Mari Lowe Center for Comparative Oncology Research

3800 Spruce Street, Philadelphia, PA 19104

Generation of canine single chain Fragment variable antibody libraries for the identification and targeting of tumor-associated antigens in the dog

 


Fig1. Cartoon of a single chain fragment variable (scFv) comprised of the VH and VL regions of immunoglobulin linked by a flexible linker. scFvs are the smallest antibody fragments that maintain antigen- binding specificity and affinity.

The use of monoclonal antibodies and antibody fragments to directly target tumors has shown promising results in clinical trials in humans. However, these exciting targeted approaches have not been possible in canine cancer patients since tumor antigens have not been identified, monoclonal antibodies of canine origin are not available and the efficacy of xenogeneic antibodies in the dog is limited by neutralizing antibody responses. Recently, it has been shown by screening combinatorial antibody libraries generated from human cancer patients that these patients break self-tolerance and generate antibody responses against their own tumors and that screening these antibody responses can lead to the identification of antibodies and antibody fragments that can be used therapeutically to target cancer in vivo. Researchers in Dr. Mason’s laboratory at PennVet have developed a system to clone the rearranged variable heavy (VH) and variable light (VL) immunoglobulin chains within circulating B lymphocytes of healthy dogs and dogs with spontaneously occurring cancers. They have used this system to generate combinatorial single chain (scFv) antibody libraries that provide a “fossil record” of the patient’s immunological repertoire (Fig. 1). Using phage display technology, these scFv libraries are being analyzed for their ability to bind specifically to tumor antigens. Tumor specific canine scFvs can then be isolated and used to target tumor antigens in vivo. Furthermore, tumor antigens that are recognized by scFvs can be isolated, analyzed and identified. Dr. Mason is currently exploring this novel targeting approach to treat canine hemangiosarcoma, a highly aggressive, fatal malignancy of vascular endothelial cells that affects large breed dogs. The overall hypothesis is that as in human cancer patients, canine cancer patients generate antibody responses against their own TAAs. These tumor-specific antibodies can be used either alone or linked to a chemotherapeutic agent to specifically target primary and metastatic neoplastic lesions in vivo. Dr. Mason’s laboratory is using standard molecular techniques to recapitulate the immune repertoire of canine patients with hemangiosarcoma and screen the resulting combinatorial antibody libraries for single chain fragments that specifically target malignant endothelial cells. These fragments are being isolated, amplified and screened in vitro for their ability to recognize and kill autologous and allogeneic canine HSA cell lines. This work aims to develop the first canine-derived, tumor-specific targeting approach for the treatment of HSA in dogs and aims to provide proof of principal for this approach that can then be employed to generate targeting reagents for many other tumor types. It is intended that canine scFvs that target malignant endothelial cells will be employed in a phase I clinical trial to evaluate the safety and efficacy of this novel approach to treat dogs with spontaneously occurring HSA. This work is generously funded by the Canine Health Foundation (American Kennel Club)  and a Chase Foundation grant from the American College of Veterinary Internal Medicine. A manuscript describing this work has recently been published:

Generation and validation of canine single chain variable fragment phage display libraries. Braganza A, Wallace K, Pell L, Parrish CR, Siegel DL, Mason NJ.  Vet Immunol Immunopathol.2010 Aug 7 [Epub]