An "off-the-shelf" fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies

Abstract

T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide-resistant, allo-tolerant "off-the-shelf" CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphoma without a requirement for autologous T cells.

Figure 1. CART7 induce fratricide

(a) Schematic of CAR constructs. (b) T cells were cultured in Xcyte media supplemented with 50 U/mL IL-2 and 10ng/ml IL15 in the presence of anti-CD3/CD28 beads (bead to cell ratio, 3:1). On day 3 the beads were removed, and 2.5×10⁴ cells were plated in each well of a 96-well plate prior to transduction with either CAR7 or CAR19. CART7 undergo fratricide and fail to demonstrate robust expansion in the days following transduction (Day 9 CART7 = 13.7% of CART19 cell count n=3 mean±s.d). (c) Editing efficiencies of gRNA targeting CD7. % NHEJ determined as a percentage of sequencing reads with indels relative to WT cells. (d) Schema of CRISPR/Cas9 gene editing of human T cells. (e). CD7 gRNA editing efficiencies in human primary T cells as determined by FACS. (f) Percentage of CD7+ T cells following gene editing with CD7g4. (n=3 mean±s.d) (i) Targeted deep-sequencing of CD7 locus following CRISPR/Cas9 gene editing with CD7g4 (n=3 mean±s.d). Viability of primary T cells following CRISPR/Cas9 gene editing with CD7g4, determined 24 hrs post electroporation (n=3 mean±s.d).

Figure 2. CD7^ΔCART7 effectively kills T-ALL cell lines in vitro and in vivo

(a) Schema of gene edited CAR-T generation. Cell counts as determined using a Nexcelom Cellometer with ViaStain™ (n=3 mean±s.d). (b) WT T cells transduced with lenti-GFP (GFP+ T cells) were used as targets and were co-incubated with CD7^ΔCART7 or CD7^ΔCART19 at a ratio of 1:1 for 24hrs. The percentage of GFP+ T cells in the population was determined by flow cytometry. (c) In vitro killing assay. CD7^ΔCART7 or CD7^ΔCART19 were cultured with [51Cr]-labeled MOLT-3, MOLT-4 or HSB-2 at various E:T ratios for 4 hours.

Figure 3. In vivo efficacy of CD7^ΔCART7

(a) Schema of xenogeneic mouse model of T-ALL. NSG mice were injected with 5×10⁵ CCRF-CEM^CBR-GFP cells on day 0, then infused with 2×10⁶ CD7^ΔCART7 or CD7^ΔCART19 on day +4. (b) Kaplan–Meier Survival curve of mice treated with CD7^ΔCART7 or CD7^ΔCART19. Median survival CD7^ΔCART19 treated mice, 31 days vs. CD7^ΔCART7 treated mice, 65 days; p=0.0003) and (c) Tumor burden as determined by BLI imaging. p values < 0.05 considered significant, *p ≤ 0.05, ** p ≤ 0.01, ***p≤ 0.001, **** p ≤ 0.0001.

Figure 4. UCART7, deficient in CD7 and TRAC, effectively kill T-ALL cell lines in vitro

(a) Multiplex gene editing results in high efficiency double deletion of TRAC and CD7 as determined by FACS and targeted deep-sequencing of the CD7 and TRAC loci. (b) CD7^ΔCART7 and UCART7 exhibit robust expansion, but yield fewer cells likely due to fratricide of both the residual non-gene edited T cells and persistent CD7 surface expression on gene edited cells (which lags genetic deletion). (c) CD7^ΔCART7, UCART7, and their respective CD19 control CAR-T cells were cultured with [51Cr]-labeled MOLT-3, HSB-2 or CCRF-CEM cells at various E:T ratios for 4 hours. UCART7 was equal to CD7^ΔCART7 in efficiency of killing the CD7+ T-ALL cell line in vitro, even at low effector to target ratios. Statistical significance was determined using one-way ANOVA, followed by a step-down Bonferroni adjustment for multiple comparisons (n=3 mean±s.d). p values < 0.05 considered significant, *p ≤ 0.05, ** p ≤ 0.01, ***p≤ 0.001, **** p ≤ 0.0001

Figure 5. UCART7 kills primary patient T-ALL blast in vitro

Primary blasts obtained from three individual patients with CD7+ T-ALL were labeled with 150 nM CFSE. Labeled cells were co-incubated at a 1:1 ratio with either CD7^ΔCART7, UCART7, or their respective CD19 controls in triplicate for 24 hours prior to FACS analysis. Accucount florescent beads were used to determine actual cell counts. Data were collected using a Gallios cytometer. (a) representative FACS plots. (b) CD7^ΔCAR7 and UCART7 effectively killed T-ALL blasts relative to CD7^ΔCAR19 and UCART19 (n=4). Data were compared using one-way ANOVA, followed by ad-hoc multiple comparisons for between-group differences, and a logarithm transformation was also performed. p values < 0.05 considered significant, *p ≤ 0.05, ** p ≤ 0.01, ***p≤ 0.001, **** p ≤ 0.0001.

Figure 6. UCART7 kills primary patient T-ALL blast in vivo without inducing xenogeneic GvHD

NSG were engrafted with 1×10⁶ PDX DFCI12 cells on day 0 followed by infusion of 2×10⁶ UCART7, UCART19, TRAC^Δ or WT T on day +1. Mice were assessed for GvHD, and blood and splenocytes analyzed by FACS 6 weeks post CAR-T injection (a) Representative flow cytometry plots of blood analysis presented to show both tumor and T cells (b) Percentage of tumor cells out of total mouse and human CD45 cells in the blood (WT n=6, UCART7 n=6, UCART19 n=6, TRAC^Δ n=4 or PDX only n=5) and spleens (n=3). Unlike TRAC^Δ T cells, WT T cells clear tumor through an alloreactive GvL effect (blood, p <0.0001; spleen, p = 0.0033). UCART7 is effective at clearing PDX relative to UCART19 (blood, p <0.0001; spleen p <0.0001). (c) Clinical GvHD scores, graded according to Cooke, et al (n=3 mean±s.d.). Unlike TRAC^Δ T cells, WT T cells induce GvHD. Representative images of mice following infusion of WT T cells, TRAC^Δ T cells, UCART7, and UCART19. *p ≤ 0.05, ** p ≤ 0.01, ***p≤ 0.001, **** p ≤ 0.0001.