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

Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinarymedicine 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 cloneswere screened and the best cloneswere selected according to productivity andhighest specificity in EGFR-coated ELISA.Uponpurificationwith ProteinG, the recombinant cetuximab-like canine IgGwas 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 antibodymediated 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 EGFRmolecule in canine cancer, on the other hand, this antibodymay be a promising research compound to establish passive immunotherapy in dog patients with cancer. Mol Cancer Ther; 13(7); 1777–90. 2014 AACR.