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CN111344019B - 调节免疫检查点作为癌症治疗的单特异性与双特异性蛋白质 - Google Patents

调节免疫检查点作为癌症治疗的单特异性与双特异性蛋白质 Download PDF

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CN111344019B
CN111344019B CN201880072318.7A CN201880072318A CN111344019B CN 111344019 B CN111344019 B CN 111344019B CN 201880072318 A CN201880072318 A CN 201880072318A CN 111344019 B CN111344019 B CN 111344019B
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游忠哲
徐静轩
黄柏霖
甘弘才
张渟宜
谢欣达
何正宏
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Yuanxiang Biotechnology Co ltd
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Abstract

提供一种单特异性与双特异性蛋白质，其特异性结合OX40及/或PD‑L1。示例的蛋白质通过PD‑L1产生抑制效果并通过OX40刺激T细胞。示例的多价蛋白质包含至少一个OX40结合位点和至少一个PD‑L1结合位点。在特定实施方式中，结合位点可以通过免疫球蛋白恒定区连接。另提供了抗OX40和抗PD‑L1抗体。

Description

调节免疫检查点作为癌症治疗的单特异性与双特异性蛋白质
【技术领域】
本发明涉及一种抗体。特别是，本发明是有关于一种用于癌症治疗的抗体。
【现有技术】
人类中两种主要类型的淋巴细胞是T淋
巴细胞(源自胸腺)和B淋巴细胞(源自骨髓)。这些细胞源自于骨髓和胎儿肝脏中的造血干细胞，并参与淋巴发育途径。这些干细胞的后代依照不同的途径成熟为B或T淋巴细胞。人类B淋巴细胞的发育完全在骨髓内发生。另一方面，T细胞从未成熟的前驱细胞发展而来，这些前驱细胞离开骨髓并通过血流到达胸腺，在那里它们增殖并分化成成熟的T淋巴细胞。
T细胞
T细胞是最丰富的(约75％的血液淋巴细胞)且有效的免疫毒杀细胞。效应T细胞(effector T-cells)在抗肿瘤免疫反应中的作用，获得体外研究和观察结果的强烈支持，CD8+ T细胞在几种类型肿瘤中的高渗透度(infiltration，又称浸润)与较佳的临床预后相关(Fridman等人，2012)。效应初始T细胞的活化需要至少三种互补信号：(i)TCR-CD3/Ag-MHC在共同受体(CD4或CD8)的辅助下的相互作用；(ii)共同刺激分子如CD80或CD86与CD28的结合，CD40与CD40L的结合；及(iii)辅助分子如细胞因子(cytokines)。
共同刺激(co-stimulation)或向T细胞提供两种不同的信号是抗原提呈细胞(antigen presenting cell，APC)对休止状态T淋巴细胞活化的广泛接受模式(Lafferty和Cunningham，1975)。此模式进一步提供了对自我与非自我和免疫耐受的区分(Bretscher和Cohn，1970；Bretscher，1999；Jenkins和Schwartz，1987)。在识别主要组织相容性复合体(major histocompatibility complex，MHC)下呈现外来抗原肽后，通过T细胞受体(TCR)转换成初级信号或抗原特异性信号。第二种或共同刺激信号藉由在抗原提呈细胞(antigen-presenting cell，APC)上表达共同刺激分子递送至T细胞，并诱导T细胞促进克隆扩充、细胞因子分泌和效应功能(effector function)(Lenschow等，1996)。在没有共同刺激的情况下，T细胞变得较不受抗原的刺激、不会产生有效的免疫反应，并且还可能导致对外来抗原无反应或具耐受性。
免疫检查点蛋白：细胞程序死亡-配体1(programmed cell death protein 1ligand，PD-L1)
免疫检查点是关于一群抑制和刺激途径，主要由配体-受体相互作用引发免疫系统僵化，特别是T细胞介导的免疫，以维持自身耐受并调节外周组织中生理反应的持续时间和幅度，通常便能减少附带组织损伤(Pardoll，2012)。肿瘤细胞将某些检查点途径作为免疫抗性的主要机制。例如，PD-L1的表达量通常在人类癌症肿瘤细胞的表面上增加。PD-L1与其受体PD-1的相互作用在肿瘤浸润的淋巴细胞(tumor infiltrated lymphocytes，TILs)上表达，特别是在T细胞上，抑制局部T细胞介导的反应以逃避免疫监视(Liang等人，2006；Sznol与Chen，2013)。因此，对癌细胞免疫抑制信号的抑制、或对T细胞直接激动性刺激，将导致和/或诱导强烈且持续性的抗肿瘤免疫反应。最近的临床研究强烈建议藉由抗体免疫检查点蛋白的阻碍、或由可溶性配体或受体的调节，是最有希望激发治疗性抗肿瘤免疫的方式(Topalian等，2014)。目前抗PD-1和抗细胞毒性T淋巴细胞相关抗原4(cytotoxic T-lymphocyte-associated antigen-4，CTLA-4)抗体已被美国食品药品监督管理局(Foodand Drug Administration，FDA)批准用于治疗诸如黑素瘤的疾病。
另一种共同刺激分子OX40受体(CD134)是TNFR超级家族的成员，其为膜结合并且主要在活化的CD4+T细胞上表达(Paterson等，1987)。通过OX40受体(以下称为“OX40”)的信息传递对效应T细胞而言是共同刺激，并引起T细胞的增殖(Watts，2005；Weinberg等人，1994)。对OX40的研究显示，其主要作用是规定在初级免疫反应中累积效应T细胞的数量，并因此控制随后发展和存活记忆T细胞的数量(Croft，2003)。已经有许多体外研究显示，OX40提供共同刺激信号，导致增强T细胞的增殖和细胞因子的产生。
双特异性(Bi-specific)/双功能性(bi-functional)抗体
在1980年代出现了使用双特异性抗体能有效地将效应免疫细胞重新以肿瘤细胞做为目标的想法(Karpovsky等人，1984；Perez等人，1985；Staerz等人，1985)。基于不存在或存在Fc片段(fragment crystallizable segment，可结晶区域片段)，双特异性支架(scaffold)通常区分为具有不同药物动力学特性的两个主要群组：类免疫球蛋白G(IgG-like)分子和小型重组双特异性形式，其中大多数衍生自单链可变片段(single chainvariable fragment，scFv)。通过其小巧的尺寸，抗体片段通常比类IgG分子更能有效地穿透肿瘤，但是这种优势将因过短的血清半衰期(几小时)而减少，并限制了它们的整体肿瘤吸收程度和停留时间(Goldenberg等，2007)。相比之下，结合新生儿Fc受体Fc片段的存在，为类IgG形式提供了长血清半衰期(>10天)并有利于被肿瘤吸收和保留，但限制了对肿瘤的穿透性。
最近的研究强调了免疫疗法的治疗功效，免疫疗法是利用患者自身免疫系统来破坏癌细胞的一类癌症治疗方法。在肿瘤内，阴性调节分子家族(统称为「检查点抑制剂」)的存在可抑制T细胞功能以抑制抗肿瘤的免疫力。检查点抑制剂，例如CTLA-4和PD-1，减弱T细胞增殖和细胞因子的产生。用拮抗剂(antagonist)单克隆抗体(monoclonal antibodies，mAbs)将CTLA-4或PD-1作为目标阻断并释放T细胞上的「剎车」，以增强抗肿瘤的免疫力。产生最佳「杀手」CD8T细胞的反应还需要T细胞受体活化加上共同刺激，如经由结合肿瘤坏死因子受体家族成员包括OX40(CD134)和4-1BB(CD137)来达成。OX40特别令人感兴趣在于其作为活性(激动剂)抗OX40单克隆抗体(mAb)的治疗时，可增强T细胞分化和细胞溶解(cytolytic)功能，从而增强针对多种肿瘤的抗肿瘤免疫力。当作为单一药物时，这些药物可以在具转移性疾病患者中诱导有效的临床和免疫反应(Linch等人，2015)。
【发明内容】
本公开内容的目的在于提供一种具有免疫调节的双特异性抗体，用于治疗患有癌症的患者进行检查，例如前列腺癌(prostate cancer)、肺癌(lung cancer)、非小细胞肺癌(Non-Small Cell Lung Cancer，NSCLC)、黑素瘤(melanoma)、淋巴瘤(lymphoma)、乳癌(breast cancer)、头颈癌(head and neck cancer)、肾细胞癌(Renal cell carcinoma，RCC)或卵巢癌(ovarian cancer)。
本公开内容提供了一种与OX40(CD134)结合的抗体或其抗原结合部分，包含：重链可变区，包含氨基酸序列，氨基酸序列与选自于由SEQ ID NO.6、SEQ ID NO.8、SEQ IDNO.10的第128-246个位置氨基酸、及SEQ ID NO.13的第124-241个位置氨基酸所组成之群组的氨基酸序列具有至少约80％的序列同源性；以及轻链可变区，包含氨基酸序列，氨基酸序列与选自于由SEQ ID NO.5的第1-108个位置氨基酸、SEQ ID NO.7的第1-108个位置氨基酸、SEQ ID NO.10的第1-112个位置氨基酸、与SEQ ID NO.13的第1-108个位置氨基酸所组成之群组的氨基酸序列具有至少约80％的序列同源性。
在一些实施方式中，抗体或其抗原结合部分系选自于由SEQ ID NOs.10、11、12及13所组成之群组的单链可变片段序列。
在一些实施方式中，抗体或其抗原结合部分是双特异性抗体。
在一些实施方式中，双特异性抗体包含免疫检查点蛋白结合位点。
在一些实施方式中，免疫检查点蛋白结合位点包含细胞程序死亡-配体1(PD-L1)结合位点、PD-1结合位点、表皮生长因子受体(epidermal growth factor receptor，EGFR)结合位点、人类表皮生长因子受体2(human epidermal growth factor receptor 2，HER2)结合位点、细胞毒性T淋巴细胞相关抗原4(cytotoxic T-lymphocyte-associatedantigen4，CTLA-4)结合位点、或淋巴细胞活化基因3(lymphocyte activation gene 3，LAG3)结合位点。
本公开内容另提供了一种与PD-L1结合的抗体或其抗原结合部分，包含：重链可变区，包含氨基酸序列，氨基酸序列与选自于由SEQ ID NO.2及SEQ ID NO.4所组成之群组的氨基酸序列具有至少约80％的序列同源性；以及轻链可变区，包含氨基酸序列，氨基酸序列与选自于由SEQ ID NO.1的第1-111个位置氨基酸及SEQ ID NO.3的第1-110个位置氨基酸所组成之群组的氨基酸序列具有至少约80％的序列同源性。
本公开内容另提供了一种双特异性抗体，包含至少一多肽链，其中所述多肽链包含：OX40结合位点以及PD-L1结合位点。OX40结合位点包含重链可变区，包含氨基酸序列，氨基酸序列与选自于由SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10的第128-246个位置氨基酸、及SEQ ID NO.13的第124-241个位置氨基酸所组成之群组的氨基酸序列具有至少约80％的序列同源性；以及轻链可变区，包含氨基酸序列，氨基酸序列与选自于由SEQ IDNO.5的第1-108个位置氨基酸、SEQ ID NO.7的第1-108个位置氨基酸、SEQ ID NO.10的第1-112个位置氨基酸、及SEQ ID NO.13的第1-108个位置氨基酸所组成之群组的氨基酸序列具有至少约80％的序列同源性。PD-L1结合位点包含重链可变区，包含氨基酸序列，氨基酸序列与选自于由SEQ ID NO.2及SEQ ID NO.4所组成之群组的氨基酸序列具有至少约80％的序列同源性；以及轻链可变区，包含氨基酸序列，氨基酸序列与选自于由SEQ ID NO.1的第1-111个位置氨基酸及SEQ ID NO.3的第1-110个位置氨基酸所组成之群组的氨基酸序列具有至少约80％的序列同源性。
在一些实施方式中，多肽链更包含Fc区、抗原结合片段(fragment-antigenbinding segment，Fab片段)以及scFv。Fab片段与Fc区的N端连接，且Fab片段包含PD-L1结合位点。scFv与Fc区的C端连接，且scFv包含OX40结合位点。
在一些实施方式中，多肽链更包含连接子界于Fc区和scFv之间。
在一些实施方式中，scFv包含氨基酸序列系选自于由SEQ ID NO.18的第455-707个位置氨基酸、SEQ ID NO.19的第455-708个位置氨基酸、SEQ ID NO.20的第455-701个位置氨基酸、SEQ ID NO.21的第455-706个位置氨基酸、SEQ ID NO.22的第455-706个位置氨基酸、SEQ ID NO.23的第455-706个位置氨基酸、SEQ ID NO.24的第455-706个位置氨基酸、SEQ ID NO.25的第455-706个位置氨基酸、SEQ ID NO.26的第455-706个位置氨基酸、SEQID NO.27的第455-706个位置氨基酸、SEQ ID NO.28的第455-706个位置氨基酸、及SEQ IDNO.29的第455-706个位置氨基酸所组成之群组。
在一些实施方式中，双特异性抗体包含一对多肽链。
在一些实施方式中，双特异性抗体为IgG、IgE、IgM、IgD、IgA、或IgY抗体。
在一些实施方式中，双特异性抗体为IgG抗体。
在一些实施方式中，IgG抗体为IgG1、IgG2、IgG3、或IgG4抗体。
本公开内容另提供了一种抗体药物复合体，包含治疗剂；以及抗体或其抗原结合部分，其系与PD-L1和/或OX40结合，其中治疗剂藉由连接子与抗体或其抗原结合部分共价接合。
在一些实施方式中，抗体或其抗原结合部分选自于如前述之抗体或其抗原结合部分。
本公开内容另提供了一种医药组成物，包含如前述之抗体、其抗原结合部分、或双特异性抗体；以及至少一药学上可接受的载剂。
本公开内容另提供了一种治疗癌症的方法，方法包含对有需要之个体施予有效剂量之如前述之抗体、其抗原结合部分或双特异性抗体。
在一些实施方式中，癌症系选自于由前列腺癌、肺癌、非小细胞肺癌、黑色素瘤、淋巴瘤、乳癌、头颈癌、肾细胞癌和卵巢癌所组成之群组。
本公开内容另提供了一种核酸分子，其编码如前述之抗体、其抗原结合部分或双特异性抗体。
应理解，前面的一般性描述和以下的详细描述都是基于实施例，并且旨在针对要求保护的本发明提供进一步的说明。
【图式简单说明】
为让本发明之上述和其他目的、特征、优点与实施例能更明显易懂，所附图式之说明如下：
图1显示免疫检查点调节T细胞介导的免疫。抗体可以依需求使用针对检查点的激动或拮抗的(agonistic or antagonistic)抗体建构双功能融合蛋白，例如抗ICOS、抗CD28、抗OX40、抗CD27、抗PD-1、抗CTLA4、抗LAG3、或抗BTLA。
图2A与图2B显示藉由直接酶联免疫分析法(direct enzyme-linkedimmunosorbent assay，direct ELISA)筛选重组PD-L1的噬菌体克隆(clone)。
图3A与图3B显示藉由基于细胞的ELISA筛选表达OX40的人类胚胎肾细胞293(human embryonic kidney 293 cells，HEK293)之噬菌体克隆。
图4显示通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfatepolyacrylamide gel electrophoresis，SDS-PAGE)以非还原试剂对PD-L1专一性之抗体先导物(antibody lead)纯化，以揭示其完整性和纯度。
图5显示通过SDS-PAGE以非还原或还原试剂对OX40专一性之抗体先导物纯化，以揭示其完整性和纯度。NR，非还原型(non-reducing)、R，还原型(reducing)。
图6显示纯化的抗免疫检查点蛋白的直接配体结合活性与抗PD-L1抗体先导物拮抗PD-L1的实施例。首先将以配体预先涂覆(pre-coated)的孔槽与所示各种浓度的抗体先导物一起培养。然后用辣根过氧化物酶(horseradish peroxidase，HRP)接合(conjugated)的山羊抗人类IgG Fab特异性抗体检测结合的蛋白质，并依OD450读值绘图。
图7显示纯化的抗免疫检查点蛋白的直接配体结合活性与抗OX40抗体先导物拮抗OX40的实施例。首先将以配体预先涂覆的孔槽与所示各种浓度的抗体先导物一起培养。然后用辣根过氧化物酶接合的山羊抗人类IgG Fab特异性抗体检测结合的蛋白质，并依OD450读值绘图。
图8显示使用表达PD-L1的293细胞的流式分析(flow analysis)。表达PD-L1的HEK293首先与纯化的抗体先导物一起培养，并用-488接合的山羊抗人类IgG(重链+轻链，H+L)检测结合的抗体，然后进行荧光活化细胞分选仪(fluorescence-activatedcell sorter，FACS)分析。
图9显示使用表达OX40的293细胞的流式分析。表达OX40的HEK293首先与纯化的抗OX40抗体先导物一起培养，并用-488接合的山羊抗人类IgG(H+L)检测结合的抗体，然后进行荧光活化细胞分选仪分析。NS：没有染色(no staining)。
图10显示以纯化的抗PD-L1抗体阻碍PD-1/PD-L1的相互作用。将所示的纯化抗体用于具有生物素的PD-L1-Fc和重组人类PD-1/His(hPD-1/His)以评估PD-1/PD-L1相互作用的抑制活性。藉由链霉亲合素(streptavidin)-HRP检测重组PD-L1-Fc与PD-1/His(hPD-1/His)的结合，并通过ELISA分析。
图11A和图11B显示在抗体治疗3天(图11A)或5天(图11B)后，具有1或10微克/毫升(μg/mL)的抗PD-L1抗体先导物于混合淋巴细胞反应(mixed lymphocyte reaction，MLR)分析中刺激T细胞的增殖与IL-2及/或IFN-γ(干扰素-γ，interferon-γ)的产生。
图12A显示抗OX40抗体先导物藉由剂量反应(dosage response)增强CD3+T细胞活化的能力，参考抗体也有相同的结果。图12B显示人类T细胞以结合于孔盘中的抗CD3抗体和几种浓度的抗OX40抗体先导物刺激3天后，存在于细胞培养基中的人类IL-2和IFN-γ浓度。
图13A和图13B显示人类T细胞以结合于孔盘中的抗CD3抗体和几种浓度的抗OX40抗体先导物经过3天的刺激后，存在于细胞培养基中人类IL-2(图13A)和IFN-γ(图13B)的浓度。
图14显示抗体重链Fc与OX40特异性scFv区融合的结构。
图15显示抗免疫检查点抗体-人类OX40融合蛋白的PAGE凝胶分析的实施例。纯化的融合蛋白-抗PD-L1-OX40 scFv融合蛋白显示分别具有约220千道尔顿(kDa)的分子量(non-reducing，NR，非还原型)，并且皆具有约85kDa的重链和约25kDa的轻链(reducing，R，还原型)。每栏注入3μg。
图16A和图16B显示在单一、结合、或抗PD-L1-OX40 scFv双特异性抗体治疗3天(图16A)或5天(图16B)后，双特异性抗体于MLR分析中协同刺激T细胞活化使IL-2及IFN-γ产生。
图17A至图17E分别显示双特异性抗体：抗PD-L1-OX40抗体、和抗PD-L1-OX40 Ab-V1至V4的聚集和纯度测定，且双特异性抗体在OX40 scFv中具有5种不同的连接子。
图18显示选自OX40克隆B17 scFv的抗PD-L1-OX40 Ab-V4至V12的序列变体(sequence variants)(SEQ ID NOS:30-38)。
图19显示抗免疫检查点抗体-人类OX40融合蛋白的PAGE凝胶分析的实施例。纯化的融合蛋白-抗PD-L1-OX40 Ab-V5融合蛋白显示分别具有约220千道尔吞(kDa)的分子量(非还原型)，并且皆具有约80kDa的重链和约30kDa的轻链(还原型)。
图20显示评估双特异性抗体变体的结合活性的ELISA方法的流程图。
图21显示双特异性抗体变体对人类PD-L1的结合活性及其EC50。
图22显示双特异性抗体变体对人类OX40的结合活性及其EC50。
图23显示双特异性抗体变体：抗PD-L1-OX40 Ab-V5的离体血清稳定性。
图24A和图24B显示经单一、结合、或抗PD-L1-OX40 Ab-V5双特异性抗体调节T细胞后，于治疗3天IL-2的产量(图24A)与5天IFN-γ的产量(图24B)。
图25为显示抗PD-L1-OX40 Ab-V5双特异性抗体和单克隆抗体治疗于Fox ChaseBeige小鼠中，PC-3肿瘤生长影响的图式。
【实施方式】
现在将参照本发明的实施方案详细地进行说明，本发明实施方案的实例将结合附图进行说明。只要有可能，在附图和说明书中使用相同的附图标记表示相同或相似的部分。
本发明描述了分离具功能性激动OX40 scFv的双功能性蛋白质的表达、纯化和特征，所述OX40 scFv与抗免疫检查点蛋白质抗体的Fc区的C端融合。这些蛋白质与其相应的检查点相互作用，并传递抑制或刺激信号以调节T细胞参与免疫。本发明中Fc融合蛋白的组成皆源自于人类，因此预期是非免疫原性的(non-immunogenic)并且可以用于人类的治疗。
双特异性分子如双特异性抗体(bispecific antibodies，BsAb)提供了用单一治疗剂、同时将相同分子目标或不同目标上的多个表位(epitopes)做为目标的手段。作为癌症疗法，它们具有潜力赋予新的或更有效的活性、降低商品成本、并促进新治疗方案的开发，而不是两种单克隆抗体的混合物(Chames和Baty，2009；Thakur和Lum，2010)。最近，catumaxomab是一种以人类上皮细胞黏附分子(human epithelial cell adhesionmolecule，EpCAM)和CD3作为目标的三功能双特异性抗体，其已显示出对上皮癌的腹膜癌(peritoneal carcinomatosis of epithelial cancers)患者有明显的临床益处(Heiss等人，2010)。对CD19和CD3具有双重特异性结合能力的双特异性T细胞结合抗体(bispecificT-cell engaging antibody，BiTE Ab)在CD19表达恶性血液病(hematologicalmalignancies)的患者中，也表现出令人鼓舞的临床活性(Bargou等人，2008)。尽管人们对开发作为癌症治疗剂的双特异性分子非常感兴趣，但是过去在生产具稳定和活性的双特异性分子技术中面临挑战，并阻碍了大多数双特异性形式的临床评价。许多工程化抗体(engineered Abs)形式，包括类IgG双特异性抗体已经克服稳定性或溶解度的问题(Bargou等人，2008；Demarest和Glaser，2008；Lu等人，2005)。此外，已经采取了几种策略来提高双特异性分子的产品质量和体内稳定性，包括PEG化(PEGylation)、与人类血清白蛋白接合、以及Fc的工程化(Muller等人，2007；Ridgway等人，1996)。上述一般形式的双特异性单链抗体具有以下优点：编码四个可变区(variable region)的核苷酸序列、两个连接子、或一个间隔子(spacer)可以在单个启动子的控制下于合适的宿主表达生物中。这增加了设计这些结构的灵活性以及实验者在其生产过程中的控制程度。此外，IgG的Fc对用于设计新疗法中是非常具有吸引力的架构，因为它包含除结合能力之外的所有抗体功能。Fc的工程对于提高双特异性抗体有效性而言是至关重要的。因此，以IgG为基础的结构在本发明中用于免疫细胞或免疫治疗目标细胞，作为两个独立的目标。
以免疫检查点蛋白为目标是活化抗肿瘤免疫中备受期待的方法。目前临床上评估了抗检查点蛋白，例如PD-1、PD-L1、CTLA-4、LAG3等(图1)。初步的实验数据已显示免疫检查点蛋白抑制剂能够增强抗肿瘤免疫力，并在临床反应上具有药效持久性的潜力。然而，尽管这些药物在许多恶性肿瘤中具有显著的临床功效，但很明显它们对许多患者来说仍不够有效。许多其他免疫调节途径，如由骨髓表达或分泌的抑制因子、以及肿瘤微环境中的基质细胞(stromal cell)，是与免疫检查点阻碍物协同作用的潜在目标。因此，结合抗癌或双特异性抗体疗法对于实现癌症患者的完全缓解和治疗是不可或缺的。
本发明描述了不同免疫检查点蛋白特异性scFv蛋白与抗免疫检查点蛋白抗体Fc融合的结构、表达和特征。如果融合对应物是免疫增强剂，例如抗EGFR、抗HER2、或抗CTLA-4抗体，则位于融合结构C端的OX40 scFv将会增强融合蛋白的免疫力，并超过单独以OX40本身活化免疫力的方式。
自OmniMab库产生的抗体
为了产生针对PD-L1或OX40具疗效的抗体，进行了OmniMab噬菌粒库(phagemidlibrary)的选择。噬菌粒库由AP Biosciences公司(APBio Inc.)从超过100个健康捐赠者B细胞收集产生。在第一轮筛选中使用之噬菌体系以Hyperphage(M13K07ΔρΙΙΙ，德国海德堡Progen公司)制备。针对PD-L1或OX40的固相筛选和细胞筛选是用于选择PD-L1或OX40特异性结合物，并从OmniMab库中分离。在第一轮选择中使用重组人类PD-L1-Fc或OX40-Fc(APBio Inc.)进行固相筛选，然后将表达PD-L1或OX40的HEK293细胞用于另外2至3轮的富集过程(enrichment)。在三轮选择后，特异性PD-L1或OX40结合物藉由使用相应的重组蛋白以直接或基于细胞ELISA筛选和分离(图2A、图2B、图3A及图3B)。预先涂覆的PD-L1-Fc重组蛋白或表达OX40的293细胞以含有补救噬菌体(rescued phages)的上清液印迹(blot)1小时，并用含有0.1％Tween-20的磷酸盐缓冲生理盐水(phosphate buffered saline，PBS)洗涤三次。噬菌体藉由辣根过氧化物酶接合的抗M13抗体(Roche)检测，并以TMB底物(3,3',5,5'-Tetramethylbenzidine substrate，TMB substrate)做为信号产生，并记录OD450读值。分离阳性结合物并送测序以确认重链和轻链的序列和多样性。对PD-L1或OX40专一的重链和轻链可变区如SEQ ID NO.1至SEQ ID NO.8所示：SEQ ID NO.1是PD-L1克隆6的轻链、SEQID NO.2是PD-L1克隆6的重链可变区、SEQ ID NO.3是PD-L1克隆32的轻链、SEQ ID NO.4是PD-L1克隆32的重链可变区、SEQ ID NO.5是OX40克隆B17的轻链、SEQ ID NO.6是OX40克隆B17的重链可变区、SEQ ID NO.7是OX40克隆B19的轻链、SEQ ID NO.8是OX40克隆B19的重链可变区。如图2A、图2B、图3A与图3B所示，与阴性对照组相比，许多克隆被分离出并且已知其特异性地识别相应的抗原。
亚克隆(subcloning)和表达/纯化作为IgG形式筛选出的PD-L1或OX40特异性结合物
为了便于快速筛选具有T细胞活化功能的特异性结合物，由ELISA筛选出PD-L1或OX40的阳性结合物的重链和轻链，接着进行扩增、分解(digest)与亚克隆到携带IgG4恒定区(constant region)的APBio特异性IgG表达载体中(SEQ ID NO.9)。在序列验证后，制备质体(plasmid)并使用293fectin转染试剂(Invitrogen)转染到HEK293细胞中以表达抗体。培养4天后，藉由蛋白G层析(Protein G chromatography)将分泌到无血清培养基的抗体从培养基上清液中亲和纯化出。纯化的抗体接着浓缩，并于PBS缓冲液中透析。透析蛋白的最终浓度藉由NanoDrop2000分光亮度计测定，且在有或没有还原剂的条件下以SDS-PAGE测定纯度和完整性，如图4及图5所示。各种纯化的抗体先导物(PD-L1专一或OX40专一)、参考抗体(MPDL3280A辨识PD-L1、GSK3174998辨识OX40)的完整性在HEK293细胞中是正常的。
在一些实施方式中，本公开内容提供一种与OX40(CD134)结合的抗体或其抗原结合部分，包含重链可变区以及轻链可变区。重链可变区包含氨基酸序列，此氨基酸序列与选自于由SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10的第128-246个位置氨基酸、与SEQ IDNO.13的第124-241个位置氨基酸所组成之群组的氨基酸序列具有至少约80％的序列同源性。在一些实施例中，重链可变区与前述的氨基酸序列具有至少约85％、90％或95％的序列同源性。轻链可变区包含一氨基酸序列，此氨基酸序列与选自于由SEQ ID NO.5的第1-108个位置氨基酸、SEQ ID NO.7的第1-108个位置氨基酸、SEQ ID NO.10的第1-112个位置氨基酸、与SEQ ID NO.13的第1-108个位置氨基酸所组成之群组的氨基酸序列具有至少约80％的序列同源性。在一些实施例中，轻链可变区包含与前述的氨基酸序列具有至少约85％、90％或95％的序列同源性。
在一些实施方式中，本公开内容提供一种与PD-L1结合的抗体或其抗原结合部分，包含重链可变区以及轻链可变区。重链可变区包含一氨基酸序列，此氨基酸序列与选自于由SEQ ID NO.2与SEQ ID NO.4所组成之群组的氨基酸序列具有至少约80％的序列同源性。在一些实施例中，重链可变区与前述的氨基酸序列具有至少约85％、90％或95％的序列同源性。轻链可变区，包含一氨基酸序列，氨基酸序列与选自于由SEQ ID NO.1的第1-111个位置氨基酸与SEQ ID NO.3的第1-110个位置氨基酸所组成之群组的氨基酸序列具有至少约80％的序列同源性。在一些实施例中，轻链可变区包含与前述的氨基酸序列具有至少约85％、90％或95％的序列同源性。
由直接ELISA测定PD-L1或OX40特异性IgG先导物的结合活性
PD-L1或OX40的纯化抗体先导物(抗PD-L1或OX40抗体先导物)于人类PD-L1-Fc或OX40-Fc直接涂覆的装置中进行ELISA结合特征分析。图6及图7分别显示抗PD-L1抗体与抗OX40抗体的ELISA结合结果。对于PD-L1特异性抗体，大多数先导物显示出与参考抗体(RefAb，MPDL3280A，Roche)相似或更好的结合活性。
纯化的人类PD-L1或OX40 IgG1 Fc嵌合体(PD-L1-Fc或OX40-Fc chimera，APBio)在PBS中透析，调整浓度至1mg/mL，然后再用PBS稀释至最终浓度为1μg/mL。将Nunc-ImmunoMaxisorp 96孔盘以每孔0.1mL重组PD-L1-Fc或OX40-Fc嵌合体涂覆，留下空孔用于非特异性结合的对照组，并在4℃隔夜培养。移除PD-L1-Fc或OX40-Fc嵌合体溶液，孔盘用0.4mL冲洗缓冲液(wash buffer，含0.1％Tween-20的PBS溶液)洗涤三次。将孔盘中所有的孔槽加入0.4mL阻断缓冲液(blocking buffer)(含5％低脂奶粉的PBS)，并在室温下培养混合1小时。移除阻断缓冲液后，用0.4mL冲洗缓冲液洗涤孔盘三次。在PBS中制备PD-L1或OX40测试抗体的连续稀释液，每孔加入0.1mL稀释的抗体。将孔盘在室温下培养1小时。移除抗体溶液，每孔用0.4mL冲洗缓冲液洗涤孔盘三次。辣根过氧化物酶标记的山羊抗人类IgG F(ab')2特异性F(ab')2抗体(Jackson Immunoresearch#109-036-097)用PBS以1：2000稀释，每孔加入0.1mL。将孔盘于室温下培养1小时，并用每孔0.4mL的冲洗缓冲液洗涤。加入0.1mL TMB试剂(Invitrogen)并在室温下培养1至5分钟。藉由加入0.05mL 1N盐酸(HCl)终止反应，并在Bio-Tek Spectra上于450nm处读取吸光值。计算抗PD-L1抗体先导物对PD-L1的EC50并显示，由直接ELISA侦测出大多数先导物具有良好的结合活性，MPDL3280A(Ref Ab)也呈现相同的结果(图6)。相对的，大多数抗OX40抗体先导物显示较参考抗体(Ref Ab，GSK3174998)低的结合活性(图7)。
通过FACS确认PD-L1与OX40特异性IgG先导物的结合活性
纯化的抗体先导物(抗PD-L1或OX40抗体先导物)也用于流式细胞技术，以确定和比较先导物与PD-L1或OX40表达的HEK293细胞的结合活性。图8及图9显示，稳定表达PD-L1或OX40的HEK293细胞经FACS可知相应抗体先导物的结合活性。
FACS分析以抗PD-L1抗体先导物将稳定表达PD-L1的293细胞进行染色，以检测PD-L1结合活性。稳定表达的293细胞与1μg/mL纯化的抗PD-L1抗体先导物、参考抗体(Ref AbMPDL3280A)、或作为阴性对照的同种型抗体在冰上培养1小时。用1倍PBS洗涤细胞三次，然后在冰上与-488接合的山羊抗人类IgG(H+L)(Invitrogen Inc.)再一起培养1小时。染色后，用1倍PBS洗涤细胞三次，并重新悬浮于1倍PBS/2％胎牛血清(Fetal BovineSerum，FBS)中，然后通过FACS Calibur(BD Biosciences，Inc)和FlowJo(TreeStar，LLC)分析。相似的情形，于图9中对于稳定表达OX40的HEK293细胞而言，抗OX40抗体先导物的结合能力也使用相同的方法分析如前所述。如图8所示，大多数抗PD-L1抗体先导物与参考抗体皆具有良好的结合活性。这显示选自OmniMab库的噬菌体克隆确实可识别细胞中原始的PD-L1。
此情形也同时在抗OX40抗体先导物中被观察到如图9所示。以FACS分析OX40稳定表达的293细胞克隆2D5用纯化的抗OX40抗体先导物染色，以检测O40的结合能力。稳定表达的细胞以2μg/mL的抗OX40参考抗体(OX40 ref.)或抗CD137参考抗体(CD137ref.)作为对照组抗体在冰上培养1小时。这些细胞以1倍的PBS洗涤3次，并以-488接合山羊抗人类IgG(H+L)(Invitrogen Inc.)于冰上再培养1小时。染色后，这些细胞以1倍的PBS洗涤3次，悬浮于含2％FBS的1倍PBS中准备以FACS Calibur(BD Biosciences，Inc)和FlowJo(TreeStar，LLC)分析。
配体竞争结合(ELISA测定)
抗体先导物所显示的结合选择性和亲和力测定，是用于评价本发明的抗PD-L1抗体先导物阻断PD-L1与PD-1结合的能力。
抗体藉由ELISA测试阻断人类PD-L1-Fc嵌合体(PD-L1-Fc)与重组人类PD-1/His(hPD-1/His)结合的能力。将纯化的重组hPD-1/His(APBio)在PBS中透析至1mg/mL，然后在与生物素(Abcam)结合。将Nunc Maxisorp 96孔盘用在PBS中的250ng hPD-1/His涂覆过夜。移除hPD-1/His溶液，用0.4mL冲洗缓冲液(0.1％Tween-20的PBS溶液)洗涤孔盘三次。所有孔槽中加入0.4mL阻断缓冲液(含5％低脂奶粉的PBS)，在室温下培养1小时并混合。在阻断步骤期间，将抗体原液(antibody stocks)在PBS中以200nM至0nM的范围以2倍连续稀释。将纯化的重组具生物素的PD-L1-Fc嵌合体在PBS中稀释至4μg/mL。用0.2mL冲洗缓冲液(0.1％Tween-20的PBS溶液)洗涤以PD-1/His涂覆的孔盘三次。将60μL抗体稀释液(抗PD-L1抗体先导物或参考抗体Ref Ab MPDL3280A)单独加入60μL具生物素的PD-L1-Fc嵌合体，并在室温下培养1小时。孔盘被洗涤的方式如前所述。将链霉亲和素-辣根过氧化物酶在PBS中以1：2000稀释，将100μL所得溶液加入到经洗涤的孔盘的孔槽中，并在室温下培养1小时。孔盘被洗涤的方式如前所述，每个孔槽中加入100μL TMB受质溶液并培养10分钟。用50μL 1N HCl终止反应，并使用Bio-Tek读数器读取450nm处的吸光值如图10所示。部分抗体先导物藉由竞争ELISA(competition ELISA)抑制PD-1-PD-L1之间的相互作用。与参考抗体(Ref AbMPDL3280A)相比，大多数抗体先导物显示出类似的阻断活性。
藉由抗PD-L1抗体抑制PD-1：PD-L1配体相互作用以增强刺激T细胞活化
PD-1信息传导途径抑制中度TCR/CD28共同刺激信号，首先减少细胞因子产生而T细胞增殖则没有减少。随着TCR/CD28共同刺激信号减弱，PD-1途径占主导地位，细胞因子的产生大幅减少，同时也伴随着增殖的减少。因此，为了藉由抑制PD-1与PD-L1的相互作用来确认PD-1的抑制情形，本发明的人类抗体增强T细胞活化，并进行混合淋巴细胞反应(MLR)。
通过RosetteSepTM人类单核细胞富集组(产品型号15068)富集来自人类全血的单核细胞，并在分化培养基含10％FBS RPMI 1640、100ng/mL(1000U/mL)GM-CSF、100ng/mL(500U/mL)中培养6天。由DC-SIGN-PE、与异硫氰酸荧光素(fluorescein isothiocyanate，FITC)抗体接合的抗CD14、与PE抗体接合的抗CD83、或与FITC抗体接合的抗CD80检查从单核细胞分化的树突细胞(dendritic cells，DC)，以在混合淋巴细胞反应中验证分化情形与验证曾经为抗原提呈细胞(antigen-presenting cell，APC)。
藉由RosetteSepTM人类CD4+T细胞富集组(商品型号15062)分离来自人类全血的异体(Allogenic)CD4+T细胞。用抗CD4接合的APC抗体检查CD4+T细胞的纯度以确保纯度高于95％，然后用1μM CFSE(CellTraceTM CFSE细胞增殖试剂盒，Life technologies，商品型号C34554)标记T细胞增殖试验。标记的CD4+T细胞用于与未成熟的树突细胞及不同的抗体先导物共同培养3天和5天，以观察抗体先导物是否可以通过阻断PD-1和PD-L1之间的相互作用来恢复T细胞活化。培养3天和5天后，收集上清液中的细胞因子，例如藉由ELISA定量的IL-2和IFN-γ。与同种型IgG(iso#1，#2)处理的培养物相比(如图11A和图11B所示)，向未成熟树突细胞和异体T细胞培养物中添加抗PD-L1抗体先导物(克隆6、32、28、51、64、27和37)预期会导致T细胞增殖和细胞因子产生增加。在第3天(图11A)或第5天(图11B)抗体施予后与同种型抗体相比，MLR中IL-2和IFN-γ产生显著增加，特别是对于抗PD-L1抗体克隆6的结果。抗体治疗5天后，细胞因子的增加仍然显著，其结果与参考抗体(ref)MPDL3280A相似。这显示抗PD-L1抗体克隆6应该是双特异性抗体复合物的潜在先导物之一。
抗OX40抗体的激动活性分析
为了活化以OX40共同刺激的T细胞增殖和细胞因子的产生，纯化的抗体先导物以增强细胞因子产生、增殖和诱导人类CD3+ T细胞增殖的能力作为功能性筛选。抗CD3抗体(OKT3，BioLegend商品型号317304)和抗OX40抗体先导物(克隆B6、B70、B120、A4、B17、B19和B30)、参考抗体(GSK3174998)或同种型抗体(iso#1号、#2)涂覆在Maxisorp 96孔盘中。同时，使用商业上可获得的RosetteSepTM人类T细胞富集组(STEMCELL商品型号15061)，从健康成人自愿者的血液中分离出天然的人类CD3+T细胞。然后通过CFSE(CellTraceTM CFSE细胞增殖试剂盒，Life technologies，商品型号C34554)标记分离的CD3+ T细胞，并以每毫升1×106个细胞接种到含有RPMI 1640培养基、10％FBS和2.5mM L-谷氨酰胺的预先涂覆抗体的孔槽中，以确定细胞增殖和细胞因子的产生。培养3天后，通过流式细胞仪收集细胞用于增殖测定，然后藉由定量ELISA分析培养基中IL-2和IFN-γ产量。
在图12A中显示对T细胞活化具有激动活性的抗OX40抗体先导物的筛选。所有抗OX40抗体先导物均显示通过剂量反应增强CD3+ T细胞活化的能力，参考抗体也呈现相同的结果。高剂量的抗体治疗显示出明显更高的T细胞活化活性。同时，细胞因子的产生(图12B)，例如IL-2和IFN-γ也显示出相似的T细胞活化反应，特别是对于抗OX40抗体先导物克隆B17。细胞因子在抗OX40抗体先导物B17施予3天后高度诱发。此增强情形远高于以参考抗体治疗的结果，这表示克隆B17应该是双特异性抗体架构的候选者之一。
如图13A及图13B的数据显示，这两个抗OX40抗体先导物克隆B17和B19经过3天的治疗后，在测定中具有更好的激动活性。IL-2的产生或IFN-γ的产生在抗体治疗后明显增加，并显示出剂量依赖的相关性。在更高剂量的抗体治疗中记录更高细胞因子的产生。
为了评估OX40抗体先导物B17和B19的激动活性，还测定了EC50以及激动活性检测，并记录细胞因子的产生作为比较。
抗PD-L1-OX40 scFv抗体的结构、表达和纯化
由于双特异性被设计为与OX40 scFv融合的IgG，因此抗免疫检查点抗体Fc端与OX40 scFv融合。抗体可以是抑制性抗免疫检查点抗体(例如抗PD-L1、抗PD-1、抗CTLA4、抗LAG3等)、或刺激性抗体(例如抗CD28、抗CD137、抗CD27、抗ICOS等)。将连接子置于抗体Fc和OX40 scFv之间以产生双特异性抗体如图14所绘示。
在一些实施方式中，抗PD-L1抗体先导物克隆6被设计为IgG形式，另一方面，抗OX40抗体先导物转形(transform)为scFv形式以与抗PD-L1抗体先导物克隆6Fc区的C端融合。由于从抗体到scFv形式的转形可能导致结合活性或特异性的降低；因此，使用几种抗OX40抗体先导物进行scFv的转形。双功能抗PD-L1抗体Fc与全长OX40 scFv(SEQ ID NO.10为克隆A4、SEQ ID NO.11为克隆B17、SEQ ID NO.12为克隆B19、或SEQ ID NO.13为克隆B120)融合建构。将短小灵活的多肽连接子(GGGGS)2(SEQ ID NO.14)置于例如Fc区的抗PD-L1抗体重链C端和OX40 scFv的N端之间，以确保正确折叠并尽量减少空间阻碍(sterichindrance)。抗PD-L1-OX40 scFv抗体的编码序列显示在SEQ ID NO.16(抗PD-L1-克隆6重链-OX40克隆B17 scFv)和SEQ ID NO.17(抗PD-L1-克隆6重链-OX40克隆B19scFv)。建构的抗体Fc融合蛋白由信号肽(SEQ ID NO.15)引导并由哺乳动物细胞表达，并通过一步蛋白G色谱法(1-step Protein G chromatography)从转染的细胞培养上清液中纯化。如图15所示，在单步纯化过程中可以获得大于90％的纯度，并且显示纯化的融合蛋白具有正确的分子量(Mw＝220kDa)。
在MLR中增强抗PD-L1-OX40 scFv双特异性抗体先导物对T细胞活化的刺激
通过抑制PD-1和PD-L1之间的相互作用与激动活化OX40的信号传导，来确定双特异性抗体在增强T细胞活化中的协同作用，双特异性抗体先导物抗PD-L1-OX40 scFv抗体应用于MLR如前所述。然后在抗体施予3或5天后记录IL-2和IFN-γ的产生。以等量或等摩尔量(mole)之单一、结合、或双特异性抗体比较T细胞活化增强中的协同效果，并将同种型IgG作为阴性对照组。如图16A和图16B所示的数据，单一抗PD-L1的抗体先导物显示在施予3天后IL-2显著诱发，参考抗体MPDL3280A亦有相同的结果；相对的，无论是施予抗体3天或5天后，抗OX40抗体先导物无法使细胞因子明显增加。GSK3174998是OX40的参考抗体。然而，抗OX40抗体和抗PD-L1抗体的组合显示出在抗体处理3和5天后，细胞因子的产生显著上升。在双特异性抗体先导物治疗中也观察到协同作用，并且细胞因子产量的增加与组合治疗有相关联。这表示抗PD-L1-OX40 scFv双特异性抗体先导物也与抗体的组合治疗有相同的效果，而在scFv转形中不损失任何结合活性。
双特异性抗体的聚集和纯度测定
由于纯化的抗PD-L1克隆6-OX40克隆B17 scFv抗体在单柱蛋白A层析纯化(singlecolumn protein A chromatography purification)后通过SEC-HPLC分析显示出较低的纯度(74.07％)，因此产生了几种抗体变体以提高纯度和减少本发明中双特异性抗体的聚集。如上所述的连接子用于替换在双特异性抗体抗PD-L1-OX40抗体(SEQ ID NO.16)中的OX40B17 scFv内的连接子，并从CHO细胞中产生出抗PD-L1-OX40 Ab-V1至V4(SEQ ID NO.18至SEQ ID NO.21)。然后纯化这些变体并通过XBridge Protein BEH SEC-HPLC柱(Waters，产品型号No.186007640)分析。数据总结如下表1，双特异性抗体变体之一：抗PD-L1-OX40 Ab-V4显示抗体纯度有显著改善。纯度从74.07％增加到92.27％。因此，抗PD-L1-OX40 Ab-V4用于进一步改进以提高抗体纯度。
表1、OX40 B17 scFv中的不同连接子
为了显示双特异性抗体的大小分布，使用Waters Alliance 2695分离模块将样品装填到XBridge Protein BEH SEC-HPLC柱(Waters，商品型号186007640)上。使用Water2996 PDA检测器在280nm处检测蛋白质峰。流动相是等强度的(isocratic)25mM磷酸钠(Sigma，商品型号04272和商品型号04269)与200mM氯化钠(AMRESCO，商品型号0241)、pH6.8、流速为0.4mL/分钟。峰值百分比由峰面积(peak area)的部分决定，如图17A至图17E所示。
抗PD-L1-OX40 Ab-V4显示出纯度有显著改善(图17E)。在OX40 B17 scFv片段中进一步将双特异性抗体工程化以再次提高纯度。图18中显示在OX40 B17 scFv中的几个残基被不同氨基酸取代，并且重链变体与抗PD-L1克隆6轻链配对，以产生来自抗PD-L1-OX40Ab-V5至V12(SEQ ID NO.22至SEQ ID NO.29)的几种双特异性抗体变体，然后如上所述方式进行表达和纯化。双特异性抗体变体的纯度总结如下表2，抗PD-L1-OX40 scFv-V5在那些抗体变体中显示出最佳纯度，纯度高达96.46％。这显示工程化双特异性抗体具有优异的纯度，并且还显示出在未来对于双特异性抗体具有良好开发能力。如图19所示，经由SDS-PAGE分析抗PD-L1-OX40 Ab-V5的完整性，并且在还原和非还原条件下显示出良好的完整性。
表2、抗体的纯度
同时，工程化的双特异性抗体变体也经由直接ELISA应用于人PD-L1和OX40的结合活性评估方法，如图20所示。所有双特异性抗体变体显示出对人类PD-L1有的相同结合活性(图21)，此结合活性与抗PD-L1克隆6抗体相似。在人类OX40结合测试中也观察到这种现象(图22)。与其他变体相比，仅抗PD-L1-OX40 Ab-V10对人类OX40显示出较弱的结合活性。这显示OX40 scFv的工程化不会影响OX40的结合活性，PD-L1或OX40保留了结合活性。由于抗PD-L1-OX40 Ab-V5显示出优异的抗体纯度和对PD-L1和OX40的结合活性，因此选择抗PD-L1-OX40 Ab-V5用于血清稳定性测试。
离体血清稳定性
在人血清(BioreclamationIVT，商品型号HMSRM)中评估稳定性，同时亦针对以及来自相关临床前物种的血清进行评估：恒河猴(rhesus monkey，BioreclamationIVT，商品型号RHSSRM)和CD1小鼠(BioreclamationIVT，商品型号MSESRM)。将样品以15μg/mL的最终浓度加入到不同物种的血清，并在37℃水浴中培养。在0、1、2、3、7、10和14天的培养时间后收集血清样品，并在-80℃下冷冻储存直至进行分析。
三明治ELISA(sandwich ELISA)定量
取100μL浓度为1μg/mL于PBS中的OX40-Fc涂覆于ELISA孔盘(NUNC，商品型号442404)，并在4℃隔夜培养。冲洗缓冲液为含有0.1％Tween-20(Sigma，商品型号P2287-500mL)的PBS，封闭缓冲液为冲洗缓冲液中添加1％牛血清蛋白(bovine serum albumin，BSA，UniRegion，商品型号AR-BSA001-100G)。在封闭缓冲液中用10倍稀释的方式连续稀释3次以制备血清样品，并在阻断缓冲液中连续稀释3次以制备出浓度为10nM的标准品。使用标准操作准则将生物素化的PD-L1-Fc以生物素快速接合试剂盒(abcam，商品型号ab201796)进行标记，并在封闭缓冲液中制备成30nM。链霉亲合素-辣根过氧化物酶(Streptavidin-HRP，Abcam公司，商品型号ab7403)在封闭缓冲液中制备成浓度为1μg/mL。以冲洗缓冲液洗涤3次后，将所有样品以每孔100μL加入孔盘中，并在室温下培养1小时。用100μL TMB溶液(Invitrogen，商品型号00-2023)进行TMB显色2分钟，并用100μL 1N HCl溶液(Merck，商品型号1.00317.1000)终止反应。经由ELISA读数器(Biotek，Powerwave XS)读取OD 450nm的吸收波长。
选择抗PD-L1-OX40 Ab-V5用于离体血清稳定性，是因为其对于PD-L1和OX40具有优异的纯度和结合活性。所述纯化的双特异性抗体与自不同物种，例如人类、小鼠或猴子的血清混合。培养数天后，取出样品并藉由三明治ELISA分析以确定抗体含量。如图23所示，经过14天在37℃的培养后，抗PD-L1-OX40 Ab-V5仍表现出良好的血清稳定性。在人类、小鼠或猴子中的抗体浓度仍高于70％，显示抗体也具有良好的血清稳定性。
为了测量抗PD-L1-OX40 Ab-V5抗体调节T细胞反应能力，将纯化的T细胞与异体的树突细胞一起培养，并藉由在GM-CSF和IL-4中培养单核细胞几天来制备。设置平行孔盘以允许在第3天和第5天收集上清液，使用市售ELISA试剂盒分别测量IL-2和IFN-γ。如图24A和图24B所示，抗体处理3或5天后，在双特异性抗体处理(V5)以及参考抗体中高度增加了IL-2和IFN-γ的产生。再者，抗PD-L1-OX40 Ab-V5抗体的增强效应，明显优于单独使用抗PD-L1抗体或抗OX40抗体的治疗。这暗示工程化的双特异性抗体V5仍然具有激动活性以及如同组合治疗般而没有丧失功能性，并且可以在未来开发为各种实体肿瘤或癌症的治疗性抗体。
双特异性抗体的抗肿瘤活性(体内模型)
由于PD-L1与OX40在双特异性抗体中缺乏啮齿动物交叉反应性，阻碍了标准鼠同源或人类异种移植肿瘤模型用于评估抗体抗人类肿瘤的功效。因此，使用SCID-Bg小鼠(CB.17/Icr.Cg PkrdcscidLystbg/CrI)产生新的huPBL-SCID-Bg异种肿瘤小鼠模型，其含有米色(beige，Bg)突变、缺乏T和B淋巴细胞、与缺乏有功能性的自然杀手细胞(naturalkiller cell，NK cell)。使用如下所述的模型评估双特异性抗体抗人肿瘤的功效。
PC-3人类前列腺取自美国典型培养物保藏中心(American Type CultureCollection，ATCC)，并在含有L-谷氨酰胺(L-glutamine)、丙酮酸钠(sodium pyruvate)、青霉素/链霉素(penicillin/streptomycin)和10％热灭活胎牛血清(heat-inactivatedFBS，Gibco商品型号10437号)的RPMI-1640(Invitrogen)中培养。细胞在T-150 Falcon烧瓶中生长至饱和。随后，用胰蛋白酶处理细胞(胰蛋白酶0.25％-EDTA；Invitrogen)并将生长放大至足够的细胞数用于接种。根据制造商的操作准则(STEMCELL Technologies Inc.)，使用LymphoprepTM将从以肝素处理之血液(heparinized blood)中分离出外周血淋巴细胞(peripheral blood lymphocytes，PBMC)。将计数的细胞悬浮液进行组合，使得每只小鼠在PBS中单次推注(single bolus injection)0.1mL时，接受0.75×106个PBMC和3×106个肿瘤细胞。为了促进在小鼠内生长的肿瘤细胞，接着将另外0.1mL基质胶(matrigel)与组合细胞悬浮液混合，然后立即注射到制备小鼠。
对于每只小鼠，将0.2mL体积的组合细胞悬浮液皮下注射到动物的右侧腹中。接种14天后，形成实体瘤并达到约250至300mm3，并且每周两次持续三周以双特异性抗体(10mg/kg Anti-PD-L1-OX40 Ab-V5)、PD-L1参考抗体(Ref Ab,MPDL3280A)或对照组抗体(同种型抗体，Isotype)以腹腔内注射(intraperitoneal injection，i.p.)的方式进行应激(challenged)。在实验期间每周两次通过Pressier卡尺进行肿瘤与测试样品的测量，并且也记录体重。肿瘤体积使用以下算式计算：长度×宽度2×0.44＝体积(mm3)并绘制在图25中。在实验终止之前，肿瘤体积达到2000mm3或动物体重减轻20％的情况时将小鼠从研究中移除。当在接种后第7天测量肿瘤时观察到类似的结果，并且根据肿瘤体积随机分配动物。对于动物研究，每组包含6只小鼠。如图25中显示的数据，双特异性抗体在PC-3异种移植小鼠模型中显示出显著的抗肿瘤成效。肿瘤大小在接种后18天有缩小，相同的结果在施予PD-L1参考抗体中也有观察到，并继续降低至100mm3以下。PC-3异种移植小鼠模型初步证明了双特异性抗体的抗肿瘤作用，并揭示了其未来治疗药物的潜力。
总体来说，这些结果表示双特异性抗体在PD-1/PD-L1信号传导中维持其免疫检查点阻断，并对OX40信号传导具激动活性。正在进行的研究将使用适当的动物模型进一步研究这些蛋白质的生物活性，例如人源化NOD.Cg-Prkdcscid II2rgtmIwjI/SzJ(NSG)模型中的PC-3肿瘤。
本发明中的Fc区可以来自任何免疫球蛋白同种型(immunoglobulin isotypes)、亚类(subclasses)、同种异型(allotypes)、或工程化突变体(engineered mutants)，例如钮与扣的Fc片段(knob and hole Fc fragment)。
实施例
以下实施例描述了产生适合于将人类PD-L1和OX40作为目标与治疗目的的单克隆抗体。从抗PD-L1抗体克隆6与OX40抗体克隆B17分别产生组合的人类抗人类PD-L1和抗OX40抗体。人类可变区(V区)序列的区段源自于与人类抗体(生殖细胞和非生殖细胞，germlineand non-germline)无关的序列数据库。
实施例1产生与PD-L1和OX40结合的IgG抗体
本发明提供的特定抗体最初由Fabs与人类PD-L1或OX40结合产生。从噬菌体展示库(phage display library)：OmniMab噬菌粒库中选择Fab，接着在交替筛选相应的Fc融合蛋白(PD-L1-Fc或OX40-Fc)和表达人类相应蛋白(PD-L1或OX40)的细胞。在直接ELISA或基于细胞的ELISA(cell-based ELISA)筛选后，对阳性克隆的重链和轻链进行测序。这些Fab包括指定为PD-L1的「OM-PD-L1-6」和「OM-PD-L1-32」等；OX40的「OM-OX40-A4」、「OM-OX40-B17」和「OM-OX40-B19」等。本案公开的PD-L1抗体：PD-L1-克隆3、PD-L1-克隆6和PD-L1-克隆32，从HEK293细胞或CHO-S细胞中的「OM-PD-L1-6」和「OM-PD-L1-32」产生。在此期间，本案公开的OX40抗体：「OX40-A4」、「OX40-B17」及「OX40-B19」从HEK293细胞或CHO-S细胞中的「OM-OX40-A4」、「OM-OX40-B17」及「OM-OX40-B19」产生。同时将PD-L1和OX40作为目标的双特异性抗体被设计为抗PD-L1 6-OX40 scFv B17抗体与抗PD-L1 6-OX40 scFv B19抗体。所提供的Fab之轻链可变区和重链可变区的氨基酸序列，分别与轻链可变区和重链可变区的氨基酸序列相同。
实施例2抗PD-L1-OX40 scFv双特异性抗体与其相应目标的体外结合
如图14所示，建构抗PD-L1-OX40双特异性抗体并在HEK293细胞或CHO-S细胞中表达。藉由蛋白G层析从培养上清液中亲和纯化含有双特异性抗体的培养基。接着浓缩经纯化的抗体，然后在PBS缓冲液中透析并藉由SDS-PAGE分析如图15所示。为了在ELISA上测试纯化的融合蛋白与PD-L1或OX40的直接结合能力，以每孔100ng浓度的重组PD-L1或OX40涂覆在ELISA 96孔盘中。然后将各种浓度的纯化抗PD-L1-OX40抗体加入每个孔槽中并培养1小时。洗涤后，每个孔槽中加入1：5000稀释的抗Fab或辣根过氧化物酶接合物(JacksonImmunochemicals)并再培养1小时。最后洗涤后，加入TMB底物(Invitrogen Inc.)并测量450nm处的OD吸光值。使用GraphPad Prism 5通过S形曲线拟合(sigmoidal curvefitting)分析数据和计算EC50。
实施例3由FACS分析抗PD-L1-OX40 scFv的抗原结合特异性
为了测试抗PD-L1-OX40 scFv抗体结合特异性，稳定表达PD-L1的293细胞、IFN-γ刺激的A549、或WiDr(结肠直肠腺癌细胞株)以1μg/mL抗PD-L1-OX40 scFv抗体在冰上染色1小时，然后用1倍PBS洗涤三次。以488接合的山羊抗人类IgG(H+L)检测结合的抗体融合蛋白，然后进行FACS分析。同种型抗体为测试的阴性对照组。结果显示，与单独的抗PD-L1相比，抗PD-L1-OX40 scFv抗体维持其抗原结合特异性。还使用稳定表达OX40的293细胞测试了抗PD-L1-OX40 scFv抗体的结合特异性。
实施例4双功能性抗体的体外免疫调节作用
为了测量抗PD-L1-OX40 scFv抗体调节T细胞反应能力，将纯化的T细胞与异体的树突细胞一起培养，藉由在GM-CSF和IL-4中培养单核细胞几天来制备。设置平行孔盘以允许在第3天和第5天收集上清液，使用市售ELISA试剂盒分别测量IL-2和IFN-γ。Genentech/Roche的人源化抗PD-L1(MPDL3280A)将出现在预定孔盘中并作为阳性对照组。如图16A和16B所示，抗体施予3或5天后，双特异性抗体处理以及组合处理下IL-2和IFN-γ的含量显著提升。特别的是，由抗PD-L1克隆6抗体和抗OX40克隆B17抗体组成的双特异性抗体(抗PD-L1-OX40 scFv B17抗体)、或组合(抗PD-L1克隆6抗体和抗OX40克隆B17抗体)显示T细胞活化的增强情形高于PD-L1和OX40参照抗体的组合(PD-L1 Ref Ab加上OX40 Ref Ab)。这显示与参考OX40抗体GSK3174998相比，抗OX40 B17抗体可进行特异性表位(epitope)结合，从而产生更好的激动活性。
实施例5体内双特异性抗体诱导的人类白血球扩增
由于缺乏可检测PD-L1或OX40抗体与鼠PD-L1或OX40的交叉反应性(cross-reactivity)、和对人免疫细胞存在的要求，因此需要开发用于双特异性抗体之体内功能评估的模型。具有严重联合免疫缺陷(severe combined immunodeficient，SCID)突变和缺乏IL-2受体共同γ链(通常称为NSG)的NOD遗传背景的小鼠，能够支持大量人类外周白血球(human peripheral blood leukocytes，huPBL)的植入并维持至少30天(King等人，2008)。此小鼠模型，也称为huPBL-NSG模型，用于评估体内全身施用抗体对人免疫细胞的效果。
具体地，通过静脉内注射将600万新鲜分离的人类PBMC转移到huPBL-NSG小鼠中。PBMC注射9天后，通过腹腔内注射给动物并施用单一1mg/kg剂量之单克隆抗体、双特异性抗体或IgG4同种型对照抗体。在PBMC植入后第24至28天，通过流式细胞仪以人和鼠CD45抗体染色评估PBMC。前方和侧向散射轮廓用于确定框选淋巴细胞范围。双特异性抗体能够增强人类白血球的扩增，这可藉由移植小鼠外周血中人类CD45+细胞比例的增加来证明。每组具有n≥6只小鼠。
实施例6由抗PD-L1-OX40 scFv抗体对huPBL-NSG中PC-3或A498肿瘤细胞生长的抑制
属于PD-L1阳性的人类前列腺癌细胞株PC-3(#CRL-1435)或肾癌细胞株A498(/>HTB-44TM)，可用于huPBL-NSG小鼠中建立异种移植模型。对于肿瘤形成，如上所述，将每只小鼠3×106个PC-3细胞(或A498细胞)以皮下注射到huPBL-NSG小鼠中。为了评估对肿瘤生长的抑制作用，小鼠肿瘤细胞植入14天后，将每周两次持续4周以静脉注射不同浓度的抗PD-L1-OX40 scFv抗体、参考抗体、或0.1至3mg/kg的同种型抗体。如Fox ChaseBeige小鼠模型中所述，肿瘤生长将每周测量两次持续5周。
实施例7于小鼠和猴子中抗PD-L1-OX40 scFv的药物动力学评估
将10mg/kg至40mg/kg双功能蛋白(抗PD-L1-OX40 scFv)通过皮下注射或静脉注射施予小鼠或猴子。在注射后不同时间点采集血清样品至15天。使用三明治ELISA测定法测定血清样品中Fc融合蛋白的浓度。
虽然本发明已以实施方式公开如上，然其并非用以限定本发明，任何熟习此技艺者，在不脱离本发明之精神和范围内，当可作各种之更动与修饰，因此本发明之保护范围当视后附之权利要求书所界定者为准。
对于本领域技术人员将显而易见的是，在不脱离本发明的范围或精神的情况下，可以对本发明的结构进行各种修改和变型。鉴于前述内容，旨在使本发明涵盖本发明的修改和变型，使它们落入所附权利要求的范围内。
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<110> 圆祥生命科技股份有限公司
<120> 调节免疫检查点作为癌症治疗的单特异性与双特异性蛋白质
<130> 9022-1808584
<140> 2018800723187
<141> 2018-12-28
<150> US 62/611,543
<151> 2017-12-29
<160> 43
<170> PatentIn version 3.5
<210> 1
<211> 217
<212> PRT
<213> 智人 (Homo sapiens)
<400> 1
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Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu
85 90 95
Asn Ala Trp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205
Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 2
<211> 118
<212> PRT
<213> 智人 (Homo sapiens)
<400> 2
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 3
<211> 216
<212> PRT
<213> 智人 (Homo sapiens)
<400> 3
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Asn Asp Val Gly Ser Tyr
20 25 30
Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Val Ile Tyr Glu Val Ala Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser
85 90 95
Ser Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 4
<211> 121
<212> PRT
<213> 智人 (Homo sapiens)
<400> 4
Gln Val Gln Leu Val Gln Ser Gly Ala Lys Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Val Pro Gly Tyr Ser Tyr Gly Pro Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 5
<211> 214
<212> PRT
<213> 智人 (Homo sapiens)
<400> 5
Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Lys
1 5 10 15
Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe
35 40 45
Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His
85 90 95
Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys
100 105 110
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
115 120 125
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly
130 135 140
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly
145 150 155 160
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala
165 170 175
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser
180 185 190
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
195 200 205
Ala Pro Thr Glu Cys Ser
210
<210> 6
<211> 126
<212> PRT
<213> 智人 (Homo sapiens)
<400> 6
Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala Leu Asp Ala
100 105 110
Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 7
<211> 214
<212> PRT
<213> 智人 (Homo sapiens)
<400> 7
Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Lys
1 5 10 15
Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe
35 40 45
Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His
85 90 95
Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys
100 105 110
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
115 120 125
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly
130 135 140
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly
145 150 155 160
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala
165 170 175
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser
180 185 190
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
195 200 205
Ala Pro Ala Glu Cys Ser
210
<210> 8
<211> 118
<212> PRT
<213> 智人 (Homo sapiens)
<400> 8
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Ser Ser Ser Arg Tyr Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr 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 Tyr Cys
85 90 95
Ala Arg Gln Asn Glu Asp Val Ala Phe Gly Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 9
<211> 327
<212> PRT
<213> 智人 (Homo sapiens)
<400> 9
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser 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 Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 10
<211> 246
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> OX40 scFv (克隆A4)
<400> 10
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Glu Val Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser
85 90 95
Asn Asn Leu Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Ser Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
130 135 140
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Glu
145 150 155 160
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
165 170 175
Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys
180 185 190
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu
195 200 205
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
210 215 220
Arg Gly Gly Val Gly Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met
225 230 235 240
Val Thr Val Ser Ser Ala
245
<210> 11
<211> 250
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> OX40 scFv (克隆B17)
<400> 11
Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Lys Thr
1 5 10 15
Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser
20 25 30
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr
35 40 45
Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn
50 55 60
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp
65 70 75 80
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val
85 90 95
Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Ser Ser Gly Gly Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
115 120 125
Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
130 135 140
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
145 150 155 160
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Leu Ile Ser Trp
165 170 175
Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
180 185 190
Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser
195 200 205
Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ser Ile Tyr Asp
210 215 220
Pro Pro Pro Gly Ser Arg Ser Ala Leu Asp Ala Phe Asp Ile Trp Gly
225 230 235 240
Gln Gly Thr Met Val Thr Val Ser Ser Ala
245 250
<210> 12
<211> 243
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> OX40 scFv (克隆B19)
<400> 12
Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Lys
1 5 10 15
Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe
35 40 45
Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His
85 90 95
Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Ser Ser Gly Gly Gly
100 105 110
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
115 120 125
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu
130 135 140
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn Trp
145 150 155 160
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser
165 170 175
Ser Ser Ser Arg Tyr Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
180 185 190
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn
195 200 205
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Asn
210 215 220
Glu Asp Val Ala Phe Gly Ile Trp Gly Gln Gly Thr Met Val Thr Val
225 230 235 240
Ser Ser Ala
<210> 13
<211> 241
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> OX40 scFv (克隆B120)
<400> 13
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Thr Leu Gly Asp Lys Tyr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Arg Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ser Tyr
85 90 95
Val Phe Gly Thr Gly Thr Lys Val Thr Val Ser Ser Gly Gly Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln
115 120 125
Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr
130 135 140
Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser Trp Ile
145 150 155 160
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His
165 170 175
Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile
180 185 190
Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val
195 200 205
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Trp Gly
210 215 220
Gly Ser Phe Ile Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235 240
Ala
<210> 14
<211> 10
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 多肽连接子 (peptide linker)
<400> 14
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 15
<211> 20
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 信号肽 (signal peptide)
<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
20
<210> 16
<211> 704
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-克隆6重链-OX40克隆B17 scFv抗体
<400> 16
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly
580 585 590
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
595 600 605
Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
610 615 620
Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
625 630 635 640
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu
645 650 655
Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr
660 665 670
Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala Leu Asp
675 680 685
Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala
690 695 700
<210> 17
<211> 696
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-克隆6重链-OX40克隆B19 scFv抗体
<400> 17
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
565 570 575
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly
580 585 590
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
595 600 605
Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
610 615 620
Val Ser Ser Ile Ser Ser Ser Ser Arg Tyr Ile Tyr Tyr Ala Asp Ser
625 630 635 640
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
645 650 655
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
660 665 670
Cys Ala Arg Gln Asn Glu Asp Val Ala Phe Gly Ile Trp Gly Gln Gly
675 680 685
Thr Met Val Thr Val Ser Ser Ala
690 695
<210> 18
<211> 707
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V1
<400> 18
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser
565 570 575
Arg Ser Ser Leu Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val
580 585 590
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
595 600 605
Phe Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
610 615 620
Leu Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr
625 630 635 640
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
645 650 655
Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala
660 665 670
Leu Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser
675 680 685
Ala Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val
690 695 700
Ser Ser Ala
705
<210> 19
<211> 708
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V2
<400> 19
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Gly Lys Gly Ser Gly Gly Lys Gly Thr Gly Gly Lys Gly Ser
565 570 575
Gly Gly Lys Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Val Val
580 585 590
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
595 600 605
Thr Phe Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys
610 615 620
Gly Leu Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr
625 630 635 640
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
645 650 655
Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr
660 665 670
Ala Leu Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg
675 680 685
Ser Ala Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr
690 695 700
Val Ser Ser Ala
705
<210> 20
<211> 701
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V3
<400> 20
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Glu Val
565 570 575
Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu
580 585 590
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met
595 600 605
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Leu
610 615 620
Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly
625 630 635 640
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln
645 650 655
Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ser
660 665 670
Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala Leu Asp Ala Phe Asp
675 680 685
Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala
690 695 700
<210> 21
<211> 706
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V4
<400> 21
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Lys Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
565 570 575
Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln
580 585 590
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
595 600 605
Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
610 615 620
Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala
625 630 635 640
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
645 650 655
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
660 665 670
Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala
675 680 685
Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
690 695 700
Ser Ala
705
<210> 22
<211> 706
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V5
<400> 22
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Gln Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
565 570 575
Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln
580 585 590
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
595 600 605
Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
610 615 620
Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala
625 630 635 640
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
645 650 655
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
660 665 670
Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala
675 680 685
Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
690 695 700
Ser Ala
705
<210> 23
<211> 706
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V6
<400> 23
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Glu Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
565 570 575
Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
580 585 590
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
595 600 605
Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
610 615 620
Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala
625 630 635 640
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
645 650 655
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
660 665 670
Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala
675 680 685
Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
690 695 700
Ser Ala
705
<210> 24
<211> 706
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V7
<400> 24
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Gln Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
565 570 575
Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
580 585 590
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
595 600 605
Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
610 615 620
Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala
625 630 635 640
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
645 650 655
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
660 665 670
Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala
675 680 685
Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
690 695 700
Ser Ala
705
<210> 25
<211> 706
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V8
<400> 25
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Ala Pro Gly Lys Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
565 570 575
Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Ile Val Val Gln
580 585 590
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
595 600 605
Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
610 615 620
Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala
625 630 635 640
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
645 650 655
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
660 665 670
Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala
675 680 685
Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
690 695 700
Ser Ala
705
<210> 26
<211> 706
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V9
<400> 26
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Ala Pro Gly Lys Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
565 570 575
Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
580 585 590
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
595 600 605
Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
610 615 620
Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala
625 630 635 640
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
645 650 655
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
660 665 670
Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala
675 680 685
Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
690 695 700
Ser Ala
705
<210> 27
<211> 706
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V10
<400> 27
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
565 570 575
Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
580 585 590
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
595 600 605
Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
610 615 620
Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala
625 630 635 640
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
645 650 655
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
660 665 670
Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala
675 680 685
Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
690 695 700
Ser Ala
705
<210> 28
<211> 706
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V11
<400> 28
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Glu Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
565 570 575
Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln
580 585 590
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
595 600 605
Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
610 615 620
Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala
625 630 635 640
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
645 650 655
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
660 665 670
Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala
675 680 685
Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
690 695 700
Ser Ala
705
<210> 29
<211> 706
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V12
<400> 29
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Arg Tyr
20 25 30
Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Val Phe Gly Ala Ala Lys Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Phe Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Ser Gly Asp Ser Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
450 455 460
Val Thr Pro Gly Gln Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu
465 470 475 480
Glu Ser Lys Ile Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
485 490 495
Val Leu Val Ile Phe Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu
500 505 510
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
515 520 525
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
530 535 540
Ser Asn Ser Asp His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
545 550 555 560
Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
565 570 575
Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln
580 585 590
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
595 600 605
Asp Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
610 615 620
Glu Trp Val Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala
625 630 635 640
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
645 650 655
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu
660 665 670
Tyr Tyr Cys Ala Ser Ile Tyr Asp Pro Pro Pro Gly Ser Arg Ser Ala
675 680 685
Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser
690 695 700
Ser Ala
705
<210> 30
<211> 176
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V4
<400> 30
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val
1 5 10 15
Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Lys Thr Ala Arg
20 25 30
Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr
50 55 60
Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly
65 70 75 80
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
85 90 95
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe
100 105 110
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly
115 120 125
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
165 170 175
<210> 31
<211> 176
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V5
<400> 31
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Glu
1 5 10 15
Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Gln Thr Ala Arg
20 25 30
Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr
50 55 60
Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly
65 70 75 80
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
85 90 95
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe
100 105 110
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly
115 120 125
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
165 170 175
<210> 32
<211> 176
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V6
<400> 32
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val
1 5 10 15
Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Glu Thr Ala Arg
20 25 30
Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr
50 55 60
Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly
65 70 75 80
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
85 90 95
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Val Phe
100 105 110
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly
115 120 125
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
165 170 175
<210> 33
<211> 176
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V7
<400> 33
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val
1 5 10 15
Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Gln Thr Ala Arg
20 25 30
Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr
50 55 60
Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly
65 70 75 80
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
85 90 95
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Val Phe
100 105 110
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly
115 120 125
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
165 170 175
<210> 34
<211> 176
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V8
<400> 34
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val
1 5 10 15
Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys Thr Ala Arg
20 25 30
Ile Thr Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr
50 55 60
Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly
65 70 75 80
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
85 90 95
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe
100 105 110
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly
115 120 125
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Ile Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
165 170 175
<210> 35
<211> 176
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V9
<400> 35
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val
1 5 10 15
Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys Thr Ala Arg
20 25 30
Ile Thr Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr
50 55 60
Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly
65 70 75 80
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
85 90 95
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Val Phe
100 105 110
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly
115 120 125
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
165 170 175
<210> 36
<211> 176
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V10
<400> 36
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val
1 5 10 15
Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg
20 25 30
Ile Thr Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr
50 55 60
Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly
65 70 75 80
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
85 90 95
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Val Phe
100 105 110
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly
115 120 125
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
165 170 175
<210> 37
<211> 176
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V11
<400> 37
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val
1 5 10 15
Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Glu Thr Ala Arg
20 25 30
Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr
50 55 60
Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly
65 70 75 80
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
85 90 95
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe
100 105 110
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly
115 120 125
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
165 170 175
<210> 38
<211> 176
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 抗PD-L1-OX40 Ab-V12
<400> 38
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Val
1 5 10 15
Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Gln Thr Ala Arg
20 25 30
Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val Ser Trp Tyr
35 40 45
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe Tyr Asp Thr
50 55 60
Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser Asn Ser Gly
65 70 75 80
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
85 90 95
Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His Val Ile Phe
100 105 110
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly
115 120 125
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Thr Met His Trp Val
165 170 175
<210> 39
<211> 15
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 连接子 (linker)
<400> 39
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 40
<211> 19
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 连接子 (linker)
<400> 40
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg
1 5 10 15
Ser Ser Leu
<210> 41
<211> 20
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 连接子 (linker)
<400> 41
Gly Gly Lys Gly Ser Gly Gly Lys Gly Thr Gly Gly Lys Gly Ser Gly
1 5 10 15
Gly Lys Gly Ser
20
<210> 42
<211> 13
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 连接子 (linker)
<400> 42
Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser
1 5 10
<210> 43
<211> 18
<212> PRT
<213> 人工序列 (Artificial Sequence)
<220>
<223> 连接子 (linker)
<400> 43
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly

Claims (30)

1.一种与OX40结合的抗体或其抗原结合部分，其特征在于，包含：
一重链可变区，其序列如SEQ ID NO.6所示，以及一轻链可变区，其序列如SEQ ID NO.5的第1-108位氨基酸所示；
一重链可变区，其序列如SEQ ID NO.8所示，以及一轻链可变区，其序列如SEQ ID NO.7的第1-108位氨基酸所示；或
一重链可变区，其序列如SEQ ID NO.10的第128-246位氨基酸所示，以及一轻链可变区，其序列如SEQ ID NO.10的第1-112位氨基酸所示。
2.如权利要求1所述之抗体或其抗原结合部分，其中该抗体或其抗原结合部分系选自于由SEQ ID NOs.10、11及12所组成之群组的单链可变片段序列。
3.如权利要求1所述之抗体或其抗原结合部分，其中该抗体或其抗原结合部分是双特异性抗体。
4.如权利要求3所述之抗体或其抗原结合部分，其中该双特异性抗体包含一免疫检查点蛋白结合位点。
5.如权利要求4所述之抗体或其抗原结合部分，其中该免疫检查点蛋白结合位点包含一细胞程序死亡-配体1结合位点、细胞程序死亡-配体1结合位点、表皮生长因子受体(EGFR)结合位点、人类表皮生长因子受体2结合位点、细胞毒性T淋巴细胞相关抗原4结合位点或淋巴细胞活化基因3结合位点。
6.一种与PD-L1结合的抗体或其抗原结合部分，其特征在于，包含：
一重链可变区，其序列如SEQ ID NO.2所示，以及一轻链可变区，其序列如SEQ ID NO.1的第1-111位氨基酸所示；或
一重链可变区，其序列如SEQ ID NO.4所示，以及一轻链可变区，其序列如SEQ ID NO.3的第1-110位氨基酸所示。
7.一种双特异性抗体，包含至少一多肽链，其特征在于，其中该多肽链包含：
一OX40结合位点，具有：
一重链可变区，该重链可变区的氨基酸序列如SEQ ID NO.6所示；以及
一轻链可变区，该轻链可变区的氨基酸序列如SEQ ID NO.5的第1-108位氨基酸所示；以及
一PD-L1结合位点，具有：
一重链可变区，该重链可变区的氨基酸序列如SEQ ID NO.2所示；以及
一轻链可变区，该轻链可变区的氨基酸序列如SEQ ID NO.1的第1-111位氨基酸所示。
8.如权利要求7所述之双特异性抗体，其中该多肽链具有：
一F c区；
一F ab片段，与该F c区的N端连接，且该F ab片段包含该P D-L 1结合位点；以及
一s cFv，与该F c区的C端连接，且该s cFv包含该OX40结合位点。
9.如权利要求8所述之双特异性抗体，其中该多肽链更包含一连接子界于该F c区和该s cFv之间。
10.如权利要求9所述之双特异性抗体，其中该s cFv包含一氨基酸序列系选自于由SEQ ID NO.18的第455-707位氨基酸、S EQ ID NO.19的第455-708位氨基酸、S EQ ID NO.20的第455-701位氨基酸、S EQ ID NO.21的第455-706位氨基酸、S EQ ID NO.22的第455-706位氨基酸、S EQ ID NO.23的第455-706位氨基酸、S EQ ID NO.24的第455-706位氨基酸、SEQ ID NO.25的第455-706位氨基酸、S EQ ID NO.26的第455-706位氨基酸、S EQ ID NO.27的第455-706位氨基酸、S EQ ID NO.28的第455-706位氨基酸、及S EQ ID NO.29的第455-706位氨基酸所组成之群组。
11.如权利要求7所述之双特异性抗体，其中该双特异性抗体包含一对多肽链。
12.如权利要求11所述之双特异性抗体，其中该双特异性抗体为Ig G、IgE、IgM、IgD、IgA或IgY抗体。
13.如权利要求12所述之双特异性抗体，其中该双特异性抗体为IgG抗体。
14.如权利要求13所述之双特异性抗体，其中该IgG抗体为IgG1、IgG2、IgG3或IgG4抗体。
15.一种抗体药物复合体，其特征在于，包含：
一治疗剂；以及
一抗体或其抗原结合部分，其系与PD-L1和/或OX40结合，其中该治疗剂藉由一连接子与该抗体或其抗原结合部分共价接合，其中该抗体或其抗原结合部分选自于以下所组成之群组选择其一：
a)与OX40结合的抗体或其抗原结合部分，具有：
一重链可变区，其序列如SEQ ID NO.6所示，以及一轻链可变区，其序列如SEQ ID NO.5的第1-108位氨基酸所示；
一重链可变区，其序列如SEQ ID NO.8所示，以及一轻链可变区，其序列如SEQ ID NO.7的第1-108位氨基酸所示；或
一重链可变区，其序列如SEQ ID NO.10的第128-246位氨基酸所示，以及一轻链可变区，其序列如SEQ ID NO.10的第1-112位氨基酸所示；
b)与PD-L1结合的抗体或其抗原结合部分，具有：
一重链可变区，其序列如SEQ ID NO.2所示，以及一轻链可变区，其序列如SEQ ID NO.1的第1-111位氨基酸所示；或
一重链可变区，其序列如SEQ ID NO.4所示，以及一轻链可变区，其序列如SEQ ID NO.3的第1-110位氨基酸所示；以及
c)一双特异性抗体，包含至少一多肽链，其中该多肽链包含：
一OX40结合位点，具有：
一重链可变区，该重链可变区的氨基酸序列如SEQ ID NO.6所示；以及
一轻链可变区，该轻链可变区的氨基酸序列如SEQ ID NO.5的第1-108位氨基酸所示；以及
一PD-L1结合位点，具有：
一重链可变区，该重链可变区的氨基酸序列如SEQ ID NO.2所示；以及
一轻链可变区，该轻链可变区的氨基酸序列如SEQ ID NO.1的第1-111位氨基酸所示。
16.一种医药组成物，其特征在于，包含：
一如权利要求1至5中任一项所述之抗体或其抗原结合部分；以及
至少一药学上可接受的载剂。
17.一种医药组成物，其特征在于，包含：
一如权利要求6所述之抗体或其抗原结合部分；
以及
至少一药学上可接受的载剂。
18.一种医药组成物，其特征在于，包含：
一如权利要求7至14中任一项所述之双特异性抗体；以及
至少一药学上可接受的载剂。
19.一种如权利要求1至5中任一项所述之抗体或其抗原结合部分在制备治疗癌症的药物中的用途。
20.如权利要求19所述之用途，其中该癌症系选自于由前列腺癌、肺癌、黑色素瘤、淋巴瘤、乳癌、头颈癌、肾细胞癌和卵巢癌所组成之群组。
21.如权利要求20所述之用途，其中该肺癌为非小细胞肺癌。
22.一种如权利要求6所述之抗体或其抗原结合部分在制备治疗癌症的药物中的用途。
23.如权利要求22所述之用途，其中该癌症系选自于由前列腺癌、肺癌、黑色素瘤、淋巴瘤、乳癌、头颈癌、肾细胞癌和卵巢癌所组成之群组。
24.如权利要求23所述之用途，其中该肺癌为非小细胞肺癌。
25.一种如权利要求7至14中任一项所述之该双特异性抗体在制备治疗癌症的药物中的用途。
26.如权利要求25所述之用途，其中该癌症系选自于由前列腺癌、肺癌、黑色素瘤、淋巴瘤、乳癌、头颈癌、肾细胞癌和卵巢癌所组成之群组。
27.如权利要求26所述之用途，其中该肺癌为非小细胞肺癌。
28.一种核酸分子，其特征在于，其编码如权利要求1至5中任一项所述之抗体或其抗原结合部分。
29.一种核酸分子，其特征在于，其编码如权利要求6所述之抗体或其抗原结合部分。
30.一种核酸分子，其特征在于，其编码如权利要求7至14中任一项所述之双特异性抗体。
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