A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR

Abstract

Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR(+) T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR(+)TCR(neg) T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.

Figure 1

ZFN pairs targeting sites within genomic loci of TCR-α and β constant region. Each exon is shown by a block. Black blocks represent coding regions. Gray columns represent noncoding regions. One ZFN pair was designed to bind exon 1 of the TCR α constant region (TRAC) and another ZFN pair binds a conserved sequence on exon 1 of the TCR β constant regions 1 (TRBC1) and 2 (TRBC2). Underlined nucleotide sequences represent the intended binding sequence of each ZFN.

Figure 2

Disruption of the TCR αβ-CD3 complex in primary T cells. (A) Schematic presentation of ZFN transfer. A pair of ZFN-encoding mRNA was electrotransferred 6 days after stimulation of CAR^neg T cells. T cells were then cultured with 50 IU/mL of IL-2 and incubated at 30°C or 37°C 5%CO₂, as indicated. CD3 expression was analyzed by flow cytometry on day 7 to 9 after electroporation. (B) Down-regulation of CD3 after electrotransfer of mRNA encoding the TCR αβ targeted ZFNs. Day 9 after electrotransfer of the indicated doses of mRNA coding for TRAC or TRBC targeted ZFN pairs, TCR αβ-CD3 expression was analyzed by costaining for CD4, CD8, and CD3ϵ. Representative flow data at day 9 after ZFN electrotransfer is shown. Flow cytometry data are gated on cells excluding PI. Numbers in the lower right quadrant represent the percentage of CD3ϵ negative cells in T-cell populations. Top panels show CD3ϵ expression in T cells cultured at 37°C after ZFN transfer and bottom panels show CD3ϵ expression in T cells transiently cultured at 30°C from day 2 to 3 after ZFN transfer. (C) Surveyor nuclease assay to detect ZFN-mediated modification of TCR target sites in T cells. Arrows indicate the fragments produced by a surveyor nuclease digest of amplicons bearing a mismatch at the intended site of ZFN cleavage in the TRAC or TRBC1 locus, respectively. Lane headings indicate both the mRNA dose, specific ZFN pair delivered via electrotransfer, and temperature of incubation for the different samples. Numbers beneath each lane indicate the percentage of modified alleles in each sample.

Figure 3

TCR^neg T cells can be enriched by depletion of CD3ϵ⁺ T cells. (A) CD3 expression before and after depletion using CD3-specific paramagnetic beads. Flow cytometry reveals expression of CD3ϵ in CD4⁺ and CD8⁺ T cells 15 days after stimulation by OKT3-loaded aAPC (9 days after ZFN transfection). Numbers in the bottom-right quadrant represent the percentage of CD3ϵ^neg T cells. Representative results using in vitro numerically expanded T cells. (B) CD3ϵ^neg T cells can be further enriched by additional round of depletion with CD3-specific paramagnetic beads. Flow cytometry revealing expression of CD3ϵ in CD4⁺ and CD8⁺ T cells after 2 rounds of depletion of CD3ϵ^pos T cells. Numbers in the lower right quadrant represents the percentage of CD3ϵ negative cells in CD4⁺ and CD8⁺ T-cell populations. (C) Vβ repertoire analysis in T cells modified with ZFNs. The Vβ usage clonogram was analyzed by a panel of TCR-specific mAbs, costained with CD4 and CD8. Percentage of specific Vβ⁺ T-cell fractions within CD4 and CD8 gating is shown. The nomenclatures of Vβ repertoire shown are based on Arden et al. Representative data from 3 independent experiments are shown.

Figure 4

Elimination of TCR αβ-CD3 complex from CD19-specific CAR⁺ T cells. (A) Schematic of electrotransfer of mRNA coding for ZFN pairs in CAR⁺ T cells. mRNA species encoding the indicated ZFN pairs were electrotransferred into CAR⁺ T cells 2 days after stimulation with CD19⁺ aAPC. After electroporation, cells were maintained with 50 IU/mL of IL-2 and incubated for 2 days at 30°C 5%CO₂. CD3ϵ expression was analyzed 9 days after electroporation by flow cytometry. (B) Disruption of TCR αβ-CD3 complex expression after electrotransfer of mRNA encoding the TCR-specific ZFNs. Flow cytometry analysis of CD3ϵ expression in T cells 9 days after electrotransfer of mRNA species encoding the indicated ZFN pairs, gated on the PI-negative population. (C) Surveyor nuclease assay. Arrows indicate the fragments produced by a surveyor nuclease digest of amplicons bearing a mismatch at the intended site of ZFN cleavage in the TRAC or TRBC loci, respectively. Samples were analyzed 9 days after electroporation. The numbers at the bottom represent percentages of modified alleles in each sample.

Figure 5

Functional consequences of ZFN-mediated TCR knockout in CAR⁺ T cells. (A) Loss of responsiveness of TCR^neg CAR⁺ T cells to TCR stimulation. Dilution of PKH26 was measured 10 days after stimulation with aAPC loaded with OKT3 (top panel) or expressing CD19 (bottom panel). Flow cytometry data were gated on CAR⁺ T cells. Parental: CAR⁺ T cells without modification; no mRNA: mock electroporated CAR⁺ T cells; TRAC CD3^neg: CAR⁺ T cells electroporated with mRNA encoding ZFN pairs specific for TRAC, and depleted CD3^pos population; TRBC CD3^neg: CAR⁺ T cells electroporated with mRNA encoding ZFN pairs specific for TRBC, and depleted for CD3^pos population. (B) Redirected specificity of TCR^neg CAR⁺ T cells. Specific lysis by CAR⁺ T cells of an EL4 (mouse T-cell line) modified to express a truncated version of human CD19 (closed symbols) was measured by standard 4 hour ⁵¹Cr release assay. Specificity is shown by lack of lysis of CD19^neg (parental) EL4 cells (open symbols). CAR⁺ T cells were modified by ZFNs (TRAC and TRBC) or unmodified CAR⁺ T cells (parental and no mRNA). The error bars represent SD. (C) Cytotoxicity by TCR^neg CAR⁺ T cells against CD19⁺ primary B-cell tumors. Specific lysis by CAR⁺ T cells of B-cell malignances derived from patients was measured by 6 hour ⁵¹Cr release assay (effector: target ratio = 30:1). DLBCL: diffuse large B-cell lymphoma, CLL: chronic lymphocytic lymphoma, and MCL: mantle cell lymphoma. The error bars represent the standard deviation.

Figure 6

Propagation of TCR^neg CAR⁺ T cells on CD19 expressing aAPC. (A) Sustained proliferation of TCR^neg CAR⁺ T cells. CAR⁺ T cells with (TRAC and TRBC) or without (parental and no mRNA) TCR modification by ZFNs were stimulated with γ-irradiated CD19⁺ aAPCs every 2 weeks. Viable T cells were enumerated every 7 days and inferred total numbers were calculated. Representative data from 3 independent experiments are shown. (B) Analysis of TCR^neg CAR⁺ T cells after propagation. Flow cytometry analysis of CD3ϵ expression (top panel), αβTCR and γδTCR expression (middle panel), and subset analysis for memory pool (bottom panel) of TCR^negCAR⁺ T cells after 28 days of propagation on aAPCs. Numbers are percent expression for the quadrant. (C) Vβ repertoire analysis in TCR^neg CAR⁺ T cells after propagation on aAPCs. The Vβ usage clonogram was analyzed by a panel of TCR-specific mAbs, costained with CD4 and CD8. Percentage of identified Vβ⁺ T-cell fractions within CD4 and CD8 flow cytometry gates is shown. The nomenclatures of Vβ repertoire shown are based on Arden et al. Representative data from 3 independent experiments are shown.