CN113045658B - 抗cll1抗体及其应用 - Google Patents
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Abstract
本发明提供了抗CLL1抗体及其应用,所述抗CLL1抗体可变区包括SEQ ID NO:1~6、SEQ ID NO:5和SEQ ID NO:7~11或SEQ ID NO:12~17的CDR。本发明的抗CLL1抗体对游离和细胞表面CLL1均具有显著的结合能力,经过人源化改造后,进一步提高了抗体与CLL1的亲和力,在肿瘤的临床诊断和/或治疗方面具有重要的应用前景。
Description
技术领域
本发明属于生物医药技术领域,涉及抗CLL1抗体及其应用。
背景技术
C型凝集素样分子1(CLL1),又称C型凝集素域家族12成员A(CLEC12A),在文献和数据库中还有DCAL-2、MICL、CD371等名称,是一种II型跨膜蛋白,全长265个氨基酸,分子量为30762道尔顿,编码基因位于chr12:9,951,268~9,995,694(GRCh38/hg38)。目前一些主流数据库中CLL1基因的识别码如下:Q5QGZ9(UniprotKB)、31713(HGNC)、160364(EntrezGene)、ENSG00000172322(Ensembl)和(612088)OMIM。
研究表明,CLL1限制性地表达于造血细胞上,主要包括外周血和骨髓中髓系来源的细胞,比如单核细胞、树突状细胞、粒细胞以及大多数急性髓细胞白血病(AML)细胞。值得注意的是,虽然CLL1大量表达于外周血和骨髓中的髓细胞上,但是在外周组织中的髓系来源的细胞上不表达,比如组织巨噬细胞和组织树突状细胞均不表达CLL1。研究还发现,CLL1表达于AML干细胞(CD34+/CD38-)以及一小部分造血祖细胞(CD34+/CD38+或者CD34+/CD33+)上,但是在正常的造血干细胞(CD34+/CD38-或者CD34+/CD33-)上不表达。由于这种特殊的表达模式,CLL1有望成为潜在的诊断和治疗AML的靶点。
目前已知的人CLL1基因包括7个转录本,其中5个编码蛋白质,人CLL1蛋白是一种膜受体,经典结构的胞外段具有C型凝集素结构域,胞内段具有免疫受体酪氨酸基序(ITIM),磷酸化ITIM与含SH2结构域的磷酸酶结合,发挥负性调节粒细胞和单核细胞的功能。
发明内容
本发明提供了抗CLL1抗体及其应用,所述抗体单独和/或与其他药物联合用于癌症和自身免疫性疾病的治疗。
为达此目的,本发明采用以下技术方案:
第一方面,本发明提供了抗CLL1抗体,所述抗CLL1抗体包括重链可变区和轻链可变区;
所述重链可变区包括SEQ ID NO:3、SEQ ID NO:9或SEQ ID NO:14所示的CDR3;
所述轻链可变区包括SEQ ID NO:6、SEQ ID NO:11或SEQ ID NO:17所示的CDR3;
所述重链可变区还包括SEQ ID NO:1、SEQ ID NO:7或SEQ ID NO:12所示的CDR1;
所述重链可变区还包括SEQ ID NO:2、SEQ ID NO:8或SEQ ID NO:13所示的CDR2;
所述轻链可变区还包括SEQ ID NO:4、SEQ ID NO:10或SEQ ID NO:15所示的CDR1;
所述轻链可变区还包括SEQ ID NO:5或SEQ ID NO:16所示的CDR2。
本发明中,抗体的重链可变区的CDR1~3和轻链可变区的CDR1~3共同决定抗体对抗原的特异性识别结合能力,含有SEQ ID NO:1~6、SEQ ID NO:5和SEQ ID NO:7~11或SEQID NO:12~17的CDR的抗体对CLL1蛋白具有显著的结合能力。
在一个具体实施例中,所述抗CLL1抗体23D7的重链可变区包括SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、SEQ ID NO:3所示的CDR3;
所述抗CLL1抗体23D7的轻链可变区包括SEQ ID NO:4所示的CDR1、SEQ ID NO:5所示的CDR2、SEQ ID NO:6所示的CDR3;
SEQ ID NO:1:RYWMH;
SEQ ID NO:2:YIYPGSGTSNYDEKFKS;
SEQ ID NO:3:EARYTMDY;
SEQ ID NO:4:SASSSVSYIY;
SEQ ID NO:5:DTSNLAS;
SEQ ID NO:6:QQWSSFP。
本发明中,包含SEQ ID NO:1~3的重链可变区CDR和SEQ ID NO:4~6的轻链可变区CDR的抗CLL1抗体23D7具有CLL1蛋白结合活性。
优选地,所述抗CLL1抗体23D7的重链可变区包括SEQ ID NO:18所示的氨基酸序列,轻链可变区包括SEQ ID NO:19所示的氨基酸序列;
SEQ ID NO:18:
QVQLQQPGSDLVRPGASVKLSCKASGYTFTRYWMHWVKQRPGHGLEWIGYIYPGSGTSNYDEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTREARYTMDYWGQGTSVTVSS;
SEQ ID NO:19:
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYIYWYQQKPGSSPGLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSFPPTFGAGTKLELK。
在一个具体实施例中,所述抗CLL1抗体19C1的重链可变区包括SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2、SEQ ID NO:9所示的CDR3;
所述抗CLL1抗体19C1的轻链可变区包括SEQ ID NO:10所示的CDR1、SEQ ID NO:5所示的CDR2、SEQ ID NO:11所示的CDR3;
SEQ ID NO:7:SYWIE;
SEQ ID NO:8:EIFPGSGSIKYNEKFKG;
SEQ ID NO:9:GGTYNDYSLFDY;
SEQ ID NO:10:SASSSVSYMY;
SEQ ID NO:11:QQWSSYP。
本发明中,包含SEQ ID NO:7~9的重链可变区CDR和SEQ ID NO:5、10~11的轻链可变区CDR的抗CLL1抗体19C1具有CLL1蛋白结合活性。
优选地,所述抗CLL1抗体19C1的重链可变区包括SEQ ID NO:20所示的氨基酸序列,轻链可变区包括SEQ ID NO:21所示的氨基酸序列;
SEQ ID NO:20:
QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEIFPGSGSIKYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVHYCARGGTYNDYSLFDYWGQGTTLTVSS;
SEQ ID NO:21:
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIFDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLELK。
在一个具体实施例中,所述抗CLL1抗体27H4的重链可变区包括SEQ ID NO:12所示的CDR1、SEQ ID NO:13所示的CDR2、SEQ ID NO:14所示的CDR3;
所述抗CLL1抗体27H4的轻链可变区包括SEQ ID NO:15所示的CDR1、SEQ ID NO:16所示的CDR2、SEQ ID NO:17所示的CDR3;
SEQ ID NO:12:GYHMH;
SEQ ID NO:13:RINPYNGAASHNQKFKD;
SEQ ID NO:14:GWDYDGGYYAMDY;
SEQ ID NO:15:KSSQSLLYSDNQKNYLA;
SEQ ID NO:16:WASTRES;
SEQ ID NO:17:QQYYTYP。
本发明中,包含SEQ ID NO:12~14的重链可变区CDR和SEQ ID NO:15~17的轻链可变区CDR的抗CLL1抗体27H4具有CLL1蛋白结合活性。
优选地,所述抗CLL1抗体27H4的重链可变区包括SEQ ID NO:22所示的氨基酸序列,轻链可变区包括SEQ ID NO:23所示的氨基酸序列;
SEQ ID NO:22:
EVQLQQSGPELVKPGASVKISCKASGYSFTGYHMHWVKQSHVKSLEWIGRINPYNGAASHNQKFKDKATLTVDKSSSTAYMELHSLTSEDSAVYYCARGWDYDGGYYAMDYWGQGTSVTVSS;
SEQ ID NO:23:
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSDNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPYTFGGGTKLEIK。
优选地,对抗CLL1抗体27H4进行人源化改造,优化27H4的框架区,获得人源化H27H4抗体具有与CLL1更强的亲和力,所述抗CLL1抗体H27H4的重链可变区包括SEQ ID NO:24所示的氨基酸序列,轻链可变区包括SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:27所示的氨基酸序列;
SEQ ID NO:24:
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSS;
SEQ ID NO:25:
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIK;
SEQ ID NO:26:
DIVMTQSPLSLPVTPGEPASISCKSSQSLLYSDNQKNYLAWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYTYPYTFGQGTKLEIK;
SEQ ID NO:27:
DIVMTQSPDSLAVSLGERATINCKSSQSLLYSDNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTYPYTFGQGTKLEIK。
优选地,所述抗CLL1抗体为单体,由一组重链和轻链形成,所述重链可变区和轻链可变区之间通过链间二硫键连接。
优选地,所述抗CLL1抗体为多聚体,由多组重链和轻链形成,所述重链可变区和轻链可变区之间通过链间二硫键连接,不同的重链可变区之间通过链间二硫键连接。
优选地,所述抗CLL1抗体还包括恒定区。
优选地,所述抗CLL1抗体修饰有糖基化基团。
第二方面,本发明提供了核酸分子,所述核酸分子包括编码第一方面所述的抗CLL1抗体的DNA片段。
第三方面,本发明提供了表达载体,所述表达载体包括第二方面所述的核酸分子。
第四方面,本发明提供了重组细胞,所述重组细胞表达第一方面所述的抗CLL1抗体。
优选地,所述重组细胞的基因组中整合有第二方面所述的核酸分子。
优选地,所述重组细胞包括第三方面所述的表达载体。
第五方面,本发明提供了一种第一方面所述的抗CLL1抗体的制备方法,所述制备方法包括以下步骤:
(1)将抗CLL1抗体的编码核酸连接入质粒,转入感受态细胞,培养后挑取单克隆细胞进行筛选;
(2)提取筛选的阳性克隆的表达载体,转入宿主细胞,培养并收集上清液,分离纯化得到所述抗体。
第六方面,本发明提供了药物组合物,所述药物组合物包括第一方面所述的抗CLL1抗体。
优选地,所述药物组合物还包括抗肿瘤药物。
优选地,所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
第七方面,本发明提供了第一方面所述的抗CLL1抗体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组细胞或第六方面所述的药物组合物在制备疾病检测试剂和/或疾病治疗药物中的应用。
优选地,所述疾病包括急性髓系白血病。
第八方面,本发明提供了一种治疗癌症的方法,所述方法包括向患者施用有效剂量的第一方面所述的抗CLL1抗体。
优选地,所述方法还包括与抗CLL1抗体同时、分开或依次施用一种或多种抗肿瘤药物。
优选地,所述癌症包括急性髓系白血病。
与现有技术相比,本发明具有如下有益效果:
(1)本发明的抗CLL1抗体23D7、27H4、19C1对CLL1具有显著的结合能力,ch23D7、ch27H4和ch19C1结合抗原CLL1的亲和力分别为2.19nM、3.83nM和10.9nM,与对照抗体1075.7相当;
(2)本发明的抗CLL1抗体23D7、27H4、19C1可以结合细胞表面的CLL1蛋白,结合力随着抗体浓度的升高而增大;
(3)本发明的抗体经过人源化改造后,进一步提高了抗体与游离和/或细胞表面CLL1的亲和力;
(4)本发明的抗体及其人源化改造抗体在治疗CLL1阳性肿瘤中具有重要的应用前景。
附图说明
图1为嵌合抗体ch23D7、ch27H4和ch19C1的ForteBIO评价结果;
图2为流式检测嵌合抗体ch23D7、ch27H4、ch19C1与CLL1抗原的结合;
图3为流式检测人源化抗体hz27H4与CLL1抗原的结合;
图4为流式检测抗体结合CLL1转瞬293T细胞的能力;
图5A为23D7-scFv-hFc与CLL1抗原的结合能力的MPA检测结果,图5B为27H4-scFv-hFc与CLL1抗原的结合能力的MPA检测结果,图5C为19C1-scFv-hFc与CLL1抗原的结合能力的MPA检测结果,图5D为Hz27H4-scFv-hFc与CLL1抗原的结合能力的MPA检测结果;
图6A为27H4-scFv-hFc与重组人源CLL-1结合的动力学拟合结果,图6B为19C1-scFv-hFc与重组人源CLL-1结合的动力学拟合结果,图6C为23D7-scFv-hFc与重组人源CLL-1结合的动力学拟合结果,图6D为Hz27H4-scFv-hFc与重组人源CLL-1结合的动力学拟合结果,图6E为h27H4H1L1与重组人源CLL-1结合的动力学拟合结果,图6F为h27H4H1L2与重组人源CLL-1结合的动力学拟合结果。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。
实施例1抗体的获取
选择10只鼠龄为7~8周的BALB/C健康雌性小鼠,采用免疫原CLL1-mFc(CLL1胞外段和鼠IgG1 Fc段的融合蛋白,CLL1胞外段的氨基酸序列如SEQ ID NO:28所示)进行免疫;第二次免疫两周后,从小鼠尾静脉取血分离血清,ELISA检测抗体效价;选取抗体效价达到可融合要求的两只小鼠,在融合前三天腹腔注射抗原50μg/100μL/只进行冲击免疫;同时,效价检测前一周复苏骨髓瘤细胞SP2/0,37℃、5%CO2培养箱培养,融合前一天对骨髓瘤细胞进行传代或更换新鲜培养基,使细胞保持最佳状态;
SEQ ID NO:28:
HVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTLQTIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKMACAAQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSAWVIRNAPDLNNMYCGYINRLYVQYYHCTYKKRMICEKMANPVQLGSTYFREA;
对冲击免疫的小鼠进行断颈取血,75%酒精消毒10min后取脾脏,去除结缔组织,制备脾细胞悬液;将脾细胞悬液转移到50mL离心管中,加入RPMI1640至30mL,1000rpm离心5min,弃上清,加入RPMI1640至30mL,进行细胞计数;取生长状态良好的骨髓瘤细胞(活细胞数>95%)转移到50mL离心管中,加入RPMI1640至30mL,1000rpm离心5min,弃上清,加入RPMI1640至30mL,进行细胞计数;
将脾细胞和骨髓瘤细胞按照4:1的比例混合,1000rpm离心5min,弃上清,将沉淀细胞块弹成糊状后置于37℃水浴中,在1min内加入1mL融合剂并搅拌均匀,置于37℃水浴45~60s,在1min内加入RPMI1640终止融合剂的融合作用,1000rpm离心5min,弃上清;
轻轻将细胞弹匀,缓缓加入含HAT的完全培养液,将细胞悬液加入到预先准备好的完全培养基中,排枪滴加入96孔板中,每孔150~200μL,37℃、CO2培养箱培养、观察;
从细胞融合后的第一天开始,观察细胞的生长状态、并确定培养液无污染,培养7~10天后将HAT培养液更换为HT培养液,继续培养3~4天,每孔取上清进行ELISA检测。
ELISA筛选步骤如下:
(1)抗原包被:将浓度为50ng/mL的纯抗原human CLL1 ECD-His(人CLL1胞外区连接His标签)用包被液稀释后,取100μL加入到聚苯乙烯酶联检测板的各孔中,4℃过夜;
(2)封闭:次日将酶标板平衡至室温,PBS清洗三次,每孔加入100μL封闭液,室温孵育一小时后PBS清洗3次,拍干;
(3)加待测样品:无菌条件下取融合细胞培养上清,35~50μL/孔加样到封闭好的酶标板中,同时,设置阴性对照孔(无细胞生长)和阳性对照孔(添加阳性血清),室温孵育1h,PBST(0.05%吐温)清洗三次,PBS清洗两次;
(4)加二抗:以50μL/孔的添加量加入稀释的酶标二抗,37℃孵育30min,PBS清洗3次,拍干;
(5)显色:加入双组分TMB显色液终止液(Solarbio,Cat#PR1210)各50μL/孔,添加前混合均匀,37℃显色15~30min,随后加入50μL/孔终止液终止反应;
(6)读数:以450nm单波长测定各孔OD值,按从高到低原则选择读值高的多个克隆进行下一步功能筛选。
选取ELISA初筛的阳性孔,并对汇合率高的阳性孔细胞传代至24孔板,进一步进行功能实验,确定进行亚克隆的克隆编号;采用有限稀释法进行细胞亚克隆,同时冻存保种,具体如下:将细胞克隆稀释后铺96孔板,每个克隆铺1个板,于HT培养基中培养7~10天,第7天后镜下观察,挑选单克隆细胞上清用ELISA进一步筛选阳性克隆;
对克隆进行测序,获得克隆23D7、19C1、27H4的氨基酸序列,其中,23D7的重链可变区如SEQ ID NO:18所示、轻链可变区如SEQ ID NO:19所示,19C1的重链可变区如SEQ IDNO:20所示、轻链可变区如SEQ ID NO:21所示,27H4的重链可变区如SEQ ID NO:22所示,轻链可变区如SEQ ID NO:23所示。
实施例2抗体的表达与纯化
本实施例根据单克隆鉴定测序结果,设计特异性引物,PCR获得抗体23D7、19C1、27H4的基因,并将基因克隆至人IgG1重链恒定区Fc段编码基因上游,构建重组真核表达载体,获得人鼠嵌合抗体表达质粒;
将所述人鼠嵌合抗体表达质粒瞬时转染293F细胞,通过瞬时表达和亲和纯化获得以23D7、19C1、27H4为亲本的嵌合抗体ch23D7、ch19C1、ch27H4。
实施例3抗体的亲和力测试
本实施例采用ForteBio亲和力测量方法(P.Estep等,High throughputsolution-based measurement of antibody-antigen affinity and epitopebinning.MAbs,2013.5(2):270-278.)对嵌合抗体ch23D7、ch19C1、ch27H4进行亲和力检测,对照抗体(Anti-CLL1-Ref)选择抗人CLL-1抗体1075.7(专利US8536310B2)的VL和VL链(SEQID NO:29)。
简言之,将抗体装载至抗人IgG捕获(AHC)生物传感器上,将传感器在测定缓冲液中离线平衡30min,在线监测60s用于确立基线;将装载抗体的传感器与100nM抗原humanCLL1 ECD-His共孵育5min,随后转移至测定缓冲液中,5min后测定解离速率;动力学分析采用1:1结合模型进行。
结果如表1和图1所示,ch23D7、ch27H4和ch19C1结合抗原CLL1的亲和力分别为2.19nM、3.83nM和10.9nM,对照抗体1075.7结合抗原CLL1的亲和力为1.08nM。
表1
样品编号 | 浓度(nM) | Response | KD(M) | Kon(1/Ms) | Kdis(1/s) | RMax |
ch23D7 | 100 | 0.6536 | 2.19E-09 | 1.30E+05 | 2.84E-04 | 0.6693 |
ch27H4 | 100 | 0.6341 | 3.83E-09 | 2.65E+05 | 1.01E-03 | 0.6237 |
ch19C1 | 100 | 0.6194 | 1.09E-08 | 1.80E+05 | 1.97E-03 | 0.6574 |
ch1075.7 | 100 | 0.2361 | 4.14E-10 | 1.08E+05 | 4.48E-05 | 0.2384 |
SEQ ID NO:29:
ENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELGGGGSGGGGSGGGGSDIQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSS。
实施例4抗体与HEK293细胞上CLL1的结合
本实施例采用流式细胞术检测抗体ch23D7、ch27H4、ch19C1与HEK293细胞上CLL1的结合,步骤如下:
采用PBS+5%BSA重悬5×105个过表达CLL1的HEK293细胞,4℃孵育30min;加入不同浓度抗体(1μg/mL~0.01μg/mL,10倍梯度稀释),4℃孵育60min;离心洗涤后加入含有FITC-标记羊抗人IgG-Fc二抗(1:200,sigma,F9512)的PBS+5%BSA溶液,冰上避光孵育30min;将细胞洗涤3次后进行流式细胞术分析;
设置对照组CLL1-RefAb(ch1075.7)、Cell+二抗和Blank。
结果如图2所示,嵌合抗体ch23D7、ch27H4、ch19C1、ch1075.7都能有效结合HEK293-CLL1细胞,并随着抗体浓度的提高,平均荧光强度(MFI)也随之升高。
实施例5抗体的人源化改造
本实施例对27H4进行人源化改造。简言之,将27H4抗体的基因序列与人抗体Germline数据库进行比对,找出同源性高的序列,同时避免不常用或小类Germline;选定人源化模板后,重排(rearranged)抗体在特定FR位点的氨基酸出现频率,并进行CDR移植,避免引入糖基化等蛋白修饰位点和易化学降解的位点;若CDR移植后序列的亲和力下降,进行回复突变,突变原则为对FR区进行逐级单点突变;若CDR区存在修饰位点或化学降解位点,且对蛋白质控有影响,对位点进行逐级单点突变;每轮突变后进行亲和力(KD/Kon/Koff)检测;最终获得的人源化序列在亲和力(与亲本比较相差3倍以内)、稳定性、蛋白质量(一步PA>90%)等方面均具有最优表现。
根据以上原则,获得人源化hz27H4抗体的一条重链可变区hz27H4H1(SEQ ID NO:24)和三条轻链可变区hz27H4L1(SEQ ID NO:25)、hz27H4L2(SEQ ID NO:26)和hz27H4L3(SEQ ID NO:27)。
实施例6 ForteBIo分析抗体人源化前后与CLL1的亲和力差异
简言之,将4μg/mL抗体装载至抗人IgG捕获(AHC)生物传感器上,将传感器在测定缓冲液中离线平衡30min,在线监测60s用于确立基线;将装载抗体的传感器与60nM抗原human CLL1 ECD-His共孵育3min,随后转移至测定缓冲液中,3min后测定解离速率;动力学分析采用1:1结合模型进行。
结果如表2所示,三个人源化抗体(hz27H4H1L1、hz27H4H1L2和hz27H4H1L3)与亲本抗体(ch27H4)相比,均保留了较高的亲和力。
表2 ForteBIo分析抗体人源化前后与CLL1的亲和力差异
样品编号 | 浓度(nM) | Response | KD(M) | Kon(1/Ms) | Kdis(1/s) |
hz27H4H1L1 | 60 | 0.6123 | 2.29E-09 | 3.77E+05 | 8.62E-04 |
hz27H4H1L2 | 60 | 0.7255 | 2.21E-09 | 4.41E+05 | 9.76E-04 |
hz27H4H1L3 | 60 | 0.7224 | 2.49E-09 | 4.38E+05 | 1.09E-03 |
ch27H4 | 60 | 0.5972 | 3.88E-09 | 3.20E+05 | 1.24E-03 |
Anti-CLL1 Ref Ab | 60 | 0.2189 | 6.76E-09 | 1.48E+05 | 9.97E-04 |
实施例7流式检测人源化抗体与CLL1的结合
本实施例采用流式细胞术检测抗体hz27H4H1L1、hz27H4H1L2与HEK293细胞上CLL1的结合,步骤如下:
采用PBS+5%BSA重悬2×105个过表达CLL1的HEK293细胞,4℃孵育30min;加入不同浓度抗体(5μg/mL~0.002286μg/mL,3倍梯度稀释),4℃孵育60min;离心洗涤后加入含有FITC-标记羊抗人IgG-Fc二抗(1:200,sigma,F9512)的PBS+5%BSA溶液,冰上避光孵育30min;将细胞洗涤3次后进行流式细胞术分析;Graphpad软件计算得出EC50。
设置对照组ch27H4、CLL1-RefAb(ch1075.7)和NC-huIgG1。
结果如图3、图4和表3所示,ch27H4以剂量依赖的方式与CLL1结合,EC50(n=1)为0.3583μg/mL,hz27H4H1L1以剂量依赖的方式与CLL1结合,EC50值(n=1)为0.2681μg/mL,hz27H4H1L2以剂量依赖的方式与CLL1结合,EC50值(n=1)为0.3214μg/mL,CLL1-refAb以剂量依赖的方式与CLL1结合,EC50值(n=1)为0.2246μg/mL,ch27H4、hz27H4H1L1和hz27H4H1L2以与CLL1-refAb类似的EC50与人CLL1结合。
表3流式检测人源化抗体与HEK293-CLL1细胞的结合(EC50)
- | ch27H4 | hz27H4H1L1 | hz27H4H1L2 | CLL1-refAb |
EC50(μg/mL) | 0.3583 | 0.2618 | 0.3214 | 0.2446 |
实施例8膜蛋白质组阵列评估抗体特异性
本实施例采用膜蛋白质组阵列(Membrane Proteome Array,MPA)验证抗体的非靶点结合的相互作用。膜蛋白质组阵列(MPA)是一个分析特异性抗体和其他配体靶向人膜蛋白的平台,可用于确定抗体靶点的特异性。
将含有约6000个膜蛋白克隆(占人膜蛋白组的94%以上)的质粒分别转染到HEK-293T细胞(ATCC,CRL-3216)或QT6细胞(ATCC,CRL-1708)中,按18000个细胞/孔的密度接种至384孔细胞培养板(Corning,3764);孵育36小时后,试验抗体以预先确定的浓度加入到膜蛋白组阵列基质板中,使用流式细胞仪直接检测抗体scFv与约6000种膜蛋白表达细胞的结合情况。所有靶蛋白都具有天然构象和适当的翻译后修饰,哺乳动物表达的单链抗体(scFv)为VL-(G4S)3-VH结构,C端融合表达人hIgG1-Fc,具体信息见表4。
测试结果如图5A、图5B、图5C和图5D所示,23D7-scFv-hFc、27H4-scFv-hFc、19C1-scFv-hFc、Hz27H4-scFv-hFc都能特异性结合CLL1靶标抗原,其中FCGR1A、FCGR2B、FCGR3B是IgG Fc受体(IgG Fc receptors)。
表4所用抗体信息
抗体 | 基本信息 | 靶抗原 | Uniprot |
23D7-scFv-hFc | Anti-CLL1 scFv-hIgG1 | CLL1 | Q5QGZ9 |
27H4-scFv-hFc | Anti-CLL1 scFv-hIgG1 | CLL1 | Q5QGZ9 |
19C1-scFv-hFc | Anti-CLL1 scFv-hIgG1 | CLL1 | Q5QGZ9 |
Hz27H4-scFv-hFc | Anti-CLL1 scFv-hIgG1 | CLL1 | Q5QGZ9 |
实施例9表面等离子体共振法测定抗体亲和力
本实施例采用表面等离子体共振技术(Surface Plasmon Resonance,SPR)检测并比较两种CLL-1抗原(重组人源CLL-1,Acro,货号:CLA-H5245,批号:3413a-9B8F1-SQ;重组食蟹猴CLL-1,Acro,货号:CLA-H5263,批号:3765-2079F1-SS)与6个抗体的亲和力,单链抗体(scFv)为VL-(G4S)3-VH结构,C端融合表达人hIgG1-Fc,样品信息表见表5。
表5样品信息表
(1)样品配制
抗体稀释液(配体):将抗体用1×HBS-EP+运行缓冲液稀释至5μg/mL;
重组人源CLL-1稀释液(分析物1):取重组人源CLL-1(250μg/mL)用运行缓冲液稀释至50nM,2倍梯度稀释得到50nM、25nM、12.5nM、6.25nM、3.125nM、0nM的重组人源CLL-1稀释液;
重组食蟹猴CLL-1稀释液(分析物2):取重组食蟹猴CLL-1(250μg/mL)用运行缓冲液稀释至50nM,2倍梯度稀释得到50nM、25nM、12.5nM、6.25nM、3.125nM、0nM的重组食蟹猴CLL-1稀释液;
(2)重组人源CLL-1抗原分析
用Protein A芯片进行检测,5μg/mL抗体稀释液以10μL/min的流速通过实验流路(Fc2、Fc4),捕获20s使捕获量约为454RU,之后将流速调节为30μL/min,依次加入不同浓度的重组人源CLL-1稀释液(0、3.125nM、6.25nM、12.5nM、25nM、50nM),同时经过实验流路(Fc2、Fc4)和参比流路(Fc1、Fc3)表面,结合时间为85s,解离时间为70s,最后加入甘氨酸液(Glycine,pH=1.5)60s,对芯片进行再生并进入下一个循环。
(3)重组食蟹猴CLL-1抗原分析
用Protein A芯片进行检测,5μg/mL抗体稀释液以10μL/min的流速通过实验流路(Fc2、Fc4),捕获20s使捕获量约为454RU,之后将流速调节为30μL/min,依次加入不同浓度的重组食蟹猴CLL-1稀释液(0、3.125nM、6.25nM、12.5nM、25nM、50nM),同时经过实验流路(Fc2、Fc4)和参比流路(Fc1、Fc3)表面,结合时间为85s,解离时间为70s,最后加入甘氨酸液(Glycine,pH=1.5)60s,对芯片进行再生并进入下一个循环。
(4)数据分析
用数据分析软件Evaluation Software3.1对试验结果进行分析,将样品试验流路采集所得传感信号进行参比流路、样品空白双扣减,并选用动力学“1:1”模型进行拟合,得出各批次样品同shTNF-α的动力学参数(ka:结合速率;kd:解离速率;kD:结合解离平衡常数)。6个抗体与重组人源CLL-1结合的动力学拟合结果如表6和图6A、图6B、图6C、图6D、图6E和图6F所示。
表6抗体同重组人CLL-1结合的动力学拟合结果统计表
样品 | ka(1/Ms) | kd(1/s) | KD(M) | Rmax(RU) | Chi<sup>2</sup>(RU<sup>2</sup>) |
27H4-scFv-hFc | 4.11E+06 | 1.26E-02 | 3.06E-09 | 249.8 | 0.839 |
19C1-scFv-hFc | 8.86E+05 | 5.09E-03 | 5.75E-09 | 273.8 | 0.11 |
23D7-scFv-hFc | 2.93E+05 | 3.22E-02 | 1.10E-07 | 272.1 | 0.232 |
Hz27H4-scFv-hFc | 4.12E+06 | 6.25E-03 | 1.52E-09 | 273.9 | 0.638 |
h27H4H1L1(全抗) | 4.91E+06 | 5.49E-03 | 1.12E-09 | 256.3 | 0.67 |
h27H4H1L2(全抗) | 5.04E+06 | 5.44E-03 | 1.08E-09 | 207.1 | 0.342 |
结果显示,除23D7-scFv-hFc与重组人源CLL-1的结合亲和力较低外,另外5个抗体与重组人源CLL-1结合的亲和力均在1nM~6nM之间;6个抗体与重组食蟹猴CLL-1均没有结合。
综上所述,本发明的抗CLL1抗体23D7、27H4、19C1对CLL1具有显著的结合能力,经过人源化改造后,进一步提高了抗体与CLL1的亲和力,在肿瘤的临床诊断和/或治疗方面具有重要的应用前景。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 广州百暨基因科技有限公司
<120> 抗CLL1抗体及其应用
<130> 20201208
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Claims (15)
1.抗CLL1抗体,其特征在于,所述抗CLL1抗体包括重链可变区和轻链可变区;
所述抗CLL1抗体的重链可变区包括SEQ ID NO: 12所示的CDR1、SEQ ID NO: 13所示的CDR2、SEQ ID NO: 14所示的CDR3;
所述抗CLL1抗体的轻链可变区包括SEQ ID NO: 15所示的CDR1、SEQ ID NO: 16所示的CDR2、SEQ ID NO: 17所示的CDR3。
2.根据权利要求1所述的抗CLL1抗体,其特征在于,所述抗CLL1抗体的重链可变区包括SEQ ID NO: 22所示的氨基酸序列,轻链可变区包括SEQ ID NO: 23所示的氨基酸序列。
3.根据权利要求1所述的抗CLL1抗体,其特征在于,所述抗CLL1抗体的重链可变区包括SEQ ID NO: 24所示的氨基酸序列,轻链可变区包括SEQ ID NO: 25、SEQ ID NO: 26或SEQID NO: 27所示的氨基酸序列。
4.根据权利要求1所述的抗CLL1抗体,其特征在于,所述抗CLL1抗体为单体,由一组重链和轻链形成,所述抗CLL1抗体的重链可变区和轻链可变区之间通过链间二硫键连接。
5.根据权利要求1所述的抗CLL1抗体,其特征在于,所述抗CLL1抗体为多聚体,由多组重链和轻链形成,所述抗CLL1抗体的重链可变区和轻链可变区之间通过链间二硫键连接,所述抗CLL1抗体的不同重链可变区之间通过链间二硫键连接。
6.根据权利要求1所述的抗CLL1抗体,其特征在于,所述抗CLL1抗体还包括恒定区。
7.根据权利要求1所述的抗CLL1抗体,其特征在于,所述抗CLL1抗体修饰有糖基化基团。
8.核酸分子,其特征在于,所述核酸分子包括编码权利要求1-7任一项所述的抗CLL1抗体的DNA片段。
9.表达载体,其特征在于,所述表达载体包括权利要求8所述的核酸分子。
10.重组细胞,其特征在于,所述重组细胞表达权利要求1-7任一项所述的抗CLL1抗体。
11.根据权利要求10所述的重组细胞,其特征在于,所述重组细胞的基因组中整合有权利要求8所述的核酸分子。
12.根据权利要求10所述的重组细胞,其特征在于,所述重组细胞包括权利要求9所述的表达载体。
13.药物组合物,其特征在于,所述药物组合物包括权利要求1-7任一项所述的抗CLL1抗体。
14.根据权利要求13所述的药物组合物,其特征在于,所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。
15.权利要求1-7任一项所述的抗CLL1抗体、权利要求8所述的核酸分子、权利要求9所述的表达载体、权利要求10~12任一项所述的重组细胞或权利要求13~14任一项所述的药物组合物在制备急性髓系白血病检测试剂和/或疾病治疗药物中的应用。
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AU2023254830A1 (en) * | 2022-04-11 | 2024-10-17 | Vor Biopharma Inc. | Binding agents and methods of use thereof |
WO2024032247A1 (zh) * | 2022-08-09 | 2024-02-15 | 合源康华医药科技(北京)有限公司 | 一种cll1抗体及其应用 |
CN115850476B (zh) * | 2022-08-09 | 2023-09-05 | 合源康华医药科技(北京)有限公司 | 一种cll1抗体及其应用 |
WO2024141063A1 (zh) * | 2022-12-30 | 2024-07-04 | 甘李药业股份有限公司 | 抗rsv病毒抗体、组合物、制剂及其应用 |
WO2024149225A1 (zh) * | 2023-01-10 | 2024-07-18 | 合源康华医药科技(北京)有限公司 | 一种人源化cll1抗体、嵌合抗原受体及其应用 |
CN116640211A (zh) * | 2023-02-28 | 2023-08-25 | 浙江康佰裕生物科技有限公司 | 特异性结合cll1蛋白的单域抗体及其应用 |
CN116462761B (zh) * | 2023-06-14 | 2023-09-08 | 浙江时迈药业有限公司 | 针对cll1的抗体及其用途 |
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EP2211903A4 (en) | 2007-10-17 | 2011-07-06 | Nuvelo Inc | CLL-1 ANTIBODY |
US9163090B2 (en) * | 2012-05-07 | 2015-10-20 | Cellerant Therapeutics, Inc. | Antibodies specific for CLL-1 |
EA201790569A1 (ru) * | 2014-09-12 | 2017-08-31 | Дженентек, Инк. | Антитела и иммуноконъюгаты против cll-1 |
JP2018504143A (ja) * | 2015-01-26 | 2018-02-15 | セレクティスCellectis | がん免疫治療のための抗hsp70特異的キメラ抗原受容体(car) |
JP6996983B2 (ja) | 2015-06-16 | 2022-02-21 | ジェネンテック, インコーポレイテッド | 抗cll-1抗体及び使用方法 |
WO2017091615A1 (en) * | 2015-11-24 | 2017-06-01 | Cellerant Therapeutics, Inc. | Humanized anti-cll-1 antibodies |
SG10201606949QA (en) * | 2016-08-19 | 2018-03-28 | Singapore Health Serv Pte Ltd | Immunosuppressive composition for use in treating immunological disorders |
CN110357960A (zh) * | 2018-04-10 | 2019-10-22 | 广州爱思迈生物医药科技有限公司 | 抗体及抗体改造方法 |
CN111116753A (zh) * | 2018-10-30 | 2020-05-08 | 上海泰因生物技术有限公司 | 一种双特异性抗体的制备方法 |
CN112961242B (zh) * | 2020-06-30 | 2022-01-04 | 广州百暨基因科技有限公司 | 抗b7h3抗体及其应用 |
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CN113045658A (zh) | 2021-06-29 |
CN113896795B (zh) | 2022-07-15 |
CN113980134A (zh) | 2022-01-28 |
EP4039708A4 (en) | 2023-11-08 |
JP2023509821A (ja) | 2023-03-10 |
AU2020480236A1 (en) | 2022-07-28 |
GB2610467A (en) | 2023-03-08 |
WO2022120943A1 (zh) | 2022-06-16 |
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EP4039708A1 (en) | 2022-08-10 |
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