CN113004406B - 抗cd47抗体及其应用 - Google Patents
抗cd47抗体及其应用 Download PDFInfo
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- CN113004406B CN113004406B CN201911335936.9A CN201911335936A CN113004406B CN 113004406 B CN113004406 B CN 113004406B CN 201911335936 A CN201911335936 A CN 201911335936A CN 113004406 B CN113004406 B CN 113004406B
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Abstract
本发明涉及抗CD47抗体、其抗原结合片段及其医药用途。还提供了包含所述抗体CDR区的嵌合抗体、人源化抗体、包含CD47抗体及其抗原结合片段的药物组合物,以及所述抗体在制备用于治疗疾病或病症的药物中的用途。
Description
技术领域
本申请属于免疫学领域。具体地,涉及抗CD47抗体及其应用。
背景技术
癌症免疫疗法是近年来生物科学领域的重头大戏,基于T细胞的CTLA4抗体、PD-1抗体、PD-L1抗体等的免疫检查点抑制剂疗法和CAR-T、TCR-T等细胞疗法皆是近年来大热的免疫疗法。这些无一例外是围绕如何恢复T细胞功能来进行,换句话说,主要围绕如何提高获得性免疫系统能力。但是以免疫检查点(checkpoint)为靶点,激活T细胞功能,从而提高获得性免疫系统能力来攻克癌症这一道路仍充满曲折。然而固有免疫系统在肿瘤免疫治疗中的作用,却长期没有得到发挥。事实上在整个肿瘤浸润区域,巨噬细胞在肿瘤组织约占50%,更重要的是巨噬细胞的数量同肿瘤的预后呈现反向联系,这进一步说明巨噬细胞在肿瘤中有很重要的作用。巨噬细胞发挥吞噬效应需要两个信号同时起作用:一个是靶向细胞表面的“吃我”信号的激活,另一个是相同目标表面“别吃我”信号的失活。任何一个信号的缺少都不足以引发吞噬效应的发生。越来越多的证据表明,CD47是一类“别吃我”信号,它通过与巨噬细胞表面的信号调节蛋白α(Signal regulatory proteinα,SIRPα)相互结合抑制巨噬细胞的吞噬作用。肿瘤细胞也可以通过CD47的表达逃避巨噬细胞吞噬作用(例如参见EP2242512及其中引用的相关文献)。
CD47也称为整联蛋白相关蛋白(IAP),是具有氨基末端免疫球蛋白结构域和羧基末端多重跨膜区的50kDa膜蛋白。它与多种配体相互作用,包括但不限于单调节蛋白α(SIRPα),SIRPγ,整联蛋白和血小板反应蛋白-1(TSP-1)。SIRPα主要在骨髓细胞上表达,包括巨噬细胞,骨髓树突细胞(DC),粒细胞,肥大细胞及其前体,包括造血干细胞。CD47/SIRPα相互作用传递“不要吃我”信号,阻止自体吞噬作用。
对患者肿瘤和匹配的邻近正常(非肿瘤)组织的分析显示CD47蛋白在癌细胞上过表达,这有效地帮助它们逃避先天免疫监视和消除。阻断CD47-SIRPα与抗CD47抗体的相互作用已经显示出有效诱导体外肿瘤细胞的吞噬作用并抑制体内各种血液和实体肿瘤的生长。因此,CD47是癌症治疗的有效靶标,并且需要其适当的拮抗剂来制备人类治疗剂。
现有技术中已知的绝大多数阻抗CD47与SIRPα结合的抗体在促进巨噬细胞的吞噬作用的同时,引起血红细胞的凝集,从而使得相应抗体的治疗效果大打折扣。因此,在针对各种肿瘤和/或癌症的治疗中,仍然急需开发具有良好的靶点特异性、疗效优异(例如改善巨噬细胞的吞噬作用,抑制肿瘤生长、甚至使得肿瘤完全消失),且副作用较小的抗CD47抗体。
发明内容
本申请提供了以下内容:
1.抗CD47抗体或其抗原结合片段,其特征在于包含以下CDRs:SEQ ID NO:2表示的HCDR1,SEQ ID NO:3表示的HCDR2,SEQ ID NO:4表示的HCDR3,SEQ ID NO:6表示的LCDR1,SEQ ID NO:7表示的LCDR2和SEQ ID NO:8表示的LCDR3;
SEQ ID NO:10表示的HCDR1,SEQ ID NO:11表示的HCDR2,SEQ ID NO:12表示的HCDR3,SEQ ID NO:14表示的LCDR1,SEQ ID NO:15表示的LCDR2和SEQ ID NO:16表示的LCDR3;
SEQ ID NO:18表示的HCDR1,SEQ ID NO:19表示的HCDR2,SEQ ID NO:20表示的HCDR3,SEQ ID NO:22表示的LCDR1,SEQ ID NO:23表示的LCDR2和SEQ ID NO:24表示的LCDR3;或
SEQ ID NO:26表示的HCDR1,SEQ ID NO:27表示的HCDR2,SEQ ID NO:28表示的HCDR3,SEQ ID NO:30表示的LCDR1,SEQ ID NO:31表示的LCDR2和SEQ ID NO:32表示的LCDR3。
2.上述1所述的抗CD47抗体或其抗原结合片段,其特征在于包含:
(1)重链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:1所示的氨基酸序列,或
与SEQ ID NO:1所示的序列具有至少60%,70%,80%,85%,优选至少86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%序列同一性的序列,和
轻链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:5所示的氨基酸序列,或
与SEQ ID NO:5所示的序列具有至少60%,70%,80%,85%,优选至少86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%序列同一性的序列;
(2)重链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:9所示的氨基酸序列,或
与SEQ ID NO:9所示的序列具有至少60%,70%,80%,85%,优选至少86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%序列同一性的序列,和
轻链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:13所示的氨基酸序列,或
与SEQ ID NO:13所示的序列具有至少60%,70%,80%,85%,优选至少86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%序列同一性的序列;
(3)重链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:17所示的氨基酸序列,或
与SEQ ID NO:17所示的序列具有至少60%,70%,80%,85%,优选至少86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%序列同一性的序列,和
轻链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:21所示的氨基酸序列,或
与SEQ ID NO:21所示的序列具有至少60%,70%,80%,85%,优选至少86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%序列同一性的序列;
(4)重链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:25所示的氨基酸序列,或
与SEQ ID NO:25所示的序列具有至少60%,70%,80%,85%,优选至少86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%序列同一性的序列,和
轻链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:29所示的氨基酸序列,或
与SEQ ID NO:29所示的序列具有至少60%,70%,80%,85%,优选至少86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%或99%序列同一性的序列。
3.上述1-2任一项所述的抗体或其抗原结合片段,其中所述抗体还包含重链恒定区和轻链恒定区,优选来自人IgG或IgM,更优选IgG1或IgG4。
4.上述1-2任一项所述的抗体或其抗原结合片段,其中所述抗原结合片段选自Fab,svFv,Fab′,dAb,F(ab′)2,Fv或Fab/c。
5.上述1-2任一项所述的抗体或其抗原结合片段,其中所述CD47为人CD47或猴CD47。
6.上述1-2任一项所述的抗体或其抗原结合片段,其中所述抗体是人源化抗体、嵌合抗体、多特异性抗体(例如双特异性抗体)。
7.编码上述1-6任一项所述的抗体或其抗原结合片段的多核苷酸。
8.药物组合物,其包含上述1-6任一项所述的抗体或其抗原结合片段;可选地,其还包括药学上可接受的载体和/或赋形剂。
9.上述1-6任一项所述的抗体或其抗原结合片段或上述8所述的药物组合物预防和/或治疗和/或辅助治疗和/或诊断治疗肿瘤疾病或在制备预防和/或治疗和/或辅助治疗和/或诊断治疗肿瘤疾病的药物中的用途,优选地,所述肿瘤是表达CD47的肿瘤,优选癌症,所述癌症选自卵巢癌、黑色素瘤、前列腺癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、子宫癌、肝癌、膀胱癌、子宫颈癌、口腔癌、脑癌、睾丸癌、皮肤癌、甲状腺癌以及血液学恶性肿瘤;优选地,所述血液学恶性肿瘤选自骨髓瘤、慢性白血病和急性白血病。
优选地,所述血液学恶性肿瘤选自骨髓瘤、慢性白血病和急性白血病。
10.上述8所述的药物组合物或上述9所述的用途,所述药物组合物或药物为适于注射的形式,优选是适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
如本发明中所使用的,术语“轻链”包括全长轻链和其具有可变区序列来赋予结合特异性的片段。全长轻链包括可变区结构域VL和恒定区结构域CL。轻链的可变区结构域在多肽的氨基末端。轻链包括κ链和λ链。
如本发明中所使用的,术语“重链”包括全长重链和其具有可变区序列来赋予结合特异性的片段。全长重链包括可变区结构域VH和3个恒定区结构域CH1、CH2和CH3。VH结构域在多肽的氨基末端,并且CH结构域在羧基末端,CH3最靠近多肽的羧基末端。重链可具有任何同种型,包括IgG(包括IgG1、IgG2、IgG3和IgG4亚型)、IgA(包括IgA1和IgA2亚型)、IgM和IgE。
如本发明中所使用的,术语“Fab片段”由一条轻链和CH1以及一条重链的可变区组成。Fab分子的重链不能与另一条重链分子形成二硫键。
如本发明中所使用的,术语“Fc”区含有两个包含抗体的CH1和CH2结构域的重链片段。两个重链片段通过两个或更多个二硫键以及通过CH3结构域的疏水相互作用保持在一起。
如本发明中所使用的,术语“Fab’片段”含有一条轻链和一条重链的部分(其含有VH结构域和CH1结构域以及还有CH1与CH2结构域之间的区域的部分),以便可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’)2分子。
如本发明中所使用的,术语“F(ab’)2片段”含有两条轻链和两条含有CH1与CH2结构域之间的恒定区的部分的重链,以便在两条重链之间形成链间二硫键。F(ab’)2片段从而由通过两条重链之间的二硫键保持在一起的两个Fab’片段组成。
如本发明中所使用的,术语“Fv区”包含来自重链和轻链的可变区,但缺乏恒定区。
如本发明中所使用的,术语“Fd”片段意指由VH和CH1结构域组成的抗体片段(Ward等人,Nature 341:544-546(1989))。
如本发明中所使用的,术语“dAb”片段(Ward等人,Nature 341:544-546(1989))由VH结构域组成。
如本发明中所使用的,术语“Fab/c”片段是免疫球蛋白经胃蛋白酶消化形成的抗体裂解中间产物,其兼有Fab和Fc区的优点,即具有扩散能力强,体内代谢清除慢的特点,并能保持高度亲和力(刘建军,《细胞与分子免疫学杂志》,1989(4):29-29)。
如本发明中所使用的,术语“单链抗体”是其中重链与轻链可变区通过柔性接头连接来形成单条多肽链(其形成抗原结合区)的Fv分子(参见,例如,Bird等人,Science.242:423-426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA.90:5879-5883(1988))。
如本发明中所使用的,术语“多特异性抗原结合蛋白”或“多特异性抗体”是靶向不止一种抗原或表位的抗原结合蛋白或抗体。
如本发明中所使用的,术语“双特异性”、“双重特异性”或“双功能性”抗原结合蛋白或抗体是分别具有两个不同的抗原结合位点的杂交抗原结合蛋白或抗体。双特异性抗体是一种多特异性抗原结合蛋白或多特异性抗体,并且可通过多种方法产生,包括,但不限于杂交瘤的融合或Fab’片段的连接。参见,例如,Songsivilai和Lachmann,1990,Clin.Exp.Immunol.79:315-321;Kostelny等人,1992,J.Immunol.148:1547-1553。双特异性抗原结合蛋白或抗体的两个结合位点将结合两个不同的表位,所述表位存在于相同或不同的蛋白质靶标上。
如本发明中所使用的,术语“人源化抗体”是指,人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段,其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如,小鼠、大鼠或兔)抗体。此外,受体抗体的框架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换,或被其他抗体的氨基酸残基替换,以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容,可参见例如,Jones et al.,Nature,321:522 525(1986);Reichmann etal.,Nature,332:323 329(1988);Presta,Curr.Op.Struct.Biol.,2:593 596(1992);和Clark,Immunol.Today 21:397 402(2000)。
如本文中使用的,术语“相似性”或“序列相似性”、“同一性”是指两个或更多个蛋白质或多肽分子的序列之间的关系,如通过比对和比较序列测定的。“百分比同一性”意指被比较的分子中的氨基酸之间的相同残基的百分比,并且可基于待比较的最小的分子的大小来计算。为了进行这些计算,必须通过特定的数学模型或计算机程序(即,“算法”)来解决比对中的缺口(如果有的话)。当用于多肽时,术语″大体上的同一性″,是指两个肽序列,当例如使用程序GAP或BESTFIT,利用程序提供的缺省缺口权重,进行最佳对齐时,共有至少70%、75%或80%的序列同一性,至少90%或95%的序列同一性,和至少97%、98%或99%的序列同一性。在某些情况下,不相同的残基位点相异在于保守氨基酸置换。″保守氨基酸置换″是这样的置换,即其中氨基酸残基被具有拥有相似化学性质(例如,电荷或正在水性)的侧链R基团的另一个氨基酸残基置换。一般地,保守氨基酸置换将基本上不改变蛋白质的功能性质。在其中两个或更多个氨基酸序列彼此相异在于保守置换的情况下,可上调百分比序列同一性以就置换的保守性质进行修正。用于进行该调整的方法对于本领域技术人员来说是熟知的。参见例如Pearson,Methods Mol.Biol.243:307-31(1994)。具有拥有相似化学性质的侧链的氨基酸组的实例包括1)脂肪族羟基侧链:甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸:2)脂肪族羟基侧链:丝氨酸和苏氨酸:3)含酰胺侧链:天冬酰胺和谷氨酰胺:4)芳香族侧链:苯丙氨酸、酪氨酸和色氨酸:5)碱性侧链:赖氨酸、精氨酸和组氨酸:6)酸性侧链:天冬氨酸和谷氨酸;和7)含硫侧链:半胱氨酸和甲硫氨酸。例如,保守氨基酸置换组是缬氨酸-亮氨酸-异亮氨酸-甘氨酸-丙氨酸、苯丙氨酸-酪氨酸、苏氨酸-丝氨酸、赖氨酸-精氨酸、谷氨酸-天冬氨酸和天冬酰胺-谷氨酰胺。
可选择地,保守置换是在Gonnet等人,Science 256:1443-45(1992)(通过引用合并入本文)中公开的PAM250对数似然矩阵(PAM250 log-likelihood matrix)中具有正值的任何变化。“中度保守的”置换是在PAM250对数似然矩阵中具有非负值的任何变化。
附图说明
图1.CD47抗体与表达人类CD47的CHO细胞的结合。使用过表达人类CD47(CHO-hCD47细胞)的CHO细胞系的基于细胞的流式测定本发明CD47鼠单抗与人类CD47的结合。
图2.CD47抗体与表达猴CD47的CHO细胞的结合。使用过表达猴CD47(CHO-cynoCD47细胞)的CHO细胞系的基于细胞的流式测定本发明CD47鼠单抗与猴CD47的结合。
图3.CD47抗体阻断SIRPα与CCRF-CEM细胞上的CD47的结合。基于细胞的流式测定。
图4.显示了人巨噬细胞与CD47抗体对肿瘤细胞的吞噬作用。
图5.CD47抗体在不同剂量下诱导红细胞(RBC)凝聚的活性。
具体实施方式
以下,通过实施例对本发明进行更具体的说明。但是,本领域技术人员可以理解的是,以下的实施例仅仅是为了说明本发明的目的,而非用于限制本发明。
实施例1鼠单抗的制备
1.hCD47-ECD-HIS重组表达质粒的构建
以GenBank提供的序列为模板(CEJ 95640.1),全基因合成人CD47胞外区(hCD47-ECD)全长编码DNA序列并且在3’端添加6个HIS标签序列,通过5’端EcolI和3’端HindIII双酶切位点克隆入表达载体pCDNA3.4(thermo公司),建立CD47胞外全长蛋白的重组真核表达质粒,即hCD47-ECD-HIS重组质粒DNA。
2.hCD47-ECD-HIS重组蛋白的表达与纯化
(1)瞬时转染前一天Expi293(ThermoFisher Scientific;A14635)细胞传代,用Dynamis培养基(gibco;A2617502)按2*106的密度接种1L摇瓶(conning;431147),放入细胞培养摇床(AdolfKuhner;ISF4-XC)中37℃;8%CO2;120rpm培养;
转染当天,Expi293细胞用细胞计数仪(Countstar;IC1000)计数,用新鲜Dynamis培养稀释调整细胞密度为2.9*106;准备转染;PEI∶DNA=3∶1;混匀5min,将二者轻柔混匀20次,静置15-30min,不要超过30min。将DNA-PEI混合物加入Expi293细胞中,混匀,放入细胞培养摇床(Adolf Kuhner;ISF4-XC)中37℃;8%CO2;120rpm培养;转染4h之后补加双抗(gibco;15140122)和抗凝剂(gibco;0010057);
(2)收获上清:转染连续培养7天,之后收样,先低速1000rpm;10min;4℃离心(湘仪H2050R),再高速12000rpm;30min;4℃;收集细胞培养上清,0.22um过滤。
(3)HisTrap亲和层析柱纯化:将上清以1mL/min速度上样HisTrap亲和层析柱,完成上样后,用5个柱体积的20mM Tris-HCl,150mM NaCl pH8.0平衡液冲洗层析柱;用5个柱体积的20mM Tris-HCl,150mM NaCl,0-500mM咪唑pH8.0洗脱液冲洗层析柱,收集洗脱峰。纯化后的CD47-ECD蛋白用聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。
3.抗CD47单克隆抗体的制备与筛选
将上述纯化的hCD47-ECD-HIS重组蛋白(以下简称为hCD47抗原)用于BALB/C小鼠(购于广东省实验动物中心)免疫。具体方法如下:
(1)动物免疫:经过纯化的hCD47抗原以完全弗氏佐剂乳化,采用皮下或腹腔注射方法免疫6-8周龄BALB/C小鼠,免疫剂量为25μg/只,每组5只;间隔1周后进行第二次免疫,以不完全弗氏佐剂乳化,免疫剂量为25μg/只。免疫4次后取尾血以ELISA法梯度稀释测定血清效价;融合前三天加强免疫,选取抗体效价最高的小鼠进行细胞融合。
(2)细胞融合:骨髓瘤细胞采用BALB/C来源的sp2/0(CRL-1581),融合时处于对数生长期;取已免疫小鼠脾脏,制成淋巴细胞单细胞悬液;小鼠脾淋巴细胞与骨髓瘤细胞以1∶5-1∶10混合,滴加37℃的50%PEG1500(pH 8.0)1mL,加入不完全培养基DMEM及其余终止液,离心弃上清后加入HAT培养基悬浮混匀,铺板96孔,置于37℃、5%CO2恒温培养箱中进行培养。培养一周后,用HT培养基进行第一次换液,再培养三天后,用HT培养基进行第二次换液。
(3)筛选和克隆:
融合2周左右,取细胞上清进行ELISA试验检测,检测细胞上清与纯化的CD47-ECD-His重组蛋白的结合情况,筛选出ELISA结果为阳性的细胞后,进行第二次ELISA试验复测;
对阳性孔细胞进行有限稀释,每次有限稀释后7天测定ELISA值,挑取OD280阳性值较高的单克隆孔进行有限稀释,直至ELISA测定96孔板全板结果为阳性。挑取阳性值高的单克隆株。
(4)细胞上清单抗的制备与纯化:将阳性克隆用含15%血清的DMEM培养基培养于T25培养瓶中培养,扩培时,800rpm/min离心5min,弃上清并将细胞转移到500ml摇瓶中,加入无血清培养基(Hybridoma-SFM Complete DPM;Gibco;12300-067),使细胞密度约为3×105个/mL。继续培养1~2周后,当细胞死亡率达到60%-70%时,收取细胞悬液8000rpm/min高速离心20min,取上清,亲和层析法进行上清纯化,采用Protein A亲和层析进行抗体纯化。上样;流洗;pH7.4、2.5M PBS流洗,冲至UV280基线为0;洗脱:pH3.5、0.1M柠檬酸溶液,每段2ml收集洗脱管,每管加入100ul 1M Tris溶液;浓缩收集液;纯化后的单抗浓度测定、SDS-PAGE胶核实抗体纯度;分装(100uL/管),保存在-80℃
(5)在Expi293细胞中表达并且纯化实施例中使用的Hu5F9对照抗体,Hu5F9是在Expi293细胞中瞬时表达的人CD47抗体,其序列与美国专利US2015/0183874 A1中的抗体“5F9”的序列相同。
对于Expi293细胞中抗体的瞬时表达,使用载体pCDNA3.4。首先将抗体的重链和轻链克隆到单独的pCDNA3.4载体中。使用化学转染的方法将带有抗体分子重链和轻链的pCDNA3.4载体转入Expi293细胞中。采用的化学转染试剂为PEI(购自Polysciences),按照生产产商提供的方案瞬时转染培养的Expi293。
瞬时转染前一天Expi293(ThermoFisher Scientific;A14635)细胞传代,用Dynamis培养基(gibco;A2617502)按2*106的密度接种1L摇瓶(conning;431147),放入细胞培养摇床(Adolf Kuhner;ISF4-XC)中37℃;8%CO2;120rpm培养;
转染当天,Expi293细胞用细胞计数仪(Countstar;IC1000)计数,用新鲜Dynamis培养稀释调整细胞密度为2.*106;准备转染;PEI∶DNA=3∶1;混匀5min,将二者轻柔混匀20次,静置15-30min,不要超过30min。将DNA-PEI混合物加入Expi293细胞中,混匀,放入细胞培养摇床(Adolf Kuhner;ISF4-XC)中37℃;8%CO2;120rpm培养;转染4h之后补加双抗(gibco;15140122)和抗凝剂(gibco;0010057);
收获上清纯化:转染连续培养7天,之后收样,先低速1000rpm;10min;4℃离心(湘仪H2050R),再高速12000rpm;30min;4℃;收集细胞培养上清,0.22um过滤。
将培养上清应用于Protein A Sepharose柱(GE Healthcare)。柱用PBS洗涤,然后用洗脱缓冲液(0.1M柠檬酸钠缓冲液,pH 3.0)洗脱蛋白质。收集的组分用1M Tris pH9.0中和。最后,将纯化的样品与PBS进行透析。在还原或非还原条件下,用10%凝胶上的十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析洗脱抗体组分的纯度。考马斯亮蓝染色显示条带。
实施例2:本发明抗CD47抗体的亲和力测定
采用生物光干涉测量(ForteBio)测定法测定本发明抗体结合人CD47(hCD47)的平衡解离常数(KD)。ForteBio亲和力测定按照现有的方法(Estep,P等人,High throughputsolution Based measurement of antibody-antigen affi nity and epitopebinning.MAbs;2013.5(2):p.270-8)进行。简言之,传感器在分析缓冲液中线下平衡30分钟,然后线上检测60秒建立基线,在线加载如上所述获得的经纯化的抗体至AHQ传感器(ForteBio)上进行ForteBio亲和测量。再将具有加载的抗体的传感器暴露于100nM的CD47抗原中作用5分钟,之后将传感器转移至分析缓冲液解离5分钟用于解离速率测量。使用1∶1结合模型进行动力学的分析。
在如以上测定法所述进行的实验中,CD47-60、CD47-50、5CD47-4、6CD47-9亲和力如表1所示。
表1:
抗体名称 | kon(1/Ms) | kdis(1/s) | KD(M) |
CD47-60 | 6.57E5 | 1.15E-6 | 1.75E-12 |
CD47-50 | 6.96E5 | 1.66E-6 | 2.38E-12 |
5CD47-4 | 5.09E5 | <1.0E-07 | <1.0E-12 |
6CD47-9 | 7.12E5 | 6.79E-5 | 9.54E-11 |
Hu5F9 | 7.44E5 | 6.36E-5 | 8.55E-11 |
可见,本发明上述示例抗体均显示极高的亲和力,与本领域已知、公认的优异CD47抗体Hu5F9具有相当或更好的的亲和力。
实施例3:本发明抗CD47抗体与人CD47的结合
在基于流式细胞术的测定法中测量本发明的上述示例抗体与人CD47的结合。
通过转染携带有全基因合成的全长人CD47(序列来源GenBank CEJ 95640.1)的pCDNA3.4载体,产生过表达人CD47的CHO细胞稳定细胞株(CHO-hCD47细胞)。将CHO-hCD47细胞(0.2×106个细胞)与不同浓度的实验抗体((本发明的上述示例抗体以及Hu5F9)最高浓度为60ug/ml,三倍稀释,总共测试了10个浓度)在含0.1%牛血清白蛋白(BSA)的PBS中,冰上孵育30分钟。然后将细胞洗涤至少两次,用FCM buffer(1XPBS+3%BSA)配制APC标记的抗鼠IgG(Fc)Ab(Biolegend)荧光二抗,按100ul/孔加入对应的96孔板中,4度冰箱孵育30min。取出96孔板,250g离心5min,小心去上清后,加入FCM buffer 200ul/孔,再次250g离心5min,小心去上清,将细胞洗涤至少两次用1xPBS 100uL/孔重悬,并通过流式细胞术进行分析,并根据其MFI用GraphPad拟合浓度依赖的曲线。本领域已知的CD47抗体Hu5F9作为阳性对照(美国专利US2015/0183874A1)。结果见表2和图1。
表2
抗体名称 | EC50(nM) |
CD47-60 | 3.298 |
CD47-50 | 2.417 |
5CD47-4 | 2.517 |
6CD47-9 | 3.534 |
Hu5F9 | 3.016 |
可见,本发明上述示例抗体均显示出很好的特异性结合能力,与本领域已知、公认的优异CD47抗体Hu5F9对照抗体相比,本发明的抗体对细胞水平的人CD47表现出类似或更高的结合能力
实施例4:本发明抗CD47抗体与猴CD47的结合
通过转染携带有全基因合成的全长猴CD47(序列来源UniParc A0A2K5X4I2-1)的pCDNA3.4载体,产生过表达猴CD47的CHO细胞稳定细胞株(CHO-cynoCD47细胞)。将CHO-cynoCD47细胞(0.2×106个细胞)与不同浓度的实验抗体((本发明的上述示例抗体以及Hu5F9)最高浓度为60ug/ml,三倍稀释,总共测试了10个浓度)在含0.1%牛血清白蛋白(BSA)的PBS中,冰上孵育30分钟。然后将细胞洗涤至少两次,用FCM buffer(1XPBS+3%BSA)配制APC标记的抗鼠IgG(Fc)Ab(Biolegend)荧光二抗,按100ul/孔加入对应的96孔板中,4℃孵育30min。取出96孔板,250g离心5min,小心去上清后,加入FCM buffer 200ul/孔,再次250g离心5min,小心去上清,将细胞洗涤至少两次用1xPBS 100uL/孔重悬,并通过流式细胞术进行分析,并根据其MFI用GraphPad拟合浓度依赖的曲线。本领域已知的CD47抗体Hu5F9作为阳性对照(美国专利US2015/0183874 A1)。结果见表3和图2。
表3.
抗体名称 | EC50(nM) |
CD47-60 | 0.9506 |
CD47-50 | 0.671 |
5CD47-4 | 0.6908 |
6CD47-9 | 0.6385 |
Hu5F9 | 0.822 |
可见,本发明上述示例抗体均显示出与猴CD47有很好的特异性结合能力,与本领域已知、公认的优异CD47抗体Hu5F9对照抗体相比,本发明的抗体对细胞水平形式的猴CD47表现出类似或更高的结合能力。实施例5.本发明抗CD47抗体对人CD47配体SIRP α与CD47相互作用的阻断
通过流式细胞术测量本发明的上述4个示例抗体阻断人CD47与SIRP α结合的能力。
抗体稀释:取出已用20nM醋酸钠溶液(pH6~8)稀释至0.25mg/ml的鼠单抗及对照单抗,用FCM buffer将鼠单抗稀释成180ug/ml(144ul样品+56ul buffer),然后3倍梯度稀释,共10个浓度梯度;将对照抗体Hu5F9也稀释成180ug/ml,同样3倍梯度稀释,共10个浓度梯度;将亚型对照mIgG1(Biolegend)稀释成60ug/ml、2.22ug/ml、0.082ug/ml;配体hSIRPα-hFC(Acro Biosystems)稀释至1ug/ml。
取CCRF-CEM(ATCC#CCL-119)离心400g 5min后弃去上清,用FCM buffer调整细胞密度为2*106,按100uL/管加入到96孔V型板,并且在不同浓度梯度的CD47抗体条件下监控hSIRP α-hFC结合。使用PE anti-human IgG Fc二抗(Biolegend)确定结合的SIRPα。本领域已知的CD47抗体Hu5F9作为阳性对照(美国专利US2015/0183874A1)。结果见表4和图3。
表4
抗体名称 | EC50(nM) |
CD47-60 | 0.5494 |
CD47-50 | 0.3689 |
5CD47-4 | 0.348 |
6CD47-9 | 0.3451 |
Hu5F9 | 0.4493 |
可见,本发明上述示例抗体均能够在细胞水平显著抑制阻断CD47与SIRPα的结合,与本领域已知、公认的优异CD47抗体Hu5F9对照抗体相比,本发明的抗体对表现出类似或更好的阻断能力。
实施例6.本发明抗CD47抗体促进巨噬细胞吞噬肿瘤细胞的能力检测
在基于流式细胞术的测定法中测量本发明的抗体促进巨噬细胞吞噬肿瘤细胞的能力。
巨噬细胞的分化:
取自捐赠者的新鲜血液,得到外周血单核细胞(PBMC)。将分离的PBMC铺板于60mm平板中,体积为8ml/板,37度静置2h,用含10%FBS的X-VIVO 15培养基(LONZA)洗涤2次,2ml/次,然后配置含rhGM-CSF(R&D;7954-GM-010)和10%FBS的X-VIVO 15培养基,rhGM-CSF终浓度为10ng/ml,按8ml/皿加入到细胞培养皿中;转移至5%CO2 37度细胞培养箱中,每3天对半更换新鲜培养基;在第5天加入IFN gamma(R&D;285-IF-100)20ng/ml刺激1h30min,然后用10%FBS的X-VIVO15培养基(含rhGM-CSF 10ng/ml和LPS(Sigma;L2630-10mg)100ng/ml)连续刺激2天。在第7天将巨噬细胞上清吸到15ml离心管中,同时加入预冷的DPBS,直接用细胞刮刀收集细胞;
将靶肿瘤细胞CCRF-CEM(购自ATCC)按照CellTraceTM CFSE(BD Bioscience)试剂盒的说明,进行荧光标记。将标记好的肿瘤细胞与上述已经完成分化的巨噬细胞按照1∶1的比例共培养,同时加入1ug/ml抗体在37℃孵育2小时。然后将细胞洗涤至少两次,将细胞小心吹下,
加入别藻青蛋白(allophycocyanin,APC)标记的CD14抗体(购自Biolegend;B259538),在含0.1%BSA的PBS中冰上(避光)孵育30分钟。将细胞洗涤至少两次并通过流式细胞术进行分析。被吞噬的细胞群体为活细胞中CD14阳性并且荧光染料CFSE(carboxyfluorescein diacetate,succinimidyl ester,羧基荧光素双乙酸盐,琥珀酰亚胺酯)也为阳性的细胞群体。结果见图4。
如图4所示,这4种被测抗体显示了很强的促进巨噬细胞吞噬肿瘤细胞的能力,且促进巨噬细胞吞噬肿瘤细胞的能力显著高于对照抗体Hu5F9的能力。
实施例7.CD47抗体对人类红细胞(RBC)血凝集分析
公开的研究表明,某些抗CD47抗体可能引起人红细胞(RBC)的血细胞凝集。因此,进行血细胞凝集分析表征抗CD47抗体促进RBC凝集的能力。通过观察抗体避免人RBC发生沉降的能力就RBC凝集对CD47抗体进行筛选。出乎意料的是,发现5CD47-4抗体是独特的,其无法促进血凝反应,同时具有高亲和力和阻断SIRPα的能力。
人红细胞在PBS中稀释到10%,与滴入的CD47抗体在圆底96孔板内在37℃孵育2小时。未沉淀的红细胞的存在是证明血细胞凝聚的证据,与未凝聚的红细胞沉淀形成的清晰的红点相比,未沉淀的红细胞呈雾状。
如图5所示,CD47抗体Hu5F9已经显示在等于或高于0.176ug/mL的浓度下,红细胞会显著凝聚,但是,5CD47-4抗体在浓度达到45ug/mL的实验中,未引起实质上的红细胞凝聚。可见,本申请公开的抗体具有显著降低的血细胞凝集作用,因此在临床治疗中具有显著降低的副作用,可以广泛应用于多种癌症的治疗中。
Claims (18)
1.抗CD47抗体或其抗原结合片段,其特征在于包含以下CDRs:
SEQ ID NO:18表示的HCDR1,SEQ ID NO:19表示的HCDR2,SEQ ID NO:20表示的HCDR3,SEQ ID NO:22表示的LCDR1,SEQ ID NO:23表示的LCDR2和SEQ ID NO:24表示的LCDR3。
2.权利要求1所述的抗CD47抗体或其抗原结合片段,其特征在于包含:
重链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:17所示的氨基酸序列,和
轻链可变区,其包含下述序列或由下述序列组成:
SEQ ID NO:21所示的氨基酸序列。
3.权利要求1-2任一项所述的抗体或其抗原结合片段,其中所述抗体还包含重链恒定区和轻链恒定区。
4.权利要求3所述的抗体或其抗原结合片段,其中所述重链恒定区和轻链恒定区来自人IgG或IgM。
5.权利要求3所述的抗体或其抗原结合片段,其中所述重链恒定区和轻链恒定区来自人IgG1或IgG4。
6.权利要求1-2任一项所述的抗体或其抗原结合片段,其中所述抗原结合片段选自Fab,svFv,Fab',dAb,F(ab')2,Fv或Fab/c。
7.权利要求1-2任一项所述的抗体或其抗原结合片段,其中所述CD47为人CD47或猴CD47。
8.权利要求1-2任一项所述的抗体或其抗原结合片段,其中所述抗体是人源化抗体、嵌合抗体、多特异性抗体。
9.权利要求1-2任一项所述的抗体或其抗原结合片段,其中所述抗体是双特异性抗体。
10.编码权利要求1-8任一项所述的抗体或其抗原结合片段的多核苷酸。
11.药物组合物,其包含权利要求1-8任一项所述的抗体或其抗原结合片段。
12.权利要求11所述的药物组合物,其还包括药学上可接受的载体和/或赋形剂。
13.权利要求1-8任一项所述的抗体或其抗原结合片段或权利要求11或12所述的药物组合物在制备预防和/或治疗和/或辅助治疗和/或诊断治疗表达CD47的肿瘤疾病的药物中的用途。
14.权利要求13所述的用途,其中所述肿瘤是癌症。
15.权利要求14所述的用途,其中所述癌症选自卵巢癌、黑色素瘤、前列腺癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、子宫癌、肝癌、膀胱癌、子宫颈癌、口腔癌、脑癌、睾丸癌、皮肤癌、甲状腺癌以及血液学恶性肿瘤。
16.权利要求15所述的用途,其中所述血液学恶性肿瘤选自骨髓瘤、慢性白血病和急性白血病。
17.权利要求11或12所述的药物组合物或权利要求13所述的用途,所述药物组合物或药物为适于注射的形式。
18.权利要求11或12所述的药物组合物或权利要求13所述的用途,所述药物组合物或药物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
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