CN108430499B - 嵌合和人源化抗人类ctla4单克隆抗体和其用途 - Google Patents
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Abstract
本发明涉及与人类CTLA4分子结合的嵌合和人源化抗体的组合物以及其在癌症免疫疗法中和用于与其它免疫治疗剂相比减少自身免疫副作用的用途。
Description
技术领域
本发明涉及与人类CTLA4分子结合的嵌合和人源化抗体以及其使用方法。
背景技术
人类和其它哺乳动物的免疫系统负责提供针对感染和疾病的保护。这类保护由体液免疫应答和细胞介导的免疫应答两者提供。体液应答导致产生能够识别和中和外来标靶(抗原)的抗体和其它生物分子。相比之下,细胞介导的免疫应答涉及巨噬细胞、嗜中性粒细胞、自然杀伤细胞(NK)和抗原特异性细胞毒性T淋巴细胞由T细胞的活化,以及各种细胞因子响应于抗原识别的释放。
T细胞最优地介导针对抗原的免疫应答的能力需要两种独特信号传导相互作用。首先,排列在抗原呈递细胞(APC)表面上的抗原必须以MHC:肽复合物形式呈递给抗原特异性原初T细胞(1,2)。这类呈递通过T细胞受体(TCR)递送信号,所述T细胞受体引导T细胞引发对所呈递的抗原具有特异性的免疫应答。第二,通过APC与独特T 细胞表面分子之间的相互作用介导的一系列共刺激信号首先触发T细胞的活化和增殖并且最终触发其抑制(3-5)。因此,第一信号赋予免疫应答特异性,而第二信号用以确定应答的性质、量值和持续时间同时将免疫性限于自身。这些第二信号分子之中特别重要的是抗原呈递细胞的B7.1(CD80)(6)和B7.2(CD86)(7-9)配体与T淋巴细胞的 CD28和CTLA4受体(10-12)之间的结合。
细胞毒性T淋巴细胞抗原-4(CTLA4)被认为是适应性免疫应答的关键调节因子,在维持外周耐受性和使出现的T细胞应答库成形方面具有重要作用,并且因此是治疗癌症和炎症的治疗标靶。用抗CTLA4抗体治疗已经在临床前模型中显示为增强抗肿瘤免疫性的强大工具(10)。用针对CTLA4的抗体进行的单一疗法促进对各种来源的可移植肿瘤的排斥。
基于有前景的临床前肿瘤模型研究,已经对针对CTLA4的抗体在不同人类恶性肿瘤中的临床潜力进行了探索。尽管抗CTLA4(伊匹单抗(Ipilimumab),以Yervoy销售) 已经在治疗黑色素瘤方面展现了功效,但CTLA4的治疗和靶向与自身免疫类毒性相关。来自CTLA4抑制的特征副作用通常被称为免疫相关不良事件(irAE),并且最常见的 irAE是皮疹、肝炎、结肠炎和内分泌病、尤其垂体机能减退。因此,需要通过增加功效同时减少相关irAE来改进抗CTLA4抗体的治疗潜力。
免疫疗法和肿瘤治疗领域的另一焦点是组合不同免疫检查抑制剂以便增强抗肿瘤活性,尤其是针对免疫原性差的肿瘤。然而,这一方法与进一步增加自身免疫副作用的风险相关,进一步突显了需要选择性调节癌症免疫性而不增强自身免疫性。
对CD28受体的配体的进一步研究已经鉴别和表征了一组相关B7分子(“B7超家族”)(32-33)。当前存在若干已知的家族成员:B7.1(CD80)、B7.2(CD86)、可诱导共刺激因子配体(ICOS-L)、程序性死亡-1配体(PD-L1;B7-H1)、程序性死亡-2配体(PD- L2;B7-DC)、B7-H3、B7-H4和B7-H6(35-36)。
B7-H1广泛表达于不同人类和小鼠组织中,如两种物种的心脏、胎盘、肌肉、胎肝、脾脏、淋巴结和胸腺以及仅小鼠的肝脏、肺脏和肾脏中(37)。B7-H1(PD-L1,CD274) 是B7超家族的特别重要的成员,因为其在使对肿瘤的免疫应答成形中起关键作用(38;美国专利第6,803,192号、第7,794,710号;美国专利申请公开第2005/0059051号、第 2009/0055944号、第2009/0274666号、第2009/0313687号;PCT公开第WO 01/39722 号、第WO 02/086083号)。
程序性死亡-1(“PD-1”)是B7-H1以及B7-DC的受体。PD-1是T细胞调节因子的延伸CD28/CTLA4家族的I型膜蛋白成员(39;美国专利申请公开第2007/0202100号、第2008/0311117号、第2009/00110667号;美国专利第6,808,710号、第7,101,550号、第7,488,802号、第7,635,757号、第7,722,868号;PCT公开第WO 01/14557号)。与 CTLA4相比,PD-1更广泛地负面调节免疫应答。PD-1表达于活化T细胞、B细胞和单核细胞上(40-41)并且以低水平表达于自然杀伤(NK)T细胞中(42-43)。
B7-H1与PD-1的相互作用已经被发现可向T和B细胞提供关键负共刺激信号(43)并且充当细胞死亡诱导因子(39)。B7-H1和PD-1在抑制T细胞活化和增殖中的作用已经表明,这些生物分子可以充当治疗炎症和癌症的治疗标靶。因此,已经提出使用抗PD1 和抗B7-H1抗体治疗感染和肿瘤以及上调适应性免疫应答,并且据证实对治疗多种人类肿瘤有效。然而,并非全部受试者都对抗PD-1或抗B7-H1治疗有应答或具有完全应答,并且因此对将抗PD-1或抗B7-H1抗体与其它免疫检查抑制剂组合以便增强抗肿瘤活性存在很大兴趣。
4-1BB(也称为CD137和TNFRSF9)是另一免疫检查点分子。CD137的最具特征的活性是其对于活化T细胞的共刺激活性。CD137的交联增强T细胞增殖、IL-2分泌、存活和溶细胞活性。此外,类似抗CTLA4,抗4-1BB抗体可以在小鼠中增强免疫活性以消除肿瘤(27-29)。然而,不同于抗CTLA4抗体有加重自身免疫疾病的倾向,癌症治疗性抗4-1BB mAb据显示可消除自身免疫疾病在易感狼疮的小鼠中的出现,其中其抑制抗dsDNA抗体产生并且减轻肾脏病理学(25,26)。先前数据已经证实,有可能通过组合抗CTLA4治疗与抗4-1BB抗体来减少抗CTLA4治疗在小鼠结肠癌肿瘤模型中的自身免疫副作用,同时增强抗肿瘤活性(19)。这证实,有可能抵消抗CTLA4肿瘤疗法的自身免疫副作用。
抗人类CTLA4抗体的临床前筛选充满了困难,因为体外免疫相关性有时具有极小价值,如抗小鼠CTLA4抗体的经验所展示。诱导体内有力抗肿瘤免疫性的相同抗小鼠 CTLA4抗体可以对体外T细胞具有可变效应。抗CTLA4抗体响应于同种异体抗原增强 T细胞增殖,但响应于抗CD 28的共刺激抑制T细胞增殖(30,31)。此外,CTLA4与抗体结合可以促进或抑制相同培养物中的不同T细胞亚组的增殖(32)。如果可以研究啮齿动物模型中的人类T细胞应答,那么这一复杂情况可以被克服。
本文描述当用以增强免疫应答和用于抗肿瘤疗法时自身免疫副作用减少的抗CTLA4抗体。此外,这些抗体可以与其它检查点抑制剂(如抗PD-1和抗4-1BB)组合用以增强抗肿瘤同时消除自身免疫副作用。
发明内容
本发明涉及与人类CTLA4分子结合的抗体组合物和其抗原结合片段以及其用于癌症免疫疗法、减少自身免疫副作用的用途。具体来说,本发明涉及对CTLA4配体B7.1 和B7.2的CTLA4阻断活性增强、效应子功能增强、或与可溶CTLA4的结合相对于膜结合或固定CTLA4减少的抗体。
所述抗体可以包含具有氨基酸序列的轻链可变氨基酸序列,所述氨基酸序列包含具有SEQ ID NO:1中所阐述的氨基酸序列的轻链可变氨基酸序列;和具有SEQ ID NO:2 中所阐述的氨基酸序列的重链可变氨基酸序列。所述抗体还可以包含具有SEQ ID NO: 27、28或29中所阐述的氨基酸序列的重链可变氨基酸序列;和具有SEQ ID NO:30、31 或32中所阐述的氨基酸序列的轻链可变氨基酸序列。所述抗体可以包含具有SEQ ID NO:21、22和23中所阐述的CDR序列的轻链可变区;和具有SEQ ID NO:24、25和26 中所阐述的CDR序列的重链可变区。更具体来说,所述抗体可以包含具有SEQ ID NO: 33、34或35中所阐述的CDR2序列的重链可变区;和具有SEQ ID NO:36、37或38中所阐述的CDR序列的轻链可变区。
所述抗体的免疫球蛋白重链恒定区可以包含SEQ ID NO:3或4中所阐述的氨基酸序列。所述抗体的免疫球蛋白重链恒定区还可以包含突变。相对于SEQ ID NO:3中的 hIgG1骨架的序列,突变可以是M135Y、S137T、T139E、S181A、E216A或K217A或其组合。优选地,所述抗体的免疫球蛋白重链恒定区可以包含全部六种突变。所述抗体可以包含具有SEQ ID NO:6中所阐述的氨基酸序列的重链氨基酸序列;和具有SEQ ID NO:8中所阐述的氨基酸序列的轻链氨基酸序列。所述抗体还可以包含具有SEQ ID NO: 9、11或13中所阐述的氨基酸序列的重链氨基酸序列;和具有SEQ ID NO:15、17或19 中所阐述的氨基酸序列的轻链氨基酸序列。所述抗体能够结合人类CTLA4。所述抗体还可以抑制人类CTLA4与B7-1或B7-2的结合。
本文进一步提供本文所描述抗体的抗原结合片段。
本文还提供一种医药组合物,其包含治疗有效量的本文所描述的抗体。所述医药组合物可以包含生理学上可接受的载剂或赋形剂。
在另一个方面,本文中呈现增强受试者的一种或多种免疫功能或应答的方法,其包含向有需要的受试者给予本文所描述的抗CTLA4抗体组合物和医药组合物。在一具体实施例中,本文中呈现预防、治疗和/或管理需要活化或增强一种或多种免疫功能或应答的疾病的方法。所述疾病可以是癌症(cancer),所述癌症可以是人类恶性肿瘤(malignancy)。具体来说,人类恶性肿瘤可以是黑色素瘤(melanoma)、肺癌(lung cancer)、乳腺癌(breastcancer)、肝细胞上皮癌(hepatocellular carcinoma)、卵巢上皮癌(ovarian carcinoma)、前列腺上皮癌(prostate carcinoma)、霍奇金氏(Hodgkin's)或非霍奇金氏淋巴瘤(non-Hodgkin’s lymphoma)、急性骨髓性白血病(acute myelogenic leukemia)、慢性骨髓性白血病(chronic myelogenic leukemia)、急性淋巴细胞性白血病(acute lymphocyticleukemia)、慢性淋巴细胞性白血病(chronic lymphocytic leukemia)或肾细胞上皮癌(renal cell carcinoma)。在另一实施例中,待治疗的疾病是传染性疾病。本文所描述的方法可以使与免疫疗法相关的自身免疫不良作用减到最少。
在其它具体实施例中,所述方法包含组合疗法,其中向受试者给予本文所描述的抗 CTLA4抗体组合物与另一疗法的组合,其可以活化或增强一种或多种免疫功能或应答。在另一实施例中,本文所描述的抗CTLA4抗体组合物以佐剂形式与抗原性组合物组合给予。在一具体实施例中,本文所描述的抗CTLA4抗体组合物与疫苗组合物组合给予以诱导或活化或增强疫苗组合物所引起的免疫应答。
在一具体实施例中,向受试者给予本文所描述的抗CTLA4抗体组合物与一种或多种靶向不同免疫调节途径的其它疗法的组合。在一优选实施例中,靶向不同免疫调节途径的疗法的活性与本文所描述的抗CTLA4抗体组合物互补或协同。在一种情况下,本文所描述的抗CTLA4抗体组合物与其它检查点抑制剂或小肿瘤免疫调节剂(如吲哚胺 2,3-双加氧酶(IDO)抑制剂)组合给予。在另一情况下,本文所描的抗CTLA4抗体组合物与免疫刺激分子组合给予。具体实施例包括将本文所描述的抗CTLA4抗体组合物与抗PD-1(派姆单抗(pembrolizumab)(Keytruda)或纳武单抗(Nivolumab)(Opdivo))、抗B7-H1(阿特珠单抗(atezolizumab)(Tecentriq)或度伐单抗(durvalumab))、抗B7- H3、抗B7-H4、抗LAG3、抗Tim3、抗CD40、抗OX40、抗BTLA、抗CD27、抗ICOS 或抗41BB组合。在另一实施例中,本文所描述的抗CTLA4抗体组合物和第二免疫刺激分子组合于单一双特异性抗体中。
在另一实施例中,本文所描述的抗人类CTLA4抗体可以相对于可溶CTLA4分子优先与表达于细胞表面上的人类CTLA-4结合。抗人类CTLA4抗体可以与人类CTLA4结合并且优先上调体内B7.1或B7.2的表达。所述抗体可以含于用于调节免疫应答(免疫疗法)和治疗癌症的组合物中。
本发明进一步涉及筛选具有优选活性的抗人类CTLA4 mAb的方法。临床前筛选抗人类CTLA4 mAb充满了困难,因为癌症免疫性与自身免疫不良作用的体外免疫相关性不确定。已经在人类抗CTLA4(伊匹单抗)的临床试验中观察到显著自身免疫副作用,尤其当与抗PD-1组合时。为了鉴别免疫相关毒性降低的抗CTLA4抗体,可以使用人类 CTLA4基因敲入小鼠筛选在人源化小鼠中展现抗肿瘤活性的抗体的减少体内自身免疫不良作用的能力。
在另一实施例中,本发明涉及一种筛选抗肿瘤效应增强的抗人类CTLA4 mAb的方法,其中所述抗体展现肿瘤环境中的Treg细胞的增强的局部耗尽。
在又一实施例中,本发明涉及通过监测B7.1和B7.2于免疫细胞(如抗原呈递细胞(APC))上的表达水平来监测体内抗CTLA4抗体的阻断效应的方法。本发明进一步涵盖通过测量B7.1和B7.2于离体免疫细胞上的表达水平来测量体内抗CTLA4抗体的生物活性和监测对抗CTLA4治疗的显然应答的生物标记。
为了对L3D10亲本抗体以及人源化变异体PP4631和PP4637的CTLA4结合表位作图,利用小鼠和人类CTLA4蛋白与B7-1具交叉反应性但与抗CTLA-4抗体不具交叉反应性的事实。因此,设计人类CTLA-4Fc蛋白的多种突变体,其中来自人类CTLA-4蛋白的氨基酸簇经来自鼠类Ctla-4蛋白的氨基酸置换。因为这一研究中使用的抗CTLA-4 抗体不与鼠类Ctla-4结合,所以当抗体结合表位的关键残基经鼠类氨基酸置换时可以消除抗人类CTLA-4抗体的结合。
附图说明
图1.在IgG1 Fc区中具有新颖突变组合的嵌合(左)和人源化(右)L3D10抗体的示意图。突变于Fc区中的位置通过其氨基酸位置编号来确定并且氨基酸通过其单字母代码来确定,编号之前的字母代表置换的氨基酸并且编号之后的字母代表引入的氨基酸。抗体的可变区以空心椭圆形描绘并且人类序列以灰色矩形描绘。V=可变区;C=恒定区;L=轻链;H=重链。
图2.嵌合L3D10和10D1与板固定CTLA4的如通过ELISA测定的CTLA4结合。将ELISA板用1μg/ml CTLA4-His蛋白(义翘神州生物(Sino Biological,China))涂布。添加既定浓度的生物素化结合蛋白并且使用HRP结合抗生蛋白链菌素测量结合。10D1- 1和-2是同一抗体的两个独立材料批次。B7.1-Fc是阳性对照并且Fc是阴性对照。
图3.L3D10竞争分析。10D1在阻断嵌合L3D10与CTLA4的结合方面不如嵌合 L3D10高效。实验如图2中那样执行,但生物素化嵌合L3D10在添加到ELISA板之前与既定浓度的未标记CTLA4-结合蛋白或CTLA4-Fc混合。注意到未标记L3D10所致的阻断与10D1相比要好得多,这表明这些抗体结合位点不一致。
图4.阻断CTLA4与板固定B7.1的结合。将B7.1Fc蛋白以0.5μg/ml涂布到ELISA 板上。在洗涤和阻断之后,在既定浓度的竞争蛋白存在下以0.25μg/ml添加生物素化 CTLA4-Fc蛋白。所示数据是405nM下的一式两份光密度的平均值。B7.1-Fc、嵌合L3D10 和CTLA4-Fc都以剂量依赖性方式阻断CTLA4:B7.1相互作用,而10D1抗体的两个单独批次在所有测试剂量下都未能阻断。CTLA4的生物素化不会毁坏CTLA4上的10D1 表位,因为10D1的两个批次都展示出与生物素化CTLA4的强结合(数据未展示)。
图5.阻断CTLA4与板固定B7.2的结合。将B7.2Fc蛋白以0.5μg/ml涂布到ELISA 板上。在洗涤和阻断之后,在既定浓度的竞争蛋白存在下以0.25μg/ml添加生物素化 CTLA4-Fc蛋白。嵌合L3D10以剂量依赖性方式阻断CTLA4:B7.2相互作用,而10D1抗体的两个单独批次即使在最高浓度下也未能完全阻断CTLA4:B7.2相互作用。
图6.10D1和L3D10两者都有力地阻断使用可溶B7-1和B7-2与固定CTLA4-Fc的B7-CTLA4相互作用。将不同剂量的抗人类CTLA4 mAb与0.25μg/ml生物素化人类 CTLA4-Fc一起添加到涂布有人类B7-1Fc的板。使用HRP结合抗生蛋白链菌素测量结合到板的CTLA4的量。所示数据是一式两份的平均值并且代表两个独立实验。
图7.阻断CTLA4与细胞表面表达的B7.1的结合。将生物素化CTLA4-Fc蛋白在既定浓度的竞争蛋白存在下以0.5μg/ml添加到表达B7.1的CHO细胞。通过流式细胞术检测生物素化融合蛋白与经小鼠或人类B7-1和B7-2转染的CHO细胞的结合。使用藻红蛋白结合抗生蛋白链菌素测量结合受体的量。所示数据是一式三份样品的平均荧光强度。嵌合L3D10以剂量依赖性方式阻断CTLA4:B7.1相互作用,而10D1抗体的两个单独批次在所有测试剂量下都未能阻断。
图8.阻断CTLA4与细胞表面表达的鼠类B7.1的结合。当mB7-1表达于CHO细胞上时,10D1对小鼠B7-1-人类CTLA4相互作用有适度但可检测的阻断。将不同剂量的抗人类CTLA4mAb与0.25μg/ml人类CTLA4-Fc一起添加到表达小鼠B7-1的CHO 细胞。所示数据是平均值和SEM或一式三份数据并且代表两个独立实验。
图9.阻断CTLA4与细胞表面表达的B7.2的结合。将生物素化CTLA4-Fc蛋白在既定浓度的竞争蛋白存在下以0.5μg/ml添加到表达B7.2的CHO细胞。嵌合L3D10以剂量依赖性方式阻断CTLA4:B7.2相互作用,而10D1抗体的两个单独批次即使在最高浓度下也未能完全阻断CTLA4:B7.2相互作用。这一图中所示的数据已经被重复至少5 次。
图10. 10D1比L3D10更好地与生物素化人类CTLA4-Fc结合。将不同剂量的抗人类CTLA4 mAb或对照IgG涂布到板上。以0.25μg/ml添加生物素化CTLA4-Fc。使用 HRP结合抗生蛋白链菌素测量结合到板的CTLA4的量。所示数据是一式两份的平均值并且代表两个独立实验。
图11.L3D10但非10D1阻断多组氨酸标记的CTLA4与表达人类B7-1的CHO细胞之间的相互作用。将表达人类B7-1的CHO细胞用多组氨酸标记的CTLA4以及既定剂量的抗体孵育,用PE-抗生蛋白链菌素检测并且通过FACSCanto II测量CTLA4-Fc的量。所示数据是一式三份样品的平均荧光强度并且代表两个独立实验。
图12.嵌合L3D10诱导同基因MC38模型中的确立的肿瘤的完全缓解。上图描绘实验设计并且下图展示接受对照IgG(左下图,n=6)或嵌合L3D10(右下图,n=5)的小鼠中的MC38肿瘤的生长动力学。
图13.嵌合L3D10和10D1在MC38肿瘤模型中的治疗效应。研究中使用体重为约 20克的人类CTLA4敲入小鼠。将1×106个MC38肿瘤细胞皮下注射到Ctla4h/h小鼠中,并且当肿瘤达到直径为0.5cm大小时,将携有肿瘤的小鼠随机分为三组,每组有5或6 只小鼠。随后如由箭头所指示在第7、10、13和16天将小鼠用100μg/注射的10D1、嵌合L3D10或对照hIgGFc处理(i.p.)。展示一式两份实验的结果(左和右图),并且所示数据是肿瘤大小的平均值和标准差(左图中n=6/组,右图中n=5/组)。L3D10和10D1 在这一模型中具有类似治疗效应并且都能够诱导确立的肿瘤的完全缓解。使用下式计算肿瘤的直径(d):D=√(ab),V=ab2/2,其中a是长径,而b是短径。通过双向重复测量 ANOVA(处理×时间)执行统计分析。在左图中:10D1对hIgGFc:P=5.71e-07;L3D10 对hIgGFc:P=5.53e-07;10D1对L3D10:P=0.869。
图14.抗CTLA-4 mAb对CTLA4h/m小鼠中的MC38的有效排斥。如图13中那样,但使用杂合CTLA4h/m小鼠。所示数据是肿瘤直径的平均值和SEM(6只小鼠/组);10D1 对hIgGFc:P=0.0011;L3D10对hIgGFc:P=5.55e-05;10D1对L3D10:P=0.0346。
图15.嵌合L3D10和10D1在B16-F1黑色素瘤肿瘤模型中的治疗效应。研究中使用体重为约20克的人类CTLA4敲入小鼠。箭头指示处理的时间(50μg/小鼠/处理)。所示数据是肿瘤大小的平均值和标准差(n=4/组)。L3D10在这一模型中具有类似治疗效应并且两者都能够延迟这一侵袭性并且免疫原性差的肿瘤模型中的肿瘤生长。
图16.用于测量体内CTLA4阻断的分析。树突状细胞上的B7.1或B7.2结合与T 细胞表面上的CTLA4结合并且由其下调。然而,阻断抗CTLA4抗体的结合防止 B7.1/B7.2与CTLA4结合并且因此防止B7.1和B7.2的下调,导致B7.1/B7.2表达的净增加。然而,在表达人类和小鼠CTLA4两者的嵌合T细胞的情况下,结合人类CTLA4 的抗体不防止B7.1/B7.2与鼠类CTLA4结合,其恢复B7.1/B7.2抑制。
图17A-F.10D1不阻断体内B7-CTLA4相互作用。使用图11中所描述的分析,将来自用抗CTLA4抗体处理的小鼠的细胞用以分析B7.1和B7.2表达。图17A展示实验设计的图。简单点说,年龄和性别匹配的小鼠腹膜内接受500μg抗体或其对照。在注射之后24小时,将小鼠处死并且将其脾细胞用抗CD11c、CD11b、抗B7-1和抗B7-2 mAb 染色。图17B展示了展示针对B7表达分析的CD11chiDC的表型的代表性数据。图17C 展示了描绘B7-1于来自接受对照IgG1-Fc、L3D10或10D1的小鼠的DC上的水平的代表性直方图。上图中的数据展示纯合敲入小鼠中的抗体效应,而下图中的数据展示杂合小鼠中的抗体效应。图17D如图17C中那样展示,但展示的是B7-2的表达。图17C和 D中展示的数据代表来自每组3只小鼠的数据,并且已经被重复一次,每组有三只小鼠。图17E显示,在人类CTLA4纯合小鼠中,L3D10但非10D1诱导B7-1(左图)和B7-2 (右图)的表达。所示数据由涉及每组总共6只小鼠的两个实验概述。在每个实验中,将对照小鼠中的平均数据人工定义为100%,并且将实验组中的平均数据针对对照标准化。图17F如图17E中那样,但使用杂合小鼠。L3D10和10D1两者都不阻断共显性表达小鼠和人类Ctla4基因两者的小鼠中的B7-CTLA4相互作用。
图18.L3D10与人类但非小鼠CTLA4结合。所示数据是在门控Cd3+Cd4+细胞之中使用来自Ctla4h/h(上)或Ctla4m/m(下)小鼠的脾细胞对CTLA4进行胞内染色的点图。抗小鼠CTLA4mAb 4F10用作对照。
图19.嵌合L3D10和10D1在CTLA4h/m小鼠中的治疗效应。上图描绘实验设计。将Ctla4h/h小鼠用结肠癌细胞系MC38攻击,并且当肿瘤达到直径为约5mm大小时,将小鼠用对照人类IgG-Fc、L3D10或10D1处理4次,并且在6周时间段内观察肿瘤大小。下图展示接受对照IgG、嵌合L3D10或10D1的小鼠(n=6/组)中的MC38肿瘤的生长动力学。尽管如图16中所展示体内CTLA4阻断活性存在明显差异,但L3D10和 10D1两者都展示针对嵌合CTLA4m/h小鼠中的MC38模型的强抗肿瘤活性。
图20A-B.10D1和L3D10对B16黑色素瘤生长具有类似治疗效应。将1×105个B16 肿瘤细胞注射(s.c.)到Ctla4h/h小鼠(n=4-5)中,并且如由箭头所指示在第11、14、17 天(图20A)或第2、5和8天(图20B)用100μg(图20A)或250μg(图20B)10D1、 L3D10或对照IgGFc处理(i.p.)。在图20A中,10D1对hIgGFc:P=0.0265;L3D10对 hIgGFc:P=0.0487;10D1对L3D10:P=0.302。在图20B中,10D1对hIgGFc:P= 0.00616;L3D10对hIgGFc:P=0.0269;10D1对L3D10:P=0.370。数据表示每组4-5 只小鼠的平均值±SEM。通过双向重复测量ANOVA执行统计分析。
图21A-B.在完成排斥之前被杀死以便评估肿瘤微环境内的Treg耗尽的Ctla4h/h(图 21A)和Ctla4m/h(图21B)小鼠中的L3D10与10D1之间的免疫治疗效应。所示数据是两个独立实验的肿瘤直径的平均值和SEM,每组涉及5只小鼠。
图22A-F.阻断B7-CTLA4相互作用不促成抗CTLA4 mAb的癌症免疫治疗活性。图22A展示两种抗CTLA4 mAb尽管阻断活性大大地不同但免疫治疗效应相当。将5×105个MC38肿瘤细胞注射(s.c.)到Ctla4h/h小鼠(n=6)中,并且如由箭头指示在第7、10、 13和16天用100μg 10D1、L3D10或对照hIgG-Fc处理(i.p.)。数据表示每组六只小鼠的平均值±SEM。通过双向重复测量ANOVA(处理×时间)执行统计分析。10D1对hIgG- Fc:P=5.71e-07;L3D10对hIgG-Fc:P=5.53e-07;10D1对L3D10:P=0.869。数据代表三个独立实验。图22B.在两种抗体都不阻断B7-CTLA4相互作用的小鼠中,两者都诱导稳定肿瘤排斥。如图22A中那样,但使用表达小鼠和人类CTLA4两者的杂合小鼠。 10D1对hIgG-Fc:P=0.0011;L3D10对hIgG-Fc:P=5.55e-05;10D1对L3D10:P= 0.0346。数据代表三个独立实验。图22C-F.阻断B7-CTLA4相互作用不促成肿瘤微环境中的选择性Treg耗尽。图22C和D.无关于其阻断B7-CTLA4相互作用的能力,L3D10 和10D1不使脾脏中的Treg缺失。所示数据是Foxp3+细胞在Ctla4h/h(图22C)和Ctla4m/h (图22D)小鼠中的脾脏CD4T细胞之中的%。n=6,e和f,L3D10和10D1两者都使Ctla4h/h(图22E)和Ctla4m/h(图22F)小鼠中的肿瘤浸润CD4T细胞之中的Treg缺失。 c-f中展示的数据是如以箭头所指示在肿瘤细胞攻击之后17(实验1)或19天(实验2) 和在起始4次抗CTLA4mAb处理之后10或12天Treg的%。
图23A-F.评估通常使用的抗小鼠CTLA4mAb 9H10和9D9的阻断活性。图23A和 B显示,如果B7-1(图23A)和B7-2(图23B)涂布到板上,那么9H10不阻断B7-CTLA4 相互作用。将生物素化小鼠CTLA4-Fc融合蛋白在既定浓度的对照IgG或抗小鼠CTLA4 mAb 9D9和9H10存在下用涂布B7的板孵育。用HRP结合抗生蛋白链菌素检测CTLA4 结合。所示数据是一式两份的平均值并且代表两个独立实验。图23C和D显示,9D9和 9H10展现出与可溶(图23C)和板结合CTLA4-Fc(图23D)的差异性结合。所示数据是一式两份的平均值并且代表至少两个独立实验。图23E和F展示在用500μg抗体腹膜内处理之后24小时抗小鼠CTLA4mAb 9D9和9H10对来自WT(Ctla4m/m)脾细胞的 CD11chi DC上的B7-1(图23E)和B7-2(图23F)水平的效应。数据在两个独立实验中由每组6只独立小鼠概述,每组各自涉及3只小鼠。
图24A-D.抗小鼠CTLA4 mAb 4F10的独特体内和体内阻断活性。图24A和B展示4F10对CTLA4-Fc与板涂布的B7-1(图24A)或B7-2(图24B)的相互作用的效应。将生物素化小鼠CTLA4-Fc融合蛋白在既定浓度的对照IgG或抗小鼠CTLA4mAb 4F10 存在下用涂布B7的板孵育。用HRP结合抗生蛋白链菌素检测CTLA4结合。所示数据是一式两份的平均值并且代表两个独立实验。图24C和D展示4F10对B7-1和B7-2表达的影响。来自每组6只小鼠的B7-1(图24C)和B7-2(图24D)水平的概要数据。将对照IgG处理组中的B7水平人工定义为100%。
图25.嵌合L3D10和10D1与抗PD-1组合的不良作用。上图描绘实验设计。研究中使用10天大的仅雌性的体重大于4克的人类CTLA4敲入小鼠。其接受指定蛋白质或其组合。箭头指示处理的时间(100μg/小鼠/处理)。所示数据是体重增加%的平均值和标准差。嵌合L3D10和10D1在成年小鼠中具有相当癌症治疗效应(图13),但当10D1 与抗PD-1 mAb组合时可见独特不良作用。
图26.嵌合L3D10和10D1与抗PD-1组合的不良作用。图展示来自图25中概述的实验的接受对照IgG、10D1+抗PD-1或嵌合L3D10+抗PD-1的小鼠(n=5/组)在第42 天时的最终体重。在抗PD1+10D1组合的情况下观察到显著体重减轻,这在抗PD-1+嵌合L3D10组合的情况下不可见。
图27.嵌合L3D10和10D1与抗PD-1组合的病理学作用。为了进一步检查当与抗 PD-1组合给予时L3D10与10D1相比的相对毒性,我们查看以上图26中描述的小鼠的大体解剖。用10D1+PD-1处理的小鼠的子宫/卵巢/膀胱和胸腺显著更小,而用L3D10+ 抗PD-1处理的小鼠中的器官与hIgG对照相当。相比之下,由用10D1处理的小鼠解剖的心脏呈现出更大的大小,外观明显更白。
图28A-D.用10D1与抗PD-1组合处理导致异常红血球生成。鉴于图27中观察到的心脏的差异,我们查看小鼠内的红血球生成,并且观察到用10D1+抗PD-1处理的小鼠相对于用L3D10+抗PD-1或对照抗体(hIgG)处理的组(其极其类似)的明显差异。用10D1+抗PD-1处理的小鼠的骨髓的颜色明显更白(图28A)并且分离的血的颜色是几乎完全白色(图28B)。根据这个,当我们使用CD119和CD71标记的分布分析红血细胞的分化时,我们观察到10D1+抗PD-1处理小鼠中经历IV期发生的细胞的数目统计上显著减少。代表性FACS分布图展示于图28C中,而概要数据呈现于图28D中。
图29.抗红血细胞抗体的流式细胞术分析。将来自NOD.SCID.I12rg-/-(NSG)小鼠的血液样品用来自在围产期期间接受抗体处理的小鼠的血浆样品染色。将来自NSG小鼠的血清和不具有血清的那些用作阴性对照。所有血清以1∶50稀释度使用。这些数据显示,无小鼠产生抗红细胞抗体。
图30.用嵌合L3D10和10D1与抗PD-1组合处理的小鼠的心脏的病理学。为了进一步确定L3D10对10D1与抗PD-1组合的毒理学,我们对图26中描述的小鼠的心脏执行组织学分析。用10D1+抗PD-1处理的小鼠展现高水平的T细胞浸润,这在用L3D10+ 抗PD-1处理的小鼠或用人类IgG对照处理的小鼠中未观察到。
图31.用嵌合L3D10和10D1与抗PD-1组合处理的小鼠的肺脏的病理学。为了进一步确定L3D10对10D1与抗PD-1组合的毒理学,我们对图26中描述的小鼠的肺脏执行组织学分析。用10D1+抗PD-1处理的小鼠展现高水平的T细胞浸润,这在用L3D10+ 抗PD-1处理的小鼠或用人类IgG对照处理的小鼠中未观察到。
图32.用嵌合L3D10和10D1与抗PD-1组合处理的小鼠的唾液腺的病理学。为了进一步确定L3D10对10D1与抗PD-1组合的毒理学,我们对图26中描述的小鼠的唾液执行组织学分析。与在用L3D10+抗PD-1处理的小鼠或用人类IgG对照处理的小鼠中所观察到的相比,用10D1+抗PD-1处理的小鼠展现高得多水平的T细胞浸润。
图33A-F.用嵌合L3D10和10D1与抗PD-1组合处理的小鼠的肾脏和肝脏的病理学。为了进一步确定L3D10对10D1与抗PD-1组合的毒理学,我们对图26中描述的小鼠的肾脏和肝脏执行组织学分析。图33A-C是来自肾脏的切片并且图33D-E是取自肝脏的切片。与在用L3D10+抗PD-1处理的小鼠或用人类IgG对照处理的小鼠中所观察到的相比,用10D1+抗PD-1处理的小鼠展现高水平的T细胞浸润。
图34.用嵌合L3D10和10D1与抗PD-1组合处理的小鼠的毒性分数。所示的这个组织数据概述图30-33,并且展示用10D1+抗PD-1处理的小鼠相对于L3D10+抗PD-1 的高毒性分数,后者的分数仅略高于hIgG对照小鼠组。
图35.10D1+抗PD-1在Ctla4h/m小鼠中不具有显著毒性,如在围产期期间接受抗体处理的小鼠的正常体重增加所证明。小鼠在第10、13、16、19和22天腹膜内接受既定抗体或组合的处理(100μg/小鼠/注射/抗体)。至少每3天一次对小鼠称重。
图36.L3D10和10D1对板固定CTLA4展示类似结合模式。将ELISA板用1μg/mlCTLA4-His蛋白(义翘神州生物)涂布。添加既定浓度的生物素化结合蛋白并且使用HRP 结合抗生蛋白链菌素测量结合。10D1-1和-2是同一抗体的两个独立材料批次。hIgG-Fc 是人类Ig阴性对照。
图37.L3D10展现减少的结合可溶CTLA4。将既定浓度的抗人类CTLA4 mAb涂布于板上过夜,在用牛血清白蛋白洗涤和阻断之后,以0.25μg/ml添加生物素化CTLA4- Fc。在孵育和洗涤之后,使用HRP标记的抗生蛋白链菌素测量捕获的CTLA4-Fc的量。
图38.人源化抗体可变区与亲本L3D10抗体序列的比对。将人源化抗体序列的重链可变区(上)(SEQ ID NO:62-64)和轻链可变区(下)(SEQ ID NO:70-72)与亲本 L3D10抗体(重链:SEQ ID NO:57;轻链:SEQ ID NO:65)和对应的人类抗体构架(重链:SEQ ID NO:58-61;轻链:SEQ ID NO:66-69)比对。小鼠亲本序列的回复突变以黄色突显。新颖氨基酸(即在亲本抗体序列或对应的人类抗体构架中不存在的氨基酸残基) 以绿色突显。引入CDR2序列中的突变以紫色展示。CDR序列基于www.bioinf.org.uk/abs/ 以红色展示。
图39A-B.人源化L3D10抗体与10D1相比的抗肿瘤活性。使用人类CTLA4敲入小鼠的MC38小鼠肿瘤模型,我们查看人源化L3D10抗体与嵌合L3D10抗体和10D1相比的抗肿瘤活性。上图展示体内实验的处理时程;从接种之后第7天开始每3天向小鼠给予总共4个剂量的抗体。所有人源化抗体(n=6/组)完全根除肿瘤并且与10D1相当 (下图)。
图40.人源化L3D10抗体在CTLA4h/m小鼠中的抗肿瘤活性。上图展示体内实验的处理时程;Ctla4h/m小鼠在MC38肿瘤注射之后的指定日期以30(-30,实线)或10(- 10,点线)mg/注射的剂量接受对照hIg或三种不同抗人类CTLA4mAb之一。每三天一次测量肿瘤大小。
图41.抗CTLA-4 mAb在最小疾病B16-F1肿瘤模型中的治疗效应。使用人类CTLA4敲入小鼠中的B16-F1小鼠肿瘤模型,我们查看人源化L3D10抗体的抗肿瘤活性。将 1×105个B16肿瘤细胞注射(s.c.)到Ctla4h/h小鼠(n=5-6)中。在第2、5和8天,将小鼠用对照Ig、10D1、嵌合L3D10或PP4637和PP4638(250μg/小鼠,i.p.)处理。每隔一天测量肿瘤发生率和大小。10D1对hIgGFc:P=0.00616;L3D10对hIgGFc:P=0.0269; 10D1对L3D10:P=0.370;PP4637对hIgGFc:P=0.0005;PP4637对10D1:P=0.805; PP4638对h1gGFc:P=0.0016;PP4638对10D1:P=0.856。数据表示每组5-6只小鼠的平均值±SEM。从未出现肿瘤的小鼠的肿瘤大小被视为0。
图42.在10D1、PP4631和PP4637雌性之中比较其与抗PD-1 mAb的组合毒性。如图例中所规定,将雌性CTLA4h/h小鼠在出生之后第10天或第11天用4次抗体(100μg/ 小鼠/注射,每三天一次)或对照Fc注射处理。每3天一次对小鼠称重。所示数据是在 30天时间段内体重增加%的平均值和SEM。在第43天将所有小鼠处死用于组织学分析。每组使用的小鼠的数目展示于标记的圆括号中。
图43.10D1和抗PD-1的组合疗法导致贫血,而PP4631+抗PD-1或PP4637+抗PD- 1的组合疗法不导致贫血。所示数据是已经在第11、14、17和20天以100μg/小鼠/抗体的剂量接受四次抗体处理的43天大的小鼠的血细胞比容。
图44A-B.10D1+抗PD-1的组合疗法导致系统T细胞活化,而PP4631+抗PD-1或PP4637+抗PD-1的组合疗法不导致系统T细胞活化。所示数据是外周血液(图44A)或脾脏(图44B)中具有原初(CD44l℃D62Lhi)、中枢记忆(CD44hiCD62Lhi)和效应记忆 (CD44hiCD62Llo)T细胞的表型的CD4(上图)和CD8T细胞(下图)的%。由已经在第11、14、17和20天以100μg/小鼠/抗体的剂量接受四次抗体处理的43天大的小鼠收集细胞。
图45.L3D10的人源化不会影响与固定CTLA4的结合。如图36中所描述测定人源化L3D10抗体结合固定CTLA4的能力。X轴指示添加到溶液中的抗CTLA-4 mAb的浓度。人源化不会影响与固定CTLA4的结合,并且全部3种人源化抗体都展现类似的与亲本嵌合L3D10抗体和10D1的结合。当使用CTLA4-Ig代替CTLA-4-his时观察到类似模式。
图46.人源化进一步减少L3D10与可溶CTLA4的结合。如图37中所描述测定人源化L3D10抗体结合可溶CTLA4的能力。X轴指示涂布到ELISA板上的抗CTLA-4 mAb的浓度。人源化相对于亲本L3D10嵌合抗体进一步减少与可溶CTLA4的结合。当使用CTLA4-Ig代替CTLA-4-his时观察到类似模式。
图47A-B.PP4631、PP4638和PP4637不阻断体外B7-CTLA-4相互作用。图47A展示抗人类CTLA-4 mAb 10D1、PP4631、PP4637和L3D10对B7-1-CTLA-4相互作用的阻断。以0.5μg/ml的浓度固定B7-1Fc。将生物素化CTLA4-Fc以0.25μg/ml与既定剂量的抗体一起添加。所示数据是405nM下的一式两份光密度的平均值。图47B展示抗人类CTLA-4 mAb 10D1和L3D10对B7-2-CTLA-4相互作用的阻断。如图47A中那样,但固定B7-2-Fc。
图48.如其缺乏对B7-1和B7-2于树突状细胞上的表达的效应所展现,PP4631和PP4637不阻断体内B7-CTLA-4相互作用。来自每组3只小鼠的B7-1(a)和B7-2(b) 水平的概要数据。将对照IgG处理组中的B7水平人工定义为100%。
图49.基于最低治疗剂量下的肿瘤排斥,与抗PD-1 mAb组合展现最佳安全概况(参看图42)的PP4637最有力地导致肿瘤排斥。Ctla4h/m小鼠在指定日期以30(-30,实线) 或10(-10,点线)μg/注射的剂量接受对照IgFc或三种不同抗人类CTLA4 mab之一。每三天一次测量肿瘤大小。在10μg/注射下,PP4637(HL32)最高效地诱导肿瘤排斥。
图50.人源化抗体纯度评估。通过蛋白A色谱法纯化瞬时表达的人源化L3D10抗体,并且通过还原和非还原SDS-PAGE评估来自全部3种抗体的样品。在还原和非还原两种条件下,纯化蛋白产生凝胶条带,大小方面指示抗体分子。“流出”泳道展示蛋白A 柱流通,表明大部分抗体蛋白粘着于蛋白A柱。
图51.瞬时表达的蛋白质的尺寸排阻色谱法(SE-HPLC)。按照单步骤蛋白A色谱法通过SE-HPLC分析每一个人源化抗体的蛋白质样品。上图:抗体PP4631。中图:抗体PP4637。下图:抗体PP4638。
图52.瞬时表达的蛋白质的CE-SDS分析。按照单步骤蛋白A色谱法通过CE-SDS 分析每一个人源化抗体的蛋白质样品。左图展示非还原条件下的结果,并且右图展示还原条件下的结果。上图:抗体PP4631。中图:抗体PP4637。下图:抗体PP4638。
图53A-C.人源化L3D10抗体如通过毛细管等电聚焦(cIEF)测定的电荷同种型分布图和脱酰胺化。通过比较在经两个不同时间段(5小时和12.5小时)的高pH应力处理前后的人源化L3D10抗体来测定高pH应力下的蛋白质脱酰胺化水平,通过cIEF分析进行分析。图53A-C分别展示抗体PP4631、PP4637和PP4638的分布图。
图54A-C.人源化L3D10抗体的差示扫描量热法(DSC)热分析。为了测定不同抗体的热稳定性和熔融温度,使其经历差示扫描量热法(DSC)热分析。图54A-C分别展示抗体PP4631、PP4637和PP4638的标准化DSC曲线。
图55.人类、猕猴和小鼠CTLA-4胞外结构域的比对。比对人类(Hm,以红色展示)(SEQ ID NO:73)、猕猴(Mk,以黑色展示)和小鼠(Ms,以绿色展示)CTLA-4蛋白胞外结构域的氨基酸序列,并且保守氨基酸(相对于人类序列)以短划线(-)展示。为了帮助比对,小鼠序列在以黄色突显的位置具有缺失和插入(相对于人类和猴序列)。已知的B7-1Ig结合位点以粗体展示并且加下划线。序列展现出,人类和猴序列高度保守,而小鼠序列具有多个氨基酸差异。基于这个序列比对,设计11种并有鼠类特异性氨基酸的突变(M1-M11)(SEQ ID NO:40-50)人类CTLA-4Fc蛋白,并入到每种突变蛋白中的氨基酸以蓝色展示。
图56A-B.WT和突变CTLA-4Fc蛋白的氨基酸序列组成。如所展示设计编码WT CTLA-4Fc蛋白(SEQ ID NO:39)和11种并有鼠类Ctla-4氨基酸的突变蛋白(SEQ ID NO:40-50)的DNA构建体。氨基酸序列针对成熟蛋白,包括IgG1 Fc部分,但不包括信号肽。已知的B7-1Ig结合位点以大写蓝色字母展示并且带双下划线。突变体中置换的鼠类氨基酸残基以小写红色展示。蛋白质的IgG1 Fc部分加下划线。
图57.M11中的突变(AA103-106,YLGI>fcGm)选择性地消除与人类CTLA-4的抗体结合。所示数据是一式两份的平均值,描绘B7-1Fc(a)、L3D10(b)、PP4631(c) 和PP4637(d)与板涂布的hCTLA4-Fc(空心圆)、mCTLA4-Fc(实心三角形)、M11(实心圆)和IgG1-Fc(空心三角形)的结合。
图58.在B7-1-CTLA4复合物的3D结构中将L3D10、PP4631和PP4637图谱定位到与B7-1结合位点相邻的表位。B7-1结合基序以红色着色,而抗体表位以紫色着色。 B7-1以空间填充的带状描绘于CTLA4上方,而CTLA-4以未填充的带状描绘。
图59.WT(SEQ ID NO:39)和突变CTLA-4Fc蛋白M12-M17(SEQ ID NO:51-56) 的氨基酸序列组成。如所展示设计编码6种并有鼠类Ctla-4氨基酸的突变CTLA-4Fc蛋白M12-M17的DNA构建体。氨基酸序列针对成熟蛋白,包括IgG1 Fc部分,但不包括信号肽。已知的B7-1Ig结合位点以大写蓝色字母展示并且带双下划线。突变体中置换的鼠类氨基酸残基以小写红色展示。蛋白质的IgG1 Fc部分加下划线。
图60A-C.突变分析展现10D1(图60A)、PP4631(图60B)和PP4637(图60C) 与CTLA-4的独特结合需求。将CTLA-4Fc突变体在4℃下以1μm/ml涂布过夜。在用 BSA阻断之后,添加既定浓度的生物素化抗CTLA-4 mAb并且孵育2小时。在洗掉未结合抗体之后,用HRP标记的抗生蛋白链菌素检测结合抗体。
图61A-B.抗4-1BB和抗CTLA-4抗体在最小疾病(图61A)和确立的肿瘤(图 61B)模型两者中的治疗效应。图61A展示最小疾病的疗法。将C57BL/6小鼠用5×105个MC38细胞皮下接种。在肿瘤细胞注射之后第2、9和16天,注射对照仓鼠和大鼠 IgG、抗CTLA-4和/或抗4-1BB抗体。通过身体检查测量肿瘤大小。所示数据是肿瘤的生长动力学,每一行代表一种小鼠中的肿瘤生长。所呈现的大小是肿瘤的长径和短径的乘积。图61B展示确立的肿瘤的疗法。如图61A中那样,但疗法在肿瘤攻击之后第14 天开始;所有小鼠在mAb处理开始之前具有大小范围介于9-60mm2的确立肿瘤。两种抗体对确立的肿瘤的组合效应已经被重复3次。
图62.CD8T细胞但非CD4或NK细胞为抗体诱导的肿瘤排斥所必需。在肿瘤细胞接种之后第9、12和16天通过三次注射对CD4、CD8或NK1.1具有特异性的抗体使携有肿瘤的小鼠耗尽CD4、CD8或NK细胞(*)。在第9、16和23天注射治疗抗体(抗 CTLA-4加抗4-1BB)(垂直箭头)。所示数据是肿瘤大小的平均值和SEM(n=3)。对于耗尽CD8的组与其它组中的每一个相比,P<0.05()。
图63A-B.组合疗法减少对抗CTLA-4抗体的宿主应答。将仓鼠抗小鼠CTLA-4(图63A)或大鼠抗小鼠4-1BB(图63B)抗体涂布于ELISA板中。将不同稀释度的来自各自5只小鼠的组的血清添加到板。使用第二步骤试剂(通过吸收耗尽对大鼠和仓鼠IgG 的反应性的生物素化山羊抗小鼠抗体)测定结合抗体的相对量。所示数据是490nm下的光密度的平均值和SEM。当用相同抗体处理无肿瘤小鼠时观察到类似的对抗CTLA-4 和4-1BB的宿主抗体应答减少(数据未展示)。
图64A-B.人类CTLA-4基因敲入小鼠中的抗4-1BB和L3D10(抗人类CTLA4)抗体的组合疗法。图64A展示治疗效应。将人类CTLA4敲入小鼠用5×105个MC38肿瘤细胞皮下接种。两天后,如由箭头所指示将7只小鼠的组用大鼠和小鼠IgG、抗4-1BB 和小鼠IgG、L3D10和大鼠IgG、或L3D10和抗4-1BB处理。所示数据是平均肿瘤体积和SEM(n=7)。所有处理都显著减少肿瘤生长(P<0.001),并且双抗体处理组与对照 (P<0.0001)或L3D10抗体(P=0.0007)或抗4-1BB抗体处理(P=0.03)相比展示出显著减小的肿瘤大小。当对照IgG处理组达到早期去除准则时,将所有携有肿瘤的小鼠处死。图64B展示接受组合疗法的小鼠中的持久免疫性。双抗体处理组中的无肿瘤小鼠产生对MC38肿瘤的持久免疫性。在第一次肿瘤细胞攻击之后第110天,将双抗体处理的无肿瘤小鼠或对照未处理小鼠用5×105个肿瘤细胞皮下攻击。通过身体检查监测肿瘤生长。注意,所有在第一轮中排斥肿瘤的小鼠都对再攻击具有完全抗性,而所有未处理小鼠都具有进行性肿瘤生长。
具体实施方式
定义
如本文所用,术语“抗体”旨在表示具有“可变区”抗原识别位点的免疫球蛋白分子。术语“可变区”旨在将这类免疫球蛋白结构域与抗体广泛共有的结构域(如抗体Fc 结构域)区分。可变区包含“高变区”,其残基负责抗原结合。高变区包含来自“互补决定区”或“CDR”(即,典型地在轻链可变结构域中的大致残基24-34(L1)、50-56(L2) 和89-97(L3)处以及在重链可变结构域中的大致残基27-35(H1)、50-65(H2)和95- 102(H3)处;参考44)的氨基酸残基和/或来自“高变环”(即,轻链可变结构域中的残基26-32(L1)、50-52(L2)和91-96(L3)以及重链可变结构域中的26-32(H1)、 53-55(H2)和96-101(H3);参考45)的那些残基。“构架区”或“FR”残基是除如本文中定义的高变区残基外的那些可变结构域残基。术语抗体包括单克隆抗体、多特异性抗体、人类抗体、人源化抗体、合成抗体、嵌合抗体、骆驼化抗体、单链抗体、二硫键连接的Fv(sdFv)、内抗体和抗个体基因型(抗Id)抗体(包括例如抗Id和抗本发明的抗体的抗Id抗体)。具体来说,这类抗体包括任何类型(例如,IgG、IgE、IgM、IgD、 IgA和IgY)、类别(例如,IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或子类别的免疫球蛋白分子。
如本文所用,术语抗体的“抗原结合片段”是指抗体的一个或多个含有抗体的互补决定区(“CDR”)和任选地构成抗体的“可变区”抗原识别位点的构架残基并且展现免疫特异性结合抗原的能力的部分。这类片段包括Fab′、F(ab′).sub.2、Fv、单链(ScFv) 和其突变体、天然存在的变异体、以及包含抗体的“可变区”抗原识别位点和异源蛋白 (例如,毒素、不同抗原的抗原识别位点、酶、受体或受体配体等)的融合蛋白。如本文所用,术语“片断”是指包含具有至少5个连续氨基酸残基、至少10个连续氨基酸残基、至少15个连续氨基酸残基、至少20个连续氨基酸残基、至少25个连续氨基酸残基、至少40个连续氨基酸残基、至少50个连续氨基酸残基、至少60个连续氨基酸残基、至少70个连续氨基酸残基、至少80个连续氨基酸残基、至少90个连续氨基酸残基、至少100个连续氨基酸残基、至少125个连续氨基酸残基、至少150个连续氨基酸残基、至少175个连续氨基酸残基、至少200个连续氨基酸残基或至少250个连续氨基酸残基的氨基酸序列的肽或多肽。
人类、嵌合或人源化抗体对于人类中的体内用途特别优选,然而,鼠类抗体或其它物种的抗体宜用于许多用途(例如,体外或原位检测分析、急性体内用途等)。
“嵌合抗体”是抗体的不同部分衍生自不同免疫球蛋白分子的分子,如具有衍生自非人类抗体的可变区和人类免疫球蛋白恒定区的抗体。包含来自非人类物种的一个或多个CDR和来自人类免疫球蛋白分子的构架区的嵌合抗体可以使用本领域中已知的多种技术制造,包括例如CDR移植(EP 239,400;国际公开第WO 91/09967号;以及美国专利第5,225,539号、第5,530,101号和第5,585,089号)、镶饰或表面重修(EP 592,106;EP 519,596;46-48)和链改组(美国专利第5,565,332号)。
本发明尤其涉及“人源化抗体”。如本文所用,术语“人源化抗体”是指包含人类构架区和一个或多个来自非人类(通常小鼠或大鼠)免疫球蛋白的CDR的免疫球蛋白。提供CDR的非人类免疫球蛋白被称为“供体”并且提供构架的人类免疫球蛋白被称为“受体”。恒定区无需存在,但如果其存在,那么其必须与人类免疫球蛋白恒定区大体上一致,即至少约85-90%、优选约95%或更多一致。因此,人源化免疫球蛋白的所有部分 (可能除CDR外)与天然人类免疫球蛋白序列的相应部分大体上一致。人源化抗体是包含人源化轻链和人源化重链免疫球蛋白的抗体。举例来说,人源化抗体将不涵盖典型嵌合抗体,因为例如,嵌合抗体的整个可变区是非人类。一种说法是,供体抗体已经通过“人源化”的过程“人源化”,因为预期所得人源化抗体与提供CDR的供体抗体相比与相同抗原结合。在很大程度上,人源化抗体是接受者的高变区残基经来自非人类物种(供体抗体)(如小鼠、大鼠、兔或非人类灵长类动物)的具有所期望的特异性、亲和力以及能力的高变区残基置换的人类免疫球蛋白(受体抗体)。在一些情况下,人类免疫球蛋白的构架区(FR)残基经相应非人类残基置换。此外,人源化抗体可以包含在接受者抗体或供体抗体中未发现的残基。进行这些修饰以进一步优化抗体性能。一般来说,人源化抗体将包含至少一个并且典型地两个可变结构域中的大体上全部,其中所有或大体上所有的高变区对应于非人类免疫球蛋白的高变区并且所有或大体上所有的FR是人类免疫球蛋白序列的FR。人源化抗体任选地还将包含已经通过引入氨基酸残基取代、缺失或添加(即,突变)而改变的免疫球蛋白恒定区(Fc)、典型地与Fc.γ.RIIB多肽免疫特异性结合的人类免疫球蛋白恒定区的至少一部分。
详细描述
针对人类CTLA4蛋白的抗体伊匹单抗据显示作为唯一免疫治疗剂或与其它治疗剂(如但不限于抗PD-1抗体)组合可增加癌症患者的存活(13-15)。然而,治疗效应与显著不良作用相关(13-18)。极其需要开发新颖抗CTLA4抗体以获得更好治疗效应和/或更少自身免疫不良作用。本发明人已经发现,抗CTLA4抗体出人意料地可以用以诱导癌症排斥同时还减少与免疫疗法相关的自身免疫不良作用。
本文提供抗体物质组合物和其抗原结合片段。本发明进一步涉及这类分子的实施例,其中分子是单克隆抗体、人类抗体、嵌合抗体或人源化抗体。
详细地说,本发明提供一种分子,其包含与CTLA4、并且尤其人类CTLA4(优选以内源或转染浓度表达于活细胞的表面上)免疫特异性结合的抗体的抗原结合片段。本发明具体来说涉及这类分子的实施例,其中抗原结合片段与CTLA4结合,并且其中活细胞是T细胞。
本发明涉及能够与CTLA4免疫特异性结合的抗体和其抗原结合片段。在一些实施例中,这类分子另外能够阻断B7.1和B7.2与CTLA4的结合。
本发明进一步涉及这类分子的实施例,其中分子是单克隆抗体、人类抗体、嵌合抗体或人源化抗体。本发明包括这类抗体具单特异性、双特异性、三特异性或多特异性的实施例。
本发明进一步涉及这类分子或抗体的与CTLA4结合的实施例,并且其中其抗原结合片段包含六个CDR,其中CDR包含抗CTLA4抗体L3D10的CDR。具体来说,抗体包含抗CTLA4抗体L3D10的三个轻链和三个重链CDR。
本发明进一步涉及上述抗体的实施例,其中抗体经可检测标记或包含结合毒素、药物、受体、酶、受体配体。
本发明进一步涉及一种医药组合物,其包含治疗有效量的上述抗体组合物中的任一者和生理学上可接受的载剂或赋形剂。优选地,本发明的组合物包含预防或治疗有效量的本发明的人源化抗体和医药学上可接受的载剂。
在一具体实施例中,术语“医药学上可接受”意指得到联邦监管机构或州政府批准或在美国药典(U.S.Pharmacopeia)或其它公认的药典中列出供用于动物,并且更确切地说用于人类。术语“载剂”是指与治疗剂一起给予的稀释剂、佐剂(例如,弗氏佐剂 (Freund′sadiuvant)(完全和不完全))、赋形剂或媒剂。这类医药载剂可以是灭菌的液体,如水和油,包括石油、动物、植物或合成来源的油,如花生油、大豆油、矿物油、芝麻油等。当静脉内给予医药组合物时,水是优选的载剂。还可以使用生理盐水溶液和右旋糖水溶液以及甘油溶液作为液体载剂,特别是用于可注射溶液。适合的医药赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、稻米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、无水脱脂奶、甘油、丙二醇、二醇、水、乙醇等。必要时,组合物还可以含有少量润湿剂或乳化剂或pH缓冲剂。这些组合物可以采取溶液、悬浮液、乳液、片剂、丸剂、胶囊、粉末、持续释放配制物等的形式。
一般来说,本发明的组合物的成分可以分别供应或一起混合于单位剂型中,例如以干燥冻干粉末或无水浓缩物形式于指示活性剂量的气密密封容器(如安瓿或药囊)中。在组合物要通过输注给予的情况下,其可以用含有灭菌医药级的水或生理盐水的输注瓶分配。在组合物通过注射给予时,可以提供一安瓿注射用无菌水或生理盐水,使得各成分可以在给予之前被混合。
本发明的组合物可以配制为中性或盐形式。医药学上可接受的盐包括但不限于由阴离子形成的盐,所述阴离子如衍生自盐酸、磷酸、乙酸、草酸、酒石酸等的那些;和由阳离子形成的盐,所述阳离子如衍生自钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、 2-乙氨基乙醇、组氨酸、普鲁卡因(procaine)等的那些。
本发明进一步涉及此处描述的抗体组合物和其医药组合物用于上调免疫应答的用途。治疗癌症和慢性感染时特别需要上调免疫系统,并且因此本发明可用于治疗这类病症。如本文所用,术语“癌症(cancer)”是指由细胞的异常不受控生长产生的赘瘤或肿瘤。如本文所用,癌症明确包括白血病(leukemias)和淋巴瘤(lymphomas)。所述术语是指涉及有潜力转移到远端位点的细胞的疾病。
因此,本发明的方法和组合物还可以适用于治疗或预防多种癌症(cancer)或其它异常增殖性疾病,包括(但不限于)以下:上皮癌(carcinoma),包括膀胱、乳腺、结肠、肾、肝、肺、卵巢、胰腺、胃、子宫颈、甲状腺和皮肤的上皮癌;包括鳞状细胞上皮癌(squamous cellcarcinoma);淋巴谱系的造血肿瘤(hematopoietic tumors of lymphoid lineage),包括白血病(leukemia)、急性淋巴细胞性白血病(acute lymphocytic leukemia)、急性成淋巴细胞性白血病(acute lymphoblastic leukemia)、B细胞淋巴瘤(B-cell lymphoma)、T细胞淋巴瘤(T-cell lymphoma)、伯基特淋巴瘤(Berketts lymphoma);骨髓谱系的造血肿瘤(hematopoietic tumors of myeloid lineage),包括急性和慢性髓系白血病(acute andchronic myelogenous leukemia)和前髓细胞性白血病(promyelocytic leukemia);间充质来源的肿瘤,包括纤维肉瘤(fibrosarcoma)和横纹肌肉瘤(rhabdomyoscarcoma);其它肿瘤,包括黑色素瘤(melanoma)、精原细胞瘤(seminoma)、畸胎上皮癌(tetratocarcinoma)、成神经细胞瘤(neuroblastoma)和神经胶质瘤(glioma);中枢和外周神经系统的肿瘤,包括星形细胞瘤(astrocytoma)、成神经细胞瘤(neuroblastoma)、神经胶质瘤(glioma)和神经鞘瘤(schwannomas);间充质来源的肿瘤,包括纤维肉瘤(fibrosarcoma)、横纹肌肉瘤(rhabdomyosarcoma)和骨肉瘤(osteosarcoma);以及其它肿瘤,包括黑色素瘤(melanoma)、着色性干皮病(xenoderma pegmentosum)、角化棘皮瘤(keratoactanthoma)、精原细胞瘤(seminoma)、甲状腺滤泡性癌(thyroid follicular cancer)和畸胎上皮癌(tetratocarcinoma)。还预期由细胞凋亡的失常所导致的癌症(cancer)也将通过本发明的方法和组合物治疗。这类癌症(cancer)可以包括但不限于滤泡性淋巴瘤(follicularlymphoma);具有p53突变的上皮癌(carcinoma);乳腺、前列腺和卵巢的激素依赖性肿瘤(hormone dependent tumor);和癌前病变(precancerous lesion),如家族性腺瘤性息肉病(familial adenomatous polyposis)和骨髓发育不良综合征(myelodysplasticsyndrome)。在具体实施例中,卵巢、膀胱、乳腺、结肠、肺、皮肤、胰腺或子宫中的恶性肿瘤(malignancy)或增殖异常变化(如组织化生和发育异常)或过度增殖性病症通过本发明的方法和组合物来治疗或预防。在其它具体实施例中,肉瘤(sarcoma)、黑色素瘤(melanoma)或白血病(leukemia)通过本发明的方法和组合物来治疗或预防。
在本发明的另一实施例中,抗体组合物和其抗原结合片段可以与其它抗肿瘤疗法一起使用,包括但不限于现行标准和实验化学疗法、激素疗法、生物疗法、免疫疗法、辐射疗法或手术。在一些实施例中,本发明的分子可以与治疗或预防有效量的一种或多种药剂、治疗抗体或本领域的技术人员已知用于治疗和/或预防癌症、自身免疫疾病、传染性疾病或中毒的其它药剂组合给予。这类药剂包括例如以上论述的生物应答调节剂、细胞毒素、抗代谢物、烷化剂、抗生素或抗有丝分裂剂以及免疫治疗剂中的任一者。
在本发明的优选实施例中,抗体组合物和其抗原结合片段可以与其它抗肿瘤免疫疗法一起使用。在这类实施例中,本发明的分子与破坏或增强替代性免疫调节途径(如TIM3、TIM4、OX40、CD40、GITR、4-1-BB、B7-H1、PD-1、B7-H3、B7-H4、LIGHT、 BTLA、ICOS、CD27或LAG3)或调节效应分子的活性(如细胞因子(例如,IL-4、IL- 7、IL-10、IL-12、IL-15、IL-17、GF-β、IFNg、Flt3、BLys)和趋化因子(例如,CCL21)) 的分子组合给予以便增强免疫调节效应。具体实施例包括双特异性抗体,其包含本文所描述的抗CTLA4抗体组合物和抗PD-1(派姆单抗(Keytruda)或纳武单抗(Opdivo))、抗B7-H1(阿特珠单抗(Tecentriq)或度伐单抗)、抗B7-H3、抗B7-H4、抗LIGHT、抗 LAG3、抗TIM3、抗TIM4、抗CD40、抗OX40、抗GITR、抗BTLA、抗CD27、抗ICOS 或抗4-1BB。在又一实施例中,本发明的分子与活化免疫应答的不同阶段或方面的分子组合给予以便实现更广免疫应答。在更优选实施例中,抗体组合物和其抗原结合片段与抗PD-1或抗4-1BB抗体组合而不加重自身免疫副作用。
本发明的另一实施例包括双特异性抗体,其包含与桥接到结合另一免疫刺激分子的抗体的CTLA4结合的抗体。具体实施例包括双特异性抗体,其包含本文所描述的抗 CTLA4抗体组合物和抗PD-1、抗B7-H1、抗B7-H3、抗B7-H4、抗LIGHT、抗LAG3、抗TIM3、抗TIM4、抗CD40、抗OX40、抗GITR、抗BTLA、抗CD27、抗ICOS或抗 4-1BB。本发明进一步涉及这类抗体用于治疗癌症的用途。
给予本发明的抗体组合物的方法包括但不限于肠胃外给予(例如,皮内、肌肉内、腹膜内、静脉内和皮下)、硬膜外和经粘膜(例如,鼻内和经口途径)。在一具体实施例中,本发明的抗体是肌肉内、静脉内或皮下给予。组合物可以通过任何方便的途径,例如通过输注或快速注射、通过上皮或粘膜皮肤内层(例如,口腔粘膜、直肠和肠道粘膜等)的吸收来给予,并且可以与其它生物活性剂一起给予。给予可以是系统或局部的。
本发明的又一实施例涉及通过监测B7.1和B7.2于免疫细胞(如抗原呈递细胞(APC))上的表达水平来监测体内抗CTLA4抗体的阻断效应。CTLA4主要表达于Treg 之中,其中其通过下调B7-1和B7-2于APC(如树突状细胞)上的表达来抑制自身免疫疾病。因此,B7分子B7.1和B7.2的上调可以作为体内阻断B7-CTLA4相互作用的读出。在一具体实施例中,在抗CTLA4治疗前后将外周或肿瘤内免疫细胞从受试者中移出并且离体分析免疫细胞表面上B7.1和/或B7.2水平的减少,其中阻断抗CTLA4抗体的存在防止B7.1/B7.2由内源CTLA4结合,这转而防止B7.1和B7.2的下调,导致 B7.1/B7.2表达的净增加。在一优选实施例中,测量抗原呈递细胞上B7.1和B7.1的水平。在一最优选实施例中,测量树突状细胞上B7.1和B7.1的水平。
在另一实施例中,在抗CTLA4治疗之后免疫细胞上B7.1和B7.2的变化(减少)用作通过测量B7.1和/或B7.2于免疫细胞上的表达水平和比较治疗前后的表达水平来测量体内抗CTLA4抗体的生物活性和监测对抗CTLA4治疗的显然应答的生物标记。在一优选实施例中,在抗CTLA4疗程期间随时间推移监测B7.1和/或B7.2表达水平。
实例
实例1.生成嵌合抗CTLA4抗体
使用人类CTLA4基因敲入小鼠和hu-PBL-Scid小鼠,先前证实小鼠抗人类CTLA4 抗体减少肿瘤生长,并且将L3D10鉴别为在所测试的mAb组之中最有效。然而,所获得的抗体中无一者能够实现完全肿瘤排斥,即使在相对高剂量(>10mg/kg)下并且在形成可触肿瘤(如早期在第2天)之前在肿瘤细胞攻击之后使用时(19-21)。
因为小鼠抗体是不具有强抗体依赖性细胞的细胞毒性(ADCC)的IgG1子类别,并且因为ADCC可以参与肿瘤排斥,所以mAb的Fc以若干方式修饰以实现更好免疫治疗效应。首先,用具有强ADCC活性的人类IgG1置换ADCC弱的小鼠IgG1以产生嵌合抗体。第二,基于文献中的已知技术(22),将三种突变(S298A、E333A和K334A)引入CH中以增加ADCC活性。第三,引入三种突变(M252Y、S254T和T256E)以增加体内抗体的半衰期(23)。新嵌合抗体的设计描绘于图1左图中。
为了工程改造抗体,首先使用本领域中已知的标准方法通过DNA测序鉴别L3D10杂交瘤的可变区。将核苷酸序列翻译到SEQ ID NO:1和SEQ ID NO:2中列出的氨基酸中。正常人类IgG1 Fc序列和突变Fc序列分别公开于SEQ ID NO:3和SEQ ID NO:4 中。重链和轻链序列的氨基酸和密码子优化的核苷酸序列公开于SEQ ID NO:5-8中。
合成对应于SEQ ID NO:5和SEQ ID NO:7的DNA并且将其插入到表达载体中,并且将载体用所设计的序列转染到HEK293细胞中。简单点说,将HEK293细胞在转染之前一天接种于摇瓶中,并且使用无血清的化学成分确定的培养基使其生长。使用瞬时转染的标准操作程序将DNA表达构建体瞬时转染到0.5升HEK293细胞悬浮液中。在 20小时之后,对细胞取样以获得生存力和活细胞计数,并且测量滴度(Octet QKe, ForteBio)。在整个瞬时转染生产运行中取另外的读数。在第5天收集培养物。通过离心和过滤从瞬时转染生产运行收集和澄清L3D10的条件培养基。使上清液在蛋白A柱上运行并且用低pH缓冲液洗脱。使用0.2μm膜滤器执行过滤,随后等分。在纯化和过滤之后,由OD280和消光系数计算蛋白浓度。由一轮转染获得总共43.2mg Ig蛋白。
实例2.嵌合L3D10抗体结合位点与10D1仅部分重叠
在临床中,抗CTLA4抗体伊匹单抗据显示可改进癌症患者的存活但诱导显著自身免疫不良作用。为了评估嵌合L3D10抗体和10D1的比较结合位点,比较与CTLA4的结合和抗体竞争与CTLA4结合的能力。虽然两种抗体都以相当效率与固定CTLA4蛋白结合(图2),但10D1不完全阻断嵌合L3D10与CTLA4的结合(图3)。正如所料,未标记的L3D10完全阻断经标记的L3D10结合,表明L3D10和10D1的抗体结合位点仅部分重叠。
实例3.嵌合L3D10抗体比10D1更高效阻断CTLA4:B7.1和CTLA4:B7.2相互作用
已经报道,如果可溶B7-1和B7-2用以与固定CTLA4相互作用,那么抗人类CTLA4mAb 10D1可以阻断B7-CTLA4相互作用(49)。因为B7-1和B7-2充当细胞表面共刺激分子,所以我们使用固定B7-1和B7-2评估抗CTLA4抗体阻断B7-CTLA4相互作用的能力。使用竞争性ELISA分析形式,L3D10和10D1阻断CTLA4融合蛋白CTLA4-Ig与板固定的和细胞膜表达的B7.1和B7.2两者结合的能力。在这些实验中,使用亲和力(2.3 nM)类似于10D1(4nM)(49)的嵌合抗人类CTLA4-mAb。在板固定分析中,将B7.1fc 或B7.2Fc以1μg/ml涂布到ELISA板上,在4℃下过夜或在37℃下2小时。将生物素化CTLA4-Fc与既定浓度的B7.1-Fc、10D1或嵌合L3D10混合。使用辣根过氧化酶结合抗生蛋白链菌素测定结合到板上的B7.1的CTLA4-Fc的量。如图4中所示,虽然嵌合 L3D10、B7.1Fc和CTLA4-Fc都高效地阻断CTLA4-Fc:B7.1相互作用,但10D1的两个单独材料批次未能阻断相互作用。L3D10在低到0.2μg/ml的浓度下展示对板固定B7.1 结合的显著阻断,在约3μg/ml下实现50%抑制(IC50)。类似地,L3D10以0.03μg/ml 的IC50阻断CTLA4-Fc与板固定B7.2的结合,而来自两个不同材料批次的10D1以约 200μg/ml的IC50展现最小阻断(图5)。然而,与先前的报道(49)相一致,当板固定 CTLA4用以与可溶B7-1相互作用时抗体10D1在反向实验中有力地抑制B7-1-CTLA4 相互作用(图6)。
在细胞膜蛋白结合实验中,当B7.1表达于CHO细胞的表面上时,L3D10阻断 CTLA4-Fc的结合,但来自两个不同材料批次的10D1即使在512μg/ml下使用时也不阻断所述结合(图7)。虽然远不如L3D10有力,但高剂量的10D1在人类CTLA4与小鼠 B7-1之间实现约25%阻断(图8)。对于表达于CHO细胞表面上的B7.2,L3D10再次阻断,而10D1仅部分阻断,即使当10D1在512μg/ml下使用时也观察到小于50%抑制 (图9)。
潜在警告是,生物素化可能影响了10D1与CTLA4-Fc的结合。为了解决这个问题,我们比较L3D10和10D1与阻断研究中使用的生物素化CTLA4-Fc的结合。如图10中所示,在结合生物素化CTLA4-Fc方面10D1比L3D10更有效。因此,10D1未能阻断并非是由于与生物素化CTLA4-Fc的不充分结合。当多组氨酸标记的CTLA4用以与人类 B7-1转染的CHO细胞相互作用时观察到类似模式(图11)。综合来看,我们的数据表明,抗体10D1阻断B7-CTLA4相互作用的能力高度取决于所采用的分析,如果B7-1和 B7-2经固定,那么有最小到无可检测阻断活性,而抗体L3D10是B7-CTLA4相互作用的稳定阻断剂,无关于B7蛋白质是否经固定。
实例4.嵌合L3D10抗体比未经修饰的L3D10更高效地导致肿瘤排斥
先前报道,小鼠L3D10未能导致MC38肿瘤的完全缓解,即使观察到显著延迟(19,20)。为了确定嵌合L3D10是否可以导致同基因小鼠中的完全缓解,将1×106个MC38 肿瘤细胞移植到同基因C57BL/6小鼠中。一周后,当肿瘤达到直径为约5mm时,将小鼠用对照IgG或嵌合L3D10 mAb以仅为小鼠L3D10情况下的先前研究中所用剂量的一半的剂量处理。如图12中所示,尽管人类Ig序列存在可能的免疫原性,但发现嵌合 L3D10导致所有测试小鼠中的完全缓解。因为治疗在大肿瘤负荷确立时开始,这比肿瘤不可触时困难得多(19),所以这些实验显示嵌合L3D10比未经修饰的L3D10更高效。
实例5.嵌合L3D10抗体与10D1在导致肿瘤排斥方面具有等效活性
人类CTLA4基因敲入小鼠的可用性(20)为用临床上使用的抗CTLA-4mAb 10D1 测试嵌合抗人类CTLA-4抗体的生物活性提供了前所未有的机会。在这种人源化小鼠模型中,在内源小鼠Ctla4基因座的控制下表达编码与人类CTLA-4蛋白具有100%一致性的产物的CTLA4基因。当在人类CTLA4敲入小鼠中的MC38肿瘤模型中直接比较嵌合 L3D10和10D1的抗肿瘤活性时,显而易见的是,两种抗体在导致肿瘤排斥方面相当,而在IgG对照组中肿瘤进行性生长。图13展示一式两份实验中抗体处理对肿瘤大小的结果。
一个有趣的问题是,抗CTLA-4 mAb是否需要与所有CTLA-4相互作用(即实现标靶饱和)以便发挥免疫治疗效应。来自CTLA4h/h和CTLA4m/m小鼠的F1小鼠以共显性方式表达小鼠和人类CTLA-4蛋白两者。有趣地,如图14中所示,嵌合L3D10和10D1 两者都有效地诱导肿瘤排斥,即使约50%的CTLA-4蛋白(即所述蛋白的鼠类形式)无法与抗人类CTLA-4 mAb结合。重要地,在这种设定下,即当基因剂量有限时,L3D10 比10D1治疗上更有效(P<0.05)。
先前研究已经证实,抗小鼠Ctla-4 mAb在不与其它治疗模式组合的情况下无法诱导黑色素瘤细胞系B16-F1的排斥。因此,还使用人类CTLA4敲入小鼠中的这种更具挑战性的B16肿瘤模型测试嵌合L3D10和10D1抗体的抗肿瘤效应。如图15中所示,L3D10 和伊匹单抗两者都不能导致确立的肿瘤的排斥,但两者都导致肿瘤生长的统计上显著延迟,而不同抗体之间的差异统计上不显著。
实例6:体内CTLA4阻断
CTLA4主要表达于Treg之中,其中其通过下调B7-1和B7-2于树突状细胞上的表达来抑制自身免疫疾病(50)。因为Ctla4的靶向突变(50)和用阻断抗CTLA4 mAb治疗(51)上调B7-1和B7-2于树突状细胞上的表达,所以已经表明,Treg上的CTLA4 的生理学功能是下调DC上的B7。因此,B7的上调用作体内阻断B7-CTLA4相互作用的读出,并且使用来自具有人类CTLA4基因的纯合敲入的Ctla4h/h小鼠的T细胞开发一种分析。
如图16中所概述,表面表达的B7.1或B7.2结合T细胞表面上的CTLA4,其导致 B7.1和B7.2表达的下调。然而,阻断抗CTLA4抗体的结合防止B7.1/B7.2结合,其防止B7.1和B7.2的下调,导致B7.1/B7.2表达的净增加。然而,在表达人类和小鼠CTLA4 两者的嵌合T细胞的情况下,结合人类CTLA4的抗体不防止B7.1/B7.2与鼠类CTLA4 结合,其恢复7.1/B7.2抑制。
已经描述了在内源小鼠Ctla4基因座的控制下表达与人类CTLA4蛋白具有100%一致性的CTLA4基因的CTLA4人源化小鼠(20)。使纯合敲入小鼠(CTLA4h/h)与C57BL/6 背景回交以获得至少10代。通过使CTLA4h/h小鼠与WT BALB/c小鼠杂交来产生杂合小鼠(CTLA4h/m)。
为了测试临床上经证实的治疗性抗CTLA4 mAb 10D1,我们注射极高剂量的抗CTLA4 mAb(500μg/小鼠,其是约25mg/kg或临床中所用最高剂量的8倍)到Ctla4h/h或Ctla4m /h小鼠中,并且在注射之后24小时收集脾细胞以测量Cd11chi DC上B7-1和B7- 2的水平(图17A-B)。如图17C-E中所示,与接受人类IgG1-Fc的Ctla4h/h小鼠相比,来自嵌合L3D10处理小鼠的DC的B7.1表达在表达人类CTLA4的T细胞中具有统计上显著增加,但在表达人类和小鼠CTLA4两者的T细胞中不具有统计上显著增加。如图17C-E中所示,对于B7.2可见类似结果。B7-2的上调的量值与在人类Treg-DC共培养物中使用阻断抗CTLA4mAb所实现相当(66)。
为了进一步证实体内分析的特异性,我们测试L3D10是否可以上调小鼠和人类CTLA4共显性表达的Ctla4m/h小鼠中的B7。因为至少50%的CTLA4不与抗人类CTLA4 抗体结合,所以预期其在阻断B7-CTLA4相互作用方面将不太有力。实际上,两种抗体都不导致来自Ctla4m/h小鼠的DC上的B7-1和B7-2的上调(图17C、D、F)。Ctla4m/h小鼠中L3D10阻断的完全缺乏表明,由小鼠等位基因编码的不与L3D10结合(图18) 的CTLA4足以下调B7表达。因此,我们的数据证实,在为临床中所用最高剂量的至少 8倍的剂量下,当B7固定于板上或锚定于细胞膜上时,10D1体内和体外都不阻断B7- CTLA4相互作用。
Ctla4m/h小鼠中L3D10阻断的完全缺乏表明,由小鼠等位基因编码的不与L3D10结合(图18)的CTLA4足以下调B7表达。相比之下,10D1不增加B7.1或B7.2表达。根据模型,这表明,L3D10阻断体内CTLA4活性而10D1不阻断所述活性。
然而,尽管阻断活性存在这些明显差异,但L3D10和10D1两者都展示针对嵌合CTLA4m/h小鼠中的MC38模型的强抗肿瘤活性,如图19中所示。虽然在对照Ig处理小鼠中肿瘤进行性生长,但可通过任一抗CTLA4 mAb实现完全排斥。在多个实验中,两种抗体在导致肿瘤排斥方面相当。在另一肿瘤模型B16黑色素瘤中,两种抗体都诱导类似肿瘤生长延迟,但不可通过任一抗体实现完全排斥(图20)。
实例7:抗肿瘤效应与肿瘤内Treg耗尽相关
体内免疫调节由免疫细胞活化与免疫检查点之间的平衡产生。具体来说,调节性T细胞(Treg)是调节免疫系统、维持对自身抗原的耐受性并且消除自身免疫疾病的T细胞亚群。最近研究已经证实,抗小鼠CTLA4 mAb的治疗功效受Fc子类别和宿主Fc受体影响,其转而选择性地影响肿瘤微环境内Treg的抗体依赖性细胞毒性(52,53)。因为体内差异性CTLA4阻断活性似乎不翻译为抗肿瘤活性的差异,所以我们尝试建立抗肿瘤发生的作用机制并且查看肿瘤微环境内的Treg。为了做到这一点,我们在排斥完成之前处死携有MC38肿瘤的小鼠(图21)并且分析接受对照Ig、10D1或L3D10的Ctla4h/h敲入小鼠中Treg的频率。虽然两种抗体都不减少脾脏中的Treg(图22C),但两者都减少肿瘤微环境中的Treg(图22E)。有趣的是,10D1但非L3D10扩增脾脏中的Treg。脾脏中因10D1所致的Treg扩增重现如下临床发现,伊匹单抗增加外周血液白细胞的 FOXP3表达(54)。因为阻断和非阻断抗体在肿瘤微环境中的Treg耗尽方面相当,所以 B7-CTLA4相互作用的阻断不促成Treg耗尽。因为10D1不阻断体内B7-CTLA4相互作用并且又在Ctla4h/h小鼠和黑色素瘤患者中赋予治疗效应,所以这种相互作用的阻断不为其治疗效应所需。此外,因为两种具有大幅不同阻断效应的mAb具有相当的治疗效应和肿瘤微环境中的选择性Treg耗尽,所以阻断CTLA4-B7相互作用不增强抗体的治疗效应。
为了证实这个观察结果,我们测试两种抗CTLA4 mAb在Ctla4m/h小鼠中的治疗效应,其中抗人类CTLA4 mAb可以与最大50%的CTLA4分子结合并且其中两种抗体都不阻断B7-CTLA4相互作用以实现树突状细胞上B7的上调(图16)。此外,两种抗体都导致MC38肿瘤的快速排斥,但L3D10比10D1稍微更有效(图22B)。相应地,两种抗体都选择性地耗尽肿瘤微环境中的Treg(图22D和21F)。这些遗传数据进一步证实,肿瘤排斥的CTLA4阻断与局部Treg耗尽无关,并且因此反驳了抗CTLA4 mAb通过阻断B7-CTLA4相互作用诱导癌症免疫性的流行假说(10)。
实例8.评估通常使用的抗小鼠CTLA4 mAb 9H10和9D9的阻断活性
基于两种抗小鼠CTLA4 mAb(30,31)4F10和9H10的整个和Fab的刺激效应提出了CTLA4是T细胞调节的细胞内在负调节因子的概念,但未呈现可证实这些抗体阻断 B7-CTLA4相互作用的数据。最近,第三抗小鼠CTLA4 mAb 9D9据报道在携有肿瘤的小鼠中具有治疗效应并且导致肿瘤微环境中的局部Treg耗尽(52)。我们因此着手测试据显示可诱导肿瘤排斥的全部三种商业上可获得的抗小鼠CTLA4 mAb在生理学上相关配置下阻断B7-CTLA4相互作用的能力。作为第一测试,我们使用增加量的抗CTLA4 mAb(相对于CTLA4-Fc高达2,000倍摩尔过量)阻断生物素化CTLA4-Fc与板固定B7- 1和B7-2的结合。如图23A中所示,抗小鼠CTLA4 mAb 9H10即使在最高测试浓度下也不阻断B7-1-CTLA4相互作用,但当9D9在极高浓度下使用时观察到适度阻断。虽然 mAb 9D9有效地阻断B7-2-CTLA4相互作用,但9H10未能阻断(图23B)。有趣的是,虽然9D9展示与可溶CTLA4-Fc的强结合,但9H10展示不良结合(图23c),即使其比 9D9在结合固定小鼠CTLA4-Fc方面更有力(图23D)。因为这个分析中9H10的任何阻断活性的缺乏可以简单地反映其与可溶CTLA4-Fc的不良结合,所以我们再次使用WT 小鼠(CTLA4m/m)中树突状细胞上B7-1和B7-2的上调来测量B7-CTLA4相互作用的体内阻断。如图23E和F中所示,9H10不上调DC上的B7-1表达,而9D9使B7-1水平增加15%(P<0.05)。有趣的是,虽然9D9明确上调DC上的B7-2,但9H10不上调。因此,第一个并且最广泛研究的肿瘤免疫治疗性抗CTLA4 mAb 9H10不阻断B7-CTLA4 相互作用。因此,阻断B7-CTLA4相互作用不促成通过抗小鼠CTLA4 mAb诱导抗肿瘤免疫性。因为两种mAb都展示相当免疫治疗效应和肿瘤微环境中的相当Treg缺失(52),所以局部Treg缺失而非B7-CTLA4相互作用的阻断向抗小鼠CTLA4 mAb的治疗效应提供了统一解释。有趣的是,虽然4F10阻断体外B7-CTLA4相互作用,但其未能诱导体内DC上B7的上调(图24)。
综合来看,我们已经证实,临床上证实的治疗性抗人类CTLA4 mAb(10D1)和两种抗小鼠CTLA4 mAb(9H10和4F10)在生理学上相关条件下在不阻断B7-CTLA4相互作用的情况下赋予免疫治疗效应。此外,这类阻断对于肿瘤排斥不是必需的,即使是对于可以有力地阻断B7-CTLA4相互作用的mAb(L3D10)。因为治疗效应就在阻断B7- CTLA4相互作用方面具有1000倍差异的抗体来说大体上相同,所以这类阻断不促成抗 CTLA4 mAb的癌症治疗效应。这些数据反驳了抗CTLA4 mAb通过检查点阻断赋予免疫治疗效应的假说(55)。通过反驳流行假说,我们的数据表明,抗CTLA4 mAb的治疗效应无法通过改进抗CTLA4 mAb的阻断活性来优化。在这种情形下,特别有趣的是注意到,在阻断B7-CTLA4相互作用方面优越(56)的曲美木单抗(Tremelimumab)在III 期临床试验中未到达临床终点(57)。同时,通过证实局部Treg耗尽的肿瘤排斥之间的强相关性和通过反驳阻断B7-CTLA4相互作用参与肿瘤免疫性,我们的研究促进了肿瘤环境内的局部Treg缺失是治疗性抗CTLA4 mAb的主要机制的假说,并且因此提出了开发下一代抗CTLA4 mAb用于癌症免疫疗法的新方法。
最终,累积小鼠中的遗传数据表明,CTLA4负调节T细胞活化和这类调节通过SHP-2实现(58,59)的初始概念(30,31)可能需要重新讨论(60)。因此,虽然Ctla4-/-小鼠中的严重自身免疫疾病已经用以支持CTLA4为T细胞活化的细胞内在负调节因子的观念(61,62),但已经出现了至少三行与这个观点不一致的遗传数据。首先,Treg中但非效应T细胞中Ctla4基因的谱系特异性缺失足以再现具有Ctla4基因的生殖系缺失的小鼠中观察到的自身免疫表型(50)。这些数据表明,Ctla4-/-小鼠中的自身免疫性不是由于在效应T细胞中缺乏细胞内在负调节因子CTLA4。第二,在由WT和Ctla4-/-T细胞两者组成的嵌合小鼠中,自身免疫表型通过WTT细胞的共存而预防(63)。这些数据再次强有力地说明,自身免疫疾病并非由缺乏细胞内在负调节因子导致。缺乏细胞内在负调节因子效应还由以下事实展现,在嵌合小鼠中,在病毒感染期间未观察到Ctla4-/-T细胞的优先扩增(64)。第三,据提出可介导负CTLA4调节(58,59)的Shp2的T细胞特异性缺失据证实可减少而非增强T细胞活化(65)。在报道的这些遗传数据的情形下,因为提议CTLA4为T细胞活化的负调节因子,本文中报道的我们的数据号召重新评估癌症免疫疗法中的CTLA4检查点阻断。
实例9.嵌合L3D10当与其它免疫治疗抗体组合使用时展现减少的免疫不良事件
最近的临床研究已经披露,抗PD-1与抗CTLA4 mAb之间的组合疗法进一步增加了末期黑色素瘤患者的存活。然而,55%的接受组合疗法的患者出现3和4级免疫相关不良事件(irAE)。因此开发具有较小毒性的抗体至关重要。我们已经开发了一种体内模型,其重现临床中观察到的与抗CTLA-4与抗PD-1 mAb的组合疗法相关的irAE。在这种模型中,我们用高剂量的抗PD-1和抗CTLA-4 mAb处理围产期期间的人类CTLA4基因敲入小鼠(CTLA4h/h)。我们发现,虽然年幼小鼠耐受单独mAb处理,但抗PD-1和 10D1的组合疗法导致严重irAE,具有多个器官炎症、贫血和如图25中所示的严重生长停滞。相比之下,当与抗PD-1组合时,嵌合L3D10如正常体重增加所展现仅展现轻微 irAE。
为了进一步检查当与抗PD-1组合给予时嵌合L3D10与10D1相比的相对毒性,我们查看在给予之后42天CTLA4h/h敲入小鼠中的病理学作用。如图26中所示,用L3D10+ 抗PD-1处理的小鼠的最终体重(第42天)类似于用hIgG阴性对照抗体处理的小鼠。然而,通过比较,用10D1+抗PD-1处理的小鼠的体重要低得多。因此,当我们查看这些小鼠的大体解剖时,用10D1+PD-1处理的小鼠的子宫/卵巢/膀胱和胸腺显著更小(图 27)。此外,用L3D10+抗PD-1处理的小鼠的器官与hIgG对照相当。相比之下,由用 10D1处理的小鼠解剖的心脏呈现出略微更大的大小,外观明显更白。因此,我们决定查看小鼠内的红血球生成,并且观察到用10D1+抗PD-1处理的小鼠相对于用L3D10+抗 PD-1或对照抗体处理的组(其极其类似)的明显差异。如图27A中所示,用10D1+抗 PD-1处理的小鼠的骨髓的颜色明显更白并且分离的血的颜色是几乎完全白色(图28b)。根据这个,当我们使用CD71和CD119标记更仔细查看经历不同血液发生阶段的细胞时。代表性FACS分布图展示于图28C中,而概要数据呈现于图28D中。这些数据展现 10D1+抗PD-1处理小鼠中经历IV期发生的细胞数目的统计上显著减少(图28D)。
为了探索10D1处理小鼠中贫血的潜在机制,我们测试10D1+PD-1处理是否诱导抗红血细胞抗体。如图29中所示,未检测到抗红血细胞抗体。因此,出现红细胞特异性自身抗体不造成抗PD-1+10D1处理小鼠中的贫血。
为了进一步确定L3D10对10D1与抗PD-1组合的毒理学,我们在固定于10%福尔马林中至少24小时之后对心脏(图30)、肺脏(图31)、唾液腺(图32)以及肾脏和肝脏(图33)执行组织学分析。在所研究的每一个组织中,用10D1+抗PD-1处理的小鼠展现高水平的T细胞浸润。基于炎症严重性的毒性分数概述于图34中,其展示用10D1+ 抗PD-1处理的小鼠相对于L3D10+抗PD-1的高毒性分数,后者的分数仅略高于hIgG对照小鼠组。
实例10:L3D10对于可溶CTLA4具有减少的结合。
L3D10和10D1对板固定CTLA4展示类似结合模式(图36)。作为L3D10相对于 10D1毒性降低、具体来说与10D1相关的T细胞浸润/活性增加的可能解释,我们决定查看与可溶CTLA4的结合。我们选择查看这个,因为CTLA4多态性与多种自身免疫疾病之间的关联涉及可溶CTLA4的缺陷产生(《自然(nature)》2003,423:506-511),并且sCTLA4同种型的遗传沉默增加了小鼠中I型糖尿病的发作(《糖尿病(Diabetes)》 2011,60:1955-1963)。此外,可溶CTLA4(阿巴西普(abatacept)和贝拉西普(belatacept)) 是广泛使用的免疫抑制药物。根据这个想法,当我们查看与可溶CTLA4的相对结合时,我们观察到L3D10结合的显著减少(图37)。
我们已经证实,抗CTLA-4 mAb在杂合Ctla4h/m小鼠中诱导稳定肿瘤注射,其中仅50%的CTLA-4分子可以与抗人类CTLA-4 mAb结合。为了确定50%的CTLA-4的参与是否足以诱导irAE,我们用抗PD-1+10D1处理Ctla4h/m小鼠。如图35中所示,抗PD- 1+10D1未诱导Ctla4h/m小鼠中的体重减轻。因此,irAE和癌症免疫性可以是遗传非偶联的。
体内活性证实,L3D10抗体保持其抗肿瘤活性但展现与其它免疫治疗抗体(如10D1) 情况下观察到相比减少的自身免疫不良作用,表明有可能增强抗肿瘤活性而不加重自身免疫不良事件。因此,自身免疫副作用不是癌症免疫性的必需代价并且有可能解偶联这两种活性。L3D10的表征证实,其阻断CTLA4与B7.1和B7.2相互作用的能力比10D1 更有效并且这涉及抗体之间的CTLA4结合位点的差异。此外,L3D10与具有赋予增强抗体的治疗效应的强ADCC活性的突变的经修饰的人类IgG1 Fc结构域融合。进一步表征证实,L3D10和10D1以类似结合概况与固定CTLA4结合。然而,L3D10展现比10D1 低得多的与可溶CTLA4的结合亲和力。综合来看,我们的数据证实,抗体L3D10在临床用于治疗具有较少严重不良事件的癌症患者方面具有极大潜力。
实例11.L3D10的人源化
人源化过程通过生成同源建模的抗体3D结构和基于结构建模创建亲本抗体的分布而开始。基于跨越构架的总体序列一致性、匹配界面位置、类似类别的CDR典型位置和将必须去除的N-糖基化位点的存在,鉴别利用的受体构架。一个轻链(LC)和一个重链(HC)构架选用于人源化设计。
通过创建使亲本抗体序列的选择部分与人类构架序列融合(包括将CDR序列移植到受体构架中)的多个杂合序列来设计人源化抗体。亲本抗体L3D10的预测CDR序列如下表1A中所指示提供为SEQ ID NO:21-26:
表1A:亲本抗体L3D10的预测CDR序列
抗体链 | CDR | SEQ ID NO |
可变轻 | 1 | 21 |
2 | 22 | |
3 | 23 | |
可变重 | 1 | 24 |
2 | 25 | |
3 | 26 |
使用3D模型,通过眼睛和计算机建模以分离最可能保留抗原结合的序列有系统地分析这些人源化序列。目标是使最终人源化抗体中的人类序列的量达到最大同时保留初始抗体特异性。
基于所选受体构架设计三个人源化轻链(LC1、LC2和LC3)和三个人源化重链(HC1、HC2和HC3)。三个HC或三个LC序列中的每一个来自相同生殖系,如图38中所示鼠类亲本序列的回复突变不同。人源化可变区氨基酸序列和其优化的编码核苷酸序列列于SEQ IDNO:9-20中。人源化重链和轻链的CDR2序列相对于亲本L3D10抗体序列都含有氨基酸变化并且如下表1B中所指示列于SEQ ID NO 33-38中。
表1B:人源化抗体可变区的CDR2序列.
抗体序列 | CDR2序列 | SEQ ID NO |
HC1 | YIWYDGNTNFHPSLKSR | 33 |
HC2 | YIWYDGNTNFHSSLKSR | 34 |
HC3 | YIWYDGNTNFHSPLKSR | 35 |
LC1 | AATNLQS | 36 |
LC2 | AATNLQD | 37 |
LC3 | AATSLQS | 38 |
轻和重人源化链现在可以组合以形成变异完全人源化抗体。测试人源化轻链和重链的所有可能组合的表达水平和抗原结合亲和力以鉴别表现得类似于亲本抗体的抗体。
使用计算单克隆抗体的人源性分数的新工具(24)。这一分数表示抗体可变区序列看起来人类样的程度,这在使抗体人源化时是重要因素。亲本和人源化抗体的人源性分数展示于下表2和3中。基于我们的方法,对于重链,79或更高的分数指示看起来人类样;对于轻链,86或更高的分数指示看起来人类样。
表2:人源化轻链信息和人源性分数.
表3:人源化重链信息和人源性分数.
通过以下方式构建全长抗体基因:首先合成可变区序列。针对在哺乳动物细胞中的表达优化序列。随后将这些可变区序列克隆到已经含有人类Fc结构域的表达载体中;对于重链,利用hIgG1(M252Y、S254T、T256E、S298A、E333A、K334A)骨架。另外,为了比较,使用相同骨架Fc序列将嵌合亲本重链和轻链的可变区构建为全长嵌合链。
全部9种人源化抗体都经历0.01升小规模生产。还按比例增大嵌合亲本抗体用于直接比较。将用于指定重链和轻链的质粒使用化学成分确定的培养基在不存在血清下转染到HEK293细胞悬浮液中以制得抗体。使用MabSelect SuRe蛋白A培养基(通用电气医疗集团(GE Healthcare))纯化条件培养基中的全抗体。10种测试的抗体展示于下表 4中。
表4:HEK293细胞中瞬时产生的十种抗体
抗体名称 | 重链 | 轻链 | PP号 | 产量(mg/L) |
人源化HC1+LC1 | H3106 | L3106 | 4630 | 54 |
人源化HC1+LC2 | H3106 | L3107 | 4631 | 50 |
人源化HC1+LC3 | H3106 | L3108 | 4632 | 45 |
人源化HC2+LC1 | H3107 | L3106 | 4633 | 37 |
人源化HC2+LC2 | H3107 | L3107 | 4634 | 44 |
人源化HC2+LC3 | H3107 | L3108 | 4635 | 40 |
人源化HC3+LC1 | H3108 | L3106 | 4636 | 46 |
人源化HC3+LC2 | H3108 | L3107 | 4637 | 55 |
人源化HC3+LC3 | H3108 | L3108 | 4638 | 53 |
嵌合亲本 | H2872 | L2872 | 4629 | 28 |
通过Octet评估9种人源化抗体组合和嵌合亲本抗体与抗原(huCTLA4)的亲和力。在Octet Red96系统(ForteBio)上执行多浓度动力学实验。将抗hIgG Fc生物传感器(ForteBio,编号18-5064)在样品稀释剂(0.1%BSA于PBS和0.02%Tween 20中)中水合并且在pH1.7甘氨酸中预调节。用样品稀释剂在600nM下开始,使用7点、2倍连续稀释来稀释抗原。将所有抗体用样品稀释剂稀释到10μg/mL,并且随后固定到抗 hIgG Fc生物传感器上120秒。在于样品稀释剂中确立基线60秒之后,将生物传感器移动到以一系列浓度含有抗原的孔以测量缔合。对于在样品稀释剂中的每种目标蛋白,观察缔合120秒并且观察解离180秒。通过将动力学传感图拟合到单价结合模型(1∶1结合)来表征结合亲和力。完全动力学测量值概述于下表5中。
表5:人源化抗体和亲本抗体的动力学测量值
实例12.人源化抗CTLA4抗体的抗肿瘤活性
基于相对结合亲和力和人源性分数,我们选择3种抗体用于进一步评估:
PP4631-高亲和力和良好表达
PP4637-高亲和力和良好表达
PP4638-略低的亲和力但最高的人源化分数
通过在HEK293细胞中在0.1升规模下瞬时生产随后蛋白A纯化来产生用于这些抗体中的每一者的材料。如下表6中所示,通过Octet分析证实纯化抗体的结合亲和力。
表6.人源化抗体和亲本抗体的动力学测量值
我们使用在以上实例5中描述的人类CTLA4敲入小鼠中的同基因MC38小鼠肿瘤模型评估这三种人源化抗体与10D1和嵌合L3D10抗体相比的抗肿瘤活性。图39A展示体内实验的处理时程;从接种之后第7天开始每3天向小鼠给予总共4个剂量的抗体。如图39B中所示,所有人源化抗体完全根除肿瘤并且与10D1相当。
在另一实验中,我们使用在实例5中描述的杂合Ctla4h/m小鼠(图14)中的同基因MC38小鼠肿瘤模型,在两种不同剂量下评估人源化抗体PP4631和PP4637与10D1和嵌合L3D10抗体相比的抗肿瘤活性。如图40中所示,当在30mcg/小鼠/注射(1.5mg/kg) 下使用时所有mAb都不可区别,而在10mcg/小鼠/注射(0.5mg/kg)下PP4637更有效, PP4631和10D1展示相当活性。
还如图41中所示使用人类CTLA4敲入小鼠中的同基因B16-F1黑色素瘤小鼠肿瘤模型证实人源化抗体与10D1和嵌合L3D10抗体相比的抗肿瘤活性。从接种之后第2天开始每3天向小鼠给予总共3个剂量的抗体。如图41中所示,L3D10和人源化抗体延迟肿瘤生长并且与10D1相当。
实例13.L3D10的人源化克隆相对于10D1维持优越安全概况.
为了测试L3D10的优越安全概况在人源化之后是否得以维持,我们针对其当与抗PD-1组合使用时的不良作用将PP4631和PP4637与10D1比较。如图42中所示,PP4631 和PP4637当与抗PD-1组合使用时毒性都小于10D1。
与图28中描述的缺陷性红血球生成相一致,如图43中所示,基于全血细胞计数(CBC),用10D1加抗PD-1处理的小鼠是贫血的,而接受抗PD-1+PP4631和抗PD- 1+PP4637的小鼠具有基本上正常CBC概况。此外,对PBL中的T细胞分布的分析揭露,接受10D1+抗PD-1的小鼠中的CD4和CD8T细胞都有稳定系统活化,但接受抗 PD-1+PP4631或抗PD-1+PP4637的小鼠中并不这样(图44),进一步支持基于L3D10的抗CTLA-4 mAb不导致系统T细胞活化的观念。
实例14.人源化抗CTLA4抗体的结合特征
为了证实人源化抗体保留其CTLA4结合特征,我们查看与固定和板结合CTLA4的结合。如图45中所示,人源化不会影响与固定CTLA4的结合,并且全部3种人源化抗体都展现类似的与亲本嵌合L3D10抗体的结合。然而,如图46中所示,人源化进一步减少L3D10与可溶CTLA4的结合。基于减少的与可溶CTLA4的结合,预期3种人源化抗体将诱导同等肿瘤排斥,自身免疫副作用比L3D10甚至更少。
我们已经证实,嵌合L3D10的阻断活性比10D1高1000倍。这显示出一个有趣的可能性,阻断B7-CTLA-4相互作用可以解释其缺乏irAE。如图47和48中所示,PP4631 和PP4637两者都不阻断体外和体内B7-CTL-A4相互作用。PP4631和PP4637展示出减少的irAE的事实进一步支持阻断B7-CTLA-4相互作用并不引起L3D10的安全性改进的观念。
鉴于所提出的CTLA-4在针对自身免疫疾病进行保护方面的作用,我们提出减少与可溶CTLA-4的结合作为改进安全概况的潜在机制。为了测试这个假说,我们使用在围产期期间接受抗PD-1+抗CTLA-4 mAb的雌性小鼠之中的生长体重增加作为irAE的基本指示。如图42中所示,在接受10D1和抗PD-1两者的小鼠中观察到体重增加的严重减少,而接受PP4637+抗PD-1的小鼠具有最低irAE,随后是PP4631,并且随后是L3D10。与减少的与sCTLA-4的结合的严格逆相关性与中心假说相一致。
实例15.人源化抗CTLA4抗体的可加工性评估
为了评估三种不同人源化抗体的开发和制造潜力,执行多种分析方法以表征不同抗体。
作为初步评估,基于氨基酸序列计算三种领先候选抗体的预测分子量和等电点。如表7中所示,所有抗体都相当类似,但抗体具有略低的PI。
表7:三种人源化抗体的理论参数
蛋白名称 | 理论MW(Da) | 理论PI |
PP4631 | (49647.8+23483.1)X 2=96614.0 | 7.9 |
PP4637 | (49644.9+23483.1)X 2=96611.1 | 7.65 |
PP4638 | (49644.9+23311.9)X 2=96568.7 | 7.9 |
产物产量评估
为了评估不同抗体的生产率,将HEK293细胞用表达不同抗体的重链和轻链的载体瞬时转染。随后使用无血清培养基将这些细胞在摇瓶中培养6天。在6天之后,收集上清液并且通过一步蛋白A色谱法纯化抗体。如下表8中所示,抗体PP4631和PP4637展现类似蛋白产量,而抗体PP4638以低得多的相对产量产生。
表8:人源化抗体生产产量评估.
抗体 | 浓度(mg/mL) | OD 260/280 | 产量(mg/L) |
PP4631 | 1.280 | 0.53 | 126 |
PP4637 | 4.532 | 0.53 | 118 |
PP4638 | 0.729 | 0.57 | 56 |
为了评估瞬时表达的抗体的纯度,通过还原和非还原SDS-PAGE分析样品。如图50中所示,来自全部3种抗体的样品产生指示抗体分子的凝胶条带,并且样品在蛋白A纯化之后相对纯。
尺寸排阻色谱法
为了进一步检查在瞬时表达之后不同抗体的纯度和聚集,我们对纯化蛋白执行尺寸排阻色谱法。简单点说,将50μg经过滤(使用0.22μm过滤器)样品用于使用TOSOH G3000SWxl 5μm柱的SE-HPLC分离。PBS pH 7.4用作流动相。如下表9中所示,所有人源化抗体在蛋白A纯化之后都展示>90%纯度。抗体PP4631和PP4637展现抗体样品存在类似低水平的较高分子量(MW)聚集和降解,大部分蛋白质在主峰内。相比之下,抗体PP4638具有较高水平的聚集和一定降解。SE-HPLC色谱图展示于图51中。
表9:尺寸排阻色谱法
抗体 | 聚集 | 主峰 | 降解 |
PP4631 | 2.6% | 97.4% | 0 |
PP4637 | 3.0% | 97.0% | 0 |
PP4638 | 6.5% | 92.4% | 1.1% |
毛细管电泳(CE)
毛细管电泳用以定量在还原和非还原条件下峰条带内的蛋白质的量以及未糖基化重链蛋白的量。简单点说,将100μg样品与碘乙酰胺(非还原条件)或β-巯基乙醇(还原条件)一起与2μL 10kDa标准蛋白一起稀释到CE-SDS样品缓冲液中。随后在70℃下处理样品10分钟。在分离中,使用PA-800,50μm I.D.裸熔融硅石毛细管;运行长度 20.2cm;分离电压15kV;OD220用于检测。如下表10中所示,全部三种蛋白质都展现高纯度水平,与SDS-PAGE相一致,并且都高度糖基化。CE-SDS色谱图展示于图52中。
表10:毛细管电泳
抗体 | 非还原% | 还原% | 未糖基化重链 |
PP4631 | 97.3 | 99.5 | 0.3 |
PP4637 | 97.2 | 99.5 | 0.4 |
PP4638 | 96.9 | 99.4 | 0.4 |
脱酰胺化:毛细管等电聚焦(cIEF)和液相色谱法-质谱分析(LC-MS)
通过比较经两个不同时间段(5小时和12.5小时)的高pH应力处理和不经所述处理的抗体,随后进行cIEF和LC-MS分析,来测定高pH应力下的蛋白质脱酰胺化水平。
通过毛细管等电聚焦(cIEF)测定不同抗体的电荷同种型分布图和等电点。简单点说,样品经历缓冲液交换到20mM Tris pH 8.0中,并且随后将100μg样品蛋白与两性电解质甲基纤维素以及PI 7.05和PI 9.77标记混合。iCE3TM用于分析,100μm I.D.毛细管;1.5kV加3kV;OD280用于检测。在脱酰胺化应力处理中,将样品用500mM NaHCO3处理5小时或12.5小时,随后用cIEF和LC-MS检查。分析的结果展示于下表11中并且LC-MS图展示于图53中。全部三种抗体都展示出在应力条件下脱酰胺物质量的预测增加和主峰的相应下降。如由氨基酸序列所预测,抗体PP4637的pI比PP4631和PP4638 低一点(表7),并且与预测pI相比更高的观察pI推测起来指示糖基化。
表11:等电聚焦和脱酰胺化
差示扫描量热法(DSC)热分析
为了测定不同抗体的热稳定性和熔融温度,使其经历差示扫描量热法(DSC)热分析。简单点说,使2mg/mL于PBS pH 7.4中的样品以1℃/min的速率经历从15℃到105 ℃的温度斜变。监测两种样品和缓冲液(作为背景)的Cp随温度变化。在背景减除下获得Cp对温度曲线,并且峰指示分析物的Tm。如下表12中所示,全部三种抗体都展现类似高的熔融温度。三种抗体的DSC曲线展示于图54中。
表12:尺寸排阻色谱法
抗体 | T<sub>M</sub>(℃) |
PP4631 | 75.6 |
PP4637 | 76.2 |
PP4638 | 76.6 |
氧化:肽作图
通过在有或无氧化应力下使用LC-MS进行肽作图来评估人源化抗体的氧化修饰。使样品在65℃下在6M GnCl和5mM β-ME存在下变性,随后用碘乙酰胺乙酰化。随后在55℃下用胰蛋白酶(普洛麦格(Promega),测序级)使加工的样品消化,并且将消化混合物在C18逆相LC柱(ACQUITY UPLC BEH130C18,2.1×100mm,1.7μm)上分离并且使用Masslynx和Biophatmlynx分析工具通过质谱分析(沃特世(Waters)XEVO- G2S QTOF)分析。在氧化应力分析中,将样品用0.05%或0.1%H2O2处理1小时,随后用LC-MS检查。结果展示于下表13-16中。
表13.人源化抗体在甲硫氨酸位点处的氧化。上图:抗体PP4631。中图:抗体PP4637。下图:抗体PP4638。
表14.人源化抗体PP4631在色氨酸位点处的氧化。红色数字指示所见片段化的证据;“-”指示无一者检测到;“0”指示在极低水平下检测到。
表15.人源化抗体PP4637在色氨酸位点处的氧化。红色数字指示所见片段化的证据;“-”指示无一者检测到;“0”指示在极低水平下检测到。
表16.人源化抗体PP4638在色氨酸位点处的氧化。红色数字指示所见片段化的证据;“-”指示无一者检测到;“0”指示在极低水平下检测到。
结合特异性
通过在两种不同浓度下相对于10D1评估检测与两种不表达CTLA4的不同细胞系(CHO和HEK293)的非特异性结合的能力来测定不同抗体的结合特异性。简单点说,将100μg/mL或20μg/mL样品(或参考mAb)于PBS中用3×10e6个细胞/毫升(CHO 或HEK293)孵育。FITC标记的兔抗人类IgG抗体(中国武汉的博士德(Boster,Wuhuan, China))用于检测并且通过FACS测量标靶mAb与细胞的结合。如下表17中所示,抗体PP4631和PP4637展现极低结合和良好特异性,而抗体PP4638展现与对照细胞系的非特异性结合活性。
表17:与CHO和HEK293细胞系的结合特异性
实例16.L3D10和人源化抗体的表位作图
为了对L3D10亲本抗体以及人源化变异体PP4631和PP4637的CTLA-4结合表位作图,我们利用小鼠和人类CTLA4蛋白与B7-1具交叉反应性但与抗CTLA-4抗体不具交叉反应性的事实。因此,我们设计人类CTLA-4Fc蛋白的多种突变体,其中来自人类 CTLA-4蛋白的氨基酸簇经来自鼠类Ctla-4蛋白的氨基酸置换。因为这一研究中使用的抗CTLA-4抗体不与鼠类Ctla-4结合,所以当抗体结合表位的关键残基经鼠类氨基酸置换时应消除抗人类CTLA-4抗体的结合。
基于野生型人类CTLA-4Fc序列构建编码11种CTLA-4Fc突变蛋白(M1-M11) (SEQID NO:40-50)的DNA载体,并且通过以0.01mL规模瞬时转染于HEK293中随后进行一步蛋白A色谱法纯化而产生蛋白质。
通过ELISA执行抗CTLA4抗体与CTLA4Fc蛋白的结合。将板用CTLA-4Fc蛋白以1μg/mL涂布,并且随后在结合分析中在可溶相中使用生物素化抗体或B7-1Fc融合蛋白,使用辣根过氧化酶(HRP)结合抗生蛋白链菌素测量结合蛋白的量。
抗人类CTLA-4抗体不与鼠类Ctla-4交叉反应,这推测起来反映了胞外结构域中的人类与小鼠CTLA-4之间的氨基酸序列的差异。图55展示人类、猕猴和小鼠CTLA-4胞外结构域的比对,并且突显人类与猕猴之间的序列保守,同时展示鼠类与灵长类动物序列之间的众多差异。归因于MYPPPY结合基序的保守,小鼠和人类CTLA4蛋白与B7- 1具交叉反应性(72)。
为了对抗人类CTLA-4抗体的结合表位作图,我们生成将鼠类特异性氨基酸簇并入到人类CTLA-4序列中的多种非重叠CTLA-4Fc突变蛋白。并入到11种突变体中的每一者中的氨基酸展示于图55中,并且WT和突变CTLA-4Fc蛋白的氨基酸序列展示于图56中。这些蛋白质通过瞬时转染于HEK293细胞中而产生,并且产量提供于表18中。相对于WT人类CTLA-4Fc蛋白,许多突变如由其产量指示呈现为影响蛋白表达。
表18:瞬时产生于HEK293细胞中的WT和突变CTLA-4Fc蛋白.
蛋白名称 | 产量(mg) |
CTLA-4Fc WT对照 | 0.72 |
突变体1 | 1.29 |
突变体2 | 0.03 |
突变体3 | 0.21 |
突变体4 | 0.11 |
突变体5 | 1.89 |
突变体6 | 0.38 |
突变体7 | 0.25 |
突变体8 | 1.61 |
突变体9 | 0.01 |
突变体10 | 0.04 |
突变体11 | 1.70 |
随后通过ELISA来测定嵌合L3D10以及人源化抗体PP4631和PP4637结合固定CTLA-4Fc突变构建体的能力,其中将板用CTLA-4突变构建体涂布,并且添加生物素化抗CTLA-4抗体或B7-1g对照蛋白,并且使用HRP结合抗生蛋白链菌素测量结合。结合分析的结果展示于表19-22中。正如所料,全部4种结合蛋白对于WT CTLA-4Fc 蛋白都展现良好的剂量依赖性结合。然而,引入到M9和M10蛋白中的突变呈现为改变总体结构并且这些突变体未能结合B7-1Fc。如由相对于WT蛋白减少的结合所指示,引入M2和M4中的突变也部分改变CTLA-4构象。与这个观念相一致,这些突变体(M2、 M4、M9和M10)中的全部4者都以低得多的产量表达(表18)。相比之下,使用与WT CTLA-4Fc蛋白的结合和B7-1Fc蛋白的结合作为参考,M11明显突显为良好表达的蛋白质、高效地结合B7-1Fc但未能结合两种人源化抗CTLA-4抗体。其与初始L3D10的结合也减少约100倍(表20)。正如所料,影响总体确认的突变也影响与抗CTLA-4抗体的结合。
表19:CTLA4Ig突变体如由其与B7-1Ig融合蛋白的结合所指示的完整性。通过ELISA执行与CTLA4Fc蛋白的结合,通过辣根过氧化酶(HRP)结合抗生蛋白链菌素测量结合的生物素化蛋白的量。所示值是OD450测量值。WT=野生型CTLA-4Fc。M1- M11是CTLA-4Fc突变蛋白。
表20:嵌合L3D10抗体的表位作图。通过ELISA执行与CTLA4Fc蛋白的结合,通过辣根过氧化酶(HRP)结合抗生蛋白链菌素测量结合的生物素化蛋白的量。所示值是OD450测量值。WT=野生型CTLA-4Fc。M1-M11是CTLA-4Fc突变蛋白
表21:人源化抗体PP4631的表位作图。通过ELISA执行与CTLA4Fc蛋白的结合,通过辣根过氧化酶(HRP)结合抗生蛋白链菌素测量结合的生物素化蛋白的量。所示值是OD450测量值。WT=野生型CTLA-4Fc。M1-M11是CTLA-4Fc突变蛋白
表22:人源化抗体PP4637的表位作图。通过ELISA执行与CTLA4Fc蛋白的结合,通过辣根过氧化酶(HRP)结合抗生蛋白链菌素测量结合的生物素化蛋白的量。所示值是OD450测量值。WT=野生型CTLA-4Fc。M1-M11是CTLA-4Fc突变蛋白
表23:来自重复研究的原始数据展示仅在M11中抗原表位有特定丢失。如同在表2-5中,但包括额外对照以展示结合的特异性。
因为L3D10保留与M11的显著结合,所以我们测试结合是否是特异性的。我们用人类CTLA4-Fc(hCTLA4Fc)、小鼠CTLA4-Fc(mCTLA4-Fc)、对照IgG1-Fc或所有突变hCTLA4-Fc涂布板并且测量其与B7-1Fc以及L3D10、PP4631和PP4637的结合。大批数据呈现于表23中。如图57中所示,生物素化B7-1同样好地结合hCTLA-4、mCTLA- 4和M11。分析的特异性由与IgG1-Fc结合的缺乏展现。有趣的是,虽然L3D10与M11 的结合比与IgG1-Fc和mCTLA4-Fc的结合更强,但与IgG1-Fc的显著结合表明嵌合抗体与M11的结合可能是非特异性的。相比之下,人源化抗体中无一者与M11、mCTLA- 4和IgG1-Fc对照结合。这些数据证实,引入M11中的突变选择性地消除L3D10、PP4631 和PP4637与CTLA-4的结合。
使用已知复合结构133,我们在3D结构中对CTLA-4表位作图。如图58中所示,由这些mAb识别的表位定位于由B7-1覆盖的区域内。因此,L3D10、PP4631和PP4637 与CTLA-4的结合将与B7-1的结合互斥。不良阻断PP4631和PP4637是由于较低亲合力而非独特结合结构域。
利用小鼠和人类CTLA4蛋白与B7-1具交叉反应性但抗人类CTLA-4抗体与鼠类Ctla-4蛋白不交叉反应的事实,我们能够通过ELISA对L3D10衍生抗体的结合表位作图。使用人类CTLA-4Fc蛋白的其中来自人类CTLA-4蛋白的氨基酸簇经来自鼠类Ctla- 4蛋白的氨基酸置换的多种突变体,我们明确证实,当我们置换紧接着CTLA-4的已知 B7-1结合结构域的4个氨基酸时,基本上消除抗体的剂量依赖性结合。结合表位紧邻 B7-1结合结构域图谱定位的事实与L3D10抗体展现的阻断体外和体内B7-CTLA-4相互作用的能力充分相关。因为可溶CTLA4通过使胞外IgV结构域的C末端氨基酸与胞内结构域融合来产生,所以很容易推测与多态C末端结构域残基(来自C末端的仅18氨基酸)结合的抗体更有可能失去与可溶CTLA-4的反应性,其中大胞内结构域与胞外结构域的C末端融合。
为了进一步研究抗CTLA4抗体的结合结构域,设计6种额外突变CTLA4-Fc融合蛋白,命名为M12-M17(SEQ ID NO:51-56)(图59),并且将其用以比较抗CTLA4抗体10D1(图60A)、PP4631(图60B)和PP4637(图60C)的结合。如图60中所示, M11中的处于位置y103L104I106处的突变消除与10D1、PP4631和PP4637的结合,证实 10D1、PP4631和PP4637的结合位点包括残基y103L104I106。重要的是,A29>Y的额外突变恢复在Y103L104I106具有突变的CTLA-4与PP4631和PP4637的结合。这些数据证实, CTLA4中的位置A29对于抗体PP4631和PP4637的结合很重要,但对于10D1不重要。
实例17.抗CTLA-4 mAb在诱导肿瘤排斥方面与抗4-1BB协同
动物模型中的研究已经表明,由抗CTLA-4单克隆抗体(mAb)引起的抗肿瘤应答至少部分是由于针对正常“自身”分化抗原的抗原特异性T细胞应答(73,74)。抗CTLA- 4抗体加重自身免疫疾病的倾向在小鼠中证据充分(75-78)。这个观念得到进一步确证并且被证明是患者具有需要中断治疗的严重自身免疫表现的更为新近的人类试验中的主要限制(79)。另一方面,癌症治疗性抗4-1BB mAb据显示可消除自身免疫疾病在易感狼疮的小鼠中的出现(24,25)。
抗4-1BB mAb可以刺激抗肿瘤应答和降低自身免疫表现的事实引发了这种抗体与抗CTLA-4 mAb的组合可以引起癌症排斥而不造成自身免疫性的令人感兴趣的可能性。在这一研究中,抗CTLA-4和抗4-1BB组合以诱导大的确立的肿瘤的排斥。
抗鼠类CTLA-4和抗鼠类4-1BB抗体在诱导CD8T细胞介导的肿瘤排斥方面的组合效
应.
两种模型(一种具有最小疾病并且一种具有大的确立的肿瘤)用以测试组合抗鼠类 4-1BB和抗鼠类CTLA-4 mAb处理的抗肿瘤效应。将C57BL/6小鼠用皮下接种MC38结肠癌细胞攻击,并且在肿瘤细胞接种之后的不同时间,将抗体注射到肿瘤攻击小鼠中,并且通过身体检查监测肿瘤大小和发生率。
在最小疾病模型中,在接种肿瘤细胞之后48小时开始,将小鼠用仓鼠IgG加大鼠IgG、抗4-1BB加仓鼠IgG(单独抗4-1BB组)、抗CTLA-4加大鼠IgG(单独抗CTLA- 4组)或抗4-1BB与抗CTLA-4的组合处理。在第2、9和16天腹膜内(i.p.)给予抗体。用单独抗4-1BB或抗CTLA-4 mAb处理导致肿瘤生长延迟,每个组中5只小鼠中有 1只排斥肿瘤,而用抗CTLA-4和抗4-1BB mAb两者处理的5只小鼠中有4只在实验结束时无肿瘤。图61A展示每只小鼠的肿瘤生长测量值。为了比较各组之间的生长速率,将线性随机效应模型应用到数据。组合疗法相对于单独抗CTLA-4使肿瘤大小的每天生长显著减少4.6mm2/天(p=0.0094)。此外,组合疗法相对于单独抗4-1BB使生长显著减少8.4mm2/天(p=0.0006)。除了生长速率之外,在初始肿瘤攻击之后六周比较处理组之间的实际肿瘤大小。六周时的平均肿瘤大小对于被给予组合疗法的小鼠(27.5mm2) 与分别被给予抗CTLA-4(137.8mm2,p=0.0251)或抗4-1BB(287.6,p=0.0006)的小鼠相比显著更小。因此,在最小肿瘤负荷设定下,抗4-1BB和抗CTLA-4 mAb的组合相对于分别给予抗4-1BB或抗CTLA-4导致显著肿瘤生长延迟。
为了确定组合mAb处理针对小肿瘤负荷的抗肿瘤效应是否可以延伸到针对较大肿瘤负荷的治疗应用,将具有确立肿瘤的小鼠用抗体处理。将野生型C57BL/6小鼠用皮下接种MC38结肠癌细胞攻击。使肿瘤生长14天,这时将具有确立肿瘤(通常直径>7 mm)的小鼠选择并且随机分为四个处理组:仓鼠IgG加大鼠IgG、抗4-1BB加仓鼠IgG、抗CTLA-4加大鼠IgG和抗4-1BB mAb与抗CTLA-4 mAb的组合。在肿瘤攻击之后第 14、21和28天腹膜内给予抗体。如图61B中所示,用抗CTLA-4 mAb处理当与对照 IgG处理相比时不阻碍肿瘤生长,但排斥可见于组中的八只小鼠之一中。用抗4-1BB mAb 处理稍微减缓肿瘤生长。但八只小鼠中仅有一只排斥肿瘤。相比之下,抗CTLA-4和抗 4-1BB mAb两者的组合疗法导致8只小鼠中有7只根除肿瘤并且预防其余小鼠中的进一步肿瘤生长。如上所述,通过将线性随机效应模型应用到数据比较各组之间的生长速率。组合疗法相对于单独抗CTLA-4使肿瘤大小的每天生长显著减少10.6mm2/天(p< 0.0001)。此外,组合疗法相对于单独抗4-1BB使生长显著减少6.2mm2/天(p=0.0002)。除了生长速率之外,在初始肿瘤攻击之后八周比较处理组之间的实际肿瘤大小。八周时的估算平均肿瘤大小对于被给予组合疗法的小鼠(-1.7mm2,95%CI:-10.8,7.5mm2) 与分别被给予抗CTLA-4(404.9mm2,95%CI:285.4,524.4mm2;p<0.0001)或抗4- 1BB(228.4mm2,95%CI:200.4,689.9mm2;p=0.0004)的小鼠相比显著更小。因此,在较大肿瘤负荷下,组合mAb相对于单独抗CTLA-4或抗4-1BB似乎也显著延迟肿瘤生长。
MC38已知会形成肝脏转移。80为了评估治疗抗体对肝脏转移的作用,通过组织学分析所有参加实验的小鼠的肝脏转移。如表24中所示,约60%的对照Ig处理小鼠在肝脏中具有微转移。用单独抗CTLA-4或抗4-1BB抗体处理稍微降低转移速率,但降低未达统计显著性。引人注目地,用两种抗体处理的组中仅1/22小鼠具有肝脏转移。使用逻辑回归模型,我们发现,被给予单独抗4-1BB的小鼠的肝脏转移的几率比被给予抗4- 1BB和抗CTLA-4两者的小鼠的几率高约4.7倍(95%CI:1.6,13.7;p=0.0050)。类似地,被给予仅抗CTLA-4的小鼠的肝脏转移的几率与被给予两种处理的小鼠相比高3.6 倍(95%CI:1.3,10.2;p=0.0174)。因此,组合疗法当与用单独任一抗体处理相比时显著减少MC38的肝脏转移。
表24:组合疗法大体上减少肝脏转移*
*数据由4个独立实验概述。在H&E染色之后检查每个肝脏的至少两个切片。
为了确定哪个免疫细胞亚组促成由组合mAb处理引起的抗肿瘤效应,用单克隆抗体耗尽主要淋巴细胞亚组。皮下注射MC38肿瘤细胞。在肿瘤可触后,将携有肿瘤的小鼠分成四个组。每个组具有一系列腹膜内抗体注射以耗尽不同免疫细胞亚组,包括正常大鼠IgG情况下的无耗尽、用抗CD4 mAb(GK 1.5)使CD4T细胞耗尽、用抗CD8 mAb (2.4.3)使CD8 T细胞耗尽和用抗NK1.1 mAb(PK136)使NK细胞耗尽。另外,每周一次持续三周将所有组中的所有小鼠用抗CTLA-4加抗4-1BB mAb处理。通过对在完成实验之前立即取自小鼠的外周血液进行流式细胞术来评估免疫细胞亚组的充足耗尽 (数据未展示)。正如所料,无免疫细胞耗尽的小鼠响应于用抗CTLA-4与抗4-1BB mAb 的组合处理(图62)。类似地,耗尽NK细胞和CD4 T细胞不影响组合抗CTLA-4加抗 4-1BB mAb疗法的抗肿瘤活性。然而,耗尽CD8 T细胞消除组合抗体疗法的抗肿瘤活性。在第28天,耗尽CD8 T细胞的小鼠的估算平均肿瘤大小(92.3mm2,95%CI:64.5, 120.1mm2)显著大于无免疫细胞耗尽的小鼠(28.7mm2,95%CI:-17.1,74.4mm2)、耗尽CD4 T细胞的小鼠(16.7mm2,95%CI:1.0,32.4mm2)和耗尽NK细胞的小鼠 (9.3mm2,95%CI:-8.3,26.9mm2)的平均肿瘤大小。这些数据证实,抗CTLA-4和抗 4-1BBmAb处理的肿瘤根除效应是CD8 T细胞依赖性的。
抗4-1BB抗体减少对异种抗CTLA-4抗体的抗体应答.
重复抗体疗法的障碍之一是对治疗抗体的宿主抗体应答的增强。81因为已知4-1BB可减少对蛋白质的抗体应答,所以我们评估抗4-1BB抗体对于对抗CTLA-4抗体的宿主应答的作用。如图63中所示,在用对照IgG或抗4-1BB处理的小鼠中检测到极少(如果存在的话)抗抗体应答。与抗CTLA-4 mAb促进CD4 T细胞应答的能力82相一致,用抗CTLA-4加大鼠IgG处理的小鼠针对给予的4F10抗体和大鼠IgG出现强宿主抗体应答(图63A-B)。当抗4-1BB与抗CTLA-4 mAb共同给予时,这个应答减少超过30 倍。这些数据表明,抗4-1BB抗体可能通过减少对治疗剂的宿主应答而增加其它共同给予的治疗蛋白的持续时间。
在人类CTLA-4敲入小鼠中,抗小鼠4-1BB和抗人类CTLA-4抗体的组合诱导肿瘤排
斥和持久癌症免疫性.
因为抗4-1BB减少抗体针对抗CTLA-4抗体的产生,所以有趣的问题是肿瘤排斥因抗4-1BB所致的增强是否仅仅归因于其在抑制抗体应答方面的作用。这种人类CTLA4 基因敲入小鼠使我们可测试抗人类CTLA4抗体的抗肿瘤效应是否可以通过抗4-1BB抗体来增强。如图64A中所示,虽然单独抗人类CTLA-4(L3D10)和抗4-1BB抗体(2A) 都导致延迟的肿瘤生长,但两种抗体的组合导致最显著肿瘤排斥。在用抗CTLA-4、4- 1BB或两种抗体处理的组中,分别有1/7、2/7、5/7小鼠从未出现肿瘤,而未处理组中的所有小鼠都出现肿瘤。因为抗人类CTLA-4抗体具有小鼠来源,所以4-1BB抗体的影响无法归于其抑制抗体于治疗抗CTLA-4抗体。此外,我们的数据还证实,组合疗法的优越效应有可能适用于基于抗人类CTLA-4抗体的免疫疗法。
为了测试双抗体处理小鼠是否对进一步肿瘤细胞攻击免疫,我们在其第一次肿瘤细胞攻击之后110天用肿瘤细胞攻击其。如图64B中所示,在第一轮中已经排斥肿瘤细胞的五只双抗体处理小鼠全部保持无肿瘤,而对照未处理小鼠具有进行性肿瘤生长。因此,组合疗法还诱导对癌细胞的持久免疫性。
基于蛋白质的免疫疗法的障碍之一是对治疗蛋白的宿主免疫性。在抗体的情况下,宿主可以将抗体安装到异种型、异型和个体基因型表位。81异种型应答可以通过完全人源化来消除,但其它抗抗体应答需要特殊考虑。对于抗CTLA-4抗体障碍更明显,因为其本身是佐剂。Mittler等人的先前研究展现T细胞依赖性体液免疫应答的显著抑制。83我们的数据证实,共同给予抗4-1BB抗体减少对抗CTLA-4抗体的宿主应答,这暗示了使用抗CTLA-4和抗4-1BB抗体的组合疗法的另一优点。
综合来看,我们的数据证实,抗CTLA-4和抗4-1BB抗体的组合疗法提供三种主要优点,即癌症免疫性效应的增加、自身免疫副作用的相互抑制和抗抗体应答的改善。
本说明书中提及的所有公开和专利都以引用的方式并入本文中,引用的程度如同每个单独公开或专利申请都经具体并且单独指示以全文引用的方式并入一般。虽然已经结合其具体实施例描述了本发明,但是应理解,能够进行进一步修改,并且本申请旨在涵盖本发明的一般来说根据本发明的原理并且包括如下与本公开的偏离的任何变化形式、用途或改编,所述偏离如在本发明所涉及领域内在已知或惯用实践内出现并且可应用于在上文中阐述的必需特征。
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序列表
<110> OncoImmune, Inc.
Liu, Yang
Zheng, Pan
Devenport, Martin
<120> 嵌合和人源化抗人类CTLA4单克隆抗体和其用途
<130> 060275.0600.03PC00
<150> 62/267,735
<151> 2015-12-15
<150> 62/359,036
<151> 2016-07-06
<150> 62/309,169
<151> 2016-03-16
<160> 73
<170> PatentIn version 3.5
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85 90 95
Lys Thr Glu Gly His Tyr Tyr Gly Ser Asn Tyr Gly Tyr Tyr Ala Leu
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120 125
<210> 3
<211> 329
<212> PRT
<213> 智人
<400> 3
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 4
<211> 329
<212> PRT
<213> 人工序列
<220>
<223> 工程改造的片段
<400> 4
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 5
<211> 1437
<212> DNA
<213> 人工序列
<220>
<223> 优化的片段
<400> 5
atggacccca agggcagcct gagctggaga atcctgctgt tcctgagcct ggccttcgag 60
ctgagctacg gccaggtgca gctgaaagag tctggccctg gcctggtggc cccttcccag 120
tccctgtcta tcacctgtac cgtgtccggc ttctccctga cctcctacgg cctgtcttgg 180
gtgcgacagc ctcctggcaa gggcctggaa tggctgggag tgatttggta cgacggcaac 240
accaacttcc actccgccct gatctcccgg ctgaccatct ccaaggacaa ctccaagtcc 300
caggtgttcc tggaactgaa ctccctgcag accgacgaca ccgccaccta ctactgcgct 360
aagaccgagg gccactacta cggctccaac tacggctact acgccctgga ctattggggc 420
cagggcacct ccgtgaccgt gtcctctgct agcaccaagg gccccagcgt gttccctctg 480
gcccccagca gcaagagcac cagcggcgga accgccgccc tgggctgcct ggtgaaggac 540
tacttccccg agcccgtgac cgtgtcctgg aacagcggcg ctctgaccag cggagtgcac 600
accttccctg ccgtgctgca gagcagcggc ctgtactccc tgagcagcgt ggtgaccgtg 660
cccagcagca gcctgggcac ccagacctac atctgcaacg tgaaccacaa gccctccaac 720
accaaggtgg acaagaaggt ggagcctaag agctgcgaca agacccacac ctgccctccc 780
tgccccgccc ccgagctgct gggcggaccc agcgtgttcc tgttccctcc caagcccaag 840
gacaccctgt acatcacccg cgaacccgag gtgacctgcg tggtggtgga cgtgagccac 900
gaggaccccg aggtgaagtt caactggtac gtggacggcg tggaggtgca caacgccaag 960
accaagcctc gggaggagca gtacaacgcc acctaccgcg tggtgagcgt gctgaccgtg 1020
ctgcaccagg actggctgaa cggcaaggag tacaagtgca aggtgagcaa caaggccctg 1080
cccgctccca tcgcagccac catcagcaag gccaagggcc agccccggga gcctcaggtg 1140
tacaccctgc cccccagccg cgacgagctg accaagaacc aggtgagcct gacctgcctg 1200
gtgaagggct tctacccctc cgacatcgcc gtggagtggg agagcaacgg ccagcctgag 1260
aacaactaca agaccacccc tcccgtgctg gacagcgacg gcagcttctt cctgtacagc 1320
aagctgaccg tggacaagtc ccggtggcag cagggcaacg tgttcagctg cagcgtgatg 1380
cacgaggccc tgcacaacca ctacacccag aagagcctga gcctgagccc cggatag 1437
<210> 6
<211> 478
<212> PRT
<213> 人工序列
<220>
<223> 优化的片段
<400> 6
Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser
1 5 10 15
Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Lys Glu Ser Gly
20 25 30
Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val
35 40 45
Ser Gly Phe Ser Leu Thr Ser Tyr Gly Leu Ser Trp Val Arg Gln Pro
50 55 60
Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Tyr Asp Gly Asn
65 70 75 80
Thr Asn Phe His Ser Ala Leu Ile Ser Arg Leu Thr Ile Ser Lys Asp
85 90 95
Asn Ser Lys Ser Gln Val Phe Leu Glu Leu Asn Ser Leu Gln Thr Asp
100 105 110
Asp Thr Ala Thr Tyr Tyr Cys Ala Lys Thr Glu Gly His Tyr Tyr Gly
115 120 125
Ser Asn Tyr Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser
130 135 140
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
145 150 155 160
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
165 170 175
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
180 185 190
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
195 200 205
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
210 215 220
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
225 230 235 240
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
260 265 270
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
275 280 285
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
290 295 300
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
305 310 315 320
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser
325 330 335
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
340 345 350
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile
355 360 365
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
370 375 380
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
385 390 395 400
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
405 410 415
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
420 425 430
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
435 440 445
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
450 455 460
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
465 470 475
<210> 7
<211> 705
<212> DNA
<213> 人工序列
<220>
<223> 优化的片段
<400> 7
atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60
gacatccaga tgacccagtc ccccgcctcc ctgtctgtgt ctgtgggcga gacagtgacc 120
atcacctgtc gggcctccga gaacatctac tccaacctgg cctggtatca gcagaagcag 180
ggcaagtccc ctcagctgct ggtgtacgcc gccaccaatc tggctgatgg cgtgccctcc 240
agattctccg gctctggctc tggcacccag tactccctga agatcaactc cctgcagtcc 300
gaggacttcg gcacctactt ttgccagcac ctgtggggca ccccctacac ctttggcggc 360
ggaacaaagc tggaaatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420
agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540
gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660
ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 705
<210> 8
<211> 234
<212> PRT
<213> 人工序列
<220>
<223> 嵌合
<400> 8
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser
20 25 30
Val Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
35 40 45
Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
50 55 60
Gln Leu Leu Val Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
85 90 95
Ser Leu Gln Ser Glu Asp Phe Gly Thr Tyr Phe Cys Gln His Leu Trp
100 105 110
Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 9
<211> 478
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体片段
<400> 9
Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser
1 5 10 15
Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Gln Glu Ser Gly
20 25 30
Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val
35 40 45
Ser Gly Phe Ser Leu Thr Ser Tyr Gly Leu Ser Trp Ile Arg Gln Pro
50 55 60
Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Trp Tyr Asp Gly Asn
65 70 75 80
Thr Asn Phe His Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp
85 90 95
Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Lys Thr Glu Gly His Tyr Tyr Gly
115 120 125
Ser Asn Tyr Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser
130 135 140
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
145 150 155 160
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
165 170 175
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
180 185 190
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
195 200 205
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
210 215 220
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
225 230 235 240
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
260 265 270
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
275 280 285
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
290 295 300
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
305 310 315 320
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser
325 330 335
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
340 345 350
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile
355 360 365
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
370 375 380
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
385 390 395 400
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
405 410 415
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
420 425 430
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
435 440 445
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
450 455 460
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
465 470 475
<210> 10
<211> 1440
<212> DNA
<213> 人工序列
<220>
<223> 人源化抗体片段
<400> 10
atggacccca agggcagcct gagctggaga atcctgctgt tcctgagcct ggccttcgag 60
ctgagctacg gccaggtgca gctgcaggaa tctggccctg gcctcgtgaa gccctccgag 120
acactgtctc tgacctgcac cgtgtccggc ttctccctga cctcctacgg cctgtcttgg 180
atcagacagc cccctggcaa gggcctggaa tggatcggct acatttggta cgacggcaac 240
accaacttcc accccagcct gaagtccaga gtgaccatct ccaaggacac ctccaagaac 300
cagttctctc tgaagctgtc ctccgtgacc gccgctgaca ccgccgtgta ctactgtgct 360
aagaccgagg gccactacta cggctccaac tacggctact acgccctgga ctattggggc 420
cagggcacct ctgtgaccgt gtcctctgct agcaccaagg gccccagcgt gttccctctg 480
gcccccagca gcaagagcac cagcggcgga accgccgccc tgggctgcct ggtgaaggac 540
tacttccccg agcccgtgac cgtgtcctgg aacagcggcg ctctgaccag cggagtgcac 600
accttccctg ccgtgctgca gagcagcggc ctgtactccc tgagcagcgt ggtgaccgtg 660
cccagcagca gcctgggcac ccagacctac atctgcaacg tgaaccacaa gccctccaac 720
accaaggtgg acaagaaggt ggagcctaag agctgcgaca agacccacac ctgccctccc 780
tgccccgccc ccgagctgct gggcggaccc agcgtgttcc tgttccctcc caagcccaag 840
gacaccctgt acatcacccg cgaacccgag gtgacctgcg tggtggtgga cgtgagccac 900
gaggaccccg aggtgaagtt caactggtac gtggacggcg tggaggtgca caacgccaag 960
accaagcctc gggaggagca gtacaacgcc acctaccgcg tggtgagcgt gctgaccgtg 1020
ctgcaccagg actggctgaa cggcaaggag tacaagtgca aggtgagcaa caaggccctg 1080
cccgctccca tcgcagccac catcagcaag gccaagggcc agccccggga gcctcaggtg 1140
tacaccctgc cccccagccg cgacgagctg accaagaacc aggtgagcct gacctgcctg 1200
gtgaagggct tctacccctc cgacatcgcc gtggagtggg agagcaacgg ccagcctgag 1260
aacaactaca agaccacccc tcccgtgctg gacagcgacg gcagcttctt cctgtacagc 1320
aagctgaccg tggacaagtc ccggtggcag cagggcaacg tgttcagctg cagcgtgatg 1380
cacgaggccc tgcacaacca ctacacccag aagagcctga gcctgagccc cggatagtaa 1440
<210> 11
<211> 478
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体片段
<400> 11
Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser
1 5 10 15
Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Gln Glu Ser Gly
20 25 30
Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val
35 40 45
Ser Gly Phe Ser Leu Thr Ser Tyr Gly Leu Ser Trp Ile Arg Gln Pro
50 55 60
Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Trp Tyr Asp Gly Asn
65 70 75 80
Thr Asn Phe His Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp
85 90 95
Thr Ser Lys Ser Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Lys Thr Glu Gly His Tyr Tyr Gly
115 120 125
Ser Asn Tyr Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu
130 135 140
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
145 150 155 160
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
165 170 175
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
180 185 190
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
195 200 205
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
210 215 220
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
225 230 235 240
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
260 265 270
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
275 280 285
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
290 295 300
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
305 310 315 320
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser
325 330 335
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
340 345 350
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile
355 360 365
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
370 375 380
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
385 390 395 400
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
405 410 415
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
420 425 430
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
435 440 445
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
450 455 460
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
465 470 475
<210> 12
<211> 1440
<212> DNA
<213> 人工序列
<220>
<223> 人源化抗体
<400> 12
atggacccca agggcagcct gagctggaga atcctgctgt tcctgagcct ggccttcgag 60
ctgagctacg gccaggtgca gctgcaggaa tctggccctg gcctcgtgaa gccctccgag 120
acactgtctc tgacctgcac cgtgtccggc ttctccctga cctcctacgg cctgtcttgg 180
atcagacagc cccctggcaa gggcctggaa tggatcggct acatttggta cgacggcaac 240
accaacttcc actcctccct gaagtccaga gtgaccatct ccaaggacac ctccaagtcc 300
caggtgtctc tgaagctgtc ctccgtgacc gccgctgaca ccgccgtgta ctactgtgct 360
aagaccgagg gccactacta cggctccaac tacggctact acgccctgga ctattggggc 420
cagggcaccc tcgtgaccgt gtcctctgct agcaccaagg gccccagcgt gttccctctg 480
gcccccagca gcaagagcac cagcggcgga accgccgccc tgggctgcct ggtgaaggac 540
tacttccccg agcccgtgac cgtgtcctgg aacagcggcg ctctgaccag cggagtgcac 600
accttccctg ccgtgctgca gagcagcggc ctgtactccc tgagcagcgt ggtgaccgtg 660
cccagcagca gcctgggcac ccagacctac atctgcaacg tgaaccacaa gccctccaac 720
accaaggtgg acaagaaggt ggagcctaag agctgcgaca agacccacac ctgccctccc 780
tgccccgccc ccgagctgct gggcggaccc agcgtgttcc tgttccctcc caagcccaag 840
gacaccctgt acatcacccg cgaacccgag gtgacctgcg tggtggtgga cgtgagccac 900
gaggaccccg aggtgaagtt caactggtac gtggacggcg tggaggtgca caacgccaag 960
accaagcctc gggaggagca gtacaacgcc acctaccgcg tggtgagcgt gctgaccgtg 1020
ctgcaccagg actggctgaa cggcaaggag tacaagtgca aggtgagcaa caaggccctg 1080
cccgctccca tcgcagccac catcagcaag gccaagggcc agccccggga gcctcaggtg 1140
tacaccctgc cccccagccg cgacgagctg accaagaacc aggtgagcct gacctgcctg 1200
gtgaagggct tctacccctc cgacatcgcc gtggagtggg agagcaacgg ccagcctgag 1260
aacaactaca agaccacccc tcccgtgctg gacagcgacg gcagcttctt cctgtacagc 1320
aagctgaccg tggacaagtc ccggtggcag cagggcaacg tgttcagctg cagcgtgatg 1380
cacgaggccc tgcacaacca ctacacccag aagagcctga gcctgagccc cggatagtaa 1440
<210> 13
<211> 478
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 13
Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser
1 5 10 15
Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Gln Glu Ser Gly
20 25 30
Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val
35 40 45
Ser Gly Phe Ser Leu Thr Ser Tyr Gly Leu Ser Trp Ile Arg Gln Pro
50 55 60
Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Trp Tyr Asp Gly Asn
65 70 75 80
Thr Asn Phe His Ser Pro Leu Lys Ser Arg Val Thr Ile Ser Val Asp
85 90 95
Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Lys Thr Glu Gly His Tyr Tyr Gly
115 120 125
Ser Asn Tyr Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu
130 135 140
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
145 150 155 160
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
165 170 175
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
180 185 190
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
195 200 205
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
210 215 220
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
225 230 235 240
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
245 250 255
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
260 265 270
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu
275 280 285
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
290 295 300
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
305 310 315 320
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser
325 330 335
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
340 345 350
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile
355 360 365
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
370 375 380
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
385 390 395 400
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
405 410 415
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
420 425 430
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
435 440 445
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
450 455 460
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
465 470 475
<210> 14
<211> 1440
<212> DNA
<213> 人工序列
<220>
<223> 人源化抗体
<400> 14
atggacccca agggcagcct gagctggaga atcctgctgt tcctgagcct ggccttcgag 60
ctgagctacg gccaggtgca gctgcaggaa tctggccctg gcctcgtgaa gccctccgag 120
acactgtctc tgacctgcac cgtgtccggc ttctccctga cctcctacgg cctgtcttgg 180
atcagacagc cccctggcaa gggcctggaa tggatcggct acatttggta cgacggcaac 240
accaacttcc actccccact gaagtccaga gtgaccatct ccgtggacac ctccaagaac 300
cagttctctc tgaagctgtc ctccgtgacc gccgctgaca ccgccgtgta ctactgtgct 360
aagaccgagg gccactacta cggctccaac tacggctact acgccctgga ctattggggc 420
cagggcaccc tcgtgaccgt gtcctctgct agcaccaagg gccccagcgt gttccctctg 480
gcccccagca gcaagagcac cagcggcgga accgccgccc tgggctgcct ggtgaaggac 540
tacttccccg agcccgtgac cgtgtcctgg aacagcggcg ctctgaccag cggagtgcac 600
accttccctg ccgtgctgca gagcagcggc ctgtactccc tgagcagcgt ggtgaccgtg 660
cccagcagca gcctgggcac ccagacctac atctgcaacg tgaaccacaa gccctccaac 720
accaaggtgg acaagaaggt ggagcctaag agctgcgaca agacccacac ctgccctccc 780
tgccccgccc ccgagctgct gggcggaccc agcgtgttcc tgttccctcc caagcccaag 840
gacaccctgt acatcacccg cgaacccgag gtgacctgcg tggtggtgga cgtgagccac 900
gaggaccccg aggtgaagtt caactggtac gtggacggcg tggaggtgca caacgccaag 960
accaagcctc gggaggagca gtacaacgcc acctaccgcg tggtgagcgt gctgaccgtg 1020
ctgcaccagg actggctgaa cggcaaggag tacaagtgca aggtgagcaa caaggccctg 1080
cccgctccca tcgcagccac catcagcaag gccaagggcc agccccggga gcctcaggtg 1140
tacaccctgc cccccagccg cgacgagctg accaagaacc aggtgagcct gacctgcctg 1200
gtgaagggct tctacccctc cgacatcgcc gtggagtggg agagcaacgg ccagcctgag 1260
aacaactaca agaccacccc tcccgtgctg gacagcgacg gcagcttctt cctgtacagc 1320
aagctgaccg tggacaagtc ccggtggcag cagggcaacg tgttcagctg cagcgtgatg 1380
cacgaggccc tgcacaacca ctacacccag aagagcctga gcctgagccc cggatagtaa 1440
<210> 15
<211> 234
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 15
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
35 40 45
Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Lys Leu Leu Leu Tyr Ala Ala Thr Asn Leu Gln Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Leu Trp
100 105 110
Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 16
<211> 705
<212> DNA
<213> 人工序列
<220>
<223> 人源化抗体
<400> 16
atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60
gacatccaga tgacccagtc cccctccagc ctgtctgcct ctgtgggcga cagagtgacc 120
atcacctgtc gggcctccga gaacatctac tccaacctgg cctggtatca gcagaagccc 180
ggcaaggccc ctaagctgct gctgtacgcc gccaccaatc tgcagtctgg cgtgccctcc 240
agattctccg gctctggctc tggcaccgac tttaccctga ccatcagctc cctgcagccc 300
gaggacttcg ccacctacta ctgccagcat ctgtggggca ccccctacac ctttggcgga 360
ggcaccaagc tggaaatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420
agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540
gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660
ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 705
<210> 17
<211> 234
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 17
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
35 40 45
Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ala Pro
50 55 60
Lys Leu Leu Leu Tyr Ala Ala Thr Asn Leu Gln Asp Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln His Leu Trp
100 105 110
Gly Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 18
<211> 705
<212> DNA
<213> 人工序列
<220>
<223> 人源化抗体
<400> 18
atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60
gacatccaga tgacccagtc cccctccagc ctgtctgcct ctgtgggcga cagagtgacc 120
atcacctgtc gggcctccga gaacatctac tccaacctgg cctggtatca gcagaagcag 180
ggcaaggccc ctaagctgct gctgtacgcc gccaccaatc tgcaggatgg cgtgccctcc 240
agattctccg gctctggctc tggcaccgac tacaccctga ccatcagctc cctgcagccc 300
gaggacttcg ccacctactt ttgccagcat ctgtggggca ccccctacac ctttggccag 360
ggcaccaagc tggaaatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420
agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540
gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660
ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 705
<210> 19
<211> 234
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 19
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
35 40 45
Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Lys Leu Leu Ile Tyr Ala Ala Thr Ser Leu Gln Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Leu Trp
100 105 110
Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 20
<211> 705
<212> DNA
<213> 人工序列
<220>
<223> 人源化抗体
<400> 20
atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60
gacatccaga tgacccagtc cccctccagc ctgtctgcct ctgtgggcga cagagtgacc 120
atcacctgtc gggcctccga gaacatctac tccaacctgg cctggtatca gcagaagccc 180
ggcaaggccc ctaagctgct gatctacgcc gccaccagtc tgcagtctgg cgtgccctcc 240
agattctccg gctctggctc tggcaccgac tttaccctga ccatcagctc cctgcagccc 300
gaggacttcg ccacctacta ctgccagcat ctgtggggca ccccctacac ctttggcgga 360
ggcaccaagg tggaaatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420
agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540
gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600
ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660
ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 705
<210> 21
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 21
Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala
1 5 10
<210> 22
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 22
Ala Ala Thr Asn Leu Ala Asp
1 5
<210> 23
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 23
Gln His Leu Trp Gly Thr Pro Tyr Thr
1 5
<210> 24
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 24
Gly Phe Ser Leu Thr Ser Tyr Gly Leu Ser
1 5 10
<210> 25
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 25
Val Ile Trp Tyr Asp Gly Asn Thr Asn Phe His Ser Ala Leu Ile Ser
1 5 10 15
Arg
<210> 26
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 26
Thr Glu Gly His Tyr Tyr Gly Ser Asn Tyr Gly Tyr Tyr Ala Leu Asp
1 5 10 15
Tyr
<210> 27
<211> 149
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 27
Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser
1 5 10 15
Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Gln Glu Ser Gly
20 25 30
Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val
35 40 45
Ser Gly Phe Ser Leu Thr Ser Tyr Gly Leu Ser Trp Ile Arg Gln Pro
50 55 60
Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Trp Tyr Asp Gly Asn
65 70 75 80
Thr Asn Phe His Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp
85 90 95
Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Lys Thr Glu Gly His Tyr Tyr Gly
115 120 125
Ser Asn Tyr Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser
130 135 140
Val Thr Val Ser Ser
145
<210> 28
<211> 149
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 28
Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser
1 5 10 15
Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Gln Glu Ser Gly
20 25 30
Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val
35 40 45
Ser Gly Phe Ser Leu Thr Ser Tyr Gly Leu Ser Trp Ile Arg Gln Pro
50 55 60
Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Trp Tyr Asp Gly Asn
65 70 75 80
Thr Asn Phe His Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp
85 90 95
Thr Ser Lys Ser Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Lys Thr Glu Gly His Tyr Tyr Gly
115 120 125
Ser Asn Tyr Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu
130 135 140
Val Thr Val Ser Ser
145
<210> 29
<211> 149
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 29
Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser
1 5 10 15
Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Gln Glu Ser Gly
20 25 30
Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val
35 40 45
Ser Gly Phe Ser Leu Thr Ser Tyr Gly Leu Ser Trp Ile Arg Gln Pro
50 55 60
Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Trp Tyr Asp Gly Asn
65 70 75 80
Thr Asn Phe His Ser Pro Leu Lys Ser Arg Val Thr Ile Ser Val Asp
85 90 95
Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Lys Thr Glu Gly His Tyr Tyr Gly
115 120 125
Ser Asn Tyr Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu
130 135 140
Val Thr Val Ser Ser
145
<210> 30
<211> 127
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 30
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
35 40 45
Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Lys Leu Leu Leu Tyr Ala Ala Thr Asn Leu Gln Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Leu Trp
100 105 110
Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
115 120 125
<210> 31
<211> 127
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 31
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
35 40 45
Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ala Pro
50 55 60
Lys Leu Leu Leu Tyr Ala Ala Thr Asn Leu Gln Asp Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln His Leu Trp
100 105 110
Gly Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
115 120 125
<210> 32
<211> 127
<212> PRT
<213> 人工序列
<220>
<223> 人源化抗体
<400> 32
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
35 40 45
Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Lys Leu Leu Ile Tyr Ala Ala Thr Ser Leu Gln Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Leu Trp
100 105 110
Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
115 120 125
<210> 33
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 33
Tyr Ile Trp Tyr Asp Gly Asn Thr Asn Phe His Pro Ser Leu Lys Ser
1 5 10 15
Arg
<210> 34
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 34
Tyr Ile Trp Tyr Asp Gly Asn Thr Asn Phe His Ser Ser Leu Lys Ser
1 5 10 15
Arg
<210> 35
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 35
Tyr Ile Trp Tyr Asp Gly Asn Thr Asn Phe His Ser Pro Leu Lys Ser
1 5 10 15
Arg
<210> 36
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 肽片段
<400> 36
Ala Ala Thr Asn Leu Gln Ser
1 5
<210> 37
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 肽
<400> 37
Ala Ala Thr Asn Leu Gln Asp
1 5
<210> 38
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 肽
<400> 38
Ala Ala Thr Ser Leu Gln Ser
1 5
<210> 39
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 39
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
35 40 45
Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp
50 55 60
Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 40
<211> 357
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 40
Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 41
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 41
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser His Gly
1 5 10 15
Leu Ala Ser Phe Pro Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
35 40 45
Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp
50 55 60
Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 42
<211> 359
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 42
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Ser His Asn Thr Asp
20 25 30
Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
35 40 45
Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
50 55 60
Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr
65 70 75 80
Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val
85 90 95
Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
100 105 110
Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 43
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 43
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Thr Asn Asp Gln Met Val Thr Glu Val
35 40 45
Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp
50 55 60
Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 44
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 44
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
35 40 45
Cys Ala Ala Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp Asp
50 55 60
Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 45
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 45
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
35 40 45
Cys Ala Thr Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp
50 55 60
Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 46
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 46
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
35 40 45
Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Tyr
50 55 60
Pro Phe Cys Ser Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 47
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 47
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
35 40 45
Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp
50 55 60
Ser Ile Cys Thr Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 48
<211> 359
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 48
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
35 40 45
Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp
50 55 60
Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Val Asp Thr Gly Leu Tyr Ile Cys Lys Val
85 90 95
Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
100 105 110
Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 49
<211> 359
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 49
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
35 40 45
Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp
50 55 60
Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Met Asp Thr Gly Leu Tyr Leu Cys Lys Val
85 90 95
Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
100 105 110
Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu
115 120 125
Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 50
<211> 358
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 50
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly
1 5 10 15
Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu
20 25 30
Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val
35 40 45
Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp
50 55 60
Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile
65 70 75 80
Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu
85 90 95
Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln
100 105 110
Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro
115 120 125
Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu
130 135 140
Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
225 230 235 240
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Pro Gly Lys
355
<210> 51
<211> 357
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 51
Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 52
<211> 357
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 52
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 53
<211> 357
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 53
Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 54
<211> 357
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 54
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 55
<211> 357
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 55
Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 56
<211> 357
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 56
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Phe Glu Gly Met Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys
115 120 125
Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu
130 135 140
Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
225 230 235 240
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Pro Gly Lys
355
<210> 57
<211> 125
<212> PRT
<213> 小鼠
<400> 57
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Leu Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Tyr Asp Gly Asn Thr Asn Phe His Ser Ala Leu Ile
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Glu Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Lys Thr Glu Gly His Tyr Tyr Gly Ser Asn Tyr Gly Tyr Tyr Ala Leu
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120 125
<210> 58
<211> 25
<212> PRT
<213> 小鼠
<400> 58
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser
20 25
<210> 59
<211> 14
<212> PRT
<213> 小鼠
<400> 59
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly
1 5 10
<210> 60
<211> 31
<212> PRT
<213> 小鼠
<400> 60
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu
1 5 10 15
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 61
<211> 11
<212> PRT
<213> 小鼠
<400> 61
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 62
<211> 125
<212> PRT
<213> 人工序列
<220>
<223> 人源化
<400> 62
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Leu Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Trp Tyr Asp Gly Asn Thr Asn Phe His Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Glu Gly His Tyr Tyr Gly Ser Asn Tyr Gly Tyr Tyr Ala Leu
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120 125
<210> 63
<211> 125
<212> PRT
<213> 人工序列
<220>
<223> 人源化
<400> 63
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Leu Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Trp Tyr Asp Gly Asn Thr Asn Phe His Ser Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Glu Gly His Tyr Tyr Gly Ser Asn Tyr Gly Tyr Tyr Ala Leu
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 64
<211> 125
<212> PRT
<213> 人工序列
<220>
<223> 人源化
<400> 64
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Leu Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Trp Tyr Asp Gly Asn Thr Asn Phe His Ser Pro Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Thr Glu Gly His Tyr Tyr Gly Ser Asn Tyr Gly Tyr Tyr Ala Leu
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 65
<211> 107
<212> PRT
<213> 小鼠
<400> 65
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Gly Thr Tyr Phe Cys Gln His Leu Trp Gly Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 66
<211> 23
<212> PRT
<213> 小鼠
<400> 66
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys
20
<210> 67
<211> 15
<212> PRT
<213> 小鼠
<400> 67
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
1 5 10 15
<210> 68
<211> 32
<212> PRT
<213> 小鼠
<400> 68
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
20 25 30
<210> 69
<211> 10
<212> PRT
<213> 小鼠
<400> 69
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
1 5 10
<210> 70
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> 人源化
<400> 70
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu
35 40 45
Tyr Ala Ala Thr Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Leu Trp Gly Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 71
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> 人源化
<400> 71
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ala Pro Lys Leu Leu Leu
35 40 45
Tyr Ala Ala Thr Asn Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln His Leu Trp Gly Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 72
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> 人源化
<400> 72
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Thr Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Leu Trp Gly Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 73
<211> 124
<212> PRT
<213> 智人
<400> 73
Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile
1 5 10 15
Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val
20 25 30
Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys
35 40 45
Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser
50 55 60
Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln
65 70 75 80
Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu
85 90 95
Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile
100 105 110
Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp
115 120
Claims (36)
1.抗CTLA4抗体,其包含(a)轻链可变区,所述轻链可变区包含(i)互补决定区CDR1区,其中所述CDR1区的氨基酸序列由SEQ ID NO:21所示,(ii) CDR2区,其中所述CDR2区的氨基酸序列由SEQ ID NO: 36、37或38所示,和(iii) CDR3区,其中所述CDR3区的氨基酸序列由SEQ ID NO: 23所示;和(b)重链可变区,所述重链可变区包含(i) CDR1区,其中所述CDR1区的氨基酸序列由SEQ ID NO: 24所示,(ii) CDR2区,其中所述CDR2区的氨基酸序列由SEQID NO:33、34或35所示,和(iii) CDR3区,其中所述CDR3区的氨基酸序列由SEQ ID NO: 26所示。
2.根据权利要求1所述的抗CTLA4抗体,其中所述重链可变区包含SEQ ID NO: 62、63或64中阐述的氨基酸序列;并且所述轻链可变区包含SEQ ID NO: 70、71或72中阐述的氨基酸序列。
3.根据权利要求1所述的抗CTLA4抗体,其中所述轻链CDR2区的氨基酸序列由SEQ IDNO: 37所示并且所述重链CDR2区的氨基酸序列由SEQ ID NO: 33所示;或所述轻链CDR2区的氨基酸序列由SEQ ID NO: 37所示并且所述重链CDR2区的氨基酸序列由SEQ ID NO: 35所示;或所述轻链CDR2区的氨基酸序列由SEQ ID NO: 38所示并且所述重链CDR2区的氨基酸序列由SEQ ID NO: 35所示。
4.根据权利要求3所述的抗CTLA4抗体,其中所述重链包含SEQ ID NO: 62中阐述的氨基酸序列并且所述轻链包含SEQ ID NO: 71中阐述的氨基酸序列。
5.根据权利要求3所述的抗CTLA4抗体,其中所述重链包含SEQ ID NO: 64中阐述的氨基酸序列并且所述轻链包含SEQ ID NO: 71中阐述的氨基酸序列。
6.根据权利要求3所述的抗CTLA4抗体,其中所述重链包含SEQ ID NO: 64中阐述的氨基酸序列并且所述轻链包含SEQ ID NO: 72中阐述的氨基酸序列。
7.根据权利要求1到6中任一项所述的抗CTLA4抗体,其中所述抗体能够结合人类CTLA4。
8.根据权利要求1到6中任一项所述的抗CTLA4抗体,其中所述抗体的特征在于与可溶CTLA4的结合减少。
9.一种根据权利要求1到8中任一项所述的抗CTLA4抗体的抗原结合片段。
10.根据权利要求9所述的抗原结合片段,其中所述片段选自下组:Fab’、F(ab’)2、Fv和ScFv。
11.一种医药组合物,其包含治疗有效量的根据权利要求1到8中任一项所述的抗CTLA4抗体或根据权利要求9或10所述的抗原结合片段和生理学上可接受的载剂或赋形剂。
12.权利要求1到8中任一项所述的抗CTLA4抗体或权利要求9或10所述的抗原结合片段或权利要求11所述的医药组合物在制备用于治疗受试者中的癌症的药物中的用途。
13.根据权利要求12所述的用途,其中所述癌症选自下组:上皮癌(carcinoma);淋巴谱系的造血肿瘤;白血病;B细胞淋巴瘤;T细胞淋巴瘤;伯基特淋巴瘤;骨髓谱系的造血肿瘤;间充质来源的肿瘤;纤维肉瘤;横纹肌肉瘤;黑色素瘤;精原细胞瘤;成神经细胞瘤;神经胶质瘤;中枢和外周神经系统的肿瘤;星形细胞瘤;神经鞘瘤;骨肉瘤;着色性干皮病;和甲状腺滤泡性癌。
14.根据权利要求13所述的用途,其中所述癌症是上皮癌,并且其中所述上皮癌是膀胱上皮癌;乳腺上皮癌;结肠上皮癌;肾上皮癌;肝上皮癌;肺上皮癌;卵巢上皮癌;胰腺上皮癌;胃上皮癌;子宫颈上皮癌;甲状腺上皮癌;皮肤上皮癌;鳞状细胞上皮癌;畸胎上皮癌;肝细胞上皮癌;前列腺上皮癌;或肾细胞上皮癌。
15.根据权利要求14所述的用途,其中所述上皮癌是结肠上皮癌。
16.根据权利要求13所述的用途,其中所述癌症是白血病,并且其中所述白血病是急性淋巴细胞性白血病;急性成淋巴细胞性白血病;急性或慢性髓系白血病(acute or chronicmyelogenous leukemia);前髓细胞性白血病;急性骨髓性白血病(acute myelogenicleukemia);或慢性淋巴细胞性白血病。
17.根据权利要求12所述的用途,其中所述癌症选自下组:卵巢癌、膀胱癌、结肠癌、皮肤癌、胰腺癌、子宫癌、肉瘤、黑色素瘤、肺癌、乳腺癌、和霍奇金氏或非霍奇金氏淋巴瘤。
18.根据权利要求17所述的用途,其中所述癌症是黑色素瘤。
19.根据权利要求12-18中任一项所述的用途,所述医药组合物进一步包含另一选自由抗PD-1抗体和抗4-1BB抗体组成的组的药剂。
20.根据权利要求19所述的用途,其中所述抗PD-1抗体或抗4-1BB抗体与所述抗CTLA4抗体以双特异性抗体形式组合于单一分子中。
21.根据权利要求12所述的用途,其中所述药物诱导肿瘤微环境中的强Treg缺失和局部T细胞活化,但诱导的系统T细胞活化是最小的。
22.根据权利要求17所述的用途,其中所述癌症是肺癌。
23.根据权利要求17所述的用途,其中所述癌症是卵巢癌。
24.抗CTLA4抗体,其包含:
包含CDR1、CDR2和CDR3的重链可变区,其中所述CDR1、CDR2和CDR3是包含SEQ ID NO:62所示的氨基酸序列的重链可变区的CDR1、CDR2和CDR3,包含SEQ ID NO: 63所示的氨基酸序列的重链可变区的CDR1、CDR2和CDR3,或包含SEQ ID NO: 64所示的氨基酸序列的重链可变区的CDR1、CDR2和CDR3;以及
包含CDR1、CDR2和CDR3的轻链可变区,其中所述CDR1、CDR2和CDR3是包含SEQ ID NO:70所示的氨基酸序列的轻链可变区的CDR1、CDR2和CDR3,包含SEQ ID NO:71所示的氨基酸序列的轻链可变区的CDR1、CDR2和CDR3,或包含SEQ ID NO:72所示的氨基酸序列的轻链可变区的CDR1、CDR2和CDR3。
25.权利要求24的抗CTLA4抗体在制备用于治疗受试者中的癌症的药物中的用途。
26.根据权利要求25所述的用途,其中所述癌症选自下组:上皮癌;淋巴谱系的造血肿瘤;白血病;B细胞淋巴瘤;T细胞淋巴瘤;伯基特淋巴瘤;骨髓谱系的造血肿瘤;间充质来源的肿瘤;纤维肉瘤;横纹肌肉瘤;黑色素瘤;精原细胞瘤;成神经细胞瘤;神经胶质瘤;中枢和外周神经系统的肿瘤;星形细胞瘤;神经鞘瘤;骨肉瘤;着色性干皮病;和甲状腺滤泡性癌。
27.根据权利要求26所述的用途,其中所述癌症是上皮癌,并且其中所述上皮癌是膀胱上皮癌;乳腺上皮癌;结肠上皮癌;肾上皮癌;肝上皮癌;肺上皮癌;卵巢上皮癌;胰腺上皮癌;胃上皮癌;子宫颈上皮癌;甲状腺上皮癌;皮肤上皮癌;鳞状细胞上皮癌;畸胎上皮癌;肝细胞上皮癌;前列腺上皮癌;或肾细胞上皮癌。
28.根据权利要求27所述的用途,其中所述上皮癌是结肠上皮癌。
29.根据权利要求26所述的用途,其中所述癌症是白血病,并且其中所述白血病是急性淋巴细胞性白血病;急性成淋巴细胞性白血病;急性或慢性髓系白血病;前髓细胞性白血病;急性骨髓性白血病;或慢性淋巴细胞性白血病。
30.根据权利要求25所述的用途,其中所述癌症选自下组:卵巢癌、膀胱癌、结肠癌、皮肤癌、胰腺癌、子宫癌、肉瘤、黑色素瘤、肺癌、乳腺癌、和霍奇金氏或非霍奇金氏淋巴瘤。
31.根据权利要求30所述的用途,其中所述癌症是黑色素瘤。
32.根据权利要求25-31中任一项所述的用途,其中所述抗CTLA4抗体与另一选自由抗PD-1抗体和抗4-1BB抗体组成的组的药剂组合。
33.根据权利要求32所述的用途,其中所述抗PD-1抗体或抗4-1BB抗体与所述抗CTLA4抗体以双特异性抗体形式组合于单一分子中。
34.根据权利要求25所述的用途,其中所述药物诱导肿瘤微环境中的强Treg缺失和局部T细胞活化,但诱导的系统T细胞活化是最小的。
35.根据权利要求30所述的用途,其中所述癌症是肺癌。
36.根据权利要求30所述的用途,其中所述癌症是卵巢癌。
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