CN114478789A - 抗pd-l1与ox40双特异性抗体及其用途 - Google Patents

抗pd-l1与ox40双特异性抗体及其用途 Download PDF

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CN114478789A
CN114478789A CN202111559140.9A CN202111559140A CN114478789A CN 114478789 A CN114478789 A CN 114478789A CN 202111559140 A CN202111559140 A CN 202111559140A CN 114478789 A CN114478789 A CN 114478789A
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刘浩
许文娟
徐婷
周伟
崔智强
叶洪涛
鲍文英
范清林
宋礼华
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Anhui Anke Biotechnology Group Co ltd
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Abstract

本发明属于生物技术领域，具体涉及抗PD‑L1与OX40双特异性抗体，可以增强T效应细胞的免疫刺激、促进细胞因子分泌的抗OX40和PD‑L1抗体，所述抗体在小鼠肿瘤模型中表现出了显著的肿瘤抑制效果。所述双特异性抗体，包括具有轻链和重链IgG结构域以及可变区部分，所述可变区部分连接到所述重链的N端或C端并串联一个或者两个纳米抗体的可变区；所述纳米抗体结构域对第一抗原OX40具有结合特异性，IgG结构域对第二抗原PD‑L1具有结合特异性。

Description

抗PD-L1与OX40双特异性抗体及其用途
技术领域
本发明属于生物技术领域，具体涉及特异性结合OX40和PD-L1的双特异性抗体和抗体片段以及含有所述抗体或抗体片段的组合物，还涉及编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途，尤其是其在治疗与OX40或PD-L1相关疾病中的用途，例如癌症等。
背景技术
OX40，也称为CD134，属于肿瘤坏死因子(TNF)受体家族的成员，是T细胞反应的重要共同刺激因子。三分子OX40与三聚体OX40L蛋白结合，激活下游NF-κB、PI3K和AKT等途径，增加效应T细胞和记忆T细胞的存活和扩增，增加细胞因子分泌。理论上，在高度免疫抑制的肿瘤微环境中，利用激动剂刺激这些共刺激受体应该能够增强抗肿瘤免疫力。大量的小鼠数据也证实了这类药物的治疗潜力。例如，基因泰克将OX40激动剂抗体和PD-L1抗体联合起来，在一种小鼠模型中，90％的结直肠癌肿瘤获得了完全缓解。
OX40是一种I型跨膜糖蛋白，主要由T细胞表达(结构性地由调节性T细胞表达，激活后由效应T细胞表达)。OX40的配体为OX40L，最初发现于HTLV-1转换的T细胞，被称为pg34，主要表达于APC，在NK细胞、肥大细胞和激活的T细胞上也有表达。OX40表达于活化的T细胞表面，且主要是CD4+T细胞，CD8+T细胞表面有少量表达，在癌症中，在肿瘤浸润淋巴细胞中发现了表达OX40的活化T细胞，而OX40及其配体OX40-L在诱导及维持T细胞反应中起关键作用，因此OX40成为肿瘤免疫治疗的一个重要靶点。
机体的免疫应答过程需要多种免疫细胞和免疫分子共同参与，其中T细胞的活化则是免疫应答的核心。通常，T细胞活化需要至少两个信号，除了第一信号T细胞识别MHC-肽化合物，识别过程中各种信号刺激也是必须的。这就是所谓的双信号假说。OX40的配体OX40L(又称CD252)以三聚体的形式和3个分子的OX40蛋白结合形成六聚体复合物，从而激活下游NF-κB、PI3K以及AKT等信号通路，这些下游信号的持续激活可以刺激细胞因子的产生，延长T细胞的存活时间，抑制调节性T细胞(Treg)的分化和活性，增强效应T细胞的杀伤能力。另外，OX40使T淋巴细胞在进行大量分裂时，将记忆T细胞的数量也进行分裂，从而利于第二次兔疫应答的顺利进行。OX40的这种生物学特性，意味着基于这一靶点开发的抗体，有望对多种免疫系统疾病带来治疗效果，包括肿瘤、器官移植、哮喘、类风湿关节炎、系统性红斑狼疮等。
在免疫原性差的肿瘤中，单一的抗OX40治疗不能提供足够的抗肿瘤免疫原性。由于TNFR的激动作用需要受体聚集，本领域仍然需要关于TNF家族(尤其是OX40分子)的具有多价抗原结合功能的、具有改善的聚集和受体激动作用的单分子实体，以满足日益增加的健康和医疗需求。本发明满足了这些和其它需求。
PD-1/PD-L1信号通路在调节免疫耐受、微生物感染及肿瘤免疫逃逸中发挥着重要作用。PD1(programmed cell death 1，程序性细胞死亡因子1)主要表达在T细胞等免疫细胞，而PD-1的配体PD-L1主要在许多人类肿瘤组织呈高表达。靶向免疫检查点分子例如PD-1或者配体PD-L1能够增加现有的肿瘤治疗方式，阻断PD-1/PD-L1信号通路可使被抑制的T细胞激活，进而攻击癌细胞。目前，已先后在乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌等数十种人类肿瘤组织中检测到PD-L1蛋白的高表达，且PD-L1的表达水平与患者的临床及预后紧密相关。
虽然PD-L1在多种肿瘤中都有表达，但是却只有不到50％的肿瘤患者对针对PD-1/PD-L1的单克隆抗体的治疗有响应，因此瞄准其他免疫检查点，例如OX40，一种能理论上有叫普遍治疗相应的靶点，能够增加并改进单克隆抗体在肿瘤治疗中的应用。
发明内容
本发明的目的在于提供一种可以增强T效应细胞的免疫刺激、促进细胞因子分泌的抗OX40和PD-L1抗体，所述抗体在小鼠肿瘤模型中表现出了显著的肿瘤抑制效果。
为实现上述目的，本发明采用的技术方案为：一种抗PD-L1与OX40双特异性抗体，包括具有轻链和重链IgG结构域以及可变区部分，所述可变区部分连接到所述重链的N端或C端并串联一个或者两个纳米抗体的可变区；所述纳米抗体结构域对第一抗原OX40具有结合特异性，IgG结构域对第二抗原PD-L1具有结合特异性。
本发明通过现有的人源化抗PD-L1单克隆抗体与全人源抗OX40单克隆抗体并以图1所示的5种结构组成形式组成了多种能够同时特异性结合PD-L1与OX40两种抗原的双特异性抗体以及OX40纳米抗体的四聚体形式。该双特异性抗体或纳米抗体四聚体(本申请包含5种，具体如下：L52-D7H232、L52-2D7H232、L52H232-D7、L52H232-2D7和2D7-Fc)都具有对PD1/PD-L1或OX40/OX40L的阻断活性。
本发明的双特异性抗体中，可变区(VHH)部分可以连接在抗PD-L1抗体重链的N端或者C端；还可以串联1个或者多个抗OX40纳米抗体VHH；所述双特异性抗体具有对OX40的第一结合特异性和对PD-L1的第二结合特异性，或，具有对PD-L1的第一结合特异性和对OX40的第二结合特异性。所获得双特异性抗体均能在ELISA水平结合人源OX40，且能结合食蟹猴子的OX40，但是不能结合鼠源OX40；能在ELISA水平结合PD-L1；能结合OX40后能激活下游信号，且具有PD-L1依赖性。
所述双特异性抗体选自全人源抗体、人源化抗体、嵌合抗体或重组抗体的一种或多种。
所述纳米抗体为全人源抗人OX40单克隆纳米抗体，包括重链可变区，所述重链可变区包含CDR1区、CDR2区和CDR3区，其中该CDR1区、CDR2区和CDR3区分别包含SEQ ID NO:34、35、36，所示氨基酸序列的任意一种或与其任意一种具有至少80％序列同一性的同源序列。
所述纳米抗体包括具有SEQ ID No:13、14、15的氨基酸序列或者具有与SEQ IDNo:13、14、15至少90％、95％、96％、97％、98％相似性的氨基酸序列。
前述的以图1所示的5种结构组成形式组成了OX40纳米抗体的四聚体形式，所述四聚体纳米抗体融合蛋白，连接在IgG1 Fc的N端，如SEQ ID NO:15所示；也可以是二聚体纳米抗体融合蛋白，连接在IgG1 Fc的N端，如SEQ ID NO:14所示。
所述重链为PD-L1抗体的IgG1重链，所述重链包括具有SEQ ID No:10、12、17、20、23、26的氨基酸序列或者具有与SEQ ID No:10、12、17、20、23、26至少90％、95％、96％、97％、98％相似性的氨基酸序列。
所述轻链为PD-L1抗体的κ轻链，所述κ轻链包括具有SEQ ID No:9、11、16、18、19、21、22、24、25、27的氨基酸序列或者具有与SEQ ID No:9、11、16、18、19、21、22、24、25、27至少90％、95％、96％、97％、98％相似性的氨基酸序列。
所述双特异性抗体还包括人IgG1框架区域的CH1、CH2、CH3。
所述IgG结构域包括IgG1恒定区，所述IgG1恒定区具有SEQ ID No:7的氨基酸序列或者具有与SEQ ID No:7至少90％、95％、96％、97％、98％相似性的氨基酸序列。
连接可变区和重链IgG结构之间的linker部分为一段氨基酸序列，其接头为(GGGGS)n，其中n＝0-4。
所述抗体是IgG1形式的抗体或IgG2形式或IgG4形式的抗体或抗原结合片段，其Fc结构域分别IgG1、IgG2、IgG3、IgG4或相应工程化亚型。
所述轻链和重链的氨基酸序列分别为：SEQ ID No:9和SEQ ID No:10(L52H232)，或SEQ ID No:16和SEQ ID No:17(L52-D7H232)，或SEQ ID No:19和SEQ ID No:20(L52-2D7H232)，或SEQ ID No:22和SEQ ID No:23(L52H232-D7)，或SEQ ID No:25和SEQ ID No:27(L52H232-2D7)。
本发明所述双特异性抗体具有较好的加速稳定性，在反复冻融以及40℃保存时能保持较高的稳定性。
在一些实施方案中，抗OX40抗体的激动剂活性由OX40信号传导(例如检测NFκB下游信号传导)来评估。因此，本发明提供了与用IgG1对照抗体相比，提高NFκB介导的转录活性水平的抗OX40抗体或其片段。与相应的对照IgG1相比，本发明的抗OX40和PD-L1双特异性抗体或其片段能够将NFκB介导的转录活性水平提高数倍。
本发明的双特异性抗体在人FcγIIb细胞的存在下，展现了显著的激动活性。
在一些实施方案中，本发明的抗OX40和PD-L1双特异性抗体相比已知的抗OX40抗体和抗PD-L1抗体具有更好的抗肿瘤活性，例如相比于已知的抗OX40抗体或PD-L1抗体，本发明的双特异性抗体能够显著降低受试者中的肿瘤体积，优选地同时不影响受试者的体重。
本发明的优选抗体在对小鼠肿瘤治疗给药时，产生的转氨酶含量变化相对较低。
本发明中OX40抗体部分是通过淘筛获得的抗体分子是纳米抗体VHH形式的，这种形式可以通过本领域的技术人员熟知的基因工程技术方便地进一步转换成Fc融合蛋白以及与共同轻链组合成IgG抗体等其他形式。
本发明中抗PD-L1抗体部分是通过兔子抗体人源化获得的，兔源抗体具有极高的亲和活性，人源化后可降低体内抗兔抗体的产生。
本发明的双特异性抗体在各项评价中均为IgG1形式，且Fc具有强ADCC活性。
本发明中优选地抗人OX40和PD-L1双特异性抗体L52-2D7H232，在小鼠的MC38肿瘤模型中展现除了强有力的肿瘤抑制活性。
本发明涉及的Fc区，以及人OX40序列是直接或间接从人体得到的。所述的直接方法包括但不限于基因组DNA克隆或cDNA文库。所述的间接方法包括但不限于Genbank或其他出版物或网站提供的生物信息为基础部分，合成或完全从头合成完整的DNA。DNA合成技术包括但不限于以PCR为基础的DNA合成方法。
全人源抗人huOX40和PD-L1双特异性抗体，所述抗体能特异性地与人huOX40和PD-L1抗原结合；所述双抗有IgG部分和VHH部分组成；IgG部分包括SEQ ID NO:16,SEQ ID NO:17,SEQ ID NO:19,SEQ ID NO:20,SEQ ID NO:22,SEQ ID NO:23,SEQ ID NO:25,SEQ IDNO:26所示氨基酸序列的任意一种或与其任意一种具有至少80％序列同一性的同源序列；即上述的4个优选的双特异性抗体：L52-D7H232、L52-2D7H232、L52H232-D7和L52H232-2D7。VHH部分包括即包括SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15所示氨基酸序列的任意一种或与其任意一种具有至少90％序列同一性的同源序列；即上述的3个纳米抗体：D7、D7-Fc和2D7-Fc。
可以利用双特异性分子、免疫交联物、嵌合抗原受体、基因改造T细胞受体或溶瘤病毒等作为宿主细胞来表达编码了所述双特异性抗体的核酸。
制备上述双特异性抗体的方法，包括培养上述宿主细胞或其他表达载体，从所述表达载体或宿主细胞中表达所述双特异性抗体。
所述双特异性抗体可以用于制备药物组合物或用于制成试剂盒或制品。。制备的药物可以用于治疗癌症，治疗方法包括向受试者施用有效量的所述双特异性抗体或其抗原结合片段或上述药物组合物；所述癌症是肺癌、结肠癌、胃癌、肾癌或肝癌。
制备药物时，除所述双特异性抗体外，还需要筛选候选药物。
进一步，筛选候选药物的步骤如下：首先加入待筛选物质处理含有CHO-PDL1-TCR和Jurkat-PD1-NFAT细胞株的体系；然后加入荧光素酶检测试剂进行酶标检测；其中，若荧光检测值随着浓度提高而增加，说明该待筛选物质为候选药物。
或，首先加入待筛选物质处理含有Jurkat-OX40-NFκB-Luc和人DG44-Fcγ-IIB细胞株的体系；然后加入荧光素酶检测试剂进行酶标检测；其中，若荧光检测值随着待测抗体浓度降低和逐渐降低，说明该待筛选物质为候选药物。
其中，上述筛选过程在培养箱中进行，培养箱中培养的时间为为5-20h，优选18h。
为了表达本发明所述的抗体或抗体的片段，可以把其相应的重链或轻链编码序列插入到表达载体的转录和翻译控制序列之间。本发明所述的表达载体包含调控序列如启动子、增强子等。所述的表达载体及其控制序列应当与受体细胞兼容。
本发明中双特异性抗体的表达还可以通过瞬时表达来实现。该表达策略包括用一种或多种带有编码抗体轻链和重链DNA片段的表达载体转染哺乳动物细胞，从而使双特异性抗体在受体细胞中表达并组装,优选的表达方式是分泌到培养基中,可以用本领域技术人员熟知的层析等方法从中回收抗体。
附图说明
图1为所述双特异性抗体和纳米抗体四聚体结构示意图；
图2为ELISA法测定抗OX40和PD-L1双特异性抗体与OX40抗原的结合活性；
图3为ELISA法测定抗OX40和PD-L1双特异性抗体与PD-L1抗原的结合活性；
图4为OX40优选抗体阻断OX40L与OX40结合的活性检测；
图5为优选双特异性抗体阻断PD-L1与PD1的结合活性检测；
图6a-b为OX40和PD-L1双特异性抗体的激动活性检测；
图7显示了优选的OX40和PD-L1双特异性抗体的激动活性具有PD-L1依赖性；
图8a-e显示了OX40和PD-L1双特异性抗体的SEC纯度检测，五幅图分别为2D7-Fc、L52-2D7H232、L52H232-2D7、L52-D7H232、L52H232-D7；
图9显示了优选OX40和PD-L1双特异性抗体的交叉结合人源、鼠源和猴子OX40抗原的活性；
图10-a展示了本发明优选的双特异性抗体L52-2D7H232在MC38肿瘤小鼠模型中具有较好的抑瘤效果；
图10b-f显示了各组小鼠肿瘤体积变化情况；
图11记录了本发明抗体在对肿瘤小鼠进行治疗的过程中，体重变化情况。
具体实施方案
以下实施例可以进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。实施例不包括对传统方法的详细描述。实施例中未注明的技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用仪器未注明生产厂商者，均为可以通过市场购买获得的常规产品。
本发明主要涉及特异性结合PD-L1和OX40的双特异性抗体，在一些实施案例中，双特异性抗体包括结合PD-L1的第一臂和结合OX40的第二臂，在一些实施案例中，双特异性抗体包括结合OX40的第一臂和结合PD-L1的第二臂；含有臂的结构域包括但不限于Fab和VHH结构，Fc结构域可以是IgG1、IgG2、IgG3、IgG4或相应工程化亚型。
实施例1：双特异性抗体分子构建
将OX40的纳米抗体D7分别以二聚体或四聚体的方式连接在PD-L1重链的N端或C端，并构建了纳米抗体D7的四聚体Fc融合形式；所形成的双特异性抗体形式均为对称型结构，Fc段为IgG1的野生型，各分子形式见图1。
实施例2：PD-L1和OX40双特异性抗体ELISA水平结合OX40检测
将表达纯化的rhOX40-Avi-His蛋白稀释至1.0μg/ml铺96孔ELISA板并4℃过夜；第二天TPBS(PBS+0.1％Tween 20)洗涤三遍，3％脱脂奶粉溶于TPBS，37℃封闭1小时；然后TPBS洗三遍，加入待测抗体(首孔0.1μM，连续5倍梯度稀释)，室温震荡温育2小时；TPBS洗三遍，加入1:5000稀释的goat-anti human Fc，每孔100μl，室温震荡温育30min；TPBS洗三遍，每孔加入混有0.1％过氧化氢的OPD邻苯二胺(购自Sigma公司，货号78412)底物工作液100μl进行显色，约3-7分钟后加入100μl 1M硫酸终止，酶标仪(购自Biotek公司，货号ELX 800)测定OD490值；结果分析见图2。
实施例3：PD-L1和OX40双特异性抗体ELISA水平结合PD-L1检测
用ELISA法检测双特异性抗体与抗原蛋白PD-L1的结合情况。将表达纯化的抗原蛋白PD-L1-avi-his 1.0μg/ml浓度铺ELISA板并4℃放置过夜，ELISA板用PBS+0.1％Tween20溶液冲洗3次，用PBS-3％脱脂奶粉溶液37℃封闭1h。用PBS+0.1％Tween20溶液冲洗3次，用浓度梯度的双特异性抗体(首孔1μg/ml，5倍梯度稀释)与抗原结合，再用羊抗人Fc-HRP的第二抗体孵育，OPD显色，1M H2SO4终止。显色ELISA板用酶标仪在OD490下读数，将读数与浓度制图(图3)。
实施例4：双特异性抗体阻断OX40与OX40L结合检测
在ELISA水平检测双特异性抗体是否可以阻断OX40L与OX40的结合。将OX40抗原以1μg/ml浓度铺板于96孔ELISA板，4℃过夜，第二天ELISA板用PBS+0.1％Tween20溶液冲洗3次，用PBS-3％脱脂奶粉溶液37℃封闭1h。用PBS+0.1％Tween20溶液冲洗3次，以20μg/ml的OX40L-mouse Fc为稀释buffer将待测抗体进行梯度稀释(首孔10μg/ml，5倍梯度稀释)，分别加入对应ELISA孔中室温2h；再用羊抗鼠Fc-HRP的第二抗体孵育1h，OPD显色，1M H2SO4终止。显色ELISA板用酶标仪在OD490下读数，利用读数与浓度制图(图4)。数据显示，纳米抗体D7-Fc和优选双特异性抗体L52-2D7H232可以在ELISA水平阻断OX40L与OX40的结合。
实施例5：双特异性抗体阻断PD1/PD-L1通路信号传导
复苏CHO-PDL1-TCR和Jurkat-PD1-NFAT细胞，用培养基使细胞混悬后，在黑色康宁96孔板中加入100μl细胞混悬液(使孔中细胞数达到50000cells/well)，并在黑色康宁96孔板四周加入80μl的PBS缓冲液，随后放入CO2培养箱培养16-24h。用稀释buffer稀释抗体浓度至最高浓度100μmol，再做3倍梯度稀释，共9个梯度备用；吸取掉CHO-PDL1-TCR细胞上清，将配制好的PDL1单抗稀释液(40μl每个孔)与铺好板的CHO-PDL1-TCR细胞室温孵育20分钟后，再用移液枪加入40μl Jurkat-PD1-NFAT(6.25x 105cells/ml)混悬液到黑色康宁96孔板中，随后放入CO2培养箱中培养6h。最后将CO2培养箱中的黑色康宁96孔板每孔中加入80ul已经达到室温的Promega Bio-GloTM Luciferase Assary System，并放置在室温用振荡器振荡10分钟后上机检测，读取RLU值并作图分析(5)。
结果显示优选抗体L52-D7H232和L52-2D7H232与对照抗体L52H232和Durvalumab能不同程度的阻断PD1/PD-L1通路信号传导。
实施例6：荧光素酶报告基因T细胞活化测定法：
可以通过在荧光素酶报告基因测定法中测量NFκB介导的转录活化的促进来评估本发明的双特异性抗体的激动剂活性。各收集一定数量的CHO-DG44-FcγIIB(44F10)和Jurkat-OX40-NFκB-Luc(21C6)细胞置于离心管中，用理论buffer(含指定终浓度的CD3或CD28抗体)重悬细胞；将混合细胞加入白底96孔板中，每孔50μL；配制抗体稀释液：用理论buffer稀释抗体浓度至首孔浓度0.48μM，再做2倍梯度稀释，共10个梯度备用，加入上述铺有细胞的96孔板中，50μL/well；将上述细胞-抗体混合悬液置于混匀仪上充分混合2min，随后放入CO2培养箱培养18h，提前1-2h取出Promega Bio-GloTM Luciferase Assary System在避光条件下平衡至室温，取出96孔板室温平衡10min后加入80μl Luciferase Assary，轻轻加入后室温避光反应3-5min，上机检测RLU信号(图6-a，6-b)。
在如上实验方法中，测得优选抗体L52-D7H232、L52-2D7H232和2D7-Fc与对照IgG1抗体相比，表现出了显著激动剂活性；但L52H232-2D7并未表现出明显的激动剂活性，而L52-2D7H232却表现出了最强的激动剂活性。
实施例7：优选双特异性抗体激动剂活性为PD-L1依赖性
CHO-DG44-PDL1、CHO-K1细胞分别胰酶消化后，分别用理论Buffer:1640+10％FBS重悬至密度1.6x106cells/ml，各25μl/孔即40000cells/孔铺于白色康宁96孔板。用1640+10％FBS培养基稀释抗体，初浓度150nM，4倍梯度稀释至10孔。用理论Buffer混悬Jurkat-OX40-NFκB-21C6细胞浓度达到8ⅹ105cells/ml，白色康宁96孔板中加入25μlJurkat-OX40-NFκB-21C6，细胞浓度20000cells/孔，25μl/孔，分别向铺板的两种细胞中加入L52H232、2D7-FC、L52-2D7H232、L52H232+2D7-FC抗体稀释液50μl每孔后于振荡器上混匀，随后放入CO2培养箱中培养15h后每孔加入100μl已经达到室温的Promega Bio-GloTM luciferaseAssary System，并放置在室温10min后上机检测。实验结果见图7，结果表明优选抗体L52-2D7H232在DG44-PDL1-FL-4B6和Jurkat-OX40-NFκB-21C6检测体系中有明显的PDL1依赖的活性。
实施例8：本发明的抗OX40和PD-L1双特异性抗体的纯度检测
用分子排阻的方式检测双特异性抗体的纯度，结果表明OX40和PD-L1双特异性抗体L52-D7H232、L52-2D7H232、L52H232-D7和L52H232-2D7纯度较高，OX40纳米抗体四聚体2D7-Fc的纯度也较高；这与对称型双特异性抗体的结果稳定性相符。
实施例9：本发明的抗PD-L1和OX40双特异性抗体与鼠源和食蟹猴OX40结合检测
在ELISA水平检测本发明的抗PD-L1和OX40双特异性抗体是否能够与鼠源和食蟹猴OX40结合。分别将人鼠猴三种来源的OX40以适当浓度铺板后，先后加入待测抗体和检测抗体，根据OD490读数来分析实验结果，见图9；实验结果表明，本发明的抗PD-L1和OX40双特异性抗体除了与人源OX40能较好结合之外，对食蟹猴OX40也具有较好的结合活性，但不特异性识别鼠源OX40。另本实验室还鉴定了优选的PD-L1和OX40双特异性抗体也不与鼠源PD-L1结合。
实施例10：本发明抗体的抗肿瘤活性
执行以下实验以确定本发明的抗PD-L1和OX40双特异性抗体是否展现强有力的抗肿瘤活性。为此，从百奥赛图基因生物技术有限公司购买PD-L1/OX40双人源化C57小鼠，获得阻断OX40/OX40L或PD1/PD-L1相互作用抗体L52-D7H232、L52-2D7H232、L52H232和Durvalumab并在小鼠皮下注射MC38-PD-L1细胞，待肿瘤体积均值100-120mm3后分组并开始IP给药，用双特异性抗体和对照抗体进行一周2次的给药处理，并一周2次测量肿瘤体积和称量动物体重，共给药四周。实验结果表明测试的双特异性抗体能够比对照单药抗体更好地抑制肿瘤的生长，见图10；优选抗体L52-2D7H232组6只小鼠的肿瘤在给药后不同时间肿瘤完全消除，见图10-e。且双特异性抗体实验组相比对照组也没有显著影响小鼠的体重；即优选抗PD-L1和OX40双特异性抗体在小鼠体内具有强有力的抗肿瘤活性。
本发明还涵盖本文所述的任何实施方案的任意组合。本文所述的任何实施方案或其任何组合适用于本文所述发明的任何PD-L1和OX40双特异性抗体或其片段、方法和用途。
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SEQUENCE LISTING
<110>安徽安科生物工程集团（股份）有限公司
<120>抗PD-L1与OX40双特异性抗体及其用途
<160> 36
<170>PatentIn version 3.5
<210> 1
<211> 277
<212> PRT
<213> Artificial Sequence
<220>
<223> Human OX40
<400> 1
Met Cys Val Gly Ala ArgArg Leu GlyArgGly Pro Cys Ala Ala Leu
1 5 10 15
Leu LeuLeuGly Leu Gly Leu Ser Thr Val ThrGly Leu His Cys Val
20 25 30
Gly Asp Thr Tyr Pro Ser Asn Asp ArgCysCys His Glu CysArg Pro
35 40 45
GlyAsnGly Met Val Ser ArgCys Ser Arg Ser Gln AsnThr Val Cys
50 55 60
Arg Pro CysGly Pro GlyPhe Tyr Asn Asp Val Val Ser Ser Lys Pro
65 70 75 80
Cys Lys Pro CysThrTrpCysAsn Leu Arg Ser Gly Ser Glu Arg Lys
85 90 95
Gln Leu CysThr Ala Thr Gln Asp Thr Val CysArgCysArg Ala Gly
100 105 110
Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys
115 120 125
Pro ProGly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp
130 135 140
ThrAsnCysThr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn
145 150 155 160
Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro
165 170 175
Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr
180 185 190
Glu Ala Trp Pro ArgThr Ser Gln Gly Pro Ser ThrArg Pro Val Glu
195 200 205
Val Pro GlyGlyArg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val
210 215 220
Leu Gly Leu LeuGly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu
225 230 235 240
ArgArg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro ProGlyGly
245 250 255
Gly Ser PheArgThr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser
260 265 270
Thr Leu Ala Lys Ile
275
<210> 2
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> BMS986178 VL
<400> 2
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 ThrCysArg Ala Ser Gln Gly Ile Ser SerTrp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser ArgPhe Ser Gly
50 55 60
Ser Gly Ser GlyThr Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr TyrCys Gln Gln Tyr Asn Ser Tyr Pro Pro
85 90 95
ThrPheGlyGlyGlyThr Lys Val Glu Ile Lys
100 105
<210> 3
<211> 118
<212> PRT
<213> Artificial Sequence
<220>
<223> BMS986178 VH
<400> 3
Asp Val Gln Leu Val Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPheThrPhe Ser Ser Tyr
20 25 30
Ser Met AsnTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser SerSerSerSerThr Ile Asp Tyr Ala Asp Ser Val
50 55 60
Lys GlyArgPheThr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr TyrCys
85 90 95
Ala Arg Glu Ser GlyTrp Tyr Leu Phe Asp Tyr TrpGly Gln GlyThr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 4
<211> 108
<212> PRT
<213> Artificial Sequence
<220>
<223> Durvalumab VL
<400> 4
Glu Ile Val Leu Thr Gln Ser Pro GlyThr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser CysArg Ala Ser Gln Arg Val Ser SerSer
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Tyr Asp Ala Ser SerArg Ala ThrGly Ile Pro Asp ArgPhe Ser
50 55 60
Gly Ser Gly Ser GlyThr Asp PheThr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr TyrCys Gln Gln Tyr Gly Ser Leu Pro
85 90 95
TrpThrPheGly Gln GlyThr Lys Val Glu Ile Lys
100 105
<210> 5
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> Durvalumab VH
<400> 5
Glu Val Gln Leu Val Glu Ser GlyGlyGly Leu Val Gln Pro GlyGly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPheThrPheSerArg Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys GlyArgPheThr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys
85 90 95
Ala Arg Glu GlyGlyTrpPheGly Glu Leu Ala Phe Asp Tyr TrpGly
100 105 110
Gln GlyThr Leu Val Thr Val Ser Ser
115 120
<210> 6
<211> 106
<212> PRT
<213> Artificial Sequence
<220>
<223> Kappa constant
<400> 6
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser GlyThr Ala Ser Val ValCys Leu LeuAsnAsnPhe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
GlyAsn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser SerThr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser PheAsnArgGly Glu Cys
100 105
<210> 7
<211> 328
<212> PRT
<213> Artificial Sequence
<220>
<223> IgG1 constant
<400> 7
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser GlyGlyThr Ala Ala Leu GlyCys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser TrpAsn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His ThrPhe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser
50 55 60
Leu Ser Ser Val ValThr Val Pro Ser SerSer Leu GlyThr Gln Thr
65 70 75 80
Tyr Ile CysAsn Val Asn His Lys Pro Ser AsnThr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrCys Pro ProCys
100 105 110
Pro Ala Pro Glu Leu LeuGlyGly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val ThrCys
130 135 140
Val ValVal Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsnTrp
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 AsnGly 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 ThrCys Leu Val Lys GlyPhe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser AsnGly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgTrp Gln GlnGlyAsn
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
325
<210> 8
<211> 230
<212> PRT
<213> Artificial Sequence
<220>
<223> IgG1 Fc
<400> 8
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys GlyPhe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu AsnAsn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro
225 230
<210> 9
<211> 217
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232 LC
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Th rCys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys ArgThr
100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
115 120 125
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
130 135 140
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
145 150 155 160
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
195 200 205
Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 10
<211> 446
<212> PRT
<213> Artificial Sequence
<220>
<223> L52-H232 HC
<400> 10
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser CysThr Val Ser Gly Ile Asp Leu Ser Ser Tyr
20 25 30
Asp Met ThrTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Ser Tyr Val Ser ArgThr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys Ala
85 90 95
Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu ThrCys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
<210> 11
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232 VL
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile ThrCys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
AsnArg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr TrpThr Ser Phe Leu Ala Ser Gly Val Pro Ser ArgPhe
50 55 60
Ser Gly Ser Gly Ser GlyThr Glu PheThr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp AspPhe Ala Thr Tyr TyrCys Ala GlyGly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 12
<211> 118
<212> PRT
<213> Artificial Sequence
<220>
<223> L52-H232 VH
<400> 12
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr
20 25 30
Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 13
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<223> D7 VHH
<400> 13
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ala Ser Ile Tyr
20 25 30
Gly Met Arg Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val
35 40 45
Ala Gly Ile Val Asp Ala Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asn His Glu Gly Glu Val Gly Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ala
115 120
<210> 14
<211> 363
<212> PRT
<213>Artificial Sequence
<220>
<223> D7-Fc
<400> 14
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ala Ser Ile Tyr
20 25 30
Gly Met Arg Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val
35 40 45
Ala Gly Ile Val Asp Ala Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asn His Glu Gly Glu Val Gly Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Thr Asp Lys Thr His Thr Cys
130 135 140
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
145 150 155 160
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
165 170 175
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
180 185 190
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
195 200 205
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
210 215 220
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
225 230 235 240
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
245 250 255
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
260 265 270
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
275 280 285
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser AsnGly Gln
290 295 300
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
305 310 315 320
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
325 330 335
Gln Gly Asn Val Ser Ser Cys Ser Val Met His Glu Ala Leu His Asn
340 345 350
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
355 360
<210> 15
<211> 728
<212> PRT
<213> Artificial Sequence
<220>
<223> 2D7-Fc
<400> 15
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ala Ser Ile Tyr
20 25 30
Gly Met Arg Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val
35 40 45
Ala Gly Ile Val Asp Ala Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asn His Glu Gly Glu Val Gly Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ala Ser Glu Phe Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Thr Gly Asp Val
130 135 140
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
145 150 155 160
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ala Ser Ile Tyr Gly Met
165 170 175
Arg Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala Gly
180 185 190
Ile Val Asp Ala Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly
195 200 205
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
210 215 220
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
225 230 235 240
Gly Asn His Glu Gly Glu Val Gly Leu Asp Tyr Trp Gly Gln Gly Thr
245 250 255
Gln Val Thr Val Ser Ala Ser Glu Phe Gly Gly Gly Gly Ser Gly Gly
260 265 270
Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Gln Val Gln Leu Val Glu
275 280 285
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
290 295 300
Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Asp Met Thr Trp Val Arg
305 310 315 320
Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly Tyr Ile Ser Tyr Val
325 330 335
Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
340 345 350
Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg
355 360 365
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Asp Gly
370 375 380
Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
385 390 395 400
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
405 410 415
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
420 425 430
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
435 440 445
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
450 455 460
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
465 470 475 480
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
485 490 495
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
500 505 510
Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
515 520 525
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
530 535 540
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
545 550 555 560
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
565 570 575
Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
580 585 590
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
595 600 605
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
610 615 620
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu
625 630 635 640
Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
645 650 655
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
660 665 670
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
675 680 685
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
690 695 700
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
705 710 715 720
Gln Lys Ser Leu Ser Leu Ser Pro
725
<210> 16
<211> 217
<212> PRT
<213>Artificial Sequence
<220>
<223> L52-D7H232 LC
<400> 16
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr
100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
115 120 125
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
130 135 140
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
145 150 155 160
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
195 200 205
Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 17
<211> 586
<212> PRT
<213> Artificial Sequence
<220>
<223> L52-D7H232 HC
<400> 17
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ala Ser Ile Tyr
20 25 30
Gly Met Arg Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val
35 40 45
Ala Gly Ile Val Asp Ala Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asn His Glu Gly Glu Val Gly Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ala Ser Glu Phe Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Gln Val Gln Leu
130 135 140
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
145 150 155 160
Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Asp Met Thr Trp
165 170 175
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly Tyr Ile Ser
180 185 190
Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
195 200 205
Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
210 215 220
Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys Ala Arg Asp Arg Pro
225 230 235 240
Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr Leu Val Thr Val
245 250 255
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
260 265 270
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
275 280 285
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
290 295 300
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
305 310 315 320
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
325 330 335
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
340 345 350
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
355 360 365
Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe
370 375 380
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
385 390 395 400
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
405 410 415
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
420 425 430
Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
435 440 445
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
450 455 460
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
465 470 475 480
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg
485 490 495
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
500 505 510
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
515 520 525
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
530 535 540
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgTrp Gln Gln
545 550 555 560
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
565 570 575
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
580 585
<210> 18
<211>111
<212> PRT
<213> Artificial Sequence
<220>
<223> L52-D7H232 VL
<400> 18
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile ThrCys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
AsnArg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 19
<211> 217
<212> PRT
<213> Artificial Sequence
<220>
<223> L52-2D7H232 LC
<400> 19
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr
100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
115 120 125
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
130 135 140
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
145 150 155 160
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
195 200 205
Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 20
<211> 722
<212> PRT
<213> Artificial Sequence
<220>
<223> L52-2D7H232 HC
<400> 20
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ala Ser Ile Tyr
20 25 30
Gly Met Arg Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val
35 40 45
Ala Gly Ile Val Asp Ala Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asn His Glu Gly Glu Val Gly Leu Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ala Ser Glu Phe Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu
130 135 140
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
145 150 155 160
Ala Ala Ser Gly Phe Thr Ala Ser Ile Tyr Gly Met Arg Trp Phe Arg
165 170 175
Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala Gly Ile Val Asp Ala
180 185 190
Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg PheThr Ile
195 200 205
Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu
210 215 220
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Asn His Glu
225 230 235 240
Gly Glu Val Gly Leu Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
245 250 255
Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
Gly Ser Leu Glu Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
275 280 285
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp
290 295 300
Leu Ser Ser Tyr Asp Met ThrTrp Val Arg Gln Ala Pro Gly Lys Gly
305 310 315 320
Leu Glu Tyr Ile Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala
325 330 335
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn
340 345 350
Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
355 360 365
Tyr Tyr Cys Ala Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp
370 375 380
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
385 390 395 400
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser GlyGlyThr
405 410 415
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
420 425 430
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
435 440 445
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val ValThr
450 455 460
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
465 470 475 480
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
485 490 495
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
500 505 510
GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
515 520 525
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
530 535 540
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
545 550 555 560
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
565 570 575
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
580 585 590
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
595 600 605
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
610 615 620
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
625 630 635 640
Ser Leu ThrCys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile Ala Val
645 650 655
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
660 665 670
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
675 680 685
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
690 695 700
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
705 710 715 720
Ser Pro
<210> 21
<211> 111
<212> PRT
<213>Artificial Sequence
<220>
<223> L52-2D7H232 VL
<400> 21
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 22
<211> 217
<212> PRT
<213>ArtificialSequence
<220>
<223> L52H232-D7 LC
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser ArgPhe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys ArgThr
100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
115 120 125
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
130 135 140
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
145 150 155 160
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
195 200 205
Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 23
<211> 582
<212> PRT
<213> Artificial Sequence
<220>
<223> L52-H232-D7 HC
<400> 23
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr
20 25 30
Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys Ala
85 90 95
Arg Asp Arg Pro Asp Gly Ala AlaThrAsn Leu TrpGly Gln GlyThr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu ThrCys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyGly
435 440 445
Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Ser Leu Glu Asp Val
450 455 460
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
465 470 475 480
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ala Ser Ile Tyr Gly Met
485 490 495
Arg Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala Gly
500 505 510
Ile Val Asp Ala Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly
515 520 525
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln
530 535 540
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
545 550 555 560
Gly Asn His Glu Gly Glu Val Gly Leu Asp Tyr Trp Gly Gln Gly Thr
565 570 575
Gln Val Thr Val Ser Ala
580
<210> 24
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232-D7 VL
<400> 24
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 25
<211> 217
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232-2D7 LC
<400> 25
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser ArgPhe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr
100 105 110
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
115 120 125
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
130 135 140
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
145 150 155 160
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
195 200 205
Thr Lys Ser PheAsnArgGly Glu Cys
210 215
<210> 26
<211> 717
<212> PRT
<213> Artificial Sequence
<220>
<223> L52-H232-2D7 HC
<400> 26
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr
20 25 30
Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys Ala
85 90 95
Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu ThrCys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyGly
435 440 445
Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Asp Val Gln Leu Val Glu
450 455 460
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
465 470 475 480
Ala Ala Ser Gly Phe Thr Ala Ser Ile Tyr Gly Met Arg Trp Phe Arg
485 490 495
Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala Gly Ile Val Asp Ala
500 505 510
Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
515 520 525
Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu
530 535 540
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Asn His Glu
545 550 555 560
Gly Glu Val Gly Leu Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
565 570 575
Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
580 585 590
Ser Leu Glu Leu Glu Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu
595 600 605
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
610 615 620
Thr Ala Ser Ile Tyr Gly Met Arg Trp Phe Arg Gln Ala Pro Gly Lys
625 630 635 640
Gly Arg Glu Leu Val Ala Gly Ile Val Asp Ala Gly Ser Ala Thr Tyr
645 650 655
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
660 665 670
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
675 680 685
Ala Val Tyr Tyr Cys Ala Arg Gly Asn His Glu Gly Glu Val Gly Leu
690 695 700
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ala
705 710 715
<210> 27
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232-2D7 VL
<400> 27
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn
20 25 30
Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser ArgPhe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly
85 90 95
Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 28
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232-CDRL1
<400> 28
Gln Ser Ser Gln Asn Val Tyr Ser AsnAsnArg Leu Ser
1 5 10
<210> 29
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232-CDRL2
<400> 29
TrpThr Ser Phe Leu Ala Ser
1 5
<210> 30
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232-CDRL3
<400> 30
AlaGlyGly Tyr Ser GlyAsn Leu Tyr Thr
1 5 10
<210> 31
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232-CDRH1
<400> 31
Gly Ile Asp Leu Ser Ser Tyr Asp Met Thr
1 5 10
<210> 32
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232-CDRH2
<400> 32
Tyr Ile Ser Tyr Val Ser ArgThr Tyr
1 5
<210> 33
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> L52H232-CDRH3
<400> 33
Asp Arg Pro Asp Gly Ala AlaThrAsn Leu
1 5 10
<210> 34
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> D7-CDRH1
<400> 34
GlyPheThr Ala Ser Ile Tyr Gly Met Arg
1 5 10
<210> 35
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> D7-CDRH2
<400> 35
Gly Ile Val Asp Ala Gly Ser Ala Thr Tyr Tyr Ala Asp
1 5 10
<210> 36
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> D7-CDRH3
<400> 36
GlyAsn His Glu Gly Glu Val Gly Leu Asp Tyr
1 5 10

Claims (21)

1.一种抗PD-L1与OX40双特异性抗体，其特征在于：所述双特异性抗体包括IgG重链、轻链、连接到重链的C端或N端的纳米抗体部分，所述纳米抗体结构域对第一抗原OX40具有结合特异性，IgG重链结构域对第二抗原PD-L1具有结合特异性。
2.根据权利要求1所述的双特异性抗体，其特征在于：所述双特异性抗体选自全人源抗体、人源化抗体、嵌合抗体或重组抗体的一种或多种。
3.根据权利要求1所述的双特异性抗体，其特征在于：所述纳米抗体为全人源抗人OX40单克隆纳米抗体，包括重链可变区，所述重链可变区包含CDR1区、CDR2区和CDR3区，其中该CDR1区、CDR2区和CDR3区分别包含SEQ ID NO:34、35、36，所示氨基酸序列的任意一种或与其任意一种具有至少80％序列同一性的同源序列。
4.根据权利要求1或3所述的双特异性抗体，其特征在于：所述纳米抗体包括具有SEQID No:13、14、15的氨基酸序列或者具有与SEQ ID No:13、14、15至少90％、95％、96％、97％、98％相似性的氨基酸序列。
5.根据权利要求1所述的双特异性抗体，其特征在于：所述纳米抗体为：四聚体纳米抗体融合蛋白，连接在IgG1 Fc的N端，如SEQ ID NO:15所示；和/或，二聚体纳米抗体融合蛋白，连接在IgG1 Fc的N端，如SEQ ID NO:14所示。
6.根据权利要求1所述的双特异性抗体，其特征在于：所述重链为PD-L1抗体的IgG1重链，所述重链包括具有SEQ ID No:10、12、17、20、23、26的氨基酸序列或者具有与SEQ IDNo:10、12、17、20、23、26至少90％、95％、96％、97％、98％相似性的氨基酸序列。
7.根据权利要求1所述的双特异性抗体，其特征在于：所述轻链为PD-L1抗体的κ轻链，所述κ轻链包括具有SEQ ID No:9、11、16、18、19、21、22、24、25、27的氨基酸序列或者具有与SEQ ID No:9、11、16、18、19、21、22、24、25、27至少90％、95％、96％、97％、98％相似性的氨基酸序列。
8.根据权利要求1所述的双特异性抗体，其特征在于：所述双特异性抗体还包括人IgG1框架区域的CH1、CH2、CH3。
9.根据权利要求1所述的双特异性抗体，其特征在于：所述IgG结构域包括IgG1恒定区，所述IgG1恒定区具有SEQ ID No:7的氨基酸序列或者具有与SEQ ID No:7至少90％、95％、96％、97％、98％相似性的氨基酸序列。
10.根据权利要求1所述的双特异性抗体，其特征在于：连接可变区和重链IgG结构之间的linker部分为一段氨基酸序列，其接头为(GGGGS)n，其中n＝0-4。
11.根据权利要求1-10任一项所述的双特异性抗体，其特征在于：所述抗体是IgG1形式的抗体或IgG2形式或IgG4形式的抗体或抗原结合片段，其Fc结构域分别IgG1、IgG2、IgG3、IgG4或相应工程化亚型。
12.根据权利要求1所述的双特异性抗体，其特征在于：所述轻链和重链的氨基酸序列分别为：SEQ ID No:9和SEQ ID No:10，或SEQ ID No:16和SEQ ID No:17，或SEQ ID No:19和SEQ ID No:20，或SEQ ID No:22和SEQ ID No:23，或SEQ ID No:25和SEQ ID No:27。
13.根据权利要求1所述的双特异性抗体，其特征在于：其分离的抗体或其抗原结合部分，在ELISA水平与人OX40或PD-L1结合。
14.一种双特异性分子、免疫交联物、嵌合抗原受体、基因改造T细胞受体或溶瘤病毒，其特征在于：包含权利要求1-10任一项所述的双特异性抗体或其抗原结合部分。
15.一种核酸，编码权利要求1-10任一项所述的双特异性抗体或其抗原结合部分。
16.一种表达载体，其特征在于：利用所述表达载体表达权利要求15所述核酸。
17.一种宿主细胞，其特征在于：所述宿主细胞包括权利要求16所述的表达载体。
18.一种制备双特异性抗体的方法，其特征在于，所述方法包括培养权利要求16中所述表达载体，或包括培养权利要求14中任意一项宿主细胞，从所述表达载体或所述宿主细胞中表达所述双特异性抗体。
19.一种药物组合物，其包含权利要求1-10任一项所述的双特异性抗体或其抗原结合片段。
20.一种治疗癌症的方法，其特征在于：所述方法包括向受试者施用有效量的权利要求1-10任一项所述的双特异性抗体或其抗原结合片段或权利要求19的药物组合物；所述癌症是肺癌、结肠癌、胃癌、肾癌或肝癌。
21.一种试剂盒或制品，其包含权利要求1-10任一项所述的双特异性抗体。
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