TY - JOUR T1 - CD3xCD19 DART molecule treatment induces non-apoptotic killing and is efficient against high-risk chemotherapy and venetoclax-resistant chronic lymphocytic leukemia cells JF - Journal for ImmunoTherapy of Cancer JO - J Immunother Cancer DO - 10.1136/jitc-2019-000218 VL - 8 IS - 1 SP - e000218 AU - Anne W J Martens AU - Susanne R Janssen AU - Ingrid A M Derks AU - Homer C Adams III AU - Liat Izhak AU - Roel van Kampen AU - Sanne H Tonino AU - Eric Eldering AU - Gerritje J W van der Windt AU - Arnon P Kater Y1 - 2020/06/01 UR - http://jitc.bmj.com/content/8/1/e000218.abstract N2 - Background Bispecific antibodies are promising new therapeutics in B cell malignancies. Whether they lead to potent T cell activation despite described T cell dysfunction in chronic lymphocytic leukemia (CLL), and are able to effectively target high-risk or venetoclax-resistant samples, is currently unknown.Methods CD19+ cell lines or primary (high-risk) CLL were cocultured in vitro with healthy donor (HD) or CLL-derived T cells in the presence of a CD3xCD19 dual affinity retargeting molecule (CD3xCD19 DART). Cell cytotoxicity, T cell activation, proliferation and effector molecule production were analyzed using flow cytometry.Results Here, we report that a bispecific CD3xCD19 DART mediates efficient killing by HD T cells of CD19+ cell-lines and primary CLL cells, regardless of immunoglobulin heavy chain variable region (IGHV) mutational status TP53 status or chemotherapy, ibrutinib or venetoclax sensitivity. Whereas TCR stimulation of CLL-derived T cells resulted in dysfunctional T cell activation and proliferation, treatment with CD3xCD19 DART led to a similar activation profile in CLL-derived and HD-derived T cells. Consistently, co-culture of CLL derived T cells with JeKo-1 or CLL cells in the presence of CD3xCD19 DART resulted in significant cytotoxicity by both CD4+ and CD8+ T cells. On stimulation of CLL cells with CD40L, CLL cells become resistant to the specific inhibitor of anti-apoptotic Bcl-2 protein venetoclax, due to upregulation of Bcl-2 family members such as Bcl-XL. Nevertheless, CD40L stimulated CLL cells were as efficiently lysed on CD3xCD19 DART treatment as unstimulated CLL cells. Further examination of the mechanism of CD3xCD19 DART mediated killing showed that lysis was dependent on granules, but was independent of BAX/BAK or caspase activity, indicating non-apoptotic cell death.Conclusions These data show that CD3xCD19 DART in CLL leads to robust T cell activation and lysis of high-risk venetoclax resistant CLL cells through a non-apoptotic mechanism.Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplementary information. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. ER -