The development of a canine single-chain phage antibody library to isolate recombinant antibodies for use in translational cancer research.

IF 4.3 Q1 BIOCHEMICAL RESEARCH METHODS

Cell Reports Methods Pub Date : 2025-03-24 DOI:10.1016/j.crmeth.2025.101008

Małgorzata Lisowska, Erin G Worrall, Filip Zavadil-Kokas, Keith Charlton, Euan Murray, M Aiman Mohtar, Radovan Krejcir, Vaclav Hrabal, Jack Brydon, Ainhoa Gonzalez Urionabarrenetxea, David G Saliba, Mariana Grima, Umesh Kalathiya, Petr Muller, Adam Krejci, Borivoj Vojtesek, Kathryn L Ball, Robin Fahraeus, David J Argyle, Maciej Parys, Ted R Hupp

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Abstract

The development of canine immunotolerant monoclonal antibodies can accelerate the invention of new medicines for both canine and human diseases. We develop a methodology to clone the naive, somatically mutated variable domain repertoire from canine B cell mRNA using 5'RACE PCR. A set of degenerate primers were then designed and used to clone variable domain genes into archival "holding" plasmid libraries. These archived variable domain genes were then combinatorially ligated to produce a scFv M13 phage library. Next-generation long-read and short-read DNA sequencing methodologies were developed to annotate features of the cloned library including CDR diversity and IGHV/IGKV/IGLV subfamily distribution. A synthetic immunoglobulin G was developed from this scFv library to the canine immune checkpoint receptor PD-1. This synthetic platform can be used to clone and annotate archived antibody variable domain genes for use in perpetuity in order to develop improved preclinical models for the treatment of complex human diseases.

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犬单链噬菌体抗体文库的建立，分离重组抗体用于转化性癌症研究。

犬免疫耐受单克隆抗体的开发可以加速犬类和人类疾病新药的发明。我们开发了一种方法，利用5'RACE PCR从犬B细胞mRNA中克隆幼稚的，体细胞突变的可变结构域库。然后设计了一套简并引物，用于将可变结构域基因克隆到存档质粒文库中。然后将这些存档的可变结构域基因组合连接以产生scFv M13噬菌体文库。开发了下一代长读和短读DNA测序方法，以注释克隆文库的特征，包括CDR多样性和IGHV/IGKV/IGLV亚家族分布。从该scFv文库中合成了犬免疫检查点受体PD-1的合成免疫球蛋白G。该合成平台可用于克隆和注释存档的抗体可变结构域基因，以便永久使用，以开发用于治疗复杂人类疾病的改进的临床前模型。

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