CN113082201B - 刺激t细胞介导的对表达抗原的细胞群的免疫应答的方法 - Google Patents
刺激t细胞介导的对表达抗原的细胞群的免疫应答的方法 Download PDFInfo
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Abstract
本公开涉及使用嵌合抗原受体(CAR)修饰的细胞治疗癌症的组合物,方法和试剂盒的组合物和方法。本公开的一些实施方案涉及编码CAR的分离的核酸序列。CAR可包括抗原结合结构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合非必需器官的抗原。
Description
本申请是名称为:嵌合抗原受体修饰细胞治疗癌症的应用,申请号为:2017800222742, 申请日为:2017年03月30日的专利申请的分案申请。
交叉引用相关专利申请
本申请要求优先权美国2016年4月1日提交的题为“使用嵌合抗原受体修饰细胞治疗癌 症”的临时专利申请,其编号为62/317,261,其全部内容通过引用并入本文。
技术领域
本公开涉及修饰的细胞和用途,特别是涉及使用嵌合抗原受体(CAR)修饰的细胞治疗 癌症的组合物和方法。
背景技术
癌症被称为涉及异常细胞生长的恶性肿瘤,可能侵入或扩散到身体的其他部位。在人体 内,有超过一百种癌症,例如发生在乳腺上皮组织中的乳腺癌。由于乳腺癌细胞失去正常细 胞的特征,乳腺癌细胞之间的联系是松散的。一旦癌细胞脱落,这些脱落的癌细胞就会通过血液或淋巴系统扩散到身体上,从而危及生命。目前,乳腺癌已成为威胁女性身心健康的常 见疾病之一。临床实验已经证明免疫疗法(例如,CAR T)对于治疗癌症是有效的。但是需 要改进免疫疗法以加强对某些癌症如实体瘤的疗效。
发明内容
1.技术方案
为了解决上述问题,本发明所采用的技术方案如下:
本公开的实施方案涉及使用嵌合抗原受体(CAR)修饰的细胞治疗癌症的组合物,方法 和试剂盒。
本公开的一些实施方案涉及编码CAR的分离的核酸序列。CAR可包括抗原结合结构域, 跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合非必需 器官的抗原。例如,抗原结合结构域结合在肿瘤微环境中存在的非必需器官细胞表面上表达 的抗原。
本公开的一些实施方案涉及分离的CAR,包括抗原结合结构域,跨膜结构域,共刺激信 号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合非必需器官的抗原。
本公开的一些实施方案涉及包含人T细胞的药物组合物。人T细胞可包括编码CAR的 核酸序列。CAR可包括抗原结合结构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合非必需器官的抗原。
本公开的一些实施方案涉及包含编码CAR的核酸序列的细胞。CAR可包括抗原结合结 构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域结合非必 需器官的抗原。例如,细胞选自T细胞,自然杀伤(NK)细胞,细胞毒性T淋巴细胞(CTL)和调节性T细胞。
本公开的一些实施方案涉及包含编码CAR的核酸序列的载体。CAR可包括抗原结合结 构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合 非必需器官的抗原。
本公开的一些实施方案涉及通过T细胞介导以刺激对受试者的非必需器官中的细胞群的 免疫应答的方法。该方法可包括向受试者施用有效量的经遗传修饰以表达CAR的细胞。CAR 可包括抗原结合结构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。选择抗原结合结构域以特异性识别非必需器官的细胞群。
本公开的一些实施方案涉及治疗患有癌症的受试者的方法。该方法可以包括向受试者施 用经遗传工程改造以表达CAR的细胞。CAR可包括抗原结合结构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合非必需器官的抗原。例如, 细胞选自T细胞,自然杀伤(NK)细胞,细胞毒性T淋巴细胞(CTL)和调节性T细胞。
在一些实施方案中,抗原结合结构域是抗体、其抗原结合片段或其配体。例如,抗原结 合片段是Fab或scFv。
在一些实施方案中,抗原在存在于肿瘤微环境中的非必需器官细胞上表达。
在一些实施方案中,肿瘤是乳腺癌。在某些实施方案中,抗原是乳腺抗原。例如,乳腺 抗原是具有SEQ ID NO:29的催乳素受体(PRLR)。在一些实施方案中,抗原结合结构域是具有SEQ ID NO:20或44的催乳素受体配体。
在一些实施方案中,肿瘤是结肠直肠癌。在某些实施方案中,抗原是结肠抗原。例如, 结肠抗原是具有SEQ ID NO:33的鸟苷酸环化酶2C(GUCY2C)。
在一些实施方案中,肿瘤是胃癌。在某些实施方案中,抗原是胃腺抗原。例如,胃腺抗 原是具有SEQ ID NO:31的粘蛋白17(Muc17)。
在一些实施方案中,肿瘤是膀胱癌。在某些实施方案中,抗原是膀胱抗原。例如,膀胱 抗原是具有SEQ ID NO:35的CD207。
在一些实施方案中,肿瘤是卵巢肿瘤。在某些实施方案中,抗原是卵巢抗原。例如,卵 巢抗原是具有SEQ ID NO:25的卷曲蛋白家族受体10(FZD10)。
在一些实施方案中,肿瘤是甲状腺肿瘤。在某些实施方案中,抗原是甲状腺抗原。例如, 甲状腺抗原是具有SEQ ID NO:27的甲状腺刺激激素受体(TSHR)。
在一些实施方案中,共刺激信号传导区可包括选自CD27,CD28,4-IBB,OX40,CD30,CD40,PD-1,ICOS,淋巴细胞功能相关抗原-1(LFA-1)的共刺激分子的细胞内结构域,CD2,CD7,LIGHT,NKG2C,B7-H3,与CD83特异性结合的配体,及其任意组合。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:2-20或44中的至少一种。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:3或4,或其组合,并且肿瘤是 卵巢肿瘤。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:6或7,或其组合,并且肿瘤是 甲状腺肿瘤。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:9或10,或其组合,并且肿瘤 是乳腺癌。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:12或13,或其组合,并且肿瘤 是胃癌。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:15或16,或其组合,并且肿瘤 是结肠直肠癌。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:18或19,或其组合,并且肿瘤 是膀胱癌。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:20或44,并且肿瘤是乳腺癌。
本公开的一些实施方案涉及选择CAR的抗原结合结构域用于治疗患有肿瘤细胞的受试 者的方法。该方法可包括确定来自肿瘤细胞的细胞器官,确定器官是相对于受试者的非必需 器官,搜索数据库以识别在器官的细胞群中表达的多种标志物,基于预定条件在多个标记物 中选择一种标记物,并使用来自受试者的细胞产生表达CAR的细胞。CAR可以包括抗原结合结构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域,并且抗原结合结构域 可以与标记物结合。
在一些实施方案中,预定条件可以包括标记物存在于细胞的细胞表面上,其中肿瘤细胞 中标记物表达至少比合适的对照细胞高1.5倍,2倍,5倍,10倍,100倍或1000倍中的至 少一种,并且器官是非必需的,使得器官的损伤不会导致受试者死亡。
在一些实施方案中,非必需器官是乳腺,并且标记物是PRLR。
在一些实施方案中,非必需器官是结肠,并且标记物是GUCY2C。
在一些实施方案中,非必需器官是胃腺,并且标志物是Muc17。
在一些实施方案中,非必需器官是膀胱,并且标志物是CD207。
在一些实施方案中,非必需器官是卵巢,并且标记物是FZD10。
在一些实施方案中,非必需器官是甲状腺,并且标记物是TSHR。
在一些实施方案中,肿瘤选自乳腺癌,甲状腺肿瘤,结肠直肠癌,卵巢肿瘤,膀胱癌和 膀胱癌。
本公开的一些实施方案涉及修饰的细胞,其包含编码具有SEQ ID NO:36-43之一的CAR 的核酸序列。例如,细胞选自T细胞,自然杀伤(NK)细胞,细胞毒性T淋巴细胞(CTL)和调节性T细胞。
在一些实施方案中,CAR具有SEQ ID NO:38,并且CAR的抗原结合结构域与具有SEQID NO:20或44的催乳素受体配体结合。
在一些实施方案中,CAR具有SEQ ID NO:40,并且CAR的抗原结合结构域结合具有SEQ ID NO:33的GUCY2C。
在一些实施方案中,CAR具有SEQ ID NO:39,并且CAR的抗原结合结构域结合具有SEQ ID NO:31的Muc17。
在一些实施方案中,CAR具有SEQ ID NO:41,并且CAR的抗原结合结构域结合具有SEQ ID NO:35的CD207。
在一些实施方案中,CAR具有SEQ ID NO:36,并且CAR的抗原结合结构域结合具有SEQ ID NO:25的FZD10。
在一些实施方案中,CAR具有SEQ ID NO:37,并且CAR的抗原结合结构域结合具有SEQ ID NO:27的TSHR。
本概述旨在提供一些概念选择,下面会有详细描写。本概述并不旨在标识所要求保护的 主题的关键特征或必要特征,也不是旨在用于限制所要求保护的主题的范围。
附图说明
图1说明了非必需器官的多种肿瘤和抗原及其相应的基因。
图2显示过表达抗原的K562细胞系的构建,并包括显示已建立的靶肿瘤细胞系的图片。
图3包括说明CAR的构造的示意图。
图4显示了证明抗CD207的CAR T细胞系被建立的测定结果。
图5显示了证明抗Muc17的CAR T细胞系被建立的测定结果。
图6显示了证明抗TSHR的CAR T细胞系被建立的测定结果。
图7显示了证明抗PRLR的CAR T细胞系被建立的测定结果。
图8显示了证明抗FZD10的CAR T细胞系被建立的测定结果。
图9显示了证明抗GUCY2C的CAR T细胞系被建立的测定结果。
图10显示共培养测定结果,证明抗FZD10的CAR T细胞能够重新定殖特定肿瘤细胞并 相应地释放IFN-γ。
图11显示共培养测定结果,证明抗GUCY2C的CAR T细胞能够重新定殖特异性肿瘤细 胞并相应地释放IFN-γ。
图12显示共培养测定结果,证明抗Muc17的CAR T细胞能够重新定殖特定肿瘤细胞并 相应地释放IFN-γ。
图13显示共培养测定结果,证明抗CD207的CAR T细胞能够重新定殖特定肿瘤细胞并 相应地释放IFN-γ。
图14显示共培养测定结果,证明抗TSHR的CAR T细胞能够重新定殖特定肿瘤细胞并 相应地释放IFN-γ。
图15显示共培养测定结果,证明抗PRLR的CAR T细胞和表达PRLR配体的CAR T细胞能够重新定殖特定肿瘤细胞并相应地释放IFN-γ。
图16显示抗THSR的CAR T细胞和TSHR-K562细胞的共培养的杀伤测定结果。
图17显示抗PRLR的CAR T细胞和PRLR-K562细胞的共培养的杀伤测定结果。
图18显示表达PRLR配体的CAR T细胞和PRLR-K562细胞的共培养的杀伤测定结果。配体1表示人野生型催乳素受体配体(SEQ ID NO:20),配体2表示经修饰的人催乳素受体配体(SEQ ID NO:44)。
图19是从表达TSHR和荧光蛋白(RFP)的3t3细胞观察到的荧光信号的图像。
图20显示抗TSHR的CAR T细胞和TSHR-3T3细胞的共培养的杀伤测定结果。
图21是从表达PRLR和荧光蛋白(RFP)的3t3细胞观察到的荧光信号的图像。
图22显示抗PRLR的CAR T细胞和PRLR-3T3细胞的共培养的杀伤测定结果。
图23显示表达PRLR配体的CAR T细胞和PRLR-3T3细胞的共培养的杀伤测定结果。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
本公开涉及用于治疗癌症以及其他疾病的组合物和方法。本公开的实施方案包括构建 CAR,所述CAR包括结合对应于靶基因的抗原的抗原结合结构域。靶基因在某些组织(例如, 一组细胞或器官)中特异性表达,或者在某些组织中表达比在其他组织中表达的更多。在一 些实施方案中,CAR在经修饰的细胞(例如,T细胞或NK细胞)中表达,其被施用于具有源自该组织细胞的肿瘤的受试者。因为抗原在肿瘤中表达,所以修饰的细胞可以识别并随后 杀死这些肿瘤细胞。经修饰的细胞能够在体内复制和扩增,因此能够长期存在于受试者体内, 并且持续地控制肿瘤。在这些情况下,表达抗原的非肿瘤细胞也可被转移的CART/NK细胞 杀死或损坏。然而,由于组织选自受试者的非必需组织,因此组织内正常细胞的死亡不会对受试者造成危及生命的事件(例如,并发症)。非必需组织包括前列腺,乳腺或含有黑素细胞 的器官。
在一些实施方案中,选择用于CAR的抗原主要在癌细胞及其主要器官(例如,非必需器 官)中表达。在某些实施方案中,在使用CAR T细胞治疗之前,可以从患有源自非必需器官 的晚期癌症的受试者中除去非必需器官。例如,IV期甲状腺癌的治疗通常是包括手术和放射 性碘治疗的治疗技术的组合。在这些情况下,整个甲状腺被移除,该过程被称为全甲状腺切 除术。由于选择的抗原主要在甲状腺正常细胞和甲状腺癌细胞中表达,因此CART细胞攻击 正常细胞的风险降低。
通常,乳腺癌的治疗包括局部治疗和全身治疗。局部治疗包括乳房切除术和放射治疗, 这给患者带来了极大的痛苦,并且只能用于早期乳腺癌的治疗。全身治疗包括化学疗法,内 分泌疗法和靶向分子疗法。化疗会使患者痛苦不堪,并且疗效很差。内分泌治疗主要适用于 绝经后妇女。分子靶向治疗是近年来最活跃的研究领域之一,但是其效率相对较低。例如, 典型的HER2单克隆抗体赫赛汀单药治疗乳腺癌的复发率为15%至30%。
卵巢癌是导致切割女性生殖器官的常见恶性肿瘤之一。在卵巢癌中,上皮癌和恶性生殖 细胞肿瘤是常见的。大多数上皮性卵巢肿瘤会扩散到子宫,双侧附件,网膜和盆腔器官,对 女性的生命造成严重威胁。不同病理类型的卵巢癌有不同的治疗选择。结合手术和化疗的疗法可用于治疗卵巢癌。对于早期卵巢癌,可以进行手术,包括综合手术和保留生育手术。由 于卵巢胚胎发育,组织解剖和内分泌功能的复杂性,早期症状不典型;因此,良性、恶性以及 组织类型的诊断是相当困难的。在盆腔肿瘤转移的情况下,尤其是晚期患者的恶性生殖细胞 肿瘤,大多数患者不能切除病变。在这些情况下,放射和化疗只能用作辅助治疗,其效果有限。
子宫内膜癌发生在一组子宫内膜肿瘤中,也称为子宫内膜癌。起源于子宫内膜腺的肿瘤 称为子宫内膜腺癌。这些癌细胞主要通过淋巴转移直接传播,而对于晚期癌细胞,癌细胞可 能通过血液转移扩散到肺,肝和骨。治疗计划可以基于临床阶段,肿瘤分化水平和患者的一般状况来决定。通常,治疗包括手术治疗以及放射疗法,激素和化学疗法。保守性手术可能 会导致复发,而根治性切除可能会导致更年期过早的症状。放射和化疗只能用作辅助治疗, 其效果有限。通过使用黄体酮药物来进行孕激素治疗以控制癌症的发展是有效的,但剂量很 大,并且长期孕激素治疗可能会损害肝功能。对于晚期肿瘤转移的患者,没有有效的治疗方法。
宫颈癌是女性生殖器恶性肿瘤的最常见形式,包括原位癌和浸润性癌。宫颈癌局限于皮 质内的粘膜,无浸润时称为原位癌。当癌症间质入侵在粘膜下时,它被称为侵袭性癌症。宫 颈癌可导致女性不孕。由于晚期癌症的侵袭和转移,症状可能也出现在身体的其他相应部位。通常,治疗计划由临床阶段、肿瘤分化水平和患者的一般状况来决定。通常,治疗包括手术 治疗,放射疗法和化学疗法作为辅助疗法。手术治疗包括根治性子宫切除术和盆腔淋巴结手 术消除,并有复发风险。放射和化疗只能用作辅助治疗,其效果有限。对于晚期肿瘤转移的 患者,目前尚无有效的治疗方法。
甲状腺癌是最常见的甲状腺恶性肿瘤。有四种常见的甲状腺癌类型:乳头状(例如,混 合乳头状滤泡状癌),滤泡状,髓质(例如甲状腺淀粉样变性实体)和未分化癌。通常,乳头 状和滤泡状癌的治疗相对有效,而髓样癌通常沿淋巴和血液路径转移,而未分化癌则较少见。 晚期甲状腺癌可能有声音嘶哑,呼吸困难,吞咽困难,以及颈丛神经受压引起的交感神经受 压,发生耳,枕,肩痛等症状,以及导致局部淋巴结和远处转移等。甲状腺癌的预后和治疗水平需要得到改善。目前甲状腺癌的治疗方式包括手术,术后放射性核素治疗和手术后内分 泌治疗,其中手术治疗是首选。在选择用于治疗分化型甲状腺癌的手术方法中,术后放射性 碘治疗和TSH(促甲状腺激素,TSH)抑制治疗等的许多方面都存在争议。此外,没有统一和标准化的治疗指南,可导致治疗不完整或过度。对于晚期分化型甲状腺癌,没有有效的治 疗方法。
结直肠癌是一种常见的胃肠癌,对生命和健康构成严重威胁。结肠直肠癌的预后和治疗 水平需要得到改善。通常,结肠直肠癌的临床治疗计划包括局部切除,化学疗法和生物疗法。 使用局部切除转移性病变时,生物学疾病的标准(例如:同步和各向异性)很重要但很难评 估。例如,75%的患者在肝转移切除后会复发。大多数复发发生在肝脏中,其功效有限。常 用的化疗方案包括FOLFOX,FOLFIRI,CapeOX和FOLFOXIRI(2B)等,但结合FOLFOX方案,7%-8%的患者可延长3年PFS,而总生存率的提高不显着。最近的实验数据表明,对于可能转移的可切除病变的患者,在FOLFOX中加入西妥昔单抗的疗法是有害的。根据目前的研究数据,结合生物制剂(如贝伐单抗,西妥昔单抗和帕尼单抗)的治疗是被推荐使用的。
然而,单克隆抗体药物通常具有诸如功效和持久性的问题。患者需要长期、重复的药物 使用。同时,由于联合治疗方法一般比单药治疗更强;因此,其他类型的药物是否会影响治疗 和预后仍是一个问题。
胃癌(胃癌)是起源于胃上皮细胞恶性肿瘤,是最常见的胃肠道恶性肿瘤之一。疼痛和 体重减轻是晚期胃癌最常见的临床症状。胃癌细胞的增殖和转移途径包括淋巴转移,直接侵 袭,血行转移和腹膜转移。胃癌的预后和治疗水平需要得到改善。通常,胃癌治疗模式包括 手术,化学疗法,放射疗法,免疫疗法等。但是大多数晚期胃癌病例都有不可切除的原发性或转移性病变。肿瘤转移和胃癌复发是预后不良的主要原因
食道癌(esophageal cancer,EC)起源于食管粘膜上皮恶性肿瘤,是一种常见的临床恶性 肿瘤。肿瘤转移和食管癌复发是预后不良的主要原因。基于发病率和死亡率,食管癌在恶性 肿瘤中名列前十。食道癌的预后和治疗水平需要得到改善。目前食道癌的治疗方式是手术联 合治疗。然而食管癌的分期、手术治疗模式选择、手术方式选择、淋巴结切除术方法、术后辅助治疗等都存在问题。
除非另有定义,本文使用的所有技术和科学术语具有如常见到的公开内容所属领域的普 通技术人员所理解的相同的含义。虽然在本公开的实验和测试中可以使用和本文描述的方法 和材料类似和等同的任何方法和材料,但是描述了优选的方法和材料。对于本公开的目的,下列术语定义如下。
本文使用冠词“一个(a)”和“一个(an)”,指的是该冠词语法对象的一个或多于一个(即,指 的是至少一个)。以例子说明,“一个元件”表示一个元件或多于一个的元件。
“约”是指数量,水平,值,数,频率,百分比,维数,大小,量,重量或长度变化多达30,25,20,15,10,9,8,7,6,5,4,3,2或1%至参考数量,水平,值,数,频率,百 分比,维数,大小,量,重量或长度。
如本文所用,术语“活化”是指已经充分刺激以诱导可检测的细胞增殖的细胞的状态。活 化还可以与诱导的细胞因子的产生和可检测的效应子功能相关。术语“活化的T细胞”特别指 经历细胞分裂的T细胞。
术语“抗体”以最广义使用,明确覆盖单克隆抗体(包括全长单克隆抗体),多特异性抗体 (例如,双特异性抗体),及抗体片段,只要它们展现出期望的生物学活性或功能。本发明中 的抗体可以以多种形式存在,包括例如,多克隆抗体、单克隆抗体、Fv,Fab和F(ab)2,以及 单链抗体和人源化抗体(Harlow等,1999,In:Using Antibodies:A LaboratoryManual,Cold Spring HarborLaboratory Press,NY;Harlow等,1989,In:Antibodies:ALaboratory Manual,Cold Spring Harbor,New York;Houston等,1988,Proc.Natl.Acad.Sci.USA 85:5879-5883;Bird等人, 1988,Science 242:423-426)。
术语“抗体片段”指的是完整抗体的一部分,通常是抗体的抗原结合区或可变区。抗体片 段的例子包括但不限于Fab,Fab’,F(ab’)2和Fv片段,双功能抗体、线性抗体、单链抗体分子和由抗体片段形成的多特异性抗体。
“Fv”是包含完整抗原识别和结合位点的最小抗体片段。此片段由紧密非共价结合的一个 重链和一个轻链可变区结构域的二聚体组成。从这两个结构域的折叠产生六个超变环(每个 来自H和L链的3个环),其贡献氨基酸残基用于抗原结合并赋予抗体结合抗原的特异性。 但是,即使(Fv的或半部分,包括只有三个对抗原特异的互补性决定区(CDR))一个单可 变结构域有能力识别和结合抗原,其亲和力依然比完整的结合位点低。“抗体重链”,如本文 所用的,指的是以它们自然发生构象存在于所有抗体分子的两种类型的多肽链中较大的链。 “抗体轻链”,如本文所用的,指的是以它们自然发生构象存在于所有抗体分子的两种类型的多肽链中较小的链,κ和λ轻链指的是两个主要的抗体轻链同种型。
如本文所用的术语“合成抗体”,指利用重组DNA技术产生的抗体,例如,本文所述的噬 菌体表达的抗体。该术语也应当被解释为由DNA分子编码产生的抗体,并且该DNA分子表 达抗体蛋白或规定抗体的氨基酸序列,其中DNA或氨基酸序列是利用本领域可用和公知的 合成DNA或氨基酸序列技术获得。
如本文所用的术语“抗原”被定义为激发免疫应答的分子,该免疫应答可涉及抗体产生, 或特异性免疫活性细胞的活化,或两者皆有。抗原可以包括任何大分子包括所有的蛋白质或 肽,此外,抗原可源自重组或基因组DNA的分子。例如,DNA包括能够编码引起免疫应答 的蛋白质的核苷酸序列或部分核苷酸序列,因此能够编码如本文使用的术语“抗原”。本领域 技术人员将理解抗原不必单独地由基因的全长核苷酸序列编码。抗原可被产生、合成或可源自生物学样本。这种生物学样本可包括但不限于组织样本、肿瘤样本、细胞或生物学流体。
如本文所用的术语“抗肿瘤效应”,指的是生物学效应,其可由肿瘤体积的减少、肿瘤细 胞数的减少、转移数的减少、预期寿命的增加或与癌性病症相关的各种生理症状的改善表示。 “抗肿瘤效应”也可由本发明的肽、多核苷酸、细胞和抗体在预防肿瘤在第一位置发生的能力表示。
术语“自体抗原”指由免疫系统错误识别为外源(foreign)的任何自身抗原。自体抗原包括但 不限于细胞蛋白、磷蛋白、细胞表面蛋白、细胞脂质、核酸、糖蛋白以及细胞表面受体。
术语“自体”指关于源自相同个体的任何物质,它随后被再次引入该个体。
“同种异基因的(allogeneic)”指的是源自相同物种的不同动物的移植物。
“异种的(xenogeneic)”指的是源自不同物种的动物的移植物。
如本文所用的术语“癌症”被定义为以畸变细胞的快速和失控生长为特征的疾病。癌症细 胞可局部蔓延或通过血流和淋巴系统蔓延至身体的其他部分。各种癌症的例子包括但不限于 乳腺癌、前列腺癌、卵巢癌、子宫颈癌、皮肤癌、胰腺癌、结肠直肠癌、肾癌、肝癌、脑癌、淋巴瘤、白血病、肺癌等等。
在整个本说明书中,除非上下文另有要求,词语“包括”,将被理解为步骤或元件以及步 骤原件组的包含,但不排除任何其他步骤或元件或步骤元件组。
由“由...组成”是指包括,并且限于任何随后的短语。因此,“由......组成”一词表示所列元 素是需要的或强制性的,并且没有其它元素存在。
由“基本上由...组成”是指包括短语后列出的任何要素,并且限于其他不干扰或有助于本公 开的所列元素中指定的活性或作用的元素。因此,“基本组成”一语表示所列元素是需要的或 强制性的,但是其它元素是可选的,并且取决于它们是否影响所列元素的作用。
术语“互补”是与多核苷酸(即核苷酸的序列)通过碱基配对规则相关的。例如,序列“AGT” 是该序列互补的“TCA”。互补性可以是“部分的”,其中仅一些核酸'碱基根据碱基配对规则相 匹配。或者,核酸之间可能“完全”或“总的”互补。核酸链之间的互补性的程度显著影响核酸 链之间杂交的效率和强度。
“相应于”或“对应于”是指(a)具有参考多核苷酸的相同或互补的序列的全部或一部分的 氨基酸序列,或者具有能编码相同核苷酸序列的多核苷酸肽或蛋白的氨基酸序列;或(b)与 参考多肽或参考蛋白质的氨基酸序列基本上相同的多肽。
本文使用的术语“共刺激配体”包括能够特异性结合T细胞上的关联(cognate)共刺激分子 的抗原呈递细胞(例如,aAPC、树突细胞、B细胞等等)上的分子,由此除了提供例如将TCR/CD3 复合物与用肽负载的MHC分子结合的初级信号之外,还提供介导T细胞应答的信号,所述T 细胞应答包括但不限于增殖、活化、分化等等。共刺激配体可包括但不限于CD7、B7-1 (CD80)、B7-2(CD86)、PD-L1、PD-L2、4-1BBL、OX40L、可诱导的共刺激配体(ICOS-L)、 细胞间粘附分子(ICAM)、CD30L、CD40、CD70、CD83、HLA-G、MICA、MICB、HVEM、 淋巴毒素β受体、3/TR6、ILT3、ILT4、HVEM、结合Toll配体受体的激动剂或抗体和与B7-H3 特异性结合的配体。共刺激配体也包括与存在于T细胞上的共刺激分子特异性结合的抗体,诸如但不限于CD27、CD28、4-1BB、0X40、CD30、CD40、PD_1、IC0S、淋巴细胞功能相 关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3和与CD83特异性结合的配体。
“共刺激分子”指的是与共刺激配体特异性结合的T细胞上的关联结合伴侣,由此介导T 细胞的共刺激应答,比如增殖。共刺激分子包括但不限于MHCI类分子、BTLA和Toll样配 体受体。
“共刺激信号”指的是与初级信号结合,如TCR/CD3的结合,导致T细胞增殖和/或关键 分子的上调或下调的信号。
如本文中所使用的,术语“疾病”和“病症”可以互换使用,或者可以不同,特定弊病或病 症可以不具有已知的致病剂(病因尚未制定出),因此它尚未确认为疾病而是仅仅作为不受欢迎的病症或综合征,其特定的症状由临床医师确定。“疾病”是受试者的一种健康状态,其中 受试者不能保持稳态,如果不改善该疾病,则受试者的健康继续恶化。与之相比,受试者中 的“紊乱”是受试者能够保持稳态的一种健康状态,但其中受试者的健康状态与它没有处于该 紊乱相比不太有利。在不治疗的情况下,紊乱不一定引起动物健康状态的进一步降低。
如本文所用,术语“有效”是指足以实现所期望的,预期的或意图的结果。如本文所用的“有效量”,指能够提供治疗性或预防性益处的量。
“编码”指的是在生物学过程中,将多核苷酸诸如基因、cDNA或mRNA中核苷酸的特异 性序列用作模板合成其他多聚体和大分子的固有性质,所述多聚体和大分子具有核苷酸(即, rRNA、tRNA和mRNA)的限定序列或氨基酸的限定序列中的任一个和由其产生的生物学性 质。因此,如果相应于那个基因的mRNA的转录和翻译在细胞或其他生物学系统中产生蛋白 质,则基因编码蛋白质。核苷酸序列等同mRNA序列并通常提供在序列表中,对于非编码链, 其被用作基因或cDNA的转录模板,可被称为编码那个基因或cDNA的蛋白质或其他产物。
关于多核苷酸,“外源”指的是不自然发生在野生型细胞或生物的多核苷酸序列,但是通 常是通过分子生物学技术引入到细胞。外源多核苷酸的实例包括载体,质粒,和/或编码所需 蛋白质的人造核酸构建体。对于多核苷酸,术语“内源性的”或“天然”是指天然存在的,可能会在给定的野生型细胞或生物中找到的多核苷酸序列。同样,从第一有机体分离和利用分子 生物学技术转移到第二有机体的特定多核苷酸序列通常被认为相对于所述第二机体的“外源” 多核苷酸。在具体的实施方案中,多核苷酸序列可以被通过分子生物学技术“引入”到已包含 这样的多核苷酸序列,例如微生物,以创建另外的天然存在的多核苷酸序列的一个或多个附加拷贝,并且由此促进所编码的多肽的过表达。
术语“表达”被定义为由它的启动子驱动的特定核苷酸序列的转录和/或翻译。
“表达载体”指的是包括重组多核苷酸的载体,所述重组多核苷酸包括可操作地连接至待 表达的核苷酸序列的表达控制序列。表达载体包括足够的用于表达的顺式作用元件;用于表 达的其他元件可由宿主细胞供应或在体外表达系统中供应。表达载体包括所有本领域已知的那些,诸如并入重组多核苷酸的粘粒、质粒(例如,裸露或包含在脂质体中)和病毒(例如,慢 病毒、逆转录病毒、腺病毒和腺伴随病毒)。
“同源的”指的是两个多肽之间或两个核酸分子之间的序列相似性或序列同一性。当两个 比较序列中的位置被相同的碱基或氨基酸单体亚单元占据时,例如,如果两个DNA分子的 某一个位置被腺嘌呤占据,则所述分子在那个位置上是同源的。两个序列之间的同源性百分比为由两个序列共有的匹配或同源的位置数除以比较的位置数X lOO的函数。例如,如果两个 序列中10个位置中的6个是匹配或同源的,则两个序列是60%同源的。以例子说明,DNA 序列ATTGCC和TATGGC享有50%的同源性。通常在两个序列有最大同源性时进行比较。
如本文所用的术语“免疫球蛋白”或“Ig”被定义为起到抗体作用的一类蛋白质。该类蛋白 质中的五个成员为IgA、IgG、IgM、IgD和IgE。IgA为存在于身体分泌物诸如唾液、泪液、 母乳、胃肠分泌物和呼吸道和泌尿生殖道的粘液分泌物中的初级抗体。IgG是最常见的循环 抗体。IgM是在多数对象的初级免疫应答中产生的主要免疫球蛋白,它在凝集反应、补体结合和其他抗体应答中是最有效的免疫球蛋白,并且在抵御细菌和病毒方面是很重要的。IgD 是不具有已知抗体功能的免疫球蛋白,但可用作抗原受体。IgE是在暴露于过敏原后,通过 引起从肥大细胞和嗜碱性粒细胞释放介体,介导速发过敏性的免疫球蛋白。
“分离的”指从自然状态改变或移出。例如,天然存在于活动物中的核酸或肽不是“分离 的”,但部分或完全与它的自然状态的共存物质分离的同一核酸或肽是“分离的”。分离的核酸 或蛋白可以以基本上纯化的形式存在,或例如,可存在于非自然环境中,诸如宿主细胞。
本发明的内容中,对于核酸碱基使用以下缩写。“A”指的是腺苷,“C”指的是胞嘧啶,“G” 指的是鸟苷,“T”指的是胸苷,和“U”指的是尿苷。
除非另有规定,“编码氨基酸序列的核苷酸序列”包括为彼此简并版本并编码相同的氨基 酸序列的所有的核苷酸序列。短语编码蛋白质或RNA的核苷酸序列也可包括内含子,其程度 为编码该蛋白质的核苷酸序列可在某些版本中包含内含子(一个或多个)。
如本文所用的“慢病毒”指的是逆转录病毒科的属。在逆转录病毒中慢病毒是唯一能够感 染非分裂细胞的;它们可传递显著量的遗传信息进入宿主细胞的DNA,因此它们是传递基因 的最有效的载体之一。HIV、SIV和FIV是所有慢病毒的例子。源自慢病毒的载体提供了在体内完成显著水平的基因转移的工具。
如本文所用的术语“调节”指与缺少治疗或化合物的对象中的应答水平相比,和/或与以其 他方式相同但未治疗的对象中的应答水平相比,介导对象中应答水平的可检测的增加或减少。 该术语包括扰乱和/或影响天然信号或应答,从而在受试者(优选人)中,介导有益的治疗性 应答。
术语“可操作地连接”指的是调节序列和异源核酸序列之间的功能连接。例如,前序列或 分泌前导的DNA是可操作地连接的DNA,如果它被表达为参与该多肽的一个前蛋白的分泌;启动子或增强子能可操作地连接于编码序列,如果它影响序列的转录;核糖体结合位点能可 操作地连接到一个编码序列,以便促进翻译。通常地,“可操作地连接”意指连接的DNA序列 是邻近的,其中在相同的阅读框中必须连接两个蛋白编码区。然而,增强子不必是连续的。 连接是通过方便的限制性位点来完成的。如果不存在此类位点,合成的寡核苷酸衔接头或接 头根据常规方法来使用。
术语“过度表达的”肿瘤抗原或肿瘤抗原的“过度表达”旨在表示来自疾病区域的细胞中肿 瘤抗原的异常表达水平,例如,患者的一个特定的组织或器官,如实体瘤,相对于该组织或 器官中正常细胞中的表达水平。具有实体瘤或恶性血液疾病的患者的具有肿瘤抗原的过量表达的特征,该表达可以通过本领域中已知的标准测定法来确定。
“肠胃外”免疫原性组合物的给药包括,例如,皮下(sc),静脉内(IV),肌内(IM),或胸骨内注射,或输注技术。
术语“患者”、“对象”、“个体”等等在本文中可交换使用,并指的是服从本文描述方法的 任何动物或其细胞,不论是体外或原位。在某些非限制性实施方案中,患者,对象或个体是 人。在一些实施方案中,术语“对象”包括免疫应答可引起活的生物体(例如,哺乳动物)。受 试者的实例包括人,狗,猫,小鼠,大鼠和转基因种物。
本文所用的叙述“多核苷酸”或“核酸”指的是mRNA,RNA,cRNA,rRNA,cDNA或DNA。该术语通常是指长度为至少10个碱基的核苷酸,或者是核糖核苷酸或脱氧核苷酸,或者是任 一类型核苷酸的修饰形式。该术语包括DNA和RNA的单链和双链形式。
术语“多核苷酸变体”和“变体”等是指与参考多核苷酸序列显示出实质序列同一性的多核 苷酸或在下文定义的严格条件下与参考序列杂交的多核苷酸。这些术语还包括通过添加,缺 失或取代一个氨基酸而与参考多核苷酸不同的多核苷酸。因此,术语“多核苷酸变体”和“变体”包括其中一个或多个核苷酸被添加或删除,或具有不同的核苷酸取代的多核苷酸。在这方面, 本领域技术人员可以理解本领域包容的突变,添加,缺失和置换某些改变可以对参照多核苷 酸,由此改变的多核苷酸保留了参考多核苷酸的生物学功能或活性进行中,或增加活性相对 于所述参考多核苷酸(即,优化)。多核苷酸变体包括,例如,具有至少50%(和至少51% 至至少99%,并在之间的所有整数百分比,例如,90%,95%或98%)序列同一性的多核苷 酸与参考多核苷酸序列本文中所描述。术语“多核苷酸变体”和“变体”还包括天然存在的等位 基因变体和编码这些酶的直系同源物。
术语“多肽”、“多肽片段”,“肽”和“蛋白质”可交换使用,并指的是由肽键共价连接的氨基 酸残基组成的化合物。因此,这些术语适用于氨基酸,其中一个或多个氨基酸残基是合成的 非天然存在的氨基酸,诸如相应的天然存在的氨基酸的化学类似物,以及适用于天然存在的氨基酸的聚合物酸的聚合物。在某些方面,多肽可以包括酶多肽,或“酶”,其典型地催化(即, 增加的速率)不同的化学反应。
多肽“变体”指的通过添加,缺失或置换至少一个氨基酸残基而区别于参考多肽序列的多 肽。在某些实施方案中,多肽变体是从参考多肽的一个或多个取代:可以是保守的或非保守 区分。在某些实施方案中,所述多肽变体包含保守性取代部分,并在这方面,它是本领域的一些氨基酸可以改变为其他具有广泛相似性质的,而不改变多肽活性的性质。多肽变体还包 括其中被添加或删除的一个或多个氨基酸,或用不同的氨基酸残基取代的多肽。
如本文所用的术语“启动子”定义为通过在细胞的合成机制,或导入的合成机制识别的 DNA序列,启动一个多核苷酸序列的特异性转录所需要。表达“控制序列”是指用于在特定宿 主生物体中表达可操作连接的编码序列所必需的DNA序列。控制序列其适于原核生物,例 如,包括启动子,任选的操纵基因序列,和核糖体结合位点。真核细胞利用启动子,多腺苷 酸化信号,和增强子。
“结合”,“绑定”,或“相互作用”是识别并附着在样品或生物体中特定第二分子的分子, 但基本上不识别或粘附到样品中的其它结构上无关的分子。关于抗体的术语“特异性结合”指 识别特异性抗原但基本上不识别或结合样本中的其他分子的抗体。例如,特异性结合来自一个物种的抗原的抗体也可结合来自一个或多个物种的抗原。但是,这种跨种反应性本身不改 变抗体的类别成为特异性的。在另一个实例中,特异性结合抗原的抗体也可结合抗原的不同 等位基因形式。然而,这种交叉反应性本身不改变抗体的类别成为特异性的。在一些例子中, 术语“特异性的结合”或“特异性地结合”可关于抗体、蛋白质或肽与第二化学种类的相互作用使用,用于指该相互作用依赖化学种类上特定结构(例如,抗原决定簇或表位)的存在;例如, 抗体通常识别和结合特异性蛋白结构而不是一般地识别和结合蛋白质。如果抗体对表位“A” 是特异性的,则在包含标记的“A”和抗体的反应中存在包含表位A(或游离的、未标记的A)的 分子,将降低结合至抗体的标记的A的量。
“可溶性受体”是不结合于细胞膜的受体多肽。可溶性受体常常缺乏跨膜和胞质结构域配 体的结合受体多肽。可溶性受体可包括附加的氨基酸残基,诸如提供多肽的纯化或提供多肽 附着到一个衬底上,或免疫球蛋白恒定区序列位点的亲和标签。许多细胞表面受体具有天然存在的由蛋白水解产生的可溶性对应物。可溶性受体多肽被认为是基本上不含或缺乏跨膜和 胞内多肽区段的足够部分以提供膜锚定或信号转导。
“统计学显著”是指该结果是不太可能是偶然发生的。统计显着性可通过本领域已知的任 何方法来确定。常用的显着性度量包括P值,即如果零假设为真,观察到的事件会发生的频 率或概率。如果获得的p值小于显着性水平,则零假设被拒绝。在简单的情况下,显着性水 平是在0.05或更小的p值定义。“下降”或“减少”或“小”量通常是“统计学显著”或生理学显著 量,并且可以包括减少是约1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2,2.5,3,3.5 4, 4.5,5,6,7,8,9,10,15,20,30,40,或50倍或更多倍(例如,100,500,1000倍) (包括介于1和1之间的所有整数和小数点,例如,1.5,1.6,1.7,1.8等)本文中描述的量 或水平。
通过术语“刺激”指通过结合刺激分子(例如,TCR/CD3复合物)与它的关联配体诱导的初 级应答,由此介导信号转导事件,例如通过TCR/CD3复合物的信号转导。诱导的初级应答刺 激可介导某些分子的改变的表达,诸如TGF-β的下调和/或细胞骨架结构的重组等。
“刺激分子”,作为本文使用的术语,指存在于抗原呈递细胞上的关联刺激配体特异性结 合的T细胞上的分子。
所用的“刺激配体”指如此配体,其当存在于抗原呈递细胞(例如,aAPC、树突细胞、B- 细胞等等)上时,可与T细胞上的关联结合配偶体(在本文中被称为“刺激分子”)特异性结合, 由此介导T细胞的初级应答,其包括但不限于,活化、免疫应答的开始、增殖等等。刺激配 体在本领域中是公知的,并包括,特别是利用肽、抗-CD3抗体、超激动剂抗-CD28抗体和超激动剂抗-CD2抗体负载的MHC I类分子。
如本文所用的“基本上纯化的”细胞为基本上不含其他细胞类型的细胞。基本上纯化的细 胞也指的是已经与在其天然发生状态中与其正常相关联的其他细胞类型分离的细胞。在一些 例子中,基本上纯化的细胞群指的是均质细胞群。在其他例子中,该术语简单地指的是已经与在其天然状态中与其正常相关联的细胞分离的细胞。在一些实施方式中,体外培养细胞。 在其他实施方式中,不在体外培养细胞细胞。
如本文所用的术语“治疗性的”表示治疗和/或预防。治疗性效应通过疾病状态的抑制、缓 和或根除获得。
术语“治疗有效量”指的是将引起由研究者、兽医、医学医生或其他临床医生正在寻找的 组织、系统或对象的生物学或医学应答的对象化合物的量。术语“治疗有效量”包括以下的化 合物的量:当给药时,其足以预防治疗的紊乱或疾病的迹象或症状中的一个或多个的发展,或以一定程度减轻治疗的紊乱或疾病的迹象或症状中的一个或多个。治疗有效量将根据化合物、 疾病和其严重性、和待治疗的对象的年龄、重量等而变化。
“治疗”疾病,作为本文使用的术语,指降低对象经历的疾病或紊乱的至少一种迹象或症状的频率或严重性。
如本文所用的术语“转染的”或“转化的”或“转导的”指的是如此过程,通过该过程外源的 核酸被转移或引入宿主细胞。“转染的”或“转化的”或“转导的”细胞是已经由外源核酸转染、 转化或转导的细胞。该细胞包括原代对象细胞和它的子代。
本文所用的短语“转录控制下”或“可操作地连接”指启动子处于与多核苷酸有关的正确的 位置和朝向,以控制通过RNA聚合酶进行的转录的开始和多核苷酸的表达。
“载体”为物质组合物,其包括分离的核酸,并且其可用于传递分离的核酸至细胞内部。 很多载体在本领域中是已知的,包括但不限于线性多核苷酸、与离子或两性分子化合物相关 的多核苷酸、质粒和病毒。因此,术语“载体”包括自主复制的质粒或病毒。该术语也应被解释为包括便于将核酸转移入细胞的非质粒和非病毒化合物,诸如例如聚赖氨酸化合物、脂质 体等等。病毒载体的例子包括但不限于,腺病毒载体、腺伴随病毒载体、逆转录病毒载体等 等例如,慢病毒是复杂的反转录病毒,其中,除了常见的反转录病毒基因gag,pol和ENV, 包含其他基因的调节或结构功能。慢病毒载体是本领域中公知的。慢病毒的一些例子包括人类免疫缺陷病毒:HIV-1,HIV-2和猴免疫缺陷病毒SIV。慢病毒载体可通过减弱HIV毒力基 因产生,例如,所述基因的env,VIF,VPR,VPU和nef被删除使得载体在生物学上是安全 的。
范围:在该公开中,本发明的多个方面可以以范围形式中示出。应当理解范围形式中的描 述仅是为了方便和简洁,并不应被解释为对本发明范围硬性的限制。因此,范围的描述应被 考虑为具有具体公开的所有可能的子范围以及处于那个范围内的单个数值。例如,范围诸如从1至6的描述应被考虑为具有具体公开的子范围诸如从1至3、从1至4、从1至5、从2 至4、从2至6、从3至6等,以及那个范围内的单个数字,例如,1、2、2.7、3、4、5、5.3 和6。不管范围的宽度如何,这一点是适用的。
本发明涉及分离的核酸序列,载体,包括分离的核酸序列,包括分离的核酸序列的细胞 和使用这些细胞治疗癌症的方法。
组合物和治疗中的应用
本公开的一些实施方案涉及编码CAR的分离的核酸序列。CAR可包括抗原结合结构域, 跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合非必需 器官的抗原。例如,抗原结合结构域结合在肿瘤微环境中存在的非必需器官细胞表面上表达 的抗原。
如本文所用,非必需器官是指受试者的器官,并且器官是非必需的,使得器官的损伤不 会导致受试者的死亡。在一些实施方案中,器官损伤不会明显影响受试者的健康。例如,前 列腺可以是雄性哺乳动物的非必需器官,而乳房可以是雌性哺乳动物的非必需器官。
在某些实施方案中,在使用CAR T细胞治疗之前,可以从患有源自非必需器官的晚期癌 症的受试者中除去非必需器官。在这些情况下,治疗靶向非必需器官的抗原的CAR T细胞对 受试者的影响显着降低。非必需器官的实例包括乳腺,结肠,胃腺,卵巢和甲状腺。
本公开的一些实施方案涉及分离的CAR,其包括抗原结合结构域,跨膜结构域,共刺激 信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合非必需器官的抗原。
本公开的一些实施方案涉及包含人T细胞的药物组合物。人T细胞可包括编码CAR的 核酸序列。CAR可包括抗原结合结构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合非必需器官的抗原。
本公开的一些实施方案涉及包含编码CAR的核酸序列的细胞。CAR可包括抗原结合结 构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域结合非必 需器官的抗原。例如,细胞选自T细胞,自然杀伤(NK)细胞,NK-92细胞,细胞毒性T 淋巴细胞(CTL)和调节性T细胞。
本公开的一些实施方案涉及包含编码CAR的核酸序列的载体。CAR可包括抗原结合结 构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合 非必需器官的抗原。
本公开的一些实施方案涉及用于刺激对受试者的非必需器官中的细胞群的T细胞介导的 免疫应答的方法。该方法可包括向受试者施用有效量的经遗传修饰以表达CAR的细胞。CAR 可包括抗原结合结构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。选择抗 原结合结构域以特异性识别非必需器官的细胞群。
在一些实施方案中,可修饰T细胞以破坏与一种或多种蛋白质的生物合成或转运途径相 关的内源基因。一种或多种蛋白质的实例包括程序性细胞死亡蛋白1(PD-1)基因和主要组 织相容性复合物I(MHC I)。例如,T细胞的主要组织相容性复合物可以通过修饰T细胞中 的β-2-微球蛋白(B2M)基因,抗原呈递1(TAP1)基因和TAP相关糖蛋白(TAPBP)基因而被破坏。在某些实施方案中,可以在用编码CAR的核酸序列转移T细胞之前或之后将破 坏引入T细胞。
本公开的一些实施方案涉及治疗患有癌症的受试者的方法。该方法可以包括向受试者施 用经遗传工程改造以表达CAR的细胞。CAR可包括抗原结合结构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域。抗原结合结构域可以结合非必需器官的抗原。例如, 细胞选自T细胞,自然杀伤(NK)细胞,NK-92细胞,细胞毒性T淋巴细胞(CTL)和调节 性T细胞。
CARs分子通常包括细胞外和细胞内结构域。胞外结构域包括靶-特异性结合元件,其另 外被称为抗原结合部分。细胞内结构域(例如,细胞质结构域)或另外的胞浆结构域包括共 刺激信号传导区和ζ链部分。刺激信号传导区指的是包括共刺激分子的细胞内结构域的一部 分CAR。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子,而不是抗原受体 或它们的配体。
在CAR的胞外结构域和跨膜结构域之间,或在CAR的胞浆结构域和跨膜结构域之间, 可并入间隔结构域。如本文所用的,术语“间隔结构域”通常指起到将跨膜结构域连接至多肽 链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。间隔结构域可包括上至300个氨基酸, 优选10至100个氨基酸,最优选25至50个氨基酸。
在一些实施方案中,在本公开内容的CAR的靶特异性结合元件可以识别肿瘤抗原。肿瘤 抗原是由引起免疫应答特别是T-细胞介导的免疫应答的肿瘤细胞产生的蛋白质。肿瘤抗原在 本领域中是公知的,并包括例如神经胶质瘤相关的抗原、癌胚抗原(CEA)、β_人绒毛膜促性 腺素、α-胎蛋白(AFP)、凝集素-反应的AFP、甲状腺球蛋白、RAGE-1,MN-CA,IX、人端粒酶反转录酶、RU1、RU2(AS)、肠羧酸酯酶、mut hsp70-2、M-CSF、前列腺酶、前列腺- 特异性抗原(PSA)、PAP、NY-ES0-l、LAGE-la、p53、蛋白质、PSMA、Her2/neu、存活素和 端粒酶、前列腺-癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、中性白细胞弹性蛋白酶、ephrinB2、 CD22、胰岛素生长因子(IGF)-1、IGF-11、IGF-1受体和间皮素。
在一些实施方案中,肿瘤抗原包括HER2,CD19,CD20,CD22,κ或轻链,CD30,CD33,CD123,CD38,ROR1,ErbB3/4,EGFR,EGFRvIII,EphA2,FAP,癌胚抗原,EGP2,EGP40, 间皮素,TAG72,PSMA,NKG2D配体,B7-H6,IL-13受体α2,IL-11受体α,MUC1,MUC16, CA9,GD2,GD3,HMW-MAA,CD171,路易斯Y,G250/CAIX,HLA-AI MAGE A1, HLA-A2 NY-ESO-1,PSC1,叶酸受体的α,CD44V7/8,8H9,NCAM,VEGF受体,5T4, 胎乙酰胆碱受体,NKG2D配体蛋白,CD44v6,TEM1,TEM8,或病毒相关的抗原由肿瘤表 达。
在一些实施方案中,CAR的结合元件可包括任何抗原结合部分,其在与其同源抗原结合 时影响肿瘤细胞,使得肿瘤细胞不能生长,或促进肿瘤的死亡或减少。
在一些实施方案中,本公开内容的CAR的抗原结合元件靶向非必需器官的抗原。在一些 情况下,本公开的CAR的抗原结合元件包括抗原,包括至少在SEQ ID NO:2-20或44之一 处列出的氨基酸序列。
在一些实施方案中,内部核糖体进入位点(IRES)元件被用于创建多基因,或多顺反子, 或信息。例如,IRES元件可以连接编码CAR的核酸序列和编码各种抗原之一的核酸序列(参 见图1和表2)。在其他实施例中,如2A等其他工具可用于创建多基因或多顺反子或信息。
编码所需的分子的核酸序列可以使用本领域中公知的重组方法,例如通过从细胞中筛选 文库表达该基因,导出包括相同基因的已知的载体的基因,或者使用标准实验方法,直接通 过从含有相同基因的细胞或组织中分离而获得。备选地,基因可以不通过克隆而合成产生。
本公开的实施方式进一步涉及将本发明的DNA插入载体中。从逆转录病毒,如慢病毒 衍生的载体是实现长期的基因转移的适合的工具,因为它们允许转基因的长期,稳定整合及 在子细胞繁殖。慢病毒载体具有在从ONCO-逆转录病毒衍生的载体的附加优点,例如,它们 能转导非增殖细胞,如肝细胞鼠白血病病毒。它们也具有低免疫原性的附加优势。
编码中天然或合成的核酸的表达通常是通过可操作地连接的编码核酸的CAR多肽或其 部分的一个或多个启动子,以及将所述构建体整合到表达载体来实现的。所述载体可以是适 合于复制和整合真核生物。典型的克隆载体包含转录和翻译终止子,起始序列和用于所希望 的核酸序列的表达的调控有用的启动子。
在一些实施方案中,抗原结合结构域是抗体,其抗原结合片段或其配体。例如,抗原结 合片段是Fab或scFv。
在一些实施方案中,抗原在存在于肿瘤微环境中的非必需器官细胞上表达。
在一些实施方案中,肿瘤是乳腺癌。在某些实施方案中,抗原是乳腺抗原。例如,乳腺 抗原是具有SEQ ID NO:29的催乳素受体(PRLR)。
如本文所用,“乳腺抗原”是指在乳腺细胞上或由乳腺细胞表达的抗原。乳腺细胞的实例 包括导管上皮细胞/泡沫细胞。
催乳素受体(PRLR)参与各种细胞的生长和分化。已在许多细胞和组织中鉴定催乳素受 体,包括乳腺,生殖系统器官,中枢神经系统,垂体,肾上腺皮质,皮肤,骨骼,肺,心脏,肝脏,胰腺,胃肠道,肾脏和淋巴组织。人生长激素(hGH),人催乳素(hPRL)和人胎盘 催乳素(hPL)都以高亲和力特异性结合催乳素受体。
如本文所用,“PRLR”是指人催乳素受体。如本文所公开的,该术语应被解释为不仅包括 人催乳素受体,还包括其变体,同源物,片段和部分,其程度,这些变体,同源物,片段和 部分保留催乳素受体结合人催乳素受体的抗体或配体的能力。
在一些实施方案中,编码至少一部分人催乳素受体的核苷酸序列显示在SEQ IDNO:28 中,并且至少一部分人催乳素受体显示在SEQ ID NO:29中。
在一些实施方案中,本公开内容特别适合于将药剂递送至差异性过表达催乳素受体的细 胞。如本文所用,当催乳素受体以统计学上显着大于合适的对照细胞(例如,脑细胞)的量存在于细胞(例如,乳腺细胞)的表面上时,其“过表达”。在一些实施方案中,催乳素受体 在细胞表面上的表达比合适的对照细胞高至少约1.5倍,2倍,5倍,10倍,100倍或1000倍。例如,超过80%的乳腺肿瘤过度表达催乳素受体,其表达量比正常乳腺组织高10至1000倍。因此,本公开的一些实施方案特别适合于将药剂(例如,CAR T/NK细胞)递送至受试者的乳腺癌细胞。在这些情况下,表达催乳素受体的非肿瘤细胞也可被转移的CAR T/NK细 胞杀死。然而,如果乳腺是关于受试者的非必需组织,则杀死乳腺的正常细胞不会对受试者 造成危及生命的事件(例如,并发症)。
在一些实施方案中,抗原结合结构域是具有SEQ ID NO:20或44的催乳素受体配体。
如本文所用,催乳素受体配体是指结合催乳素受体的实体,无论是否观察到催乳素受体 结合的下游生物学效应。如将理解的,不需要特定水平的结合特异性,并且可接受的特异性 水平将取决于应用。合适的催乳素受体配体包括人胎盘催乳素及其变体(包括截短的或修饰 的形式),催乳素及其变体(包括截短的或修饰的形式)和人生长激素(hGH)(包括截短的或修饰的形式)。合适的催乳素受体配体的选择将取决于例如待递送的试剂,所用的偶联策略 和所需的受体活化程度(如果有的话)。
在一些实施方案中,催乳素受体配体的至少一部分显示于SEQ ID NO:20或44中。
在一些实施方案中,肿瘤是结肠直肠癌。在某些实施方案中,抗原是结肠抗原。例如, 结肠抗原是具有SEQ ID NO:33的鸟苷酸环化酶2C(GUCY2C)。
如本文所用,“结肠抗原”是指在结肠细胞上或由结肠细胞表达的抗原。结肠细胞的实例 包括杯状细胞和肠细胞。
鸟苷酸环化酶2C(GUCY2C)主要在肠上皮细胞中表达。GUCY2C是腹泻细菌肠毒素(STs)和肠道旁分泌激素,鸟苷素和尿鸟苷素的受体。这些配体调节肠和肾上皮细胞中的水和电解质转运,并且最终导致急性分泌性腹泻。
如本文所用,“GUCY2C”是指人鸟苷酸环化酶2C。该术语应被解释为不仅包括人鸟苷酸 环化酶2C,还包括其变体,同源物,片段和部分,其程度为这些变体,同源物,片段和部分 保留鸟苷酸环化酶2C结合抗体或配体的能力。如本文所公开的人Guanylyl环化酶2C。
在一些实施方案中,编码至少一部分GUCY2C的核苷酸序列显示于SEQ ID NO:32,并 且至少一部分GUCY2C的氨基酸序列显示于SEQ ID NO:33。
在一些实施方案中,本公开内容特别适合于将药剂递送至差异性过表达GUCY2C的细 胞。如本文所用,当GUCY2C以高于合适的对照细胞(例如,脑细胞和胰腺细胞)的统计学显著性的量存在于细胞(例如,结肠细胞)的表面上时,其是“过表达的”。在一些实施方案中,GUCY2C在细胞表面上的表达比合适的对照细胞高至少约1.5倍,2倍,5倍,10倍, 100倍或1000倍。因此,本公开的一些实施方案特别适合于将药剂(例如,CAR T/NK细胞)递送至结肠直肠癌细胞。在这些情况下,表达GUCY2C的非肿瘤细胞也可被转移的CAR T/ NK细胞杀死。然而,如果结肠是关于受试者的非必需组织,则结肠的正常细胞的死亡不会 对受试者造成危及生命的事件(例如,并发症)。
在一些实施方案中,肿瘤是胃癌。在某些实施方案中,抗原是胃腺抗原。例如,胃腺抗 原是具有SEQ ID NO:31的粘蛋白17(Muc17)。
如本文所用,“胃腺抗原”是指在胃腺细胞上或由胃腺细胞表达的抗原。胃腺细胞的实例 包括胃壁细胞,粘液细胞和表面上皮细胞。
Muc17是1型膜蛋白,包含4,493个氨基酸。Muc17属于膜形式的粘蛋白家族,其细胞外结构域的大部分包含富含丝氨酸,苏氨酸和脯氨酸的59-mer序列的串联重复序列并且是糖 基化的。
如本文所用,“Muc17”是指人粘蛋白17。如本文所公开的,该术语应被解释为不仅包括 人黏蛋白17,还包括其变体,同源物,片段和部分,其程度,这些变体,同源物,片段和部 分保留黏蛋白17结合人黏蛋白17的抗体或配体的能力。
在一些实施方案中,编码至少一部分Muc17的核苷酸序列显示在SEQ ID NO:30中,并且至少一部分Muc17的氨基酸序列显示在SEQ ID NO:31中。
在一些实施方案中,本公开内容特别适合于将药剂递送至差异性过表达Muc17的细胞。 如本文所用,当Muc17存在于细胞(例如,胃腺细胞)的表面上时,其“过表达”,其表达量 在统计学上显着高于合适的对照细胞(例如,脑细胞和胰腺细胞)。在一些实施方案中,Muc17 在细胞表面上的表达比合适的对照细胞高至少约1.5倍,2倍,5倍,10倍,100倍或1000 倍。因此,本公开的一些实施方案特别适合于将药剂递送至胃癌细胞。在这些情况下,表达 Muc17的非肿瘤细胞也可被转移的CAR T/NK细胞杀死。然而,如果胃腺是关于受试者的 非必需组织,则胃腺的正常细胞的死亡不会对受试者造成危及生命的事件(例如,并发症)。
在一些实施方案中,肿瘤是膀胱癌。在某些实施方案中,抗原是膀胱抗原。例如,膀胱 抗原是具有SEQ ID NO:35的CD207。
如本文所用,“膀胱抗原”是指在膀胱细胞上或由膀胱细胞表达的抗原。膀胱细胞的实例 包括过渡细胞和粘膜上皮细胞。
CD207(langerin或Cluster of Differentiation 207)是一种蛋白质,其在人体中由CD207 基因编码。CD207是朗格汉斯细胞上的II型跨膜,C型凝集素受体。CD207定位于Birbeck 颗粒,存在于朗格汉斯细胞的细胞质中的细胞器,包括叠加和拉链的膜。
如本文所用,“CD207”是指人CD207。该术语应被解释为不仅包括人CD207,还包括其 变体,同源物,片段和部分,其程度为这些变体,同源物,片段和部分保留人CD207结合人CD207的抗体或配体的能力,如本文所公开的。
在一些实施方案中,编码至少一部分人CD207的核苷酸序列显示于SEQ ID NO:34,并 且至少一部分人CD207的氨基酸序列显示于SEQ ID NO:35。
在一些实施方案中,本公开内容特别适合于将药剂递送至差异性过表达CD207的细胞。 如本文所用,当CD207以统计学上显着高于合适的对照细胞(例如,内分泌组织细胞)的量 存在于细胞(例如膀胱细胞)的表面上时,其“过表达”。在一些实施方案中,CD207在细胞 表面上的表达比合适的对照细胞高至少约1.5倍,2倍,5倍,10倍,100倍或1000倍。因此,本公开的一些实施方案特别适合于将药剂递送至膀胱癌细胞。在这些情况下,表达CD207 的非肿瘤细胞也可被转移的CAR T/NK细胞杀死。然而,如果膀胱是关于受试者的非必需组 织,则结肠的正常细胞的死亡不会对受试者造成危及生命的事件(例如,并发症)。
在一些实施方案中,肿瘤是卵巢肿瘤。在某些实施方案中,抗原是卵巢抗原。例如,卵 巢抗原是具有SEQ ID NO:25的卷曲蛋白家族受体10(FZD10)。
如本文所用,“卵巢抗原”是指在卵巢细胞上或由卵巢细胞表达的抗原。卵巢细胞的实例 包括滤泡细胞,颗粒细胞和生发上皮细胞。
已经克隆并表征了人Fz基因家族成员Frizzled-10(FZD10)。对FZD10核苷酸序列的分 析显示,人FZD10基因编码581个氨基酸的七跨膜受体,包括富含氨基末端的半胱氨酸结构 域和羧基末端Ser/Thr-Xxx-Val基序。在胎盘,胎儿肾,胎儿肺和脑中检测到编码FZD10的 mRNA(4.0kb)。在成人脑中,FZD10 mRNA在小脑中很丰富。FZD10基因定位于人染色体 12q24.33。FZD10与Frizzled-9(FZD9)具有65.7%的氨基酸同一性。FZD10和FZD9构成Frizzled基因的亚家族。FZD10是Wnt配体蛋白WNT7a和WNT7b的受体。小鼠和人FZD10 之间有93%的同一性。
如本文所用,“FZD10”是指人FZD10。该术语应被解释为不仅包括人FZD10,还包括其 变体,同源物,片段和部分,其程度为这些变体,同源物,片段和部分保留人FZD10与人FZD10的抗体或配体结合的能力。
在一些实施方案中,编码至少一部分人FZD10的核苷酸序列显示于SEQ ID NO:24,并 且至少一部分人FZD10的氨基酸序列显示于SEQ ID NO:25。
在一些实施方案中,本发明特别适合于将药剂递送至差异性过表达FZD10的细胞。如本 文所用,当FZD10以统计学上显着高于合适的对照细胞(例如,内分泌组织细胞)的量存在 于细胞(例如,卵巢细胞)的表面上时,其“过表达”。在一些实施方案中,FZD10在细胞表 面上的表达比合适的对照细胞高至少约1.5倍,2倍,5倍,10倍,100倍或1000倍。因此,本公开的一些实施方案特别适合于将药剂递送至卵巢癌细胞。在这些情况下,表达FZD10的非肿瘤细胞也可被转移的CAR T/NK细胞杀死。然而,如果卵巢是关于受试者的非必需组织,则卵巢的正常细胞的死亡不会对受试者造成危及生命的事件(例如,并发症)。
在一些实施方案中,肿瘤是甲状腺肿瘤。在某些实施方案中,抗原是甲状腺抗原。例如, 甲状腺抗原是具有SEQ ID NO:27的甲状腺刺激激素受体(TSHR)。
如本文所用,“甲状腺抗原”是指在甲状腺细胞上或由甲状腺细胞表达的抗原。甲状腺细 胞的实例包括滤泡细胞和滤泡旁细胞。
人TSHR是甲状腺刺激素(TSH)的受体,其存在于甲状腺膜上。当脑垂体分泌的TSH与甲状腺滤泡细胞膜上的TSHR结合时,甲状腺分泌具有代谢功能的T3和T4。TSHR是七- 跨膜受体,分子量为约95,000至100,000。据报道,人促甲状腺激素受体(TSHR)包括三个 结构域:富含亮氨酸的结构域(LRD;氨基酸36-281),切割结构域(CD;氨基酸282-409)和跨膜结构域(TMD;氨基酸)酸410-699)。发现人促甲状腺激素(hTSH)α链与LRD表面和CD表面上的许多氨基酸接触。
如本文所用,“TSHR”是指人促甲状腺激素受体。该术语应被解释为不仅包括人促甲状腺 激素受体,还包括其变体,同源物,片段和部分,其程度,其变体,同源物,片段和部分保 留人促甲状腺激素受体与抗体结合的能力。或如本文所公开的人促甲状腺激素受体的配体。
在一些实施方案中,编码至少一部分人促甲状腺激素受体的核苷酸序列显示于SEQ ID NO:26,并且至少一部分人促甲状腺激素受体的氨基酸序列显示于SEQ ID NO::27。
在一些实施方案中,本公开内容特别适合于将药剂递送至差异性过表达TSHR的细胞。 如本文所用,当TSHR存在于细胞(例如,甲状腺细胞)的表面上时,其在统计学上显着高于合适的对照细胞(例如,脑细胞或胰腺细胞)的量时“过表达”。在一些实施方案中,TSHR在细胞表面上的表达比合适的对照细胞高至少约1.5倍,2倍,5倍,10倍,100倍或1000 倍。因此,本公开的一些实施方案特别适合于将药剂递送至甲状腺癌细胞。在这些情况下, 表达TSHR的非肿瘤细胞也可被转移的CAR T/NK细胞杀死。然而,如果甲状腺是关于受试者的非必需组织,则甲状腺的正常细胞的死亡不会对受试者造成危及生命的事件(例如, 并发症)。
在一些实施方案中,共刺激信号传导区可包括选自CD27,CD28,4-1BB,OX40,CD30,CD40,PD-1,ICOS,淋巴细胞功能相关抗原-1的共刺激分子的细胞内结构域(LFA-1), CD2,CD7,LIGHT,NKG2C,B7-H3,与CD83特异性结合的配体,及其任何组合。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:2-20或44中的至少一种。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:3或4,或其组合,并且肿瘤是 卵巢肿瘤。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:6或7,或其组合,并且肿瘤是 甲状腺肿瘤。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:9或10,或其组合,并且肿瘤 是乳腺癌。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:12或13,或其组合,并且肿瘤 是胃癌。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:15或16,或其组合,并且肿瘤 是结肠直肠癌。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:18或19,或其组合,并且肿瘤 是膀胱癌。
在一些实施方案中,抗原结合结构域可包括SEQ ID NO:20或44,并且肿瘤是乳腺癌。
本公开的一些实施方案涉及选择CAR的抗原结合结构域用于治疗患有肿瘤细胞的受试 者的方法。该方法可包括确定来自肿瘤细胞的细胞器官,确定器官是相对于受试者的非必需 器官,搜索数据库以识别在器官的细胞群中表达的多种标志物,基于预定条件从多个标记中选择一个标记,并使用来自受试者的细胞产生包含CAR的细胞。CAR可以包括抗原结合结 构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域,并且抗原结合结构域可以 与标记物结合。器官的例子包括乳腺,胃腺等。在某些实施方案中,器官不包括血液组织。
在一些实施方案中,预定条件可以包括标记物存在于细胞的细胞表面上,其中肿瘤细胞 的标记物表达量至少比合适的对照细胞高至1.5倍,2倍,5倍,10倍,100倍或1000倍中 的至少一种。并且器官是非必需的,使得器官的损伤不会导致受试者死亡。
在一些实施方案中,非必需器官是乳腺,并且标记物是PRLR。
在一些实施方案中,非必需器官是结肠,并且标记物是GUCY2C。
在一些实施方案中,非必需器官是胃腺,并且标志物是Muc17。
在一些实施方案中,非必需器官是膀胱,并且标志物是CD207。
在一些实施方案中,非必需器官是卵巢,并且标记物是FZD10。
在一些实施方案中,非必需器官是甲状腺,并且标记物是TSHR。
在一些实施方案中,肿瘤选自乳腺癌,甲状腺肿瘤,结肠直肠癌,卵巢肿瘤,膀胱癌, 并且是膀胱癌。
本公开的一些实施方案涉及修饰的细胞,其包含编码具有SEQ ID NO:36-43之一的CAR 的核酸序列。例如,细胞选自T细胞,自然杀伤(NK)细胞,细胞毒性T淋巴细胞(CTL)和调节性T细胞。
在一些实施方案中,CAR具有SEQ ID NO:38,并且CAR的抗原结合结构域与具有SEQID NO:20或44的催乳素受体配体结合。
在一些实施方案中,CAR具有SEQ ID NO:40,并且CAR的抗原结合结构域结合具有SEQ ID NO:33的GUCY2C。
在一些实施方案中,CAR具有SEQ ID NO:39,并且CAR的抗原结合结构域结合具有SEQ ID NO:31的Muc17。
在一些实施方案中,CAR具有SEQ ID NO:41,并且CAR的抗原结合结构域结合具有SEQ ID NO:35的CD207。
在一些实施方案中,CAR具有SEQ ID NO:36,并且CAR的抗原结合结构域结合具有SEQ ID NO:25的FZD10。
在一些实施方案中,CAR具有SEQ ID NO:37,并且CAR的抗原结合结构域结合具有SEQ ID NO:27的TSHR。
在美国提供了与编码CAR和基因转移到哺乳动物细胞中的表达合成核酸有关的其他信 息专利No.8,906,682的描述,将其全部引入作为参考。
实施方式还涉及到的方法用于治疗患者的一种疾病,包括给患者施用有效量的本公开内 容的工程细胞。各种疾病包括癌症,诸如卵巢癌,乳腺癌,结肠癌,胶质母细胞瘤,前列腺 癌和白血病可根据本发明的方法治疗。在一些实施方案中,该方法包括向人类患者施用包含有效抗肿瘤量的人T细胞群的药物组合物,其中该群的人T细胞包括人T细胞,其包含如上 所述的核酸序列。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤,以及血管化的肿瘤。 癌症可包括非实体瘤(诸如血液学肿瘤,例如白血病和淋巴瘤)或可包括实体瘤。用本公开的CAR治疗的癌症的类型包括但不限于癌,胚细胞瘤和肉瘤,以及某些白血病或淋巴恶性肿瘤,良性和恶性肿瘤和恶性肿瘤,例如肉瘤,癌和黑素瘤。,也包括成人肿瘤/癌症和儿童肿瘤/癌 症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病,包括急性 白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前 髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性) 白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏 疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血 症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同 类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤和其他肉瘤、滑膜瘤、间皮瘤、 尤因氏肿瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、淋巴恶性肿瘤、胰腺癌、乳腺癌、肺癌、 卵巢癌、前列腺癌、肝细胞癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、甲状腺髓样癌、 乳头状甲状腺癌、嗜铬细胞瘤皮脂腺癌、乳头状癌、乳头状腺癌、髓样癌、支气管癌、肾细胞癌、肝细胞瘤、胆管癌、绒膜癌、维尔姆斯氏肿瘤、子宫颈癌、睾丸肿瘤、精原细胞瘤、膀胱癌、黑素瘤和CNS肿瘤(诸如神经胶质瘤(诸如脑干神经胶质瘤和混合神经胶质瘤)、成胶 质细胞瘤(也已知为多形性成胶质细胞瘤)星形细胞瘤、CNS淋巴瘤、生殖细胞瘤、成神经管 细胞瘤、神经鞘瘤、颅咽管瘤、室管膜瘤、松果体瘤、成血管细胞瘤、听神经瘤、少突神经胶质瘤、脑膜瘤、神经细胞瘤、视网膜母细胞瘤和脑转移)。
通常,本文所述的活化以及增值的细胞可以治疗和预防个体出现的免疫受损疾病。特别 是,本发明的工程细胞可以使用在癌症的治疗中。在某些实施方案中,本公开的细胞用于治 疗患有癌症风险的患者。因此,实施方式提供方法用于治疗患者的一种疾病,包括给患者施用有效量的本公开内容的工程T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分 诸如IL-2或其他细胞因子或细胞群结合施用。简单地说,本发明的药物组合物可包括如本文 所述的靶细胞群,与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的 组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等;碳水化合物诸如葡萄糖、甘 露糖、蔗糖或葡聚糖、甘露醇;蛋白质;多肽或氨基酸诸如甘氨酸;抗氧化剂;螯合剂诸如 EDTA或谷胱甘肽;佐剂(例如,氢氧化铝);和防腐剂。本发明的组合物优选配制用于静脉内 施用。本公开的组合物优选配制成用于静脉内给药。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率 将由这样的因素确定,如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由 临床试验确定。
当被指示“免疫学有效量”,“抗肿瘤有效量”,“肿瘤抑制有效量”或“治疗量”,本公开内容 待施用的组合物的精确量可通过考虑的个体的年龄差异,体重,肿瘤大小,感染或转移的程 度,以及测试患者(受试者)的状态的医生来确定。可通常指出:包括本文描述的T细胞的药 物组合物可以以1O4至1O9个细胞/kg体重的剂量,优选1O5至106个细胞/kg体重的剂量—— 包括那些范围内的所有整数值——施用。T细胞组合物也可以以这些剂量多次施用。细胞注 入可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等,New Eng.J.0fMed.319:1676,1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗,其可由医学领域技术人员确定。
在某些实施方式中,可期望向对象施用活化的T细胞,并随后重新抽取(redraw)血液(或 实施单采血液成分术),根据本发明活化其中的T细胞,和用这些活化和扩展的T细胞再注入 该患者。该过程可每几个周进行多次。在某些实施方式中,T细胞可从1Occ至400cc的血液 抽取中进行活化。在一些实施方式中,T细胞从20cc、30cc、40cc、50cc、60cc、70cc、80cc、 90cc或100cc的血液抽取中进行活化。不被理论所束缚,多份血液抽取/多个再输注方案可用 于选出某些T细胞群。
对象组合物的施用可以以任何方便的方式进行,包括通过喷雾法、注射、吞咽、输液、 植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。v.)。在一个实施方式中,本发明的T细胞组合物通过皮内 或皮下注射被施用给患者。在另一个实施方式中,本发明的T细胞组合物优选通过i.v.注射施 用。T细胞的组合物可被直接注入肿瘤,淋巴结或感染位置。
在本发明的某些实施方式中,利用本文描述的方法或本领域已知的其他将T细胞扩展至 治疗性水平的方法活化和扩展的细胞,与任何数量的治疗形式结合(例如,之前、同时或之后) 施用给患者,所述治疗形式包括但不限于用以下试剂进行治疗:抗病毒疗法、西多福韦和白细 胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄 法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中,本发明的T细胞可与以 下治疗方式结合使用:化疗、辐射、免疫抑制剂如环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯 和FK506,抗体或其他免疫烧蚀剂诸如CAM PATH、抗-CD3抗体或其他抗体疗法、细胞毒 素、氟达拉滨、环孢菌素、FK506、雷帕霉素、麦考酚酸、类固醇类、FR901228、细胞因子 和照射。这些药物抑制钙依赖性磷酸酶钙调神经磷酸酶(环孢菌素和FK506)或抑制对生长 因子诱导的信号传导(雷帕霉素)重要的p70S6激酶。(Liu et al.,Cell 66:807-815,1991; Henderson et al.,Immun 73:316-321,1991;Bierer et al.,Curr.Opin.Immun 5:763-773,1993; Isoniemi(supra))。在进一步的实施方式中,本发明的细胞组合物与骨髓移植、化疗剂诸如氟 达拉滨、外部光束放射疗法(XRT)、环磷酰胺或抗体诸如0KT3或CAMPATH的T细胞烧蚀 疗法结合(例如,之前、同时或之后)而施用给患者。在另一个实施方式中,本发明的细胞组 合物在B-细胞烧蚀疗法诸如与CD20反应的试剂例如Rituxan后进行施用。例如,在一个实 施方案中,受试者可以接受高剂量化学疗法的标准治疗,然后进行外周血干细胞移植。在一些实施方式中,在移植后,对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方 式中,扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施 用的剂量比例可根据本领域接受的实践实施。例如,对于成年患者,CAMPATH的剂量通常为1至约100mg,通常施用1至30天。尽管在一些例子中可使用上至40mg每天的较大剂量, 但优选的日剂量为1至1Omg每天。(美国专利号6120766中描述,其全部内容通过引用并入本文)。
使用工程T细胞治疗癌症的方法的其他信息包括美国专利No.8,906,682的描述,其全部 内容通过引用并入本文。
本公开的一些实施方案涉及编码CAR的核酸序列。例如,CAR包括抗原结合结构域,跨膜结构域和细胞内结构域。抗原结合结构域与器官谱系抗原结合,并且器官谱系抗原在肿 瘤和衍生肿瘤的正常组织中表达。
如本文所用,术语“肿瘤相关抗原”是指与正常成人组织相比由恶性细胞选择性表达或过 表达的抗原。肿瘤相关抗原包括各种组,例如肿瘤特异性抗原,肿瘤抗原,癌基因产物,器 官谱系抗原,病毒抗原等。例如,癌基因和抑制基因产物,例如非突变的HER-2/neu和p53, 与癌胚抗原是类似的,它们可以在肿瘤中过表达并且可以在一些胎儿组织中表达。
本文所用的术语“肿瘤特异性抗原”是指在肿瘤中独特表达的抗原,例如点突变的ras癌基 因,p53突变,以及抗独特型抗体(Abs)和核糖核酸(RNA)剪接变体和基因易位的产物。
本文所用的术语“器官谱系抗原”定义为在给定类型的肿瘤和肿瘤来源的正常器官中表达 的抗原。器官谱系抗原的实例包括前列腺特异性抗原(PSA)和黑素细胞抗原,例如MART-1/Melan A,酪氨酸酶,gp100和TRP-1/gp75。如果表达器官谱系抗原的正常器官不是必需的, 器官谱系抗原可以是免疫疗法的靶标,例如前列腺,乳腺或黑素细胞。如本文所用,器官是 指具有共同结构,细胞间材料和/或功能的整合的细胞群。
本公开的一些实施方案涉及包含核酸序列的载体。在一些实施方案中,载体是表达载体。
本公开的一些实施方案涉及药物组合物,其包含有效抗肿瘤量的人T或NK细胞群。群 体的人T或NK细胞包括人T或NK细胞,其包括如上所述的核酸序列。
本公开的一些实施方案涉及治疗人患者中的肿瘤的方法,该方法包括向人患者施用如上 所述的药物组合物。
在一些实施方案中,肿瘤是乳腺肿瘤,器官谱系抗原包含PRLR。
在一些实施方案中,肿瘤是结肠直肠肿瘤,并且器官谱系抗原包含CLCA1,MUC12,GUCY2C或GPR35中的至少一种。
在一些实施方案中,肿瘤是胃肿瘤,并且器官谱系抗原包含CR1L和/或MUC17。
在一些实施方案中,肿瘤是食道肿瘤,并且器官谱系抗原包含TMPRSS11B,MUC21或TMPRSS11E中的至少一种。
在一些实施方案中,肿瘤是膀胱癌,并且器官谱系抗原包含CD207。
在一些实施方案中,肿瘤是胰腺肿瘤,并且器官谱系抗原包含SLC30A8和/或CFC1。
在一些实施方案中,肿瘤是宫颈肿瘤,器官谱系抗原包含SLC12A3和/或SSTR1。
在一些实施方案中,肿瘤是卵巢肿瘤,器官谱系抗原包含GPR27和/或FZD10。
在一些实施方案中,肿瘤是甲状腺肿瘤,器官谱系抗原包含TSHR。
下表1列出了多种肿瘤和器官谱系抗原以及相应的基因。
参考以下实施例进一步描述本发明。提供这些实施例仅用于说明的目的,除非另有说明, 否则不是限制性的。因此,本公开内容不应当以任何方式被解释为限于以下实施例,而是应 该被解释为由本文所提供的教导的结果而变得明显的所有变化的案例。
实施例1
构建抗原表达的K562细胞系
用慢病毒转导K562细胞,包括编码各种抗原的核酸序列(图1)以建立靶肿瘤细胞系。 慢病毒包括IRES-mCherry(红色)构建体,其编码红色荧光以确认抗原表达。从这些细胞系 观察到红色荧光信号,表明成功建立了靶实体瘤细胞系(图2)。构建细胞系的技术可以在“含 有CD137信号转导结构域的嵌合受体介导T细胞的增强的存活和体内分子治疗的增加的抗白 血病功效”2009年8月第17卷第8期,1453–1464页中找到,通过引用并入本文。K562细 胞获自美国典型培养物保藏中心(ATCC)。
CAR T细胞的构建
用包括各种CAR的慢病毒转导原代T细胞,以建立靶向图1中列出的不同抗原的不同 CAR T细胞系。这些细胞来自健康的人类供体。如图3所示,慢病毒包括分别编码CAR分子的核酸序列,并且还包括IRES-mCherry(绿色)构建体,其编码绿色荧光以确认CAR表 达。以抗CD207/抗Muc17 CAR T细胞作为图3中的实例,测量CAR的表达以确认CAR T 细胞系表达的特异性抗原分子(参见上文方框302)图4-9.与细胞培养,慢病毒载体构建和 流式细胞术相关的技术可见于“用mRNA工程化T细胞治疗小鼠晚期白血病,人类基因治疗 22:1575-1586(2011年12月)”,其内容通过引用并入本文。
下表2列出了各种序列标识符及其用于建立各种抗原CAR T细胞的序列。
序列号: | 标识符 | 靶肿瘤 |
2 | scFv FZD10 | 卵巢肿瘤 |
5 | scFv TSHR | 甲状腺肿瘤 |
8 | scFv PRLR | 乳腺癌 |
11 | scFv Muc 17 | 胃癌 |
14 | scFv GUCY2C | 结直肠癌 |
17 | scFv CD207 | 膀胱癌 |
36 | CARFZD10 | 卵巢肿瘤 |
37 | CARTSHR | 甲状腺肿瘤 |
38 | CARPRLR | 乳腺癌 |
39 | CARMuc 17 | 胃癌 |
40 | CARGUCY2C | 结直肠癌 |
41 | CARCD207 | 膀胱癌 |
42 | CARProlactin | 乳腺癌 |
44 | CARmodified Prolactin | 乳腺癌 |
共培养测定中的IFN-γ释放
共培养各种类型的CAR T细胞和相应类型的抗原表达的K562细胞(参见表2),并测量 由抗原表达的K562细胞诱导的CAT T细胞应答。将CAR T细胞和靶肿瘤细胞以E:T 1:1/3: 1/10:1/30:1(即,CAR T细胞:靶肿瘤细胞)的比例共培养24小时。然后收集上清液, 测量IFN-γ的释放。当CAR T细胞与其相应的抗原表达的K562细胞共培养时,观察到各种 水平的IFN-γ释放。当CAR T细胞和野生型K562共培养时,IFN-γ释放不明显。该观察结果 表明,CAR T细胞特异性地识别相应的抗原表达的K562细胞,并通过释放IFN-γ攻击这些 细胞(参见图10-15)。如图15的框404所示,包括催乳素-CAR的T细胞还识别表达PRLR 的靶肿瘤细胞,并响应于共催化催乳素-CAR T细胞和抗原表达的K562细胞而释放IFN-γ。 与图15中的框402相比(scFv抗PRLR CAR T细胞),催乳素-CAR T细胞达到类似的效果。 与细胞培养相关的技术,细胞毒性T淋巴细胞测定的构建可以在“用含有CD28和CD137结 构域的遗传重新定向的人T细胞的大型,已建立的肿瘤异种移植物的控制”2009年3月3日 的第106卷第9期的3360–3365PNAS中找到,其通过引用并入本文。
CAT T细胞杀伤试验
进行CAR T细胞杀伤测定以测量CAR T细胞的有效性。从健康人供体的血液样品中获 得原代T细胞。用编码各种CAR的核酸序列(参见表2和图1)分别转导这些T细胞,并使 用流式细胞术技术测量T细胞上的CAR表达。
用编码相应人抗原的核酸序列(参见图1)分别转导K562细胞,并使用流式细胞术技术 测量抗原表达。用编码荧光蛋白(RFP)的核酸序列转导其他抗原表达K562细胞用于杀伤测 定分析。将各种CAR T细胞与相应的K562细胞以各种E:T比例(30:1,10:1,3:1,1:1)温育24小时,并观察来自共培养细胞的红色荧光信号。例如,将表达抗THSR CAR的CAR T细胞(SEQ ID NO:5)与表达人THSR的K562(SEQ ID NO:26)共培养至少5天。与 正常T细胞相比,CAR T细胞显着减少了抗原表达K562细胞的数量。图16-18中提供了抗 PRLR CAR,抗THSR CAR和催乳素CAR T细胞的实例。在这些实施例中,在共培养CAR T 细胞和相应的抗原表达K562细胞后第5天观察到共培养细胞的红色荧光信号。
使用3T3鼠成纤维细胞(ATCC)进一步进行CAR T细胞杀伤分析。用各种人抗原转导3T3细胞(参见图1),并使用流式细胞术技术测量3T3细胞上的抗原表达。此外,用编码荧 光蛋白(RFP)的核酸序列转导3T3细胞用于杀伤测定分析。将表达人靶抗原/RFP的3T3 细胞和相应的CAR T细胞分别以E:T 30:1或10:1共培养,然后从共培养细胞中观察荧 光信号至少5天。与正常T细胞相比,CAR T细胞显着减少了抗原表达3T3细胞的数量。图 19-23中提供了抗PRLR CAR,抗PRLR CAR和催乳素CAR T细胞的实例。在这些实施例中, 在共培养CAR T细胞和相应的抗原表达3T3细胞后第5天观察到共培养细胞的红色荧光信 号。
下表3列出了各种序列标识符及其序列。
序列表
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35 40 45
Ile Tyr Asp Asn Asn Lys Arg Pro Ser Ala Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln
65 70 75 80
Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Arg Leu
85 90 95
Gly Ile Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
115 120 125
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gln Ser Leu Lys
130 135 140
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Asp Asn Trp Ile Gly
145 150 155 160
Trp Val Arg Gln Lys Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile
165 170 175
Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln
180 185 190
Val Thr Ile Ser Ala Asp Lys Ser Ile Asn Thr Ala Tyr Leu Gln Trp
195 200 205
Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys Val Gly Leu
210 215 220
Asp Trp Asn Tyr Asn Pro Leu Arg Tyr Trp Gly Pro Gly Thr Leu Val
225 230 235 240
Thr Val Ser Ser
<210> 6
<211> 110
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln
1 5 10 15
Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asp Ile Gly Ser Asn
20 25 30
Tyr Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Asp Asn Asn Lys Arg Pro Ser Ala Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln
65 70 75 80
Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Arg Leu
85 90 95
Gly Ile Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu
100 105 110
<210> 7
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gln
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Asp Asn
20 25 30
Trp Ile Gly Trp Val Arg Gln Lys Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Asn Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Val Gly Leu Asp Trp Asn Tyr Asn Pro Leu Arg Tyr Trp Gly Pro Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 8
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Gly Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Gly
85 90 95
Glu Leu Pro Pro Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
130 135 140
Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr
145 150 155 160
Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val
165 170 175
Ala Thr Val Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val
180 185 190
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
195 200 205
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Ser Ala Met Tyr Tyr Cys
210 215 220
Ala Arg His Arg Gly Asn Tyr Tyr Ala Thr Tyr Tyr Tyr Ala Met Asp
225 230 235 240
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
245 250
<210> 9
<211> 113
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Gly Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Gly
85 90 95
Glu Leu Pro Pro Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Ala
<210> 10
<211> 124
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Val Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Ser Ala Met Tyr Tyr Cys
85 90 95
Ala Arg His Arg Gly Asn Tyr Tyr Ala Thr Tyr Tyr Tyr Ala Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 11
<211> 243
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly
1 5 10 15
Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His His Glu Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln
115 120 125
Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys
130 135 140
Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Trp Met Asn Trp Val Lys
145 150 155 160
Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Tyr
165 170 175
Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu
180 185 190
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu
195 200 205
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Pro Tyr Tyr
210 215 220
Gly Thr Asn Pro Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser
<210> 12
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly
1 5 10 15
Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His His Glu Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 13
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Pro Tyr Tyr Gly Thr Asn Pro Trp Phe Pro Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 14
<211> 241
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Gly Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Lys Thr Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln
115 120 125
Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
130 135 140
Ala Val Phe Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His Arg
165 170 175
Gly Asn Thr Asn Asp Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser
180 185 190
Val Asp Thr Ser Lys Asn Gln Phe Ala Leu Lys Leu Ser Ser Val Thr
195 200 205
Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Arg Gly Tyr Thr
210 215 220
Tyr Gly Asn Phe Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser
225 230 235 240
Ser
<210> 15
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Gly Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Lys Thr Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Lys
100 105
<210> 16
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Phe Gly Gly Ser Phe Ser Gly Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn His Arg Gly Asn Thr Asn Asp Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ala Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Arg Gly Tyr Thr Tyr Gly Asn Phe Asp His Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 17
<211> 246
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Arg Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gln Val Gln Leu Arg Gln Ser Gly Pro Glu Leu Val Lys Pro Gly
130 135 140
Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp
165 170 175
Ile Gly Asp Ile Tyr Pro Gly Ser Gly Tyr Ser Phe Tyr Asn Glu Asn
180 185 190
Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala
195 200 205
Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
210 215 220
Cys Ala Thr Tyr Tyr Asn Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr
225 230 235 240
Leu Val Thr Val Ser Ala
245
<210> 18
<211> 114
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Arg Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Ala
<210> 19
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Gln Val Gln Leu Arg Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Asp Ile Tyr Pro Gly Ser Gly Tyr Ser Phe Tyr Asn Glu Asn Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Thr Tyr Tyr Asn Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ala
115
<210> 20
<211> 199
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Leu Pro Ile Cys Pro Gly Gly Ala Ala Arg Cys Gln Val Thr Leu Arg
1 5 10 15
Asp Leu Phe Asp Arg Ala Val Val Leu Ser His Tyr Ile His Asn Leu
20 25 30
Ser Ser Glu Met Phe Ser Glu Phe Asp Lys Arg Tyr Thr His Gly Arg
35 40 45
Gly Phe Ile Thr Lys Ala Ile Asn Ser Cys His Thr Ser Ser Leu Ala
50 55 60
Thr Pro Glu Asp Lys Glu Gln Ala Gln Gln Met Asn Gln Lys Asp Phe
65 70 75 80
Leu Ser Leu Ile Val Ser Ile Leu Arg Ser Trp Asn Glu Pro Leu Tyr
85 90 95
His Leu Val Thr Glu Val Arg Gly Met Gln Glu Ala Pro Glu Ala Ile
100 105 110
Leu Ser Lys Ala Val Glu Ile Glu Glu Gln Thr Lys Arg Leu Leu Glu
115 120 125
Gly Met Glu Leu Ile Val Ser Gln Val His Pro Glu Thr Lys Glu Asn
130 135 140
Glu Ile Tyr Pro Val Trp Ser Gly Leu Pro Ser Leu Gln Met Ala Asp
145 150 155 160
Glu Glu Ser Arg Leu Ser Ala Tyr Tyr Asn Leu Leu His Cys Leu Arg
165 170 175
Arg Asp Ser His Lys Ile Asp Asn Tyr Leu Lys Leu Leu Lys Cys Arg
180 185 190
Ile Ile His Asn Asn Asn Cys
195
<210> 21
<211> 69
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
1 5 10 15
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
20 25 30
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
50 55 60
Ser Leu Val Ile Thr
65
<210> 22
<211> 42
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 23
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 24
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
atgcagcgcc cgggcccccg cctgtggctg gtcctgcagg tgatgggctc gtgcgccgcc 60
atcagctcca tggacatgga gcgcccgggc gacggcaaat gccagcccat cgagatcccg 120
atgtgcaagg acatcggcta caacatgact cgtatgccca acctgatggg ccacgagaac 180
cagcgcgagg cagccatcca gttgcacgag ttcgcgccgc tggtggagta cggctgccac 240
ggccacctcc gcttcttcct gtgctcgctg tacgcgccga tgtgcaccga gcaggtctct 300
acccccatcc ccgcctgccg ggtcatgtgc gagcaggccc ggctcaagtg ctccccgatt 360
atggagcagt tcaacttcaa gtggcccgac tccctggact gccggaaact ccccaacaag 420
aacgacccca actacctgtg catggaggcg cccaacaacg gctcggacga gcccacccgg 480
ggctcgggcc tgttcccgcc gctgttccgg ccgcagcggc cccacagcgc gcaggagcac 540
ccgctgaagg acgggggccc cgggcgcggc ggctgcgaca acccgggcaa gttccaccac 600
gtggagaaga gcgcgtcgtg cgcgccgctc tgcacgcccg gcgtggacgt gtactggagc 660
cgcgaggaca agcgcttcgc agtggtctgg ctggccatct gggcggtgct gtgcttcttc 720
tccagcgcct tcaccgtgct caccttcctc atcgacccgg cccgcttccg ctaccccgag 780
cgccccatca tcttcctctc catgtgctac tgcgtctact ccgtgggcta cctcatccgc 840
ctcttcgccg gcgccgagag catcgcctgc gaccgggaca gcggccagct ctatgtcatc 900
caggagggac tggagagcac cggctgcacg ctggtcttcc tggtcctcta ctacttcggc 960
atggccagct cgctgtggtg ggtggtcctc acgctcacct ggttcctggc cgccggcaag 1020
aagtggggcc acgaggccat cgaagccaac agcagctact tccacctggc agcctgggcc 1080
atcccggcgg tgaagaccat cctgatcctg gtcatgcgca gggtggcggg ggacgagctc 1140
accggggtct gctacgtggg cagcatggac gtcaacgcgc tcaccggctt cgtgctcatt 1200
cccctggcct gctacctggt catcggcacg tccttcatcc tctcgggctt cgtggccctg 1260
ttccacatcc ggagggtgat gaagacgggc ggcgagaaca cggacaagct ggagaagctc 1320
atggtgcgta tcgggctctt ctctgtgctg tacaccgtgc cggccacctg tgtgatcgcc 1380
tgctactttt acgaacgcct caacatggat tactggaaga tcctggcggc gcagcacaag 1440
tgcaaaatga acaaccagac taaaacgctg gactgcctga tggccgcctc catccccgcc 1500
gtggagatct tcatggtgaa gatctttatg ctgctggtgg tggggatcac cagcgggatg 1560
tggatttgga cctccaagac tctgcagtcc tggcagcagg tgtgcagccg taggttaaag 1620
aagaagagcc ggagaaaacc ggccagcgtg atcaccagcg gtgggattta caaaaaagcc 1680
cagcatcccc agaaaactca ccacgggaaa tatgagatcc ctgcccagtc gcccacctgc 1740
gtgtga 1746
<210> 25
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Gln Arg Pro Gly Pro Arg Leu Trp Leu Val Leu Gln Val Met Gly
1 5 10 15
Ser Cys Ala Ala Ile Ser Ser Met Asp Met Glu Arg Pro Gly Asp Gly
20 25 30
Lys Cys Gln Pro Ile Glu Ile Pro Met Cys Lys Asp Ile Gly Tyr Asn
35 40 45
Met Thr Arg Met Pro Asn Leu Met Gly His Glu Asn Gln Arg Glu Ala
50 55 60
Ala Ile Gln Leu His Glu Phe Ala Pro Leu Val Glu Tyr Gly Cys His
65 70 75 80
Gly His Leu Arg Phe Phe Leu Cys Ser Leu Tyr Ala Pro Met Cys Thr
85 90 95
Glu Gln Val Ser Thr Pro Ile Pro Ala Cys Arg Val Met Cys Glu Gln
100 105 110
Ala Arg Leu Lys Cys Ser Pro Ile Met Glu Gln Phe Asn Phe Lys Trp
115 120 125
Pro Asp Ser Leu Asp Cys Arg Lys Leu Pro Asn Lys Asn Asp Pro Asn
130 135 140
Tyr Leu Cys Met Glu Ala Pro Asn Asn Gly Ser Asp Glu Pro Thr Arg
145 150 155 160
Gly Ser Gly Leu Phe Pro Pro Leu Phe Arg Pro Gln Arg Pro His Ser
165 170 175
Ala Gln Glu His Pro Leu Lys Asp Gly Gly Pro Gly Arg Gly Gly Cys
180 185 190
Asp Asn Pro Gly Lys Phe His His Val Glu Lys Ser Ala Ser Cys Ala
195 200 205
Pro Leu Cys Thr Pro Gly Val Asp Val Tyr Trp Ser Arg Glu Asp Lys
210 215 220
Arg Phe Ala Val Val Trp Leu Ala Ile Trp Ala Val Leu Cys Phe Phe
225 230 235 240
Ser Ser Ala Phe Thr Val Leu Thr Phe Leu Ile Asp Pro Ala Arg Phe
245 250 255
Arg Tyr Pro Glu Arg Pro Ile Ile Phe Leu Ser Met Cys Tyr Cys Val
260 265 270
Tyr Ser Val Gly Tyr Leu Ile Arg Leu Phe Ala Gly Ala Glu Ser Ile
275 280 285
Ala Cys Asp Arg Asp Ser Gly Gln Leu Tyr Val Ile Gln Glu Gly Leu
290 295 300
Glu Ser Thr Gly Cys Thr Leu Val Phe Leu Val Leu Tyr Tyr Phe Gly
305 310 315 320
Met Ala Ser Ser Leu Trp Trp Val Val Leu Thr Leu Thr Trp Phe Leu
325 330 335
Ala Ala Gly Lys Lys Trp Gly His Glu Ala Ile Glu Ala Asn Ser Ser
340 345 350
Tyr Phe His Leu Ala Ala Trp Ala Ile Pro Ala Val Lys Thr Ile Leu
355 360 365
Ile Leu Val Met Arg Arg Val Ala Gly Asp Glu Leu Thr Gly Val Cys
370 375 380
Tyr Val Gly Ser Met Asp Val Asn Ala Leu Thr Gly Phe Val Leu Ile
385 390 395 400
Pro Leu Ala Cys Tyr Leu Val Ile Gly Thr Ser Phe Ile Leu Ser Gly
405 410 415
Phe Val Ala Leu Phe His Ile Arg Arg Val Met Lys Thr Gly Gly Glu
420 425 430
Asn Thr Asp Lys Leu Glu Lys Leu Met Val Arg Ile Gly Leu Phe Ser
435 440 445
Val Leu Tyr Thr Val Pro Ala Thr Cys Val Ile Ala Cys Tyr Phe Tyr
450 455 460
Glu Arg Leu Asn Met Asp Tyr Trp Lys Ile Leu Ala Ala Gln His Lys
465 470 475 480
Cys Lys Met Asn Asn Gln Thr Lys Thr Leu Asp Cys Leu Met Ala Ala
485 490 495
Ser Ile Pro Ala Val Glu Ile Phe Met Val Lys Ile Phe Met Leu Leu
500 505 510
Val Val Gly Ile Thr Ser Gly Met Trp Ile Trp Thr Ser Lys Thr Leu
515 520 525
Gln Ser Trp Gln Gln Val Cys Ser Arg Arg Leu Lys Lys Lys Ser Arg
530 535 540
Arg Lys Pro Ala Ser Val Ile Thr Ser Gly Gly Ile Tyr Lys Lys Ala
545 550 555 560
Gln His Pro Gln Lys Thr His His Gly Lys Tyr Glu Ile Pro Ala Gln
565 570 575
Ser Pro Thr Cys Val
580
<210> 26
<211> 2295
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atgaggccgg cggacttgct gcagctggtg ctgctgctcg acctgcccag ggacctgggc 60
ggaatggggt gttcgtctcc accctgcgag tgccatcagg aggaggactt cagagtcacc 120
tgcaaggata ttcaacgcat ccccagctta ccgcccagta cgcagactct gaagcttatt 180
gagactcacc tgagaactat tccaagtcat gcattttcta atctgcccaa tatttccaga 240
atctacgtat ctatagatgt gactctgcag cagctggaat cacactcctt ctacaatttg 300
agtaaagtga ctcacataga aattcggaat accaggaact taacttacat agaccctgat 360
gccctcaaag agctccccct cctaaagttc cttggcattt tcaacactgg acttaaaatg 420
ttccctgacc tgaccaaagt ttattccact gatatattct ttatacttga aattacagac 480
aacccttaca tgacgtcaat ccctgtgaat gcttttcagg gactatgcaa tgaaaccttg 540
acactgaagc tgtacaacaa tggctttact tcagtccaag gatatgcttt caatgggaca 600
aagctggatg ctgtttacct aaacaagaat aaatacctga cagttattga caaagatgca 660
tttggaggag tatacagtgg accaagcttg ctggacgtgt ctcaaaccag tgtcactgcc 720
cttccatcca aaggcctgga gcacctgaag gaactgatag caagaaacac ctggactctt 780
aagaaacttc cactttcctt gagtttcctt cacctcacac gggctgacct ttcttaccca 840
agccactgct gtgcttttaa gaatcagaag aaaatcagag gaatccttga gtccttgatg 900
tgtaatgaga gcagtatgca gagcttgcgc cagagaaaat ctgtgaatgc cttgaatagc 960
cccctccacc aggaatatga agagaatctg ggtgacagca ttgttgggta caaggaaaag 1020
tccaagttcc aggatactca taacaacgct cattattacg tcttctttga agaacaagag 1080
gatgagatca ttggttttgg ccaggagctc aaaaaccccc aggaagagac tctacaagct 1140
tttgacagcc attatgacta caccatatgt ggggacagtg aagacatggt gtgtaccccc 1200
aagtccgatg agttcaaccc gtgtgaagac ataatgggct acaagttcct gagaattgtg 1260
gtgtggttcg ttagtctgct ggctctcctg ggcaatgtct ttgtcctgct tattctcctc 1320
accagccact acaaactgaa cgtcccccgc tttctcatgt gcaacctggc ctttgcggat 1380
ttctgcatgg ggatgtacct gctcctcatc gcctctgtag acctctacac tcactctgag 1440
tactacaacc atgccatcga ctggcagaca ggccctgggt gcaacacggc tggtttcttc 1500
actgtctttg caagcgagtt atcggtgtat acgctgacgg tcatcaccct ggagcgctgg 1560
tatgccatca ccttcgccat gcgcctggac cggaagatcc gcctcaggca cgcatgtgcc 1620
atcatggttg ggggctgggt ttgctgcttc cttctcgccc tgcttccttt ggtgggaata 1680
agtagctatg ccaaagtcag tatctgcctg cccatggaca ccgagacccc tcttgctctg 1740
gcatatattg tttttgttct gacgctcaac atagttgcct tcgtcatcgt ctgctgctgt 1800
tatgtgaaga tctacatcac agtccgaaat ccgcagtaca acccagggga caaagatacc 1860
aaaattgcca agaggatggc tgtgttgatc ttcaccgact tcatatgcat ggccccaatc 1920
tcattctatg ctctgtcagc aattctgaac aagcctctca tcactgttag caactccaaa 1980
atcttgctgg tactcttcta tccacttaac tcctgtgcca atccattcct ctatgctatt 2040
ttcaccaagg ccttccagag ggatgtgttc atcctactca gcaagtttgg catctgtaaa 2100
cgccaggctc aggcataccg ggggcagagg gttcctccaa agaacagcac tgatattcag 2160
gttcaaaagg ttacccacga gatgaggcag ggtctccaca acatggaaga tgtctatgaa 2220
ctgattgaaa actcccatct aaccccaaag aagcaaggcc aaatctcaga agagtatatg 2280
caaacggttt tgtaa 2295
<210> 27
<211> 764
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Met Arg Pro Ala Asp Leu Leu Gln Leu Val Leu Leu Leu Asp Leu Pro
1 5 10 15
Arg Asp Leu Gly Gly Met Gly Cys Ser Ser Pro Pro Cys Glu Cys His
20 25 30
Gln Glu Glu Asp Phe Arg Val Thr Cys Lys Asp Ile Gln Arg Ile Pro
35 40 45
Ser Leu Pro Pro Ser Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu
50 55 60
Arg Thr Ile Pro Ser His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg
65 70 75 80
Ile Tyr Val Ser Ile Asp Val Thr Leu Gln Gln Leu Glu Ser His Ser
85 90 95
Phe Tyr Asn Leu Ser Lys Val Thr His Ile Glu Ile Arg Asn Thr Arg
100 105 110
Asn Leu Thr Tyr Ile Asp Pro Asp Ala Leu Lys Glu Leu Pro Leu Leu
115 120 125
Lys Phe Leu Gly Ile Phe Asn Thr Gly Leu Lys Met Phe Pro Asp Leu
130 135 140
Thr Lys Val Tyr Ser Thr Asp Ile Phe Phe Ile Leu Glu Ile Thr Asp
145 150 155 160
Asn Pro Tyr Met Thr Ser Ile Pro Val Asn Ala Phe Gln Gly Leu Cys
165 170 175
Asn Glu Thr Leu Thr Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val
180 185 190
Gln Gly Tyr Ala Phe Asn Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn
195 200 205
Lys Asn Lys Tyr Leu Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val
210 215 220
Tyr Ser Gly Pro Ser Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala
225 230 235 240
Leu Pro Ser Lys Gly Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn
245 250 255
Thr Trp Thr Leu Lys Lys Leu Pro Leu Ser Leu Ser Phe Leu His Leu
260 265 270
Thr Arg Ala Asp Leu Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn
275 280 285
Gln Lys Lys Ile Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser
290 295 300
Ser Met Gln Ser Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser
305 310 315 320
Pro Leu His Gln Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly
325 330 335
Tyr Lys Glu Lys Ser Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr
340 345 350
Tyr Val Phe Phe Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln
355 360 365
Glu Leu Lys Asn Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His
370 375 380
Tyr Asp Tyr Thr Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro
385 390 395 400
Lys Ser Asp Glu Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr Lys Phe
405 410 415
Leu Arg Ile Val Val Trp Phe Val Ser Leu Leu Ala Leu Leu Gly Asn
420 425 430
Val Phe Val Leu Leu Ile Leu Leu Thr Ser His Tyr Lys Leu Asn Val
435 440 445
Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Ala Asp Phe Cys Met Gly
450 455 460
Met Tyr Leu Leu Leu Ile Ala Ser Val Asp Leu Tyr Thr His Ser Glu
465 470 475 480
Tyr Tyr Asn His Ala Ile Asp Trp Gln Thr Gly Pro Gly Cys Asn Thr
485 490 495
Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu
500 505 510
Thr Val Ile Thr Leu Glu Arg Trp Tyr Ala Ile Thr Phe Ala Met Arg
515 520 525
Leu Asp Arg Lys Ile Arg Leu Arg His Ala Cys Ala Ile Met Val Gly
530 535 540
Gly Trp Val Cys Cys Phe Leu Leu Ala Leu Leu Pro Leu Val Gly Ile
545 550 555 560
Ser Ser Tyr Ala Lys Val Ser Ile Cys Leu Pro Met Asp Thr Glu Thr
565 570 575
Pro Leu Ala Leu Ala Tyr Ile Val Phe Val Leu Thr Leu Asn Ile Val
580 585 590
Ala Phe Val Ile Val Cys Cys Cys Tyr Val Lys Ile Tyr Ile Thr Val
595 600 605
Arg Asn Pro Gln Tyr Asn Pro Gly Asp Lys Asp Thr Lys Ile Ala Lys
610 615 620
Arg Met Ala Val Leu Ile Phe Thr Asp Phe Ile Cys Met Ala Pro Ile
625 630 635 640
Ser Phe Tyr Ala Leu Ser Ala Ile Leu Asn Lys Pro Leu Ile Thr Val
645 650 655
Ser Asn Ser Lys Ile Leu Leu Val Leu Phe Tyr Pro Leu Asn Ser Cys
660 665 670
Ala Asn Pro Phe Leu Tyr Ala Ile Phe Thr Lys Ala Phe Gln Arg Asp
675 680 685
Val Phe Ile Leu Leu Ser Lys Phe Gly Ile Cys Lys Arg Gln Ala Gln
690 695 700
Ala Tyr Arg Gly Gln Arg Val Pro Pro Lys Asn Ser Thr Asp Ile Gln
705 710 715 720
Val Gln Lys Val Thr His Glu Met Arg Gln Gly Leu His Asn Met Glu
725 730 735
Asp Val Tyr Glu Leu Ile Glu Asn Ser His Leu Thr Pro Lys Lys Gln
740 745 750
Gly Gln Ile Ser Glu Glu Tyr Met Gln Thr Val Leu
755 760
<210> 28
<211> 1869
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
atgaaggaaa atgtggcatc tgcaaccgtt ttcactctgc tactttttct caacacctgc 60
cttctgaatg gacagttacc tcctggaaaa cctgagatct ttaaatgtcg ttctcccaat 120
aaggaaacat tcacctgctg gtggaggcct gggacagatg gaggacttcc taccaattat 180
tcactgactt accacaggga aggagagaca ctcatgcatg aatgtccaga ctacataacc 240
ggtggcccca actcctgcca ctttggcaag cagtacacct ccatgtggag gacatacatc 300
atgatggtca atgccactaa ccagatggga agcagtttct cggatgaact ttatgtggac 360
gtgacttaca tagttcagcc agaccctcct ttggagctgg ctgtggaagt aaaacagcca 420
gaagacagaa aaccctacct gtggattaaa tggtctccac ctaccctgat tgacttaaaa 480
actggttggt tcacgctcct gtatgaaatt cgattaaaac ccgagaaagc agctgagtgg 540
gagatccatt ttgctgggca gcaaacagag tttaagattc tcagcctaca tccaggacag 600
aaataccttg tccaggttcg ctgcaaacca gaccatggat actggagtgc atggagtcca 660
gcgaccttca ttcagatacc tagtgacttc accatgaatg atacaaccgt gtggatctct 720
gtggctgtcc tttctgctgt catctgtttg attattgtct gggcagtggc tttgaagggc 780
tatagcatgg tgacctgcat ctttccgcca gttcctgggc caaaaataaa aggatttgat 840
gctcatctgt tggagaaggg caagtctgaa gaactactga gtgccttggg atgccaagac 900
tttcctccca cttctgacta tgaggacttg ctggtggagt atttagaagt agatgatagt 960
gaggaccagc atctaatgtc agtccattca aaagaacacc caagtcaagg tatgaaaccc 1020
acatacctgg atcctgacac tgactcaggc cgggggagct gtgacagccc ttcccttttg 1080
tctgaaaagt gtgaggaacc ccaggccaat ccctccacat tctatgatcc tgaggtcatt 1140
gagaagccag agaatcctga aacaacccac acctgggacc cccagtgcat aagcatggaa 1200
ggcaaaatcc cctattttca tgctggtgga tccaaatgtt caacatggcc cttaccacag 1260
cccagccagc acaaccccag atcctcttac cacaatatta ctgatgtgtg tgagctggct 1320
gtgggccctg caggtgcacc ggccactctg ttgaatgaag caggtaaaga tgctttaaaa 1380
tcctctcaaa ccattaagtc tagagaagag ggaaaggcaa cccagcagag ggaggtagaa 1440
agcttccatt ctgagactga ccaggatacg ccctggctgc tgccccagga gaaaaccccc 1500
tttggctccg ctaaaccctt ggattatgtg gagattcaca aggtcaacaa agatggtgca 1560
ttatcattgc taccaaaaca gagagagaac agcggcaagc ccaagaagcc cgggactcct 1620
gagaacaata aggagtatgc caaggtgtcc ggggtcatgg ataacaacat cctggtgttg 1680
gtgccagatc cacatgctaa aaacgtggct tgctttgaag aatcagccaa agaggcccca 1740
ccatcacttg aacagaatca agctgagaaa gccctggcca acttcactgc aacatcaagc 1800
aagtgcaggc tccagctggg tggtttggat tacctggatc ccgcatgttt tacacactcc 1860
tttcactga 1869
<210> 29
<211> 622
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Met Lys Glu Asn Val Ala Ser Ala Thr Val Phe Thr Leu Leu Leu Phe
1 5 10 15
Leu Asn Thr Cys Leu Leu Asn Gly Gln Leu Pro Pro Gly Lys Pro Glu
20 25 30
Ile Phe Lys Cys Arg Ser Pro Asn Lys Glu Thr Phe Thr Cys Trp Trp
35 40 45
Arg Pro Gly Thr Asp Gly Gly Leu Pro Thr Asn Tyr Ser Leu Thr Tyr
50 55 60
His Arg Glu Gly Glu Thr Leu Met His Glu Cys Pro Asp Tyr Ile Thr
65 70 75 80
Gly Gly Pro Asn Ser Cys His Phe Gly Lys Gln Tyr Thr Ser Met Trp
85 90 95
Arg Thr Tyr Ile Met Met Val Asn Ala Thr Asn Gln Met Gly Ser Ser
100 105 110
Phe Ser Asp Glu Leu Tyr Val Asp Val Thr Tyr Ile Val Gln Pro Asp
115 120 125
Pro Pro Leu Glu Leu Ala Val Glu Val Lys Gln Pro Glu Asp Arg Lys
130 135 140
Pro Tyr Leu Trp Ile Lys Trp Ser Pro Pro Thr Leu Ile Asp Leu Lys
145 150 155 160
Thr Gly Trp Phe Thr Leu Leu Tyr Glu Ile Arg Leu Lys Pro Glu Lys
165 170 175
Ala Ala Glu Trp Glu Ile His Phe Ala Gly Gln Gln Thr Glu Phe Lys
180 185 190
Ile Leu Ser Leu His Pro Gly Gln Lys Tyr Leu Val Gln Val Arg Cys
195 200 205
Lys Pro Asp His Gly Tyr Trp Ser Ala Trp Ser Pro Ala Thr Phe Ile
210 215 220
Gln Ile Pro Ser Asp Phe Thr Met Asn Asp Thr Thr Val Trp Ile Ser
225 230 235 240
Val Ala Val Leu Ser Ala Val Ile Cys Leu Ile Ile Val Trp Ala Val
245 250 255
Ala Leu Lys Gly Tyr Ser Met Val Thr Cys Ile Phe Pro Pro Val Pro
260 265 270
Gly Pro Lys Ile Lys Gly Phe Asp Ala His Leu Leu Glu Lys Gly Lys
275 280 285
Ser Glu Glu Leu Leu Ser Ala Leu Gly Cys Gln Asp Phe Pro Pro Thr
290 295 300
Ser Asp Tyr Glu Asp Leu Leu Val Glu Tyr Leu Glu Val Asp Asp Ser
305 310 315 320
Glu Asp Gln His Leu Met Ser Val His Ser Lys Glu His Pro Ser Gln
325 330 335
Gly Met Lys Pro Thr Tyr Leu Asp Pro Asp Thr Asp Ser Gly Arg Gly
340 345 350
Ser Cys Asp Ser Pro Ser Leu Leu Ser Glu Lys Cys Glu Glu Pro Gln
355 360 365
Ala Asn Pro Ser Thr Phe Tyr Asp Pro Glu Val Ile Glu Lys Pro Glu
370 375 380
Asn Pro Glu Thr Thr His Thr Trp Asp Pro Gln Cys Ile Ser Met Glu
385 390 395 400
Gly Lys Ile Pro Tyr Phe His Ala Gly Gly Ser Lys Cys Ser Thr Trp
405 410 415
Pro Leu Pro Gln Pro Ser Gln His Asn Pro Arg Ser Ser Tyr His Asn
420 425 430
Ile Thr Asp Val Cys Glu Leu Ala Val Gly Pro Ala Gly Ala Pro Ala
435 440 445
Thr Leu Leu Asn Glu Ala Gly Lys Asp Ala Leu Lys Ser Ser Gln Thr
450 455 460
Ile Lys Ser Arg Glu Glu Gly Lys Ala Thr Gln Gln Arg Glu Val Glu
465 470 475 480
Ser Phe His Ser Glu Thr Asp Gln Asp Thr Pro Trp Leu Leu Pro Gln
485 490 495
Glu Lys Thr Pro Phe Gly Ser Ala Lys Pro Leu Asp Tyr Val Glu Ile
500 505 510
His Lys Val Asn Lys Asp Gly Ala Leu Ser Leu Leu Pro Lys Gln Arg
515 520 525
Glu Asn Ser Gly Lys Pro Lys Lys Pro Gly Thr Pro Glu Asn Asn Lys
530 535 540
Glu Tyr Ala Lys Val Ser Gly Val Met Asp Asn Asn Ile Leu Val Leu
545 550 555 560
Val Pro Asp Pro His Ala Lys Asn Val Ala Cys Phe Glu Glu Ser Ala
565 570 575
Lys Glu Ala Pro Pro Ser Leu Glu Gln Asn Gln Ala Glu Lys Ala Leu
580 585 590
Ala Asn Phe Thr Ala Thr Ser Ser Lys Cys Arg Leu Gln Leu Gly Gly
595 600 605
Leu Asp Tyr Leu Asp Pro Ala Cys Phe Thr His Ser Phe His
610 615 620
<210> 30
<211> 1212
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atgcctaggc caggcacaat ggctctgtgc ctgctgaccc tggtgctgtc cctgctgcca 60
cctcaggctg ctgccacaaa caccacaatc aagtccaacc ccacatctac cccaacagtg 120
cccaggacca caacctgctt cggcgatgga tgtcagaaca cagcttctag atgcaagaac 180
ggcggaacct gggacggcct gaagtgccag tgtcctaacc tgtactacgg agagctgtgc 240
gaggaggtgg tgagctccat cgatatcggc ccacccgaga caatctctgc ccagatggag 300
ctgaccgtga cagtgaccag cgtgaagttc accgaggagc tgaagaacca ctctagccag 360
gagttccagg agtttaagca gaccttcacc gagcagatga acatcgtgta ctctggcatc 420
cctgagtacg tgggagtgaa catcaccaag ctgcggctgg gaagcgtggt ggtggagcac 480
gatgtgctgc tgcgcacaaa gtacacccca gagtacaaga cagtgctgga caacgctacc 540
gaggtggtga aggagaagat cacaaaggtg acaacccagc agatcatgat caacgatatc 600
tgcagcgaca tgatgtgctt caacacaacc ggaacccagg tgcagaacat cacagtgacc 660
cagtacgatc cagaggagga ctgccggaag atggccaagg agtacggcga ttacttcgtg 720
gtggagtacc gcgaccagaa gccctactgc atctcccctt gtgagccagg attttccgtg 780
tctaagaact gcaacctggg caagtgtcag atgagcctgt ccggaccaca gtgcctgtgc 840
gtgacaaccg agacacactg gtactccggc gagacatgta accagggaac ccagaagtct 900
ctggtgtacg gcctggtggg cgctggagtg gtgctgatgc tgatcatcct ggtggccctg 960
ctgatgctgg tgtttaggtc taagagagag gtgaagaggc agaagtacag actgagccag 1020
ctgtacaagt ggcaggagga ggattccgga ccagctcctg gaacattcca gaacatcgga 1080
tttgacatct gtcaggacga tgacagcatc cacctggagt ctatctacag caacttccag 1140
ccctccctga gacacatcga ccctgagaca aagatccgga tccagcgccc tcaggtcatg 1200
acaacctcct tt 1212
<210> 31
<211> 404
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Met Pro Arg Pro Gly Thr Met Ala Leu Cys Leu Leu Thr Leu Val Leu
1 5 10 15
Ser Leu Leu Pro Pro Gln Ala Ala Ala Thr Asn Thr Thr Ile Lys Ser
20 25 30
Asn Pro Thr Ser Thr Pro Thr Val Pro Arg Thr Thr Thr Cys Phe Gly
35 40 45
Asp Gly Cys Gln Asn Thr Ala Ser Arg Cys Lys Asn Gly Gly Thr Trp
50 55 60
Asp Gly Leu Lys Cys Gln Cys Pro Asn Leu Tyr Tyr Gly Glu Leu Cys
65 70 75 80
Glu Glu Val Val Ser Ser Ile Asp Ile Gly Pro Pro Glu Thr Ile Ser
85 90 95
Ala Gln Met Glu Leu Thr Val Thr Val Thr Ser Val Lys Phe Thr Glu
100 105 110
Glu Leu Lys Asn His Ser Ser Gln Glu Phe Gln Glu Phe Lys Gln Thr
115 120 125
Phe Thr Glu Gln Met Asn Ile Val Tyr Ser Gly Ile Pro Glu Tyr Val
130 135 140
Gly Val Asn Ile Thr Lys Leu Arg Leu Gly Ser Val Val Val Glu His
145 150 155 160
Asp Val Leu Leu Arg Thr Lys Tyr Thr Pro Glu Tyr Lys Thr Val Leu
165 170 175
Asp Asn Ala Thr Glu Val Val Lys Glu Lys Ile Thr Lys Val Thr Thr
180 185 190
Gln Gln Ile Met Ile Asn Asp Ile Cys Ser Asp Met Met Cys Phe Asn
195 200 205
Thr Thr Gly Thr Gln Val Gln Asn Ile Thr Val Thr Gln Tyr Asp Pro
210 215 220
Glu Glu Asp Cys Arg Lys Met Ala Lys Glu Tyr Gly Asp Tyr Phe Val
225 230 235 240
Val Glu Tyr Arg Asp Gln Lys Pro Tyr Cys Ile Ser Pro Cys Glu Pro
245 250 255
Gly Phe Ser Val Ser Lys Asn Cys Asn Leu Gly Lys Cys Gln Met Ser
260 265 270
Leu Ser Gly Pro Gln Cys Leu Cys Val Thr Thr Glu Thr His Trp Tyr
275 280 285
Ser Gly Glu Thr Cys Asn Gln Gly Thr Gln Lys Ser Leu Val Tyr Gly
290 295 300
Leu Val Gly Ala Gly Val Val Leu Met Leu Ile Ile Leu Val Ala Leu
305 310 315 320
Leu Met Leu Val Phe Arg Ser Lys Arg Glu Val Lys Arg Gln Lys Tyr
325 330 335
Arg Leu Ser Gln Leu Tyr Lys Trp Gln Glu Glu Asp Ser Gly Pro Ala
340 345 350
Pro Gly Thr Phe Gln Asn Ile Gly Phe Asp Ile Cys Gln Asp Asp Asp
355 360 365
Ser Ile His Leu Glu Ser Ile Tyr Ser Asn Phe Gln Pro Ser Leu Arg
370 375 380
His Ile Asp Pro Glu Thr Lys Ile Arg Ile Gln Arg Pro Gln Val Met
385 390 395 400
Thr Thr Ser Phe
<210> 32
<211> 3222
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atgaagacgt tgctgttgga cttggctttg tggtcactgc tcttccagcc cgggtggctg 60
tcctttagtt cccaggtgag tcagaactgc cacaatggca gctatgaaat cagcgtcctg 120
atgatgggca actcagcctt tgcagagccc ctgaaaaact tggaagatgc ggtgaatgag 180
gggctggaaa tagtgagagg acgtctgcaa aatgctggcc taaatgtgac tgtgaacgct 240
actttcatgt attcggatgg tctgattcat aactcaggcg actgccggag tagcacctgt 300
gaaggcctcg acctactcag gaaaatttca aatgcacaac ggatgggctg tgtcctcata 360
gggccctcat gtacatactc caccttccag atgtaccttg acacagaatt gagctacccc 420
atgatctcag ctggaagttt tggattgtca tgtgactata aagaaacctt aaccaggctg 480
atgtctccag ctagaaagtt gatgtacttc ttggttaact tttggaaaac caacgatctg 540
cccttcaaaa cttattcctg gagcacttcg tatgtttaca agaatggtac agaaactgag 600
gactgtttct ggtaccttaa tgctctggag gctagcgttt cctatttctc ccacgaactc 660
ggctttaagg tggtgttaag acaagataag gagtttcagg atatcttaat ggaccacaac 720
aggaaaagca atgtgattat tatgtgtggt ggtccagagt tcctctacaa gctgaagggt 780
gaccgagcag tggctgaaga cattgtcatt attctagtgg atcttttcaa tgaccagtac 840
tttgaggaca atgtcacagc ccctgactat atgaaaaatg tccttgttct gacgctgtct 900
cctgggaatt cccttctaaa tagctctttc tccaggaatc tatcaccaac aaaacgagac 960
tttgctcttg cctatttgaa tggaatcctg ctctttggac atatgctgaa gatatttctt 1020
gaaaatggag aaaatattac cacccccaaa tttgctcatg ctttcaggaa tctcactttt 1080
gaagggtatg acggtccagt gaccttggat gactgggggg atgttgacag taccatggtg 1140
cttctgtata cctctgtgga caccaagaaa tacaaggttc ttttgaccta tgatacccac 1200
gtaaataaga cctatcctgt ggatatgagc cccacattca cttggaagaa ctctaaactt 1260
cctaatgata ttacaggccg gggccctcag atcctgatga ttgcagtctt caccctcact 1320
ggagctgtgg tgctgctcct gctcgtcgct ctcctgatgc tcagaaaata tagaaaagat 1380
tatgaacttc gtcagaaaaa atggtcccac attcctcctg aaaatatctt tcctctggag 1440
accaatgaga ccaatcatgt tagcctcaag atcgatgatg acaaaagacg agatacaatc 1500
cagagactac gacagtgcaa atacgacaaa aagcgagtga ttctcaaaga tctcaagcac 1560
aatgatggta atttcactga aaaacagaag atagaattga acaagttgct tcagattgac 1620
tattacaacc tgaccaagtt ctacggcaca gtgaaacttg ataccatgat cttcggggtg 1680
atagaatact gtgagagagg atccctccgg gaagttttaa atgacacaat ttcctaccct 1740
gatggcacat tcatggattg ggagtttaag atctctgtct tgtatgacat tgctaaggga 1800
atgtcatatc tgcactccag taagacagaa gtccatggtc gtctgaaatc taccaactgc 1860
gtagtggaca gtagaatggt ggtgaagatc actgattttg gctgcaattc cattttacct 1920
ccaaaaaagg acctgtggac agctccagag cacctccgcc aagccaacat ctctcagaaa 1980
ggagatgtgt acagctatgg gatcatcgca caggagatca tcctgcggaa agaaaccttc 2040
tacactttga gctgtcggga ccggaatgag aagattttca gagtggaaaa ttccaatgga 2100
atgaaaccct tccgcccaga tttattcttg gaaacagcag aggaaaaaga gctagaagtg 2160
tacctacttg taaaaaactg ttgggaggaa gatccagaaa agagaccaga tttcaaaaaa 2220
attgagacta cacttgccaa gatatttgga ctttttcatg accaaaaaaa tgaaagctat 2280
atggatacct tgatccgacg tctacagcta tattctcgaa acctggaaca tctggtagag 2340
gaaaggacac agctgtacaa ggcagagagg gacagggctg acagacttaa ctttatgttg 2400
cttccaaggc tagtggtaaa gtctctgaag gagaaaggct ttgtggagcc ggaactatat 2460
gaggaagtta caatctactt cagtgacatt gtaggtttca ctactatctg caaatacagc 2520
acccccatgg aagtggtgga catgcttaat gacatctata agagttttga ccacattgtt 2580
gatcatcatg atgtctacaa ggtggaaacc atcggtgatg cgtacatggt ggctagtggt 2640
ttgcctaaga gaaatggcaa tcggcatgca atagacattg ccaagatggc cttggaaatc 2700
ctcagcttca tggggacctt tgagctggag catcttcctg gcctcccaat atggattcgc 2760
attggagttc actctggtcc ctgtgctgct ggagttgtgg gaatcaagat gcctcgttat 2820
tgtctatttg gagatacggt caacacagcc tctaggatgg aatccactgg cctccctttg 2880
agaattcacg tgagtggctc caccatagcc atcctgaaga gaactgagtg ccagttcctt 2940
tatgaagtga gaggagaaac atacttaaag ggaagaggaa atgagactac ctactggctg 3000
actgggatga aggaccagaa attcaacctg ccaacccctc ctactgtgga gaatcaacag 3060
cgtttgcaag cagaattttc agacatgatt gccaactctt tacagaaaag acaggcagca 3120
gggataagaa gccaaaaacc cagacgggta gccagctata aaaaaggcac tctggaatac 3180
ttgcagctga ataccacaga caaggagagc acctattttt aa 3222
<210> 33
<211> 1073
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Met Lys Thr Leu Leu Leu Asp Leu Ala Leu Trp Ser Leu Leu Phe Gln
1 5 10 15
Pro Gly Trp Leu Ser Phe Ser Ser Gln Val Ser Gln Asn Cys His Asn
20 25 30
Gly Ser Tyr Glu Ile Ser Val Leu Met Met Gly Asn Ser Ala Phe Ala
35 40 45
Glu Pro Leu Lys Asn Leu Glu Asp Ala Val Asn Glu Gly Leu Glu Ile
50 55 60
Val Arg Gly Arg Leu Gln Asn Ala Gly Leu Asn Val Thr Val Asn Ala
65 70 75 80
Thr Phe Met Tyr Ser Asp Gly Leu Ile His Asn Ser Gly Asp Cys Arg
85 90 95
Ser Ser Thr Cys Glu Gly Leu Asp Leu Leu Arg Lys Ile Ser Asn Ala
100 105 110
Gln Arg Met Gly Cys Val Leu Ile Gly Pro Ser Cys Thr Tyr Ser Thr
115 120 125
Phe Gln Met Tyr Leu Asp Thr Glu Leu Ser Tyr Pro Met Ile Ser Ala
130 135 140
Gly Ser Phe Gly Leu Ser Cys Asp Tyr Lys Glu Thr Leu Thr Arg Leu
145 150 155 160
Met Ser Pro Ala Arg Lys Leu Met Tyr Phe Leu Val Asn Phe Trp Lys
165 170 175
Thr Asn Asp Leu Pro Phe Lys Thr Tyr Ser Trp Ser Thr Ser Tyr Val
180 185 190
Tyr Lys Asn Gly Thr Glu Thr Glu Asp Cys Phe Trp Tyr Leu Asn Ala
195 200 205
Leu Glu Ala Ser Val Ser Tyr Phe Ser His Glu Leu Gly Phe Lys Val
210 215 220
Val Leu Arg Gln Asp Lys Glu Phe Gln Asp Ile Leu Met Asp His Asn
225 230 235 240
Arg Lys Ser Asn Val Ile Ile Met Cys Gly Gly Pro Glu Phe Leu Tyr
245 250 255
Lys Leu Lys Gly Asp Arg Ala Val Ala Glu Asp Ile Val Ile Ile Leu
260 265 270
Val Asp Leu Phe Asn Asp Gln Tyr Phe Glu Asp Asn Val Thr Ala Pro
275 280 285
Asp Tyr Met Lys Asn Val Leu Val Leu Thr Leu Ser Pro Gly Asn Ser
290 295 300
Leu Leu Asn Ser Ser Phe Ser Arg Asn Leu Ser Pro Thr Lys Arg Asp
305 310 315 320
Phe Ala Leu Ala Tyr Leu Asn Gly Ile Leu Leu Phe Gly His Met Leu
325 330 335
Lys Ile Phe Leu Glu Asn Gly Glu Asn Ile Thr Thr Pro Lys Phe Ala
340 345 350
His Ala Phe Arg Asn Leu Thr Phe Glu Gly Tyr Asp Gly Pro Val Thr
355 360 365
Leu Asp Asp Trp Gly Asp Val Asp Ser Thr Met Val Leu Leu Tyr Thr
370 375 380
Ser Val Asp Thr Lys Lys Tyr Lys Val Leu Leu Thr Tyr Asp Thr His
385 390 395 400
Val Asn Lys Thr Tyr Pro Val Asp Met Ser Pro Thr Phe Thr Trp Lys
405 410 415
Asn Ser Lys Leu Pro Asn Asp Ile Thr Gly Arg Gly Pro Gln Ile Leu
420 425 430
Met Ile Ala Val Phe Thr Leu Thr Gly Ala Val Val Leu Leu Leu Leu
435 440 445
Val Ala Leu Leu Met Leu Arg Lys Tyr Arg Lys Asp Tyr Glu Leu Arg
450 455 460
Gln Lys Lys Trp Ser His Ile Pro Pro Glu Asn Ile Phe Pro Leu Glu
465 470 475 480
Thr Asn Glu Thr Asn His Val Ser Leu Lys Ile Asp Asp Asp Lys Arg
485 490 495
Arg Asp Thr Ile Gln Arg Leu Arg Gln Cys Lys Tyr Asp Lys Lys Arg
500 505 510
Val Ile Leu Lys Asp Leu Lys His Asn Asp Gly Asn Phe Thr Glu Lys
515 520 525
Gln Lys Ile Glu Leu Asn Lys Leu Leu Gln Ile Asp Tyr Tyr Asn Leu
530 535 540
Thr Lys Phe Tyr Gly Thr Val Lys Leu Asp Thr Met Ile Phe Gly Val
545 550 555 560
Ile Glu Tyr Cys Glu Arg Gly Ser Leu Arg Glu Val Leu Asn Asp Thr
565 570 575
Ile Ser Tyr Pro Asp Gly Thr Phe Met Asp Trp Glu Phe Lys Ile Ser
580 585 590
Val Leu Tyr Asp Ile Ala Lys Gly Met Ser Tyr Leu His Ser Ser Lys
595 600 605
Thr Glu Val His Gly Arg Leu Lys Ser Thr Asn Cys Val Val Asp Ser
610 615 620
Arg Met Val Val Lys Ile Thr Asp Phe Gly Cys Asn Ser Ile Leu Pro
625 630 635 640
Pro Lys Lys Asp Leu Trp Thr Ala Pro Glu His Leu Arg Gln Ala Asn
645 650 655
Ile Ser Gln Lys Gly Asp Val Tyr Ser Tyr Gly Ile Ile Ala Gln Glu
660 665 670
Ile Ile Leu Arg Lys Glu Thr Phe Tyr Thr Leu Ser Cys Arg Asp Arg
675 680 685
Asn Glu Lys Ile Phe Arg Val Glu Asn Ser Asn Gly Met Lys Pro Phe
690 695 700
Arg Pro Asp Leu Phe Leu Glu Thr Ala Glu Glu Lys Glu Leu Glu Val
705 710 715 720
Tyr Leu Leu Val Lys Asn Cys Trp Glu Glu Asp Pro Glu Lys Arg Pro
725 730 735
Asp Phe Lys Lys Ile Glu Thr Thr Leu Ala Lys Ile Phe Gly Leu Phe
740 745 750
His Asp Gln Lys Asn Glu Ser Tyr Met Asp Thr Leu Ile Arg Arg Leu
755 760 765
Gln Leu Tyr Ser Arg Asn Leu Glu His Leu Val Glu Glu Arg Thr Gln
770 775 780
Leu Tyr Lys Ala Glu Arg Asp Arg Ala Asp Arg Leu Asn Phe Met Leu
785 790 795 800
Leu Pro Arg Leu Val Val Lys Ser Leu Lys Glu Lys Gly Phe Val Glu
805 810 815
Pro Glu Leu Tyr Glu Glu Val Thr Ile Tyr Phe Ser Asp Ile Val Gly
820 825 830
Phe Thr Thr Ile Cys Lys Tyr Ser Thr Pro Met Glu Val Val Asp Met
835 840 845
Leu Asn Asp Ile Tyr Lys Ser Phe Asp His Ile Val Asp His His Asp
850 855 860
Val Tyr Lys Val Glu Thr Ile Gly Asp Ala Tyr Met Val Ala Ser Gly
865 870 875 880
Leu Pro Lys Arg Asn Gly Asn Arg His Ala Ile Asp Ile Ala Lys Met
885 890 895
Ala Leu Glu Ile Leu Ser Phe Met Gly Thr Phe Glu Leu Glu His Leu
900 905 910
Pro Gly Leu Pro Ile Trp Ile Arg Ile Gly Val His Ser Gly Pro Cys
915 920 925
Ala Ala Gly Val Val Gly Ile Lys Met Pro Arg Tyr Cys Leu Phe Gly
930 935 940
Asp Thr Val Asn Thr Ala Ser Arg Met Glu Ser Thr Gly Leu Pro Leu
945 950 955 960
Arg Ile His Val Ser Gly Ser Thr Ile Ala Ile Leu Lys Arg Thr Glu
965 970 975
Cys Gln Phe Leu Tyr Glu Val Arg Gly Glu Thr Tyr Leu Lys Gly Arg
980 985 990
Gly Asn Glu Thr Thr Tyr Trp Leu Thr Gly Met Lys Asp Gln Lys Phe
995 1000 1005
Asn Leu Pro Thr Pro Pro Thr Val Glu Asn Gln Gln Arg Leu Gln Ala
1010 1015 1020
Glu Phe Ser Asp Met Ile Ala Asn Ser Leu Gln Lys Arg Gln Ala Ala
1025 1030 1035 1040
Gly Ile Arg Ser Gln Lys Pro Arg Arg Val Ala Ser Tyr Lys Lys Gly
1045 1050 1055
Thr Leu Glu Tyr Leu Gln Leu Asn Thr Thr Asp Lys Glu Ser Thr Tyr
1060 1065 1070
Phe
<210> 34
<211> 987
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
atgactgtgg agaaggaggc ccctgatgcg cacttcactg tggacaaaca gaacatctcc 60
ctctggcccc gagagcctcc tcccaagtcc ggtccatctc tggtcccggg gaaaacaccc 120
acagtccgtg ctgcattaat ctgcctgacg ctggtcctgg tcgcctccgt cctgctgcag 180
gccgtccttt atccccggtt tatgggcacc atatcagatg taaagaccaa tgtccagttg 240
ctgaaaggtc gtgtggacaa catcagcacc ctggattctg aaattaaaaa gaatagtgac 300
ggcatggagg cagctggcgt tcagatccag atggtgaatg agagcctggg ttatgtgcgt 360
tctcagttcc tgaagttaaa aaccagtgtg gagaaggcca acgcacagat ccagatctta 420
acaagaagtt gggaagaagt cagtacctta aatgcccaaa tcccagagtt aaaaagtgat 480
ttggagaaag ccagtgcttt aaatacaaag atccgggcac tccagggcag cttggagaat 540
atgagcaagt tgctcaaacg acaaaatgat attctacagg tggtttctca aggctggaag 600
tacttcaagg ggaacttcta ttacttttct ctcattccaa agacctggta tagtgccgag 660
cagttctgtg tgtccaggaa ttcacacctg acctcggtga cctcagagag tgagcaggag 720
tttctgtata aaacagcggg gggactcatc tactggattg gcctgactaa agcagggatg 780
gaaggggact ggtcctgggt ggatgacacg ccattcaaca aggtccaaag tgtgaggttc 840
tggattccag gtgagcccaa caatgctggg aacaatgaac actgtggcaa tataaaggct 900
ccctcacttc aggcctggaa tgatgcccca tgtgacaaaa cgtttctttt catttgtaag 960
cgaccctatg tcccatcaga accgtga 987
<210> 35
<211> 328
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Met Thr Val Glu Lys Glu Ala Pro Asp Ala His Phe Thr Val Asp Lys
1 5 10 15
Gln Asn Ile Ser Leu Trp Pro Arg Glu Pro Pro Pro Lys Ser Gly Pro
20 25 30
Ser Leu Val Pro Gly Lys Thr Pro Thr Val Arg Ala Ala Leu Ile Cys
35 40 45
Leu Thr Leu Val Leu Val Ala Ser Val Leu Leu Gln Ala Val Leu Tyr
50 55 60
Pro Arg Phe Met Gly Thr Ile Ser Asp Val Lys Thr Asn Val Gln Leu
65 70 75 80
Leu Lys Gly Arg Val Asp Asn Ile Ser Thr Leu Asp Ser Glu Ile Lys
85 90 95
Lys Asn Ser Asp Gly Met Glu Ala Ala Gly Val Gln Ile Gln Met Val
100 105 110
Asn Glu Ser Leu Gly Tyr Val Arg Ser Gln Phe Leu Lys Leu Lys Thr
115 120 125
Ser Val Glu Lys Ala Asn Ala Gln Ile Gln Ile Leu Thr Arg Ser Trp
130 135 140
Glu Glu Val Ser Thr Leu Asn Ala Gln Ile Pro Glu Leu Lys Ser Asp
145 150 155 160
Leu Glu Lys Ala Ser Ala Leu Asn Thr Lys Ile Arg Ala Leu Gln Gly
165 170 175
Ser Leu Glu Asn Met Ser Lys Leu Leu Lys Arg Gln Asn Asp Ile Leu
180 185 190
Gln Val Val Ser Gln Gly Trp Lys Tyr Phe Lys Gly Asn Phe Tyr Tyr
195 200 205
Phe Ser Leu Ile Pro Lys Thr Trp Tyr Ser Ala Glu Gln Phe Cys Val
210 215 220
Ser Arg Asn Ser His Leu Thr Ser Val Thr Ser Glu Ser Glu Gln Glu
225 230 235 240
Phe Leu Tyr Lys Thr Ala Gly Gly Leu Ile Tyr Trp Ile Gly Leu Thr
245 250 255
Lys Ala Gly Met Glu Gly Asp Trp Ser Trp Val Asp Asp Thr Pro Phe
260 265 270
Asn Lys Val Gln Ser Val Arg Phe Trp Ile Pro Gly Glu Pro Asn Asn
275 280 285
Ala Gly Asn Asn Glu His Cys Gly Asn Ile Lys Ala Pro Ser Leu Gln
290 295 300
Ala Trp Asn Asp Ala Pro Cys Asp Lys Thr Phe Leu Phe Ile Cys Lys
305 310 315 320
Arg Pro Tyr Val Pro Ser Glu Pro
325
<210> 36
<211> 480
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu
20 25 30
Ser Val Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu
35 40 45
Asn Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser
50 55 60
Pro Gln Leu Leu Val Tyr Val Ala Thr Asn Leu Ala Asp Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile
85 90 95
Asn Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe
100 105 110
Trp Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln
130 135 140
Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser
145 150 155 160
Cys Thr Ala Ser Gly Phe Asn Ile Asn Asp Thr Tyr Met His Trp Val
165 170 175
Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro
180 185 190
Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr
195 200 205
Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser
210 215 220
Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala Arg
225 230 235 240
Gly Ser Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
245 250 255
Ala Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
260 265 270
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
275 280 285
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
290 295 300
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
305 310 315 320
Leu Ser Leu Val Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
325 330 335
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
340 345 350
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
355 360 365
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
370 375 380
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
385 390 395 400
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
405 410 415
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
420 425 430
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
435 440 445
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
450 455 460
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475 480
<210> 37
<211> 488
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
20 25 30
Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser
35 40 45
Asp Ile Gly Ser Asn Tyr Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Ala Ile
65 70 75 80
Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly
85 90 95
Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr
100 105 110
Trp Asp Ser Arg Leu Gly Ile Ala Val Phe Gly Gly Gly Thr Gln Leu
115 120 125
Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
145 150 155 160
Gly Gln Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr
165 170 175
Asp Asn Trp Ile Gly Trp Val Arg Gln Lys Pro Gly Lys Gly Leu Glu
180 185 190
Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro
195 200 205
Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Asn Thr
210 215 220
Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr
225 230 235 240
Tyr Cys Val Gly Leu Asp Trp Asn Tyr Asn Pro Leu Arg Tyr Trp Gly
245 250 255
Pro Gly Thr Leu Val Thr Val Ser Ser Ala Lys Pro Thr Thr Thr Pro
260 265 270
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
275 280 285
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
290 295 300
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
305 310 315 320
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg
325 330 335
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
340 345 350
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
355 360 365
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
370 375 380
Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
385 390 395 400
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
405 410 415
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210> 38
<211> 496
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
20 25 30
Ala Val Ser Leu Gly Gln Gly Ala Thr Ile Ser Cys Arg Ala Ser Lys
35 40 45
Ser Val Ser Thr Ser Gly Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys
50 55 60
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu
65 70 75 80
Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
85 90 95
Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr
100 105 110
Cys Gln His Ser Gly Glu Leu Pro Pro Ser Phe Gly Gly Gly Thr Lys
115 120 125
Leu Glu Ile Lys Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu
145 150 155 160
Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe
165 170 175
Thr Phe Ser Ser Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys
180 185 190
Arg Leu Glu Trp Val Ala Thr Val Ser Ser Gly Gly Thr Tyr Thr Tyr
195 200 205
Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
210 215 220
Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Ser
225 230 235 240
Ala Met Tyr Tyr Cys Ala Arg His Arg Gly Asn Tyr Tyr Ala Thr Tyr
245 250 255
Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
260 265 270
Ser Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
275 280 285
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
290 295 300
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
305 310 315 320
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
325 330 335
Leu Ser Leu Val Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
340 345 350
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
355 360 365
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
370 375 380
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
385 390 395 400
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
405 410 415
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
420 425 430
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
435 440 445
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
450 455 460
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
465 470 475 480
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490 495
<210> 39
<211> 487
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu
20 25 30
Ala Ala Ser Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys
35 40 45
Ser Ile Ser Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr
50 55 60
Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His
100 105 110
His Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
130 135 140
Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser
145 150 155 160
Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Trp
165 170 175
Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly
180 185 190
Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys
195 200 205
Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
210 215 220
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
225 230 235 240
Arg Gly Pro Tyr Tyr Gly Thr Asn Pro Trp Phe Pro Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ser Ala Lys Pro Thr Thr Thr Pro Ala
260 265 270
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
305 310 315 320
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg Gly
325 330 335
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
340 345 350
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
355 360 365
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
370 375 380
Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
385 390 395 400
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
405 410 415
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
420 425 430
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
450 455 460
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
465 470 475 480
Met Gln Ala Leu Pro Pro Arg
485
<210> 40
<211> 485
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
20 25 30
Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
35 40 45
Ser Val Ser Arg Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
85 90 95
Gly Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
100 105 110
Lys Thr Trp Pro Arg Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Lys
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
130 135 140
Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr
145 150 155 160
Leu Ser Leu Thr Cys Ala Val Phe Gly Gly Ser Phe Ser Gly Tyr Tyr
165 170 175
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly
180 185 190
Glu Ile Asn His Arg Gly Asn Thr Asn Asp Asn Pro Ser Leu Lys Ser
195 200 205
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ala Leu Lys
210 215 220
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg
225 230 235 240
Glu Arg Gly Tyr Thr Tyr Gly Asn Phe Asp His Trp Gly Gln Gly Thr
245 250 255
Leu Val Thr Val Ser Ser Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg
260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
275 280 285
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
305 310 315 320
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg Gly Arg Lys
325 330 335
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
340 345 350
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
355 360 365
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
370 375 380
Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
385 390 395 400
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
405 410 415
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
420 425 430
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
435 440 445
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
450 455 460
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
465 470 475 480
Ala Leu Pro Pro Arg
485
<210> 41
<211> 490
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu
20 25 30
Pro Val Arg Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
35 40 45
Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln
50 55 60
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg
65 70 75 80
Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn
85 90 95
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr
100 105 110
Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr
115 120 125
Lys Leu Glu Ile Lys Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Arg Gln Ser Gly Pro Glu
145 150 155 160
Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
165 170 175
Tyr Thr Phe Thr Asp Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly
180 185 190
Gln Gly Leu Glu Trp Ile Gly Asp Ile Tyr Pro Gly Ser Gly Tyr Ser
195 200 205
Phe Tyr Asn Glu Asn Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys
210 215 220
Ser Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
225 230 235 240
Ser Ala Val Tyr Phe Cys Ala Thr Tyr Tyr Asn Tyr Pro Phe Ala Tyr
245 250 255
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Pro Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380
Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
420 425 430
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
435 440 445
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
450 455 460
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
465 470 475 480
Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 42
<211> 443
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Leu Pro Ile Cys Pro Gly Gly Ala Ala Arg Cys
20 25 30
Gln Val Thr Leu Arg Asp Leu Phe Asp Arg Ala Val Val Leu Ser His
35 40 45
Tyr Ile His Asn Leu Ser Ser Glu Met Phe Ser Glu Phe Asp Lys Arg
50 55 60
Tyr Thr His Gly Arg Gly Phe Ile Thr Lys Ala Ile Asn Ser Cys His
65 70 75 80
Thr Ser Ser Leu Ala Thr Pro Glu Asp Lys Glu Gln Ala Gln Gln Met
85 90 95
Asn Gln Lys Asp Phe Leu Ser Leu Ile Val Ser Ile Leu Arg Ser Trp
100 105 110
Asn Glu Pro Leu Tyr His Leu Val Thr Glu Val Arg Gly Met Gln Glu
115 120 125
Ala Pro Glu Ala Ile Leu Ser Lys Ala Val Glu Ile Glu Glu Gln Thr
130 135 140
Lys Arg Leu Leu Glu Gly Met Glu Leu Ile Val Ser Gln Val His Pro
145 150 155 160
Glu Thr Lys Glu Asn Glu Ile Tyr Pro Val Trp Ser Gly Leu Pro Ser
165 170 175
Leu Gln Met Ala Asp Glu Glu Ser Arg Leu Ser Ala Tyr Tyr Asn Leu
180 185 190
Leu His Cys Leu Arg Arg Asp Ser His Lys Ile Asp Asn Tyr Leu Lys
195 200 205
Leu Leu Lys Cys Arg Ile Ile His Asn Asn Asn Cys Ala Lys Pro Thr
210 215 220
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
225 230 235 240
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
245 250 255
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
260 265 270
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
275 280 285
Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
290 295 300
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
305 310 315 320
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
325 330 335
Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr
340 345 350
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
355 360 365
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
370 375 380
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
385 390 395 400
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
405 410 415
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
420 425 430
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
435 440
<210> 43
<211> 434
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Arg Cys Gln Val Thr Leu Arg Asp Leu Phe Asp
20 25 30
Arg Ala Val Val Leu Ser His Tyr Ile His Asn Leu Ser Ser Glu Met
35 40 45
Phe Ser Glu Phe Asp Lys Arg Tyr Thr His Gly Arg Gly Phe Ile Thr
50 55 60
Lys Ala Ile Asn Ser Cys His Thr Ser Ser Leu Ala Thr Pro Glu Asp
65 70 75 80
Lys Glu Gln Ala Gln Gln Met Asn Gln Lys Asp Phe Leu Ser Leu Ile
85 90 95
Val Ser Ile Leu Arg Ser Trp Asn Glu Pro Leu Tyr His Leu Val Thr
100 105 110
Glu Val Arg Gly Met Gln Glu Ala Pro Glu Ala Ile Leu Ser Lys Ala
115 120 125
Val Glu Ile Glu Glu Gln Thr Lys Arg Leu Leu Glu Arg Met Glu Leu
130 135 140
Ile Val Ser Gln Val His Pro Glu Thr Lys Glu Asn Glu Ile Tyr Pro
145 150 155 160
Val Trp Ser Gly Leu Pro Ser Leu Gln Met Ala Asp Glu Glu Ser Arg
165 170 175
Leu Ser Ala Tyr Tyr Asn Leu Leu His Cys Leu Arg Arg Asp Ser His
180 185 190
Lys Ile Asp Asn Tyr Leu Lys Leu Leu Lys Cys Arg Ile Ile His Asn
195 200 205
Asn Asn Cys Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
210 215 220
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
225 230 235 240
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
245 250 255
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
260 265 270
Leu Leu Leu Ser Leu Val Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu
275 280 285
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
290 295 300
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
305 310 315 320
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
325 330 335
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
340 345 350
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
355 360 365
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
370 375 380
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
385 390 395 400
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
405 410 415
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
420 425 430
Pro Arg
<210> 44
<211> 190
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
Arg Cys Gln Val Thr Leu Arg Asp Leu Phe Asp Arg Ala Val Val Leu
1 5 10 15
Ser His Tyr Ile His Asn Leu Ser Ser Glu Met Phe Ser Glu Phe Asp
20 25 30
Lys Arg Tyr Thr His Gly Arg Gly Phe Ile Thr Lys Ala Ile Asn Ser
35 40 45
Cys His Thr Ser Ser Leu Ala Thr Pro Glu Asp Lys Glu Gln Ala Gln
50 55 60
Gln Met Asn Gln Lys Asp Phe Leu Ser Leu Ile Val Ser Ile Leu Arg
65 70 75 80
Ser Trp Asn Glu Pro Leu Tyr His Leu Val Thr Glu Val Arg Gly Met
85 90 95
Gln Glu Ala Pro Glu Ala Ile Leu Ser Lys Ala Val Glu Ile Glu Glu
100 105 110
Gln Thr Lys Arg Leu Leu Glu Arg Met Glu Leu Ile Val Ser Gln Val
115 120 125
His Pro Glu Thr Lys Glu Asn Glu Ile Tyr Pro Val Trp Ser Gly Leu
130 135 140
Pro Ser Leu Gln Met Ala Asp Glu Glu Ser Arg Leu Ser Ala Tyr Tyr
145 150 155 160
Asn Leu Leu His Cys Leu Arg Arg Asp Ser His Lys Ile Asp Asn Tyr
165 170 175
Leu Lys Leu Leu Lys Cys Arg Ile Ile His Asn Asn Asn Cys
180 185 190
Claims (5)
1.人T细胞在制备治疗结肠直肠癌药物中的用途,所述人T细胞包含编码嵌合抗原受体(CAR)的核酸序列,所述CAR包含抗原结合结构域,跨膜结构域,共刺激信号传导区和CD3ζ信号传导结构域;所述抗原结合结构域与在肿瘤微环境中存在的非必需器官细胞表面上表达的抗原鸟苷酸环化酶2C(GUCY2C)结合,所述抗原结合结构域的氨基酸序列如SEQ ID NO:14所示。
2.根据权利要求1所述的用途,所述CAR的氨基酸序列如SEQ ID NO:40所示。
3.根据权利要求1所述的用途,所述鸟苷酸环化酶2C(GUCY2C)具有SEQ ID NO:33所示的氨基酸序列。
4.根据权利要求1所述的用途,其中抗原GUCY2C表达量至少比合适的对照细胞高至1.5倍,2倍,5倍,10倍,100倍或1000倍中的至少一种。
5.根据权利要求1所述的用途,其中共刺激信号传导区选自CD27,CD28,4-1BB,OX40,CD30,CD40,PD-1,ICOS,淋巴细胞功能相关抗原-1的共刺激分子的细胞内结构域LFA-1,CD2,CD7,LIGHT,NKG2C,B7-H3,与CD83特异性结合的配体,及其任何组合。
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WO2015142675A2 (en) * | 2014-03-15 | 2015-09-24 | Novartis Ag | Treatment of cancer using chimeric antigen receptor |
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WO2015142675A2 (en) * | 2014-03-15 | 2015-09-24 | Novartis Ag | Treatment of cancer using chimeric antigen receptor |
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