BARs (B -cell receptor a ntigens for r everse targeting): A Novel and Ultimately Specific Treatment Concept for B-Cell Neoplasms

Introduction: The major task of a B-cell receptor is the binding and internalization of its antigenic target, and its processing for antigen presentation to T-cells. Chronic antigenic stimulation has been discussed to play a role in the pathogenesis of malignant B-cell lymphomas. We therefore systematically searched for the antigenic targets of BCRs from various B-cell neoplasms.

Methods: Recombinant BCRs were expressed as recombinant Fabs (rFabs) based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA lymphoma biopsies. Whenever possible, "natural" Fabs (nFabs) were also obtained by papain digestion of fresh or cultured lymphoma cells. Both nFab and rFab were used to screen for binding to proteins expressed on macroarrays derived from human cDNA expression libraries and identical binding pattern of nFabs and rFabs was demonstrated by an antigen competition assay.

Results: Two antigens (paratarg-7 and sumoylated HSP-90 which are hyperphosphorylated and sumoylated, respectively, in patients compared to healthy controls) are the targets of paraproteins from (depending on ethnicity) 30-50% of all multiple myeloma patients; the BCR from 67% of patients with primary CNS lymphoma target hyperglycosylated neurabin, 26% of the BCR from ABC-type DLBCL target hypophosphorylated ARS2 and 45% of all mantle cell BCR target LRPAP1; optineurin is the BCR target of 12% follicular lymphomas and various autoantigens have been identified as the targets of roughly 30% of all CLL cases. For all autoantigens binding to its specific BCR, rapid internalization and induction of proliferation was demonstrated, indicating partial dependence on antigenic stimulation even in cell lines that had been in culture for years. Most importantly, BCR-specific cytotoxicity of recombinant pseudomonas-exotoxin conjugated ARS2 against an ABC-DLBCL cell line with BCR specific for ARS2 (OCI-Ly3) was demonstrated in vitro and in vivo after establishment of OCI-Ly3 lymphomas in SCID beige mice.

Conclusions: Assuming that only a minority of BCR targets have been identified to date, the prevalence of posttranslationally modified autoantigens strongly supports a role of chronic antigenic stimulation in many B-cell neoplasms. Due to the predominance of a single or few BCR antigens in each malignant B-cell entity studied, BARs represent an attractive and novel therapeutic concept for a broad spectrum of B-cell neoplasms and are the first therapeutic approach in oncology that targets exclusively the malignant cells. BARs can be used for conjugation with toxins, radionuclides and small molecules as well as for bispecific constructs (e. g. with CD3 or CD16) and CAR T-cells, the toxicity of which should be drastically reduced due to the ultimate specificity of BARs that spares normal B-cells. Supported by Wilhelm-Sander-Stiftung