Identification of cytomegalovirus (CMV)pp65 antigen-specific human monoclonal antibodies using single B cell-based antibody gene cloning from melanoma patients
Identification of cytomegalovirus (CMV)pp65 antigen-specific human monoclonal antibodies using single B cell-based antibody gene cloning from melanoma patients
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DOI：
10.1016/j.imlet.2010.09.013
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发表时间：
2011-03-30
影响因子：
4.4
通讯作者：
Akiyama, Yasuto
中科院分区：
文献类型：
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作者：
Iizuka, Akira;Komiyama, Masaru;Akiyama, Yasuto
近年来，由于高度先进的蛋白质工程技术，除了嵌合抗体之外，高度人源化和全人源抗体的开发在医学领域变得至关重要。近十年来，人们已经进行了利用临床样本生产全人源抗体的研究方法，但仍存在效率和准确性问题亟待解决。本研究基于新型IgG抗体测量ELISA和抗体基因拷贝数定量PCR，建立了人单B细胞RT-PCR介导的IgG单克隆抗体（mAb）基因克隆方法，并成功鉴定了CMVpp65特异性人mAb。每个细胞的人IgG mRNA拷贝数的定量PCR表明检测范围为10-250拷贝/细胞。 CMVpp65（+）表面IgG（+）B细胞是从表现出高滴度血清抗CMVpp65 IgG抗体的黑色素瘤患者中收集的。 RT-PCR 在 88 个单个 B 细胞中的 64% (IGH) 和 84% (β-肌动蛋白) 中获得成功。最终在21个单细胞中同一细胞中IGH和IGL基因均扩增成功，克隆出18个针对CMVpp65抗原特异性的IgG抗体基因。 13 种重组人单链片段可变 (scFv) 抗体中的 4 种对全长 CMVpp65 蛋白表现出强烈的反应。这些结果表明，目前通过ELISA进行抗体效价筛选、基于单B细胞RT-PCR的抗体基因克隆以及scFv重组抗体制备的全人单克隆抗体生产程序是治疗性抗体开发的有效方法。 (C) 2010 Elsevier B.V. 保留所有权利。
Recently, because of highly advanced protein engineering technology, beyond the chimeric antibody, highly humanized and fully human antibody development is becoming crucial in the medical field. In the last decade, investigational approaches using clinical samples for fully human antibody production have been performed, but there are still problems with efficiency and accuracy, which should be solved.In the present study, based on novel IgG antibody-measuring ELISA and antibody gene copy number-quantitative PCR, a human single B cell RT-PCR-mediated IgG monoclonal antibody (mAb) gene cloning method was established, and CMVpp65-specific human mAbs were successfully identified. Quantitative PCR for the human IgG mRNA copy number per cell demonstrated that the detection range was 10-250 copies/cell. CMVpp65(+) surfaceIgG(+) B cells were collected from melanoma patients who showed high titers of serum anti-CMVpp65 IgG antibody. RT-PCR was successful in 64% (IGH) and 84% (beta-actin) of 88 single B cells. Finally, both IGH and IGL gene amplifications in the same cell were successful in 21 single cells, and 18 IgG antibody genes specific for CMVpp65 antigen were cloned. Four of 13 recombinant human single-chain fragment variable (scFv) antibodies showed strong responses to full-length CMVpp65 protein. These results suggested that the current fully human mAb production procedure through antibody-titer screening by ELISA, single B cell RT-PCR-based antibody gene cloning, and the making of scFv recombinant antibody is an efficient method of therapeutic antibody development. (C) 2010 Elsevier B.V. All rights reserved.