The B-Cell Receptor Antigen ARS2 Can be Integrated into a BAR-Body Format to Treat Diffuse Large B-Cell Lymphomas in Xenograft Mouse Models

Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2, LRPAP1 and Neurabin-I as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type, 45% of mantle cell lymphomas (MCLs) and 66% of primary CNS lymphomas, respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. This ARS2 containing BAR-body showed promising efficacy in in-vitro experiments. Here, we report the results of the initial in-vivo experiments using lymphoma xenograft mouse models.

To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector and transfected into HEK293 cells for production. Flow cytometry was used for binding experiments of the ARS2 BAR-body to lymphoma cell lines. For mouse experiments we inoculated 1x10⁷ cells of the human DLBCL cell line U2932 (expresses a BCR with reactivity for ARS2) subcutaneously into the left flank of 12 NOD SCID mice. Tumor volume was calculated from day 14 measuring the long and short diameters of the tumor mass in millimeters. 6 mice were treated with 60 mg/kg ARS2 BAR-body intraperitoneally on days 23, 25, 30, 32, 37, 39, 44 and 46 after tumor inoculation. 6 control mice were mock-treated with PBS following the same time schedule. Tumor growth was evaluated by calculating the change in tumor volume from the first measurement at day 14. We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis. Flow cytometric binding assays confirmed specific binding to the DLBCL cell line U2932 which expresses a BCR receptor with reactivity for ARS2 while control cell lines could not be stained by the ARS2 BAR-body. U2932 cells could successfully be transplanted subcutaneously into the flank of NOD SCID mice to generate a xenograft mouse model. Tumors in the treatment group increased their mean volume 14.1 times while tumors in the control group grew by a factor of 18.3 as compared to the initial mean tumor volume analyzed at day 14.

Approaches using the cognate antigen of B-cell receptors to target malignant B cells have an exclusive specificity for the BCR of the malignant clone and can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells. The ARS2 BAR-body shows promising activity against ARS2 reactive B-cell lymphoma cells in in-vivo mouse experiments. Toxic effects using the ARS2 BAR-body for the first time in-vivo were not observed. Further studies are necessary to reproduce and optimize the current experiments to form the basis for the translational development of BAR-bodies.