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.

Engineered cross‐reacting nanobodies simplify comparative oncology between humans and dogs

“…Comparative studies would be critically simplified by the identification of new antibodies suitable for the characterization of the canine model . Ideally, such binders should be able to: (1) cross‐react with disease‐related molecular biomarkers shared between humans and dogs, and (2) be engineered into formats more adapted to final applications than conventional immunoglobulin G (IgG) antibodies . Nanobodies are antibody fragments which correspond to the variable region of the heavy‐chain‐only antibodies (VHH) of Camelidae origin.…”

Canine and feline lymphoma: challenges and opportunities for creating a paradigm shift

“…The use of ‘human’ monoclonal antibodies in veterinary oncology is usually not feasible due to the development of an immune response to foreign protein but species‐specific (e.g. caninized) monoclonal antibodies are being developed for canine lymphoma . This is a truly exciting prospect, as it will deliver new and affordable reagents to the veterinary oncology community …”

“…caninized) monoclonal antibodies are being developed for canine lymphoma. 34 This is a truly exciting prospect, as it will deliver new and affordable reagents to the veterinary oncology community. 35,36…”

Canine cancer immunotherapy studies: linking mouse and human

“…Multi-agent chemotherapy is the recommended treatment for high-grade lymphomas (most commonly diffuse large B-cell lymphoma) in the dog [66]. In addition, since the chimeric mAb rituximab binds an epitope of human CD20 that is not conserved in dogs, numerous canine specific anti-CD20 mAbs are in various stages of development for the study and treatment of dog lymphoid neoplasms [66–70]. …”