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

## TL;DR

This study shows that Fab antibody fragments targeting DLA-DR can directly suppress canine lymphoma growth, both in lab tests and in mice, without needing immune system involvement.

## Contribution

The study reveals a direct tumor-suppressing mechanism of Fab fragments targeting DLA-DR, independent of immune-mediated effects.

## Key findings

- E11 Fab fragments suppressed canine lymphoma cell growth in vitro and in vivo as effectively as full antibodies.
- Treatment with E11 Fab fragments significantly delayed tumor progression in mice compared to control antibodies.
- The cytotoxic effect of E11 Fab fragments does not rely on MHC II crosslinking or Fc engagement.

## Abstract

Antibodies targeting pan-MHC class II (MHC II) epitopes have demonstrated efficacy in suppressing the growth of B-cell lymphoma in immunodeficient mouse models. However, the precise delineation between direct cellular mechanisms and immune-mediated effects responsible for this therapeutic efficacy has often remained poorly defined. Here, we present evidence that monovalent Fab fragments targeting dog leukocyte antigen DR (DLA-DR) elicit direct tumor-suppressing effects towards canine lymphoma cells. Remarkably, the magnitude of this suppression is comparable to that achieved by divalent F(ab′)2 fragments and full monoclonal antibody (mAb) counterparts. Therefore, our data reveal an antibody cytotoxicity mechanism that operates independently of cell surface MHC II crosslinking or Fc engagement and is intrinsically potent. Given their inherent advantages, including reduced immunogenicity and enhanced tissue penetrability, Fab fragments derived from therapeutic anti-pan MHC II monoclonal antibodies (mAbs) represent a compelling option for further clinical development.

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 observed to promote death of cDLBCL cells in vitro and in vivo. Consequently, DLA-DR antigens are considered a prospective target for passive immunotherapy aside from CD20. 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 1.5 × 107 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.