B-Cell Receptor Antigens As the Targeting Moiety for Bispecific Constructs - a Novel Treatment Approach for B-Cell Lymphomas

Background

Chronic antigenic stimulation of the B-cell receptor (BCR) has been suggested to play a role in the pathogenesis of B-cell lymphomas, however only few B-cell receptor antigens have been identified until recently, when we identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors from approximately 25% of DLBCLs of the ABC type and 45% of mantle cell lymphomas, respectively. These BCR antigens can be used to therapeutically target lymphoma cells in an approach we designated as BARs (B-cell receptor antigens for reverse targeting). To test whether BARs can substitute for the B-cell targeting antibody CD19, we designed T-cell engaging anti-CD3 single chain fragments (scFv) and NK-cell engaging anti-CD16 scFv with the respective BAR as the targeting moiety.

Material and methods

Variable heavy- and light- chain genes of the anti-CD3 OKT3 hybridoma and the anti-CD16 3G8 hybridoma were cloned into a pcDNA 3.1 vector by standard cloning techniques, followed by DNA sequences of the autoantigens ARS2 or LRPAP1. VH and VL were linked by a glycine-serine linker, as was VL to the autoantigenic epitope resulting in a VH-(Gly₄Ser₁)ⁿ-VL-(Gly₄Ser₁)ⁿ-ARS2/LRPAP1 DNA sequence. The final cloning product was transfected in HEK 293 cells for production of bispecific constructs. Binding capacity to lymphoma cell lines (DLBCL: OCI-Ly3, U2932, HBL-1; MCL : MAVER-1, GRANTA-519) and PBMCs was assessed by flow cytometry. Western blot analysis was used for detection of bispecific constructs after incubation with the monoclonal anti-His Tag antibody. Cytotoxicity was evaluated by LDH release assay.

Results

We cloned, expressed, characterized and tested 4 bispecific constructs: αCD3-ARS2, αCD3-LRPAP1, αCD16-ARS2 and αCD16-LRPAP1. αCD3 bispecific constructs bound specifically to CD3 on T cells whereas αCD16 constructs bound exclusively to CD16-positive cells. Bispecific ARS2 constructs bound specifically to DLBCL cells with a B-cell receptor specific for ARS2, while LRPAP1 containing constructs bound exclusively to MCL with a B-cell receptor specific for LRPAP1. αCD3-ARS2 and αCD3-LRPAP1 induced dose-dependent T-cell mediated, rapid cytotoxicity exclusively in lymphoma cell lines with B-cell receptors specific for ARS2 and LRPAP1, respectively, starting at concentrations as low as 0,1 µg/ml. NK-cell mediated cytotoxicity against the respective DLBCL and MCL cell lines was observed with αCD16 constructs αCD16-ARS2 and αCD16-LRPAP1 at concentrations starting from 1 µg/ml. Control lymphoma cell lines with BCR specificities other than for ARS2 or LRPAP1 were not affected.

Conclusion

BARs can substitute for B-cell-directed antibodies (e. g. CD19) as the targeting moiety in bispecific constructs for the therapeutic targeting of B-cell lymphomas. Because approaches using the BCR antigen for targeting the malignant B cells have an exclusive specificity for the cells with the specific surface BCR (which are exclusively the respective lymphoma cells), they do not only represent a sensu stricto tumor-specific approach, they can also be expected to be more effective (due to the higher density of BCR molecules on the lymphoma cell surface), but less toxic than the currently available bispecific T-cell and NK-cell engaging antibodies, because they leave all other cells, in particular normal B-lymphocytes, unaffected. Supported by Wilhelm-Sander-Stiftung