CN114656566A - 一种靶向cd47的抗体及其应用 - Google Patents
一种靶向cd47的抗体及其应用 Download PDFInfo
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- CN114656566A CN114656566A CN202111582135.XA CN202111582135A CN114656566A CN 114656566 A CN114656566 A CN 114656566A CN 202111582135 A CN202111582135 A CN 202111582135A CN 114656566 A CN114656566 A CN 114656566A
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Abstract
本发明涉及生物医药领域，具体而言，涉及一种靶向CD47的抗体及其应用。本发明提供的抗CD47抗体能特异性结合肿瘤细胞，并阻断人SIRPa与人CD47信号，可以促进巨噬细胞对肿瘤细胞的吞噬作用，该抗体的亲和力高、特异性强，安全性好，不会引起红细胞凝集，且与RBC、血小板表现出极弱的结合。
Description
技术领域
本发明属于生物医药技术领域。具体地，涉及一种靶向CD47的抗体及其应用，更具体地，涉及抗CD47抗体或其抗原结合片段、核酸、载体、细胞、组合物、应用以及试剂盒。
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，以提高获得性免疫系统的能力，进而攻克癌症的道路仍充满曲折。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。
发明内容
CD47单克隆抗体与红细胞有较高的靶向结合性，易引发红细胞凝集，使相应抗体在治疗效果上大打折扣、并引发药物副作用，本发明旨在提供一种靶向CD47抗体，所述抗体能够阻断CD47与SIRPα结合，促进巨噬细胞发挥吞噬作用，更难能可贵的是，能在一定程度上避免红细胞血发生凝集，安全性好。
本发明的目的是提供抗CD47抗体或其抗原结合片段，所述抗体含有以下CDRs：
如SEQ ID NO:1或与SEQ ID NO:1具有至少95％同源性的氨基酸序列所示的LCDR1，如SEQ ID NO:2或与SEQ ID NO:2具有至少95％同源性的氨基酸序列所示的LCDR2，如SEQ ID NO:3或与SEQ ID NO:3具有至少95％同源性的氨基酸序列所示的LCDR3；
如SEQ ID NO:10或与SEQ ID NO:10具有至少95％同源性的氨基酸序列所示的HCDR1，如SEQ ID NO:11或与SEQ ID NO:11具有至少95％同源性的氨基酸序列所示的HCDR2，如SEQ ID NO:12或与SEQ ID NO:12具有至少95％同源性的氨基酸序列所示的HCDR3。
本发明的另一目的是提供所述抗CD47抗体或其抗原结合片段相关的核酸、载体、细胞或药物组合物。
本发明还涉及所述抗CD47抗体或其抗原结合片段、及其相关的核酸、载体、细胞或药物组合物在制备治疗和/预防CD47阳性肿瘤的药物中的应用。
本发明还涉及所述抗CD47抗体或其抗原结合片段，及其相关的核酸、载体或细胞在制备检测CD47或诊断CD47相关疾病的试剂盒中的应用。
本发明还提供了一种检测CD47的试剂盒，所述试剂盒含有所述抗CD47抗体或其抗原结合片段。
本发明还提供了一种诊断CD47相关疾病的试剂盒，所述试剂盒含有所述抗CD47抗体或其抗原结合片段。
附图说明
图1是CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与人CD47结合的平均荧光强度。
图2是CD47抗体(7A11H52、7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与人CD47结合的平均荧光强度。
图3是CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、7A11H52、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与猴CD47结合的平均荧光强度。
图4是CD47抗体(7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与猴CD47结合的平均荧光强度。
图5是CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42和hIgG4-同型对照)对人CD47的CD47/SIRPα结合抑制结果。
图6是CD47抗体(7A11H52、7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55)对人CD47的CD47/SIRPα结合抑制结果。
图7是CD47抗体促进巨噬细胞吞噬肿瘤细胞的能力测定结果。
图8是CD47抗体的RBC凝集能力测定结果。
图9是CD47抗体与人类红细胞结合能力的测定结果。
图10是CD47抗体与人类血小板结合分析结果。
图11是CD47抗体对激活巨噬细胞吞噬红细胞的作用分析结果。
图12是CD47抗体对人B淋巴细胞皮下移植瘤模型的抗肿瘤结果。
图13是CD47抗体对人恶性黑色素瘤模型的抗肿瘤结果。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
本发明涉及抗CD47抗体或其抗原结合片段，所述抗体含有以下CDRs：
如SEQ ID NO:1或与SEQ ID NO:1具有至少95％同源性的氨基酸序列所示的LCDR1，如SEQ ID NO:2或与SEQ ID NO:2具有至少95％同源性的氨基酸序列所示的LCDR2，如SEQ ID NO:3或与SEQ ID NO:3具有至少95％同源性的氨基酸序列所示的LCDR3；
如SEQ ID NO:10或与SEQ ID NO:10具有至少95％同源性的氨基酸序列所示的HCDR1，如SEQ ID NO:11或与SEQ ID NO:11具有至少95％同源性的氨基酸序列所示的HCDR2，如SEQ ID NO:12或与SEQ ID NO:12具有至少95％同源性的氨基酸序列所示的HCDR3。
该抗体或其抗原结合片段的一个重要优点在于其与CD47具有高亲和力。
该抗体或其抗原结合片段的一个重要优点在于其具有阻断CD47与SIRPα结合的活性。
该抗体或其抗原结合片段的一个重要优点在于其具有促进巨噬细胞吞噬肿瘤细胞的能力。
该抗体或其抗原结合片段的一个重要优点在于其具有显著降低红细胞血凝聚的作用。
该抗体或其抗原结合片段的一个重要优点在于其与红细胞表现出极弱水平的低结合或不结合。
因具有上述特性，该抗体或其抗原结合片段优选可作为抗体药物使用。
在本发明中，“抗体或其抗原结合片段”此技术术语是结合特定抗原的蛋白，其泛指包含互补决定区(CDR区)的一切蛋白及蛋白片段。“抗体”特别指全长抗体。“全长抗体”此用语包括多克隆抗体及单克隆抗体。
术语“抗原结合片段”是包含抗体CDR的一部分或全部的物质，其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合至抗原。此类片段具生物活性，因为其结合至靶抗原，且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。在一些实施方式中，抗原结合片段具有特异性识别并结合CD47的作用。在一些实施方式中，抗原结合片段是具有阻断CD47与其配体结合，激活免疫细胞功能的片段，在一个方面中，此类片段选自Fab(由完整的轻链和Fd构成)，Fv(由VH和VL构成)，ScFv(单链抗体，VH和VL之间由一连接肽连接而成)或单域抗体(仅由VH组成)。所述片段可通过重组核酸技术产生，或可通过抗原结合分子(包括完整抗体)的酶裂解或化学裂解产生。
术语“互补性决定区”或“CDR”是指免疫球蛋白的重链和轻链的高度可变区。有三种重链CDR和三种轻链CDR。此处，取决于情况，术语“CDR”和“CDRs”用于指包含一种或多种或者甚至全部的对抗体或其抗原结合片段与其识别的抗原或表位的结合亲和力起作用的主要氨基酸残基的区域。在另一具体实施方式中，CDR区或CDR是指IMGT定义的免疫球蛋白的重链和轻链的高度可变区。
在本发明中，重链的互补决定区用HCDR表示，轻链的互补决定区用LCDR表示。本领域常用的CDR标示方法包括：Kabat编号方案、Chothia和Lesk编号方案以及1997年Lefranc等人为免疫球蛋白超家族的所有蛋白质序列引入的新的标准化编号系统。Kabat等人是第一个为免疫球蛋白可变区提出标准化编号方案的人。在他们的“免疫学蛋白质序列”(Sequences of Proteins of Immunological Interest)的汇编中，轻链(λ，κ)可变区和抗体重链的氨基酸序列，以及T细胞受体的可变区(α，β，γ，δ)对齐并编号。在过去的几十年中，序列的积累导致了KABATMAN数据库的创建，Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。本发明采用Kabat注释标准标示CDR区，但其他方法标示的CDR区也属于本发明的保护范围。
术语“特异性识别”、“选择性结合”、“选择性地结合”和“特异性地结合”或其类似表述是指抗体或其抗原结合片段对预先确定的抗原上的表位的结合。通常，抗体或其抗原结合片段以大约小于10-6M，例如大约小于10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合。
在本发明中，所提供的抗体或其抗原结合片段特异性识别的对象可以为多种属来源的CD47，例如人、鼠、猴(如食蟹猴cynomolgus monkey)。
在一些实施方式中，所述抗体含有重链可变区序列和轻链可变区序列的至少之一，所述重链可变区序列和轻链可变区序列的至少之一的至少一部分来自鼠源抗体、人源化抗体、灵长目源抗体或其突变体的至少之一。
在一些实施方式中，所述轻链可变区的氨基酸序列如SEQ ID NO:5～9任一项所示或与SEQ ID NO:5～9任一项所示具有至少95％同源性的序列。
在一些实施方式中，所述重链可变区的氨基酸序列为SEQ ID NO:14～18任一项所示或与SEQ ID NO:14～18任一项所示具有至少95％同源性的序列。
在一些实施方式中，所述轻链可变区的氨基酸序列如SEQ ID NO:5所示、重链可变区的氨基酸序列如SEQ ID NO:14所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:6所示、重链可变区的氨基酸序列如SEQ ID NO:14所示；或所述轻链可变区的氨基酸序列如SEQID NO:6所示、重链可变区的氨基酸序列如SEQ ID NO:15所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:6所示、重链可变区的氨基酸序列如SEQ ID NO:16所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:6所示、重链可变区的氨基酸序列如SEQ ID NO:17所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:6所示、重链可变区的氨基酸序列如SEQ ID NO:18所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:8所示、重链可变区的氨基酸序列如SEQ ID NO:14所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:9所示、重链可变区的氨基酸序列如SEQ ID NO:14所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:7所示、重链可变区的氨基酸序列如SEQ ID NO:16所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:8所示、重链可变区的氨基酸序列如SEQ ID NO:16所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:9所示、重链可变区的氨基酸序列如SEQ ID NO:16所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:9所示、重链可变区的氨基酸序列如SEQ ID NO:18所示。
在一些实施方式中，所述抗体具有恒定区，重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD中的任一种；轻链恒定区为κ或λ链。
在一些实施方式中，所述恒定区的种属来源选自鼠、兔、羊、猴、或人。
在一些实施方式中，所述抗体为CDR移植抗体、多聚体抗体或双特异性抗体中的任一种或几种。
在一些实施方式中，所述抗原结合片段为F(ab’)2、Fab、scFv、Fv及单域抗体中的任一种或几种。
在一些实施方式中，所述CD47为人CD47、鼠CD47或猴CD47。
本发明还涉及核酸，所述核酸编码所述抗CD47抗体或其抗原结合片段。
核酸通常是RNA或DNA，核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。当其连入载体时优选采用DNA核酸。
此外，鉴于抗体为膜蛋白，所以核酸通常带有信号肽序列。
本发明还涉及载体，所述载体携带上述核酸。
术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。在一些实施方式中，本发明所述载体中包含基因工程中常用的调控元件，例如增强子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号，或者多腺苷酸化信号和多聚U序列等)。
本发明还涉及细胞，所述细胞携带上述核酸或含有上述载体。利用编码抗CD47抗体或其抗原结合片段的核酸与载体连接后，在细胞表达能够获得相应抗体。可以将上述载体导入到真核细胞、尤其是哺乳动物细胞中，构建获得能够表达本发明所述抗CD47抗体或其抗原结合片段的细胞。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本发明还涉及药物组合物，所述组合物含有所述抗CD47抗体或其抗原结合片段、所述核酸、所述载体或所述细胞。
在一些实施方式中，所述组合物还含有药学可接受的载体。“药学可接受的载体”可以包括生理学上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等，用来延长抗体的保存限期或效力。
所述抗CD47抗体或其抗原结合片段、所述核酸、所述载体、所述细胞或所述组合物在制备治疗和/预防CD47阳性肿瘤的药物中的应用，也应在本发明的保护范围之内。
所述药物能够降低红细胞血凝集、降低其与红细胞的结合活性。
所述抗CD47抗体或其抗原结合片段、所述核酸、所述载体或所述细胞在制备检测CD47或诊断CD47相关疾病的试剂盒中的应用，也应在本发明的保护范围之内。
本发明还提供了一种检测CD47的试剂盒，所述试剂盒含有所述抗CD47抗体或其抗原结合片段。
本发明还提供了一种诊断CD47相关疾病的试剂盒，所述试剂盒含有所述抗CD47抗体或其抗原结合片段。
本发明具有以下有益效果：
本发明提供了抗CD47抗体或其抗原结合片段，该抗体在体外未发生红细胞血凝集，更难能可贵的是与红细胞表现出极弱水平的低结合或不结合；能够有效地阻断CD47与SIRPα结合、激活介导巨噬细胞对肿瘤细胞的吞噬活性，展现出了显著的与CD47阳性肿瘤细胞的靶向特异性，亲和力高、特异性强，不会引起红细胞凝集，且与人类红细胞、血小板表现出极弱的结合，安全性好；因此，所述抗CD47抗体或其抗原结合片段可以作为肿瘤免疫治疗系统中非常有前景的靶点，在人类肿瘤治疗方面发挥强有力作用，在制备治疗CD47阳性肿瘤的药物中具有广泛的应用前景。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1抗CD47鼠单抗的筛选
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
hCD47-ECD-HIS重组蛋白的表达与纯化，包括以下步骤：
(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(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2.9*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。将DNA-PEI混合物加入Expi293细胞中，混匀，放入细胞培养摇床(AdolfKuhner；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鼠单抗的筛选
将步骤2纯化得到的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，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和纯化：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100uL1MTris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℃。
实施例2鼠单抗人源化设计及组合
鼠抗体轻链CDR的氨基酸序列如SEQ ID NO:1～3所示：
| 7A11D3D3-LCDR1 | 7A11D3D3-LCDR2 | 7A11D3D3-LCDR3 |
| SEQ ID NO:1 | SEQ ID NO:2 | SEQ ID NO:3 |
| KSSQSLLNSRTRKNYLA | WASTRES | KQSYNLRT |
鼠抗体重链CDR的氨基酸序列如SEQ ID NO:10～12所示：
| 7A11D3D3-HCDR1 | 7A11D3D3-HCDR2 | 7A11D3D3-HCDR3 |
| SEQ ID NO:10 | SEQ ID NO:11 | SEQ ID NO:12 |
| SNWMN | MIHPSDSETRLNQKFKD | GTTVVDAFAY |
鼠抗体轻链可变区(>7A11D3D3-VL)的氨基酸序列如SEQ ID NO:4所示。
鼠抗体重链可变区(>7A11D3D3-VH)的氨基酸序列如SEQ ID NO:13所示。
对鼠抗体进行人源化设计，7A11D3D3鼠单抗轻链设计5条人源化序列，分别为7A11-L01～05，人源化轻链可变区(7A11-L01～05)的氨基酸序列如SEQ ID NO:5～9所示；重链设计5条人源化序列，分别为7A11-H01～05，人源化重链可变区(7A11-H01～05)的氨基酸序列如SEQ ID NO:14～18所示。
对以上氨基酸序列进行组合，所得抗体及其重链可变区(VH)和轻链可变区(VL)对应的序列如表1所示。
表1抗体及其重链可变区和轻链可变区对应的序列
| 抗体名称 | VH | VL |
| 7A11H11 | SEQ ID NO：14 | SEQ ID NO：5 |
| 7A11H12 | SEQ ID NO：14 | SEQ ID NO：6 |
| 7A11H22 | SEQ ID NO：15 | SEQ ID NO：6 |
| 7A11H32 | SEQ ID NO：16 | SEQ ID NO：6 |
| 7A11H42 | SEQ ID NO：17 | SEQ ID NO：6 |
| 7A11H52 | SEQ ID NO：18 | SEQ ID NO：6 |
| 7A11H14 | SEQ ID NO：14 | SEQ ID NO：8 |
| 7A11H15 | SEQ ID NO：14 | SEQ ID NO：9 |
| 7A11H33 | SEQ ID NO：16 | SEQ ID NO：7 |
| 7A11H34 | SEQ ID NO：16 | SEQ ID NO：8 |
| 7A11H35 | SEQ ID NO：16 | SEQ ID NO：9 |
| 7A11H55 | SEQ ID NO：18 | SEQ ID NO：9 |
实施例3CD47抗体的生产
将人源化可变结构域与分泌信号和人类κ以及人类FcIgG4S228P恒定结构域组合，将其克隆到哺乳动物表达系统中，并且转染到293细胞中以生成人源化mAb。将人源化变体表达为全长IgG分子、分泌到培养基中并使用蛋白质A纯化。
在Expi293细胞中瞬时表达并纯化人源化抗体和阳性对照抗体Hu5F9-G4(Hu5F9-G4序列已在美国专利US2015/0183874A1中公开)。
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PEI(购自Polysciences)化学转染试剂、按照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，按照生产商提供的方案瞬时转染培养的Expi293细胞。
瞬时转染前一天Expi293(ThermoFisher Scientific；A14635)细胞传代，用Dynamis培养基(gibco；A2617502)按2E6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2.9E6；准备转染；PEI：DNA＝3:1；混匀5min，将二者轻柔混匀20次，静置15～30min。将DNA-PEI混合物加入Expi293细胞中，混匀，放入细胞培养摇床(AdolfKuhner；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。
实施例4CD47抗体亲和力测定
1、实验方法
采用生物膜干涉技术(ForteBio)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 affinity and epitopebinning.MAbs；2013.5(2)：p.270-8)进行，具体为：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，在线加载实施例3获得的经纯化的抗体至AHC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。
2、实验结果
CD47抗体亲和力测定结果如表2所示，结果显示CD47抗体具有高亲和力。
表2CD47抗体亲和力测定结果
| 抗体名称 | KD(M) | kon(1/Ms) | kd(1/s) |
| 7A11H11 | 5.90E-10 | 5.90E-10 | 5.90E-10 |
| 7A11H12 | 4.59E-10 | 4.59E-10 | 4.59E-10 |
| 7A11H14 | 5.62E-10 | 5.62E-10 | 5.62E-10 |
| 7A11H15 | 2.12E-10 | 1.48E+06 | 3.13E-04 |
| 7A11H35 | 8.81E-10 | 8.81E-10 | 8.81E-10 |
| 7A11H22 | 7.40E-10 | 7.40E-10 | 7.40E-10 |
| 7A11H33 | 3.60E-10 | 3.60E-10 | 3.60E-10 |
| 7A11H42 | 3.09E-10 | 3.09E-10 | 3.09E-10 |
实施例5CD47抗体与人CD47的结合
1、实验方法
在基于流式细胞术的测定法中测量本发明CD47抗体与人CD47的结合。具体步骤为：
采用表达人CD47的癌细胞系CCRF-CEM(上海中国科学院细胞库)属于人急性淋巴细胞白血病T淋巴细胞。将CCRF-CEM细胞(0.1×106个细胞)与不同浓度(最高浓度为30ug/mL，三倍稀释，共10个浓度)的实验抗体(本发明CD47抗体以及Hu5F9-G4抗体)，在含3％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PE Goat anti human IgG Fc(1:500x稀释)荧光二抗，按100uL/孔加入对于的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FCMbuffer 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。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体与人CD47结合的EC50结果如表3所示，CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与人CD47结合的平均荧光强度如图1所示，CD47抗体(7A11H52、7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与人CD47结合的平均荧光强度如图2所示，结果显示，本发明CD47抗体与阳性对照抗体对细胞水平的人CD47具有相当的特异性结合能力。
表3CD47抗体与人CD47的结合能力测定结果
| 抗体名称 | EC50(nM) |
| 7A11H11 | 0.8218 |
| 7A11H12 | 0.7157 |
| 7A11H22 | 0.8202 |
| 7A11H32 | 0.7172 |
| 7A11H42 | 0.7269 |
| 7A11H52 | 0.9148 |
| 7A11H14 | 0.9334 |
| 7A11H15 | 0.7234 |
| 7A11H33 | 0.7598 |
| 7A11H34 | 1.195 |
| 7A11H35 | 0.8623 |
| 7A11H55 | 1.209 |
| Hu5F9-G4 | 0.7289 |
实施例6CD47抗体与猴CD47的结合
1、实验方法
通过转染携带全长猴CD47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(0.1×106个细胞)与不同浓度(最高浓度为10ug/mL，三倍稀释，共10个浓度)的实验抗体(本发明CD47抗体以及Hu5F9-G4抗体)，在含3％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FCMbuffer(1XPBS+3％BSA)配制PE Goat anti human IgG Fc(1:500x稀释)荧光二抗，按100uL/孔加入对于的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1x PBS 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。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体与猴CD47结合的EC50结果如表4所示，CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、7A11H52、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与猴CD47结合的平均荧光强度如图3所示，CD47抗体(7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55、阳性对照抗体Hu5F9-G4和hIgG4-同型对照)与猴CD47结合的平均荧光强度如图4所示，结果显示，相比，本发明抗体与阳性对照抗体对细胞水平形式猴具有相当的特异性结合能力。
表4CD47抗体与猴CD47结合的EC50结果
| 抗体名称 | EC50(nM) |
| 7A11H11 | 8.932 |
| 7A11H12 | 7.353 |
| 7A11H22 | 8.546 |
| 7A11H32 | 17.12 |
| 7A11H42 | 5 |
| 7A11H52 | 4.506 |
| 7A11H14 | 4.957 |
| 7A11H15 | 7.271 |
| 7A11H33 | 4.847 |
| 7A11H34 | 8.609 |
| 7A11H35 | 4.379 |
| 7A11H55 | 4.616 |
| Hu5F9-G4 | 5.123 |
实施例7CD47抗体对人CD47配体SIRPα与CD47相互作用的阻断
1、实验方法
通过流式细胞术测定本发明CD47抗体阻断人CD47与SIRPα的结合能力。具体步骤为：
抗体稀释：用FCM buffer(1XPBS+3％BSA)将本发明CD47抗体和对照抗体Hu5F9-G4稀释成90ug/mL，然后3倍梯度稀释成10个浓度，将亚型对照hIgG4稀释成30ug/mL、1.1ug/mL、0.04ug/mL，配体hSIRPα-mFC(AcroBiosystems)稀释至10ug/mL。
将CCRF-CEM(上海中国科学院细胞库)细胞按0.1×106个细胞/孔加入到96孔V型板，并且在CD47抗体量增加的条件下监控hSIRPα-mFC结合。使用PE Goat anti mouse IgGFc二抗(Biolegend)确定结合的SIRPα。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体(7A11H11、7A11H12、7A11H22、7A11H32、7A11H42、Hu5F9-G4和hIgG4-同型对照)对人CD47的CD47/SIRPα结合抑制结果如图5所示，CD47抗体(7A11H52、7A11H14、7A11H15、7A11H33、7A11H34、7A11H35、7A11H55)对人CD47的CD47/SIRPα结合抑制结果如图6所示，结果显示，本发明CD47抗体能够在细胞水平显著抑制阻断CD47与SIRPα的结合，与阳性对照抗体相比，本发明CD47抗体表现出相当的阻断能力。
实施例8CD47抗体促进巨噬细胞吞噬肿瘤细胞能力的检测
1、实验方法
在基于流式细胞术的测定法中测定本发明CD47抗体促进巨噬细胞吞噬肿瘤细胞的能力。具体步骤为：
取捐赠者的新鲜血液，分离得到外周血单核细胞(PBMC)，并通过hCD14磁珠(Miltenyi/130-050-201)从PBMC中分离CD14阳性单核细胞，配置含rhGM-CSF(R&D；7954-GM-010)的完全培养基，rhGM-CSF终浓度为50ng/mL，CD14阳性单核细胞的浓度为5E5/mL，按20mL/皿加入到细胞培养皿中；转移至5％CO2 37℃细胞培养箱中，每3天对半更换新鲜培养基；(含50ng/mLGM-CSF)继续培养4天。在第8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；
选择人CD47高表达的肿瘤细胞系Jurkat(上海中国科学院细胞库)作为靶细胞类型，将靶肿瘤细胞按照CellTraceTM CFSE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，同时加入终浓度10ug/mL、1ug/mL、0.1ug/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。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体促进巨噬细胞吞噬肿瘤细胞的能力测定结果如图7所示，结果显示，本发明CD47抗体具有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与阳性对照抗体Hu5F9-G4的能力相当。
实施例9CD47抗体对人类红细胞(RBC)血凝集分析
1、实验方法
进行红细胞血凝集分析来表征CD47抗体的RBC凝集能力。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。具体方法为：
人类红细胞在PBS中稀释到2％，与滴入的CD47抗体(浓度依次为200ug/mL、100ug/mL、50ug/mL、25ug/mL、12.5ug/mL、6.25ug/mL、1.5625ug/mL、0.78125ug/mL、0.390625ug/mL、0.195313ug/mL、0.097656ug/mL)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红细胞血凝聚的证据，与未凝聚的红细胞沉淀形成清晰的红点相比，未沉淀的红细胞呈雾状。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体的RBC凝集能力测定结果如图8所示，结果显示，当CD47抗体浓度达到200ug/mL时均未引起细胞凝聚，表明本发明CD47抗体具有显著降低红细胞血凝聚的作用，CD47抗体在临床治疗癌症中能够显著降低其副作用、安全性好。
实施例10CD47抗体与人类红细胞(RBC)结合分析
1、实验方法
CD47单抗存在与人类红细胞结合的特性，对CD47抗体抑制剂来说，存在药效受红细胞干扰及肿瘤脱靶的潜在风险。若能筛选到与红细胞结合活性低的抗体则能够降低脱靶的风险，提高其安全性。具体步骤如下：
(1)抗体稀释：用FACS buffer将CD47抗体稀释成初始浓度20μg/mL，体积180uL，3倍梯度稀释(60+120)，11个浓度；
(2)细胞计数并铺板：将RBC细胞离心250g 5min后弃去上清，用FACS buffer调整细胞密度为2E+06，按100uL/管均分到96孔V型板中；
(3)将上述稀释好的抗体加入细胞中，100uL/孔，2℃～8℃孵育0.5h；
(4)取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200uL/孔，再次250g离心5min，小心去上清；
(5)用FACS buffer配制PE goat anti-human IgG Fc(biolegend)荧光二抗(1:500稀释)，按100uL/孔加入对应的96孔板中，重悬细胞，2℃～8℃孵育30min；
(6)取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200uL/孔，再次250g离心5min，小心去上清；
(7)用1xPBS 100uL/孔重悬，FACS检测。使用流式细胞仪(Beckman，cytoflex)分析数据，GraphPad Prism作图。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体与人类红细胞结合能力的测定结果如图9所示，结果显示，与阳性对照抗体相比，本发明CD47抗体与人类红细胞的结合活性显著降低，其作为药物时的安全性提高。
实施例11CD47抗体与血小板结合分析
1、实验方法
同CD47单抗结合人类红细胞一样，CD47单抗存在与血小板结合的活性特征，存在血小板降低带来的诸多副作用。与血小板结合低的抗体能够降低脱靶的风险，提高其安全性。具体方法如下：
抗体稀释：用FACS buffer将抗体稀释成20μg/ml，体积240ul；
细胞计数并铺板：将全血细胞稀释20倍，按100uL/管均分到96孔V型板中；
将上述稀释好的抗体加入到细胞中，100uL/孔，2℃-8℃孵育0.5h；
取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200ul/孔，再次250g离心5min，小心去上清；
用FACS buffer配制PE荧光二抗1:500稀释(PE goat anti-human IgG Fc；biolegend；409304)，按100ul/孔加入对于的96孔板中，同时每孔中加入1ul APC antihuman CD61(biolegend；336411),重悬细胞，2℃-8℃孵育30min；
取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200ul/孔，再次250g离心5min，小心去上清；
用1xPBS 200uL/孔重悬，FACS检测。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体与血小板结合分析结果如图10所示，结果显示本发明的CD47抗体与血小板的结合活性显著低于阳性对照抗体，显示出了更优的安全性。
实施例12CD47抗体对激活巨噬细胞吞噬红细胞的作用分析
1、实验方法
检测方法同实施例8，将红细胞替换为肿瘤细胞作为靶细胞，检测本发明的CD47抗体是否对巨噬细胞吞噬红细胞存在激活作用。以Hu5F9-G4抗体作为阳性对照抗体。
2、实验结果
CD47抗体对激活巨噬细胞吞噬红细胞的作用分析结果如图11所示，结果显示本发明的CD47抗体极低介导巨噬细胞对红细胞的吞噬，且介导作用明显低于阳性对照抗体，显示出了更优的安全性。
实施例13抗肿瘤活性分析
1、实验方法
选用70只NOD SCID雌性小鼠(购于浙江维通利华实验动物技术有限公司)构建人B淋巴细胞皮下移植瘤模型(Raji)和人恶性黑色素瘤模型(A375)，评估本发明的CD47抗体的抗肿瘤活性。以Hu5F9-G4抗体和TJC-4抗体作为阳性对照抗体，hIgG4为同型对照抗体。
2、实验结果
CD47抗体对人B淋巴细胞皮下移植瘤模型的抗肿瘤结果如图12所示，结果显示本发明的CD47抗体在抗人B淋巴细胞皮下移植瘤效果上显著好于阳性对照抗体。
CD47抗体对人恶性黑色素瘤模型的抗肿瘤结果如图13所示，结果显示与阳性对照抗体相比，本发明的CD47抗体在抗人恶性黑色素瘤效果上更加有优势。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
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<120> 一种靶向CD47的抗体及其应用
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Claims (14)
1.抗CD47抗体或其抗原结合片段，其特征在于，所述抗体含有以下CDRs：
如SEQ ID NO:1或与SEQ ID NO:1具有至少95％同源性的氨基酸序列所示的LCDR1，如SEQ ID NO:2或与SEQ ID NO:2具有至少95％同源性的氨基酸序列所示的LCDR2，如SEQ IDNO:3或与SEQ ID NO:3具有至少95％同源性的氨基酸序列所示的LCDR3；
如SEQ ID NO:10或与SEQ ID NO:10具有至少95％同源性的氨基酸序列所示的HCDR1，如SEQ ID NO:11或与SEQ ID NO:11具有至少95％同源性的氨基酸序列所示的HCDR2，如SEQID NO:12或与SEQ ID NO:12具有至少95％同源性的氨基酸序列所示的HCDR3。
2.根据权利要求1所述抗CD47抗体或其抗原结合片段，其特征在于，所述抗体含有重链可变区序列和轻链可变区序列的至少之一，所述重链可变区序列和轻链可变区序列的至少之一的至少一部分来自鼠源抗体、人源化抗体、灵长目源抗体或其突变体的至少之一。
3.根据权利要求2所述抗CD47抗体或其抗原结合片段，其特征在于，所述轻链可变区的氨基酸序列如SEQ ID NO:5～9任一项所示或与SEQ ID NO:5～9任一项所示具有至少95％同源性的序列；
优选地，所述重链可变区的氨基酸序列为SEQ ID NO:14～18任一项所示或与SEQ IDNO:14～18任一项所示具有至少95％同源性的序列。
4.根据权利要求2-3任一项权利要求所述抗CD47抗体或其抗原结合片段，其特征在于，所述轻链可变区的氨基酸序列如SEQ ID NO:5所示、重链可变区的氨基酸序列如SEQ IDNO:14所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:6所示、重链可变区的氨基酸序列如SEQ ID NO:14所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:6所示、重链可变区的氨基酸序列如SEQ ID NO:15所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:6所示、重链可变区的氨基酸序列如SEQ ID NO:16所示；或所述轻链可变区的氨基酸序列如SEQ IDNO:6所示、重链可变区的氨基酸序列如SEQ ID NO:17所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:6所示、重链可变区的氨基酸序列如SEQ ID NO:18所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:8所示、重链可变区的氨基酸序列如SEQ ID NO:14所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:9所示、重链可变区的氨基酸序列如SEQ ID NO:14所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:7所示、重链可变区的氨基酸序列如SEQ ID NO:16所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:8所示、重链可变区的氨基酸序列如SEQ ID NO:16所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:9所示、重链可变区的氨基酸序列如SEQ ID NO:16所示；或所述轻链可变区的氨基酸序列如SEQ ID NO:9所示、重链可变区的氨基酸序列如SEQ ID NO:18所示。
5.根据权利要求1-4任一项权利要求所述抗CD47抗体或其抗原结合片段，其特征在于，所述抗体具有恒定区，重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD中的任一种；轻链恒定区为κ或λ链；
优选地，所述恒定区的种属来源选自鼠、兔、羊、猴、或人。
6.根据权利要求1-5任一项权利要求所述抗CD47抗体或其抗原结合片段，其特征在于，所述抗体为CDR移植抗体、多聚体抗体或双特异性抗体中的任一种或几种；
优选地，所述抗原结合片段为F(ab’)2、Fab、scFv、Fv及单域抗体中的任一种或几种；
优选地，所述CD47为人CD47、鼠CD47或猴CD47。
7.核酸，其特征在于，所述核酸编码权利要求1～6任一所述抗CD47抗体或其抗原结合片段。
8.载体，其特征在于，所述载体包含权利要求7所述核酸。
9.细胞，其特征在于，所述细胞包含权利要求7所述核酸或权利要求8所述载体。
10.药物组合物，其特征在于，所述组合物含有权利要求1～6任一所述抗CD47抗体或其抗原结合片段、权利要求7所述核酸、权利要求8所述载体或权利要求9任一所述细胞；
优选地，所述组合物还含有药学可接受的载体。
11.权利要求1～6任一所述抗CD47抗体或其抗原结合片段、权利要求7所述核酸、权利要求8所述载体、权利要求9所述细胞或权利要求10所述组合物在制备治疗和/预防CD47阳性肿瘤的药物中的应用；
优选地，所述药物能够降低红细胞血凝集；
优选地，所述药物能够降低其与红细胞的结合活性。
12.权利要求1～6任一所述抗CD47抗体或其抗原结合片段、权利要求7所述核酸、权利要求8所述载体或权利要求9所述细胞在制备检测CD47或诊断CD47相关疾病的试剂盒中的应用。
13.一种检测CD47的试剂盒，其特征在于，所述试剂盒含有权利要求1～6任一所述抗CD47抗体或其抗原结合片段。
14.一种诊断CD47相关疾病的试剂盒，其特征在于，所述试剂盒含有权利要求1～6任一所述抗CD47抗体或其抗原结合片段。
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| CN111253488A (zh) * | 2018-12-03 | 2020-06-09 | 上海开拓者生物医药有限公司 | Cd47抗体及其制备方法和应用 |
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| SG11201705310TA (en) * | 2014-12-30 | 2017-07-28 | Celgene Corp | Anti-cd47 antibodies and uses thereof |
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