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

M. Lisowska, EG. Worrall, F. Zavadil-Kokas, K. Charlton, E. Murray, MA. Mohtar, R. Krejcir, V. Hrabal, J. Brydon, AG. Urionabarrenetxea, DG. Saliba, M. Grima, U. Kalathiya, P. Muller, A. Krejci, B. Vojtesek, KL. Ball, R. Fahraeus, DJ. Argyle, M....

. 2025 ; 5 (3) : 101008. [pub] 20250324

Language English Country United States

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc25009442

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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$a The development of canine immunotolerant monoclonal antibodies can accelerate the invention of new medicines for both canine and human diseases. We develop a methodology to clone the naive, somatically mutated variable domain repertoire from canine B cell mRNA using 5'RACE PCR. A set of degenerate primers were then designed and used to clone variable domain genes into archival "holding" plasmid libraries. These archived variable domain genes were then combinatorially ligated to produce a scFv M13 phage library. Next-generation long-read and short-read DNA sequencing methodologies were developed to annotate features of the cloned library including CDR diversity and IGHV/IGKV/IGLV subfamily distribution. A synthetic immunoglobulin G was developed from this scFv library to the canine immune checkpoint receptor PD-1. This synthetic platform can be used to clone and annotate archived antibody variable domain genes for use in perpetuity in order to develop improved preclinical models for the treatment of complex human diseases.
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$a Zavadil-Kokas, Filip $u Research Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
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$a Charlton, Keith $u University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen, UK
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$a Murray, Euan $u University of Edinburgh, Institute of Genetics and Cancer, Edinburgh, Scotland, UK; Inserm UMRS1131, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St Louis, Paris, France
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$a Mohtar, M Aiman $u University of Edinburgh, Institute of Genetics and Cancer, Edinburgh, Scotland, UK
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$a Krejcir, Radovan $u Research Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
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$a Hrabal, Vaclav $u Research Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
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$a Brydon, Jack $u University of Edinburgh, Institute of Genetics and Cancer, Edinburgh, Scotland, UK
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$a Urionabarrenetxea, Ainhoa Gonzalez $u University of Edinburgh, Institute of Genetics and Cancer, Edinburgh, Scotland, UK
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$a Saliba, David G $u Faculty of Health Sciences/Department Applied Biomedical Science, Centre for Molecular Medicine and Biobanking, University of Malta, Msida, Malta
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$a Kalathiya, Umesh $u International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland
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$a Ball, Kathryn L $u University of Edinburgh, Institute of Genetics and Cancer, Edinburgh, Scotland, UK
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$a Fahraeus, Robin $u Inserm UMRS1131, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St Louis, Paris, France
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$a Argyle, David J $u The University of Edinburgh, Royal (Dick) School of Veterinary Studies and Roslin Institute, Edinburgh, UK
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$a Parys, Maciej $u The University of Edinburgh, Royal (Dick) School of Veterinary Studies and Roslin Institute, Edinburgh, UK
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$a Hupp, Ted R $u International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland; University of Edinburgh, Institute of Genetics and Cancer, Edinburgh, Scotland, UK
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