CN111875704A - 一种egfr抗体及其应用 - Google Patents
一种egfr抗体及其应用 Download PDFInfo
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- CN111875704A CN111875704A CN202010577007.5A CN202010577007A CN111875704A CN 111875704 A CN111875704 A CN 111875704A CN 202010577007 A CN202010577007 A CN 202010577007A CN 111875704 A CN111875704 A CN 111875704A
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- antigen
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- binding fragment
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- egfr
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Abstract
本公开提供一种EGFR抗体及其应用。具体的,本公开在尼妥珠单抗的基础上通过构建突变库筛淘获得,得到一个具有更高亲和力、更高ADCC杀伤活性的抗EGFR抗体或其抗原结合片段并将其用于癌症治疗。
Description
技术领域
本公开属于生物技术领域,具体地,本公开提供一种EGFR抗体及其应用。
背景技术
表皮生长因子受体EGFR是细胞表面酪氨酸激酶家族的一员,广泛表达于表皮、多能细胞以及神经组织表面,在发育和分化过程中起关键作用。EGFR,或称HER1,c-erbB-1,是一个170kDa分子量的I型跨膜糖蛋白(ΜLlrich et al.,Nature,Vol.309,418-25,1986),其配体包括EGF,转化生长因子TGFa,肝素结合活性的类EGF生长因子以及其他多种激素及细胞生长因子(Singh,A.and Harris,R.,2005,CellμLar Signaling 17:1183-1193)。配体结合引起EGFR二聚化及磷酸化,并诱导酪氨酸激酶信号级联反应,DNA合成及细胞分裂,因此EGFR信号通路是细胞存活于增殖的重要环节。
EGFR在多种上皮肿瘤中异常激活,包括非小细胞肺癌,乳腺癌,结肠癌,头颈部癌症以及前列腺癌等(Adams,G.and Weiner,L.,2005,Nature Biotechnology,23:1147-1157)。 EGFR受体异常激活的机制包括受体过度高表达,基因扩增,激活性突变,受体高表和/或调控缺失(Baselga,J.and Arteaga,C.,2005,J.of Clin.Oncol.23:2445-2459)。EGFR的异常激活成为多种肿瘤发生发展的驱动因素之一,因此,抑制与降低EGFR活性成为一种重要的抗肿瘤的治疗策略。
尽管EGFR高表和/或活性异常是多种肿瘤的驱动因素,但单抗药物获准治疗的适应症范围却相对有限,这与药物活性和病人耐受均有关系。如何提高药物有效性,同时降低毒副作用,仍是这一领域的待解决问题。
发明内容
本发明提供一种抗EGFR抗体或其抗原结合片段。具体的,本公开在尼妥珠单抗(Nimotuzumab,或mR3)的基础上通过构建突变库筛淘获得,得到一个具有更高亲和力、更高ADCC杀伤活性的抗EGFR抗体或其抗原结合片段。尼妥珠单抗(商品名泰欣生)因其与西妥昔单抗相比独特的靶点结合位点,临床应用中表现出优越的安全性;然而,相应的,其药效也具备可以提升的空间。
所述抗EGFR抗体或其抗原结合片段,其包含重链可变区和轻链可变区,所述重链可变区的氨基酸序列为SEQ ID NO:3;所述轻链可变区的氨基酸序列为SEQ ID NO:4。
进一步地,所述重链可变区包含:如SEQ ID NO:7、8、9所述的重联HCDR1、 HCDR2、HCDR3;和/或
所述轻链可变区包含:如SEQ ID NO:10、11、12所述的轻链LCDR1、LCDR2、LCDR3。
在一些实施方式中,上述可变区序列通过设计构建突变库并淘选获得。具体步骤如下:
步骤一:设计突变库以尼妥珠单抗重链为母本序列,设计方案将其抗原互补决定区(CDR)CDR1、CDR2、 CDR3进行有偏向的突变,同时将可变区固定序列肽段也反向翻译为核苷酸序列,与突变区一起拆分为多段寡聚核苷酸(oligos),在前后相连的oligo间设计反向互补序列,分别于前后衔接的正义编码oligos有17-25个核苷酸的互补区段,这些反向互补序列将作为连接反应 (ligation)的模板。
设计尼妥珠单抗抗体重链基因突变库的编码DNA如SEQ ID NO:13所示。
正义编码寡核苷酸包含SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、所示序列。
合成反向互补序列(连接反应模板)包含如SEQ ID NO:21、SEQ ID NO:22、SEQ IDNO:23、SEQ ID NO:24、SEQ ID NO:25如SEQ ID NO:26所示序列。
上述寡核苷酸由生工生物工程(上海)股份有限公司合成。
步骤二:突变库的合成
将正义编码寡核苷酸和反向互补序列混合,并建立连接反应体系,依赖反向互补序列作为模板,将所有正义编码寡核苷酸连接,得到完整的突变库编码DNA(部分单链)。接下来,通过PCR扩增获得轻链可变区编码DNA,并通过重叠PCR的方法与突变抗体重链基因库进行PCR反应,上下游引物均带有SfiI内切酶位点,从而有助于使用SfiⅠ酶进行酶切与处理好的pComb3XSS载体进行连接,获得pComb3XSS-scFv突变抗体库。
将含有突变抗体库编码DNA的质粒经电转导入大肠杆菌TG1感受态细胞,培养扩增后,即可以保存菌种,做成细胞库。
步骤三:噬菌体抗体库包装和淘选
含有噬菌体抗体库大肠杆菌接菌至2xYT液体培养基,摇至对数期后加入M13 KO7辅助噬菌体,在合适的条件,如30℃,180rpm孵育过夜,则可以诱导噬菌体包装,同时scFv突变抗体整合进噬菌体颗粒,并展示在表面,每个噬菌体颗粒只展示一种独特型的scFv。收集噬菌体颗粒与抗原孵育,进而用磁珠抓取抗原-噬菌体抗体复合物,即可以富集具有结合活性的scFv噬菌体,这些噬菌体又可以侵染TG1细胞,制备成亚库。多次淘选后,得到亲和力较强的scFv集合。
步骤四:单克隆噬菌体抗体筛选
(1)单克隆噬菌体抗体包装
将多轮淘选后的大肠杆菌亚库涂平板,并从平板中挑取单克隆,在96孔圆底培养板中加入 300μL 2×YTAG培养基(含有100μg/ml氨苄青霉素ampicillin(A)和1%葡糖糖glucose (G))并以合适的方式诱导,则获得单克隆scFv噬菌体抗体,即培养板中每孔只含有一种特殊序列的scFv,以ELISA方法进行筛选。
(2)ELISA筛选
以EGFR胞外区包被96孔ELISA板,用正常的ELISA实验方法检测单克隆的scFv-噬菌体抗体。
经单克隆噬菌体抗体筛选,获得多个阳性序列,其中24H7的结合反应活性最高。
在一些实施方式中,所述抗体是人IgG1、IgG2、IgG3或IgG4形式的抗体或其抗原结合片段;
优选地,所述抗体是人IgG1形式的抗体或其抗原结合片段。
在一些实施方式中,所述抗体及其抗原结合片段是人源化的。
在一些实施方式中,所述抗原结合片段包括Fv、Fab、Fab’、F(ab’)2、双特异性抗体、多肽中的一种或几种组合。
在一些实施方式中,所述的抗EGFR抗体或其抗原结合片段是双特异性抗体,其包含:特异性结合EGFR的抗原结合部分,和特异性结合第二抗原的抗原结合部分;
优选的,所述第二抗原结合部分选自:PD-1、PD-L1、CD3、VEGF。
一方面,本公开还提供一种分离的抗体或其抗原结合片段,所述抗体与前述任一项所述的抗EGFR抗体或其抗原结合片段竞争性结合人EGFR,或与前述任一项所述的抗EGFR抗体或其抗原结合片段结合相同的人EGFR抗原表位。
在一些实施例中,所述抗体及其抗原结合片段Fc区是经过无岩藻糖修饰的。
进一步地,所述抗体及其抗原结合片段是经过N297无岩藻糖修饰的。
在一些实施方式中,本发明提供一种核苷酸序列或组合,所述核苷酸序列或组合编码前述的抗EGFR单克隆抗体或其抗原结合片段。
在一些实施方式中,本发明提供一种重组DNA表达载体,所述重组DNA表达载体包含如前所述的多核苷酸序列或组合。
在一些实施方式中,本发明提供一种转染如前所述的重组DNA表达载体的宿主细胞,所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞;
优选地,所述宿主细胞为哺乳动物细胞,所述哺乳动物细胞为HEK293F细胞、CHO细胞或CHO-K1细胞;
更优选地,所述宿主细胞为FUT8基因敲除的CHO-K1细胞。
具体地,所述FUT8基因敲除的CHO-K1细胞是基于CRISPR技术敲除CHO-K1细胞中FUT8基因的方法获得的。
本发明同时提供一种药物或药物组合物,其含有如前所述的抗EGFR单克隆抗体或其抗原结合片段,以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明提供一种包含如前所述的抗EGFR单克隆抗体或其抗原结合片段,或包如前所述的药物组合物,或如前所述的多核苷酸序列或组合在制备治疗肿瘤的药物中的用途;优选地,所述癌症疾病包括皮肤鳞癌、鳞状非小细胞肺癌、非鳞状非小细胞肺癌、鳞状小细胞肺癌、结直肠癌、头颈部癌、头神经胶质瘤、食管癌、胃癌、胰腺癌。
有益效果
本发明提供的EGFR抗体或其抗原结合片段,是在尼妥珠单抗的序列基础上,通过构建突变库筛淘获得,筛淘获得的EGFR抗体序列仅在重链可变区第37位氨基酸产生突变,该突变位点不属于互补决定区(complementarity-determining region,CDR),且仅仅发生了一个位点的突变,但亲和力比mR3提高了10倍,产生了预料不到的技术效果。
另一方面,本公开通过去除岩藻糖糖基化技术增强抗体的ADCC(抗体依赖的细胞毒性)活性。
附图说明
图1:ELISA检测24H7与EGFR结合活性
图2:ELISA检测EGFR/EGF结合阻断活性
图3:FACS检测24H7与A431结合活性
图4:FITC-LCA流式检测加压恢复的多克隆细胞群
图5:FITC-LCA流式检测阳性克隆株
图6:24H7-AF基于PBMC的ADCC活性
图7:报告基因法测定24H7-AF的ADCC活性
图8:报告基因法测定24H7与24H7-AF按不同比例混合稀释的ADCC活性
图9:24H7-AF在小鼠移植瘤模型中的抑瘤活性图
图10:实施例1突变库理论氨基酸多样性及频次
图11:实施例8SPR法测定24H7与EGFR的亲和力
图12:实施例9酶切lentiCRISPRv2质粒体系
图13:实施例9磷酸化并退火单链oligos体系
图14:实施例9连接反应体系
具体实施例:
一、术语说明
以下对本发明做进一步描述:
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时,为了更好地理解本发明,本公开提供相关术语的定义和解释如下:
在本发明中,术语“抗体”,指单克隆抗体。是指通常由两对相同的多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε,并且分别将抗体的同种型定义为IgM、IgD、IgG、 IgA和IgE。进一步被划分为亚类(同种型),例如,IgG1,IgG2,IgG3,IgG4,IgA1和 IgA2。“IgG形式的抗体”是指抗体的重链恒定区所属于的IgG形式。所有同一型的抗体的重链恒定区都是相同的,不同型的抗体之间的重链恒定区不同。例如,IgG1形式的抗体是指其重链恒定区Ig结构域为IgG1的Ig结构域。
在一些情况下,“抗原结合片段”也称抗体片段,指与完整抗体不同的分子,其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fv、Fab、Fab’、F(ab’)2;双抗体;线性抗体;单链抗体(例如scFv);单结构域抗体;双价或双特异性抗体或其片段;多肽;和由抗体片段形成的双特异性抗体或多特异性抗体及其片段。
在本发明中,术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段;术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段;术语Fab’片段是指抗体的单价抗原结合片段,其由一条轻链(包括可变区和恒定区)和一条重链的可变区和第一恒定区经二硫键结合组成。术语“F(ab')2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。“scFv”是指由轻链可变区与重链可变区直接相连或通过一个多肽连接子序列连接而成的工程化抗体。
“双特异性抗,指其能够分别和两种抗原或抗原表位结合,其包括特异性结合第一抗原的抗体的轻链、重链或其抗原结合部分,以及特异性结合第二抗原的抗体的轻链、重链或其抗原结合部分。在本发明的实施方案中,所述双特异性抗体中结合第一抗原的抗体的轻链、重链或其抗原结合部分可以为本发明任一项的抗体或其抗原结合部分,所述特异性结合第二抗原的抗体的轻链、重链或其抗原结合部分可以为其它抗EGFR抗体或其抗原结合部分或者针对其它抗原的抗体或其抗原结合部分。
在本发明中,术语“载体”指的是,可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞,使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说,载体包括:质粒;噬菌粒;柯斯质粒;
在本发明中,术语“宿主细胞”指的是导入载体的细胞,所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞;所述哺乳动物细胞为HEK293F细胞、CHO细胞或CHO- K1细胞;CHO-K1 FUT8-/-细胞为FUT8基因敲除的CHO-K1细胞。
在本发明中,“特异性结合”指的是,指两分子间的非随机结合反应,如抗体和产生该抗体的抗原间的反应。此处,结合第一种抗原的抗体对第二种抗原的结合亲和力是检测不到或很弱。在某些实施方式中,一个某抗原特异性抗体是指以亲和力(KD)≤10-5M(如10-6M、10-7M、10-8M、10-9M、10-10M等)结合该抗原,其中KD指解离率与结合率的比值 (koff/kon),其可以采用本领域技术人员熟悉的方法进行测定。
在本发明中,所述ADCC活性是指表达IgG Fc受体的NK细胞、巨噬细胞和中性粒细胞等效应细胞,通过与已结合在病毒感染细胞和肿瘤细胞等靶细胞表面的IgG抗体的Fc 段结合,而杀伤这些靶细胞的作用。
在本发明中,所述对细胞的驯化培养是指使细胞能够在新培养基中生长繁殖的过程。具体的,本公开所述的“渐进式驯化”即连续驯化,细胞先在含10%FBS的F-12K培养基中培养,每隔2代降低一次血清浓度(5%,2.5%,1%)。FBS浓度降低至1%时,再按照F- 12K+1%FBS:CD CHO Medium=8:2、6:4、4:6、2:8、0:10每个梯度传代2次(期间根据情况补加L-谷氨酰胺),最后转移至摇瓶进行适应性传代培养
在本发明中,20种常规氨基酸和其缩写遵从常规用法。
二、实施例
实施例1突变库设计
以尼妥珠单抗重链为母本序列,设计方案将其抗原互补决定区(CDR)CDR1、CDR2、 CDR3进行有偏向的突变,突变频次参考Sidhu SS等(J.Mol.Biol.(2004)338,299–310)。具体的,按照Kobat抗体序列氨基酸编码法则中的第31-35氨基酸(CDR1),第37氨基酸(二级结构转折氨基酸),51-55(CDR2,部分)以及101-105(CDR3,部分)分别按表中所列分配编码核苷酸序列,其理论氨基酸多样性及频次亦如图10所示。
*氨基酸种类以标准命名规则中的单字母表示。
**V=A,C或G,R=A或G,D=A,G或T,M=A或C,K=G或T,N=A,G,C或T。
将可变区固定序列肽段也反向翻译为核苷酸序列,与突变区一起拆分为多段寡聚核苷酸(oligos),在前后相连的oligo间设计反向互补序列,分别于前后衔接的正义编码oligos有17-25个核苷酸的互补区段,这些反向互补序列将作为连接反应(ligation)的模板。
设计尼妥珠单抗抗体重链基因突变库的编码DNA如SEQ ID NO:13所示。
正义编码寡核苷酸包含SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、所示序列。
合成反向互补序列(连接反应模板)包含如SEQ ID NO:21、SEQ ID NO:22、SEQ IDNO:23、SEQ ID NO:24、SEQ ID NO:25如SEQ ID NO:26所示序列。
上述寡核苷酸由生工生物工程(上海)股份有限公司合成。
实施例2突变库的合成
2.1、DNA片段连接
将正义编码寡核苷酸和反向互补序列混合,并建立连接反应体系(50μL)如下:
组成成分 | 体系 |
全部寡核苷酸(每种1微升) | 13μL |
T4 DNA ligase | 2.5μL |
10x T4 DNA连接酶反应缓冲液 | 4.5μL |
水 | 30μL |
总体积 | 50μL |
反应条件 | 16℃过夜连接 |
热失活 | 60℃加热30分钟 |
2.2、构建抗体片段突变库
通过PCR扩增获得轻链可变区编码DNA,并通过重叠PCR的方法与突变抗体重链基因库进行PCR反应体系如下。
将产物取1μL进行电泳用Axygen AP-PCR-250G纯化回收到的片段,测得回收片段浓度为:152ng/μL。
2.3、用SfiⅠ酶切构建好的ScFv突变抗体库DNA和pComb 3XSS载体并将其回收,ScFv突变抗体库DNA用Axygen AP-PCR-250G进行纯化回收,回收后取1μL跑电泳,载体跑胶回收。
2.4、将ScFv突变DNA库与载体pComb 3xss16℃过夜连接,连接体系如下:
将连接产物进行PCR进行琼脂糖凝胶电泳回收。
实施例3突变库转化大肠杆菌
3.1制备电转感受态
从M9平板上挑取TG1单克隆接入40ml 2TY培养基中,37OC 250rpm培养过夜。
将5ml菌液接种500ml 2×YT培养基,37℃250rpm振荡培养。
待TG1菌液OD600约为0.5左右,将其从摇床中取出,冰浴1小时。
2200×g,4℃,菌液离心12分钟。
弃上清,用500ml预冷的去离子水重悬沉淀。2200×g,4℃,离心12分钟。
弃上清,用250ml预冷的去离子水重悬沉淀。2200×g,4℃,离心12分钟。
弃上清,用30ml预冷的10%的甘油重悬沉淀。2200×g,4℃,离心12分钟。
弃上清,用1ml预冷的10%的甘油重悬沉淀。重悬后总体积约为2ml。
3.2往100μL感受态加入100ng的ScFv突变抗体库DNA和pComb3XSS载体连接产物;混匀冰上放置10分钟后开始电转。电转完立即加入1ml预热的SOC培养液,将完成电转的感受态全部加入到SOC培养液中(总共2ml)摇1h。取20μL梯度稀释,稀释到 106倍涂板,37℃生化培养箱内过夜培养。
实施例4噬菌体抗体库包装和淘选
第一轮噬菌体抗体库淘选
4.1噬菌体包装
将10个平板的菌全部刮下来混匀在一起总体积240ml,取2ml接入到300ml中,长至OD600=0.5左右加入300μL的M13K07辅助噬菌体侵染30min(37℃水浴静置)。
将溶液倒入离心管中,3300Xg离心10分钟,弃上清。
使用300ml 2×YT培养基(含100μg/ml ampicillin,50μg/ml kanamycin,0.1%glucose)重悬,30℃180rpm孵育过夜,以获得scFv-phage。
4.2噬菌体第一轮筛选
将过夜的菌收集在50ml离心管中,3300g离心30min,收集上清。
向上清中加入60mlPEG800和60mlNaCl冰上轻摇1h,4℃3300g离心30min。倒掉上清,将管子倒置或者再次离心2min并小心吸去全部PEG/NaCl。
离心下来的噬菌体最后用8ml的PBS重悬。11600Xg离心10min(取10μg梯度10 倍稀释然后侵染准备的TG1菌30min,37℃过夜培养计数)。
Tube1:在1.5ml EP管中加入100μL链霉亲和素包被的磁珠和1ml MPBS。室温下摇晃(end over end)1h。
Tube2:在1.5ml EP管中加入scFv-phage和100μL链霉亲和素包被的磁珠至1mlMPBS溶液中(900μL phage+100μL PBS-20%milk)。室温下摇晃1h。(900μL噬菌体来自洗脱下来的噬菌体中)
Tube3:在1.5ml EP管中加入1ml 2%MPBS以封闭EP管,减少非特异性结合。室温下摇晃(end over end)1h,弃MPBS。
取Tube2,放置在磁力架上,取上清加入至Tube3中。
取Tube3,向其中加入2μg生物素化抗原(带有his标签),室温下摇晃1h。
取Tube1,取100μL磁珠加入Tube3中,室温下摇晃15min。
取Tube3,放置在磁力架上,吸去上清。加入1ml PBST(含0.1%Tween-20),翻转多次(10s),重复10次。
加入500μL的trypsin stock solution(浓度1mg/ml),室温下摇晃15min。放置在磁力架上,吸出上清至一新EP管中。
取洗下来的噬菌体250μL侵染提前准备的TG1(OD=0.5左右)1.75ml在37℃中静置30min,然后全部涂在大平板上。(含有100μg/ml氨苄青霉素)
取洗脱下来的噬菌体,梯度10倍稀释,然后侵染准备的TG1菌30min,涂板37℃过夜孵育以测试滴度。
第一轮滴度计算,结果如下:
洗脱下来的噬菌体计数约为8×1014,第一轮筛选下来的噬菌体数为1×108,对照TG1在平板上没有菌落,无污染。
第二轮噬菌体抗体库淘选与第一轮操作步骤相同,但起始库采用是第一轮淘选富集的亚库。
实施例5.单克隆噬菌体抗体筛选
5.1单克隆噬菌体抗体包装
从平板中挑取单克隆,在96孔圆底培养板中加入300μL 2×YTAG培养基(containing100μ g/ml ampicillin,1%glucose),再挑取单克隆菌落加入各孔中,共挑取92个,37℃180rpm过夜孵育。
取一块新的96孔圆底培养板,并加入100μL的2×YTAG培养基,使用多通道移液器,取2μL中培养液加入新培养基中,37℃180rpm孵育2h。
取12μL辅助噬菌体加入到3.6ml的2×YTAG培养基中,每孔加入30μL。静止孵育1h。
加入400μL induction medium(2TY containing 100μg/ml ampicillin,50μg/mlkanamycin),30℃180rpm孵育过夜。
第二天将96孔培养的菌4000rpm离心10min用于ELISA检测。
5.2ELISA检测
1μg/ml抗原向96孔板中加入50μL,37℃包被2h,PBST洗3遍。
250μL/孔5%脱脂牛奶37℃封闭2h,PBST洗3遍。
培养液上清用1%BSA进行1:1稀释,然后将此稀释液加入板孔,60μL/孔,37℃孵育2h,PBST洗5遍。
加入50μL/孔1:4000Anti-M13 HRP(1%BSA稀释),37℃孵育1h,PBST洗5遍。
加入50μL/孔TMB显色5min,加入50μL/孔2摩尔硫酸终止显色,读取OD450吸光值。
经单克隆噬菌体抗体筛选,获得多个阳性序列,其中24H7的结合反应活性最高。
经测序获得24H7的核酸序列如SEQ ID NO.1所示。
翻译获得其氨基酸序列如SEQ IDNO.2所示。
其中,24H7重链可变区序列为SEQ ID NO.3,轻链可变区序列为SEQ ID NO.4。
所述重链可变区的HCDR1序列为SEQ ID NO:7、HCDR2序列为SEQ ID NO:8、 HCDR3序列为SEQ ID NO:9;所述轻链可变区的LCDR1序列为SEQ ID NO:10、LCDR2序列为SEQ IDNO:11、LCDR3序列为SEQ ID NO:12。
具体地,
SEQ ID NO.2: QVQLQQPGAELVKPGASVKLSCKASGYTFTNYYIYWFKQRPGQGLEWIGGINPTSGGSNFN EKFKTKATLTVDESSTTAYMQLSSLTSEDSAVYYCTRQGLWFDSDGRGFDFWGQGTTLTVS SGGGGSGGGGSGGGGSDVLMTQIPLSLPVSLGDQASISCRSSQNIVHSNGNTYLDWYLQKP GQSPNLLIYKVSNRFSGVPDRFRGSGSGTDFTLKISRVEAEDLGVYYCFQYSHVPWTFGGG TKLEIKRGQAGQHHHHHH
SEQ ID NO.3:
QVQLQQPGAELVKPGASVKLSCKASGYTFTNYYIYWFKQRPGQGLEWIGGINPTSGGSNFN EKFKTKATLTVDESSTTAYMQLSSLTSEDSAVYYCTRQGLWFDSDGRGFDFWGQGTTLTVS S
SEQ ID NO.4:
DVLMTQIPLSLPVSLGDQASISCRSSQNIVHSNGNTYLDWYLQKPGQSPNLLIYKVSNRFSG VPDRFRGSGSGTDFTLKISRVEAEDLGVYYCFQYSHVPWTFGGGTKLEIK
SEQ ID NO.7:GYTFTNYY
SEQ ID NO.8:INPTSGGS
SEQ ID NO.9:TRQGLWFDSDGRGFDF
SEQ ID NO.10:QNIVHSNGNTY
SEQ ID NO.11:KVS
SEQ ID NO.12:FQYSHVPWT
实施例6全长抗体构建
本实施例中的抗体重链可变区可与选自人IgG1、IgG2、IgG3或IgG4、或其变体的重链恒定区连接形成抗体全长重链;抗体轻链可变区可与选自人κ链或λ链、或其变体的轻链恒定区连接形成抗体全长轻链。
另一方面,本实施例还提供一种分离的抗体或其抗原结合片段,所述抗体与前述任一项所述的抗EGFR抗体或其抗原结合片段竞争性结合人EGFR,或与前述任一项所述的抗EGFR抗体或其抗原结合片段结合相同的人EGFR抗原表位。
示例性的,本公开抗体可选自如下所示轻/重链恒定区:
IgG1重链恒定区(SEQ ID NO:27) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
IgG4重链恒定区(SEQ ID NO:28):
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK
kappa轻链恒定区(SEQ ID NO:29) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
下面以IgG1为重链恒定区和kappa轻链恒定区为示例进行详细说明。
在构建全长24H7抗体时,用基因工程的方法将全长重链编码基因(如SEQ ID NO.5所示)构建到表达载体pHr并与带有轻链编码基因(如SEQ ID NO.6所示)的表达载体共转染入293F细胞,进行表达。全长抗体表达完成后分泌到细胞培养上清,收获后以Protein A进行亲和纯化。
实施例7:24H7生化活性测定
7.1、ELISA检测24H7与EGFR结合活性
用50μL 1μg/mlEGFR包被酶标板,37℃孵育2h,弃去孔内液体,用200μL PBST(含0.05%Tween20)漂洗5次;每孔加入200μL封闭液(含0.05%tween20的PBST配制的5%牛奶),37℃封闭2h;弃去孔内液体,漂洗5次。将24H7和对照抗体稀释(0.05%PBST配制1%BSA稀释液)到10μg/ml作为起始浓度,5倍梯度稀释8个浓度点(包含0点),取各浓度点50μL加入酶标板中,37℃孵育1h后漂洗5次;每孔加入50μL Anti-human IgG Fc- HRP(0.05%PBST配制1%BSA稀释液),37℃孵育1h后漂洗5次;每孔加入50μL TMB 显色液,37℃避光孵育10min;每孔加入50μL 2M H2SO4终止反应。读取紫外波长450nm 下的光吸收。结果如图1:ELISA检测24H7与EGFR结合活性。
7.2、ELISA检测EGFR/EGF结合阻断活性
用50μL 10μg/ml EGFR包被酶标板,37℃孵育2h;弃去孔内液体,用200μL PBST(含0.05%Tween20)漂洗5次;每孔加入200μL封闭液(含0.05%tween20的PBST配制的5%牛奶),37℃封闭2h;弃去孔内液体后漂洗5次。将24H7和对照抗体稀释(0.05%PBST配制1%BSA稀释液)到20μg/ml,作为起始浓度,5倍梯度稀释8个浓度点(包括0点),取各浓度孔50μL到酶标板孔中,37℃预孵育30min后漂洗5次。
将EGF稀释(0.05%PBST配制1%BSA稀释液)到4μg/ml,同时将抗体稀释 (0.05%PBST配制1%BSA稀释液)到40μg/ml作为起始浓度,5倍梯度稀释8个浓度点 (包含0点)。将EGF与各浓度抗体等体积混合,使EGF工作浓度为2μg/ml,待测抗体起始浓度为20μg/ml。取各浓度混合液50μL加入到酶标板相应孔中。37℃孵育1h后漂洗5 次;每孔加入50μL Anti-Mouse IgG-HRP(0.05%PBST配制1%BSA稀释液),37℃孵育 1h后漂洗5次;每孔加入50μLTMB显色液,37℃避光孵育10min;每孔加入50μL 2M H2SO4终止反应。读取紫外波长450nm下的光吸收。结果如图2:ELISA检测EGFR/EGF 结合阻断活性。
7.3、FACS检测24H7与A431结合活性
胰酶消化A431细胞,收集细胞离心去上清,重悬计数,将细胞密度调整到1x106/ml;按100 μL细胞悬液每管分装到离心管,1000rpm离心2min,去上清,用buffer(1xPBS,1%BSA)重悬,离心去上清。将待测抗体稀释到20μg/ml作为起始浓度,5倍梯度稀释8个浓度点 (包含0点),取各浓度50μL加到细胞沉淀中,重悬细胞,混匀,4℃孵育30min; 1000rpm离心2min,buffer洗一次,去上清;加人50μL PE-GoatAntiHumanIgG到细胞沉淀中重悬细胞,4℃孵育30min后离心,buffer洗一次,约200μL buffer重悬。用流式细胞仪检测PE荧光强度结果如图3所示。
实施例8SPR法测定24H7与EGFR的亲和力
使用BIACORE X100 PLUS对24H7与EGFR胞外区的亲和活性进行定量测定。实验中,使用CM5芯片偶联山羊抗人IgG(Fc)抗体,加载24H7之后,将不同浓度的带有His6标签的EGFR保外区蛋白作为分析物,进行结合和解离实验。最后模拟得到二者的亲和力KD数值为5.686E-9摩尔,比mR3(KD数值为5.92E-8)提高10倍。具体数据见图11
实施例9:CHO-K1FUT8-/-细胞构建方法及24H7-AF分子的制备
本专利提供一种基于CRISPR技术敲除CHO-K1细胞中FUT8基因的方法。
9.1:针对FUT8基因的序列,设计引导CAS9核酸内切酶切割的sgRNA序列针对FUT8基因第一个外显子的sgRNA有2个:1- AATGAGCATAATCCAACGCCAGG(SEQ ID NO:27)2-AATGGCTGAGTCTCTCCGGTAGG(SEQ ID NO:28),针对FUT8基因第二个外显子的 sgRNA有1个:TAGCTGTCCCCTGATCAATAGGG(SEQ ID NO:29)。
9.2:将上述sgRNA构建至lentiCRISPRv2质粒中
9.2.1酶切lentiCRISPRv2质粒体系如图12所示。
9.2.2凝胶纯化回收目的大片段。
9.2.3磷酸化并退火单链oligos体系如图13所示。
9.2.4用无菌水将2.3中的退火产物按照1:200的比例稀释。
9.2.5连接反应体系如图14所示
9.2.6取5μl 2.5中连接产物进行转化(stbl3),待抗性基因表达后,将菌液均匀地涂于含有氨苄抗生素的LB固体平板上,放于37℃细菌培养箱中过夜培养16-20h。挑取3-6个单克隆菌落转入含有氨苄抗生素的LB液体培养基中,37℃,200rpm,摇床过夜培养16-20h。提取菌体中的质粒,进行测序,验证之后质粒保存待用。
9.3三种sgRNA质粒共转染CHO-K1细胞,筛选阳性单克隆按照1.3x105/ml将贴壁培养的CHO-K1细胞接种于六孔板中,每孔中细胞悬液的体积为 2.5ml。按照3μg质粒(三种sgRNA质粒各1μg):9μl PEI溶液(sigma,1mg/ml)进行转染。转染48h后,换液加压(2.5ml完全培养液+8μg/ml puromycin),根据细胞的状态每隔2天换液一次,约7-10天后多克隆细胞长出,扩大培养后,流式检测(FITC-LCA)阳性率(83.86%),如图4。通过有限稀释法获得的单克隆细胞转移至96孔板中培养2周左右时间,流式检测多个单克隆细胞后确定阳性细胞(图5)。将上述细胞扩大培养后,提取基因组DNA,针对sgRNA作用区域的上下游设计引物进行常规PCR,将产物进行T7E1酶切以及测序,最终确定CHO-K1 FUT8-17细胞实现了FUT8基因的敲除。
9.4利用渐进式的方式将CHO-K1 FUT8-17细胞株驯化至CD CHO Medium中悬浮培养。
瞬转表达获得24H7-AF分子,具体方法如下:常规培养CHO-K1 FUT8-/-细胞(即上述成功构建的CHO-K1 FUT8-17细胞),传代并扩增CHO-K1 FUT8-/-细胞,待细胞长到对数生长期,此时细胞密度为1.5-3×106cells/ml,细胞活率在98%以上。利用新鲜预热至37℃的CD CHO Medium(添加终浓度为8mM的L-谷氨酰胺)按照2×106cells/ml的密度稀释上述细胞。严格按照Thermo ExpiCHO表达系统(A29133)试剂盒中的标准滴度方案进行瞬时转染以及补料的添加,转染6-7天后收集细胞培养上清用Protein A进行亲和纯化后获得 24H7-AF抗体分子,经SDS-PAGE以及HPLC检测其纯度>95%。
糖谱测定证实,岩藻糖糖基化类型为N297无岩藻糖修饰。
实施例10:24H7-AF抗体介导的细胞毒杀伤活性检测
10.1、测定24H7-AF基于PBMC的ADCC活性
从供血志愿者采集血液50ml,并以Ficoll法分离制备人外周血单核细胞(peripheralblood monocyte cells,PBMC),以添加血清的1640培养基进行培养。第二天将A431用胰酶消化,用培养基清洗细胞1次,用培养基将细胞密度调整到1x106/ml,向1ml细胞悬液中加入2μ L DELFIA BATDA,37℃孵育25min。用培养基清洗细胞3次,重悬,调整细胞密度到5x104/ml,100μL每孔加入到96孔细胞培养板中。将24H7-AF和对照抗体用培养基稀释到200μg/ml、40μg/ml和8μg/ml,各加入50μl到相应孔中。将50μL2.5×106/ml浓度的 PBMC细胞(E:T=25:1)加到各孔中,自发释放量和最大释放量孔用培养基补到200μL,同时靶细胞最大释放量孔再加入10μL裂解液,37℃孵育2h。孔板放进孔板离心机中离心 500rpm 5min;每孔取20μL上清转至另一个96不透明平底白板,并加入200μL铕溶液,室温在摇板机上振动孵育15min后测量荧光强度(TRF),结果如图6。
10.2、报告基因法测定24H7-AF的ADCC活性24H7-AF基于PBMC的ADCC活性胰酶消化A431细胞,收集细胞离心去上清,重悬计数,将细胞密度调整到8.3x104/ml,100 μL每孔铺到96孔不透明细胞培养板中,第二天,去上清,PBS清洗一次,加入25μL培养基。将24H7-AF和对照抗体用培养基稀释到60μg/ml作为起始浓度,5倍梯度稀释8个浓度点,取各浓度点25μL加入到96孔板中,37℃5%CO2培养箱孵育45min。收集ADCC Report Cell,按照效靶比3:1取25μL 1x106/ml密度的细胞加入孔板中。37℃5%CO2培养箱孵育6h后,室温平衡15min。每孔加入荧光检测试剂75μL,室温避光孵育5min,检测荧光信号,结果如图7。
将24H7与24H7-A按不同比例混合稀释,按照上述方法进行检测,结果如图8。
实施例11:24H7-AF在小鼠移植瘤模型中的抑瘤活性
为了探索该分子在动物体内的抑瘤活性,我们采用了在免疫缺陷小鼠体内移植A431肿瘤细胞的方法,构建了肿瘤模型。并在肿瘤体积为430mm3的时候,给小鼠注入人PBMC,同时以10mg/Kg的剂量给小鼠进行腹腔注射抗体药物或对照品。PBMC和抗体药物的注射频率均为每周两次。
受试药TA1(IgG control),TA2(西妥昔抗体),TA3(24H7-AF)在10mg/kg给药剂量,单独用药在PBMC NK人源化小鼠模型上的药效研究显示:TA2,TA3显现出良好的、具备可比性的药效,如图9所示。
本发明不局限于上述最佳实施方式,任何人在本发明的启示下都可得出其他各种形式的产品,但不论在其形状或结构上作任何变化,凡是具有与本申请相同或相近似的技术方案,均落在本发明的保护范围之内。
序列表
<110> 白先宏
<120> 一种EGFR抗体及其应用
<160> 29
<170> SIPOSequenceListing 1.0
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atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtggcccag 60
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aagctgagct gtaaggcctc cggctatact ttcaccaatt attacatcta ctggttcaag 180
cagagacctg gccagggcct ggaatggatc gggggaatca accccacatc tggcgggtcc 240
aacttcaacg agaagtttaa aaccaaggcc accctgaccg tggacgagag ttccaccacc 300
gcctacatgc agctgtcttc cctgacctcc gaggactccg ccgtctacta ttgcaccagg 360
cagggcctgt ggttcgactc cgacggacgc ggcttcgact tctgggggca gggcaccacc 420
ctgaccgtct cctccggtgg tggcggttca ggcggaggtg gctctggcgg tggcggatcg 480
gacgtgctga tgacccagat ccccctgtcc ctgcccgtga gcctgggcga tcaggcctcc 540
atctcttgcc ggtcctccca gaacattgtg cactccaacg ggaacacata cctggactgg 600
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gcctccatct cttgccggtc ctcccagaac attgtgcact ccaacgggaa cacatacctg 180
gactggtacc tgcagaaacc cggccagtcc cccaacctgc tgatctacaa ggtgtccaac 240
cggttctccg gggtccccga caggttcaga gggtccggct ccggaaccga cttcaccctg 300
aaaatctcca gagtggaagc tgaggacctg ggcgtgtact actgcttcca gtactcccat 360
gtgccctgga cctttggagg cggtaccaag ctggaaatca aaagaaccgt ggccgccccc 420
tccgtgttca ttttcccccc ctccgacgag cagctgaaat ccggcaccgc ctccgtcgtg 480
tgcctgctga acaattttta ccccagggaa gccaaggtcc agtggaaagt ggataacgcc 540
ctgcagtccg gaaactccca ggaaagcgtc accgagcagg attccaaaga ctccacctac 600
tccctgtcct ccaccctgac cctgtccaag gccgattacg agaaacacaa agtctatgcc 660
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tgctga 726
<210> 7
<211> 8
<212> PRT
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Gly Tyr Thr Phe Thr Asn Tyr Tyr
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<210> 8
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<213> 人工序列(Artificial Sequence)
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Ile Asn Pro Thr Ser Gly Gly Ser
1 5
<210> 9
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<213> 人工序列(Artificial Sequence)
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Thr Arg Gln Gly Leu Trp Phe Asp Ser Asp Gly Arg Gly Phe Asp Phe
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Gln Asn Ile Val His Ser Asn Gly Asn Thr Tyr
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Lys Val Ser
1
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Phe Gln Tyr Ser His Val Pro Trp Thr
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
caggtgcagc tgcagcagcc cggcgctgag ctggtgaagc ccggagcttc cgtgaagctg 60
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cctggccagg gcctggaatg gatcggggga dmtnmydmkd mkdmtggcgg gtccaacttc 180
aacgagaagt ttaaaaccaa ggccaccctg accgtggacg agagttccac caccgcctac 240
atgcagctgt cttccctgac ctccgaggac tccgccgtct actattgcac caggcagggc 300
dvkdvkdvkd vkdvkgacgg acgcggcttc gacttctggg ggcagggcac caccctgacc 360
gtctcctcc 369
<210> 14
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caggtgcagc tgcagcagcc cggcgctgag ctggtgaagc ccggagcttc cgtgaagct 59
<210> 15
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gagctgtaag gcctccggct atactttcac cavtrvmrvm wmykvktggn nkaagcaga 59
<210> 16
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gacctggcca gggcctggaa tggatcgggg gadmtnmydm kdmkdmtggc gggtccaac 59
<210> 17
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ttcaacgaga agtttaaaac caaggccacc ctgaccgtgg acgagagttc caccaccgc 59
<210> 18
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ctacatgcag ctgtcttccc tgacctccga ggactccgcc gtctactat 49
<210> 19
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tgcaccaggc agggcdvkdv kdvkdvkdvk gacggacgcg gcttcgactt ctgggggca 59
<210> 20
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gggcaccacc ctgaccgtct cctcc 25
<210> 21
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
agccggaggc cttacagctc agcttcacgg aagctccggg 40
<210> 22
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ccctggccag gtctctgctt gaccca 26
<210> 23
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gttttaaact tctcgttgaa gttggacccg cca 33
<210> 24
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ggaagacagc tgcatgtagg cggtggtgga actctcg 37
<210> 25
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ccctgcctgg tgcaatagta gacggcgg 28
<210> 26
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gacggtcagg gtggtgccct gcccccagaa gtcgaagc 38
<210> 27
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
aatgagcata atccaacgcc agg 23
<210> 28
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
aatggctgag tctctccggt agg 23
<210> 29
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tagctgtccc ctgatcaata ggg 23
Claims (14)
1.一种抗 EGFR 抗体或其抗原结合片段,其特征在于,所述抗 EGFR抗体或其抗原结合片段包括:
(1)如SEQ ID NO:3所示的重链可变区,和/或
(2)如SEQ ID NO:4所示的轻链可变区。
2.如权利要求1所述的抗EGFR抗体或其抗原结合片断,其特征在于,
所述重链可变区包含分别如SEQ ID NO:7、8、9所示的重链HCDR1、HCDR2、HCDR3;和/或
所述轻链可变区包含分别如SEQ ID NO:10、11、12所示的轻链LCDR1、LCDR2、LCDR3。
3.如权利要求1-2任一项所述的抗 EGFR 抗体或其抗原结合片段,其特征在于,所述重链可变区序列通过设计突变库并淘选获得。
4.如权利要求1-3任一项所述的抗 EGFR 抗体或其抗原结合片段,其中所述抗体是IgG1、IgG2、IgG3或IgG4形式的抗体或其抗原结合片段;
优选地,所述抗体是IgG1形式的抗体或其抗原结合片段。
5.如权利要求1-4任一项所述的抗 EGFR 抗体或其抗原结合片段,其特征在于,所述抗体抗体或其抗原结合片段是人源化的。
6.如权利要求1-5任一项所述的抗 EGFR 抗体或其抗原结合片段,其特征在于,所述抗原结合片段包括Fv、Fab、Fab’、F(ab’)2、双特异性抗体、多肽中的一种或几种组合。
7.如权利要求6所述的抗 EGFR抗体或其抗原结合片段,其特征在于,所述双特异性抗体包含:特异性结合EGFR 的抗原结合部分,和特异性结合第二抗原的抗原结合部分;
优选的,所述第二抗原结合部分选自: PD-1、PD-L1、CD3、VEGF。
8.如权利要求1-7任一项所述的抗 EGFR抗体或其抗原结合片段,其特征在于,所述抗体及其抗原结合片段Fc区是经过无岩藻糖修饰的;
优选的,所述抗体及其抗原结合片段是经过N297无岩藻糖修饰的。
9.一种分离的抗 EGFR 抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段与权利要求1-8任一项所述的抗体或其抗原结合片段竞争性结合人 EGFR ,或与权利要求1-8任一项所述的抗体或其抗原结合片段结合相同的人 EGFR 抗原表位。
10.一种多核苷酸序列或组合,其特征在于,所述多核苷酸序列或组合编码权利要求1-9 任一项所述的抗 EGFR抗体或其抗原结合片段的氨基酸序列。
11.一种重组DNA表达载体,其特征在于,所述重组DNA表达载体包含权利要求10所述的多核苷酸序列或组合。
12.一种转染如权利要求11所述的重组DNA表达载体的宿主细胞,其特征在于,所述宿主细胞为包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞,优选地,所述哺乳动物细胞为HEK293F细胞、CHO细胞或CHO-K1细胞;更优选地,所述宿主细胞为FUT8基因敲除的CHO-K1细胞。
13.一种药物或药物组合物,其含有如权利要求1-9任一项所述的抗EGFR单克隆抗体或其抗原结合片段,以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
14.一种根据权利要求1-9任一项所述的抗 EGFR 抗体或其抗原结合片段,或包含权利要求13所述的药物组合物,或权利要求10所述的多核苷酸序列或组合在制备治疗肿瘤疾病的药物中的用途;
优选的,所述肿瘤疾病包括皮肤鳞癌、鳞状非小细胞肺癌、非鳞状非小细胞肺癌、鳞状小细胞肺癌、结直肠癌、头颈部癌、头神经胶质瘤、食管癌、胃癌、胰腺癌。
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