CN116396388A - 一种抗b7-h3抗体及其应用 - Google Patents
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Abstract
本发明公开了一种抗B7‑H3抗体及其应用,涉及生物医药技术领域,本发明的抗B7‑H3抗体及其功能性片段包括选自以下各氨基酸序列或任何所述序列之变体的重链CDR:SEQ ID NO:8n+2,SEQ ID NO:8n+3和SEQ ID NO:8n+4;和/或选自以下各氨基酸序列或任何所述序列之变体的轻链CDR:SEQ ID NO:8n+6,SEQ ID NO:8n+7和SEQ ID NO:8n+8,其中,各n独立地为0、1、2、3、4、5、6、7、8、9或10。本发明通过体外杀伤实验,验证了本发明B7‑H3‑CD3双特异性抗体的抗肿瘤作用,结果表明,B7‑H3‑CD3双特异性抗体对A375‑lucf肿瘤细胞具有显著的杀伤效应。本发明可应用于制备诊断,预防和治疗人类肿瘤的药物。
Description
技术领域
本发明属于生物医药技术领域,涉及一种抗B7-H3抗体及其应用,特别涉及能与人B7-H3特异性结合的抗体或其功能性片段,及其在肿瘤治疗中的用途。
背景技术
肿瘤免疫治疗作为一种创新治疗方式已成为肿瘤治疗研究领域的一大热点,有望为患者带去新的治疗选择。通过阻断PD-L1/PD-1通路或者CTLA-4信号传导通路,可以逆转T细胞的免疫抑制状态,提高患者自身免疫系统抑制或杀伤肿瘤的能力。但是目前PD-L1/PD-1通路或者CTLA-4通路的阻断剂在临床上仅对少部分患者有效,仍需探寻新的免疫治疗靶点或新的免疫治疗策略。
B7-H3(CD276)是B7家族成员之一,位于人类第15号染色体。已有研究表明小鼠B7-H3是一个T细胞活化的负调控因子(Prasad DV,et al.,Murine B7-H3 is a negativeregulator of T cells.J Immunol,2004,173:2500–2506),与正常小鼠相比,B7-H3敲除鼠会发生更严重的呼吸道炎症,实验性自身免疫性脑脊髓炎发生的也更早(Suh WK,et al.,The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses.Nat Immunol,2003,4:899–906)。在人体中,B7-H3起初被作为一个T细胞共刺激分子报道(Chapoval AI,et al.,B7-H3:a costimulatory moleculefor T cell activation and IFN-gamma production.Nat Immunol,2001,2:269–274)。但近期研究表明人B7-H3能够抑制T细胞激活过程中IFN-γ,IL-2,IL-10和IL-13的产生,对T细胞的活化具有抑制作用(Leitner J,et al.,B7-H3is a potent inhibitor of humanT-cell activation:No evidence for B7-H3 and TREML2 interaction.Eur J Immunol,2009,39:1754-1764)。至今,B7-H3的受体或配体仍未探明。
通过检测各种临床样本,诸多研究表明B7-H3在大部分正常组织呈低表达,而在多种肿瘤组织中呈高表达,且其表达水平与不良预后相关,如肺癌(Zhang G,et al.,Diagnosis value of serum B7-H3expression in non-small cell lung cancer.LungCancer,2009,66:245-249),结直肠癌(Sun J,et al.,Clinical significance andregulation of the costimulatory molecule B7-H3 in human colorectalcarcinoma.Cancer Immunol Immunother,2010,59:1163-1171),前列腺癌(Zang X,etal.,B7-H3 and B7x are highly expressed in human prostate cancer andassociated with disease spread and poor outcome.Proc Natl Acad Sci U S A,2007,104:19458-19463),膀胱癌(Xu Z,et al.,High expression of B7-H3and CD163 incancer tissues indicates malignant clinicopathological status and poorprognosis of patients with urothelial cell carcinoma of the bladder.OncolLett,2018,15:6519-6526),以及髓母细胞瘤(Purvis IJ,et al.,Role of MYC-miR-29-B7-H3 in Medulloblastoma Growth and Angiogenesis.J Clin Med,2019,8,pii:E1158)。另外,B7-H3也被报道在其它多种肿瘤中呈现高表达,如胃癌、卵巢癌,胶质母细胞瘤,颅咽管瘤,宫颈癌,骨肉瘤,血液瘤,头颈部肿瘤,胰腺癌,皮肤癌,肾癌,脑膜瘤和乳腺癌。
另外,相关研究表明B7-H3在多种自身免疫性疾病的发病中起作用,对多种自身免疫性疾病具有潜在诊治价值,如红斑狼疮,脓毒症,关节炎,胰腺炎和I型糖尿病。
综上所述,由于B7-H3在多种肿瘤组织中广谱高表达,正常组织低表达,故被认为是极具潜力的肿瘤治疗靶点。
发明内容
本发明的一方面是提供能够与B7-H3(CD276)特异性结合的抗B7-H3抗体或其功能性片段。
本发明提供了一种抗体或其功能性片段,包括选自以下各氨基酸序列或任何所述序列之变体的重链CDR:SEQ ID NO:8n+2,SEQ ID NO:8n+3和SEQ ID NO:8n+4;和/或选自以下各氨基酸列或任何所述序列之变体的轻链CDR:SEQ ID NO:8n+6,SEQ ID NO:8n+7和SEQID NO:8n+8,其中,各n独立地为0、1、2、3、4、5、6、7、8、9或10。
在一些具体的实施方案中,本发明提供一种抗B7-H3抗体或其抗原结合片段,其中所述重链CDR包括以下三个互补决定区:
SEQ ID NO:8n+2所示的CDR1,
SEQ ID NO:8n+3所示的CDR2,和
SEQ ID NO:8n+4所示的CDR3;
所述轻链CDR包括以下三个互补决定区:
SEQ ID NO:8n+6所示的CDR1,
SEQ ID NO:8n+7所示的CDR2,和
SEQ ID NO:8n+8所示的CDR3;
其中,各n独立地为0、1、2、3、4、5、6、7、8、9或10。
在一些具体的实施方案中,本发明提供一种抗B7-H3抗体或其抗原结合片段,其包含选自氨基酸序列SEQ ID NO:8n+1或任何所述序列之变体的重链可变区,和/或选自氨基酸序列SEQ ID NO:8n+5或任何所述序列之变体的轻链可变区,其中,各n独立地为0、1、2、3、4、5、6、7、8、9或10。
在一些具体的实施方案中,本发明提供一种抗B7-H3抗体或其抗原结合片段,其中所述抗体或其功能性片段为嵌合抗体、单链抗体或双特异性抗体。
在一些具体的实施方案中,本发明提供一种抗B7-H3抗体或其抗原结合片段,其中所述单链抗体的结构表示式为:VH-(G4S)3-VL-huIgG1Fc,其中,VH为重链可变区,VL为轻链可变区,(G4S)3为肽接头,huIgG1Fc为人IgG1抗体的恒定区。所述双特异性抗体(B7-H3-CD3)的结构表示式为:VL1-(G4S)3-VH1-G4S-VH2-(G4S)3-VL2,其中,VH1为B7-H3重链可变区,VL1为B7-H3轻链可变区,VH2为CD3重链可变区,VL2为CD3轻链可变区,G4S和(G4S)3为肽接头,VH2-(G4S)3-VL2为CD3scFv,其采用的是OKT3的氨基酸序列(如下所示):
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS(VH2)GGGGSGGGGSGGGGS((G4S)3)DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(VL2)。
在一些具体的实施方案中,本发明提供一种抗B7-H3抗体或其抗原结合片段,其中所述功能性片段包括Fab,Fv,sFv,F(ab')2、单链抗体、纳米抗体、结构域抗体和多特异抗体。
本发明的另一方面提供包含所述抗体的表达载体以及包含所述表达载体的宿主细胞。
本发明的另一方面提供包含本发明抗体或其功能性片段、或包含所述核酸分子及表达载体或宿主细胞,或它们的任意组合,以及可药用载体的药物组合物。
本发明的另一方面提供包含与治疗剂缀合的本发明抗体或其功能性片段的免疫缀合物。在一些实施方案中,所述治疗剂为毒素、放射性同位素、药物或细胞毒剂。所述免疫缀合物包括抗体或功能性片段与因子的融合蛋白,以及抗体或功能性片段与毒素、反射性同位素、药物或细胞毒性剂的偶联物。
本发明的另一方面提供制备抗B7-H3抗体或其功能性片段的方法,包括:在允许产生(表达)所述抗体或其功能性片段的条件下培养本发明所述的宿主细胞,以及从所述宿主细胞中回收所产生的所述抗体或其功能性片段。
本发明的另一方面提供用于靶向B7-H3疗法来诊断、预防或治疗疾病或病症的方法,其包括向有此需要的对象施用治疗有效量的本发明抗体或其功能性片段,表达载体,宿主细胞,药物组合物或免疫缀合物。其中,所述疾病选自肿瘤或炎性疾病,所述肿瘤优包括脑胶质瘤、神经母细胞瘤、髓母细胞瘤、脑膜瘤、肺癌、食管癌、胰腺癌、肝癌、胆管癌、肾癌、膀胱癌、尿管癌、前列腺癌、皮肤癌、黑色素瘤、卵巢癌、子宫内膜癌、宫颈癌、软组织肉瘤、急性及慢性白血病、霍奇金及非霍奇金淋巴瘤,胃癌和头颈部肿瘤;所述炎性疾病包括红斑狼疮、强直性脊柱炎、多发性硬化、银屑病、抗磷脂抗体综合征、特发性血小板减少性紫癜、自身免疫性溶血性贫血、自身免疫性肝炎、关节炎、类风湿关节炎、天疱疮、吉兰巴雷综合、克罗恩病,血管炎和自身免疫性糖尿病。
本发明具备如下有益效果:
本发明通过传统杂交瘤技术制备了B7-H3的特异性单克隆抗体,并对其进行了单链抗体(scFv)和双特异性抗体(B7-H3-CD3)的构建,同时通过体外杀伤实验,验证了本发明B7-H3-CD3双特异性抗体的抗肿瘤作用,结果表明,B7-H3-CD3双特异性抗体对A375-lucf肿瘤细胞具有显著的杀伤效应。本发明可应用于制备预防、诊断和治疗人类肿瘤的药物。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为免疫荧光分析重组鼠单抗1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7对细胞过表达的B7-H3分子的结合结果;
图2为FACS分析单链抗体1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7分别对细胞内源B7-H3(2a)和细胞敲除内源B7-H3(2b)的结合结果;
图3为1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7分别与CD3scfv串联的双特异性抗体的体外杀伤肿瘤结果;
图4为使用NSG鼠模型评价1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7分别与CD3scfv串联的双特异性抗体的体内抗肿瘤分析结果。
具体实施方式
以下描述中,为了说明而不是为了限定,提出了诸如特定系统结构、技术之类的具体细节,以便透彻理解本发明实施例。然而,本领域技术人员应当清楚,在没有这些具体细节的其它实施例中也可以实现本发明。除非有特定指示,本发明使用的所有科技用语为本领域技术人员所理解的相同含义。氨基酸的缩写采用本领域所通用的20种常用氨基酸之一的标准3字母或1字母代码,如J.biol.chem,243,p3558(1968)中所述。
本领域技术人员可以对本发明的序列替换、添加和/或缺失一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸,以及在可变区将具有类似性质的氨基酸进行替换,而不实质性影响抗体的结合活性,从而获得所述抗体或其功能性片段之序列的变体。它们都被视为包括在本发明保护的范围内。
术语
本发明所述的“功能性片段”主要指的是抗体中的能够特异结合抗原的功能片段如scFv(单链抗体)、Fv、Fab、F(ab’)2、Fab’、scFv-Fc、单域抗体、类抗体、双特异性抗体、多特异抗体,或者通过化学修饰以增加半衰期的任何抗体片段。
本发明所述的“药物组合物”表示组合在一起以实现某种特定目的的至少一种药物以及任选地可药用载体或辅料的组合。在某些实施方案中,所述药物组合物包括在时间和/或空间上分开的组合,只要其能够共同作用以实现本发明的目的。一些药物组合物是通过联合施用一些可药用成分或化合物,达到增强本发明的生物功效或减小药物副作用(例如,可以和其他抗肿瘤药物联合使用,增强抗肿瘤效果)。另一些药物组合物的目的是促进对生物体的给药,利于活性成分的吸收,增强稳定性或靶向性,延长半衰期,进而更好的发挥本发明的生物功效。
本发明所述的“宿主细胞”包括原核宿主细胞、真核宿主细胞以及噬菌体。所述的原核宿主细胞可以为大肠杆菌、链霉菌或枯草杆菌等。所述真核宿主细胞可以为293细胞、293T细胞、293FT细胞、CHO细胞、COS细胞、Per6,酿酒酵母、毕赤酵母、汉森酵母、假丝酵母、部分昆虫细胞以及植物细胞。293系列细胞,Per6细胞和CHO细胞是用于生产制备抗体或重组蛋白的常用哺乳动物细胞,为本领域普通技术人员所熟知。
本发明所述的“嵌合抗体”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体,可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体,要先建立分泌鼠源性特异性单抗的杂交瘤,然后从小鼠杂交瘤细胞中克隆可变区基因,再根据需要克隆人抗体的恒定区基因,将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中,最后在真核工业系统或原核工业系统中表达嵌合抗体分子。
本发明所述的“表达载体”是指任何重组的多核苷酸构建体,该构建体可通过转化,转染或转导的方式将目的DNA片段直接或间接(如包装成病毒)导入宿主细胞内,进行目的基因表达。其中一种类型的载体是质粒,即环状双链DNA分子,可将目的DNA片段连接至质粒环中。另一种类型的载体为病毒载体,其可将目的DNA片段连接包装至病毒基因组中(如腺病毒,腺相关病毒,逆转录病毒,慢病毒,溶瘤病毒)。这些载体进入宿主细胞后,可以进行目的基因的表达。
本领域技术人员也可以通过体外转录的方式,以本发明所述核酸序列为模板,转录成RNA,进一步通过转染,转导或转化该RNA到宿主细胞,也可以表达本发明抗体或其功能性片段,发挥本发明的生物功效。
本发明所述的“有效量”是指足以显示其对于所施用对象益处的剂量。施用的实际量,以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方最终由医生来做决定,通常会考虑患者的个体情况,递送部位,施用方法,疾病严重程度以及对于医生来说其它常规因素。
本发明所述的“对象”是指哺乳动物,如人类,但也可以是其它动物,如野生动物,家畜或实验动物。
本发明提供了能够特异性结合B7-H3的抗B7-H3抗体及其功能片段。本发明的抗体或其功能性片段能够与人源B7-H3特异性结合,而不与鼠源B7-H3发生结合。
本发明的抗体可以是全长的(例如,IgG1或IgG4抗体)或可仅包含抗原结合部分(例如,Fab,F(ab')2或scFv片段),所述抗体可作为单链抗体(scFv)和双特异性抗体的功能性片段。
本发明提供了可用于肿瘤治疗的应用方法。所述方法包括运用本发明抗体或其功能性片段的细胞治疗产品(如嵌合抗原受体T细胞)、双特异抗体(如B7-H3/CD3双特异抗体),免疫缀合物(如抗体偶联美登素),以及包含本发明抗体及其功能片段和可药用载体的药物组合物。
本发明可以用于对B7-H3抗原进行定性或定量检测,例如可以将本发明抗体或其功能性片段应用于ELISA,免疫组织化学,免疫荧光和流式检测。也可以用于细胞,组织或活体的抗原示踪,例如可以将本发明抗体或其功能性片段进行荧光或同位素标记从而进行抗原示踪。
具体提供以下实施例用以证明并进一步解释本发明的一些优选的实施方式和方面,但不应被解释为限制其范围。本发明实施例中未注明具体条件的实验方法,通常按照常规条件,如冷泉港的抗体技术实验手册,分子克隆手册;或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂,为市场购买的常规试剂。
实施例1
(1)B7-H3重组蛋白的制备:编码人源B7-H3的核酸序列(NM_001024736.2)由安徽通用生物公司合成。PCR扩增并亚克隆至pcDNA3.1表达载体中(Invitrogen)。然后,将B7-H3的胞外域分别亚克隆到C端携带Fc或His标签的pcDNA3.1表达载体中。其中Fc标签包括人源Fc(hFc)和鼠源Fc(mFc)。通过瞬时转染293FT,使用FreeStyleTM无血清培养基(LifeTechnologies)摇瓶培养5-7天,收集上清,经过离心超滤,然后通过ProteinA/G或镍柱亲和层析以及分子筛色谱柱纯化携带Fc或His标签的重组B7-H3蛋白。
(2)表达人B7-H3抗原的稳转细胞株制备:将编码人源B7-H3的全长序列构建到携带IRES-EGFP的pcDNA3.1表达载体中。CHO-S(ATCC)细胞于CD-CHO培养基(LifeTechnologies)内培养;Hela细胞于含10%胎牛血清的DMEM中培养。采用LipofectamineLTX(Life Technologies)转染试剂进行细胞转染,48小时之后进行流式分选,培养至96孔板,进行单克隆稳定细胞株筛选和鉴定,并对稳定表达B7-H3的CHO(CHO.B7-H3-EGFP)和Hela(Hela.B7-H3-EGFP)细胞进行保种。
实施例2
1.抗B7-H3单克隆抗体的制备:
(1)动物免疫
使用5-6周龄的Balb/c雌性小鼠作为被免疫动物,免疫剂量为100μg/只。首次免疫采用100μl弗氏完全佐剂(Sigma)与等体积重组B7-H3蛋白混合,充分乳化后进行皮下多点注射。每隔2周,用等体积弗氏不完全佐剂(Sigma)与重组蛋白混合,充分乳化后进行皮下多点注射。加强免疫共4次,最后一次加强免疫后第10天,割尾采血检测小鼠抗体效价。细胞融合前3天,100μg重组蛋白腹腔冲击一次。
(2)细胞融合与杂交瘤筛选
无菌条件下,取小鼠脾脏,制备富含B细胞的悬液,按经典的PEG(Sigma)法,与SP2/0进行细胞融合。融合后的细胞重悬于HAT培养基进行培养。融合后第5天和第10天使用新鲜的HAT培养基进行半换液培养。融合后第11-15天进行ELISA,免疫荧光和流式分析,筛选阳性克隆。
ELISA筛选采用96孔板进行,简言之,将B7-H3重组蛋白按100ng/孔的量4度过夜包被到孔板底部,使用HRP偶联抗小鼠IgG抗体和化学发光试剂(碧云天生物科技公司)进行显色,并于酶标仪在450nm波长读值。免疫荧光染色使用稳定表达B7-H3的Hela细胞株,简言之,将细胞株在96孔板贴壁培养,加入50μl杂交瘤上清作为一抗,4度孵育2小时,PBS清洗3次,Cy3标记的Goat Anti-Mouse IgG(Proteintech)作为二抗,常温孵育1小时,PBS清洗3次,使用荧光显微镜采集图像。流式细胞术分析采用稳定表达B7-H3的CHO细胞,简言之,离心收集细胞重悬于含有50μl杂交瘤上清的PBS缓存液中,4度孵育2小时,PBS清洗3次,CY3标记的Goat Anti-Mouse IgG(碧云天生物科技公司)作为二抗,常温孵育1小时,PBS清洗3次,上流式细胞仪(Agilent-Novosampler Pro)进行分析。
根据上述ELISA分析,免疫荧光分析和流式细胞术分析结果,最终可确定十一个最优的杂交瘤克隆(分别命名为1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7),用于后续的序列克隆和亲和力分析等实验。
实施例3
杂交瘤抗体可变区序列克隆:收集对数生长期杂交瘤细胞,用Trizol(Invitrogen)提取RNA并反转录(PrimeScriptTM Reverse Transcriptase,Takara)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen)进行PCR扩增后测序,最终获得十一种单克隆抗体的重链和轻链可变区序列。其中重链和轻链的可变区CDR序列如表1所示。
表1.鼠单抗的重链和轻链可变区含有的CDR序列
实施例4
重组嵌合抗体对hela宫颈癌细胞过表达B7-H3的结合分析
将人(IgG1)和鼠(IgG2a)重链恒定区,以及人/鼠轻链恒定区,克隆入pcDNA3.1(Invitrogen)质粒载体,然后将杂交瘤克隆1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7的VH和VL基因片段分别构到有人IgG1重链恒定区和人IgGκ轻链恒定区的基因重组载体上,得到重组嵌合抗体重链表达载体和轻链表达载体,通过瞬时转染293FT,使用FreeStyleTM无血清培养基(Life Technologies)摇瓶培养5-7天,收集上清,经过离心超滤,然后通过Protein A/G亲和层析以及分子筛色谱柱纯化获得相应类型抗B7-H3重组单克隆抗体。将Hela.B7-H3-EGFP细胞铺于24孔细胞培养皿中,第二天将重组嵌合抗体1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7作为一抗,CY3标记的Goat Anti-Mouse IgG(碧云天生物科技公司)作为二抗,并用荧光共聚焦对其进行观察拍照。如图1所示,由图1结果表明:重组嵌合抗体1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7可以和细胞Hela.B7-H3-EGFP发生特异性结合。
实施例5
单链抗体对细胞内源的B7-H3的结合分析
选取B7-H3表达阳性的人皮肤癌细胞A375,CRISPR/Cas9靶向敲除B7-H3的A375细胞,按实施例2中所述方法,进行流式分析。
其中,靶向敲除B7-H3的A375细胞的制备采用经典的CRISPR/Cas9基因编辑方法。靶向B7-H3的gRNA设计采用CHOPCHOP(http://chopchop.cbu.uib.no)软件。然后将gRNA亚克隆至lentiCRISPR V2慢病毒载体(Addgene:#52961)。将慢病毒载体和包装质粒共转染HEK293T细胞,收获病毒上清。离心浓缩后,感染A375细胞,并于48小时之后添加1μg/ml的嘌呤霉素进行筛选。筛选3-5天之后,收集细胞进行流式分析,实验结果如图2所示。
由图2a结果表明:B7-H3表达阳性的A375细胞能够被1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7鼠单链抗体染成几乎100%的阳性,其中1H9S、2H2、5A3、5H5、7H7、8F3、G10H鼠单链抗体染色显示更强。由图2b结果表明:B7-H3靶向敲除的A375细胞染色呈阴性。说明1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7鼠单链抗体能够识别细胞内源表达的B7-H3分子。
实施例6
杂交瘤抗体对鼠源B7-H3的结合分析
鼠源B7-H3的cDNA克隆购自义翘神州,然后按照实施例2构建鼠源B7-H3的稳定表达细胞株CHO-mB7-H3。然后取杂交瘤1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7上清,按照实施例2进行流式细胞分析。结果表明单克隆抗体不能够与鼠源B7-H3结合。
实施例7
体外结合亲和力和动力学实验
本实施例采用表面等离子共振(SPR)方法测定,使用GE公司Biacore 8K仪器进行分析。利用由Biacore提供的试剂盒,采用标准氨基偶联法将B7-H3-His重组蛋白共价连接至CM5(GE)芯片上,然后将待测单链抗体按不同浓度梯度稀释于同样缓冲液中进样,进样后均以试剂盒内配再生试剂再生。数据的分析和采集使用Biacore 8K配套分析软件进行。所得结果如下表2。
表2.抗体抗原体外结合亲和力和动力学分析
实施例8
B7-H3-CD3双特异性抗体的体外杀伤实验
效应细胞和靶细胞的准备:抽取健康捐献者外周血,分离PBMC,采用T细胞分离试剂盒(Miltenyi T Cell Isolation Kit)分离T细胞,分离后的T细胞培养用含5%AB血清的X-VIVO(LONZA)培养基,预先用1ml含50ng/ml抗人CD3抗体(PeproTech)和50ng/ml CD28抗体(PeproTech)的包被液37℃2h孵育包被TC处理的6孔板,使用前除去包被液。将细胞以1ml/孔接种到已包被抗体的6孔板中,刺激培养48小时后,补充添加IL-2(100U/mL)和IL-15(10ng/mL)作为活化因子继续培养。靶细胞选用B7-H3高表达的人皮肤癌细胞A375,培养于添加了10%胎牛血清(Gibco)的DMEM高糖培养液中。
荧光素酶报告基因是指以荧光素(luciferin)为底物来检测萤火虫荧光素酶(fireflyluciferase)活性的一种报告系统。荧光素酶可以催化luciferin氧化成oxyluciferin,在luciferin氧化的过程中,会发出生物荧光(bioluminescence)。将携带luciferase基因的慢病毒质粒(Addgene:#72486)和包装质粒共转染HEK293T细胞,收获病毒上清。离心浓缩后,感染A375细胞,并于48小时之后进行流式分选,最终得到A375-luciferase细胞(A375-lucf)用于后续共培养实验。
细胞共培养实验:将对数生长期的A375-lucf肿瘤细胞加入96孔板中,过夜培养后,添加0ng/ml(Control)或者10ng/ml的双特异性抗体,同时按照T细胞和肿瘤细胞的效靶比分别为1:1、2:1、4:1和8:1的比例加入T细胞,24小时后,加入荧光素(luciferin),通过读取生物荧光值来测量细胞存活率,公式如下:
细胞存活率%=实验组生物荧光值/阴性对照组生物荧光值×100%
阴性对照组指的是不添加双特异性抗体和T细胞的正常生长的A375-lucf组。显示的数据为平均值±s.d.P值采用未配对双尾学生t检验获得。采用来自不同捐献者的T细胞重复实验三次,获得了相似的结果,代表性结果如图3所示。
由图3结果可知,与不添加双特异性抗体组(Control)相比,十一种B7-H3-CD3双特异性抗体(1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7)对A375-lucf肿瘤细胞具有显著的杀伤效应(*P<0.05、**P<0.01、***P<0.001)。
实施例9
异种移植物小鼠模型抗肿瘤实验
本实施例采用异种移植物小鼠模型来评估B7-H3靶向的双特异性抗体(1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7)的体内抗肿瘤活性。采用一种免疫缺陷鼠模型进行评估。
NCG重症免疫缺陷鼠模型:NCG重症免疫缺陷鼠购自南京大学模式动物所,将2×106个对数生长期的A375细胞接种于NCG鼠右后背部皮下。待6天左右肿瘤长至200mm3后,将荷瘤体积均匀的小鼠随机分组,每组5只小鼠。设置与给药等体积的生理盐水为对照组。双特异性抗体的给药方式为腹腔给药,30μg/只,每3天给药1次,共计给药4次。每3天进行小鼠称重并测量肿瘤大小。移植瘤平均体积按照公式V=1/2(L×W2)计算,其中L代表瘤体的长度,W代表瘤体的宽度。当小鼠肿瘤体积达到2000mm3或者肿瘤表面出现明显溃破,则处死小鼠,结束动物实验。所有数据均为平均值±s.d.P值采用未配对双尾学生t检验获得。实验结果如图4所示。
由图4结果可知,与不给药的对照组相比,十一种B7-H3-CD3双特异性抗体(1H9S、2H2、3HS、5A3、5H5、7H7、8F3、8HS、G10H、10HS、15D7)对A375肿瘤细胞的生长均具有显著的抑制效果,其中3HS/8HS/10HS/15D7-CD3组**P<0.01,1H9S/7H7/8F3-CD3组***P<0.001,2H2/5A3/5H5/G10H-CD3组抑制效果最佳****P<0.0001。
以下为本发明涉及的序列:
以上具体实施例仅仅是对本发明的解释,其并不是对本发明的限制,本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改,但只要在本发明的权利要求范围内都受到专利法的保护。
Claims (10)
1.一种抗体或其功能性片段,其特征在于,包括选自以下各氨基酸序列或任何所述序列之变体的重链CDR:SEQ ID NO:8n+2,SEQ IDNO:8n+3和SEQ ID NO:8n+4;和/或选自以下各氨基酸列或任何所述序列之变体的轻链CDR:SEQ ID NO:8n+6,SEQ ID NO:8n+7和SEQ IDNO:8n+8,其中,各n独立地为0、1、2、3、4、5、6、7、8、9或10。
2.根据权利要求1所述的抗体或其功能性片段,其特征在于,所述重链CDR包括以下三个互补决定区:
SEQ ID NO:8n+2所示的CDR1,
SEQ ID NO:8n+3所示的CDR2,和
SEQ ID NO:8n+4所示的CDR3;
所述轻链CDR包括以下三个互补决定区:
SEQ ID NO:8n+6所示的CDR1,
SEQ ID NO:8n+7所示的CDR2,和
SEQ ID NO:8n+8所示的CDR3;
其中,各n独立地为0、1、2、3、4、5、6、7、8、9或10。
3.根据权利要求1所述的抗体或其功能性片段,其特征在于,包含选自氨基酸序列SEQID NO:8n+1或任何所述序列之变体的重链可变区,和/或选自氨基酸序列SEQ ID NO:8n+5或任何所述序列之变体的轻链可变区,其中,各n独立地为0、1、2、3、4、5、6、7、8、9或10。
4.根据权利要求1或3所述的抗体或其功能性片段,其特征在于,所述抗体或其功能性片段为嵌合抗体、单链抗体或双特异性抗体。
5.一种包含权利要求4所述核酸分子的表达载体。
6.一种包含权利要求5所述的表达载体的宿主细胞。
7.药物组合物,其特征在于,包含权利要求1至4中任一项所述的抗体或其功能性片段,包含权利要求5所述的表达载体,包含权利要求6所述的宿主细胞,或它们的任意组合,以及可药用的载体。
8.免疫缀合物,其特征在于,包含与治疗剂缀合的权利要求1至4中任一项所述的抗体或其功能性片段;优选地,所述治疗剂为毒素、药物、放射性同位素或细胞毒剂。
9.一种制备权利要求1-4任一项所述的抗B7-H3抗体或其功能性片段的方法,包括在如权利要求6所述的宿主细胞中表达抗体或其功能性片段,以及从所述宿主细胞中回收分离所述抗体或其功能性片段的步骤。
10.权利要求1-4中任一项所述的抗体或其功能性片段,或权利要求5所述的表达载体,或权利要求6所述的宿主细胞,权利要求7所述的药物组合物,或权利要求8所述的免疫缀合物在制备用于靶向B7-H3诊断,预防或治疗炎性疾病或肿瘤的试剂或药物中的用途。
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