CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies

Abstract

Extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T-cell fratricide. CD7 is a transmembrane protein highly expressed in acute T-cell leukemia (T-ALL) and in a subset of peripheral T-cell lymphomas. Normal expression of CD7 is largely confined to T cells and natural killer (NK) cells, reducing the risk of off-target-organ toxicity. Here, we show that the expression of a CD7-specific CAR impaired expansion of transduced T cells because of residual CD7 expression and the ensuing fratricide. We demonstrate that targeted genomic disruption of the CD7 gene prevented this fratricide and enabled expansion of CD7 CAR T cells without compromising their cytotoxic function. CD7 CAR T cells produced robust cytotoxicity against malignant T-cell lines and primary tumors and were protective in a mouse xenograft model of T-ALL. Although CD7 CAR T cells were also toxic against unedited (CD7+) T and NK lymphocytes, we show that the CD7-edited T cells themselves can respond to viral peptides and therefore could be protective against pathogens. Hence, genomic disruption of a target antigen overcomes fratricide of CAR T cells and establishes the feasibility of using CD7 CAR T cells for the targeted therapy of T-cell malignancies.

© 2017 by The American Society of Hematology.

Figures

Figure 1.

T cells expressing CD7 CARs fail to expand. (A) Schematic diagram of control (ΔCAR) and full-length CD7 CAR constructs used in the study. (B) Surface expression of the CD7 CAR constructs on retrovirally transduced T cells measured by flow cytometry using anti-human IgG Fc antibodies on day 6 posttransduction. (C) Expansion of T cells transduced with truncated (ΔCAR) of full-length CD7 CAR in vitro for 14 days. (D) Viability of CD7 CAR T cells at days 2 and 6 posttransduction measured by flow cytometry. (E) Expression of CD7 in nontransduced or CD7 CAR-transduced activated T cells. A CD7-negative cell line Raji was used as a negative control. Data represent 2 to 5 independent experiments with n = 3 donors in each. Ctrl, control; Neg., negative; ns, not significant; NTR, nontransduced. *P < .05; **P < .001.

Figure 2.

Disruption of CD7 expression with CRISPR/Cas9 restores expansion of CD7 CAR T cells. (A) Representative histogram showing ablation of CD7 expression in T cells after electroporation with CRISPR/cas9 and CD7-specific gRNAs 3 days after electroporation. Numbers denote frequency of CD7-negative cells. T cells electroporated with Cas9 only were used as a negative control. (B) Downregulation of surface CD7 expression in T cells after electroporation with CRISPR/cas9 and gRNA-85. A CD7-negative cell line Raji was used as a negative control. (C) Schematic outline of the optimized protocol for generating CD7-knockout (CD7KO) CD7 CAR T cells. (D) Representative dot plots showing expression of CD7 and CD7 CAR in T cells generated with the optimized protocol. Numbers indicate percentage of cells in each quadrant. (E) Total expansion of CD7 CAR T cells with and without CD7 knockout after 14 days of in vitro culture. (F) Viability of CD7 CAR T cells with and without CD7 gene disruption measured at day 6 after transduction by flow cytometry. Lines denote individual donors. Data represent 3 independent experiments with 3 donors in each. h, hour. *P < .05.

Figure 3.

Loss of CD7 does not alter phenotype or effector function of CAR T cells. (A) T cells were electroporated with Cas9 complexed with CD7-specific or control (CD19 specific) gRNA and transduced with CD19 CAR. Representative dot plots show expression of CD7 and CD19 CAR in T cells electroporated with Cas9+gRNA 7 days posttransduction. Nontreated activated T cells were used as control. Numbers denote frequency of cells in corresponding quadrants. (B) Frequency of naïve-like cells (naïve; CCR7+CD45RA−), central memory (CM; CCR7+CD45RA−), effector memory (EM; CCR7−CD45RA−), and effector memory RA (EMRA; CCR7−CD45RA+) in CD19 CAR T cells assessed by flow cytometry on day 7 posttransduction. (C) Frequency of CD4+ and CD8+ CD19 CAR T cells 7 days posttransduction. (D) CD19 CAR T cells were incubated with CD19+ NALM6 cells, and production of TNFα and IFNγ in CD4+ cells was assessed by intracellular cytokine staining. Dot plots represent cytokine production in CD19 CAR T cells in the presence of NALM6 or in media alone. Summarized data from 3 donors are shown on the right. (E) Control or CD7KO CD19 CAR T cells or control nontransduced T cells were cocultured with GFP+ Raji cells at the effector-to-target ratio 1:1 for 72 hours. Dot plots show representative frequency of gated CAR T cells and GFP+ tumor cells at the end of coculture. Total numbers of live tumor cells (F) and CD19 CAR T cells (G) were counted by flow cytometry at 72 hours using counting beads. Lines denote individual donors. Data represent 2 independent experiments with n = 3 donors in each. *P < .05; **P < .01; ****P < .0001.

Figure 4.

Expanded CD7KOCD7 CAR T cells eradicate T-ALL and T lymphoma cell lines. (A) Surface expression of CD7 (solid histograms) in T-ALL and T-lymphoma cell lines measured by flow cytometry in comparison with isotype control (open histograms). (B) Tumor cell lines were labeled with eFluor670 and cocultured with CD7KO CD7 CAR T cells at the effector-to-target ratio 1:4 for 3 days. Dot plots show frequency of gated live tumor cells (CCRF) at the end of coculture. (C) Absolute counts of live tumor cells were measured by flow cytometry using counting beads at the end of coculture with CD7KO CD7 CAR T cells. CD7− cell line NALM6 was used as a negative control. Dashed lines represent the initial number of tumor cells on plating. (D) Representative dot plots showing intracellular cytokine staining for TNFα and IFNγ in CD7KO CD7 CAR T cells upon coculture with CCRF cells or media alone. (G) Mean frequencies of cytokine-positive CD4+ (top) and CD8+ (bottom) CD7KO CD7 CAR T cells on coculture with indicated CD7+ cell lines or media alone. Data represent 2 independent experiments with n = 3 donors in each. **P < .01; ***P < .001; ****P < .0001.

Figure 5.

Antitumor activity of CD7KOCD7 CAR T cells against primary T-ALL blasts. (A) Surface expression of CD7 in peripheral blasts in 4 T-ALL patient samples. (B) Representative dot plots showing production of TNFα and IFNγ by CD7 CAR T cells on coculture with allogeneic primary T-ALL blasts. (C) Mean frequencies of cytokine-positive CD7KO CD7 CAR T cells on coculture with individual T-ALL samples. (D) Peripheral blasts were freshly isolated from a T-ALL patient by leukapheresis, labeled with eFluor670 and cocultured with control or CD7KO CD7 CAR T cells for 48 hours. Dot plots show the frequency of live tumor cells at the end of coculture. (E) Absolute counts of live tumor cells and CD7KO CD7 CAR T cells quantified by flow cytometry at the end of coculture. Data represent 1 to 2 independent with n = 3 donors in each. *P < .05; **P < .01.

Figure 6.

CD7KOCD7 CAR T cells are cytotoxic against normal T cells and NK cells but can themselves respond to viral antigens. (A) Control or full-length CD7KO CD7 CAR T cells were cocultured with eFluor670-labeled autologous PBMC for 24 hours, and the frequency of T and NK cells was measured by flow cytometry. Dot plots show the frequency of gated CD19+ B cells (top) and CD56+CD3− NK cells and CD3+ T cells (bottom) at the end of coculture. (B) Total numbers of autologous B cells, NK cells, and T cells at the end of coculture with autologous CD7KO CD7 CAR T cells was quantified by flow cytometry using counting beads. Data represent 2 independent experiments with n = 3 donors in each. (C) Nontransduced, CD7KO and CD7KO CD7 CAR T cells were stimulated with pepmixes from cytomegalovirus, Epstein-Barr virus, and adenovirus, and the number of IFNγ+ spot-forming cells was measured by ELISPOT. Individual data from 3 donors are shown as a means of triplicate determinations. (D) CD4+ and CD8+ T cells were MACS-purified and separately assayed for IFNγ production in response to pepmixes. AdV, adenovirus; CMV, cytomegalovirus; EBV, Epstein-Barr virus; Neg., negative; Pos., positive; SFC, spot-forming cells. ***P < .001.

Figure 7.

CD7KOCD7 CAR T cells control the progression of systemic T-ALL in the mouse xenograft model. (A) Schematic outline of the experiment. NSG mice (n = 5 per group) were injected intravenously with 1 × 106 GFP-FFluc CCRF cells followed by a single intravenous injection of 2 × 106 of control or CD7KO CD7 CAR T cells 3 days later. (B) Tumor burden was monitored weekly by measuring luminescence using IVIS imaging. (C) Overall kinetics of systemic tumor progression in mice. Each line denotes an individual animal. (D) Kaplan-Meier survival curve of mice injected with control or CD7KO CD7 CAR T cells. (E) Relative frequency of CAR T cells (hCD45+ GFP−) in peripheral blood of mice in stable remission (top) or during early stages of relapse (bottom) on day 34 after CAR T-cell injection. (F) Surface expression of CD7 on relapsed CCRF tumor cells in peripheral blood of 3 relapsed mice (CCRF m1-m3, blue histograms) in comparison with control in vitro propagated cells (red histogram). Open histogram denotes CD7-negative cell line Raji. Data represent 2 independent experiments. (G) CCRF GFP+ blasts were isolated from spleens of 3 relapsed mice and cocultured with CD7KO control or CD7 CAR T cells from 3 donors for 24 hours at a 1:1 E:T ratio. The numbers of viable tumor cells were counted at the end of coculture by flow cytometry using counting beads. *P < .05; **P < .01, by log-rank Mantel-Cox test; ****P < .0001.