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首页> 外文期刊>BMC Biotechnology >Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies
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Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies

机译：免疫球蛋白人源化大鼠的抗原特异性单B细胞分选和表达克隆：一种快速，通用的产生高亲和力和区分性人单克隆抗体的方法

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BackgroundThere is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by reconstruction of human mAbs by RT-PCR and expression cloning. ResultsThis strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbs against major histocompatibility complex molecules loaded with peptides (pMHC). We then implemented this technology using human immunoglobulin transgenic rats, which after immunization with an antigen of interest express high affinity-matured antibodies with human idiotypes. Using rapid immunization, followed by tetramer-based B-cell sorting and expression cloning, we generated several fully humanized mAbs with strong affinities, which could discriminate between highly homologous proteins (eg. different pMHC complexes). ConclusionsTherefore, we describe a versatile and more effective approach as compared to hybridoma generation or phage or yeast display technologies for the generation of highly specific and discriminative fully human mAbs that could be useful both for basic research and immunotherapeutic purposes.
机译：背景技术对于生物医学应用，单克隆抗体（mAb）的需求不断增长，由于它们降低了患者的免疫原性，因此特别需要完全人结合的抗体。我们已应用四聚体蛋白和单细胞分选方法分离抗原特异性B细胞的策略，然后通过RT-PCR和表达克隆重建人mAb。结果该策略利用人类外周血B细胞，能够产生针对载有肽（pMHC）的主要组织相容性复合物分子的低亲和力人类mAb。然后，我们使用人类免疫球蛋白转基因大鼠实施了该技术，该大鼠在用目标抗原免疫后表达具有人类独特型的高亲和力成熟抗体。使用快速免疫，然后进行基于四聚体的B细胞分选和表达克隆，我们生成了几种具有很强亲和力的完全人源化单克隆抗体，可以区分高度同源的蛋白质（例如，不同的pMHC复合物）。结论因此，与杂交瘤产生或噬菌体或酵母展示技术相比，我们描述了一种通用且更有效的方法，可用于产生高度特异性和区分性的完全人mAb，可用于基础研究和免疫治疗目的。

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