通过融合白蛋白结合域延长抗CD47纳米抗体的半衰期  

Improved Pharmacokinetics of Nanobody Against CD47 by Fusion to Albumin Binding Domains

作　　者：高晓娟 娜斯拜·阿卜杜瓦哈普 马晓玲 王鲁娟 王飞 李江伟[1] GAO Xiaojuan;Nasiba Abuduwahapu;MA Xiaolin;WANG Lujuan;WANG Fei;LI Jiangwei(School of Life Science and Technology,Xinjiang University,Urumqi Xinjiang 830046,China;Xinjiang Unique Mab Biotech Co.,Ltd,Urumqi Xinjiang 830011,China)

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源基因工程重点实验室,新疆乌鲁木齐830046 [2]新疆优迈生物技术有限公司,新疆乌鲁木齐830011

出　　处：《新疆大学学报（自然科学版）（中英文）》2021年第1期69-75,共7页Journal of Xinjiang University(Natural Science Edition in Chinese and English)

基　　金：国家自然科学基金面上项目(315709357)。

摘　　要：纳米抗体是一类在生物医药中具有重要应用前景的蛋白药物,然而由于其分子过小极易被清除.为了获得更好的体内代谢,我们通过CD47纳米抗体融合表达白蛋白结合域(ABD),从而提高其体内半衰期.使用分子克隆技术合成构建带有CD47-ABD基因的原核表达载体pET22b-3D3-2-ABD,转入BL21(DE3)中进行表达及Ni~+亲和层析柱纯化,并使用酶联免疫吸附剂(ELISA)法测定其生物学活性.给小鼠尾静脉注射融合蛋白后在不同时间点采血,用双抗体夹心ELISA测定融合蛋白的血浆浓度,并计算药代动力学参数.获得了pET22b-3D3-2-ABD重组质粒,并在BL21(DE3)表达了CD47-ABD融合蛋白.通过Ni~+亲和柱纯化,获得了较高纯度和浓度的CD47-ABD融合蛋白;改造后的CD47-ABD融合蛋白仍保留抗原结合活性,与CD47抗原及人血清白蛋白(HSA)的结合活性均具有剂量依赖性.CD47-ABD融合蛋白的半衰期从改造前的35.82 min延长至了501.52 min.本研究证明了融合ABD能够显著延长CD47纳米抗体体内半衰期而不影响其抗原结合,为解决纳米抗体自身半衰期短的缺点提供了理论和技术参考.Nanobody is a promising class of biopharmaceuticals with high potential for future therapeutic applications.However,due to their small size they are rapidly cleared from circulation.To improve its pharmacokinetics properties,a CD47 binding nanobody was fused with albumin binding domain(ABD)for extending the half-life in vivo.The prokaryotic expression vector pET22b-3D3-2-ABD with CD47-ABD gene was synthesized and constructed by molecular cloning technology.It was transferred into BL21(DE3)for expression and purified by Ni^+affinity chromatography,and its biological activity was determined by enzyme-linked immunosorbent assay(ELISA).Blood was collected at different time points after mice were injected with the fusion protein into the tail vein.The plasma concentration of the fusion protein was measured by a double antibody sandwich ELISA,and the pharmacokinetic parameters were calculated.The recombinant plasmid pET22b-3D3-2-ABD was obtained and CD47-ABD fusion protein was expressed in BL21(DE3).The high purity and concentration of CD47-ABD fusion protein were obtained by Ni^+affinity column purification.The modified CD47-ABD fusion protein have similar antigen binding activity compare with CD47 nanobody,and possess serum albuminis binding activity in dosedependent manner.The half-life of CD47-ABD fusion protein was extended from 35.82 min to 501.52 min.The experiment demonstrate that the ABD fusion is a promising approach to increase the half-life of CD47 nanobody without affecting their antigen binding efficacy.It strongly suggests that the ABD fusion strategy can be potentially used as a universal method to improve the pharmokinetics properties of nanobodies.

关 键 词：CD47 纳米抗体 白蛋白结合域 融合蛋白 半衰期 

分 类 号：Q789[生物学—分子生物学]