Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells

Abstract

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19⁺ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19⁺ tumors in an in vivo mouse model.

Keywords: chimeric antigen receptor; gene editing; homology-directed repair.

Figure 1

Characterization of TRC1-2 Nuclease Activity in T Cells (A) Diagram of the TRC1-2 nuclease and recognition site within the TRAC locus. The TRC1-2 nuclease is a single-chain protein consisting of an N-terminal domain (N-domain) and C-terminal domain (C-domain) connected by a flexible linker. The recognition site consists of 9-bp half-sites recognized by each of the two nuclease domains, separated by a 4-bp central sequence. A broken white line in the recognition sequence denotes the overhangs generated following cleavage by the TRC1-2 nuclease. (B) A T7 endonuclease (T7E) assay was performed on mock-electroporated T cells and T cells treated with TRC1-2 nuclease on day 8 post-electroporation to confirm editing at the TRAC locus. (C) Flow cytometry staining of CD3 expression in CD4⁺ and CD8⁺ T cells on day 8 post-electroporation with TRC1-2 nuclease. Reduction of cell surface expression of CD3, a component of the TCR complex, is a functional marker of disruption of TCRα expression.

Figure 2

Stable Expression of GFP following Integration into the T Cell Genome by HDR (A) Diagrams of AAV vectors used to transduce cells. AAV:GFP is a vector enabling transient expression of GFP in transduced cells driven by a cytomegalovirus (CMV) promoter. AAV:TRAC:GFP contains a GFP transgene with expression driven by the JeT promoter and flanked by homology arms on the 5′ (L TRAC HA) and 3′ (R TRAC HA) sides to enable targeted integration. (B and C) T cells were mock-electroporated or electroporated with TRC1-2 mRNA and then mock transduced (No AAV) (B) or immediately transduced with AAV:GFP or AAV:TRAC:GFP at an MOI of 1e5 vector genomes (vg)/cell (C). Cells were cultured with 10 ng/mL IL-2 until 7 days post-electroporation, at which point they were cultured in medium containing 10 ng/mL IL-7 and 10 ng/mL IL-15 for the duration of the experiment. Flow cytometry was used to evaluate GFP expression 3, 11, and 21 days post-transduction.

Figure 3

Combining TRC1-2 and an AAV Donor Template Results in Highly Efficient HDR-Mediated Insertion of an Anti-CD19 CAR into the TRAC Locus and Simultaneous Disruption of TCR Expression (A) Diagram of the AAV vector used to transduce cells. AAV:TRAC:CAR contains a CD19 CAR transgene with expression driven by the JeT promoter and flanked by homology arms on the 5′ (L TRAC HA) and 3′ (R TRAC HA) sides to enable targeted integration. (B) Diagram of the PCR used to confirm CAR integration by amplification with one primer located within the CAR and one primer in TRAC outside of the homology arms at both the 5′ and 3′ ends to generate 1,872-bp and 1107-bp products, respectively. (C and D) T cells were mock-electroporated or electroporated with TRC1-2 mRNA and then immediately transduced with the indicated amounts of AAV:TRAC:CAR. (C) PCR was used to confirm the presence of the CAR transgene integrated in the TRAC locus on day 3 post-electroporation and transduction as outlined in (B). (D) CAR and CD3 expression were evaluated by flow cytometry on days 3 and 8 post-electroporation and transduction.

Figure 4

Confirmation of Targeted Insertion of the CAR Transgene by Digital Droplet PCR (A) Diagram showing digital PCR strategy. Two primer pairs and probes are used: one to detect the CAR transgene inserted in TRAC and another to detect FXN and serve as a reference standard for genomic DNA. (B) Activated CD3⁺ T cells were mock-electroporated, electroporated with TRC1-2 nuclease mRNA, or mock-electroporated and transduced with 25,000 vg/cell AAV:TRAC:CAR. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC 11 days post-electroporation/transduction. (C) Activated CD3⁺ T cells were electroporated with TRC1-2 mRNA and transduced with 50,000 vg/cell AAV:TRAC:CAR. CD3⁺ and CD3⁻ groups were magnetically separated on day 8 post-transduction. Cells were stained for CD3 expression and CAR expression in the pre-separation samples and CD3 expression post-separation to confirm purity. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC in pre-separation, CD3⁺, and CD3⁻ populations.

Figure 5

In Vitro Activity of Gene-Edited TCR Knockout Anti-CD19 CAR⁺ Cells (A) Cells were either mock-electroporated or electroporated with TRC1-2 mRNA (TRC1-2) and then immediately split into two groups; one was mock-transduced, and one was transduced with AAV:TRAC:CAR (AAV) at an MOI of 50,000 vg/cell. CD3⁺ cells were depleted from both TRC1-2 mRNA-treated groups post-electroporation or post-electroporation and transduction with AAV. T cells from all four groups were labeled with Cell Trace Violet and then cultured alone or co-cultured at a ratio of 1:1 with control CD19⁻ U937 cells or CD19⁺ Raji or Daudi cells. All cell lines were pre-treated with Mitomycin C to arrest cell growth and washed extensively prior to co-culture. After 3 days of co-culture in medium in the absence of exogenous cytokines, proliferation (dilution of Cell Trace Violet) was assessed by flow cytometry. (B and C) TCR knockout CAR⁺ T cells were produced by electroporation of T cells with TRC1-2 mRNA, followed immediately by transduction with AAV:TRAC:CAR at an MOI of 400,000. Cells were depleted of CD3⁺ cells 5 days post-electroporation and transduction. (B) TCR knockout CAR⁺ T incubated with Raji (CD19⁺), NALM-6 (CD19⁺), or U937 (CD19⁻) cells at various effector:target ratios. Cytolytic activity of the CAR T cells against the Raji, NALM-6, or U937 targets was measured by assessment of LDH release. Data are from n = 7 individual wells per sample per time point, mean ± SEM, ****p < 0.0001, *p < 0.05, two-way ANOVA with Tukey multiple comparisons test comparing Raji or NALM-6 to U937 samples at the same E:T ratio. (C) CAR T cells were incubated alone or co-cultured at a ratio of 10:1 with control CD19⁻ U937 or K562 cells or CD19⁺ Raji, Daudi, or IM-9 cells for 24 hr in medium in the absence of exogenous cytokines. Cytokine production and release were quantified from culture supernatants (n = 3, mean ± SEM).

Figure 6

Gene-Edited TCR Knockout Anti-CD19 CAR⁺ Cells Are Highly Efficacious in a Murine Model of Disseminated Lymphoma (A) Activated T cells were electroporated with TRC1-2 mRNA and transduced with AAV:TRAC:CAR at an MOI of 400,000 vg/cell and cultured for 5 days in the presence of IL-2. Five days post-transduction, cells were stained for expression of the CAR using a biotinylated CD19-Fc reagent and CD3, with TRC1-2-treated, mock-transduced cells used as a control for gating of CAR expression. CD3⁺ cells were then depleted. Enriched CD3⁻ cells were cultured for 3 additional days in the presence of IL-15 and IL-21 and then analyzed again by flow cytometry for CD3 and CAR expression and CD4 and CD8 expression. Total T cells (gated on CD4⁺ and CD8⁺) were further analyzed for CD62L and CD45RO expression. (B) 2 × 10⁵ Raji-ffluc cells were injected i.v. into 5- to 6-week-old female NSG mice on day 1. On day 4, mice were injected i.v. with 0.2 mL PBS (PBS), 0.2 mL of PBS containing 5 × 10⁶ gene-edited, mock-transduced TCR⁻ cells (TCR⁻ control), or 0.2 mL PBS containing 1 × 10⁶, 2.5 × 10⁶, or 5 × 10⁶ gene-edited, AAV-transduced TCR⁻ CAR⁺ (CAR⁺) T cells produced as described in (A). (C) Mice were monitored throughout the course of the study and euthanized according to pre-defined criteria. Kaplan-Meier survival curves are displayed. (D and E) On the indicated days, luciferin substrate (150 mg/kg in saline) was administered to live mice by intraperitoneal injection, mice were anesthetized, and luciferase activity measured using IVIS SpectrumCT (PerkinElmer). Bioluminescence images (D) are displayed for all mice in the study at the indicated time points, with bioluminescence values for each mouse displayed as individual curves plotted over time (E).