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The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine
Saurabh Jain1, Luca Aresu2, Stefano Comazzi3
1University of Edinburgh, Institute of Genetic and Molecular Medicine and School of Veterinary Medicine, Edinburgh, EH4 2XR, United Kingdom.
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.
Insights
Researchers developed a novel canine anti-CD20 antibody (NCD1.2) that targets both human and canine B-cell lymphomas. This antibody and its derived single-chain variable fragments (scFv) show bioactivity, offering a promising tool for comparative immunotherapy research.
Area of Science:
- Immunology
- Comparative Medicine
- Biotechnology
Background:
- Monoclonal antibodies are crucial for treating human diseases but lack sufficient clinical models for validation.
- Canine tumors share similarities with human cancers, making them valuable models for disease research.
- Developing canine immunotherapeutics can advance the study of human disease responses.
Purpose of the Study:
- To develop a novel anti-CD20 monoclonal antibody (NCD1.2) for a comparative canine-human model system.
- To assess the binding and bioactivity of NCD1.2 and its derived single-chain variable fragments (scFv) for potential therapeutic applications.
Main Methods:
- Developed a novel anti-CD20 monoclonal antibody (NCD1.2) with sub-nanomolar binding affinity (Kd).
- Utilized flow cytometry (FACS) and immunohistochemistry to confirm NCD1.2 binding to canine B-cells and lymphomas.
- Cloned heavy and light chains from hybridomas to create phage-display libraries and isolated bioactive scFv (scFv-3, scFv-7).
Main Results:
- NCD1.2 demonstrated high binding affinity to both human and canine CD20, validating its use in comparative models.
- NCD1.2 successfully bound to B-cells in peripheral blood and various canine B-cell lymphoma tissues.
- Two recombinant scFv (scFv-3 and scFv-7) were identified with retained bioactivity against CD20, even when expressed in bacterial systems.
Conclusions:
- NCD1.2 serves as a valuable tool for comparative canine-human CD20-targeted immunotherapies.
- The identified recombinant anti-CD20 scFv are promising candidates for bioconjugate-directed immunotherapies.
- This research establishes a foundation for utilizing canine models in the development of human cancer therapeutics.
