Generation of a Canine Anti-EGFR (ErbB-1) Antibody for Passive Immunotherapy in Dog Cancer Patients

Abstract

Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a "caninized" version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer.

Eradication of Canine Diffuse Large B-Cell Lymphoma in a Murine Xenograft Model with CD47 Blockade and Anti-CD20

“…Therefore, as with the human CD47/SIRPα interaction, the principles established by this study may be generalizable to other types of canine cancer and other canine antibodies to tumors. A popular and successful strategy in immuno-oncology is to combine treatments; CD47-blocking therapies may thus complement emerging treatment modalities using passive immunotherapy for canine diseases (56). Furthermore, anti-CD20 is used for inflammatory or autoimmune disorders, and similar spectrums of disease exist in canine models that could also benefit from B-cell depletion in combination with CD47-blocking therapies.…”

A Comparative Approach of Tumor-Associated Inflammation in Mammary Cancer between Humans and Dogs

“…The great homology in the EGFR family among humans and dogs [126] prompted us recently to produce a recombinant canine anti-EGFR antibody for canine mammary tumor treatment [125]. …”

Recent Advances in Immunotherapy for the Treatment of Cancer

“…These therapies in canines are directed against the same molecular targets for which biologic or biosimilar drugs are currently available for treating lymphoma in human (http://www.aratana.com). Similarly, a "caninized" version of anti-EGFR antibody, can225IgG, generated by fusing the variable region gene of 225 (murine precursor of Cetuximab) with canine constant heavy gamma and kappa chain genes is also under development for clinical application (Singer et al, 2014). …”