CN116284393A - 一种靶向lilrb4的单克隆抗体 - Google Patents
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Abstract
本发明涉及生物医药领域,具体提供了一种靶向LILRB4的单克隆抗体,HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID No:1、SEQ ID No:2和SEQ ID No:3所示,LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ IDNo:4、SEQ ID No:5和SEQ ID No:6所示,本发明提供的单克隆抗体与LILRB4抗原具有较高的结合能力,能够有效抑制LILRB4抗原与其配体复合物的结合,从而阻断下游NF‑κB信号通路的激活和ARG1的释放,促进T细胞增殖,及抑制组织浸润,有效用于治疗癌症。
Description
技术领域
本发明涉及生物医药技术领域,特别涉及一种靶向LILRB4的单克隆抗体。
背景技术
急性髓系白血病(AML)是一种造血干细胞恶性肿瘤,是最常见的白血病类型,在欧洲的5年相对生存率为19%。慢性粒细胞白血病(CML)是一种缓慢进展的克隆性恶性疾病,是一种源自双相造血干细胞但由祖细胞驱动的骨髓增殖性疾病。CML后的AML很常见,这可能是由先前的髓系恶性肿瘤、致白血病治疗或没有可识别的前驱症状或暴露于细胞毒性药物引起的。
在最近研究中发现,LILRB家族的几个成员在AML细胞上高度表达,并且表达与人AML患者的总体存活呈负相关,其中,白细胞免疫球蛋白样受体亚家族B成员4(LILRB4)作为LILRB家族成员,其特异性高表达在M4和M5型AML细胞上,并且高表达LILRB4的病人生存率显著降低。载脂蛋白E(ApoE)结合并激活LILRB4,募集SHP-2,调控核因子κB(NF-κB)信号通路,从而刺激尿激酶受体(uPAR)和精氨酸酶1(ARG1)的表达,进一步抑制T细胞增殖,促进组织浸润。抗LILRB4抗体能够阻断LILRB4与ApoE的相互作用,抑制NF-κB信号通路,降低ARG1表达,从而促进T细胞增殖并减少组织浸润。为此,抗LILRB4单克隆抗体的研究作为首款有望用于AML的T细胞激活剂,将为全球AML和慢性粒单核细胞白血病(CMML)病患带来创新治疗方案,具有非常大的市场潜力。为此,本发明提供了一种靶向LILRB4的单克隆抗体。
发明内容
通过对免疫文库的筛选,得到了可以与LILRB4特异性结合,且具有较高生物学活性的靶向LILRB4的单克隆抗体。
本发明具体技术方案如下:
本发明提供了一种靶向LILRB4的单克隆抗体,包括:
(1)氨基酸序列如SEQ ID No:1所示的重链互补决定区HCDR1;
(2)氨基酸序列如SEQ ID No:2所示的重链互补决定区HCDR2;
(3)氨基酸序列如SEQ ID No:3所示的重链互补决定区HCDR3;
(4)氨基酸序列如SEQ ID No:4所示的轻链互补决定区LCDR1;
(5)氨基酸序列如SEQ ID No:5所示的轻链互补决定区LCDR2;
(6)氨基酸序列如SEQ ID No:6所示的轻链互补决定区LCDR3。
进一步的,靶向LILRB4的单克隆抗体包括氨基酸序列如SEQ ID No:7所示的重链可变区和氨基酸序列如SEQ ID No:8所示的轻链可变区。
进一步的,靶向LILRB4的单克隆抗体还包括重链恒定区和轻链恒定区,所述重链恒定区选自鼠的IgG1型、IgG2a型、IgG2b型或IgG3型的恒定区的一种,所述轻链恒定区为氨基酸序列如SEQ ID No:13所示的鼠源Ck型的恒定区;所述IgG1型的恒定区的氨基酸序列如SEQ ID No:9所示,所述IgG2a型的恒定区的氨基酸序列如SEQ ID No:10所示,所述IgG2b型的恒定区的氨基酸序列如SEQ ID No:11所示,所述IgG3型的恒定区的氨基酸序列如SEQ IDNo:12所示。
优选的,所述单克隆抗体为Fab、F(ab)2、Fv或ScFv中的一种或几种组合。
本发明还提供了一种蛋白,所述蛋白包含所述的靶向LILRB4的单克隆抗体。
本发明还提供了一种核苷酸分子,所述核苷酸分子编码所述的靶向LILRB4的单克隆抗体。
本发明还提供了一种重组DNA表达载体,所述重组DNA表达载体包含所述的核苷酸分子。
本发明还提供了一种转染所述的重组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;
优选的,所述宿主细胞为哺乳动物细胞,所述哺乳动物细胞为HEK293细胞、CHO细胞或NS0细胞。
还提供了一种药物,所述药物包含所述的靶向LILRB4的单克隆抗体。
本发明还提供了所述的靶向LILRB4的单克隆抗体在制备治疗癌症药物中的应用;
所述癌症包括急性髓系白血病、急性成淋巴细胞性白血病、慢性淋巴细胞白血病、多发性骨髓瘤、母细胞性浆细胞样树状突细胞肿瘤、乳腺癌、肺癌或前列腺癌中的一种或多种。
本发明的有益效果如下:本发明提供的靶向LILRB4的单克隆抗体与LILRB4抗原具有较高的结合能力,能够有效抑制LILRB4抗原与其配体复合物的结合,从而阻断下游NF-κB信号通路的激活和ARG1的释放,促进T细胞增殖,及抑制组织浸润。此外,本发明筛选得到的单克隆抗体能够用于治疗癌症,癌症包括但不限于急性髓系白血病(AML)、急性成淋巴细胞性白血病(ALL)、慢性淋巴细胞白血病(CLL)、多发性骨髓瘤(MM)、母细胞性浆细胞样树状突细胞肿瘤(BPDCN)、乳腺癌、肺癌或前列腺癌。
附图说明
图1为本发明实施例2中pScFv-Disb-HS载体的质粒图谱;
图2为本发明实施例3中梯度稀释ELISA单克隆抗体相对亲和力图;
图3为本发明实施例5中载体pTSE的图谱;
图4为本发明实施例5中单克隆抗体分子的变性聚丙烯酰胺凝胶电泳图;
图5为本发明实施例6中单克隆抗体与LILRB4的结合能力图;
图6为本发明实施例8中单克隆抗体与人单核细胞白血病细胞(THP-1)表面LILRB4的结合能力图;
图7本发明实施例9中单克隆抗体与ApoE竞争结合LILRB4实验图;
图8为本发明实施例10中单克隆抗体抑制THP-1分泌ARG1实验图。
具体实施方式
为了更加容易理解本发明,描述实施例之前,先对本发明某些技术和科学术语作以下说明:
本文所使用的术语“抗体”,包含全抗体及其任一抗原结合片段,抗体包括鼠源抗体、人源化抗体、双特异抗体或嵌合抗体,抗体也可以是Fab、F(ab)2、Fv或ScFv(单链抗体),抗体可以是天然存在的抗体也可以是通过改变(例如突变、缺失、置换等)的抗体。
本文所使用的术语“可变区”和“恒定区”,即为抗体重链和轻链靠近N段的序列区为可变区(V区),靠近C段的其余氨基酸序列相对稳定,为恒定区(C区),可变区包括3个互补性决定区(CDR)和4个框架区(FR),每条轻链可变区和重链可变区均有3个CDR区和4个FR区组成,重链的3个CDR区分别通过HCDR1、HCDR2和HCDR3表示,轻链的3个CDR区分别通过LCDR1、LCDR2和LCDR3表示。
术语“CHO细胞”为中国仓鼠卵巢细胞(chinese hamster ovary cell);术语“HEK293细胞”为人胚肾293细胞(human embryonic kidney 293cell),术“NS0细胞”为小鼠NS0胸腺瘤细胞。
下面结合以下实施例对本发明作进一步详细说明。
实施例1
本发明实施例1提供了一种靶向LILRB4的单克隆抗体,其特征在于,包括:
(1)氨基酸序列如SEQ ID No:1所示的重链互补决定区HCDR1;
(2)氨基酸序列如SEQ ID No:2所示的重链互补决定区HCDR2;
(3)氨基酸序列如SEQ ID No:3所示的重链互补决定区HCDR3;
(4)氨基酸序列如SEQ ID No:4所示的轻链互补决定区LCDR1;
(5)氨基酸序列如SEQ ID No:5所示的轻链互补决定区LCDR2;
(6)氨基酸序列如SEQ ID No:6所示的轻链互补决定区LCDR3。
重链互补决定区HCDR1 | 重链互补决定区HCDR2 | 重链互补决定区HCDR3 | 轻链互补决定区LCDR1 | 轻链互补决定区LCDR2 | 轻链互补决定区LCDR3 |
SNTMS | TISSGGSYIYYRDSVKG | GRSRDPYYYSMDY | RSSQSLVHSNGNTYLH | KVSNRFS | SQSTHNPT |
(SEQ ID NO:1) | (SEQ ID NO:2) | (SEQ ID NO:3) | (SEQ ID NO:4) | (SEQ ID NO:5) | (SEQ ID NO:6) |
实施例2单克隆抗体分子筛选
本发明通过用LILRB4抗原(LILRB4蛋白胞外段,后续实验所使用的LILRB4抗原、蛋白均为LILRB4胞外段)免疫小鼠,优化免疫方法,创建噬菌体展示库,具体噬菌体展示库的构建与筛选鉴定如下:
步骤一:LILRB4抗原免疫小鼠
1、实验动物:
种属品系:BALB/c,雌性,小鼠;
体重:18-20g;
实验动物提供商:亦康(北京)医药科技有限公司。
2、免疫:对小鼠进行免疫,免疫抗原为人LILRB4(南京金斯瑞生物科技有限公司合成基因,本公司构建载体并表达纯化)。
步骤二:噬菌体抗体库的构建
取效价较高的小鼠脾细胞,利用Trizol试剂(购买自Ambion,货号:15596026),提取小鼠脾细胞中的总RNA,RT-PCR获得cDNA,以cDNA为模板,采用简并引物(所用简并引物参考文献:Journal of Immunological Methods 233(2000)167-177)进行PCR扩增,从而获得免疫小鼠抗体重链可变区(VH)基因库及轻链可变区(VL)基因库,轻重链分别双酶切,连接至同样分步骤酶切处理过的载体上,构建pScFv-Disb-HS-VH-VL基因库,pScFv-Disb-HS载体是采用一系列基因克隆的方法对载体pComb3载体(购自中国质粒载体菌株细胞株基因保藏中心)进行改造,使之用于噬菌体单链抗体库的构建和表达。改造后的载体命名pScFv-Disb-HS载体,获得其质粒图谱如图1所示,并以此载体为基础,构建小鼠免疫噬菌体抗体库。
步骤三:以LILRB4为抗原包被免疫管,抗原包被量为5μg/500μL/管,4℃包被过夜,再用4%脱脂奶粉/PBST分别封闭免疫管和免疫噬菌体抗体库,室温封闭1小时。封闭后的免疫噬菌体抗体库加入免疫管中进行抗原抗体结合,噬菌体投入量约为109~1012个,室温反应1小时后,使用PBST-PBS洗去未结合的噬菌体,通过0.1M pH 2.2的Glycine-HCl洗脱,最后使用1.5M pH 8.8的Tris-HCl中和洗脱下来的噬菌体抗体溶液至pH 7.0左右。
步骤四:将上述中和后的噬菌体感染10mL生长至对数期的TG1菌液,37℃培养箱中静置30分钟,取出部分菌液进行梯度稀释,涂布于2YTAG平板上,用于计算噬菌体产出量。剩余的菌液离心弃上清,将菌体沉淀重悬于少量培养基,吸出后涂布于2YTAG大平板,为下一轮筛选做准备。
步骤五:将上述感染后涂板的菌体从大平板上刮下,接菌至2YTAG液体培养基,摇至对数期后加入M13KO7辅助噬菌体超感染,在28℃条件下,220rpm培养过夜制备噬菌体,PEG/NaCl沉降纯化噬菌体用于下一轮筛选,共进行一轮噬菌体库富集筛选。
步骤六:LILRB4噬菌体单链抗体阳性克隆的筛选:经过一轮筛选后,挑取分隔良好的单克隆菌落,接种于加有2YTAG液体培养基的96孔深孔板,在37℃条件下,220rpm的条件下培养至其对数生长期,每孔加入约1010的辅助噬菌体M13KO7,在37℃的温度条件下静止感染30分钟。4000rpm,离心15分钟,弃去上清,菌体用2YTAK重悬沉淀,在28℃及220rpm的条件下培养过夜。4000rpm,4℃的条件下离心15分钟后,吸取扩增后的噬菌体上清进行ELISA鉴定,最终筛选得到亲和力较高的抗LILRB4单克隆抗体候选分子,命名为MG-Ⅰ,将上述得到的单克隆抗体进行基因测序确定为正确的抗体序列,经过测序,上述筛选到的1个单克隆抗体序列如下:
单克隆抗体分子 | 重链可变区序列 | 轻链可变区序列 |
MG-Ⅰ | SEQ ID No:7 | SEQ ID No:8 |
具体的,SEQ ID No:7(MG-Ⅰ的重链可变区的氨基酸序列):
EVKLEESGGGLVKPGGSLKLSCAASGFTFSSNTMSWVRQTPEKRLEWVATISSGGSYIYYRDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTSEGRSRDPYYYSMDYWGQGTSVTVSS;
SEQ ID No:8(MG-Ⅰ的轻链可变区的氨基酸序列):
DIVLTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPTFGAGTKLELK。
实施例3梯度稀释ELISA检测抗LILRB4单克隆抗体的亲和力
将实施例2中获得的单克隆抗体分子MG-Ⅰ进行单克隆噬菌体的展示和纯化,然后进行噬菌体梯度稀释ELISA实验鉴定亲和力,具体方法如下:
用pH 9.6的碳酸盐缓冲液包被LILRB4,100ng/孔/100μL,在4℃温度条件下包被过夜,使用PBST洗涤三次,将实施例2中筛选得到的单克隆抗体MG-Ⅰ用PBST四倍梯度稀释,每孔加入100μL稀释后的样品,在室温下静置1小时。用PBST洗涤ELISA板,将PBST稀释后的HRP-anti-M13(购买自Bio-viewshine,货号:GE27-9421-01)单克隆抗体加入ELISA板中,在室温放置1小时。TMB显色试剂盒显色,室温显色10分钟,用2M H2SO4终止后,酶标仪在450nm/630nm下读数,并计算对应的半最大效应浓度(EC50)值,具体数据如下:
克隆 | MG-Ⅰ |
EC50 | 8.541 |
通过上述数据及如图2所示,实施例2筛选出的单克隆抗体候选分子MG-Ⅰ可以与LILRB4结合。
实施例4
本发明实施例4在实施例2的基础上进一步限定了单克隆抗体分子还包括重链恒定区和轻链恒定区,重链恒定区选自鼠的IgG1型、IgG2a型、IgG2b型或IgG3型的恒定区的一种,轻链恒定区为氨基酸序列如SEQ ID No:13所示的鼠源Ck型的恒定区;IgG1型的恒定区的氨基酸序列如SEQ ID No:9所示,IgG2a型的恒定区的氨基酸序列如SEQ ID No:10所示,IgG2b型的恒定区的氨基酸序列如SEQ ID No:11所示,IgG3型的恒定区的氨基酸序列如SEQID No:12所示;具体序列如下:
SEQ ID No:9(鼠的IgG1型的重链恒定区的氨基酸序列):
AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;
SEQ ID No:10(鼠的IgG2a型的重链恒定区的氨基酸序列):
AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;
SEQ ID No:11(鼠的IgG2b型的重链恒定区的氨基酸序列):
AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;
SEQ ID No:12(鼠的IgG3型的重链恒定区的氨基酸序列):
ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA;
SEQ ID No:13(鼠的Ck型的轻链恒定区的氨基酸序列):
ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。
实施例5单克隆抗体分子制备
本发明实施例5在实施例4的基础上优选的限定了单克隆抗体分子包括鼠的IgG1型的重链恒定区(其氨基酸序列如SEQ ID No:9所示)和鼠的Ck型的轻链恒定区(其氨基酸序列如SEQ ID No:13所示)。抗体制备方法具体如下:
1、在将实施例2筛选出来的MG-Ⅰ单克隆抗体的VH和VL的编码基因分别克隆至装有重链和轻链恒定区基因的载体pTSE(如图3所示),优选的重链恒定区为鼠的IgG1型恒定区(氨基酸序列如SEQ ID No:9所示),轻链恒定区为鼠源Ck链(氨基酸序列如SEQ ID No:13所示),pTSE载体结构如图3所示(pTSE载体制备过程参见CN103525868A说明书第3页第[0019]段)。
2、瞬时转染HEK293细胞(购自中国医学科学院基础医学研究所,货号为GNHu43),进行抗体表达,使用AKTA仪器通过protein A亲和柱纯化获得MG-Ⅰ单克隆抗体,同时使用BCA试剂盒(购买自:北京汇天东方科技有限公司,货号:BCA0020)进行蛋白浓度测定,之后通过SDS-PAGE鉴定蛋白大小,结果如图4所示,从左侧到右侧依次为非还原MG-Ⅰ,还原单克隆抗体MG-Ⅰ,及蛋白质分子量Marker,每条带的分子量大小与理论一致。
实施例6单克隆抗体与LILRB4的结合实验
用pH 9.6的碳酸盐缓冲液包被LILRB4,100ng/孔/100μL,在4℃的温度条件下过夜包被。用300μL/孔PBST洗涤五次,再加入1%BSA-PBST在37℃温度条件下封闭1小时,加入不同稀释浓度的单克隆抗体MG-Ⅰ,起始最高浓度为50μg/mL,经过5倍梯度稀释,共稀释12个梯度,在37℃温度条件下孵育1小时。用300μL/孔PBST洗涤五次,再加入用1%BSA-PBST 1:2000稀释的Goat Anti-Mouse IgG-HRP(购买自solarbio,货号:SE131),在37℃温度条件下孵育1小时。TMB显色试剂盒显色,100μL/孔,室温显色8分钟,然后用2M H2SO4终止显色。酶标仪在450nm/630nm下读数,并计算对应的EC50值,具体数据如下:
克隆 | MG-Ⅰ |
EC50(ng/mL) | 188.3 |
通过上述数据及如图5所示,筛选出的单克隆抗体MG-Ⅰ可以与LILRB4结合。
实施例7单克隆抗体与LILR家族蛋白的结合实验
用pH 9.6的碳酸盐缓冲液包被LILRA1,LILRA2,LILRA3,LILRA4,LILRA5,LILRA6,LILRB1,LILRB2,LILRB3,LILRB4,LILRB5,100ng/孔/100μL,在4℃的温度条件下过夜包被。用300μL/孔PBST洗涤五次,再加入1%BSA-PBST在37℃温度条件下封闭1小时,随后加入100μL单克隆抗体MG-Ⅰ,浓度为50μg/mL。在37℃温度条件下孵育1小时。用300μL/孔PBST洗涤五次,再加入用1%BSA-PBST 1:2000稀释的Goat Anti-Mouse IgG-HRP(购买自solarbio,货号:SE131),在37℃温度条件下孵育1小时。TMB显色试剂盒显色,100μL/孔,室温显色8分钟,然后用2M H2SO4终止显色。酶标仪在450nm/630nm下读数,具体数据如下:
LILRA1 | LILRA2 | LILRA3 | LILRA4 | LILRA5 | LILRA6 | LILRB1 | LILRB2 | LILRB3 | LILRB4 | LILRB5 | |
MG-Ⅰ | 0.103 | 0.046 | 0.057 | 0.077 | 0.066 | 0.053 | 0.045 | 0.04 | 0.05 | 2.721 | 0.176 |
通过上述数据所示,筛选出的单克隆抗体MG-Ⅰ可以与LILRB4特异性结合,且不与LILR家族其它蛋白结合。
实施例8单克隆抗体与人单核细胞白血病细胞(THP-1)表面LILRB4的结合实验
取50μL不同稀释浓度的单克隆抗体MG-Ⅰ,起始工作浓度为30μg/mL,3倍梯度稀释,共稀释10个梯度,加入96孔板V底板中。随后向孔中添加50μL THP-1细胞悬浮液,浓度为2×106cells/mL,混匀。4℃条件下孵育1小时。随后每孔加入100μL PBS缓冲液,3000rpm离心5分钟弃上清。再次添加100μL/孔异硫氰酸荧光素(FITC)标记山羊抗鼠IgG(购买自北京中杉金桥生物技术有限公司,货号:ZF-0312)(1:100稀释)。混匀后,4℃避光孵育30分钟。每孔加入100μL PBS缓冲液,3000rpm离心5分钟弃上清。再次添加100μL PBS缓冲液重悬细胞,流式细胞仪上机检测。收集数据并计算对应的EC50值,具体数据如下:
克隆 | MG-Ⅰ |
EC50(ng/mL) | 4306 |
通过上述数据及如图6所示,筛选出的单克隆抗体MG-Ⅰ可以与THP-1细胞表面的LILRB4结合。
实施例9单克隆抗体与ApoE竞争结合LILRB4实验
取THP-1细胞,浓度为2×106cells/mL,铺于V底96孔板中,每孔添加50μL细胞悬浮液。随后向孔中添加50μL不同稀释浓度的单克隆抗体MG-Ⅰ,起始工作浓度为400μg/mL,5倍梯度稀释,共10个梯度。另外向孔中添加100μL 0.4μg/mL FITC标记的ApoE蛋白。4℃条件下避光孵育1.5小时。随后每孔加入200μL PBS缓冲液,3000rpm离心5分钟弃上清。再次添加100μL PBS缓冲液重悬细胞,流式细胞仪上机检测。收集数据并计算对应的IC50值,具体数据如下:
克隆 | MG-Ⅰ |
IC50(μg/mL) | 7.179 |
通过上述数据及如图7所示,筛选出的单克隆抗体MG-Ⅰ可以与ApoE竞争结合THP-1细胞表面的LILRB4。
实施例10单克隆抗体抑制THP-1分泌ARG1
取THP-1细胞,浓度为1×107cells/mL,铺于V底96孔板中,每孔添加50μL细胞悬浮液。先将不同浓度的单克隆抗体MG-Ⅰ与ApoE按照1:1比例混匀。单克隆抗体MG-Ⅰ的起始工作浓度为400μg/mL,2倍梯度稀释,共8个梯度。ApoE的工作浓度为0.5μg/mL。随后向孔中添加50μL单克隆抗体MG-Ⅰ与ApoE混合物。37℃条件下孵育20小时后,3000rpm离心5分钟。每孔取出40μL上清加入96孔平底板中,随后按照精氨酸酶活性检测试剂盒(购买自Sigma-Aldrich,货号:MAK112-1KT)配置并预热反应底物,每孔添加10μL反应底物混匀,37℃条件下反应1小时。每孔再次添加200μL反应终止液,并使用多功能酶标仪于450nm处读吸光度值。收集数据并计算对应的IC50值,具体数据如下:
克隆 | MG-Ⅰ |
IC50(μg/mL) | 257.4 |
通过上述数据及如图8所示,筛选出的单克隆抗体MG-Ⅰ可以抑制THP-1细胞分泌ARG1。
实施例11
本发明实施例11在上述实施例1-10的基础上限定了靶向LILRB4的单克隆抗体为Fab、F(ab)2、Fv或ScFv中的一种或几种组合。
本发明提供了一种蛋白,所述蛋白包含靶向LILRB4的单克隆抗体MG-Ⅰ。
本发明进一步提供了一种核苷酸分子,核苷酸分子编码上述实施例提供的靶向LILRB4的单克隆抗体MG-Ⅰ。
本发明还提供了一种重组DNA表达载体,重组DNA表达载体包含核苷酸分子。
本发明还提供了一种转染重组DNA表达载体的宿主细胞,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;
优选的,宿主细胞为哺乳动物细胞,哺乳动物细胞为HEK293细胞、CHO细胞或NS0细胞。
实施例12
本发明实施例12提供了一种药物,药物包含实施例1-10提供的靶向LILRB4的单克隆抗体MG-Ⅰ。
本发明还提供了靶向LILRB4的单克隆抗体MG-Ⅰ在制备治疗癌症药物中的应用;
癌症包括急性髓系白血病、急性成淋巴细胞性白血病、慢性淋巴细胞白血病、多发性骨髓瘤、母细胞性浆细胞样树状突细胞肿瘤、乳腺癌、肺癌或前列腺癌中的一种或多种。
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。
Claims (10)
1.一种靶向LILRB4的单克隆抗体,其特征在于,包括:
(1)氨基酸序列如SEQ ID No:1所示的重链互补决定区HCDR1;
(2)氨基酸序列如SEQ ID No:2所示的重链互补决定区HCDR2;
(3)氨基酸序列如SEQ ID No:3所示的重链互补决定区HCDR3;
(4)氨基酸序列如SEQ ID No:4所示的轻链互补决定区LCDR1;
(5)氨基酸序列如SEQ ID No:5所示的轻链互补决定区LCDR2;
(6)氨基酸序列如SEQ ID No:6所示的轻链互补决定区LCDR3。
2.如权利要求1所述的靶向LILRB4的单克隆抗体,其特征在于,包括氨基酸序列如SEQID No:7所示的重链可变区和氨基酸序列如SEQ ID No:8所示的轻链可变区。
3.如权利要求2所述的靶向LILRB4的单克隆抗体,其特征在于,还包括重链恒定区和轻链恒定区,所述重链恒定区选自鼠的IgG1型、IgG2a型、IgG2b型或IgG3型的恒定区的一种,所述轻链恒定区为氨基酸序列如SEQ ID No:13所示的鼠源Ck型的恒定区;所述IgG1型的恒定区的氨基酸序列如SEQ ID No:9所示,所述IgG2a型的恒定区的氨基酸序列如SEQ ID No:10所示,所述IgG2b型的恒定区的氨基酸序列如SEQ ID No:11所示,所述IgG3型的恒定区的氨基酸序列如SEQ ID No:12所示。
4.如权利要求1所述的靶向LILRB4的单克隆抗体,其特征在于,所述单克隆抗体为Fab、F(ab)2、Fv或ScFv中的一种或几种组合。
5.一种蛋白,其特征在于,所述蛋白包含权利要求1-4任一项所述的靶向LILRB4的单克隆抗体。
6.一种核苷酸分子,其特征在于,所述核苷酸分子编码权利要求1-4任一项所述的靶向LILRB4的单克隆抗体。
7.一种重组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6所述的核苷酸分子。
8.一种转染权利要求7所述的重组DNA表达载体的宿主细胞,其特征在于,所述宿主细胞包括原核细胞、酵母细胞、昆虫细胞或哺乳动物细胞;
优选的,所述宿主细胞为哺乳动物细胞,所述哺乳动物细胞为HEK293细胞、CHO细胞或NS0细胞。
9.一种药物,其特征在于,所述药物包含权利要求1-4任一项所述的靶向LILRB4的单克隆抗体。
10.权利要求1-4任一项所述的靶向LILRB4的单克隆抗体在制备治疗癌症药物中的应用;
所述癌症包括急性髓系白血病、急性成淋巴细胞性白血病、慢性淋巴细胞白血病、多发性骨髓瘤、母细胞性浆细胞样树状突细胞肿瘤、乳腺癌、肺癌或前列腺癌中的一种或多种。
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