CN105753986B - 一类抗cd20靶向抗体及用途 - Google Patents
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Abstract
本发明公开了一类新型CD20抗体及其在制备抗体肿瘤药物中的应用。更具体地,本发明公开了兼具I型和II型CD20抗体优势的新型CD20抗体,其核酸序列、氨基酸序列及其在制备抗肿瘤药物中的应用,既保留了I型CD20抗体诱导强烈CDC杀伤的功能,又兼具II型CD20抗体的优势,能够诱导强烈的细胞死亡,具有比亲本I型CD20抗体Rituximab和II型CD20抗体更好的抗肿瘤活性。
Description
技术领域
本发明属于生物技术领域,更具体地,本发明公开了一类抗CD20靶向抗体、其制备方法及其在制备抗体肿瘤药物中的应用。本发明公开的抗CD20靶向抗体,既具有I型CD20抗体抗肿瘤杀伤的优势,诱导CDC杀伤肿瘤细胞,又具有II型CD20抗体抗肿瘤的优势,能够诱导强烈的细胞死亡杀伤肿瘤细胞。表现出比I型CD20抗体Rituximab更好的抗肿瘤活性。
背景技术
白血病非霍奇金氏淋巴瘤(Non Hodgkin’s lymphoma , NHL)是临床最常见的血液系统和淋巴系统恶性肿瘤,好发于青壮年,发病率和死亡率呈逐年上升趋势,其中85%为B细胞来源。CD20 抗原是一种B 细胞分化抗原,仅位于前B 细胞、不成熟B 细胞和成熟B细胞,它在95 %以上的B 细胞性淋巴瘤中表达,而在造血干细胞、浆细胞和其他正常组织细胞中不表达。更为重要的是,CD20 分子在膜上为四次跨膜蛋白,与靶向抗体结合后无显著内吞及脱落,也不会因为与抗体的结合而发生抗原调变,故成为治疗B 细胞淋巴瘤的理想靶点。
CD20单抗疗法在B细胞淋巴瘤治疗中已取得了令人满意的成果。 CD20抗体Rituximab已经作为一线用药,广泛应用于B淋巴瘤的临床治疗。虽然Rituximab在B淋巴瘤的临床治疗取得突破性的进展,其具有近50%的治疗反应率和大约10%的完全反应率,被认为是最有效的肿瘤靶向抗体药物之一。但是大部分患者在治疗过程中会逐渐产生复发和抵制。因此,迫切需要设计更为有效的CD20抗体用于B淋巴瘤的治疗。
目前认为,CD20靶向抗体的抗肿瘤作用机制主要包括:补体介导的细胞溶解作用(CDC作用)、抗体依赖细胞介导的细胞毒作用(ADCC)和对肿瘤细胞的诱导死亡作用(CellDeath)。根据CD20靶向抗体作用机制的不同可以将其分为两种类型:I 型CD20抗体,包括Rituximab和大部分CD20抗体(1F5、2H7、2F2等),能够诱导强烈的CDC杀伤而诱导细胞死亡作用较弱;II型CD20抗体包括B1、11B8和Obinutuzumab,能够诱导强烈的细胞死亡而CDC杀伤较弱。目前已经批准用于临床治疗的CD20抗体,除了II型抗体B1与131I偶联药物Tositumomab和近期刚获批的Obinutuzumab外,主要为I 型CD20抗体(Rituximab和2F2)。I型CD20抗体在B淋巴瘤的临床治疗中取得良好的治疗效果,表明I型CD20抗体诱导的强烈的CDC杀伤在B淋巴瘤的临床治疗中具有非常重要的作用。然而随着研究的深入,II型CD20抗体诱导的强烈的非caspase依赖细胞死亡在淋巴瘤治疗中的重要作用逐渐显现。II型CD20抗体Obinutuzumab在临床前和临床试验中都表现出较好的肿瘤杀伤效果。虽然Obinutuzumab是经过糖基化修饰的II型CD20抗体,但是进一步的研究表明,糖基化修饰增强其ADCC和抗体依赖细胞吞噬作用(ADCP)的Obinutuzumab与未经过糖基化修饰的Obinutuzumab在小鼠体内清除B细胞中并未有显著差异。更重要的是,II型CD20抗体Obinutuzumab在淋巴瘤的临床试验中表现出比I型CD20抗体Rituximab更好的治疗效果,表明II型CD20抗体诱导的强烈的细胞死亡在B淋巴瘤治疗中发挥更加重要的作用。目前,美国FDA已经批准Obinutuzumab用于慢性淋巴细胞白血病的临床治疗。这些研究结果不仅表明,CD20靶向抗体诱导的CDC杀伤和细胞死亡在靶向抗体有效杀伤淋巴瘤细胞中都具有非常重要的作用,而且CD20靶向抗体诱导的非caspase依赖细胞死亡对杀伤淋巴瘤更加重要。然而目前,尚未有兼具I型和II型CD20抗体优势的新型CD20抗体被报道。因此,如何设计出兼具I型和II型CD20抗体抗肿瘤优势,能够同时诱导强烈CDC杀伤和细胞死亡的新型CD20抗体是目前靶向抗体研究领域的热点和难点。
发明内容
为了解决上述问题,本发明的发明人进行了长期研究,经大量试验,利用基因工程技术构建了一类兼具I型CD20抗体CDC杀伤肿瘤优势和II型CD20抗体诱导细胞死亡优势的新型CD20抗体。该抗体是在I型CD20抗体Rituximab的基础上,通过一个氨基酸的突变,进而获得了兼具I型和II型CD20抗体优势的新型CD20抗体。表现出比I型CD20抗体Rituximab更好的抗肿瘤活性。
本发明公开了:
1.一种兼具I型和II型CD20抗体抗肿瘤优势的新型CD20抗体,其特征是既能够既诱导强烈CDC杀伤,又能够诱导强烈细胞死亡来杀伤CD20阳性的B淋巴瘤细胞。
2.上述的新型CD20抗体,由四条肽链构成,分别为两条Rituximab重链突变体和两条Rituximab轻链。
3.一种分离的核苷酸分子,编码组成上述新型CD20抗体的四条肽链,分别为Rituximab重链突变体及Rituximab轻链所述核苷酸序列。
4.一种载体,含有上所述的新型CD20抗体或者新型CD20抗体核苷酸分子和所述核苷酸分子的序列操作性相连的表达调控序列,其中载体可以为pDR1,pcDNA3.1(+),pcDNA3.1/ZEO(+),pDHFR之一。
5.上述载体,为pDR1,pcDNA3.1(+),pcDNA3.1/ZEO(+),pDHFR之一。
6.一种宿主细胞,含有上述的载体,为真核细胞。
7.上述宿主细胞,为哺乳动物细胞。
8.上述宿主细胞,为CHO细胞。
9.一种组合物,含有上述新型CD20抗体,和药学上可接受的载体。
10. 上述新型CD20抗体在制备抗体肿瘤药物中的用途。
11. 上述组合物在制备抗肿瘤药物中的用途。
12.上述任一用途,还包括和其他的抗肿瘤药物联合使用。
本发明的目的是提供一类兼具I型和II型CD20抗体抗肿瘤优势的新型CD20抗体。该类抗体既保留了I型CD20抗体Rituximab诱导强烈的CDC杀伤作用,又兼具II型CD20抗体优势,能诱导强烈细胞死亡的新型CD20抗体,表现出比单药亲本抗体Rituximab更好的抗肿瘤活性。
本发明对CD20抗体结构与功能进行了深入理解,在此基础上,设计出兼具I型和II型CD20抗体优势的新型CD20抗体。为了证明该方法具有比CD20抗体Rituximab单药用药更好的疗效,本发明进行后续蛋白抗肿瘤作用的实验。
本发明中,任何合适的载体都可以使用,可以为pDR1,pcDNA3.1(+),pcDNA3.1/ZEO(+),pDHFR之一,表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列。
哺乳动物或昆虫宿主细胞培养系统可用于本发明的新型CD20抗体的表达,COS ,CHO,NS0,sf9及sf21等细胞均可适用于本发明。
可用的宿主细胞为含有上述载体的原核细胞,可以为DH5a,BL21(DE3),TG1之一。
本发明中公开的新型CD20抗体在表达条件下,培养上述的宿主细胞,从而表达,分离或纯化所述的新型CD20抗体。
可以利用亲和层析的方法对本发明公开的新型CD20抗体进行分离纯化,根据所利用的亲和柱的特性,可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的新型CD20抗体。
利用上述方法,可以将新型CD20抗体纯化为基本均一的物质,例如在SDS-PAGE电泳上为单一条带。
上述新型CD20抗体或制剂在制备抗癌的药物中应用,还包括和其它的抗肿瘤药物联合使用。
本发明公开上述新型CD20抗体,可以和药学上可以接受的辅料一起组成药物制剂组合物从而更稳定地发挥疗效,这些制剂可以保证本发明公开的新型CD20抗体氨基酸核心序列的构像完整性,同时还要保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。通常情况下,对于液体制剂,通常可以在2°C-8°C条件下保存至少稳定一年,对于冻干制剂,在30°C至少六个月保持稳定。在这里制剂可为制药领域常用的混悬、水针、冻干等制剂,优选水针或冻干制剂,对于本发明公开的上述新型CD20抗体的水针或冻干制剂,药学上可以接受的辅料包括表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合,其中表面活性剂包括非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80);poloxamer(如poloxamer 188);Triton;十二烷基硫酸钠(SDS);月桂硫酸钠;十四烷基、亚油基或十八烷基肌氨酸;Pluronics;MONAQUATTM等,其加入量应使双功能融合蛋白的颗粒化趋势最小,溶液稳定剂可以为糖类,包括还原性糖和非还原性糖,氨基酸类包括谷氨酸单钠或组氨酸,醇类包括三元醇、高级糖醇、丙二醇、聚乙二醇之一或其组合,溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态,等渗调节剂可以为氯化钠、甘露醇之一,缓冲液可以为TRIS、组氨酸缓冲液、磷酸盐缓冲液之一。
上述制剂为包含新型CD20抗体的组合物,在对包括人在内的动物给药后,抗肿瘤效果明显。具体来讲,对肿瘤的预防和/或治疗有效,可以作为抗肿瘤药物使用。
本发明中双靶向抗体及其组合物在对包括人在内的动物给药是,给药剂量因病人的年龄和体重,疾病特性和严重性,以及给药途径而异,可以参考动物实验的结果和种种情况,总给药量不能超过一定范围。具体讲静脉注射的剂量是0.1~3000mg/天。
本发明所称的抗肿瘤药物,指具有抑制和/或治疗肿瘤的药物,可以包括伴随肿瘤生长相关症状发展的延迟和/或这些症状严重程度的降低,它进一步还包括已存在的肿瘤生长伴随症状的减轻并防止其他症状的出现,还也减少或防止转移。
本发明公开的新型CD20抗体及其组合物还可以和其他的抗肿瘤药联合给药,用于肿瘤的治疗,这些抗肿瘤药包括1、细胞毒类药物(1)作用于DNA化学结构的药物:烷化剂如氮芥类、亚硝尿类、甲基磺酸酯类;铂类化合物如顺铂、卡铂和草酸铂等;丝裂霉素(MMC);(2)影响核酸合成的药物:二氢叶酸还原酶抑制剂如甲氨喋呤(MTX)和Alimta等;胸腺核苷合成酶抑制剂如氟尿嘧啶类(5FU、FT-207、卡培他滨)等;嘌呤核苷合成酶抑制剂如6-巯基嘌呤(6-MP)和6-TG等;核苷酸还原酶抑制剂如羟基脲(HU)等;DNA多聚酶抑制剂如阿糖胞苷(Ara-C)和健择(Gemz)等;(3)作用于核酸转录的药物:选择性作用于DNA模板,抑制DNA依赖RNA聚合酶,从而抑制RNA合成的药物如:放线菌素D、柔红霉素、阿霉素、表阿霉素、阿克拉霉素、光辉霉素等;(4)主要作用于微管蛋白合成的药物:紫杉醇、泰索帝、长春花碱、长春瑞滨、鬼臼硷类、高三尖杉酯碱;(5)其他细胞毒药:门冬酰胺酶主要抑制蛋白质的合成; 2、激素类抗雌激素:三苯氧胺、屈洛昔芬、依西美坦等;芳香化酶抑制剂:氨鲁米特、兰特隆、来曲唑、瑞宁德等;抗雄激素:氟它氨 RH-LH激动剂/拮抗剂:诺雷德、依那通等;3、生物反应调节剂:主要通过机体免疫功能抑制肿瘤干扰素;白细胞介素-2 ;胸腺肽类;4、单克隆抗体:美罗华(MabThera) ;Cetuximab (C225) ;赫赛汀(Trastuzumab) Bevacizumab (Avastin);5、其他括一些目前机制不明和有待进一步研究的药物;细胞分化诱导剂如维甲类;细胞凋亡诱导剂。本发明公开的双靶向抗体及其组合物可以和上述的抗肿瘤药物之一或其组合联合用药。
附图说明
图1. 新型CD20抗体的抗原结合活性。
图2. 新型CD20抗体的CDC杀伤活性。
图3. 新型CD20抗体诱导细胞死亡活性。
图4. 新型CD20抗体的ADCC杀伤活性。
图5. 新型CD20抗体的小鼠体内抗肿瘤活性。
具体实施方式
实施例 1. 新型CD20抗体重链可变区、轻链可变区基因的构建
以Rituximab抗体重链为模板,采用overlap PCR的方法在重链可变区第102位的位置引入突变点,分别突变为His、Leu、Met、Asn、Gln、Arg、Ser、Thr、Val、Trp,然后将Rituximab重链可变区突变体与抗体恒定区连接后,装入表达载体。SEQ ID NO:1显示Rituximab抗体重链可变区氨基酸序列,其核苷酸序列为SEQ ID NO:2;SEQ ID NO:3显示Rituximab抗体重链突变体H102YH可变区氨基酸序列,其核苷酸序列为SEQ ID NO:4;SEQID NO:5显示Rituximab抗体重链突变体H102YL可变区氨基酸序列,其核苷酸序列为SEQ IDNO:6;SEQ ID NO:7显示Rituximab抗体重链突变体H102YM可变区氨基酸序列,其核苷酸序列为SEQ ID NO:8;SEQ ID NO:9显示Rituximab抗体重链突变体H102YN可变区氨基酸序列,其核苷酸序列为SEQ ID NO:10;SEQ ID NO:11显示Rituximab抗体重链突变体H102YQ可变区氨基酸序列,其核苷酸序列为SEQ ID NO:12;SEQ ID NO:13显示Rituximab抗体重链突变体H102YR可变区氨基酸序列,其核苷酸序列为SEQ ID NO:14;SEQ ID NO:15显示Rituximab抗体重链突变体H102YS可变区氨基酸序列,其核苷酸序列为SEQ ID NO:16;SEQ ID NO:17显示Rituximab抗体重链突变体H102YT可变区氨基酸序列,其核苷酸序列为SEQ ID NO:18;SEQ ID NO:19显示Rituximab抗体重链突变体H102YV可变区氨基酸序列,其核苷酸序列为SEQ ID NO:20;SEQ ID NO:21显示Rituximab抗体重链突变体H102YW可变区氨基酸序列,其核苷酸序列为SEQ ID NO:22; SEQ ID NO:23显示Rituximab抗体轻链可变区氨基酸序列,其核苷酸序列为SEQ ID NO:24。
实施例2. 抗体重链恒定区、轻链恒定区的克隆
用淋巴细胞分离液(鼎国生物技术发展公司产品)分离健康人淋巴细胞,用Trizol试剂(Invitrogen公司产品)提取总RNA,根据文献(Cloned human and mouse kappaimmunoglobulin constant and J region genes conserve homology in functionalsegments. Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P. Cell. 1980Nov;22(1 Pt 1):197-207.)和文献(The nucleotide sequence of a humanimmunoglobulin C gamma1 gene. Ellison JW, Berson BJ, Hood LE. Nucleic AcidsRes. 1982 Jul 10;10(13):4071-9.)采用RT-PCR反应扩增抗体重链恒定区和轻链恒定区基因。PCR产物经琼脂糖凝胶电泳纯化回收并克隆到pGEM-T载体中,测序验证后确认获得了正确的克隆。SEQ ID NO:25显示重链恒定区氨基酸序列,其核苷酸序列为SEQ ID NO:26;SEQ ID NO:27显示轻链恒定区氨基酸序列,其核苷酸序列为SEQ ID NO:28。
实施例3.新型CD20抗体轻链的构建
以实施例1获得的Rituximab轻链可变区核苷酸序列(SEQ ID NO:24)和实施例2获得的抗体轻链恒定区核苷酸序列(SEQ ID NO:28)为模板,采用overlap PCR的方法将Rituximab轻链可变区核苷酸序列与抗体轻链恒定区核苷酸序列进行融合,然后装入表达载体。SEQ ID NO:29显示新型CD20抗体轻链氨基酸序列,其核苷酸序列为SEQ ID NO:30。
实施例4.新型CD20抗体重链的构建
以实施例1获得的新型CD20抗体重链可变区核苷酸序列(SEQ ID NO:4、6、8、10、12、14、16、18、20、22)和实施例2获得的抗体重链恒定区核苷酸序列(SEQ ID NO:25)为模板,采用overlap PCR的方法将新型CD20抗体重链可变区核苷酸序列与抗体重链恒定区核苷酸序列进行融合,然后装入表达载体。SEQ ID NO:31显示新型CD20抗体H102YH重链氨基酸序列,其核苷酸序列为SEQ ID NO:32。SEQ ID NO:33显示新型CD20抗体H102YL重链氨基酸序列,其核苷酸序列为SEQ ID NO:34。SEQ ID NO:35显示新型CD20抗体H102YM重链氨基酸序列,其核苷酸序列为SEQ ID NO:36。SEQ ID NO:37显示新型CD20抗体H102YN重链氨基酸序列,其核苷酸序列为SEQ ID NO:38。SEQ ID NO:39显示新型CD20抗体H102YQ重链氨基酸序列,其核苷酸序列为SEQ ID NO:40。SEQ ID NO:41显示新型CD20抗体H102YR重链氨基酸序列,其核苷酸序列为SEQ ID NO:42。SEQ ID NO:43显示新型CD20抗体H102YS重链氨基酸序列,其核苷酸序列为SEQ ID NO:44。SEQ ID NO:45显示新型CD20抗体H102YT重链氨基酸序列,其核苷酸序列为SEQ ID NO:46。SEQ ID NO:47显示新型CD20抗体H102YV重链氨基酸序列,其核苷酸序列为SEQ ID NO:48。SEQ ID NO:49显示新型CD20抗体H102YW重链氨基酸序列,其核苷酸序列为SEQ ID NO:50。
实施例5. 新型CD20抗体的表达与纯化
于3.5cm 组织培养皿中接种 3 × 105 CHO-K1 细胞(ATCC CRL-9618),细胞培养至90%-95% 融合时进行转染:取质粒10μg 新型CD20抗体重链(SEQ ID NO:32、34、36、38、40、42、44、46、48、50)以及4μg Rituximab轻链(SEQ ID NO:30)和20μl Lipofectamine2000Reagent(Invitrogen公司产品)分别溶于500μl无血清DMEM培养基,室温静置5分钟,将以上2种液体混合,室温孵育20分钟以使DNA-脂质体复合物形成,其间用3ml无血清的DMEM培养基替换培养皿中的含血清培养基,然后将形成的DNA-脂质体复合物加入到板中,CO2孵箱培养4小时后补加2ml含10%血清的DMEM完全培养基,置于CO2孵箱中继续培养。转染进行 24h后细胞换含 600μg/ml G418选择培养基筛选抗性克隆。取细胞培养上清用ELISA检测筛选高表达克隆:羊抗人IgG (Fc)包被于ELISA板,4°C过夜,用2%BSA-PBS于37°C封闭2h,加入待测的抗性克隆培养上清或标准品(Human myeloma IgG1,κ),37°C 温育2h,加入HRP-羊抗人IgG(κ)进行结合反应,37°C 温育1h,加入TMB于37°C作用5min,最后用H2SO4终止反应,测A450值。将筛选得到的高表达克隆用无血清培养基扩大培养,用Protein A亲和柱(GE公司产品)分离纯化新型CD20抗体。将纯化抗体用PBS进行透析,最后以紫外吸收法定量确定纯化后抗体的浓度。
实验例1. 新型CD20抗体结合活性检测
所有抗体亲和力通过流式细胞术的方法进行测定【Li B, Zhao L, Wang C, GuoH, Wu L, Zhang X, Qian W, Wang H, Guo Y. The protein-protein interfaceevolution acts in a similar way to antibody affinity maturation. J BiolChem2010; 285:3865–71.】。简要的说,纯化后的荧光标记的抗体按照浓度等比例稀释后,与CD20蛋白阳性的淋巴瘤细胞Raji (ATCC CCL-86)进行冰上孵育45分钟,洗涤两遍后进行流式细胞仪检测。根据不同浓度的抗体结合细胞后荧光强度的变化,进而评价抗体的结合活性。如图1所示,这些Rituximab的突变体表现出与亲本Rituximab抗体相似的结合活性。
实验例2. 新型CD20抗体的CDC杀伤活性
Raji细胞(ATCC CCL-86)与10μg/ml开始等两倍比稀释的11B8、Rituximab或其突变体于无酚红培养基在37℃培养1小时。按10%体积比加入人血清,于37℃继续培养4小时后,用非同位素细胞毒性试验检测试剂盒按产品说明书提供的方法进行分析,计算裂解细胞的百分数。其中未加入抗体的处理组作为实验阴性对照。如图2所示,Rituximab及其突变体都能够诱导相同程度的强烈的CDC杀伤。
实验例3. 新型CD20抗体诱导细胞死亡活性
Raji细胞(ATCC CCL-86)与10μg/ml开始等两倍比稀释的11B8、Rituximab抗体及其突变体在37℃孵箱培养48小时,洗细胞两遍后用Annexin V/PI试剂盒(BD公司产品)按照产品说明书检测细胞死亡百分率。其中,未加入抗体处理组作为实验阴性对照,11B8处理组作为实验阳性对照。如图3所示,II型CD20抗体11B8能够诱导强烈的细胞死亡,而I型CD20抗体不能够诱导强烈的细胞死亡。虽然亲本抗体Rituximab不能够诱导强烈的细胞死亡,这几个Rituximab的突变体都表现出强烈的细胞死亡诱导能力。
实验例4. 新型CD20抗体的ADCC杀伤活性
Raji细胞(ATCC CCL-86)和不同稀释度的11B8、Rituximab及其突变体、PBS或者同型对照抗体于无酚红培养基中在室温孵育1小时。按效靶比50:1加入新鲜分离的人外周血单个核细胞,于37℃继续孵育1小时后,最后采用CytoTox 96非同位素细胞毒性实验试剂盒(Promega公司产品)按产品说明书提供的方法进行分析,计算裂解细胞的百分数。其中PBS处理、同型对照抗体处理为实验阴性对照组。如图4所示,I型和II型CD20抗体都能够诱导强烈的ADCC杀伤作用。
实验例5. 新型CD20抗体的小鼠体内抗肿瘤活性
选取6到8周的雌性SCID小鼠,然后于0天时分别尾静脉接种CD20阳性的B淋巴瘤细胞Raji(ATCC CCL-86),5天后给荷瘤小鼠分别尾静脉注射PBS 或者10μg 、100μg、500μg的11B8、Rituximab、Rituximab突变体及无关抗体对照Her2抗体Trastuzumab。其中注射PBS的SCID小鼠分组作为实验对照组。定期观察各组小鼠的体重变化和肢体麻痹情况的出现与否,对小鼠的生存率进行分析,并对不同抗体的抗肿瘤疗效进行评价。如图5所示,兼具I型和II型CD20抗体优势的新型CD20抗体表现出比亲本I型CD20抗体Rituximab和II型CD20抗体更好的抗肿瘤活性。
Claims (12)
1.一种兼具I型和II型CD20抗体优势的CD20抗体,其特征是,所述抗体既保留了I型CD20抗体Rituximab诱导强烈的CDC杀伤作用,又兼具了II型CD20抗体诱导强烈的细胞死亡的作用,并且表现出比I型CD20抗体Rituximab和II型CD20抗体11B8更好的肿瘤杀伤效果;
并且,所述的抗体由四条肽链构成,分别为二条Rituximab重链突变体以及二条Rituximab轻链,并且所述的Rituximab重链突变体为Rituximab抗体重链突变体H102YQ或Rituximab抗体重链突变体H102YV;
其中,Rituximab抗体重链突变体H102YQ的重链可变区的氨基酸序列SEQ ID NO:11所示;
Rituximab抗体重链突变体H102YV的重链可变区的氨基酸序列如SEQ ID NO:19所示;
并且,所述的CD20抗体具有Rituximab抗体的轻链,其轻链可变区氨基酸序列如SEQ IDNO:23显示。
2.如权利要求1所述的CD20抗体,其特征在于,所述的CD20具有选自下组的重链突变体:
Rituximab抗体重链突变体H102YQ,其重链的氨基酸序列如SEQ ID NO:39所示;
Rituximab抗体重链突变体H102YV,其重链的氨基酸序列如SEQ ID NO:47所示;
并且,所述的CD20抗体具有Rituximab抗体的轻链,其轻链氨基酸序列如SEQ ID NO:29所示。
3.一种分离的核酸分子,其特征在于,所述核酸分子编码权利要求1所述的兼具I型和II型CD20抗体优势的CD20抗体,并且所述的核酸分子包括:
(a)选自下组的编码抗体轻链的核酸序列:SEQ ID NO:30;和
(b)选自下组的编码抗体重链的核酸序列:SEQ ID NO:40、48。
4.一种载体,其特征在于,所述载体中含有权利要求3所述的核酸分子和所述核酸分子的序列操作性相连的表达调控序列,其中载体为pcDNA3.1(+)、pcDNA3.1/ZEO(+)或pDHFR。
5.权利要求4所述的载体,其特征在于,所述载体为pcDNA3.l(+)或pcDNA3.1/ZEO(+)。
6.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求4所述的载体,并且所述宿主细胞为真核细胞。
7.权利要求6所述的宿主细胞,其特征在于,所述宿主细胞为哺乳动物细胞。
8.权利要求6所述的宿主细胞,其特征在于,所述宿主细胞为CHO细胞。
9.一种组合物,含有权利要求1所述的CD20抗体,和药学上可接受的载体。
10.权利要求1所述的CD20抗体在制备抗肿瘤药物中的用途。
11.一种如权利要求9所述的组合物在制备抗肿瘤药物中的用途。
12.权利要求11所述的用途,其特征在于,所述组合物还和其他的抗肿瘤药物联合使用。
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