Mantle cell lymphoma (MCL) accounts for 5%–10% of all lymphomas. The disease’s genetic hallmark is the t(11; 14)(q13; q32) translocation. In younger patients, the first-line treatment is chemoimmunotherapy followed by autologous stem cell transplantation. Upon disease progression, novel and targeted agents such as the BTK inhibitor ibrutinib, the BCL-2 inhibitor venetoclax, or the combination of both are increasingly used, but even after allogeneic stem cell transplantation or CAR T-cell therapy, MCL remains incurable for most patients. Chronic antigenic stimulation of the B-cell receptor (BCR) is thought to be essential for the pathogenesis of many B-cell lymphomas. LRPAP1 has been identified as the autoantigenic BCR target in about 1/3 of all MCLs. Thus, LRPAP1 could be used to target MCL cells, however, there is currently no optimal therapeutic format to integrate LRPAP1. We have therefore integrated LRPAP1 into a concept termed BAR, for B-cell receptor antigens for reverse targeting. A bispecific BAR body was synthesized consisting of the lymphoma-BCR binding epitope of LRPAP1 and a single chain fragment targeting CD3 or CD16 to recruit/engage T or NK cells. In addition, a BAR body consisting of an IgG1 antibody and the lymphoma-BCR binding epitope of LRPAP1 replacing the variable regions was synthesized. Both BAR bodies mediated highly specific cytotoxic effects against MCL cells in a dose-dependent manner at 1–20 µg/mL. In conclusion, LRPAP1 can substitute variable antibody regions in different formats to function in a new therapeutic approach to treat MCL.
About 30% of patients suffering from the chronic autoimmune liver disease primary biliary cirrhosis produce autoantibodies against Sp100, a protein migrating in SDS-PAGE at a position corresponding to 100 kDa and located on discrete dot-shaped nuclear structures. The human Sp100 cDNA has recently been cloned and the deduced amino acid sequence was found to contain similarities to several transcriptional regulatory proteins; the biologic function of the Sp100 protein, however, is still unknown. In this study we present data which show that infection of HEp2 cells with influenza A virus, transformation of glial cells with SV40 DNA, and stimulation of PBL with mitogens affect the expression of the Sp100 autoantigen. These observations prompted us to investigate whether expression of the Sp100 protein is modulated by the action of IFN. Immunofluorescence staining of HEp2 and HeLa cells grown in the presence of IFN-alpha, IFN-beta, or IFN-gamma revealed an increase both in size and number of the Sp100 protein-containing nuclear dots, whereas no such effect was observed with cells treated with TNF-alpha. As measured by an immunoblot-based ELISA the amount of Sp100 protein in INF-beta-treated cells (1000 IU/ml, 18 h) was eight to nine times higher than in untreated cells. The enhanced protein expression was accompanied by an accumulation of the Sp100-specific mRNA (13-fold increase of the normal level after 10 h of INF-beta treatment of HEp2 cells). These findings characterize the Sp100 protein as a new member of IFN-modulated proteins and raise the question whether cytokine-mediated increase of Sp100 protein expression plays a role in induction of anti-Sp100 autoantibodies.
Autoantibodies to a novel nuclear Ag, Sp100, have recently been described that recognize a nuclear protein with an apparent molecular mass of 95 to 100 kDa and a dot-like distribution within cell nuclei. By immunoscreening of a lambda gt11 cDNA expression library derived from HeLa cells with an anti-Sp100 autoimmune serum a 0.7-kb cDNA (Sp26) coding for a fragment of Sp100 was isolated. Expression of this cDNA and use of the recombinant protein in ELISA revealed that the fragment carries major Sp100 autoepitopes and that anti-Sp100 autoantibodies predominantly occur in patients suffering from primary biliary cirrhosis (50/184). The Sp26 cDNA was used as hybridization probe for isolation of longer cDNA from human liver- and placenta-derived lambda gt10 cDNA libraries. Overlapping fragments were assembled to generate a full length cDNA coding for a protein with a molecular mass of 53 kDa and an isoelectric point of 4.7. The Sp100 autoantigen expressed in vitro from this cDNA and authenticated by a capture immunoblot assay, comigrated in SDS-PAGE with the authentic HeLa autoantigen of 95 to 100 kDa and thus showed an aberrant electrophoretic mobility. Computer based protein sequence analysis of the Sp100 autoantigen revealed regions of striking sequence similarities to the alpha 1 and alpha 2 domains of various human and non-human MHC class I Ag and to several transacting transcriptional regulatory proteins.
Background 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 and LRPAP1 as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type and 45% of mantle cell lymphomas (MCLs), 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). The optimal therapeutic format BARs can be integrated in has yet to be found. Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed and tested an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. Material and methods 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 for fusion with the FLAG tag and transfected into HEK293 cells for production. Purification of the BAR-body was performed via anti-FLAG antibody affinity chromatography. The BAR-body was detected by western blot analysis and binding capacity to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and the not ARS2-reactive control DLBCL cell line TMD8 was assessed by flow cytometry. ARS2 BAR-body induced cytotoxicity of lymphoma cells with an ARS2 reactive BCR was measured by LDH release assays with human PBMCs as effector cells at an E:T ratio of 10:1. Results 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, which showed the construct at approximately 150 kD as was to be expected. The BAR-body bound specifically to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and did not bind to the DLBCL cell line TMD8, which has a B-cell receptor of different specificity or to lymphoma cell lines of different entities. In LDH release assays with 5 x 104 PBMCs and 5 x 103 lymphoma cells (E:T ratio of 10:1) the ARS2 BAR-body induced PBMC mediated specific lysis of the ARS2 reactive lymphoma cell lines U2932 and OCI-Ly3 but not the control DLBCL cell line TMD8 starting at a concentration of 0,1µg/ml. Cytotoxic effects were dose dependent, reached a maximum of 50% specific lysis at a concentration of 1µg/ml and did not increase at concentrations of 10µg/ml. Conclusion Here, we show that BARs can substitute for the variable domains as binding moiety in antibody like constructs to target the BCR of B-cell lymphomas. Because approaches using their specific cognate antigen for targeting the malignant B cells have an exclusive specificity for the BCR of the malignant clone, they can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells, because they leave normal B-lymphocytes unaffected. By incorporating BARs into the well-known format of an antibody we hope to capitalize on years of experience with this therapeutic format from conducting and interpreting in vivo experiments to the translation of the BAR approach into the clinic. Disclosures Stilgenbauer: Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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