CN117186230B - 包含亲水性氨基酸的抗人bcma纳米抗体的双特异性抗体及应用 - Google Patents
包含亲水性氨基酸的抗人bcma纳米抗体的双特异性抗体及应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及包含亲水性氨基酸的抗人BCMA纳米抗体的双特异性抗体及应用。本发明提供的抗人BCMA纳米抗体包括重链可变区,所述重链可变区包括骨架区和互补决定区,所述互补决定区氨基酸序列的第37‑47位中包含多个由疏水性氨基酸突变为亲水性氨基酸的残基。本发明提供的纳米抗体及制备的双抗能在多发性骨髓瘤等BCMA阳性的肿瘤治疗中发挥稳定的抗肿瘤效果。
Description
本申请要求2022年12月6提交的中国发明专利申请【2022115574272】“具有亲水性氨基酸残基的抗人BCMA纳米抗体及其制备方法和应用”,以及,【2022115801736】“基于具有亲水性氨基酸的抗BCMA纳米抗体的双特异性抗体、CAR-T制剂及应用”的优先权,两个优先权发明专利申请以引用方式全文并入。
技术领域
本发明属于生物技术领域,具体涉及包含亲水性氨基酸的抗人BCMA纳米抗体的双特异性抗体及应用。
背景技术
B细胞成熟抗原(B-cell maturation antigen,BCMA),也称为肿瘤坏死因子受体超家族成员17(TNFRS17)或CD269,BCMA是在浆细胞和已分化的浆细胞表面特异性表达的蛋白,由185个氨基酸残基组成了其胞外结构域,在B细胞成熟和分化为浆细胞中起主要作用。BCMA是肿瘤坏死因子超家族的增殖诱号配体(Aproliferation-inducing ligand,APRIL)和B细胞活化因子(B-cell activating factor,BAFF)的受体。二者均可触发NF-κB信号传导,增加促存活的BCL-2家族基因的表达和促凋亡基因的下调,此外BCMA可激活JNK、MAPK8信号通路,这些信号通路参与调节体液免疫,B细胞发育和稳态,促进B细胞在不同发育阶段的存活,是调节B细胞增殖、成熟和分化成浆细胞的关键。
多发性骨髓瘤(Multiple myeloma,MM)是仅次于非霍奇金淋巴瘤的第二大血液恶性肿瘤,其特征是骨髓中恶性浆细胞异常增生和溶骨性骨病变。相关的临床症状包括溶骨性病变、贫血、肾功能不全、高钙血症、感染和其他相关的器官功能障碍。在正常人体组织中,BCMA蛋白和mRNA几乎只发现在浆细胞上,但其在在浆细胞恶性转化过程中选择性地过度表达,高表达于多发性骨髓瘤细胞表面,并且MM患者的血清中BCMA水平升高,促进肿瘤细胞生长、存活和耐药性的发生,主要通过激活NF-κB、AKT、磷脂酰肌醇3激酶(PI3K)、STAT3和MAPK细胞内信号转导级联反应。目前主要的治疗方案包括化疗、自体干细胞移植(ASCT)和新型药物(蛋白酶体抑制剂、免疫调节药物和单克隆抗体(如daratumumab或elotuzumab),这些治疗方案虽然能延缓该病的进展,但MM在很大程度上仍然无法治愈,大多数患者在多线治疗后复发。
1993年在骆驼科动物和鲨鱼的血清中分离出一种仅有重链可变区(variabledomain of the heavy chain of HCAb,VHH)和两个CH2和CH3区的抗体,该抗体被称为单域抗体(singledomain antibody,sdAb),又称纳米抗体,它在分子大小、稳定性、溶解度、穿透性等方面都优于常规抗体。这种纳米抗体单独表达VHH结构,但仍然保留了对原始重链完整的抗原特异性和结合亲和力,是目前已知的可结合抗原的最小抗体片段。因此纳米抗体可在疫苗研发、疾病诊断和治疗中发挥优势。虽然纳米抗体相较于常规抗体具有众多的优点已被广泛证实,但纳米抗体如何在实际临床治疗和应用中发挥更好更稳定的作用,目前还有众多疑问未被完全掌握。以及什么类型的纳米抗体具有好的效果目前没有准确的答案,依然需要研究人员付出大量的时间和精力去探索。
综上所述,本领域亟需开发抗BCMA的纳米抗体及相关应用,以缓解目前医疗技术在治疗多发性骨髓瘤中存在的不足。
发明内容
有鉴于此,本发明的目的在于提供一系列包含亲水性氨基酸的抗人BCMA纳米抗体的双特异性抗体及应用,具体技术方案如下。
一种抗人BCMA纳米抗体,所述抗人BCMA纳米抗体包括重链可变区,所述重链可变区包括骨架区和互补决定区,所述互补决定区氨基酸序列的第37-47位中包含多个由疏水性氨基酸突变为亲水性氨基酸的残基,所述重链可变区的序列为SEQ ID NO.1~SEQ IDNO.41所示。
进一步,所述互补决定区氨基酸序列的第37位、第44位、第45位和第47位同时发生亲水性突变(在常规抗体中,上述位点通常为疏水性氨基酸,通常包括V37、G44、L45和W47),突变为的亲水性氨基酸包括F37(苯丙氨酸)或Y37(酪氨酸)、E44(谷氨酸)或Q44(谷氨酰胺)、R45(精氨酸)和G47(甘氨酸)。
进一步,所述抗人BCMA纳米抗体的亲和力KD值范围为1.5×10-9~10.0×10-12。
一种双特异性抗体,所述双特异性抗体包含特异性靶向CD3分子的第一结合域和特异性靶向BCMA蛋白的第二结合域;所述靶向BCMA蛋白的第二结合域为上述的抗人BCMA纳米抗体。
进一步,所述第一结合域与所述第二结合域通过(G4S)3linker连接。
进一步,所述靶向CD3分子的第一结合域为OKT3抗体。
上述双特异性抗体的制备方法,包括以下步骤:
步骤1:以包含人BCMA基因全长序列的质粒为模板,利用真核表达体系制备特异性靶向BCMA的重组蛋白;
步骤2:用步骤1制备的重组蛋白来免疫动物,提取免疫后动物外周血淋巴细胞的总RNA,将所述总RNA反转录为cDNA,再用巢式PCR进行两轮扩增,第一轮巢式PCR扩增的引物如SEQ ID NO.42和SEQ ID NO.43所示;第二轮巢式PCR扩增的引物如SEQ ID NO.44和SEQID NO.45所示,获得纳米抗体基因并克隆至噬菌体展示载体构建噬菌体纳米抗体文库;
步骤3:利用噬菌体展示技术,使用步骤1制备的特异性靶向BCMA的重组蛋白淘选步骤2中构建的纳米抗体,进一步淘选出能特异性结合BCMA蛋白的纳米抗体;
步骤4:将淘选出的特异性结合BCMA蛋白的纳米抗体通过(G4S)3linker与靶向CD3分子的OKT3抗体进行连接;
步骤5:对构建出的双特异性抗体进行亲和力检测。
进一步,检测步骤3中淘选出的特异性结合BCMA的纳米抗体,筛选出互补决定区氨基酸序列的第37位、第44位、第45位和第47位同时发生亲水性突变的纳米抗体。
上述双特异性抗体在制备靶向多发性骨髓瘤的药物中的应用。
上述双特异性抗体在制备B淋巴细胞恶性肿瘤、慢性淋巴细胞白血病、弥漫性大B细胞淋巴瘤、视神经脊髓炎谱系疾病、类风湿性关节炎、多发性硬化症或系统性红斑狼疮的药物中的应用。
有益技术效果
与常规抗体VH相比较,在互补决定区,本发明的VHH氨基酸序列中第37-47位中有四个疏水性氨基酸同时发生亲水性突变。常规抗体中V37、G44、L45和W47这4个疏水性氨基酸残基在进化中相当保守,在本发明VHH中,这四个位点同时突变为亲水性的氨基酸残基F37或Y37、E44或Q44、R45和G47,VHH的水溶性相应升高,因此更容易进入实体瘤内部。本发明实验进一步证明,用这种发生了亲水性突变的VHH制备的双特异性抗体都具有靶向实体瘤细胞的杀伤效果。特别的是以SEQ ID NO.25所示的VHH制备的双抗为例,当其第44位氨基酸突变为Q时,其肿瘤细胞靶向杀伤能力最强。
最后,本发明方法制备得到的纳米抗体,还具有良好的亲和力,能在多发性骨髓瘤及自发性免疫疾病的治疗中发挥巨大的临床潜力。但本发明实验证实,纳米抗体的亲和力与其种类杀伤能力并不是呈正相关。当纳米抗体亲和力过高的时候,可能会与单个肿瘤细胞表面抗原紧密结合,导致其不能灵活分开再去杀伤其他肿瘤细胞,进而导致其最后呈现的肿瘤杀伤效力一般。因此,需要筛选出对肿瘤细胞具有较好亲和力,但亲和力适中的VHH才能在实际临床治疗和应用中发挥更好更稳定的作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其它的附图。
图1为通过亲和层析的方式纯化获得高纯度的重组蛋白BCMA-mFc电泳图;
图2为免疫后提取的骆驼血淋巴细胞总RNA,PCR扩增产物电泳图(A:第一轮扩增产物;B:第二轮扩增产物);
图3为利用亲和层析的方式纯化获得重组纳米抗体电泳图;
图4为间接ELISA的方式对获得的纳米抗体与包被于酶标板中的BCMA重组蛋白及其他无关抗原共孵育测定OD450 nm的吸光值图;
图5为双特异性抗体Nbs 18-OKT3对骨髓瘤细胞的体外杀伤活性实验图;
图6为双特异性抗体Nbs 23-OKT3对骨髓瘤细胞的体外杀伤活性实验图;
图7为双特异性抗体Nbs 25-OKT3对骨髓瘤细胞的体外杀伤活性实验图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述。显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本文中“和/或”包括任何和所有一个或多个列出的相关项的组合。
本文中“多个”意指两个或两个以上,即其包含两个、三个、四个、五个等。
需要说明的是,在本文中,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。
如在本说明书中使用的,术语“大约”,典型地表示为所述值的+/-5%,更典型的是所述值的+/-4%,更典型的是所述值的+/-3%,更典型的是所述值的+/-2%,甚至更典型的是所述值的+/-1%,甚至更典型的是所述值的+/-0.5%。
在本说明书中,某些实施方式可能以一种处于某个范围的格式公开。应该理解,这种“处于某个范围”的描述仅仅是为了方便和简洁,且不应该被解释为对所公开范围的僵化限制。因此,范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如,范围的描述应该被看作已经具体地公开了子范围如从1到3,从1到4,从1到5,从2到4,从2到6,从3到6等,以及此范围内的单独数字,例如1,2,3,4,5和6。无论该范围的广度如何,均适用以上规则。
实施例1
本实施例提供BCMA重组蛋白表达载体的构建及其表达、纯化的方法
1.1真核表达载体构建
将人BCMA基因全长序列合成到pMD19-T载体中,以含有BCMA全长基因的质粒为模板,设计引物扩增获得BCMA胞外区(ECD)基因,并利用酶切连接的方式将其克隆至经限制性内切酶PstⅠ和XbaⅠ双酶切后的pVax-mFc载体中,转化至DH5α感受态细胞中并通过卡那霉素抗性平板进行筛选,挑取单克隆测序鉴定,构建成功的载体命名为pVax-mFc-BCMA-His。
1.2重组蛋白表达及纯化
将构建成功的重组质粒pVax-mFc-BCMA-His利用转染试剂PEI转染至HEK293T细胞中,表达5天后收集上清,利用Ni胶通过亲和层析的方式纯化获得高纯度的重组蛋白BCMA-mFc,结果如图1所示。所述重组蛋白具有特异性靶向BCMA靶点的作用。
实施例2
抗BCMA蛋白纳米抗体的筛选与制备方法
2.1蛋白乳化及动物免疫
将实施例1中纯化获得的BCMA重组蛋白(以1mg为例)与等体积的氢氧化铝佐剂混合后对骆驼通过颈部皮下进行第一次免疫,然后每隔2周连续免疫4次,冲击免疫后的第7天采集外周抗凝血。
2.2噬菌体抗体文库的构建及抗体筛选
2.2.1外周血淋巴细胞的分离
从骆驼颈部静脉无菌采集200mL外周血,其中包含大量B淋巴细胞,先用等体积的PBS缓冲液稀释。再用Ficoll-Paque Plus淋巴细胞分离液分离获得外周血淋巴细胞,得到的淋巴细胞可直接提取总RNA或冻存于-80℃备用。
2.2.2纳米抗体基因扩增
首先提取淋巴细胞总RNA,以RNA为模板反转录获得cDNA,再以cDNA为模板,利用巢式PCR扩增VHH基因,第一轮扩增引物为CALL001、CALL002(见表1),利用琼脂糖凝胶电泳分离并回收约700bp片段(图2A);然后以回收的700bp产物为模板进行第二轮PCR扩增,第二轮扩增引物为VHH-FOR、VHH-REV(见表1),利用琼脂糖凝胶电泳分离并回收400bp片段(图2B)。
表1 VHH基因扩增所需引物序列
引物名称 | 引物序列(5’-3’) | 序列编号 |
CALL001 | GTCCTGGCTGCTCTTCTACAAGG | 42 |
CALL002 | GGTACGTGCTGTTGAACTGTTCC | 43 |
VHH-FOR | CAGGTGCAGCTGCAGGAGTCTGGGGGAG | 44 |
VHH-REV | CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT | 45 |
2.2.3噬菌体展示载体的构建
将上述步骤中扩增回收的PCR产物和噬菌体展示载体pMECS均利用限制性内切酶Pst I和Not I双酶切,然后利用T4 DNA连接酶连接。
2.2.4电转及噬菌体抗体文库的收获
将上述步骤中制备获得的连接产物加入TG1感受态细胞中并利用电转仪转化使其进入TG1中,电转完成后加入SOC培养基并于37℃、220rpm培养活化1h,再将上述培养物均匀的涂布于LB/Amp-Glu平板,37℃培养6~8h后,收集菌体并加入无菌甘油使其终浓度为15%,将制备的噬菌体文库保存于-80℃或直接用于后续抗体筛选。
2.2.5特异性纳米抗体的筛选
以制备的噬菌体文库为抗体来源,取20倍库容量的噬菌体文库,加入辅助噬菌体M13K07从而获得表达抗体的重组噬菌体,将其与包被于酶标板中的BCMA重组蛋白孵育后用0.1M Glycine(pH 1.5)进行洗脱,将洗脱液再次感染TG1菌株并于37℃、220rpm培养至对数生长期,加入辅助噬菌体使得抗体展示在其表面。按照上述步骤反复进行3轮筛选后从第三轮筛选制备的菌落平板中挑取单克隆菌落扩大培养,利用单克隆ELISA鉴定其中能够特异性结合BCMA蛋白的纳米抗体,并对其菌落进行测序分析,获得41株抗BCMA的特异性纳米抗体,如表2所示。
表2纳米抗体全序列
2.2.6纳米抗体的制备
以2.2.5中含有纳米抗体基因的重组质粒pMECS-Nbs为模板扩增VHH基因并通过酶切连接的方式克隆至真核表达载体pcDNA3.1-hFc中,构建成功后提取质粒并将其转染至HEK293T细胞中,表达5天后收集上清,利用Ni柱通过亲和层析的方式纯化获得重组纳米抗体。
实施例3
3.1Biacore检测纳米抗体亲和力
将该批纳米抗体分别与包被于CM5芯片上的抗原BCMA-mFc利用Biacore 8k仪器检测结合亲和力,结果如表3所示。本发明中筛选到41株存在特定位点亲水性突变的靶向BCMA的纳米抗体,KD值范围在1.5×10-9~10.0×10-12之间,KD值越大亲和力越低。
表3纳米抗体亲和力数据
备注:1E+04=1×104;1E-04=1×10-4;其他的以此类推
3.2抗BCMA蛋白纳米抗体的特异性分析
通过间接ELISA的方式对纳米抗体与BCMA抗原亲和力进行比较,首先将BCMA重组蛋白包被于96孔酶标板中,封闭完成后将制备的纳米抗体(0.1μg/mL)加入酶标板中,并利用HRP标记的山羊抗人二抗进行检测,结果如图4所示,获得的41株纳米抗体与BCMA蛋白均能够特异性结合,且不与其他无关抗原反应,表明上述纳米抗体均具有良好的特异结合活性。
实施例4
双特异性抗体Nbs-OKT3对骨髓瘤细胞的体外杀伤活性分析以SEQ ID NO.18、NO.23和NO.25纳米抗体为例,分别取以上3段纳米抗体序列与抗CD3分子的抗体OKT3利用(G4S)3linker串联构建至真核表达载体中并转染HEK293T细胞,转染5天后收集上清并利用Ni柱纯化。将上述纯化的不同浓度的双特异性抗体(1μg/mL、0.1μg/mL、0.01μg/mL、0.001μg/mL)与多发性骨髓瘤细胞系MM.1S-luciferase及激活的T细胞按照效靶比3:1(激活的T细胞:多发性骨髓瘤细胞系MM.1S-luciferase)共孵育24h后,通过酶标仪检测荧光强度评价其杀伤活性,结果如图5-7所示,上述双特异性抗体对BCMA阳性的天然MM细胞系均具有较强的体外杀伤活性。本实施例中,对照抗体为一个非靶向BCMA的双特异性抗体,设置其作为对照的目的是排除本发明所提供的双特异性抗体中CD3分子的作用。结果数据见表4。
表4双特异性抗体对骨髓瘤细胞的体外杀伤活性实验数据
上面结合附图对本发明的实施例进行了描述,但是本发明并不局限于上述的具体实施方式,上述的具体实施方式仅仅是示意性的,而不是限制性的,本领域的普通技术人员在本发明的启示下,在不脱离本发明宗旨和权利要求所保护的范围情况下,还可做出很多形式,这些均属于本发明的保护之内。
Claims (4)
1.一种双特异性抗体,其特征在于,所述双特异性抗体包含特异性靶向CD3分子的第一结合域和特异性靶向BCMA蛋白的第二结合域;所述靶向BCMA蛋白的第二结合域为抗人BCMA纳米抗体,所述抗人BCMA纳米抗体包括重链可变区,所述重链可变区包括骨架区和互补决定区,所述互补决定区氨基酸序列的第37位、第44位、第45位和第47位的疏水性氨基酸同时突变为亲水性氨基酸;所述重链可变区的序列如SEQ ID NO.18、SEQ ID NO.23或SEQ IDNO.25所示。
2.如权利要求1所述的双特异性抗体,其特征在于,所述第一结合域与所述第二结合域通过(G4S)3linker连接。
3.如权利要求1所述的双特异性抗体,其特征在于,所述靶向CD3分子的第一结合域为OKT3抗体。
4.权利要求1所述的双特异性抗体在制备靶向多发性骨髓瘤的药物中的应用。
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