title: The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.
abstract: Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.
authors.affiliation: University of Edinburgh, Institute of Genetic and Molecular Medicine and School of Veterinary Medicine, Edinburgh, EH4 2XR, United Kingdom.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Jain
authors.affiliation: Dipartimento di Biomedicina Comparata e Alimentazione (BCA) Department of Comparative Biomedicine and Food Science, Università di Padova 35020 Legnaro (PD), Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Aresu
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, via Celoria 10, 20133 Milano, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Comazzi
authors.affiliation: University of Edinburgh, Institute of Genetic and Molecular Medicine and School of Veterinary Medicine, Edinburgh, EH4 2XR, United Kingdom.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Shi
authors.affiliation: University of Edinburgh, Institute of Genetic and Molecular Medicine and School of Veterinary Medicine, Edinburgh, EH4 2XR, United Kingdom.
authors.forename: Erin
authors.initials: E
authors.lastname: Worrall
authors.affiliation: Mologic, Ltd, Bedford Technology Park, Thurleigh, Bedford, MK44 2YP, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Clayton
authors.affiliation: Mologic, Ltd, Bedford Technology Park, Thurleigh, Bedford, MK44 2YP, United Kingdom.
authors.forename: William
authors.initials: W
authors.lastname: Humphries
authors.affiliation: Mologic, Ltd, Bedford Technology Park, Thurleigh, Bedford, MK44 2YP, United Kingdom.
authors.forename: Sandra
authors.initials: S
authors.lastname: Hemmington
authors.affiliation: Mologic, Ltd, Bedford Technology Park, Thurleigh, Bedford, MK44 2YP, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Davis
authors.affiliation: University of Edinburgh, Institute of Genetic and Molecular Medicine and School of Veterinary Medicine, Edinburgh, EH4 2XR, United Kingdom.|INSERM Unité 940, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St Louis, 27 rue Juliette Dodu, Paris, France.
authors.forename: Euan
authors.initials: E
authors.lastname: Murray
authors.affiliation: Bahit Dar University College of Medicine and Health Sciences Department of Medical Biochemistry and Molecular Biology, Bahir Dar, Ethiopia.
authors.forename: Asmare A
authors.initials: AA
authors.lastname: Limeneh
authors.affiliation: University of Edinburgh, Institute of Genetic and Molecular Medicine and School of Veterinary Medicine, Edinburgh, EH4 2XR, United Kingdom.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Ball
authors.affiliation: Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Ruckova
authors.affiliation: Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Muller
authors.affiliation: Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic.
authors.forename: Borek
authors.initials: B
authors.lastname: Vojtesek
authors.affiliation: INSERM Unité 940, Institut de Génétique Moléculaire, Université Paris 7, Hôpital St Louis, 27 rue Juliette Dodu, Paris, France.
authors.forename: Robin
authors.initials: R
authors.lastname: Fahraeus
authors.affiliation: University of Edinburgh, Institute of Genetic and Molecular Medicine and School of Veterinary Medicine, Edinburgh, EH4 2XR, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Argyle
authors.affiliation: University of Edinburgh, Institute of Genetic and Molecular Medicine and School of Veterinary Medicine, Edinburgh, EH4 2XR, United Kingdom.
authors.forename: Ted R
authors.initials: TR
authors.lastname: Hupp
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D018951:Antigens, CD20 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004285:Dogs; D000939:Epitopes / Q000276:immunology; D015870:Gene Expression; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000276:immunology / Q000494:pharmacology*
chemical_list: D018951:Antigens, CD20; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies