Selection of novel affinity-matured human chondroitin sulfate proteoglycan 4 antibody fragments by yeast display.
Selection of novel affinity-matured human chondroitin sulfate proteoglycan 4 antibody fragments by yeast display.
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通过酵母展示选择新型亲和力成熟的人硫酸软骨素蛋白聚糖 4 抗体片段。
DOI：
10.1093/protein/gzx038
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Chandramohan,Vidyalakshmi
中科院分区：
文献类型：
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作者：
Yu,Xin;Qu,Liang;Bigner,DarellD;Chandramohan,Vidyalakshmi
硫酸软骨素蛋白聚糖4（Chondroitin sulfate proteoglycan 4，CSPG 4）在多种恶性肿瘤中高水平表达，在肿瘤的生长和发展中起重要作用，是肿瘤免疫治疗的一个很有前途的靶点。靶向CSPG 4的免疫疗法的临床应用由于缺乏全人高亲和力CSPG 4抗体或抗体片段而受到阻碍。为了克服这一局限性，我们进行亲和力成熟的一种新的人类CSPG 4单链抗体片段（scFv）使用随机诱变方法和筛选改进的变体从酵母展示库使用改良的全细胞淘选方法，然后通过荧光激活细胞分选。在六轮淘选和分选后，分离前七个突变体scFv，并通过流式细胞术和表面等离子体共振表征其结合亲和力。这些高度特异性、亲和力成熟的变体显示出对CSPG 4抗原的纳摩尔至皮摩尔结合亲和力。虽然每种突变体仅具有2至6个氨基酸取代，但与亲本克隆相比，它们表现出约270-3000倍的亲和力改善。我们的研究已经产生了亲和力成熟的scFv，用于开发靶向CSPG 4表达肿瘤的基于抗体的临床治疗剂。
Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human high-affinity CSPG4 antibodies or antibody fragments. To overcome this limitation, we performed affinity maturation on a novel human CSPG4 single-chain Fv fragment (scFv) using the random mutagenesis approach and screened for improved variants from a yeast display library using a modified whole-cell panning method followed by fluorescence-activated cell sorting. After six rounds of panning and sorting, the top seven mutant scFvs were isolated and their binding affinities were characterized by flow cytometry and surface plasmon resonance. These highly specific, affinity-matured variants displayed nanomolar to picomolar binding affinities to the CSPG4 antigen. While each of the mutants harbored only two to six amino acid substitutions, they represented ~270–3000-fold improvement in affinity compared to the parental clone. Our study has generated affinity-matured scFvs for the development of antibody-based clinical therapeutics targeting CSPG4-expressing tumors.