Development of novel monoclonal antibodies to dog leukocyte antigen DR displaying direct and immune‐mediated cytotoxicity toward canine lymphoma cell lines

Abstract

Spontaneous canine lymphoma (CL) has become a promising, nonrodent model for advancing the therapeutic strategies of human hematological malignancies. As new resources for veterinary and comparative studies on CL-associated antigens, we developed 2 novel mouse monoclonal antibodies, denoted B5 and E11, that recognized the canine major histocompatibility Class II DR antigens (dog leukocyte antigen DR). Using flow cytometry and solid phase immunoenzymatic assays, we showed that the antigens recognized by B5 and E11 were strongly expressed in several CL cell lines and the ex vivo canine neoplastic cells of B and mixed B/T immunophenotypes. Additionally, we evaluated a minimal cross-reactivity of B5 and E11 with the human B-cell line, Raji. By the ectopic expression of the hybrid murine/canine I-E/DR dimers in the HEK293 cells, we demonstrated that the epitope of B5 was localized to the invariant DRα chain, whereas the epitope of E11 was collectively formed by the DRα and DRβ chains. Both epitopes were conformational and conserved in all the tested unrelated individuals of different dog breeds. In vitro treatment of 2 CL B-cell lines (CLBL1 and CLB70) with B5 and E11 rapidly induced a direct apoptotic cell death. Similarily, both mouse monoclonal antibodies efficiently killed the above cell lines through the mechanisms of complement-dependent and antibody-mediated cellular phagocytosis. Collectively, our data support the further development of B5 and E11 as novel tools for dog leukocyte antigen DR-targeted, preclinical trials involving CL.

An Antibody Specific for the Dog Leukocyte Antigen DR (DLA-DR) and Its Novel Methotrexate Conjugate Inhibit the Growth of Canine B Cell Lymphoma

“…To evaluate the cytotoxicity of B5 and B5-MTX, canine B cell lymphoma/leukemia cell lines expressing DLA-DR (CLBL1 and CLB70) and not expressing DLA-DR (GL-1) were exposed to 2 μg/mL of both preparations for 48 h. As previously reported [19], several hallmarks of direct apoptotic cell death, including caspase 3/7 activation, annexin V binding, DNA fragmentation (subG1 DNA content), were observed upon B5 treatment of DLA-DR expressing cell lines (Supplementary Figure S1). In comparison with B5, the B5-MTX conjugate exerted more potency, but also cell-specific cytotoxic effects at the same time, as no significant increase in toxicity against the DLA-DR non-expressing GL-1 cell line was detected.…”

“…We sought for more sensitive methods than bioluminescence to quantify tumor cell burden in bone marrows and other tissues of CLBL1-Luc-infused mice. For this purpose, Western blotting and flow cytometry with an anti-pan-DLA-DR antibody, E11 [19], was used. This antibody was chosen because it recognized a different epitope of DLA-DR than B5 and therefore did not interfere with mAbs infused for therapeutic treatment.…”

“…A good safety profile of IMMU-114 has been reported both in human and canine patients [14]. However, due to the limited cross-reactivity of IMMU-114 with canine DLA-DR [19], a thorough assessment of the full therapeutic potential of this target in dogs is difficult.…”

“…This antibody binds strongly to a conformational epitope of canine DR alpha chain (DLA-DRα), but shows only minimal cross-reactivity with HLA-DRα. We have previously shown that B5 exerts immune-dependent and direct cytotoxic effects in vitro [19].…”

Session 6: Advances in Anticancer Therapy

“…However, studies on novel target molecules like Class II MHC antigens are under way (6). A recent paper reports novel monoclonal antibodies against dog leukocyte antigen DR (DLA-DR) that exhibits direct and immune-mediated cytotoxicity towards canine lymphoma cell lines (7). Additionally, the antigens recognized by these antibodies are strongly expressed in several canine lymphoma cell lines and ex vivo canine neoplastic cells of B and mixed B/T immunophenotypes, which demonstrates their diagnostic potential.…”