P611: cd37 as a molecular target for car-t cells against chronic lymphocytic leukemia

Background: CD37 is a tetraspanin predominantly expressed on the surface of mature B cells. Although the role of CD37 in B-cell biology has not been fully understood, recent studies demonstrate its involvement in the regulation of B-cell survival and proliferation, apoptotic signaling, as well as tumor evasion. CD37 is a promising target for immunotherapies such as monoclonal antibodies (mAbs) and antibody-drug conjugates, some of which are currently in clinical trials. Recently, CD37 has also gained attention as a target for chimeric antigen receptor (CAR)-T cells and has proven efficacy in preclinical studies in B- and T-cell-derived malignancies. CD37 is stably expressed across multiple B-cell malignancies, however, its levels are exceptionally high on the surface of chronic lymphocytic leukemia (CLL) cells. CLL is an incurable disease but in the majority of cases can be managed thanks to the advent of novel targeted therapies. However, a small population of CLL patients does not achieve complete response following targeted therapies. For those patients, CAR-T cells offers an attractive therapeutic option, as illustrated by the recent clinical trial combining CD19 CAR-T cells with ibrutinib. Moreover, the longest remission has been observed in a CLL patient following CAR-T cell infusion, which supports the feasibility of CAR-T cell application in a selected population of CLL patients. Aims: To investigate the utility of CD37 as a target for CAR-T cells to be used in CLL. Methods: We performed quantitation of surface expression of B-cell specific proteins CD19, CD20, and CD37 using BD Quantibrite™ Beads in CLL cell lines – HG-3, Mec-1, PGA, and on 20 primary CLL samples. Further, we evaluated the sensitivity of CLL cell lines and patient-derived CLL cells to anti-CD19, anti-CD20, and anti-CD37 CAR-T cells. For this purpose, healthy donor peripheral blood mononuclear cells stimulated with anti-CD3/CD28 mAbs were used as a source of T cells for transduction with second-generation CD19-, CD20-, and CD37-CAR constructs. CAR-modified T cells were then expanded with Dynabeads™ Human T-Activator CD3/CD28. In cytotoxicity assays, CLL cell lines or primary CLL cells were stained with CellTrace™ Violet and incubated for 16h with different effector to target (E:T) ratios. Cell viability was assessed using flow cytometry upon propidium iodide staining. Results: By analyzing Leukemia MILE study data we observed that across multiple hematological malignancies, CD37 levels are exceptionally high in CLL cells. Moreover, by phenotyping CLL cell lines we demonstrate that CD37 expression is high in HG-3, Mec-1, and PGA cells and quantitatively superior to CD19 and CD20. Similarly, by quantifying surface CD37 in primary CLL cells, we confirmed that it predominates over the expression of other antigens. We then investigated the potency of CAR-T cells against CLL cell lines. Among the three CARs tested, CD20 CAR-T cells were the least effective (HG-3 – 31%, Mec-1 – 45%, PGA – 26% of cytotoxicity). In contrast, CD37 CAR-T cells demonstrated profound cytolytic activity against CLL cell lines (HG-3 – 79%, Mec-1 – 67%, PGA – 45% of cytotoxicity) comparable to the efficacy of CD19 CAR-T cells currently used in clinics (FMC-63-based construct). Finally, we show that patient-derived CLL cells are notably sensitive to CD37 CAR-T cell-mediated cytotoxicity.Summary/Conclusion: Targeting CD37 via CAR-T cells is a promising therapeutic option for CLL patients and an attractive alternative to CD19-directed CAR-based therapies. Further in vivo studies are warranted. Funding: Polish National Science Centre 2019/35/D/NZ5/01191 Keywords: Tetraspanin, CAR-T, Chronic lymphocytic leukemia, Cytotoxicity