Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli
Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli
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DOI：
10.1016/j.pep.2013.10.023
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发表时间：
2014-02-01
影响因子：
1.6
通讯作者：
Saltzman, Daniel A.
中科院分区：
文献类型：
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作者：
Drees, Jeremy J.;Augustin, Lance B.;Saltzman, Daniel A.
程序性死亡配体I(PD-L1)是T细胞活化的重要调节因子，已成为肿瘤免疫治疗的重要靶点。单链可变区(ScFv)具有一些理想的特性，是一种有吸引力的替代单抗的实验或治疗目的。以小鼠PD-L1为抗原免疫3只鸡，提取其脾组织中的mRNA，构建免疫球蛋白可变区文库。利用剪接-重叠延伸聚合酶链式反应，将可变区cDNA组合成全长单链抗体。M13噬菌体展示鉴定了抗PD-L1(αPD-L1 scFv)的功能单链抗体。将单链抗体以可溶性蛋白的形式在重组大肠杆菌的周质和培养上清液中表达，并用6x-His标签用固定化金属亲和层析纯化。αPD-L1单链抗体的解离常数为7.11×10(-10)M，具有与拮抗单抗相当的抑制生物活性，为阻断PD-1/PD-L1信号通路提供了一种替代试剂。(C)2013 Elsevier Inc.保留所有权利。
Programmed death ligand I (PD-L1), is an important regulator of T-cell activation and has emerged as an important target for cancer immunotherapy. Single chain variable fragments (scFvs) have several desirable characteristics and are an attractive alternative to monoclonal antibodies for experimental or therapeutic purposes. Three chickens were immunized against murine PD-L1, and mRNA isolated from their spleens was used to generate an immunized immunoglobulin variable region library. Using splice-overlap extension PCR, variable region cDNAs were combined to generate full-length scFvs. M13 phage display of the resulting scFv library identified a functional scFv against PD-L1 (alpha PD-L1 scFv). The scFv was expressed as soluble protein in the periplasm and culture supernatant of recombinant Escherichia coli and purified with a 6x-His tag using immobile metal affinity chromatography. The dissociation constant of alpha PD-L1 scFv was determined to be 7.11 x 10(-10) M, and the scFv demonstrated inhibitory biological activity comparable to an antagonistic monoclonal antibody, providing an alternative agent for blocking PD-1/PD-L1 signaling. (C) 2013 Elsevier Inc. All rights reserved.