A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies

Abstract

Options for targeted therapy of T-cell malignancies remain scarce. Recent clinical trials demonstrated that chimeric antigen receptors (CARs) can effectively redirect T lymphocytes to eradicate lymphoid malignancies of B-cell origin. However, T-lineage neoplasms remain a more challenging task for CAR T cells due to shared expression of most targetable surface antigens between normal and malignant T cells, potentially leading to fratricide of CAR T cells or profound immunodeficiency. Here, we report that T cells transduced with a CAR targeting CD5, a common surface marker of normal and neoplastic T cells, undergo only limited fratricide and can be expanded long-term ex vivo. These CD5 CAR T cells effectively eliminate malignant T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma lines in vitro and significantly inhibit disease progression in xenograft mouse models of T-ALL. These data support the therapeutic potential of CD5 CAR in patients with T-cell neoplasms.

Figure 1

CD5 CAR T cells expand and downregulate CD5. (A) Schematic structure of CD5 CAR and transduction efficiency of primary activated T cells. (B) Expansion of activated T cells transduced with either Ctrl CAR or CD5 CAR. Data denote mean ± SD from 4 donors. (C) Surface expression of CD5 on NT T cells or T cells transduced with Ctrl CAR or CD5 CAR at 7 days post-activation. (D) Relative expression of CD5 messenger RNA in NT activated T cells or T cells transduced with CD5 CAR at 7 days post-stimulation. Ctrl CAR, control CAR; NT, nontransduced.

Figure 2

CD5 CAR T cells produce limited fratricide and spare VSTs. (A) Autologous GFP⁺ T cells were mixed with T cells transduced with Ctrl CAR, truncated CD5 CAR (ΔCD5 CAR, without intracellular signaling domains), or full length CD5 CAR at 1:2 E:T ratio and cocultured for 7 days. Numbers in dot plots denote cell counts of gated GFP⁺ autologous T cells per well at indicated time points (left). Graph (right) summarizes data from 4 donors ± SD. (B) Phenotype of activated T cells 10 days after transduction with Ctrl CAR or CD5 CAR. Naïve T cells (T_NAIVE, CD45RA⁺ CCR7⁺), central memory (T_CM, CD45RA⁻ CCR7⁺), effector and effector-memory (T_EFF/T_EM, CD45RA⁻ CCR7⁻), and T_EMRA (CD45RA⁺ CCR7⁻) subsets are shown on representative dot plots with gating strategy (left) and as mean data from 3 donors (right). (C) Phenotype of autologous GFP⁺ T cells after coculture with Ctrl CAR- or CD5 CAR-transduced T cells for 24 hours. Data shown as mean average from 3 donors. (D) Autologous GFP⁺ T cells were cocultured with Ctrl CAR T or CD5 CAR T cells for 72 hours and purified by cell sorting. Frequency of T cells specific for cytomegalovirus, Epstein-Barr virus, and adenovirus among sorted cells was measured by IFN-γ ELISPOT.

Figure 3

CD5 CAR T cells eliminate malignant T cells in vitro. (A) Cytotoxicity of CD19 CAR- and CD5 CAR-transduced T cells against T-ALL and T-lymphoma cell lines was assessed in a 5-hour Cr release assay. CD19⁺CD5⁻ Raji cells (bottom right panel) were used as a negative control for CD5 CAR and positive control for CD19 CAR T cells. (B) Panel i: production of IFN-γ and TNF-α by CD4⁺ (top) and CD8⁺ (bottom) T cells transduced with CD19 CAR or CD5 CAR was measured by intracellular cytokine staining. Panel ii: bar graphs show mean ± SD from 3 donors. (C) Long-term coculture of CAR T cells with GFP⁺ target cell lines Jurkat, CCRF, and MOLT4 at an initial E:T ratio 1:4. Numbers in dot plots denote percentage of target GFP⁺ cells at indicated time points. (D) Sequential killing of GFP⁺ Jurkat cells by CD5 CAR T cells. Graph indicates number of target Jurkat cells per well at the beginning and the end of each cycle of cell killing. Data from 3 individual donors are shown.

Figure 4

Multiple mechanisms contribute to resistance to fratricide. (A) Inhibition of cytotoxicity of CD5 CAR T cells against autologous T cells and Jurkat cells by blocking either FasL (brefeldin A + aFasL), perforin (CMA + EGTA), or both pathways. Cell death was measured by Annexin V after 2 hours of coculture. (B) Expression of PI-9 protein in CD5 CAR T cells and malignant T-cell lines was measured by intracellular staining and flow cytometry (left). Bar graphs show MFI of PI-9 (right). (C) Expression of cathepsin B transcript in CD5 CAR T cells and target cell lines was measured by quantitative polymerase chain reaction. (D) Levels of Bcl-2 transcript in CD5 CAR T cells and target cell lines. (E) Protein expression of Bcl-2 was measured by intracellular staining and flow cytometry. (F) Bid expression in CD5 CAR T cells and target cell lines was measured by quantitative polymerase chain reaction. Error bars denote SD for 3 different T cell donors. MFI, mean fluorescence intensity.

Figure 5

CD5 CAR T cells recognize and kill primary T-ALL cells. (A) Production of IFN-g upon coculture with different primary T-ALL samples was assessed by intracellular cytokine staining. Numbers indicate percent or CAR⁺ T cells positive for IFN-g. (B) Production of IFN-g (left), TNFa (middle), and expression of CD107a (right) by CD5 CAR T cells upon mixing with thawed T-ALL blasts from 2 patients (T-ALL #295 and #315). Bar graphs depict frequency of cytokine-producing CD4⁺ and CD8⁺ T cells as average ± SEM from 4 donors. (C) Cytotoxicity of CD5 CAR T cells against fresh primary T-ALL blasts isolated from peripheral blood mononuclear cells of a T-ALL patient #394 was measured in a 5-hour Cr release assay. (D) Protein expression of PI-9 and (E) Bcl-2 in T-ALL blasts from donor #394 was measured by intracellular staining and flow cytometry. Expression histogram in Jurkat cells is shown with a dotted line (left). Bar graphs depict corresponding MFI compared with CD5 CAR T cells (mean ± SD from 3 donors) and Jurkat T-ALL cell line (right).

Figure 6

CD5 CAR T cells control progression of T-ALL in xenograft mouse models. (A) Jurkat-FFluc cells (3 × 10⁶ per mouse) were IV injected followed by IV injection of CAR T cells (10 × 10⁶ per mouse) on days 3 and 6 postimplantation. Tumor burden was assessed by IVIS imaging at indicated time points. (B) Kaplan–Meier survival curve; mice were euthanized after developing hind limb paralysis. (C) Eradication of systemic disease by CD5 CAR T cells. Mice were engrafted with Jurkat-FFluc cells, which established systemic disease by day 6. (D) Total luminescence from Jurkat cells recorded on day 6 (prior to CAR T-cell injection) and day 12. (E) Kaplan–Meier survival curve for eradication of systemic disease. (F) CCRF-CEM–FFluc cells (1 × 10⁶ per mouse) were IV injected followed by IV injection of CAR T cells (10 × 10⁶ per mouse) on day 3 and 6 post-implantation. Tumor burden was assessed by IVIS imaging at indicated time points. (G) Relative frequency CCRF-GFP in peripheral blood of mice on day 18 post-engraftment is shown on representative dot plots. (H) Kaplan–Meier survival curve for the CCRF model. P values are shown for each experiment.