CN113621065A - 靶向4-1bb的全人源抗体及其制备方法和应用 - Google Patents
靶向4-1bb的全人源抗体及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及靶向4‑1BB的全人源抗体及其制备方法和应用，属于生物技术领域。提供了两种特异性结合人4‑1BB的重组单克隆抗体及其制备方法与应用，还提供了编码所述抗体的核酸分子。本发明通过重组抗体技术，从噬菌体展示全人源抗体文库中获得了两种能特异性结合人4‑1BB、且具有较好的T细胞激活作用的抗体。所述抗体在制备通过激活4‑1BB以增强机体免疫能力的肿瘤免疫治疗药物及联合药物开发方面有良好的应用前景。
Description
技术领域
本发明属于生物技术领域，更具体地，本发明涉及两种靶向4-1BB的全人源抗体及其制备方法和应用。
背景技术
4-1BB又名CD137，是一种分子量约为30kDa的I型跨膜糖蛋白分子，属于肿瘤坏死因子受体家族成员(TNFR)，由肿瘤坏死因子受体超家族成员9(TNFRSF9)基因编码，是T细胞表面一种重要的激活型免疫检查点分子。在T细胞和NK细胞等被激活后，4-1BB会有很高的表达，能够活化细胞毒性T细胞和记忆T细胞，促进T细胞增殖和存活，有助于对肿瘤细胞进行杀伤。人4-1BB全长255个氨基酸，包含23aa信号肽(ss)、163aa的胞外区(ECD)、27aa的跨膜区域(187-213)和42aa的胞内区域(ICD)。除了表达于CD4+和CD8+T细胞表面，4-1BB也可表达于NK细胞、NKT细胞、树突状细胞(DC)、Tregs和部分B细胞等。
4-1BB的配体为4-1BBL(又被称为CD137L)，是一种TNF家族的II型跨膜蛋白分子，表达于各种活化的抗原递呈细胞(APC)表面，如IFN-γ活化的巨噬细胞、CD40配体活化的B细胞、单核细胞、树突状细胞等。
4-1BB/4-1BBL是除CD28/B7之外的另一组重要的共刺激分子，为T细胞活化提供第二信号，它们的协同刺激信号主要作用于活化后期。4-1BB与4-1BBL作用模式属于经典的TNF-TNFR结合模式，三个4-1BB单体分子(mono-4-1BB)结合三聚体化的4-1BBL，达到成簇的目的，促进后续信号通路的传导。采用激活型的抗体，也可以实现4-1BB成簇目的。这种结合可以导致TNFR相关因子TRAF1和TRAF2的快速招募，TRAF介导的NF-kB和MAPK细胞内信号的激活导致T细胞分裂和增殖，增加细胞存活率，增强CD4+和CD8+T细胞的效应器功能。此外，4-1BB还能够刺激T细胞分泌IL-2、IFN-γ和TNF-α等细胞因子，并通过与CD28共刺激分子的协同作用减少免疫调节因子IL-4和TGF-β的产生。
鉴于4-1BB对T细胞的激活的功能，开发4-1BB的激活型抗体可以激活T细胞，达到肿瘤免疫治疗的目的。百时美施贵宝的Urelumab和辉瑞的Utomilumab两种抗体在临床研究阶段的进展相对较快。Urelumab(BMS-663513)是一种IgG4单克隆抗体，有着令人鼓舞的抗肿瘤活性，但是显示出较严重的副作用(肝脏毒性、血小板减少等)。Utomilumab(PF-05082566)是人源化IgG2单克隆抗体，比Urelumab的激动活性弱，具有良好的安全性，但是抗肿瘤活性相对较弱。因此，开发出安全性和有效性更好的4-1BB抗体，并在药物设计和联合用药方面进一步探索，对通过调节机体免疫力的肿瘤治疗具有积极意义。
发明内容
本发明的目的在于提供两种靶向4-1BB的全人源抗体及其制备方法和应用。
在一个方面，本发明涉及两种分离的单克隆抗体(例如，人抗体)，或其抗原结合部分，其具有包含CDR1区、CDR2区和CDR3区的重链可变区，其中CDR1区、CDR2区和CDR3区包含与下述具有至少80％、85％、90％、95％、98％、99％或100％同一性的氨基酸序列
(1)分别为SEQ ID NO:1、2和3；或
(2)分别为SEQ ID NO:4、5和6；
其中所述抗体或其抗原结合片段结合4-1BB。这些氨基酸序列可分别由SEQ IDNO：17至22的核酸序列编码。
在一个方面，本发明的分离的单克隆抗体(例如，人抗体)或其抗原结合部分包含重链可变区，所述重链可变区包含与SEQ ID NO：13和14中的任一个具有至少80％、85％、90％、95％、98％、99％或100％同一性的氨基酸序列，其中所述抗体或其抗原结合片段结合4-1BB。这些氨基酸序列可以分别由SEQ ID NO：29和30的核酸序列编码。
在一个实施方式中，本发明的单克隆抗体或其抗原结合部分包含轻链可变区，所述轻链可变区包含CDR1区、CDR2区和CDR3区，其中所述CDR1区、CDR2区和CDR3区包含与下述具有至少80％、85％、90％、95％、98％、99％或100％同一性的氨基酸序列
(1)分别为SEQ ID NO:7、8和9；或
(2)分别为SEQ ID NO:10、11和12；
其中所述抗体或其抗原结合片段结合4-1BB。这些氨基酸序列可以分别由SEQIDNO：23至28的核酸序列编码。
在一个方面，本发明的分离的单克隆抗体(例如，人抗体)或其抗原结合部分包含轻链可变区，所述轻链可变区包含与SEQ ID NO：15和16中的任一个具有至少80％、85％、90％、95％、98％、99％或100％同一性的氨基酸序列，其中所述抗体或其抗原结合片段结合4-1BB。这些氨基酸序列可以分别由SEQ ID NO：31和32的核酸序列编码。
在一个方面，本发明的分离的单克隆抗体或其抗原结合部分包含各自含有CDR1区、CDR2区和CDR3区的重链可变区和轻链可变区，其中所述重链可变区CDR1、CDR2和CDR3以及所述轻链可变区CDR1、CDR2和CDR3包含(1)分别与SEQ ID NO：1、2、3、7、8和9；或(2)分别与SEQ ID NO：4、5、6、10、11和12具有至少80％、85％、90％、95％、98％、99％或100％同一性的氨基酸序列，其中所述抗体或其抗原结合片段结合4-1BB。
本发明在一个实施方案中，抗体或其抗原结合部分包含重链可变区和轻链可变区，重链可变区和轻链可变区包含与以下具有至少80％、85％、90％、95％、98％、99％或100％同一性的氨基酸序列：(1)分别为SEQ ID NO：13和15；(2)分别为SEQ ID NO：14和16；其中抗体或其抗原结合片段结合4-1BB。
本发明的有益效果是：与现有技术的抗4-1BB抗体诸如Utomilumab相比，本发明的抗体或其抗原结合部分具有相当的或甚至更好的对人4-1BB的结合亲和力/能力，能够有效阻断4-1BB与配体4-1BBL的结合，能够刺激人外周血淋巴细胞增殖和分泌更高水平的IFN-γ细胞因子。这些实验结果表明，本发明提供的抗体或其抗原结合部分，在开发调节4-1BB对机体免疫激活的药物、激活T淋巴细胞的药物方向有良好的应用前景。
附图说明
图1为在单噬菌体ELISA实验中抗体8F5和13G4对人4-1BB和对照BSA的结合情况。抗体8F5和13G4对人4-1BB和对照BSA的光吸收值表明筛选到的两个抗体能特异性结合人4-1BB。
图2为抗体8F5和13G4对不同种属4-1BB的结合情况。IgG抗体8F5和13G4能结合hu4-1BB和cyno4-1BB蛋白，不能结合mouse4-1BB蛋白。
图3为4-1BB抗体阻断配体4-1BBL与4-1BB的结合情况。
图4为抗体8F5特异性结合细胞表面表达的4-1BB蛋白。
图5为抗体13G4特异性结合细胞表面表达的4-1BB蛋白。
图6为8F5和13G4比Utomilumab具有更好的刺激CD4+T细胞分泌IFN-γ细胞因子活性。
具体实施方式
为了可以更容易地理解本发明，首先定义某些术语。在整个详细描述中阐述了另外的定义。
术语“抗体”是指表现所需生物学活性的抗体的任何形式，通常包含完整抗体IgG或抗体片段。“抗体片段”和“抗原结合片段”是指抗体的抗原结合片段及抗体片段，其通常包括至少部分母抗体的抗原结合区或可变区。抗体片段保留亲本抗体的至少某些结合特异性。抗体片段的例子包括但不限于：scFv、Fab、Fab′、F(ab′)2等。
“单链Fv抗体”(或“scFv抗体”)是指包含抗体的VH和VL结构域的抗体片段，这些结构域存在于一条多肽链中。一般而言，Fv多肽在VH和VL结构域之间包含额外的多肽接头，该接头使得scFv能形成用于抗原结合的所需结构。“Fab′片段”含有一条轻链和包含VH结构域和CH1结构域及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′)2分子。“Fc”区通常指含有抗体的CH2和CH3结构域的两个重链片段。
“全人源抗体”为其氨基酸序列对应于由人产生的抗体的氨基酸序列的抗体，和/或业已用任何用于制备本文所示的人抗体的技术制备的抗体。该定义明确排除了包含非人类抗原结合残基的人源化抗体。
术语“EC50”也被称为半数最大有效浓度，指的是在指定的暴露时间之后引起基线与最大值之间的一半响应的抗体浓度。
术语″IC50″也称为半数最大抑制浓度，是指相对于抗体的缺失，抑制特定生物或生物化学功能50％的抗体的浓度。
术语“抗体衍生物”指的是抗体的任何修饰形式，例如抗体与另一种药剂或抗体的缀合物。
术语“药物组合物”指用于人的药物制剂。该药物组合物包含本发明的抗体或其抗原结合片段以及载体、稳定剂和/或赋形剂的合适制剂。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1结合4-1BB的抗体的产生
利用噬菌体展示技术，从HSA(TM)全人源天然抗体库中筛选人4-1BB胞外区蛋白(hu4-1BB ECD，以下简称hu4-1BB)特异性抗体。为此目的，在400ml2×YT/氨苄青霉素培养基接种噬菌体展示全人源单链抗体天然库的甘油菌，使细胞密度达到OD600＝0.1，在37℃和200rpm条件下振荡培养直至细胞密度达到OD600＝0.5。用1012pfu的M13KO7辅助噬菌体(购自Invitrogen)感染和培养30分钟。加入50mg/l卡那霉素后在37℃和200rpm条件下振荡培养30分钟后，通过离心分离沉淀，重悬于400ml 2×YT/氨苄青霉素/卡那霉素培养基，在37℃和200rpm条件下振荡培养16小时。最后细胞通过离心(20分钟，5000×g，4℃)分离沉淀并丢弃，上清用0.45μm规格滤膜过滤后，加入1/4体积20％(w/v)PEG8000、2.5M NaCl溶液并在冰浴中保温1小时沉淀噬菌体颗粒。随后离心沉淀(20分钟，8000×g，4℃)，弃上清，将噬菌体重悬于25ml预冷PBS(137mM NaCl，2.7mM KCl，8mM Na2HPO4，2mM KH2PO4)中，离心(5分钟，20000×g，4℃)。向上清液加入1/4体积20％(w/v)PEG8000、2.5M NaCl溶液，并冰浴30分钟再次沉淀噬菌体颗粒。离心沉淀(30分钟，20000×g，4℃)，再次将噬菌体沉淀重悬于2ml预冷PBS中，在冰上保持30分钟并离心(30分钟，17000×g，4℃)。上清液与含4％(w/v)BSA的PBS溶液以1:1混合，置于旋转混合器上，室温下保温30分钟，然后直接用于筛选。
利用上述噬菌体抗体库，针对生物素标记的人4-1BB重组蛋白，实施了四轮定向筛选，筛选方案如下：该噬菌体抗体库与生物素标记的抗原4-1BB，在室温下保温2小时，然后与经封闭液2％(w/v)BSA封闭过的链霉亲和素磁珠MyOne C1在室温下保温30分钟。随后用PBST(含0.1％吐温-20)缓冲液洗涤磁珠,除去非特异性结合或结合能力较弱的噬菌体。结合能力强的噬菌体，则用甘氨酸-盐酸(pH 2.2)从磁珠上洗脱下来，用Tris中和液(pH 9.1)中和后，用于感染处于对数生长中期的大肠杆菌ER2738，并被用于下一轮筛选。在四轮筛选中，磁珠的用量分别为50μl、20μl、10μl和10μl，生物素标记的抗原4-1BB浓度分别为200nM、10nM、5nM和1nM,PBST的洗涤次数分别为10次、10次、15次和20次。
从第四轮筛选所得的克隆中随机挑选96个，并用单噬菌体ELISA(酶联免疫吸附实验)分析其与人4-1BB的结合能力。为此目的，每个单菌落接种300μl2×YT/氨苄青霉素培养基(含2％葡萄糖)于96孔深孔培养板，并在37℃和250rpm下振荡培养16小时。用20μl培养物接种到500μl 2×YT/氨苄青霉素培养基(含0.1％葡萄糖)，在37℃和250rpm下振荡培养1.5小时。准备辅助噬菌体溶液，取75μl的M13KO7(滴度为3×1012pfu/ml)混入到15ml 2×YT培养基中，50μl/孔加到培养板中。在37℃和150rpm条件培养30分钟，然后加入准备好的卡那霉素溶液50μl/孔(取180μl的50mg/ml卡那霉素，加入到15ml 2×YT培养基)，在37℃和250rpm下振荡培养16小时。最后离心沉淀细胞(30分钟，5000×g，4℃)，将上清转移到新的96孔深孔培养板。
为进行单噬菌体ELISA，在96孔MediSorp ELISA板(购自Nunc)上分别使用100ng/well抗原4-1BB以及阴性对照蛋白BSA(100μl/孔)，在4℃包被过夜。每个孔用含2％BSA(w/v)的PBST封闭。随后用PBST清洗孔3次并排净。然后加入100μl/孔上面制备的每种噬菌体溶液到板上各孔中。37℃保温2小时后，用PBST洗涤3次。为了检测结合的噬菌体，将抗M13抗体过氧化物歧化酶偶联物(购自GE Healthcare)以1:5000稀释于PBST中，并取100μl加到每个孔中。37℃保温1小时后用PBST漂洗孔3次，然后用PBS漂洗3次。最后吸取50μl TMB底物加入到孔中，并在室温下显色10分钟，随后加入每孔50μl的2M H2SO4终止显色反应。用酶联免疫检测仪(Bio-Rad)在450nm测量消光值。结合测序分析，观察到多个不同的抗体克隆，其中两个抗体(scFv)8F5(SEQ ID NO：33(核苷酸)，34(氨基酸))和13G4(SEQ ID NO：35(核苷酸)，36(氨基酸))，在ELISA实验中对人4-1BB结合信号较强，对BSA无结合(图1)，且在随后的分析种表现出配体4-1BBL抑制活性和对4-1BB的激活活性。
实施例2 4-1BB抗体对不同种属蛋白的结合分析
将上述两个抗体分别构建成human IgG2完整抗体，通过转染CHO悬浮细胞进行表达，随后通过Protein A进行亲和纯化，获得纯化抗体，用于下述分析鉴定。通过在4℃孵育过夜将人4-1BB、cyno4-1BB、mouse 4-1BB蛋白固定到96孔板上。然后将所述板通过在37℃与于含2％BSA的PBS一起孵育2小时来封闭。在封闭之后，将所述板用PBST(含有0.05％Tween20的PBS)洗涤3次。用结合缓冲液(含有0.05％Tween20和1％BSA的PBS)稀释抗4-1BB抗体至500ng/ml，并且在37℃将其与固定的蛋白质一起孵育1小时。在结合之后，将所述板用PBST洗涤3次，在37℃与在结合缓冲液中1/15,000稀释的过氧化物酶标记的驴抗人IgG((JacksonIm.Research)一起孵育1小时，再次洗涤，用TMB显影并且使用2M H2SO4停止反应。两种抗体与三种不同种属的4-1BB蛋白的结合如图2所示。结果表明，两种抗体均可以特异性结合hu4-1BB和cyno4-1BB，而不能结合mouse4-1BB。
实施例3 4-1BB抗体阻断4-1BBL的结合活性分析
为了检测两种4-1BB抗体是否能够阻断配体4-1BBL对4-1BB的结合，进行了以下抑制ELISA实验。通过在4℃孵育过夜将人4-1BBL配体蛋白固定到96孔板上。然后将所述板孔通过在37℃与于含2％BSA的PBS一起孵育2小时来封闭。在封闭之后，将所述板用PBST(含有0.05％Tween20的PBS)洗涤3次。用结合缓冲液(含有0.05％Tween20和1％BSA的PBS)稀释抗体至不同浓度。在不同浓度的4-1BB抗体存在的条件下，将4-1BB蛋白(带6His tag)加入并且在37℃将其与固定的配体蛋白一起孵育1小时。在结合之后，将所述板用PBST洗涤3次，在37℃与在结合缓冲液中1/8,000稀释的anti-6His mAb((Proteintech)一起孵育1小时。再次洗涤后，加入1/10,000稀释的氧化物酶标记的羊抗鼠多克隆抗体(JacksonIm.Research)孵育1小时。最后洗涤6次后，用TMB显影并且使用2M H2SO4停止反应。两种抗体对4-1BBL结合4-1BB的抑制情况如图3所示。结果表明，两种抗体均可以竞争结合的方式抑制4-1BBL对4-1BB的结合。
实施例4 4-1BB抗体的亲和力SPR测定
为定量分析两种抗体与抗原蛋白人4-1BB的结合，使用Biacore T200系统通过多循环动力学测定方法，测量抗体的亲和力和动力学参数。根据制造商的说明，用NHS/EDC偶联通过伯氨基将Anti-human IgG(Fc)antibody(购自GE)偶联到传感器芯片CM5的羧甲基葡聚糖表面，最终偶联量为8300-8794RU。将两种4-1BB抗体和Utomilumab对照抗体稀释0.1μg/mL，分别捕获到芯片上，随后与不同浓度的4-1BB蛋白相互作用。动力学测量在25℃、30μl/min、1×HBS-EP+工作缓冲液中进行，再生条件为3M氯化镁、10μl/min作用30秒。在每一个测量循环中，特定浓度的4-1BB流过芯片表面，抗原与被捕获在芯片上的IgG抗体相互作用而导致传感片表面分子浓度的变化将由SPR信号的改变而得到测定,并以共振单位(RU)表示。以时间对共振单位(RU)连续作图,并扣除了未固定抗原的参比通道的RU数值后，得到的传感图记录了整个反应过程包括结合和解离过程。用Biacore T200 evaluation software评估所得作用曲线，并计算亲和力KD值。两种4-1BB抗体和Utomilumab分别对人4-1BB蛋白的结合数据总结在表1中，两种抗体显示出优异的4-1BB蛋白结合能力。
表1.两种4-1BB抗体与人4-1BB蛋白的亲和力测定结果
| mAb | KD(M) | Ka(M<sup>-1</sup>s<sup>-1</sup>) | Kd(s<sup>-1</sup>) |
| 8F5 | 5.18E-10 | 4.09E+05 | 2.12E-04 |
| 13G4 | 1.13E-09 | 1.49E+05 | 1.68E-04 |
| Utomilumab | 1.25E-08 | 1.16E+06 | 1.45E-02 |
实施例5 4-1BB抗体的细胞结合活性的FACS分析
通过CytoFLEX流失细胞分析仪，分析抗体8F5和13G4与稳转表达4-1BB的HEK293T细胞的结合能力。取对数生长期的细胞HEK293T_4-1BB和HEK293T，分别接种到6cm平皿，接种细胞密度为90％，37℃、5％CO2培养箱过夜培养。次日细胞经消化和离心后，以1×106～1×107/mL的浓度重悬于1％含小牛血清的磷酸盐缓冲液(PBS-NBS)中，按100ul/管的量加入流式专用管中。分别加入8F5和13G4抗体20μg/ml，每管加入100μl，冰浴45分钟；以PBS-NBS洗涤两次后，加入1:100稀释的羊抗人-FITC，冰浴45分钟。最后以PBS-NBS洗涤两次，弃上清，重悬于300μl PBS-NBS缓冲液，上机读取细胞染色的荧光信号。结果如图4和5所示，8F5和13G4抗体均可以特异性识别细胞表面表达的4-1BB蛋白。
实施例6 4-1BB抗体促进人CD4+T细胞分泌细胞因子IFN-γ
从捐献者提供的新鲜血液中分离PBMC细胞，用PBS+2％FBS缓冲液洗涤后，重悬细胞并计数。进一步使用CD4+T细胞分离kit，从前述PBMC细胞中分离出CD4+T细胞群，用RPM1640+10％FBS完全培养基重悬细胞。在96孔细胞培养板底部预包被2μg/ml羊抗鼠Fc多克隆抗体，经PBS+2％BSA封闭后，加入50ng/ml OKT-3鼠单抗进行捕获，反应1小时后，用PBS洗涤一次，随后加入前述分离的CD4+T细胞和不同浓度的4-1BB抗体及Utomilumab，将所述细胞培养板置于37℃、5％CO2培养箱中培养48-72小时，离心收集培养上清，用IFN-γELISA检测试剂盒检测细胞因子的浓度。结果如图6所示，两种4-1BB抗体8F5和13G4均可以有效刺激CD4+T细胞分泌IFN-γ因子，且比Utomilumab具有更强的活性。
序列表
<110> 武汉海沙百得生物技术有限公司
<120> 靶向4-1BB的全人源抗体及其制备方法和应用
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gtcctaggt 729
<210> 34
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115 120 125
Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser
130 135 140
Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val
145 150 155 160
Gly Gly Tyr Asp Phe Val Ser Trp Tyr Gln His His Pro Gly Lys Ala
165 170 175
Pro Lys Leu Met Ile Tyr Gly Val Ala Asn Arg Pro Ser Gly Val Ser
180 185 190
Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
195 200 205
Ser Gly Leu Gln Ala Glu Asp Asp Ala Asp Tyr Tyr Cys Ser Ser Tyr
210 215 220
Ser Ser Thr Ser Thr Arg Tyr Val Phe Gly Thr Gly Thr Lys Val Thr
225 230 235 240
Val Leu Gly
<210> 35
<211> 738
<212> DNA
<213> homo sapiens
<400> 35
caggtgcagc tgcaggagtc ggggggaggc ttggtccagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagt agctattgga tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtggccaac ataaagcaag atggaagtga gaaatactat 180
gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagtcgat 300
agcagtggct ggggtgcttt tgatatctgg gggcaaggga ccacggtcac cgtctcgagt 360
ggtggaggcg gttcaggcgg aggtggttct ggcggtggcg gatcgtcttc tgagctgact 420
caggaccctg ctgtgtctgt ggccttggga cagacagtca ggatcacatg ccaaggagac 480
agcctcagaa ggtattatgc aagctggtac cagcagaagc caggacaggc gcctttactt 540
gtcatctatg gtaacagcaa ccggccctca gggatcccag accgattctc tggctccagt 600
tcaggggaca cagattcctt gaccatcact ggggctcagg cggaagatga ggctgactat 660
tactgtaact cccgggacag cagtggtaac catccttatg tggtattcgg cggagggacc 720
cagctcaccg ttttaagt 738
<210> 36
<211> 246
<212> PRT
<213> homo sapiens
<400> 36
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys 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 Val Asp Ser Ser Gly Trp Gly Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Ser Ser Glu Leu Thr Gln Asp Pro Ala
130 135 140
Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp
145 150 155 160
Ser Leu Arg Arg Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln
165 170 175
Ala Pro Leu Leu Val Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Ile
180 185 190
Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asp Thr Asp Ser Leu Thr
195 200 205
Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser
210 215 220
Arg Asp Ser Ser Gly Asn His Pro Tyr Val Val Phe Gly Gly Gly Thr
225 230 235 240
Gln Leu Thr Val Leu Ser
245
Claims (9)
1.两种分离的单克隆抗体或其抗原结合部分，包含重链可变区，所述重链可变区包含CDR1区、CDR2区和CDR3区，其中所述CDR1区、所述CDR2区和所述CDR3区分别包含如下的氨基酸序列：
(1)分别为SEQ ID NO:1、2和3；或
(2)分别为SEQ ID NO:4、5和6；
其中所述抗体或其抗原结合片段结合4-1BB。
2.根据权利要求1所述的抗体或其抗原结合部分，其进一步包含轻链可变区，所述轻链可变区包含CDR1区、CDR2区和CDR3区，其中所述CDR1区、所述CDR2区和所述CDR3区分别包含如下的氨基酸序列：
(1)分别为SEQ ID NO:7、8和9；或
(2)分别为SEQ ID NO:10、11和12。
3.根据权利要求1所述的抗体或其抗原结合部分，其包含重链可变区，所述重链可变区包含与SEQ ID NO:13和14中任一项具有至少80％、85％、90％、95％、98％、99％或100％同一性的氨基酸序列。
4.权利要求1的抗体或其抗原结合部分，其包含轻链可变区，所述轻链可变区包含与SEQ ID NO:15和16具有至少80％、85％、90％、95％、98％、99％或100％同一性的氨基酸序列。
5.权利要求1的抗体或其抗原结合部分，其(a)结合人4-1BB；(b)结合猴4-1BB；(c)不与小鼠4-1BB结合；(d)抑制4-1BB/4-1BBL相互作用；(e)诱导活化T细胞释放IFN-γ。
6.权利要求1的抗体或其抗原结合部分，其中所述抗原结合片段选自抗体的scFv、(scFv)2、Fab、Fab’或F(ab’)2。
7.一种药物组合物，其包含权利要求1的抗体或其抗原结合部分，以及抗体任何形式的修饰、药学上可接受的载体。
8.权利要求7的药物组合物，其还包含抗肿瘤剂。
9.权利要求1的抗体或其抗原结合部分在制备用于抑制肿瘤生长的药物中的用途。
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| WO2023093899A1 (zh) * | 2021-11-29 | 2023-06-01 | 江苏恒瑞医药股份有限公司 | 经修饰的蛋白或多肽 |
| CN116554324A (zh) * | 2022-01-28 | 2023-08-08 | 三优生物医药(上海)有限公司 | 一种特异性识别4-1bb的抗体、其制备方法及其用途 |
Citations (2)
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|---|---|---|---|---|
| US20180344870A1 (en) * | 2017-04-13 | 2018-12-06 | Agenus Inc. | Anti-cd137 antibodies and methods of use thereof |
| US20200362051A1 (en) * | 2017-11-13 | 2020-11-19 | Crescendo Biologics Limited | Molecules that bind to cd137 and psma |
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|---|---|---|---|---|
| US20180344870A1 (en) * | 2017-04-13 | 2018-12-06 | Agenus Inc. | Anti-cd137 antibodies and methods of use thereof |
| US20200362051A1 (en) * | 2017-11-13 | 2020-11-19 | Crescendo Biologics Limited | Molecules that bind to cd137 and psma |
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| SUN K HO ET AL: "Epitope and Fc-Mediated Cross-linking, but Not High Affinity, Are Critical for Antitumor Activity of CD137 Agonist Antibody with Reduced Liver Toxicity" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023093899A1 (zh) * | 2021-11-29 | 2023-06-01 | 江苏恒瑞医药股份有限公司 | 经修饰的蛋白或多肽 |
| CN116554324A (zh) * | 2022-01-28 | 2023-08-08 | 三优生物医药(上海)有限公司 | 一种特异性识别4-1bb的抗体、其制备方法及其用途 |
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