Fab Antibody Fragments to Dog Leukocyte Antigen DR (DLA-DR) Directly Suppress Canine Lymphoma Cell Line Growth in Vitro and in Murine Xenotransplant Model

Background: /Objectives: Canine Diffuse Large B-cell Lymphoma (cDLBCL) is characterized by a high prevalence of MHC II DR (DLA-DR) antigen overexpression. Murine anti-pan-DLA-DR monoclonal antibodies (mAbs) B5 and E11 have been previously shown to induce cDLBCL cell death in vitro and in vivo. Consequently, DLA-DR antigens are considered a promising alternative target to CD20 for passive immunotherapy. While infusion of anti-pan MHC II mAbs has demonstrated tumor suppression in cDLBCL xenografted immunodeficient mice, the relative contributions of direct cellular versus immune-mediated mechanisms to this therapeutic effect remain undefined. This study aimed to dissect these potential mechanisms of mAb E11. Methods: Canine lymphoma and leukemia cell lines CLBL1 and CLB70 were incubated with full E11 antibody or its F(ab’)2 and Fab fragments and cell viability was assessed with sub-G1 assay then, NOD-SCID mice were xenotransplanted with 15×106 canine CLBL1 cells expressing nanoluciferase and were infused either with mAb E11 or its fragments, each at 1 mg/kg body mass, twice weekly for three consecutive weeks. Tumor burden was monitored by assessing body weight, nanoluciferase activity in blood, and by flow cytometric analyses of bone marrow tumor cell content. Time to tumor progression (TTP) was calculated based on weight loss and luminescence measurements. Results: We observed cytotoxic activity of monovalent E11-Fab fragments in vitro and in vivo. The mean TTP for mice treated with irrelevant mouse IgG antibodies was 9.8±4.65 days. In contrast, treatment with E11 Fab fragments resulted in a TTP of 19.1±2.67 days, which was similar to that achieved with the full E11 mAb (19.5±1.73 days) and E11 F(ab')2 fragments (18.1±2.9 days). Conclusions: Our findings demonstrate a potent antibody cytotoxicity mechanism that operates in vivo and is independent of cell surface MHC II crosslinking or Fc engagement. These data support the promising potential of E11-Fab fragments for further clinical development as a therapeutic agent in canine lymphoma.