Cytotoxic activity of monoclonal antibodies to cell surface antigens of canine B cell neoplasms

Introduction: Spontaneous canine lymphoma (CL) has become a promising, non-rodent model for advancing the therapeutic strategies of human hematological malignancies. Therapy of CL, unlike human non-Hodgkin’s lymphoma, lacks modern biological medicines that are able to significantly prolong the lifespan of patients. Here, we evaluate two proprietary mAbs to DLA-DR as tools for development of a rapid screening test and experimental therapy of CL.

Materials and methods: Two murine mAbs (B5 and E11) that recognize conformational, monomorphic and non-overlapping epitopes present uniquely on the DLA-DR dimers were produced. These mAbs were analyzed by flow cytometry, ELISA and lateral flow tests (LFT) using a panel of canine lymphoma cell lines, biopsies from lymph nodes of dogs with confirmed CL or enlarged for reasons other than CL and peripheral blood mononuclear cells (cPBMCs) from healthy dogs. The ELISA and LTF methods were statistically evaluated for sensitivity and specificity of CL detection. The mAbs were also tested for their direct and complement mediated cytotoxicity towards CL cell lines.

Results:Two hybridoma clones (B5.1 and E11.6) were chosen based on their strong and selective immunoreactivity towards canine B cell neoplasm (CD21⁺/CD79a⁺), T cell lymphoma (CD3⁺/CD5⁺) and mixed T/B (CD3⁺/CD79a⁺) cell neoplasms but not for normal cPBMCs. In vitro treatment of two canine lymphoma B-cell lines (CLBL1 and CLB70) with B5 and E11 rapidly induced a direct apoptotic cell death. Similarily, both mAbs efficiently killed the above cell lines through the mechanisms of complement-dependent and antibody-mediated cellular phagocytosis. Interestingly, simultaneous use of both antibodies produces a strong synergistic effect on CL cell lines.

Conclusions: Our data show that DLA-DR epitopes recognized by the B5 and E11 mAbs are promising targets for development of rapid screening tests and experimental therapy of primary CL and possibly other conditions manifesting an increase in the expression of DLA-DR antigen.