Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia

Abstract

BackgroundWhile achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy.MethodsFirst, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable (IGHV)-unmutated (9ATM-mutated, 8TP53-mutated, and 9 without mutations inATM,TP53,NOTCH1orSF3B1) and 6IGHV-mutated samples without mutations in the above-mentioned genes. Then, we mimicked the leukemic microenvironment in the primary cells by ‘2S stimulation’ through interleukin-2 and nuclear factor kappa B. Finally, CRISPR/Cas9-generatedATM-knockout andTP53-knockout clones (four and seven, respectively) from CLL-derived cell lines MEC1 and HG3 were used. All these samples were exposed to CAR T cells. In vivo survival study in NSG mice using HG3 wild-type (WT),ATM-knockout orTP53-knockout cells was also performed.ResultsPrimary unstimulated CLL cells were specifically eliminated after >24 hours of coculture with CAR T cells. ‘2S’ stimulated cells showed increased survival when exposed to CAR T cells compared with unstimulated ones, confirming the positive effect of this stimulation on CLL cells’ in vitro fitness. After 96 hours of coculture, there was no difference in survival among the genetic classes. Finally, CAR T cells were specifically activated in vitro in the presence of target knockout cell lines as shown by the production of interferon-γ when compared with control (CTRL) T cells (p=0.0020), but there was no difference in knockout cells’ survival. In vivo, CAR T cells prolonged the survival of mice injected with WT,TP53-knockout andATM-knockout HG3 tumor cells as compared with CTRL T cells (p=0.0485, 0.0204 and <0.0001, respectively). When compared withATM-knockout,TP53-knockout disease was associated with an earlier time of onset (p<0.0001), higher tumor burden (p=0.0002) and inefficient T-cell engraftment (p=0.0012).ConclusionsWhile in vitro no differences in survival of CLL cells of various genetic backgrounds were observed, CAR T cells showed a different effectiveness at eradicating tumor cells in vivo depending on the driver mutation. Early disease onset, high-tumor burden and inefficient T-cell engraftment, associated withTP53-knockout tumors in our experimental setting, ultimately led to inferior performance of CAR T cells.

Hypermethylation of CD19 promoter enables antigen-negative escape to CART-19 in vivo and in vitro

“…CAR T cell preparation, quality assessment and in vivo studies were previously described. 15 Briefly, T cells were isolated from buffy coats of healthy donors purchased from the Department of Transfusion and Tissues of University Hospital Brno using the RosetteSep Human T cell enrichment Cocktail (StemCell). Immediately following isolation, T cells were activated using IL-2 (50 U/mL; Miltenyi Biotec) and Dynabeads Human T-activator CD3/CD28 (ratio 1:3 bead:cell; ThermoFisher Scientific).…”

Current State of CAR T-Cell Therapy in Chronic Lymphocytic Leukemia

“…This may be partially remedied by concurrent ibrutinib treatment, which is known to cause an efflux of tumor cells from the tissue compartments into the blood [ 61 ]. Additionally, some preclinical evidence exists showing that TP53 -mutated CLL gives rise to early disease onset, high tumor burden, and inefficient T-cell engraftment in a mouse model, leading to inferior performance of anti-CD19 CAR T cells [ 62 ]. When we closely inspected patient lists from anti-CD19 clinical trials ( Table 1 ), only 3 of 20 cases unequivocally bearing del17p ( TP53 genetic locus) reached complete remissions.…”

Humanized Mice Are Precious Tools for Preclinical Evaluation of CAR T and CAR NK Cell Therapies

“…In contrast to CD19-CAR T cells that only targeted CD19 + leukemic cells, the bispecific CD19/CD20-CAR T cells also eradicated all leukemic cells, even those that lost CD19 expression at the surface in a xenograft NSG model [ 86 ]. Interestingly, anti-CD19 CAR T cell therapy was evaluated in a B-CLL xenograft mice model by injecting into mice B cell lines carrying individual KOs representative for the mutational landscape in B-CLL [ 87 ]. In vivo, they confirmed that anti-CD19 CAR T cells prolonged survival of the different genetic classes of B-CLL tumor cells and revealed a differential anti-tumor efficacy according to the mutation introduced [ 87 ].…”

“…Interestingly, anti-CD19 CAR T cell therapy was evaluated in a B-CLL xenograft mice model by injecting into mice B cell lines carrying individual KOs representative for the mutational landscape in B-CLL [ 87 ]. In vivo, they confirmed that anti-CD19 CAR T cells prolonged survival of the different genetic classes of B-CLL tumor cells and revealed a differential anti-tumor efficacy according to the mutation introduced [ 87 ]. This emphasizes the need for more personalized and optimized CAR design in treatment of B-CLL.…”