggaagggggtctaaggaatcttctcttctcttttacagtatggtgattgaactatgattcctagacaattcttgatcactattcttatctgcctcctccaagtctgtgccaccctcgctctggtggccaacgccagtccagactgtattgggcccttcgcctcctacgtgctctttgccttcaccacctgcatctgctgctgtagcatagtctgcctgcttatcaccttcttccagttcattgactggatctttgtgcgcatcgcctacctgcgccaccacccccagtaccgcgaccagcgagtggcgcggctgctcaggctcctctgataagcatgcgggctctgctacttctcgcgcttctgctgttagtgctcccccgtcccgtcgacccccggtcccccacccagtcccccgaggaggtccgcaaatgcaaattccaagaaccctggaaattcctcaaatgctaccgccaaaaatcagacatgcatcccagctggatcatgatcattgggatcgtgaacattctggcctgcaccctcatctcctttgtgatttacccctgctttgactttggttggaactcgccagaggcgctctatctcccgcctgaacctgacacaccaccacagcaacctcaggcacacgcactaccaccactacagcctaggccacaatacatgcccatattagactatgaggccgagccacagcgacccatgctccccgctattagttacttcaatctaaccggcggagatgactgacccactggccaacaacaacgtcaacgaccttctcctggacatggacggccgcgcctcggagcagcgactcgcccaacttcgcattcgccagcagcaggagagagccgtcaaggagctgcaggatgcggtggccatccaccagtgcaagagaggcatcttctgcctggtgaaacaggccaagatctcctacgaggtcactccaaacgaccatcgcctctcctacgagctcctgcagcagcgccagaagttcacctgcctggtcggagtcaaccccatcgtcatcacccagcagtctggcgataccaaggggtgcatccactgctcctgcgactcccccgactgcgtccacactctgatcaagaccctctgcggcctccgcgacctcctccccatgaactaatcacccccttatccagtgaaataaagatcatattgatgatgattttacagaaataaaaaataatcatttgatttgaaataaagatacaatcatattgatgatttgagtttaacaaaaaaataaagaatcacttacttgaaatctgataccaggtctctgtccatgttttctgccaacaccacttcactcccctcttcccagctctggtactgcaggccccggcgggctgcaaacttcctccacacgctgaaggggatgtcaaattcctcctgtccctcaatcttcattttatcttctatcagatgtccaaaaagcgcgtccgggtggatgatgacttcgaccccgtctacccctacgatgcagacaacgcaccgaccgtgcccttcatcaacccccccttcgtctcttcagatggattccaagagaagcccctgggggtgttgtccctgcgactggccgaccccgtcaccaccaagaacggggaaatcaccctcaagctgggagagggggtggacctcgattcctcgggaaaactcatctccaacacggccaccaaggccgccgcccctctcagtttttccaacaacaccatttcccttaacatggatcaccccttttacactaaagatggaaaattatccttacaagtttctccaccattaaatatactgagaacaagcattctaaacacactagctttaggttttggatcaggtttaggactccgtggctctgccttggcagtacagttagtctctccacttacatttgatactgatggaaacataaagcttaccttagacagaggtttgcatgttacaacaggagatgcaattgaaagcaacataagctgggctaaaggtttaaaatttgaagatggagccatagcaaccaacattggaaatgggttagagtttggaagcagtagtacagaaacaggtgttgatgatgcttacccaatccaagttaaacttggatctggccttagctttgacagtacaggagccataatggctggtaacaaagaagacgataaactcactttgtggacaacacctgatccatcaccaaactgtcaaatactcgcagaaaatgatgcaaaactaacactttgcttgactaaatgtggtagtcaaatactggccactgtgtcagtcttagttgtaggaagtggaaacctaaaccccattactggcaccgtaagcagtgctcaggtgtttctacgttttgatgcaaacggtgttcttttaacagaacattctacactaaaaaaatactgggggtataggcagggagatagcatagatggcactccatataccaatgctgtaggattcatgcccaatttaaaagcttatccaaagtcacaaagttctactactaaaaataatatagtagggcaagtatacatgaatggagatgtttcaaaacctatgcttctcactataaccctcaatggtactgatgacagcaacagtacatattcaatgtcattttcatacacctggactaatggaagctatgttggagcaacatttggggctaactcttataccttctcatacatcgcccaagaatgaacactgtatcccaccctgcatgccaacccttcccaccccactctgtggaacaaactctgaaacacaaaataaaataaagttcaagtgttttattgattcaacagttttacaggattcgagcagttatttttcctccaccctcccaggacatggaatacaccaccctctccccccgcacagccttgaacatctgaatgccattggtgatggacatgcttttggtctccacgttccacacagtttcagagcgagccagtctcgggtcggtcagggagatgaaaccctccgggcactcccgcatctgcacctcacagctcaacagctgaggattgtcctcggtggtcgggatcacggttatctggaagaagcagaagagcggcggtgggaatcatagtccgcgaacgggatcggccggtggtgtcgcatcaggccccgcagcagtcgctgccgccgccgctccgtcaagctgctgctcagggggtccgggtccagggactccctcagcatgatgcccacggccctcagcatcagtcgtctggtgcggcgggcgcagcagcgcatgcggatctcgctcaggtcgctgcagtacgtgcaacacagaaccaccaggttgttcaacagtccatagttcaacacgctccagccgaaactcatcgcgggaaggatgctacccacgtggccgtcgtaccagatcctcaggtaaatcaagtggtgccccctccagaacacgctgcccacgtacatgatctccttgggcatgtggcggttcaccacctcccggtaccacatcaccctctggttgaacatgcagccccggatgatcctgcggaaccacagggccagcaccgccccgcccgccatgcagcgaagagaccccgggtcccggcaatggcaatggaggacccaccgctcgtacccgtggatcatctgggagctgaacaagtctatgttggcacagcacaggcatatgctcatgcatctcttcagcactctcaactcctcgggggtcaaaaccatatcccagggcacggggaactcttgcaggacagcgaaccccgcagaacagggcaatcctcgcacagaacttacattgtgcatggacagggtatcgcaatcaggcagcaccgggtgatcctccaccagagaagcgcgggtctcggtctcctcacagcgtggtaagggggccggccgatacgggtgatggcgggacgcggctgatcgtgttcgcgaccgtgtcatgatgcagttgctttcggacattttcgtacttgctgtagcagaacctggtccgggcgctgcacaccgatcgccggcggcggtctcggcgcttggaacgctcggtgttgaaattgtaaaacagccactctctcagaccgtgcagcagatctagggcctcaggagtgatgaagatcccatcatgcctgatggctctgatcacatcgaccaccgtggaatgggccagacccagccagatgatgcaattttgttgggtttcggtgacggcgggggagggaagaacaggaagaaccatgattaacttttaatccaaacggtctcggagtacttcaaaatgaagatcgcggagatggcacctctcgcccccgctgtgttggtggaaaataacagccaggtcaaaggtgatacggttctcgagatgttccacggtggcttccagcaaagcctccacgcgcacatccagaaacaagacaatagcgaaagcgggagggttctctaattcctcaatcatcatgttacactcctgcaccatccccagataattttcatttttccagccttgaatgattcgaactagttcgtgaggtaaatccaagccagccatgataaagagctcgcgcagagcgccctccaccggcattcttaagcacaccctcataattccaagatattctgctcctggttcacctgcagcagattgacaagcggaatatcaaaatctctgccgcgatccctgagctcctccctcagcaataactgtaagtactctttcatatcctctccgaaatttttagccataggaccaccaggaataagattagggcaagccacagtacagataaaccgaagtcctccccagtgagcattgccaaatgcaagactgctataagcatgctggctagacccggtgatatcttccagataactggacagaaaatcgcccaggcaatttttaagaaaatcaacaaaagaaaaatcctccaggtggacgtttagagcctcgggaacaacgatgaagtaaatgcaagcggtgcgttccagcatggttagttagctgatctgtagaaaaaacaaaaatgaacattaaaccatgctagcctggcgaacaggtgggtaaatcgttctctccagcaccaggcaggccacggggtctccggcgcgaccctcgtaaaaattgtcgctatgattgaaaaccatcacagagagacgttcccggtggccggcgtgaatgattcgacaagatgaatacacccccggaacattggcgtccgcgagtgaaaaaaagcgcccgaggaagcaataaggcactacaatgctcagtctcaagtccagcaaagcgatgccatgcggatgaagcacaaaattctcaggtgcgtacaaaatgtaattactcccctcctgcacaggcagcaaagcccccgatccctccaggtacacatacaaagcctcagcgtccatagcttaccgagcagcagcacacaacaggcgcaagagtcagagaaaggctgagctctaacctgtccacccgctctctgctcaatatatagcccagatctacactgacgtaaaggccaaagtctaaaaatacccgccaaataatcacacacgcccagcacacgcccagaaaccggtgacacactcaaaaaaatacgcgcacttcctcaaacgcccaaaactgccgtcatttccgggttcccacgctacgtcatcaaaacacgactttcaaattccgtcgaccgttaaaaacgtcacccgccccgcccctaacggtcgcccgtctctcagccaatcagcgccccgcatccccaaattcaaacacctcatttgcatattaacgcgcacaaaaagtttgaggtatattattgatgatgg
SEQ ID NO:58.AdC68-734载体的完整序列
TTAATTAAccatcttcaataatatacctcaaactttttgtgcgcgttaatatgcaaatgaggcgtttgaatttggggaggaagggcggtgattggtcgagggatgagcgaccgttaggggcggggcgagtgacgttttgatgacgtggttgcgaggaggagccagtttgcaagttctcgtgggaaaagtgacgtcaaacgaggtgtggtttgaacacggaaatactcaattttcccgcgctctctgacaggaaatgaggtgtttctgggcggatgcaagtgaaaacgggccattttcgcgcgaaaactgaatgaggaagtgaaaatctgagtaatttcgcgtttatggcagggaggagtatttgccgagggccgagtagactttgaccgattacgtgggggtttcgattaccgtgtttttcacctaaatttccgcgtacggtgtcaaagtccggtgtttttactactgtaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctgtccctatcagtgatagagatctccctatcagtgatagagagtttagtgaaccgtcagatccgctagggtaccaacATGGCTAGCATCGTCGGAGGGTGGGAGTGCGAAAAGCACTCACAGCCATGGCAGGTCCTGGTCGCCTCGCGCGGACGCGCCGTGTGTGGAGGTGTGCTGGTCCACCCGCAGTGGGTGTTGACTGCGGCCCATTGCATCAGAAATAAGTCCGTGATCCTCTTGGGGAGACATTCCCTGTTTCACCCCGAAGATACTGGACAGGTGTTCCAAGTGAGCCACTCCTTCCCGCATCCACTGTACGACATGAGCCTGCTGAAGAACCGCTTTCTGCGGCCAGGGGACGACTCATCACACGATTTGATGCTGCTTCGGCTCTCGGAACCGGCCGAGCTCACCGACGCAGTGAAGGTCATGGACCTCCCTACGCAAGAGCCTGCTCTCGGTACCACTTGTTACGCATCGGGATGGGGCTCCATCGAGCCGGAAGAATTCCTGACCCCGAAAAAGCTGCAGTGCGTGGATCTGCACGTGATTTCGAATGACGTGTGCGCGCAAGTGCATCCACAAAAGGTCACTAAGTTCATGCTGTGCGCCGGAAGGTGGACCGGCGGAAAATCGACCTGTTCCGGCGACAGCGGAGGCCCACTCGTGTGCAACGGTGTGCTGCAGGGCATCACTAGCTGGGGATCAGAACCGTGCGCGCTTCCGGAGCGGCCCTCGCTCTACACGAAGGTGGTGCACTACCGCAAATGGATTAAAGATACCATCGTCGCAAACCCTggatccgaaggtaggggttcattattgacctgtggagatgtcgaagaaaacccaggacccGCTAGCAAAGCAGTGCTGCTGGCGCTCCTGATGGCTGGACTCGCGCTGCAGCCTGGAACCGCCCTGCTCTGTTACTCGTGCAAGGCCCAAGTCTCGAATGAGGACTGTTTGCAAGTGGAAAACTGCACCCAGCTCGGAGAACAATGCTGGACTGCACGGATCCGCGCTGTCGGCCTGCTGACCGTGATCTCCAAAGGGTGCTCATTGAACTGCGTGGACGATAGCCAGGACTACTACGTGGGAAAGAAGAATATCACTTGTTGCGACACGGATCTTTGCAACGCGTCCGGAGCGCACGCCCTGCAGCCAGCAGCCGCCATTCTGGCCCTGCTTCCGGCCCTGGGGTTGCTGCTCTGGGGTCCGGGCCAGCTCggatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaATGGCTAGCGCTCGCAGACCGCGGTGGCTGTGTGCAGGGGCGCTCGTCCTGGCGGGTGGCTTCTTTTTGCTCGGCTTTCTTTTCGGATGGTTCATCAAATCGTCAAACGAAGCTACCAATATCACCCCGAAGCACAACATGAAGGCCTTTCTGGATGAGCTGAAGGCTGAGAACATTAAGAAGTTCCTCTACAACTTCACCCAGATCCCACATTTGGCGGGCACTGAGCAGAACTTTCAGTTGGCTAAGCAGATCCAGAGCCAGTGGAAGGAATTCGGCCTGGACTCCGTCGAGCTGGCGCATTACGATGTGCTGCTGAGCTACCCTAATAAGACTCATCCGAACTATATCTCGATTATCAATGAGGACGGAAACGAAATCTTTAACACGTCCCTCTTCGAGCCGCCACCGCCTGGATACGAGAACGTGTCAGATATCGTGCCTCCGTTCTCGGCCTTCTCGCCCCAGGGAATGCCCGAAGGGGACCTGGTGTACGTGAACTACGCAAGGACCGAGGACTTCTTCAAGTTGGAGCGGGATATGAAGATCAATTGCAGCGGAAAGATCGTCATCGCCCGCTACGGCAAAGTGTTCCGCGGCAACAAGGTGAAGAATGCACAGTTGGCAGGCGCCAAGGGCGTCATCCTCTACTCGGATCCTGCCGACTACTTCGCTCCTGGCGTGAAATCCTACCCTGATGGTTGGAATCTGCCAGGAGGAGGGGTGCAGAGGGGAAATATCCTGAACCTGAACGGTGCCGGTGACCCACTTACTCCGGGTTACCCGGCCAACGAATACGCGTACAGGCGGGGTATCGCGGAAGCCGTCGGACTGCCGTCCATCCCGGTCCATCCGATTGGTTACTACGACGCCCAGAAGCTCCTCGAAAAGATGGGAGGCAGCGCCCCTCCGGACTCGTCATGGAGAGGCTCGCTGAAGGTGCCATACAACGTGGGACCCGGATTCACTGGAAATTTCAGCACTCAAAAAGTGAAGATGCACATTCACTCCACTAACGAAGTCACCAGGATCTACAACGTCATCGGAACCCTCCGGGGAGCGGTGGAACCGGACCGCTACGTGATCCTCGGTGGACACCGGGATAGCTGGGTGTTCGGAGGAATCGATCCTCAATCGGGCGCAGCCGTCGTCCATGAAATCGTCAGGTCCTTTGGTACTCTTAAGAAGGAGGGCTGGCGCCCTAGACGCACTATTCTGTTCGCCTCGTGGGATGCCGAAGAATTTGGTCTGCTCGGCAGCACCGAATGGGCTGAGGAAAACTCCCGCCTGCTCCAAGAACGCGGAGTGGCGTACATCAATGCCGACTCATCCATCGAAGGAAACTACACGCTGCGGGTGGACTGCACTCCACTGATGTACTCGCTCGTGCACAACCTGACCAAAGAACTCAAATCCCCAGACGAAGGATTCGAGGGAAAATCGCTGTACGAGTCGTGGACCAAGAAGAGCCCATCCCCGGAGTTCAGCGGGATGCCGCGGATCTCAAAGCTCGGATCAGGAAATGATTTCGAAGTGTTCTTTCAGAGGCTGGGAATTGCGTCGGGAAGGGCTCGGTACACGAAAAACTGGGAAACTAACAAGTTCTCGGGATACCCGCTGTACCACTCGGTGTATGAAACTTACGAACTGGTGGAGAAATTCTACGATCCTATGTTTAAGTACCACCTGACTGTGGCCCAAGTGAGAGGCGGAATGGTGTTCGAGTTGGCCAATTCAATTGTGCTGCCATTCGATTGCCGCGACTACGCCGTGGTGCTGAGAAAGTACGCAGACAAAATCTACTCAATCAGCATGAAGCACCCACAAGAGATGAAAACCTACTCAGTCTCCTTCGACTCCCTCTTCTCCGCGGTGAAGAACTTCACCGAGATCGCGAGCAAATTCTCGGAGCGCCTTCAAGATTTTGACAAATCCAATCCGATCGTCCTCCGCATGATGAATGACCAGCTCATGTTTCTCGAACGGGCCTTCATCGATCCACTGGGACTTCCGGACCGGCCGTTTTACCGCCACGTGATCTACGCGCCCTCGTCGCATAACAAGTATGCTGGAGAGAGCTTCCCGGGTATCTACGACGCATTGTTCGACATTGAGTCCAAGGTGGATCCGTCCAAAGCCTGGGGTGAAGTGAAGCGCCAAATCTACGTGGCGGCCTTTACCGTCCAGGCGGCAGCAGAAACCTTGAGCGAGGTGGCTTGActcgagcctaagcttctagataagatatccgatccaccggatctagataactgatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatatgctggccaccgtacatgtggcttcccatgctcgcaagccctggcccgagttcgagcacaatgtcatgaccaggtgcaatatgcatctggggtcccgccgaggcatgttcatgccctaccagtgcaacctgaattatgtgaaggtgctgctggagcccgatgccatgtccagagtgagcctgacgggggtgtttgacatgaatgtggaggtgtggaagattctgagatatgatgaatccaagaccaggtgccgagcctgcgagtgcggagggaagcatgccaggttccagcccgtgtgtgtggatgtgacggaggacctgcgacccgatcatttggtgttgccctgcaccgggacggagttcggttccagcggggaagaatctgactagagtgagtagtgttctggggcgggggaggacctgcatgagggccagaataactgaaatctgtgcttttctgtgtgttgcagcagcatgagcggaagcggctcctttgagggaggggtattcagcccttatctgacggggcgtctcccctcctgggcgggagtgcgtcagaatgtgatgggatccacggtggacggccggcccgtgcagcccgcgaactcttcaaccctgacctatgcaaccctgagctcttcgtcgttggacgcagctgccgccgcagctgctgcatctgccgccagcgccgtgcgcggaatggccatgggcgccggctactacggcactctggtggccaactcgagttccaccaataatcccgccagcctgaacgaggagaagctgttgctgctgatggcccagctcgaggccttgacccagcgcctgggcgagctgacccagcaggtggctcagctgcaggagcagacgcgggccgcggttgccacggtgaaatccaaataaaaaatgaatcaataaataaacggagacggttgttgattttaacacagagtctgaatctttatttgatttttcgcgcgcggtaggccctggaccaccggtctcgatcattgagcacccggtggatcttttccaggacccggtagaggtgggcttggatgttgaggtacatgggcatgagcccgtcccgggggtggaggtagctccattgcagggcctcgtgctcgggggtggtgttgtaaatcacccagtcatagcaggggcgcagggcatggtgttgcacaatatctttgaggaggagactgatggccacgggcagccctttggtgtaggtgtttacaaatctgttgagctgggagggatgcatgcggggggagatgaggtgcatcttggcctggatcttgagattggcgatgttaccgcccagatcccgcctggggttcatgttgtgcaggaccaccagcacggtgtatccggtgcacttggggaatttatcatgcaacttggaagggaaggcgtgaaagaatttggcgacgcctttgtgcccgcccaggttttccatgcactcatccatgatgatggcgatgggcccgtgggcggcggcctgggcaaagacgtttcgggggtcggacacatcatagttgtggtcctgggtgaggtcatcataggccattttaatgaatttggggcggagggtgccggactgggggacaaaggtaccctcgatcccgggggcgtagttcccctcacagatctgcatctcccaggctttgagctcggagggggggatcatgtccacctgcggggcgataaagaacacggtttccggggcgggggagatgagctgggccgaaagcaagttccggagcagctgggacttgccgcagccggtggggccgtagatgaccccgatgaccggctgcaggtggtagttgagggagagacagctgccgtcctcccggaggaggggggccacctcgttcatcatctcgcgcacgtgcatgttctcgcgcaccagttccgccaggaggcgctctccccccagggataggagctcctggagcgaggcgaagtttttcagcggcttgagtccgtcggccatgggcattttggagagggtttgttgcaagagttccaggcggtcccagagctcggtgatgtgctctacggcatctcgatccagcagacctcctcgtttcgcgggttgggacggctgcgggagtagggcaccagacgatgggcgtccagcgcagccagggtccggtccttccagggtcgcagcgtccgcgtcagggtggtctccgtcacggtgaaggggtgcgcgccgggctgggcgcttgcgagggtgcgcttcaggctcatccggctggtcgaaaaccgctcccgatcggcgccctgcgcgtcggccaggtagcaattgaccatgagttcgtagttgagcgcctcggccgcgtggcctttggcgcggagcttacctttggaagtctgcccgcaggcgggacagaggagggacttgagggcgtagagcttgggggcgaggaagacggactcgggggcgtaggcgtccgcgccgcagtgggcgcagacggtctcgcactccacgagccaggtgaggtcgggctggtcggggtcaaaaaccagtttcccgccgttctttttgatgcgtttcttacctttggtctccatgagctcgtgtccccgctgggtgacaaagaggctgtccgtgtccccgtagaccgactttatgggccggtcctcgagcggtgtgccgcggtcctcctcgtagaggaaccccgcccactccgagacgaaagcccgggtccaggccagcacgaaggaggccacgtgggacgggtagcggtcgttgtccaccagcgggtccaccttttccagggtatgcaaacacatgtccccctcgtccacatccaggaaggtgattggcttgtaagtgtaggccacgtgaccgggggtcccggccgggggggtataaaagggtgcgggtccctgctcgtcctcactgtcttccggatcgctgtccaggagcgccagctgttggggtaggtattccctctcgaaggcgggcatgacctcggcactcaggttgtcagtttctagaaacgaggaggatttgatattgacggtgccggcggagatgcctttcaagagcccctcgtccatctggtcagaaaagacgatctttttgttgtcgagcttggtggcgaaggagccgtagagggcgttggagaggagcttggcgatggagcgcatggtctggtttttttccttgtcggcgcgctccttggcggcgatgttgagctgcacgtactcgcgcgccacgcacttccattcggggaagacggtggtcagctcgtcgggcacgattctgacctgccagccccgattatgcagggtgatgaggtccacactggtggccacctcgccgcgcaggggctcattagtccagcagaggcgtccgcccttgcgcgagcagaaggggggcagggggtccagcatgacctcgtcgggggggtcggcatcgatggtgaagatgccgggcaggaggtcggggtcaaagtagctgatggaagtggccagatcgtccagggcagcttgccattcgcgcacggccagcgcgcgctcgtagggactgaggggcgtgccccagggcatgggatgggtaagcgcggaggcgtacatgccgcagatgtcgtagacgtagaggggctcctcgaggatgccgatgtaggtggggtagcagcgccccccgcggatgctggcgcgcacgtagtcatacagctcgtgcgagggggcgaggagccccgggcccaggttggtgcgactgggcttttcggcgcggtagacgatctggcggaaaatggcatgcgagttggaggagatggtgggcctttggaagatgttgaagtgggcgtggggcagtccgaccgagtcgcggatgaagtgggcgtaggagtcttgcagcttggcgacgagctcggcggtgactaggacgtccagagcgcagtagtcgagggtctcctggatgatgtcatacttgagctgtcccttttgtttccacagctcgcggttgagaaggaactcttcgcggtccttccagtactcttcgagggggaacccgtcctgatctgcacggtaagagcctagcatgtagaactggttgacggccttgtaggcgcagcagcccttctccacggggagggcgtaggcctgggcggccttgcgcagggaggtgtgcgtgagggcgaaagtgtccctgaccatgaccttgaggaactggtgcttgaagtcgatatcgtcgcagcccccctgctcccagagctggaagtccgtgcgcttcttgtaggcggggttgggcaaagcgaaagtaacatcgttgaagaggatcttgcccgcgcggggcataaagttgcgagtgatgcggaaaggttggggcacctcggcccggttgttgatgacctgggcggcgagcacgatctcgtcgaagccgttgatgttgtggcccacgatgtagagttccacgaatcgcggacggcccttgacgtggggcagtttcttgagctcctcgtaggtgagctcgtcggggtcgctgagcccgtgctgctcgagcgcccagtcggcgagatgggggttggcgcggaggaaggaagtccagagatccacggccagggcggtttgcagacggtcccggtactgacggaactgctgcccgacggccattttttcgggggtgacgcagtagaaggtgcgggggtccccgtgccagcgatcccatttgagctggagggcgagatcgagggcgagctcgacgagccggtcgtccccggagagtttcatgaccagcatgaaggggacgagctgcttgccgaaggaccccatccaggtgtaggtttccacatcgtaggtgaggaagagcctttcggtgcgaggatgcgagccgatggggaagaactggatctcctgccaccaattggaggaatggctgttgatgtgatggaagtagaaatgccgacggcgcgccgaacactcgtgcttgtgtttatacaagcggccacagtgctcgcaacgctgcacgggatgcacgtgctgcacgagctgtacctgagttcctttgacgaggaatttcagtgggaagtggagtcgtggcgcctgcatctcgtgctgtactacgtcgtggtggtcggcctggccctcttctgcctcgatggtggtcatgctgacgagcccgcgcgggaggcaggtccagacctcggcgcgagcg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gtcatcacccagcagtctggcgataccaaggggtgcatccactgctcctgcgactcccccgactgcgtccacactctgatcaagaccctctgcggcctccgcgacctcctccccatgaactaatcacccccttatccagtgaaataaagatcatattgatgatgattttacagaaataaaaaataatcatttgatttgaaataaagatacaatcatattgatgatttgagtttaacaaaaaaataaagaatcacttacttgaaatctgataccaggtctctgtccatgttttctgccaacaccacttcactcccctcttcccagctctggtactgcaggccccggcgggctgcaaacttcctccacacgctgaaggggatgtcaaattcctcctgtccctcaatcttcattttatcttctatcagatgtccaaaaagcgcgtccgggtggatgatgacttcgaccccgtctacccctacgatgcagacaacgcaccgaccgtgcccttcatcaacccccccttcgtctcttcagatggattccaagagaagcccctgggggtgttgtccctgcgactggccgaccccgtcaccaccaagaacggggaaatcaccctcaagctgggagagggggtggacctcgattcctcgggaaaactcatctccaacacggccaccaaggccgccgcccctctcagtttttccaacaacaccatttcccttaacatggatcaccccttttacactaaagatggaaaattatccttacaagtttctccaccattaaatatactgagaacaagcattctaaacacactagctttaggttttggatcaggtttaggactccgtggctctgccttggcagtacagttagtctctccacttacatttgatactgatggaaacataaagcttaccttagacagaggtttgcatgttacaacaggagatgcaattgaaagcaacataagctgggctaaaggtttaaaatttgaagatggagccatagcaaccaacattggaaatgggttagagtttggaagcagtagtacagaaacaggtgttgatgatgcttacccaatccaagttaaacttggatctggccttagctttgacagtacaggagccataatggctggtaacaaagaagacgataaactcactttgtggacaacacctgatccatcaccaaactgtcaaatactcgcagaaaatgatgcaaaactaacactttgcttgactaaatgtggtagtcaaatactggccactgtgtcagtcttagttgtaggaagtggaaacctaaaccccattactggcaccgtaagcagtgctcaggtgtttctacgttttgatgcaaacggtgttcttttaacagaacattctacactaaaaaaatactgggggtataggcagggagatagcatagatggcactccatataccaatgctgtaggattcatgcccaatttaaaagcttatccaaagtcacaaagttctactactaaaaataatatagtagggcaagtatacatgaatggagatgtttcaaaacctatgcttctcactataaccctcaatggtactgatgacagcaacagtacatattcaatgtcattttcatacacctggactaatggaagctatgttggagcaacatttggggctaactcttataccttctcatacatcgcccaagaatgaacactgtatcccaccctgcatgccaacccttcccaccccactctgtggaacaaactctgaaacacaaaataaaataaagttcaagtgttttattgattcaacagttttacaggattcgagcagttatttttcctccaccctcccaggacatggaatacaccaccctctccccccgcacagccttgaacatctgaatgccattggtgatggacatgcttttggtctccacgttccacacagtttcagagcgagccagtctcgggtcggtcagggagatgaaaccctccgggcactcccgcatctgcacctcacagctcaacagctgaggattgtcctcggtggtcgggatcacggttatctggaagaagcagaagagcggcggtgggaatcatagtccgcgaacgggatcggccggtggtgtcgcatcaggccccgcagcagtcgctgccgccgccgctccgtcaagctgctgctcagggggtccgggtccagggactccctcagcatgatgcccacggccctcagcatcagtcgtctggtgcggcgggcgcagcagcgcatgcggatctcgctcaggtcgctgcagtacgtgcaacacagaaccaccaggttgttcaacagtccatagttcaacacgctccagccgaaactcatcgcgggaaggatgctacccacgtggccgtcgtaccagatcctcaggtaaatcaagtggtgccccctccagaacacgctgcccacgtacatgatctccttgggcatgtggcggttcaccacctcccggtaccacatcaccctctggttgaacatgcagccccggatgatcctgcggaaccacagggccagcaccgccccgcccgccatgcagcgaagagaccccgggtcccggcaatggcaatggaggacccaccgctcgtacccgtggatcatctgggagctgaacaagtctatgttggcacagcacaggcatatgctcatgcatctcttcagcactctcaactcctcgggggtcaaaaccatatcccagggcacggggaactcttgcaggacagcgaaccccgcagaacagggcaatcctcgcacagaacttacattgtgcatggacagggtatcgcaatcaggcagcaccgggtgatcctccaccagagaagcgcgggtctcggtctcctcacagcgtggtaagggggccggccgatacgggtgatggcgggacgcggctgatcgtgttcgcgaccgtgtcatgatgcagttgctttcggacattttcgtacttgctgtagcagaacctggtccgggcgctgcacaccgatcgccggcggcggtctcggcgcttggaacgctcggtgttgaaattgtaaaacagccactctctcagaccgtgcagcagatctagggcctcaggagtgatgaagatcccatcatgcctgatggctctgatcacatcgaccaccgtggaatgggccagacccagccagatgatgcaattttgttgggtttcggtgacggcgggggagggaagaacaggaagaaccatgattaacttttaatccaaacggtctcggagtacttcaaaatgaagatcgcggagatggcacctctcgcccccgctgtgttggtggaaaataacagccaggtcaaaggtgatacggttctcgagatgttccacggtggcttccagcaaagcctccacgcgcacatccagaaacaagacaatagcgaaagcgggagggttctctaattcctcaatcatcatgttacactcctgcaccatccccagataattttcatttttccagccttgaatgattcgaactagttcCtgaggtaaatccaagccagccatgataaagagctcgcgcagagcgccctccaccggcattcttaagcacaccctcataattccaagatattctgctcctggttcacctgcagcagattgacaagcggaatatcaaaatctctgccgcgatccctgagctcctccctcagcaataactgtaagtactctttcatatcctctccgaaatttttagccataggaccaccaggaataagattagggcaagccacagtacagataaaccgaagtcctccccagtgagcattgccaaatgcaagactgctataagcatgctggctagacccggtgatatcttccagataactggacagaaaatcgcccaggcaatttttaagaaaatcaacaaaagaaaaatcctccaggtggacgtttagagcctcgggaacaacgatgaagtaaatgcaagcggtgcgttccagcatggttagttagctgatctgtagaaaaaacaaaaatgaacattaaaccatgctagcctggcgaacaggtgggtaaatcgttctctccagcaccaggcaggccacggggtctccggcgcgaccctcgtaaaaattgtcgctatgattgaaaaccatcacagagagacgttcccggtggccggcgtgaatgattcgacaagatgaatacacccccggaacattggcgtccgcgagtgaaaaaaagcgcccgaggaagcaataaggcactacaatgctcagtctcaagtccagcaaagcgatgccatgcggatgaagcacaaaattctcaggtgcgtacaaaatgtaattactcccctcctgcacaggcagcaaagcccccgatccctccaggtacacatacaaagcctcagcgtccatagcttaccgagcagcagcacacaacaggcgcaagagtcagagaaaggctgagctctaacctgtccacccgctctctgctcaatatatagcccagatctacactgacgtaaaggccaaagtctaaaaatacccgccaaataatcacacacgcccagcacacgcccagaaaccggtgacacactcaaaaaaatacgcgcacttcctcaaacgcccaaaactgccgtcatttccgggttcccacgctacgtcatcaaaacacgactttcaaattccgtcgaccgttaaaaacgtcacccgccccgcccctaacggtcgcccgtctctcagccaatcagcgccccgcatccccaaattcaaacacctcatttgcatattaacgcgcacaaaaagtttgaggtatattattgatgatggTTAATTAA
SEQ ID NO:59:优选的EMCV IRES(pIRES)的核苷酸序列
TAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATG
(最小EMCV IRES(mIRES)缺少下划线的15个核苷酸)
SEQ ID NO:60.包含免疫原性PSA、PSCA和PSMA多肽的氨基酸序列(由质粒916以及载体AdC68-734和AdC68W-734编码)
MASIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANPGSEGRGSLLTCGDVEENPGPASKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQLGSQTLNFDLLKLAGDVESNPGPMASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA
SEQ ID NO:61.编码SEQ ID NO:60的氨基酸序列的核苷酸序列
atggctagcatcgtcggagggtgggagtgcgaaaagcactcacagccatggcaggtcctggtcgcctcgcgcggacgcgccgtgtgtggaggtgtgctggtccacccgcagtgggtgttgactgcggcccattgcatcagaaataagtccgtgatcctcttggggagacattccctgtttcaccccgaagatactggacaggtgttccaagtgagccactccttcccgcatccactgtacgacatgagcctgctgaagaaccgctttctgcggccaggggacgactcatcacacgatttgatgctgcttcggctctcggaaccggccgagctcaccgacgcagtgaaggtcatggacctccctacgcaagagcctgctctcggtaccacttgttacgcatcgggatggggctccatcgagccggaagaattcctgaccccgaaaaagctgcagtgcgtggatctgcacgtgatttcgaatgacgtgtgcgcgcaagtgcatccacaaaaggtcactaagttcatgctgtgcgccggaaggtggaccggcggaaaatcgacctgttccggcgacagcggaggcccactcgtgtgcaacggtgtgctgcagggcatcactagctggggatcagaaccgtgcgcgcttccggagcggccctcgctctacacgaaggtggtgcactaccgcaaatggattaaagataccatcgtcgcaaaccctggatccgaaggtaggggttcattattgacctgtggagatgtcgaagaaaacccaggacccgctagcaaagcagtgctgctggcgctcctgatggctggactcgcgctgcagcctggaaccgccctgctctgttactcgtgcaaggcccaagtctcgaatgaggactgtttgcaagtggaaaactgcacccagctcggagaacaatgctggactgcacggatccgcgctgtcggcctgctgaccgtgatctccaaagggtgctcattgaactgcgtggacgatagccaggactactacgtgggaaagaagaatatcacttgttgcgacacggatctttgcaacgcgtccggagcgcacgccctgcagccagcagccgccattctggccctgcttccggccctggggttgctgctctggggtccgggccagctcggatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaatggctagcgctcgcagaccgcggtggctgtgtgcaggggcgctcgtcctggcgggtggcttctttttgctcggctttcttttcggatggttcatcaaatcgtcaaacgaagctaccaatatcaccccgaagcacaacatgaaggcctttctggatgagctgaaggctgagaacattaagaagttcctctacaacttcacccagatcccacatttggcgggcactgagcagaactttcagttggctaagcagatccagagccagtggaaggaattcggcctggactccgtcgagctggcgcattacgatgtgctgctgagctaccctaataagactcatccgaactatatctcgattatcaatgaggacggaaacgaaatctttaacacgtccctcttcgagccgccaccgcctggatacgagaacgtgtcagatatcgtgcctccgttctcggccttctcgccccagggaatgcccgaaggggacctggtgtacgtgaactacgcaaggaccgaggacttcttcaagttggagcgggatatgaagatcaattgcagcggaaagatcgtcatcgcccgctacggcaaagtgttccgcggcaacaaggtgaagaatgcacagttggcaggcgccaagggcgtcatcctctactcggatcctgccgactacttcgctcctggcgtgaaatcctaccctgatggttggaatctgccaggaggaggggtgcagaggggaaatatcctgaacctgaacggtgccggtgacccacttactccgggttacccggccaacgaatacgcgtacaggcggggtatcgcggaagccgtcggactgccgtccatcccggtccatccgattggttactacgacgcccagaagctcctcgaaaagatgggaggcagcgcccctccggactcgtcatggagaggctcgctgaaggtgccatacaacgtgggacccggattcactggaaatttcagcactcaaaaagtgaagatgcacattcactccactaacgaagtcaccaggatctacaacgtcatcggaaccctccggggagcggtggaaccggaccgctacgtgatcctcggtggacaccgggatagctgggtgttcggaggaatcgatcctcaatcgggcgcagccgtcgtccatgaaatcgtcaggtcctttggtactcttaagaaggagggctggcgccctagacgcactattctgttcgcctcgtgggatgccgaagaatttggtctgctcggcagcaccgaatgggctgaggaaaactcccgcctgctccaagaacgcggagtggcgtacatcaatgccgactcatccatcgaaggaaactacacgctgcgggtggactgcactccactgatgtactcgctcgtgcacaacctgaccaaagaactcaaatccccagacgaaggattcgagggaaaatcgctgtacgagtcgtggaccaagaagagcccatccccggagttcagcgggatgccgcggatctcaaagctcggatcaggaaatgatttcgaagtgttctttcagaggctgggaattgcgtcgggaagggctcggtacacgaaaaactgggaaactaacaagttctcgggatacccgctgtaccactcggtgtatgaaacttacgaactggtggagaaattctacgatcctatgtttaagtaccacctgactgtggcccaagtgagaggcggaatggtgttcgagttggccaattcaattgtgctgccattcgattgccgcgactacgccgtggtgctgagaaagtacgcagacaaaatctactcaatcagcatgaagcacccacaagagatgaaaacctactcagtctccttcgactccctcttctccgcggtgaagaacttcaccgagatcgcgagcaaattctcggagcgccttcaagattttgacaaatccaatccgatcgtcctccgcatgatgaatgaccagctcatgtttctcgaacgggccttcatcgatccactgggacttccggaccggccgttttaccgccacgtgatctacgcgccctcgtcgcataacaagtatgctggagagagcttcccgggtatctacgacgcattgttcgacattgagtccaaggtggatccgtccaaagcctggggtgaagtgaagcgccaaatctacgtggcggcctttaccgtccaggcggcagcagaaaccttgagcgaggtggct
SEQ ID NO:62.质粒916的核苷酸序列
ggcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcaaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacaggtcgacaatattggctattggccattgcatacgttgtatctatatcataatatgtacatttatattggctcatgtccaatatgaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttacgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacaccaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggattccccgtgccaagagtgactcaccgtccggatctcagcaagcaggtatgtactctccagggtgggcctggcttccccagtcaagactccagggatttgagggacgctgtgggctcttctcttacatgtaccttttgcttgcctcaaccctgactatcttccaggtcaggatcccagagtcaggggtctgtattttcctgctggtggctccagttcaggaacagtaaaccctgctccgaatattgcctctcacatctcgtcaatctccgcgaggactggggaccctgtgacgaacatggctagcatcgtcggagggtgggagtgcgaaaagcactcacagccatggcaggtcctggtcgcctcgcgcggacgcgccgtgtgtggaggtgtgctggtccacccgcagtgggtgttgactgcggcccattgcatcagaaataagtccgtgatcctcttggggagacattccctgtttcaccccgaagatactggacaggtgttccaagtgagccactccttcccgcatccactgtacgacatgagcctgctgaagaaccgctttctgcggccaggggacgactcatcacacgatttgatgctgcttcggctctcggaaccggccgagctcaccgacgcagtgaaggtcatggacctccctacgcaagagcctgctctcggtaccacttgttacgcatcgggatggggctccatcgagccggaagaattcctgaccccgaaaaagctgcagtgcgtggatctgcacgtgatttcgaatgacgtgtgcgcgcaagtgcatccacaaaaggtcactaagttcatgctgtgcgccggaaggtggaccggcggaaaatcgacctgttccggcgacagcggaggcccactcgtgtgcaacggtgtgctgcagggcatcactagctggggatcagaaccgtgcgcgcttccggagcggccctcgctctacacgaaggtggtgcactaccgcaaatggattaaagataccatcgtcgcaaaccctggatccgaaggtaggggttcattattgacctgtggagatgtcgaagaaaacccaggacccgctagcaaagcagtgctgctggcgctcctgatggctggactcgcgctgcagcctggaaccgccctgctctgttactcgtgcaaggcccaagtctcgaatgaggactgtttgcaagtggaaaactgcacccagctcggagaacaatgctggactgcacggatccgcgctgtcggcctgctgaccgtgatctccaaagggtgctcattgaactgcgtggacgatagccaggactactacgtgggaaagaagaatatcacttgttgcgacacggatctttgcaacgcgtccggagcgcacgccctgcagccagcagccgccattctggccctgcttccggccctggggttgctgctctggggtccgggccagctcggatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaatggctagcgctcgcagaccgcggtggctgtgtgcaggggcgctcgtcctggcgggtggcttctttttgctcggctttcttttcggatggttcatcaaatcgtcaaacgaagctaccaatatcaccccgaagcacaacatgaaggcctttctggatgagctgaaggctgagaacattaagaagttcctctacaacttcacccagatcccacatttggcgggcactgagcagaactttcagttggctaagcagatccagagccagtggaaggaattcggcctggactccgtcgagctggcgcattacgatgtgctgctgagctaccctaataagactcatccgaactatatctcgattatcaatgaggacggaaacgaaatctttaacacgtccctcttcgagccgccaccgcctggatacgagaacgtgtcagatatcgtgcctccgttctcggccttctcgccccagggaatgcccgaaggggacctggtgtacgtgaactacgcaaggaccgaggacttcttcaagttggagcgggatatgaagatcaattgcagcggaaagatcgtcatcgcccgctacggcaaagtgttccgcggcaacaaggtgaagaatgcacagttggcaggcgccaagggcgtcatcctctactcggatcctgccgactacttcgctcctggcgtgaaatcctaccctgatggttggaatctgccaggaggaggggtgcagaggggaaatatcctgaacctgaacggtgccggtgacccacttactccgggttacccggccaacgaatacgcgtacaggcggggtatcgcggaagccgtcggactgccgtccatcccggtccatccgattggttactacgacgcccagaagctcctcgaaaagatgggaggcagcgcccctccggactcgtcatggagaggctcgctgaaggtgccatacaacgtgggacccggattcactggaaatttcagcactcaaaaagtgaagatgcacattcactccactaacgaagtcaccaggatctacaacgtcatcggaaccctccggggagcggtggaaccggaccgctacgtgatcctcggtggacaccgggatagctgggtgttcggaggaatcgatcctcaatcgggcgcagccgtcgtccatgaaatcgtcaggtcctttggtactcttaagaaggagggctggcgccctagacgcactattctgttcgcctcgtgggatgccgaagaatttggtctgctcggcagcaccgaatgggctgaggaaaactcccgcctgctccaagaacgcggagtggcgtacatcaatgccgactcatccatcgaaggaaactacacgctgcgggtggactgcactccactgatgtactcgctcgtgcacaacctgaccaaagaactcaaatccccagacgaaggattcgagggaaaatcgctgtacgagtcgtggaccaagaagagcccatccccggagttcagcgggatgccgcggatctcaaagctcggatcaggaaatgatttcgaagtgttctttcagaggctgggaattgcgtcgggaagggctcggtacacgaaaaactgggaaactaacaagttctcgggatacccgctgtaccactcggtgtatgaaacttacgaactggtggagaaattctacgatcctatgtttaagtaccacctgactgtggcccaagtgagaggcggaatggtgttcgagttggccaattcaattgtgctgccattcgattgccgcgactacgccgtggtgctgagaaagtacgcagacaaaatctactcaatcagcatgaagcacccacaagagatgaaaacctactcagtctccttcgactccctcttctccgcggtgaagaacttcaccgagatcgcgagcaaattctcggagcgccttcaagattttgacaaatccaatccgatcgtcctccgcatgatgaatgaccagctcatgtttctcgaacgggccttcatcgatccactgggacttccggaccggccgttttaccgccacgtgatctacgcgccctcgtcgcataacaagtatgctggagagagcttcccgggtatctacgacgcattgttcgacattgagtccaaggtggatccgtccaaagcctggggtgaagtgaagcgccaaatctacgtggcggcctttaccgtccaggcggcagcagaaaccttgagcgaggtggcttaaagatctgggccctaacaaaacaaaaagatggggttattccctaaacttcatgggttacgtaattggaagttgggggacattgccacaagatcatattgtacaaaagatcaaacactgttttagaaaacttcctgtaaacaggcctattgattggaaagtatgtcaaaggattgtgggtcttttgggctttgctgctccatttacacaatgtggatatcctgccttaatgcctttgtatgcatgtatacaagctaaacaggctttcactttctcgccaacttacaaggcctttctaagtaaacagtacatgaacctttaccccgttgctcggcaacggcctggtctgtgccaagtgtttgctgacgcaacccccactggctggggcttggccataggccatcagcgcatgcgtggaacctttgtggctcctctgccgatccatactgcggaactcctagccgcttgttttgctcgcagccggtctggagcaaagctcataggaactgacaattctgtcgtcctctcgcggaaatatacatcgtttcgatctacgtatgatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggaattctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactc
SEQ ID NO:63.AdC68W-734载体的完整序列
ccatcttcaataatatacctcaaactttttgtgcgcgttaatatgcaaatgaggcgtttgaatttggggaggaagggcggtgattggtcgagggatgagcgaccgttaggggcggggcgagtgacgttttgatgacgtggttgcgaggaggagccagtttgcaagttctcgtgggaaaagtgacgtcaaacgaggtgtggtttgaacacggaaatactcaattttcccgcgctctctgacaggaaatgaggtgtttctgggcggatgcaagtgaaaacgggccattttcgcgcgaaaactgaatgaggaagtgaaaatctgagtaatttcgcgtttatggcagggaggagtatttgccgagggccgagtagactttgaccgattacgtgggggtttcgattaccgtgtttttcacctaaatttccgcgtacggtgtcaaagtccggtgtttttactactgtaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctgtccctatcagtgatagagatctccctatcagtgatagagagtttagtgaaccgtcagatccgctagggtaccaacATGGCTAGCATCGTCGGAGGGTGGGAGTGCGAAAAGCACTCACAGCCATGGCAGGTCCTGGTCGCCTCGCGCGGACGCGCCGTGTGTGGAGGTGTGCTGGTCCACCCGCAGTGGGTGTTGACTGCGGCCCATTGCATCAGAAATAAGTCCGTGATCCTCTTGGGGAGACATTCCCTGTTTCACCCCGAAGATACTGGACAGGTGTTCCAAGTGAGCCACTCCTTCCCGCATCCACTGTACGACATGAGCCTGCTGAAGAACCGCTTTCTGCGGCCAGGGGACGACTCATCACACGATTTGATGCTGCTTCGGCTCTCGGAACCGGCCGAGCTCACCGACGCAGTGAAGGTCATGGACCTCCCTACGCAAGAGCCTGCTCTCGGTACCACTTGTTACGCATCGGGATGGGGCTCCATCGAGCCGGAAGAATTCCTGACCCCGAAAAAGCTGCAGTGCGTGGATCTGCACGTGATTTCGAATGACGTGTGCGCGCAAGTGCATCCACAAAAGGTCACTAAGTTCATGCTGTGCGCCGGAAGGTGGACCGGCGGAAAATCGACCTGTTCCGGCGACAGCGGAGGCCCACTCGTGTGCAACGGTGTGCTGCAGGGCATCACTAGCTGGGGATCAGAACCGTGCGCGCTTCCGGAGCGGCCCTCGCTCTACACGAAGGTGGTGCACTACCGCAAATGGATTAAAGATACCATCGTCGCAAACCCTggatccgaaggtaggggttcattattgacctgtggagatgtcgaagaaaacccaggacccGCTAGCAAAGCAGTGCTGCTGGCGCTCCTGATGGCTGGACTCGCGCTGCAGCCTGGAACCGCCCTGCTCTGTTACTCGTGCAAGGCCCAAGTCTCGAATGAGGACTGTTTGCAAGTGGAAAACTGCACCCAGCTCGGAGAACAATGCTGGACTGCACGGATCCGCGCTGTCGGCCTGCTGACCGTGATCTCCAAAGGGTGCTCATTGAACTGCGTGGACGATAGCCAGGACTACTACGTGGGAAAGAAGAATATCACTTGTTGCGACACGGATCTTTGCAACGCGTCCGGAGCGCACGCCCTGCAGCCAGCAGCCGCCATTCTGGCCCTGCTTCCGGCCCTGGGGTTGCTGCTCTGGGGTCCGGGCCAGCTCggatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaATGGCTAGCGCTCGCAGACCGCGGTGGCTGTGTGCAGGGGCGCTCGTCCTGGCGGGTGGCTTCTTTTTGCTCGGCTTTCTTTTCGGATGGTTCATCAAATCGTCAAACGAAGCTACCAATATCACCCCGAAGCACAACATGAAGGCCTTTCTGGATGAGCTGAAGGCTGAGAACATTAAGAAGTTCCTCTACAACTTCACCCAGATCCCACATTTGGCGGGCACTGAGCAGAACTTTCAGTTGGCTAAGCAGATCCAGAGCCAGTGGAAGGAATTCGGCCTGGACTCCGTCGAGCTGGCGCATTACGATGTGCTGCTGAGCTACCCTAATAAGACTCATCCGAACTATATCTCGATTATCAATGAGGACGGAAACGAAATCTTTAACACGTCCCTCTTCGAGCCGCCACCGCCTGGATACGAGAACGTGTCAGATATCGTGCCTCCGTTCTCGGCCTTCTCGCCCCAGGGAATGCCCGAAGGGGACCTGGTGTACGTGAACTACGCAAGGACCGAGGACTTCTTCAAGTTGGAGCGGGATATGAAGATCAATTGCAGCGGAAAGATCGTCATCGCCCGCTACGGCAAAGTGTTCCGCGGCAACAAGGTGAAGAATGCACAGTTGGCAGGCGCCAAGGGCGTCATCCTCTACTCGGATCCTGCCGACTACTTCGCTCCTGGCGTGAAATCCTACCCTGATGGTTGGAATCTGCCAGGAGGAGGGGTGCAGAGGGGAAATATCCTGAACCTGAACGGTGCCGGTGACCCACTTACTCCGGGTTACCCGGCCAACGAATACGCGTACAGGCGGGGTATCGCGGAAGCCGTCGGACTGCCGTCCATCCCGGTCCATCCGATTGGTTACTACGACGCCCAGAAGCTCCTCGAAAAGATGGGAGGCAGCGCCCCTCCGGACTCGTCATGGAGAGGCTCGCTGAAGGTGCCATACAACGTGGGACCCGGATTCACTGGAAATTTCAGCACTCAAAAAGTGAAGATGCACATTCACTCCACTAACGAAGTCACCAGGATCTACAACGTCATCGGAACCCTCCGGGGAGCGGTGGAACCGGACCGCTACGTGATCCTCGGTGGACACCGGGATAGCTGGGTGTTCGGAGGAATCGATCCTCAATCGGGCGCAGCCGTCGTCCATGAAATCGTCAGGTCCTTTGGTACTCTTAAGAAGGAGGGCTGGCGCCCTAGACGCACTATTCTGTTCGCCTCGTGGGATGCCGAAGAATTTGGTCTGCTCGGCAGCACCGAATGGGCTGAGGAAAACTCCCGCCTGCTCCAAGAACGCGGAGTGGCGTACATCAATGCCGACTCATCCATCGAAGGAAACTACACGCTGCGGGTGGACTGCACTCCACTGATGTACTCGCTCGTGCACAACCTGACCAAAGAACTCAAATCCCCAGACGAAGGATTCGAGGGAAAATCGCTGTACGAGTCGTGGACCAAGAAGAGCCCATCCCCGGAGTTCAGCGGGATGCCGCGGATCTCAAAGCTCGGATCAGGAAATGATTTCGAAGTGTTCTTTCAGAGGCTGGGAATTGCGTCGGGAAGGGCTCGGTACACGAAAAACTGGGAAACTAACAAGTTCTCGGGATACCCGCTGTACCACTCGGTGTATGAAACTTACGAACTGGTGGAGAAATTCTACGATCCTATGTTTAAGTACCACCTGACTGTGGCCCAAGTGAGAGGCGGAATGGTGTTCGAGTTGGCCAATTCAATTGTGCTGCCATTCGATTGCCGCGACTACGCCGTGGTGCTGAGAAAGTACGCAGACAAAATCTACTCAATCAGCATGAAGCACCCACAAGAGATGAAAACCTACTCAGTCTCCTTCGACTCCCTCTTCTCCGCGGTGAAGAACTTCACCGAGATCGCGAGCAAATTCTCGGAGCGCCTTCAAGATTTTGACAAATCCAATCCGATCGTCCTCCGCATGATGAATGACCAGCTCATGTTTCTCGAACGGGCCTTCATCGATCCACTGGGACTTCCGGACCGGCCGTTTTACCGCCACGTGATCTACGCGCCCTCGTCGCATAACAAGTATGCTGGAGAGAGCTTCCCGGGTATCTACGACGCATTGTTCGACATTGAGTCCAAGGTGGATCCGTCCAAAGCCTGGGGTGAAGTGAAGCGCCAAATCTACGTGGCGGCCTTTACCGTCCAGGCGGCAGCAGAAACCTTGAGCGAGGTGGCTTGActcgagcctaagcttctagataagatatccgatccaccggatctagataactgatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatatgctggccaccgtacatgtggcttcccatgctcgcaagccctggcccgagttcgagcacaatgtcatgaccaggtgcaatatgcatctggggtcccgccgaggcatgttcatgccctaccagtgcaacctgaattatgtgaaggtgctgctggagcccgatgccatgtccagagtgagcctgacgggggtgtttgacatgaatgtggaggtgtggaagattctgagatatgatgaatccaagaccaggtgccgagcctgcgagtgcggagggaagcatgccaggttccagcccgtgtgtgtggatgtgacggaggacctgcgacccgatcatttggtgttgccctgcaccgggacggagttcggttccagcggggaagaatctgactagagtgagtagtgttctggggcgggggaggacctgcatgagggccagaataactgaaatctgtgcttttctgtgtgttgcagcagcatgagcggaagcggctcctttgagggaggggtattcagcccttatctgacggggcgtctcccctcctgggcgggagtgcgtcagaatgtgatgggatccacggtggacggccggcccgtgcagcccgcgaactcttcaaccctgacctatgcaaccctgagctcttcgtcgttggacgcagctgccgccgcagctgctgcatctgccgccagcgccgtgcgcggaatggccatgggcgccggctactacggcactctggtggccaactcgagttccaccaataatcccgccagcctgaacgaggagaagctgttgctgctgatggcccagctcgaggccttgacccagcgcctgggcgagctgacccagcaggtggctcagctgcaggagcagacgcgggccgcggttgccacggtgaaatccaaataaaaaatgaatcaataaataaacggagacggttgttgattttaacacagagtctgaatctttatttgatttttcgcgcgcggtaggccctggaccaccggtctcgatcattgagcacccggtggatcttttccaggacccggtagaggtgggcttggatgttgaggtacatgggcatgagcccgtcccgggggtggaggtagctccattgcagggcctcgtgctcgggggtggtgttgtaaatcacccagtcatagcaggggcgcagggcatggtgttgcacaatatctttgaggaggagactgatggccacgggcagccctttggtgtaggtgtttacaaatctgttgagctgggagggatgcatgcggggggagatgaggtgcatcttggcctggatcttgagattggcgatgttaccgcccagatcccgcctggggttcatgttgtgcaggaccaccagcacggtgtatccggtgcacttggggaatttatcatgcaacttggaagggaaggcgtgaaagaatttggcgacgcctttgtgcccgcccaggttttccatgcactcatccatgatgatggcgatgggcccgtgggcggcggcctgggcaaagacgtttcgggggtcggacacatcatagttgtggtcctgggtgaggtcatcataggccattttaatgaatttggggcggagggtgccggactgggggacaaaggtaccctcgatcccgggggcgtagttcccctcacagatctgcatctcccaggctttgagctcggagggggggatcatgtccacctgcggggcgataaagaacacggtttccggggcgggggagatgagctgggccgaaagcaagttccggagcagctgggacttgccgcagccggtggggccgtagatgaccccgatgaccggctgcaggtggtagttgagggagagacagctgccgtcctcccggaggaggggggccacctcgttcatcatctcgcgcacgtgcatgttctcgcgcaccagttccgccaggaggcgctctccccccagggataggagctcctggagcgaggcgaagtttttcagcggcttgagtccgtcggccatgggcattttggagagggtttgttgcaagagttccaggcggtcccagagctcggtgatgtgctctacggcatctcgatccagcagacctcctcgtttcgcgggttgggacggctgcgggagtagggcaccagacgatgggcgtccagcgcagccagggtccggtccttccagggtcgcagcgtccgcgtcagggtggtctccgtcacggtgaaggggtgcgcgccgggctgggcgcttgcgagggtgcgcttcaggctcatccggctggtcgaaaaccgctcccgatcggcgccctgcgcgtcggccaggtagcaattgaccatgagttcgtagttgagcgcctcggccgcgtggcctttggcgcggagcttacctttggaagtctgcccgcaggcgggacagaggagggacttgagggcgtagagcttgggggcgaggaagacggactcgggggcgtaggcgtccgcgccgcagtgggcgcagacggtctcgcactccacgagccaggtgaggtcgggctggtcggggtcaaaaaccagtttcccgccgttctttttgatgcgtttcttacctttggtctccatgagctcgtgtccccgctgggtgacaaagaggctgtccgtgtccccgtagaccgactttatgggccggtcctcgagcggtgtgccgcggtcctcctcgtagaggaaccccgcccactccgagacgaaagcccgggtccaggccagcacgaaggaggccacgtgggacgggtagcggtcgttgtccaccagcgggtccaccttttccagggtatgcaaacacatgtccccctcgtccacatccaggaaggtgattggcttgtaagtgtaggccacgtgaccgggggtcccggccgggggggtataaaagggtgcgggtccctgctcgtcctcactgtcttccggatcgctgtccaggagcgccagctgttggggtaggtattccctctcgaaggcgggcatgacctcggcactcaggttgtcagtttctagaaacgaggaggatttgatattgacggtgccggcggagatgcctttcaagagcccctcgtccatctggtcagaaaagacgatctttttgttgtcgagcttggtggcgaaggagccgtagagggcgttggagaggagcttggcgatggagcgcatggtctggtttttttccttgtcggcgcgctccttggcggcgatgttgagctgcacgtactcgcgcgccacgcacttccattcggggaagacggtggtcagctcgtcgggcacgattctgacctgccagccccgattatgcagggtgatgaggtccacactggtggccacctcgccgcgcaggggctcattagtccagcagaggcgtccgcccttgcgcgagcagaaggggggcagggggtccagcatgacctcgtcgggggggtcggcatcgatggtgaagatgccgggcaggaggtcggggtcaaagtagctgatggaagtggccagatcgtccagggcagcttgccattcgcgcacggccagcgcgcgctcgtagggactgaggggcgtgccccagggcatgggatgggtaagcgcggaggcgtacatgccgcagatgtcgtagacgtagaggggctcctcgaggatgccgatgtaggtggggtagcagcgccccccgcggatgctggcgcgcacgtagtcatacagctcgtgcgagggggcgaggagccccgggcccaggttggtgcgactgggcttttcggcgcggtagacgatctggcggaaaatggcatgcgagttggaggagatggtgggcctttggaagatgttgaagtgggcgtggggcagtccgaccgagtcgcggatgaagtgggcgtaggagtcttgcagcttggcgacgagctcggcggtgactaggacgtccagagcgcagtagtcgagggtctcctggatgatgtcatacttgagctgtcccttttgtttccacagctcgcggttgagaaggaactcttcgcggtccttccagtactcttcgagggggaacccgtcctgatctgcacggtaagagcctagcatgtagaactggttgacggccttgtaggcgcagcagcccttctccacggggagggcgtaggcctgggcggccttgcgcagggaggtgtgcgtgagggcgaaagtgtccctgaccatgaccttgaggaactggtgcttgaagtcgatatcgtcgcagcccccctgctcccagagctggaagtccgtgcgcttcttgtaggcggggttgggcaaagcgaaagtaacatcgttgaagaggatcttgcccgcgcggggcataaagttgcgagtgatgcggaaaggttggggcacctcggcccggttgttgatgacctgggcggcgagcacgatctcgtcgaagccgttgatgttgtggcccacgatgtagagttccacgaatcgcggacggcccttgacgtggggcagtttcttgagctcctcgtaggtgagctcgtcggggtcgctgagcccgtgctgctcgagcgcccagtcggcgagatgggggttggcgcggaggaaggaagtccagagatccacggccagggcggtttgcagacggtcccggtactgacggaactgctgcccgacggccattttttcgggggtgacgcagtagaaggtgcgggggtccccgtgccagcgatcccatttgagctggagggcgagatcgagggcgagctcgacgagccggtcgtccccggagagtttcatgaccagcatgaaggggacgagctgcttgccgaaggaccccatccaggtgtaggtttccacatcgtaggtgaggaagagcctttcggtgcgaggatgcgagccgatggggaagaactggatctcctgccaccaattggaggaatggctgttgatgtgatggaagtagaaatgccgacggcgcgccgaacactcgtgcttgtgtttatacaagcggccacagtgctcgcaacgctgcacgggatgcacgtgctgcacgagctgtacctgagttcctttgacgaggaatttcagtgggaagtggagtcgtggcgcctgcatctcgtgctgtactacgtcgtggtggtcggcctggccctcttctgcctcgatggtggtcatgctgacgagcccgcgcgggaggcaggtccagacctcggcgcgagcgggtcggag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ccagcagtctggcgataccaaggggtgcatccactgctcctgcgactcccccgactgcgtccacactctgatcaagaccctctgcggcctccgcgacctcctccccatgaactaatcacccccttatccagtgaaataaagatcatattgatgatgattttacagaaataaaaaataatcatttgatttgaaataaagatacaatcatattgatgatttgagtttaacaaaaaaataaagaatcacttacttgaaatctgataccaggtctctgtccatgttttctgccaacaccacttcactcccctcttcccagctctggtactgcaggccccggcgggctgcaaacttcctccacacgctgaaggggatgtcaaattcctcctgtccctcaatcttcattttatcttctatcagatgtccaaaaagcgcgtccgggtggatgatgacttcgaccccgtctacccctacgatgcagacaacgcaccgaccgtgcccttcatcaacccccccttcgtctcttcagatggattccaagagaagcccctgggggtgttgtccctgcgactggccgaccccgtcaccaccaagaacggggaaatcaccctcaagctgggagagggggtggacctcgattcctcgggaaaactcatctccaacacggccaccaaggccgccgcccctctcagtttttccaacaacaccatttcccttaacatggatcaccccttttacactaaagatggaaaattatccttacaagtttctccaccattaaatatactgagaacaagcattctaaacacactagctttaggttttggatcaggtttaggactccgtggctctgccttggcagtacagttagtctctccacttacatttgatactgatggaaacataaagcttaccttagacagaggtttgcatgttacaacaggagatgcaattgaaagcaacataagctgggctaaaggtttaaaatttgaagatggagccatagcaaccaacattggaaatgggttagagtttggaagcagtagtacagaaacaggtgttgatgatgcttacccaatccaagttaaacttggatctggccttagctttgacagtacaggagccataatggctggtaacaaagaagacgataaactcactttgtggacaacacctgatccatcaccaaactgtcaaatactcgcagaaaatgatgcaaaactaacactttgcttgactaaatgtggtagtcaaatactggccactgtgtcagtcttagttgtaggaagtggaaacctaaaccccattactggcaccgtaagcagtgctcaggtgtttctacgttttgatgcaaacggtgttcttttaacagaacattctacactaaaaaaatactgggggtataggcagggagatagcatagatggcactccatataccaatgctgtaggattcatgcccaatttaaaagcttatccaaagtcacaaagttctactactaaaaataatatagtagggcaagtatacatgaatggagatgtttcaaaacctatgcttctcactataaccctcaatggtactgatgacagcaacagtacatattcaatgtcattttcatacacctggactaatggaagctatgttggagcaacatttggggctaactcttataccttctcatacatcgcccaagaatgaacactgtatcccaccctgcatgccaacccttcccaccccactctgtggaacaaactctgaaacacaaaataaaataaagttcaagtgttttattgattcaacagttttacaggattcgagcagttatttttcctccaccctcccaggacatggaatacaccaccctctccccccgcacagccttgaacatctgaatgccattggtgatggacatgcttttggtctccacgttccacacagtttcagagcgagccagtctcgggtcggtcagggagatgaaaccctccgggcactcccgcatctgcacctcacagctcaacagctgaggattgtcctcggtggtcgggatcacggttatctggaagaagcagaagagcggcggtgggaatcatagtccgcgaacgggatcggccggtggtgtcgcatcaggccccgcagcagtcgctgccgccgccgctccgtcaagctgctgctcagggggtccgggtccagggactccctcagcatgatgcccacggccctcagcatcagtcgtctggtgcggcgggcgcagcagcgcatgcggatctcgctcaggtcgctgcagtacgtgcaacacagaaccaccaggttgttcaacagtccatagttcaacacgctccagccgaaactcatcgcgggaaggatgctacccacgtggccgtcgtaccagatcctcaggtaaatcaagtggtgccccctccagaacacgctgcccacgtacatgatctccttgggcatgtggcggttcaccacctcccggtaccacatcaccctctggttgaacatgcagccccggatgatcctgcggaaccacagggccagcaccgccccgcccgccatgcagcgaagagaccccgggtcccggcaatggcaatggaggacccaccgctcgtacccgtggatcatctgggagctgaacaagtctatgttggcacagcacaggcatatgctcatgcatctcttcagcactctcaactcctcgggggtcaaaaccatatcccagggcacggggaactcttgcaggacagcgaaccccgcagaacagggcaatcctcgcacagaacttacattgtgcatggacagggtatcgcaatcaggcagcaccgggtgatcctccaccagagaagcgcgggtctcggtctcctcacagcgtggtaagggggccggccgatacgggtgatggcgggacgcggctgatcgtgttcgcgaccgtgtcatgatgcagttgctttcggacattttcgtacttgctgtagcagaacctggtccgggcgctgcacaccgatcgccggcggcggtctcggcgcttggaacgctcggtgttgaaattgtaaaacagccactctctcagaccgtgcagcagatctagggcctcaggagtgatgaagatcccatcatgcctgatggctctgatcacatcgaccaccgtggaatgggccagacccagccagatgatgcaattttgttgggtttcggtgacggcgggggagggaagaacaggaagaaccatgattaacttttaatccaaacggtctcggagtacttcaaaatgaagatcgcggagatggcacctctcgcccccgctgtgttggtggaaaataacagccaggtcaaaggtgatacggttctcgagatgttccacggtggcttccagcaaagcctccacgcgcacatccagaaacaagacaatagcgaaagcgggagggttctctaattcctcaatcatcatgttacactcctgcaccatccccagataattttcatttttccagccttgaatgattcgaactagttcCtgaggtaaatccaagccagccatgataaagagctcgcgcagagcgccctccaccggcattcttaagcacaccctcataattccaagatattctgctcctggttcacctgcagcagattgacaagcggaatatcaaaatctctgccgcgatccctgagctcctccctcagcaataactgtaagtactctttcatatcctctccgaaatttttagccataggaccaccaggaataagattagggcaagccacagtacagataaaccgaagtcctccccagtgagcattgccaaatgcaagactgctataagcatgctggctagacccggtgatatcttccagataactggacagaaaatcgcccaggcaatttttaagaaaatcaacaaaagaaaaatcctccaggtggacgtttagagcctcgggaacaacgatgaagtaaatgcaagcggtgcgttccagcatggttagttagctgatctgtagaaaaaacaaaaatgaacattaaaccatgctagcctggcgaacaggtgggtaaatcgttctctccagcaccaggcaggccacggggtctccggcgcgaccctcgtaaaaattgtcgctatgattgaaaaccatcacagagagacgttcccggtggccggcgtgaatgattcgacaagatgaatacacccccggaacattggcgtccgcgagtgaaaaaaagcgcccgaggaagcaataaggcactacaatgctcagtctcaagtccagcaaagcgatgccatgcggatgaagcacaaaattctcaggtgcgtacaaaatgtaattactcccctcctgcacaggcagcaaagcccccgatccctccaggtacacatacaaagcctcagcgtccatagcttaccgagcagcagcacacaacaggcgcaagagtcagagaaaggctgagctctaacctgtccacccgctctctgctcaatatatagcccagatctacactgacgtaaaggccaaagtctaaaaatacccgccaaataatcacacacgcccagcacacgcccagaaaccggtgacacactcaaaaaaatacgcgcacttcctcaaacgcccaaaactgccgtcatttccgggttcccacgctacgtcatcaaaacacgactttcaaattccgtcgaccgttaaaaacgtcacccgccccgcccctaacggtcgcccgtctctcagccaatcagcgccccgcatccccaaattcaaacGcctcatttgcatattaacgcgcacaaaaagtttgaggtatattattgatgatgg
SEQ ID NO:64.包含免疫原性PSA、PSMA和PSCA多肽的氨基酸序列(由质粒457和载体AdC68X-733编码)
MASIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANPGSQTLNFDLLKLAGDVESNPGPMASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVAGSEGRGSLLTCGDVEENPGPASKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQL
SEQ ID NO:65.编码SEQ ID NO:64的氨基酸序列的核苷酸序列
ATGGCTAGCATCGTCGGAGGGTGGGAGTGCGAAAAGCACTCACAGCCATGGCAGGTCCTGGTCGCCTCGCGCGGACGCGCCGTGTGTGGAGGTGTGCTGGTCCACCCGCAGTGGGTGTTGACTGCGGCCCATTGCATCAGAAATAAGTCCGTGATCCTCTTGGGGAGACATTCCCTGTTTCACCCCGAAGATACTGGACAGGTGTTCCAAGTGAGCCACTCCTTCCCGCATCCACTGTACGACATGAGCCTGCTGAAGAACCGCTTTCTGCGGCCAGGGGACGACTCATCACACGATTTGATGCTGCTTCGGCTCTCGGAACCGGCCGAGCTCACCGACGCAGTGAAGGTCATGGACCTCCCTACGCAAGAGCCTGCTCTCGGTACCACTTGTTACGCATCGGGATGGGGCTCCATCGAGCCGGAAGAATTCCTGACCCCGAAAAAGCTGCAGTGCGTGGATCTGCACGTGATTTCGAATGACGTGTGCGCGCAAGTGCATCCACAAAAGGTCACTAAGTTCATGCTGTGCGCCGGAAGGTGGACCGGCGGAAAATCGACCTGTTCCGGCGACAGCGGAGGCCCACTCGTGTGCAACGGTGTGCTGCAGGGCATCACTAGCTGGGGATCAGAACCGTGCGCGCTTCCGGAGCGGCCCTCGCTCTACACGAAGGTGGTGCACTACCGCAAATGGATTAAAGATACCATCGTCGCAAACCCTggatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaATGGCTAGCGCTCGCAGACCGCGGTGGCTGTGTGCAGGGGCGCTCGTCCTGGCGGGTGGCTTCTTTTTGCTCGGCTTTCTTTTCGGATGGTTCATCAAATCGTCAAACGAAGCTACCAATATCACCCCGAAGCACAACATGAAGGCCTTTCTGGATGAGCTGAAGGCTGAGAACATTAAGAAGTTCCTCTACAACTTCACCCAGATCCCACATTTGGCGGGCACTGAGCAGAACTTTCAGTTGGCTAAGCAGATCCAGAGCCAGTGGAAGGAATTCGGCCTGGACTCCGTCGAGCTGGCGCATTACGATGTGCTGCTGAGCTACCCTAATAAGACTCATCCGAACTATATCTCGATTATCAATGAGGACGGAAACGAAATCTTTAACACGTCCCTCTTCGAGCCGCCACCGCCTGGATACGAGAACGTGTCAGATATCGTGCCTCCGTTCTCGGCCTTCTCGCCCCAGGGAATGCCCGAAGGGGACCTGGTGTACGTGAACTACGCAAGGACCGAGGACTTCTTCAAGTTGGAGCGGGATATGAAGATCAATTGCAGCGGAAAGATCGTCATCGCCCGCTACGGCAAAGTGTTCCGCGGCAACAAGGTGAAGAATGCACAGTTGGCAGGCGCCAAGGGCGTCATCCTCTACTCGGATCCTGCCGACTACTTCGCTCCTGGCGTGAAATCCTACCCTGATGGTTGGAATCTGCCAGGAGGAGGGGTGCAGAGGGGAAATATCCTGAACCTGAACGGTGCCGGTGACCCACTTACTCCGGGTTACCCGGCCAACGAATACGCGTACAGGCGGGGTATCGCGGAAGCCGTCGGACTGCCGTCCATCCCGGTCCATCCGATTGGTTACTACGACGCCCAGAAGCTCCTCGAAAAGATGGGAGGCAGCGCCCCTCCGGACTCGTCATGGAGAGGCTCGCTGAAGGTGCCATACAACGTGGGACCCGGATTCACTGGAAATTTCAGCACTCAAAAAGTGAAGATGCACATTCACTCCACTAACGAAGTCACCAGGATCTACAACGTCATCGGAACCCTCCGGGGAGCGGTGGAACCGGACCGCTACGTGATCCTCGGTGGACACCGGGATAGCTGGGTGTTCGGAGGAATCGATCCTCAATCGGGCGCAGCCGTCGTCCATGAAATCGTCAGGTCCTTTGGTACTCTTAAGAAGGAGGGCTGGCGCCCTAGACGCACTATTCTGTTCGCCTCGTGGGATGCCGAAGAATTTGGTCTGCTCGGCAGCACCGAATGGGCTGAGGAAAACTCCCGCCTGCTCCAAGAACGCGGAGTGGCGTACATCAATGCCGACTCATCCATCGAAGGAAACTACACGCTGCGGGTGGACTGCACTCCACTGATGTACTCGCTCGTGCACAACCTGACCAAAGAACTCAAATCCCCAGACGAAGGATTCGAGGGAAAATCGCTGTACGAGTCGTGGACCAAGAAGAGCCCATCCCCGGAGTTCAGCGGGATGCCGCGGATCTCAAAGCTCGGATCAGGAAATGATTTCGAAGTGTTCTTTCAGAGGCTGGGAATTGCGTCGGGAAGGGCTCGGTACACGAAAAACTGGGAAACTAACAAGTTCTCGGGATACCCGCTGTACCACTCGGTGTATGAAACTTACGAACTGGTGGAGAAATTCTACGATCCTATGTTTAAGTACCACCTGACTGTGGCCCAAGTGAGAGGCGGAATGGTGTTCGAGTTGGCCAATTCAATTGTGCTGCCATTCGATTGCCGCGACTACGCCGTGGTGCTGAGAAAGTACGCAGACAAAATCTACTCAATCAGCATGAAGCACCCACAAGAGATGAAAACCTACTCAGTCTCCTTCGACTCCCTCTTCTCCGCGGTGAAGAACTTCACCGAGATCGCGAGCAAATTCTCGGAGCGCCTTCAAGATTTTGACAAATCCAATCCGATCGTCCTCCGCATGATGAATGACCAGCTCATGTTTCTCGAACGGGCCTTCATCGATCCACTGGGACTTCCGGACCGGCCGTTTTACCGCCACGTGATCTACGCGCCCTCGTCGCATAACAAGTATGCTGGAGAGAGCTTCCCGGGTATCTACGACGCATTGTTCGACATTGAGTCCAAGGTGGATCCGTCCAAAGCCTGGGGTGAAGTGAAGCGCCAAATCTACGTGGCGGCCTTTACCGTCCAGGCGGCAGCAGAAACCTTGAGCGAGGTGGCTggatccgaaggtaggggttcattattgacctgtggagatgtcgaagaaaacccaggacccGCTAGCAAAGCAGTGCTGCTGGCGCTCCTGATGGCTGGACTCGCGCTGCAGCCTGGAACCGCCCTGCTCTGTTACTCGTGCAAGGCCCAAGTCTCGAATGAGGACTGTTTGCAAGTGGAAAACTGCACCCAGCTCGGAGAACAATGCTGGACTGCACGGATCCGCGCTGTCGGCCTGCTGACCGTGATCTCCAAAGGGTGCTCATTGAACTGCGTGGACGATAGCCAGGACTACTACGTGGGAAAGAAGAATATCACTTGTTGCGACACGGATCTTTGCAACGCGTCCGGAGCGCACGCCCTGCAGCCAGCAGCCGCCATTCTGGCCCTGCTTCCGGCCCTGGGGTTGCTGCTCTGGGGTCCGGGCCAGCTC
SEQ ID NO:66.并入质粒459和载体AdC68X-735的多抗原构建体(PSCA-F2A-PSMA-mIRES-PSA)的核苷酸序列
ATGGCTAGCAAAGCAGTGCTGCTGGCGCTCCTGATGGCTGGACTCGCGCTGCAGCCTGGAACCGCCCTGCTCTGTTACTCGTGCAAGGCCCAAGTCTCGAATGAGGACTGTTTGCAAGTGGAAAACTGCACCCAGCTCGGAGAACAATGCTGGACTGCACGGATCCGCGCTGTCGGCCTGCTGACCGTGATCTCCAAAGGGTGCTCATTGAACTGCGTGGACGATAGCCAGGACTACTACGTGGGAAAGAAGAATATCACTTGTTGCGACACGGATCTTTGCAACGCGTCCGGAGCGCACGCCCTGCAGCCAGCAGCCGCCATTCTGGCCCTGCTTCCGGCCCTGGGGTTGCTGCTCTGGGGTCCGGGCCAGCTCggatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaATGGCTAGCGCTCGCAGACCGCGGTGGCTGTGTGCAGGGGCGCTCGTCCTGGCGGGTGGCTTCTTTTTGCTCGGCTTTCTTTTCGGATGGTTCATCAAATCGTCAAACGAAGCTACCAATATCACCCCGAAGCACAACATGAAGGCCTTTCTGGATGAGCTGAAGGCTGAGAACATTAAGAAGTTCCTCTACAACTTCACCCAGATCCCACATTTGGCGGGCACTGAGCAGAACTTTCAGTTGGCTAAGCAGATCCAGAGCCAGTGGAAGGAATTCGGCCTGGACTCCGTCGAGCTGGCGCATTACGATGTGCTGCTGAGCTACCCTAATAAGACTCATCCGAACTATATCTCGATTATCAATGAGGACGGAAACGAAATCTTTAACACGTCCCTCTTCGAGCCGCCACCGCCTGGATACGAGAACGTGTCAGATATCGTGCCTCCGTTCTCGGCCTTCTCGCCCCAGGGAATGCCCGAAGGGGACCTGGTGTACGTGAACTACGCAAGGACCGAGGACTTCTTCAAGTTGGAGCGGGATATGAAGATCAATTGCAGCGGAAAGATCGTCATCGCCCGCTACGGCAAAGTGTTCCGCGGCAACAAGGTGAAGAATGCACAGTTGGCAGGCGCCAAGGGCGTCATCCTCTACTCGGATCCTGCCGACTACTTCGCTCCTGGCGTGAAATCCTACCCTGATGGTTGGAATCTGCCAGGAGGAGGGGTGCAGAGGGGAAATATCCTGAACCTGAACGGTGCCGGTGACCCACTTACTCCGGGTTACCCGGCCAACGAATACGCGTACAGGCGGGGTATCGCGGAAGCCGTCGGACTGCCGTCCATCCCGGTCCATCCGATTGGTTACTACGACGCCCAGAAGCTCCTCGAAAAGATGGGAGGCAGCGCCCCTCCGGACTCGTCATGGAGAGGCTCGCTGAAGGTGCCATACAACGTGGGACCCGGATTCACTGGAAATTTCAGCACTCAAAAAGTGAAGATGCACATTCACTCCACTAACGAAGTCACCAGGATCTACAACGTCATCGGAACCCTCCGGGGAGCGGTGGAACCGGACCGCTACGTGATCCTCGGTGGACACCGGGATAGCTGGGTGTTCGGAGGAATCGATCCTCAATCGGGCGCAGCCGTCGTCCATGAAATCGTCAGGTCCTTTGGTACTCTTAAGAAGGAGGGCTGGCGCCCTAGACGCACTATTCTGTTCGCCTCGTGGGATGCCGAAGAATTTGGTCTGCTCGGCAGCACCGAATGGGCTGAGGAAAACTCCCGCCTGCTCCAAGAACGCGGAGTGGCGTACATCAATGCCGACTCATCCATCGAAGGAAACTACACGCTGCGGGTGGACTGCACTCCACTGATGTACTCGCTCGTGCACAACCTGACCAAAGAACTCAAATCCCCAGACGAAGGATTCGAGGGAAAATCGCTGTACGAGTCGTGGACCAAGAAGAGCCCATCCCCGGAGTTCAGCGGGATGCCGCGGATCTCAAAGCTCGGATCAGGAAATGATTTCGAAGTGTTCTTTCAGAGGCTGGGAATTGCGTCGGGAAGGGCTCGGTACACGAAAAACTGGGAAACTAACAAGTTCTCGGGATACCCGCTGTACCACTCGGTGTATGAAACTTACGAACTGGTGGAGAAATTCTACGATCCTATGTTTAAGTACCACCTGACTGTGGCCCAAGTGAGAGGCGGAATGGTGTTCGAGTTGGCCAATTCAATTGTGCTGCCATTCGATTGCCGCGACTACGCCGTGGTGCTGAGAAAGTACGCAGACAAAATCTACTCAATCAGCATGAAGCACCCACAAGAGATGAAAACCTACTCAGTCTCCTTCGACTCCCTCTTCTCCGCGGTGAAGAACTTCACCGAGATCGCGAGCAAATTCTCGGAGCGCCTTCAAGATTTTGACAAATCCAATCCGATCGTCCTCCGCATGATGAATGACCAGCTCATGTTTCTCGAACGGGCCTTCATCGATCCACTGGGACTTCCGGACCGGCCGTTTTACCGCCACGTGATCTACGCGCCCTCGTCGCATAACAAGTATGCTGGAGAGAGCTTCCCGGGTATCTACGACGCATTGTTCGACATTGAGTCCAAGGTGGATCCGTCCAAAGCCTGGGGTGAAGTGAAGCGCCAAATCTACGTGGCGGCCTTTACCGTCCAGGCGGCAGCAGAAACCTTGAGCGAGGTGGCTTGAagatctgaccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatATGGCTAGCATCGTCGGAGGGTGGGAGTGCGAAAAGCACTCACAGCCATGGCAGGTCCTGGTCGCCTCGCGCGGACGCGCCGTGTGTGGAGGTGTGCTGGTCCACCCGCAGTGGGTGTTGACTGCGGCCCATTGCATCAGAAATAAGTCCGTGATCCTCTTGGGGAGACATTCCCTGTTTCACCCCGAAGATACTGGACAGGTGTTCCAAGTGAGCCACTCCTTCCCGCATCCACTGTACGACATGAGCCTGCTGAAGAACCGCTTTCTGCGGCCAGGGGACGACTCATCACACGATTTGATGCTGCTTCGGCTCTCGGAACCGGCCGAGCTCACCGACGCAGTGAAGGTCATGGACCTCCCTACGCAAGAGCCTGCTCTCGGTACCACTTGTTACGCATCGGGATGGGGCTCCATCGAGCCGGAAGAATTCCTGACCCCGAAAAAGCTGCAGTGCGTGGATCTGCACGTGATTTCGAATGACGTGTGCGCGCAAGTGCATCCACAAAAGGTCACTAAGTTCATGCTGTGCGCCGGAAGGTGGACCGGCGGAAAATCGACCTGTTCCGGCGACAGCGGAGGCCCACTCGTGTGCAACGGTGTGCTGCAGGGCATCACTAGCTGGGGATCAGAACCGTGCGCGCTTCCGGAGCGGCCCTCGCTCTACACGAAGGTGGTGCACTACCGCAAATGGATTAAAGATACCATCGTCGCAAACCCT
Claims (24)
1.一种复制缺陷型C68载体,其包含:
(a)复制缺陷型C68核苷酸序列;以及
(b)多抗原构建体,其包含至少一个编码免疫原性PSA多肽的核苷酸序列、至少一个编码免疫原性PSCA多肽的核苷酸序列和至少一个编码免疫原性PSMA多肽的核苷酸序列,并且还包含分别位于所述编码免疫原性PSA多肽的核苷酸序列、所述编码免疫原性PSCA多肽的核苷酸序列和所述编码免疫原性PSMA多肽之间的分隔物序列,
其中所述免疫原性PSA多肽包含SEQ ID NO:15的氨基酸27-263或SEQ ID NO:17的氨基酸4-240,其中所述免疫原性PSCA多肽包含SEQ ID NO:21的氨基酸序列或SEQ ID NO:21的氨基酸4-125。
2.权利要求1的复制缺陷型C68载体,其中所述复制缺陷型C68核苷酸序列是缺少选自E1A、E1B、E2A、E2B、E3、E4、L1、L2、L3、L4和L5基因的至少一个基因的SEQ ID NO:57的序列,并且其中所述免疫原性PSMA多肽选自:
1)包含SEQ ID NO:1的氨基酸15-750的多肽;
2)包含SEQ ID NO:3的氨基酸序列的多肽;
3)包含SEQ ID NO:5的氨基酸序列的多肽;
4)包含SEQ ID NO:7的氨基酸序列的多肽;
5)包含SEQ ID NO:9的氨基酸4–739的多肽;
6)包含SEQ ID NO:3的氨基酸4–739的多肽;
7)包含SEQ ID NO:5的氨基酸4-739的多肽;
8)包含SEQ ID NO:7的氨基酸4-739的多肽;以及
9)包含SEQ ID NO:9的氨基酸序列的多肽。
3.权利要求2的复制缺陷型C68载体,其中所述复制缺陷型C68核苷酸序列是缺少E1A、E1B和E3基因的SEQ ID NO:57的序列,并且其中所述多抗原构建体在编码两个不同免疫原性多肽的两个核苷酸序列之间还包含分隔物序列,而且具有式(I)的结构:
PAA1-SS1-PAA2-SS2-PAA3 (I)
其中在式(I)中:
(i)PAA1、PAA2和PAA3各自为编码免疫原性PSA多肽的核苷酸序列、编码免疫原性PSCA多肽的核苷酸序列或编码免疫原性PSMA多肽的核苷酸序列,条件是PAA1、PAA2和PAA3编码不同的PAA多肽,以及
(ii)SS1和SS2为分隔物序列,并且可以相同或不同。
4.权利要求3的复制缺陷型C68载体,其中所述分隔物序列选自2A肽序列和IRES。
5.权利要求4的复制缺陷型C68载体,其中所述2A肽序列为选自以下的2A-肽序列:FMDV、ERAV、PTV1、EMC-B、EMCV、TME-GD7、ERBV、TaV、DrosC、CrPV、ABPV、IFV、猪轮状病毒、人轮状病毒、布氏锥虫(T brucei)TSR1和克氏锥虫(T cruzi)AP核酸内切酶;并且其中所述IRES为EMCV IRES。
6.权利要求5的复制缺陷型C68载体,其中式(I)的PAA1为编码免疫原性PSA多肽的核苷酸序列或编码免疫原性PSCA多肽的核苷酸序列。
7.权利要求6的复制缺陷型C68载体,其中:
(i)PAA1为编码免疫原性PSA多肽的核苷酸序列;
(ii)PAA2为编码免疫原性PSCA或PSMA多肽的核苷酸序列;
(iii)SS1为2A-肽序列;以及
(iv)SS2为2A-肽序列或EMCV IRES。
8.权利要求7的复制缺陷型C68载体,其中所述2A-肽序列为FMDV2A-肽序列或TAV 2A-肽序列。
9.权利要求8的复制缺陷型C68载体,其中:
(1)编码免疫原性PSA多肽的核苷酸序列选自:(i)SEQ ID NO:18的核苷酸序列;(ii)包含SEQ ID NO:18的核苷酸10-720的核苷酸序列;(iii)包含SEQ ID NO:58的核苷酸1115-1825的核苷酸序列;以及(iv)包含SEQ ID NO:58的核苷酸1106-1825的核苷酸序列;
(2)编码免疫原性PSCA多肽的核苷酸序列选自:(i)SEQ ID NO:22的核苷酸序列;(ii)包含SEQ ID NO:22的核苷酸10-372的核苷酸序列;(iii)包含SEQ ID NO:58的核苷酸1892-2257的核苷酸序列;以及(iv)包含SEQ ID NO:58的核苷酸1886-2257的核苷酸序列;并且
(3)编码免疫原性PSMA多肽的核苷酸序列选自:(i)包含SEQ ID NO:2的核苷酸43-2250的核苷酸序列;(ii)SEQ ID NO:4的核苷酸序列;(iii)SEQ ID NO:6的核苷酸序列;(iv)SEQID NO:8的核苷酸序列;(v)SEQ ID NO:10的核苷酸序列;(vi)包含SEQ ID NO:4的核苷酸10–2217的核苷酸序列;(vii)包含SEQ ID NO:6的核苷酸10–2217的核苷酸序列;(viii)包含SEQ ID NO:8的核苷酸10–2217的核苷酸序列;(ix)包含SEQ ID NO:10的核苷酸10–2217的核苷酸序列;(x)包含SEQ ID NO:58的核苷酸2333-4543的核苷酸序列;以及(xi)包含SEQID NO:58的核苷酸2324-4543的核苷酸序列。
10.权利要求3的复制缺陷型C68载体,其中在式(I)中:
(i)PAA1为编码免疫原性PSA多肽的核苷酸序列,并且包含SEQ ID NO:58的核苷酸1115-1825或包含SEQ ID NO:58的1106-1114;
(2)PAA2为编码免疫原性PSCA多肽的核苷酸序列,并且包含SEQ ID NO:58的核苷酸1892-2257或包含SEQ ID NO:58的1886-2257;
(3)PAA3为编码免疫原性PSMA多肽的核苷酸序列,并且包含SEQ ID NO:58的核苷酸2333-4543或包含SEQ ID NO:58的2324-4543;
(4)SS1为编码T2A的核苷酸序列;以及
(5)SS2为编码F2A的核苷酸序列。
11.权利要求1的复制缺陷型C68载体,其中所述多抗原构建体包含编码SEQ ID NO:60或SEQ ID NO:64的氨基酸序列的核苷酸序列。
12.权利要求1的复制缺陷型C68载体,其中所述多抗原构建体包含SEQ ID NO:61、SEQID NO:65、SEQ ID NO:66的核苷酸序列,或者SEQ ID NO:61、SEQ ID NO:65、SEQ ID NO:66的核苷酸序列的简并变体。
13.权利要求11或权利要求12的复制缺陷型C68载体,其还包含CMV启动子。
14.权利要求1的复制缺陷型C68载体,其包含SEQ ID NO:58的核苷酸序列、SEQ ID NO:58的核苷酸9-34811或SEQ ID NO:63的核苷酸序列。
15.一种组合物,其包含权利要求1-14中任一项的复制缺陷型C68载体。
16.一种细胞,其包含权利要求1-14中任一项的复制缺陷型C68载体。
17.一种药物组合物,其包含权利要求1-14中任一项的载体和药学可接受的赋形剂。
18.权利要求1-14中任一项的载体在制备用于治疗人中的前列腺癌的药物中的用途。
19.权利要求18的用途,其中所述药物与免疫调节剂组合施用。
20.权利要求18的用途,其中所述药物与(a)免疫抑制细胞抑制剂和(b)免疫效应细胞增强剂组合施用。
21.权利要求20的用途,其中所述免疫抑制细胞抑制剂选自蛋白激酶抑制剂、COX-2抑制剂和PDE5抑制剂,并且其中所述免疫效应细胞增强剂选自CTLA-4抑制剂、CD40激动剂、TLR激动剂、4-1BB激动剂、OX40激动剂、GITR激动剂、PD-1拮抗剂和PD-L1拮抗剂。
22.权利要求21的用途,其中:
(1)所述蛋白激酶抑制剂选自:伊马替尼、索拉菲尼、拉帕替尼、zactima MP-412、达沙替尼、来妥替尼、苹果酸舒尼替尼、阿昔替尼、厄洛替尼、吉非替尼、波舒替尼、坦罗莫司以及尼洛替尼;
(2)所述CTLA-4抑制剂选自依匹木单抗和曲美木单抗;
(3)所述CD40激动剂为选自G28-5、mAb89、EA-5、2C6、CP870893和达西珠单抗的抗CD40抗体;并且
(4)所述TLR激动剂为选自CpG 24555、CpG 10103、CpG7909和CpG1018的CpG寡核苷酸。
23.权利要求22的用途,其中所述免疫抑制细胞抑制剂为选自索拉菲尼、达沙替尼、伊马替尼、阿昔替尼和苹果酸舒尼替尼的蛋白激酶抑制剂,并且其中所述免疫效应细胞增强剂为曲美木单抗。
24.权利要求22的用途,其中所述免疫抑制细胞抑制剂为选自索拉菲尼、达沙替尼、伊马替尼、阿昔替尼和苹果酸舒尼替尼的蛋白激酶抑制剂,并且其中所述免疫效应细胞增强剂为选自CpG24555、CpG10103、CpG7909和CpG1018的CpG寡核苷酸。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361898966P | 2013-11-01 | 2013-11-01 | |
US61/898,966 | 2013-11-01 | ||
PCT/IB2014/065419 WO2015063647A1 (en) | 2013-11-01 | 2014-10-17 | Vectors for expression of prostate-associated antigens |
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