Identification of the Antigenic Targets of the Major CLL-Derived Stereotyped BCRs and First Demonstration of Sub-Subsets
Authors: Preuss Klaus-Dieter , Fadle Natalie , Regitz Evi , Kemele Maria , Wölfle Manuela , Stilgenbauer Stephan , Pfreundschuh Michael 
Published: October 2019

Abstract
Background: The B-cell receptor (BCR) is the hallmark of mature B cells and a clonal BCR is expressed on the neoplastic cells of B-cell malignancies. The BCR mediated signalling provides proliferative and anti-apoptotic signals for the neoplastic B cells and represents an attractive therapeutic target. The BCRs of about 1/3 of CLL cases display highly homologous complementary determining regions 3 (CDR3), on the basis of which stereotyped subsets have been defined. The homologies of the BCR of a given subset suggest selection by shared antigen(s), which might play an important role in the pathogenesis of the respective B-cell neoplasm by chronic antigenic stimulation.
Methods: Tumor cells from CLL patients belonging to subsets 2, 3, 4, 5, 6, 8 were studied. Natural Fabs (nFab) were obtained by papain digestion of membrane-bound Igs. Subset-derived nFabs were used to screen for reactivity with proteins represented in two high-density protein macroarrays, one derived from human fetal brain cDNA and a second derived from a mixture of expression libraries derived from activated T-cells, lung and colon. Identified antigens were analysed by standard techniques. All methods used (nFab generation by papain digestion, BCR cloning, membrane screening, epitope mapping etc.) are described in detail in Zwick C et al., Blood 2013;121(23):4708-4717.
Results: Our ongoing systematic search for CLL subset-specific antigens together with our previously published data (Zwick et al) led to the identification and epitope characterization of the antigenic targets for the 6 most common CLL subsets: MARK3 as antigen for subset 2, NCOR2 as antigen for subset 3, CaCyBP as antigen for subset 4, FAM32A protein as antigen for subset 5 and GLDC as antigen for subset 8. In addition we could confirm MYHIIA as antigen for subset 6 (in accordance with Chu CC et al, Blood 2010;115(19):3907-3915). The analysis of the BCR binding revealed (except for oxLDL) in minimum 2 different epitopes for each subset-specific antigen, thus defining sub-subsets with BCRs binding to distinct epitopes. Sequence analysis of the respective BCR suggests that this epitope-spreading is due to antigen-driven mutation/maturation of BCRs with a highly homologous CDR3.
Conclusions: The antigenic targets (and stimulus) for the clonal BCRs of the major sterotyped subsets have been identified providing for the first time experimental evidence for the hypothesis that BCRs of a given subset indeed target the identical antigen. Moreover, based on the BCR-binding epitopes, sub-subsets of stereotypes have been defined by BCRs obviously originating from the same germline, but having undergone different antigen-driven mutations.
Supported by Deutsche Forschungsgemeinschaft DFG, Deutsche José Carreras Leukämie Stiftung, Wilhelm-Sander-Stiftung, Deutsche Krebshilfe e.V.

Disclosures
Stilgenbauer: Pharmacyclics, Janssen: Honoraria, Research Funding.